Gene Therapy - Progress and Prospects Review Series

Each Gene Therapy Progress and Prospects review provides a succinct summary of the last 2 years of progress in a specific aspect of gene therapy research and will highlight prospects for the next 2 years. Written by the leaders in the field, the concise, targeted content will cover the most significant as well as the 'hottest' topics. From identifying potential target diseases to the vectors, technologies and systems being developed to detect efficiency, the whole range of the field will be covered. The Progress and Prospects format has been specifically designed to be reader-friendly, including a bulleted section that allows a quick snapshot of the Progress and Prospects as the expert authors see them.

Cancer gene therapy using tumour suppressor genesI A McNeish, S J Bell and N R Lemoine

Noninvasive imaging of gene therapy in living subjectsJ J Min and S S Gambhir

Gene therapy for severe combined immunodeficiencyH B Gaspar, S Howe and A J Thrasher

Parkinson's diseaseE A Burton, J C Glorioso and D J Fink

Gene therapy of lysosomal storage disordersS H Cheng and A E Smith

Adenoviral vectorsJ A St George

Gene therapy for the hemophiliasC E Walsh

Gene therapy in organ transplantationJ Bagley and J Iacomini

Naked DNA gene transfer and therapyH Herweijer and J A Wolff

Therapeutic angiogenesis for limb and myocardial ischemiaT A Khan, F W Sellke and R J Laham

Alpha-1 antitrypsinA A Stecenko and K L Brigham

Nonviral vectorsT Niidome and L Huang

Post-intervention vessel remodelingJ Rutanen, H Puhakka and S Ylä-Herttuala

Cystic fibrosisU Griesenbach, S Ferrari, D M Geddes and E W F W Alton

March 2004, Volume 11, Number 6, Pages 497-503

Table of contents    Previous  Article  Next   PDF

ReviewGene Therapy Progress and Prospects: cancer gene therapy using tumour suppressor genes

I A McNeish1, S J Bell1 and N R Lemoine1

1Cancer Research UK Molecular Oncology Unit, Imperial College

School of Medicine, Hammersmith Hospital, London, UK

Correspondence to: Dr IA McNeish, Cancer Research UK Molecular Oncology Unit, MRC Cyclotron Building, Hammersmith Hospital, London W12 0HS, UK

Abstract

Targeting tumour suppressor gene pathways is an attractive therapeutic strategy in cancer. Since the first clinical trial took place in 1996, at least 20 other trials have investigated the possibility of restoring p53 function, either alone or in combination with chemotherapy, but with limited success. Other recent clinical trials have sought to harness abnormalities in the p53 pathway to permit tumour-selective replication of adenoviral vectors such as dl1520 (Onyx-015). Other tumour suppressor genes, such as retinoblastoma (Rb) and PTEN (phosphatase, tensin homologue, deleted on chromosome 10), are the targets for imminent clinical trials, while microarray technologies are revealing multiple new genes that are potential targets for future gene therapy.

Gene Therapy (2004) 11, 497503. doi:10.1038/sj.gt.3302238Published online 5 February 2004

Keywords

tumour suppressor gene; p53; Rb; PTEN; cinical trial

In brief

Progress

Clinical trials of p53 gene replacement have had limited success Replicating adenoviral vectors targeting abnormal p53 function

have also had limited success in clinical trials Targeting the Rb pathway: Rb mutants may be more potent

tumour suppressors than wild-type Rb New oncolytic adenoviruses also target the Rb pathway The INK4ARF locus provides two potential targets for gene

therapy PTEN expression alters metastatic potential and reduces

neovascularization Multiple new tumour suppressor genes offer new therapeutic

possibilities, especially mda-7 and OPCML

Prospects

The ability to induce growth arrest and apoptosis in vitro does not guarantee clinical success.

Fuller understanding of downstream targets of p53 and Rb is

necessary. Clinical trials of second-generation oncolytic viruses targeting

Rb pathway will be eagerly awaited Combinations of tumour suppressor genes may offer new greater

therapeutic potential

New tumour suppressor genes will be discovered

Introduction

It has long been recognized that the development of invasive malignancy requires multiple genetic events, and modern technologies now suggest that tens, if not hundreds, of genes may be aberrantly expressed in malignant cells.1,2 In the last decade, studies on p53 replacement have dominated the literature and it remains the only tumour suppressor gene to be evaluated formally in clinical trials. Here, we review the progress that has been made in the past 2 years in the field of tumour suppressor gene therapy and the future prospects for utilizing pathways other than p53, including the well characterized, such as retinoblastoma (Rb) and PTEN (phosphatase, tensin homologue, deleted on chromosome 10), as well as those described more recently, such as melanoma differentiation associated gene-7 (mda-7) and opioid binding proteincell adhesion molecule-like gene (OPCML).

Clinical trials of p53 gene replacement have had limited success

After the promise of the first clinical trial of p53 gene replacement in non-small-cell lung carcinoma in 1996, those published in the past 2 years3,4,5,6,7 have been somewhat disappointing. In a neoadjuvant bladder carcinoma trial5, 12 patients received either intratumoral or intravesical injections of an adenovirus-encoding wild-type p53 (Ad p53) 3 days prior to radical cystectomy. Interestingly, transfection efficiency was much greater following intravesical administration and, overall, 711 (64%) evaluable patients (including 79 of the intravesical cohort) had evidence of transgene expression by vector-specific reverse-transcriptase PCR (RT-PCR), as well as some evidence of increased expression (both mRNA and protein) of p21Waf1Cip1, a p53 target gene. By contrast, in patients with locally advanced bladder cancer treated with intravesical Ad p53 at comparable doses,6 only 27 (29%) tumours demonstrated p53 transgene expression, with no detectable changes in the expression of either p21Waf1Cip1 or Bax. When comparing transgene expression in these two bladder cancer trials, it is possible that the larger instillation volume (120 ml) and the use of a transfection-enhancing agent in the neoadjuvant trial 5 contributed to the higher transfection rates.

In a phase I recurrent glioma trial, 12 patients received intratumoral Ad p53 at doses between 3 1010 to 3 1012 particles, followed by tumour resection, at which time more Ad p53 was injected into the tumour bed.7 Before Ad p53 injection, only one of eight assessed tumours was p53 positive (by immunohistochemistry), while 1012 showed nuclear p53 staining after injection and 78 showed positive staining for p21Waf1Cip1. However, the zone of transfected cells extended no more than 8 mm from the injection site and the median overall survival for the whole cohort was only 43 weeks.

In non-small-cell lung cancer, intratumoral injection of 7.5 1012 particles of Ad p53 every 21 or 28 days produced transgene expression in 1725 (68%) tumours. Patients also received chemotherapy (either carboplatin and paclitaxel or cisplatin and vinorelbine), but the frequency of overall tumour response was the same in Ad p53-injected lesions and noninjected lesions (52 versus 48%, respectively). However, there was a suggestion that the Ad p53-treated lesions reduced in size by a greater amount than the noninjected controls.

Ovarian cancer is traditionally thought to be an appealing target for clinical gene therapy because the disease tends to remain localized within the abdominal cavity, so that intraperitoneal vector delivery is a rational strategy. The extensive experience of p53 gene therapy in this disease culminated in a randomized phase III trial in which women with p53-null or p53 mutant tumours were randomized to chemotherapy alone or chemotherapy plus intraperitoneal Ad p53 following optimum debulking primary surgery. However, the first interim analysis indicated that not only did Ad p53 fail to improve effectiveness but was also associated with increased toxicity. As a result, the study has been abandoned (reported in Zeimet and Marth8).

Despite the limited clinical efficacy, some positive factors have emerged from these trials. Firstly, it is noticeable that the trials have been designed with credible scientific as well as clinical end points. Secondly, except for the experience in the ovarian phase III trial, of which few details are available,

treatment has largely been well tolerated with minimal toxicity. However, one must address why the trials were relatively unsuccessful and two broad possibilities emerge. Firstly, there remains the perennial problem of optimizing gene transfer. Improving gene transfer in the clinical setting with delivery of vectors to tumours disseminated throughout the body is a huge problem and lies outside the scope of this review. Secondly, there remains the possibility that p53 is the 'wrong' transgene. Although p53 mutations are found in many malignancies and defective p53 function may be causally linked to chemotherapy resistance,9 many aspects of p53 biology remain unanswered, especially what determines whether cells undergo apoptosis or cell cycle arrest in response to p53 activation.10 There is some evidence that low-level p53 expression, such as is likely to result from adenoviral gene transfer, causes cell cycle arrest rather than cell death. Also, the proapoptotic function of p53 depends upon transactivation of genes such as Bax, Apaf-1, Fas and PTEN, whose own expression or activity may be abnormal in tumour cells.11 It is known that mutant p53 can act in a dominant-negative manner in p53 tetramers,12 which could negate the effect of ectopically expressed wild-type protein. Finally, there is evidence that polymorphisms of the p53 gene (especially codon 72 arginine versus proline) can determine the responsiveness of tumours to chemo- and radiotherapy by influencing inhibition of p73.13 Only once all these issues have been addressed is there likely to be any advance in the field of p53 gene replacement.

Replicating adenoviral vectors targeting abnormal p53 function have also had limited success in clinical trials

The adenovirus E1B 55 kDa protein suppresses p53 function in infected cells and E1B 55K-deleted adenoviral vectors may be able to replicate within and cause cytolysis of tumours with defective p53 function. In the past 2 years, six separate phase III trials of such a virus (variously known as dl1520, Onyx-015 and CI-1042) have been published, in a range of tumour types, including colorectal,14,15 ovarian 16 and pancreatic carcinomas,17,18 and in patients with liver metastases from gastrointestinal malignancies.19 A total of 93 patients received doses of up to 2 1012 viral particles per injection with no objective clinical responses seen in any patient treated with dl1520 as a single agent. However, in combination with chemotherapy, some responses were seen; with 5-FU, eight patients with colorectal liver metastases demonstrated either partial or minor responses, at least five of whom had previously been refractory to 5-FU.14,19 In primary pancreatic carcinoma, two patients had partial responses in combination with gemcitabine.17

One complexity in analysing these results is that it is now apparent that cellular p53 status is not the only determinant of the replication of this virus.20 There have been many reports of replication within cells that are p53 wild type and there is contradictory evidence on the possible importance of the mdm-2hdm-2 inhibitor p14Arf.21,22 Similarly, E1B 55K almost certainly has functions in addition to p53 suppression, including modulating viral and cellular mRNA nuclear transport and stimulating late viral mRNA translation. Given this, the results of the trials and the uncertainties over p53 replacement, it seems unlikely that any further significant progress will be made with dl1520.

Targeting the Rb pathway: Rb mutants may be more potent tumour suppressors than wild-type Rb

Rb is the paradigmatic tumour suppressor gene, originally postulated in 1971. It is the target for transforming viral proteins such as HPV E7 and adenovirus E1A, inactivation of the Rb and p53 genes alone can induce malignancy in mouse models23 and abnormalities in the Rb pathway and the G1S checkpoint probably exist in all malignancies.24 The pathway has many components that are potential targets for therapy (see Figure 1). Upon growth stimulation, cyclin D expression increases and it forms complexes with cyclin-dependent kinase 4 (cdk4) or cdk6 and these complexes sequester the cdk inhibitor p27Kip1 from cyclin Ecdk2. The cyclin Dcdk4 and cyclin Ecdk2 complexes are now able to phosphorylate Rb and this phosphorylated form of Rb can no longer bind the E2F family of transcription factors, freeing E2F to transactivate the genes necessary for S-phase entry. The activity of cyclin Dcdk is also controlled by the INK4 family of inhibitors, of which p16INK4A is perhaps the best known.

Rather surprisingly, there have been many fewer studies on replacement of Rb family members than p53. Early reports suggested that the ability of Rb expression alone to inhibit tumour cell growth is variable and Rb expression may, paradoxically, inhibit p53-induced apoptosis.25 Rb phosphorylation mutants and truncated variants may have enhanced tumour suppressor function compared to the wild-type protein. One such derivative is Rb94, in which translation is initiated from a second AUG codon in the Rb mRNA and

which lacks the N-terminal 112 amino acids of the full-length protein. There is evidence that Rb94 has a longer half-life than Rb itself and remains in the hypophosphorylated form for extended periods. Two recent reports suggest that adenovirus-mediated Rb94 gene transfer can induce apoptosis in models of head and neck26 and bladder27 cancers, with minimal effects on nonimmortalized normal cells. Of note, Rb94 appears able to induce cell death regardless of the Rb status of tumours, unlike the full-length protein, which is not effective in tumours bearing wild-type Rb. One potential explanation for this is that Rb94, in addition to generating caspase-mediated apoptosis, appeared to induce cell cycle blockade at G2M (rather than G1) and also rapid telomere erosion with ensuing chromosomal instability. Although it had previously been reported that full-length Rb could inhibit telomerase, the cell cycle findings are novel and as yet unexplained.

Another Rb variant, Rb56, is also a C-terminal derivative. It contains the regions necessary for E2F binding and may be capable of inhibiting E2F-mediated transcription more efficiently than full-length Rb. Recent work on Rb56 has demonstrated the ability of a fusion protein, consisting of Rb56 and the DP-1 binding domains of E2F to induce cell cycle arrest in vascular smooth muscle cells and inhibit smooth muscle cell hyperplasia in response to intimal injury.28 Taken together, these reports suggest that Rb mutants and splice variants may be more potent tumour suppressors than Rb. Data on the potential of the other two members of the Rb family, p107 and p130, in gene therapy are very limited, but retrovirus-mediated transfer of the p130 gene can suppress the growth of lung carcinoma cells in vitro and in vivo.29

New oncolytic adenoviruses also target the Rb pathway

Following on from dl1520, a second generation of selectively replicating adenoviral vectors has now been developed. The viruses nearest to clinical trial specifically target Rb function. The adenoviral E1A protein contains two conserved regions, CR-1 (amino acids 3060) and CR-2 (amino acids 120127), the latter critical for binding to and inactivating Rb and whose deletion prevents formation of E1ARb complexes. Two similar mutants have been described recently; dl922947 is deleted in amino acids 122129,30 while 24 is deleted in amino acids 121128.31 Both have been assessed in in vitro and in vivo models of cancer and dl922947 is capable of replicating with much greater efficiency within a panel of tumour cell lines than dl1520, with minimal S-phase induction in quiescent nonimmortalized cells.30 Most recently, 24 has been modified further to include a RGD-4C peptide into the adenoviral fibre, which permits infection of cells independent of the normal coxsackie adenovirus receptor that is frequently expressed at very low levels on tumour cells.32 24-RGD is capable of lysing ovarian carcinoma and glioma cells in vitro, as well as extending the survival of mice-bearing xenografts of both tumour types.32,33 Of note, 24-RGD appeared to have a significantly greater cytopathic effect than 24 and its replication on normal human astrocytes was at least 3 log scales lower than a wild-type adenovirus.33 Clinical trials of both 24-RGD and dl922947 are imminent.

Further adenoviral mutants also explore targeting of the Rb pathway. Ar6pAE2fF34 and Onyx-41135 both have an E2F promoter in place of the adenoviral E1A promoter. In addition, Onyx-411 has a second E2F promoter to drive the expression of the E4 region and is also deleted in the E1A-CR-2 region, like dl922947. The rationale behind these modifications is that the E2F promoter is selectively activated in the presence of a defective Rb pathway and E4 gene products, especially E4 orf46, cooperate with E1A and E1B proteins to create a cellular environment that permits efficient expression of viral genes and thus productive viral infection.36 Both Ar6pAE2fF and Onyx-411 demonstrate tumour-specific replication with minimal effect upon normal cells, including proliferating epithelial cells, and both were more potent and tumour selective than dl1520.

INK4ARF locus provides two potential targets for gene therapy

The INK4ARF locus on chromosome 9 encodes two separate tumour suppressor genes from alternative reading frames, p16INK4A and p14Arf (also known as p19Arf in mice), which serve to highlight the close link between the p53 and Rb pathways.24 p16INK4A is a potent inhibitor of the cyclin Dcdk4 complex that phosphorylates Rb, while p14Arf inhibits hdm-2 (mdm-2 in mice), whose functions are to prevent p53-mediated transcription and to promote p53 ubiquitination. Homozygous deletions of the INK4ARF locus are seen in many malignancies especially melanoma.37

Adenoviral delivery of the p16INK4A gene is able to induce cell cycle arrest in vitro and, in cooperation with adenoviral p53 expression, induce apoptosis and inhibit tumour growth in vivo. It appears that p16INK4A may be a more effective inducer of apoptosis than other members of the INK4 family (p15INK4B, p18INK4C) or the Waf1Cip1 family (p21, p27). Interestingly, it appears that p16INK4A is able to induce apoptosis in cells lacking Rb, suggesting that it may have alternative functions. This has been reiterated by more recent work in which the effectiveness of p16INK4A and p53 gene delivery was compared in ovarian carcinoma models with varied p16INK4A and p53 status (wild type, null and mutant).38 In all cell lines, p16INK4A appears

as a more efficient inducer of growth arrest, but not apoptosis, than p53. In vivo, however, adenoviral p16INK4A (Ad p16) produces statistically greater survival in p16INK4A- and p53-null or wild-type models than Ad p53 alone or even Ad p16 and Ad p53 combined. Clearly, as has been mentioned above, the limited efficacy of Ad p53 could result from abnormalities in downstream effectors of p53-mediated apoptosis. However, the same may be true of pathways downstream of p16INK4A. Therefore, it remains possible that p16INK4A has additional functions, of which downregulation of vascular endothelial growth factor (VEGF) is one possibility. Other groups have recently demonstrated the efficacy of adenoviral p16INK4A delivery in lymphoma39 and glioma40 models, but one note of caution is necessary. There is evidence that ectopic overexpression of p16INK4A can produce resistance to some chemotherapy drugs, possibly by inducing G1 cell cycle arrest, as many chemotherapy drugs are at their most effect in S phase.41

In the past 2 years, more interest has focused on p14Arf. Several reports have demonstrated that adenoviral delivery of the p14Arf gene is capable of inducing cell cycle arrest and apoptosis in a wide variety of tumour models42,43,44,45,46,47 and can sensitize cells to chemotherapy.48 Initial reports suggested that intact p53 pathways were required for p14Arf-mediated cytotoxicity47,48,49 and that cotransfection with wild-type p53 could enhance the p14Arf effect.46 It now appears that p14Arf is capable of affecting proteins other than p53, such as E2F-1,50 HIF1 51 and topoisomerase I.52 Cluster analysis of gene expression patterns in mouse embryo fibroblasts indicates that p19ARF induces expression of both p53-dependent and -independent genes, the latter including members of the B-cell translocation family (BtgTob) that can inhibit proliferation in cells regardless of p53 status.43 Recently, p14Arf has shown itself capable of inducing apoptosis in p53- and Bax-null DU145 prostate carcinoma cells.42 In p53-null H358 lung carcinoma cells, p14Arf induces arrest in G2 phase, followed by apoptosis. This G2 arrest correlates with inhibition of CDC2, inactivation of CDC25C and induction of p21Waf1. Of note, p14Arf is capable of inducing tumour regression in H358 xenografts.53 One possible explanation for the discrepancy between these results and earlier studies that suggested p53 was an absolute requirement for p14Arf-mediated cell death is timing.49 In p53-null cells, it takes up to 6 days for G2 arrest to take place, in contrast to only 2448 h in p53-positive cells.53

PTEN expression alters metastatic potential and reduces neovascularization

PTEN, also known as MMAC1 and TEP-1, is a phosphatase whose importance as a tumour suppressor gene is being increasingly recognized. Although PTEN can dephosphorylate proteins such as focal adhesion kinase, its primary function is to degrade the products of phosphatidylinositol 3'-kinase (PI-3kinase) by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4,-bisphosphate at the 3' position. One of the main downstream targets of the PI-3kinase pathway is the kinase Akt, also known as PKB (protein kinase B), which, in turn, can activate a wide range of signals that lead to cell proliferation and decreased apoptosis (see Figure 2). Thus, the loss of PTEN activity, which is seen in up to 40% of all malignancies,54,55 can have diverse effects on cell growth and differentiation.

In the past 2 years, there have been a number of studies investigating PTEN gene replacement, many of which have focused on prostate cancer. In PTEN-null prostate cancer, expression of PTEN causes a decrease in Bcl-2 expression and sensitizes cells to doxorubicin and vincristine chemotherapy,56 and also sensitizes cells to death receptor-mediated apoptosis that could be overcome with Bcl-2 overexpression.57 In another prostate model, adenoviral PTEN (Ad PTEN) delivery to PC3 cells in vitro leads to G1 arrest, but not apoptosis.58 Interestingly, when the PC3 cells are transfected with Ad PTEN and then implanted orthotopically into mice, there is no reduction in tumorigenicity, but a significant reduction in the development of lymph node metastases, implying that PTEN may not be a critical regulator of tumour formation and growth, but a controller of dissemination. When Ad PTEN is injected directly into pre-existing prostate xenografts, there is no tumour regression, which further underlines this point.

By contrast, injection of Ad PTEN into bladder xenografts produced demonstrable tumour regression and induction of apoptosis, but only in PTEN-null UM-UC-3 tumours. In tumours that are PTEN wild type, Ad PTEN injection produced only transient growth inhibition.59 Alongside reduction in phosphorylated Akt expression, another observation from the UM-UC-3 tumours is a reduction in VEGF expression both in vitro and in vivo, the latter accompanied by a reduction in tumour vessel formation. VEGF is known to be an AktPTEN target,60 and neovascularization is a marker of transformation from low- to high-grade gliomas in humans with PTEN mutations seen almost exclusively in high-grade tumours. Further indication of the potential of PTEN to influence angiogenesis in glioma is shown with U87MG xenografts in mice. In the presence of PTEN expression, in vivo growth is reduced, with marked reduction in angiogenic activity.61 Even in the presence of proangiogenic signals such as constitutive EGFR activation andor p53 inactivation, Ad PTEN delivery to glioma xenografts in mice produces a marked reduction in tumour vascularity.62 Therefore, there may be a differential role for PTEN in different tumour types, reducing invasion and metastatic potential in some models and inhibiting tumour vascularization in others.

Multiple new tumour suppressor genes offer new therapeutic possibilities, especially mda-7 and OPCML

Mda-7 (also known as IL-24) is a member of the IL-10 family of cytokines and was first described as a potential tumour suppressor gene, when shown to be expressed on differentiated melanocytes but not melanoma cells. Subsequently, it was shown that adenoviral delivery of the mda-7 gene (Ad mda-7) is able to induce apoptosis in malignant cells but not normal epithelial cells in both melanoma63 and NSCLC.64

Work in the past 2 years has extended knowledge on this gene. Expression is downregulated in a wide variety of malignancies,65 while restoration of expression via Ad mda-7 can also induce growth arrest in vivo.66 The mechanisms via which mda-7 induces growth arrest and apoptosis are complex. It appears to upregulate the expression of TRAIL and its receptors DR45, which could sensitize tumour cells to death receptor-mediated apoptosis.66 There is also evidence that mda-7 can increase the expression of the RNA-dependent protein kinase PKR in some NSCLC cells.67 The normal role of PKR is to limit viral infection by inhibiting protein synthesis and hence block viral protein production, but it may also function as a regulator of tumorigenesis. Recently, microarray analysis suggests that Ad mda-7 transfection can alter expression of members of both the -catenin and PI3kinase signalling pathways in some breast and NSCLC cell lines.68 Curiously, this analysis was performed on the same NSCLC line (H1299) as had been studied previously,67 but PKR was not one of the genes whose expression was upregulated. Finally, several reports suggest that mda-7 may have a role in angiogenesis. Ad mda-7 is able to inhibit endothelial cell differentiation and reduce tumour vascularity in human lung cancer xenografts in mice,66 and purified mda-7 protein is capable of inhibiting endothelial cell differentiation and migration more effectively than endostatin.69

Finally, another potential tumour suppressor gene has been identified in ovarian cancer that may have therapeutic potential. OPCML is a member of the family of Ig domain-containing glycosylphosphatidylinositol-anchored cell adhesion molecules and its expression is completely absent in over 80% of ovarian carcinomas, including both established cell lines and primary tumours.70 Interestingly, the downregulation appears due mainly to CpG island methylation, and restoration of OPCML expression was able to impair ovarian carcinoma cell growth both in vitro and in vivo.70 Clearly, more work will be required to evaluate the pathways via which OPCML functions in ovarian carcinoma.

Conclusions and prospects

Although targeting tumour suppressor gene pathways is an attractive and logical strategy for cancer gene therapy, results from clinical trials have not mirrored the preclinical studies. Clearly, the ability to induce cell cycle arrest and apoptosis in vitro or growth arrest in mouse xenografts does not guarantee responses in clinical trials. Several specific hurdles must be overcome if such therapies are to become routine. Firstly, a greater understanding of the biology of the ubiquitous p53 and Rb tumour suppressor genes pathways is vital, especially an understanding of their own downstream targets and how these may be altered in malignancy. Secondly, other pathways need to be thoroughly evaluated, especially those that appear to be tumour-type specific. Surprisingly, little gene therapy work has been published on restoring well-known tumour suppressor genes such as BRCA1 in breast cancer and APC in colon cancer. The novel genes, OPCML and mda-7, may offer new disease-specific pathways to target in ovarian and melanomalung carcinoma, respectively. Thirdly, restoring tumour suppressor gene function alone may be insufficient and combination treatments, either with multiple genes (eg one disease-specific and one ubiquitous gene) or a tumour suppressor gene with an apoptosis inducer such as chemotherapy or activated caspases, may be required. However, one thing is certain: extending our knowledge of tumour suppressor genes and their normal roles must ultimately lead to improved therapies for all malignancies.

References

1 Rosenwald A et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 19371947. Article PubMed

2 Khan J et al. Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat

Med 2001; 7: 673679. Article PubMed

3 Buller RE et al. A phase III trial of rAdp53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Ther 2002; 9: 553566. Article PubMed

4 Schuler M et al. Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung cancer: results of a multicenter phase II study. J Clin Oncol 2001; 19: 17501758. PubMed

5 Kuball J et al. Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation. J Clin Oncol 2002; 20: 957965. Article PubMed

6 Pagliaro LC et al. Repeated intravesical instillations of an adenoviral vector in patients with locally advanced bladder cancer: a phase I study of p53 gene therapy. J Clin Oncol 2003; 21: 22472253. Article PubMed

7 Lang FF et al. Phase I trial of adenovirus-mediated p53 gene therapy for recurrent glioma: biological and clinical results. J Clin Oncol 2003; 21: 25082518. Article PubMed

8 Zeimet AG, Marth C. Why did p53 gene therapy fail in ovarian cancer? Lancet Oncol 2003; 4: 415422. Article PubMed

9 Reles A et al. Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer. Clin Cancer Res 2001; 7: 29842997. PubMed

10 Vousden KH, Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer 2002; 2: 594604. Article PubMed

11 Wolf BB et al. Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity. J Biol Chem 2001; 276: 3424434251. Article PubMed

12 de Vries A et al. Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function. Proc Natl Acad Sci USA 2002; 99: 29482953. Article PubMed

13 Bergamaschi D et al. p53 polymorphism influences response in cancer chemotherapy via modulation of p73-dependent apoptosis. Cancer Cell 2003; 3: 387402. PubMed

14 Reid T et al. Intra-arterial administration of a replication-selective adenovirus (dl1520) in patients with colorectal carcinoma metastatic to the liver: a phase I trial. Gene Therapy 2001; 8:

16181626. Article PubMed

15 Hamid O et al. Phase II trial of intravenous CI-1042 in patients with metastatic colorectal cancer. J Clin Oncol 2003; 21: 14981504. Article PubMed

16 Vasey PA et al. Phase I trial of intraperitoneal injection of the E1B-55-kDa-gene-deleted adenovirus ONYX-015 (dl1520) given on days 1 through 5 every 3 weeks in patients with recurrentrefractory epithelial ovarian cancer. J Clin Oncol 2002; 20: 15621569. Article PubMed

17 Hecht JR et al. A phase III trial of intratumoral endoscopic ultrasound injection of ONYX-015 with intravenous gemcitabine in unresectable pancreatic carcinoma. Clin Cancer Res 2003; 9: 555561. PubMed

18 Mulvihill S et al. Safety and feasibility of injection with an E1B-55 kDa gene-deleted, replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase I trial. Gene Therapy 2001; 8: 308315. Article PubMed

19 Reid T et al. Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral, immunologic, and clinical endpoints. Cancer Res 2002; 62: 60706079. PubMed

20 Dix BR et al. Does the antitumor adenovirus ONYX-015dl1520 selectively target cells defective in the p53 pathway? J Virol 2001; 75: 54435447. Article PubMed

21 Ries SJ et al. Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015). Nat Med 2000; 6: 11281133. Article PubMed

22 Edwards SJ et al. Evidence that replication of the antitumor adenovirus ONYX-015 is not controlled by the p53 and p14(ARF) tumor suppressor genes. J Virol 2002; 76: 1248312490. Article PubMed

23 Flesken-Nikitin A et al. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. Cancer Res 2003; 63: 34593463. PubMed

24 Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002; 2: 103112. Article PubMed

25 Ip SM et al. pRb-expressing adenovirus Ad5-Rb attenuates the p53-induced apoptosis in cervical cancer cell lines. Eur J Cancer 2001; 37: 24752483. Article PubMed

26 Li D et al. The role of adenovirus-mediated retinoblastoma 94 in the treatment of head and neck cancer. Cancer Res 2002; 62: 46374644. PubMed

27 Zhang X et al. Adenoviral-mediated retinoblastoma 94 produces rapid telomere erosion, chromosomal crisis and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells. Cancer Res 2003; 63: 760765. PubMed

28 Wills KN et al. Tissue-specific expression of an anti-proliferative hybrid transgene from the human smooth muscle alpha-actin promoter suppresses smooth muscle cell proliferation and neointima formation. Gene Therapy 2001; 8: 18471854. Article PubMed

29 Claudio PP et al. Mutations in the retinoblastoma-related gene RB2p130 in lung tumors and suppression of tumor growth in vivo by retrovirus- mediated gene transfer. Cancer Res 2000; 60: 372382. PubMed

30 Heise C et al. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med 2000; 6: 11341139. Article PubMed

31 Fueyo J et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000; 19: 212. Article PubMed

32 Bauerschmitz GJ et al. Treatment of ovarian cancer with a tropism modified oncolytic adenovirus. Cancer Res 2002; 62: 12661270. PubMed

33 Fueyo J et al. Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway. J Natl Cancer Inst 2003; 95: 652660. Article PubMed

34 Jakubczak JL et al. An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy. Cancer Res 2003; 63: 14901499. PubMed

35 Johnson L et al. Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell 2002; 1: 325337. Article PubMed

36 Branton PE, Roopchand DE. The role of adenovirus E4orf4 protein in viral replication and cell killing. Oncogene 2001; 20: 78557865. Article PubMed

37 Sharpless NE et al. Both products of the mouse Ink4aArf locus suppress melanoma formation in vivo. Oncogene 2003; 22: 50555059. Article PubMed

38 Modesitt SC et al. In vitro and in vivo adenovirus-mediated p53 and p16 tumor suppressor therapy in ovarian cancer. Clin Cancer Res 2001; 7: 17651772. PubMed

39 Turturro F et al. Effects of adenovirus-mediated expression of p27Kip1, p21Waf1 and p16INK4A in cell lines derived from t(2;5) anaplastic large cell lymphoma and Hodgkin's disease. Leukemia Lymphoma 2002; 43: 13231328. PubMed

40 Simon M et al. Conditional expression of the tumor suppressor p16 in a heterotopic glioblastoma model results in loss of pRB expression. J Neurooncol 2002; 60: 112. Article PubMed

41 Kawakami Y et al. Adenovirus-mediated p16 gene transfer changes the sensitivity to taxanes and Vinca alkaloids of human ovarian cancer cells. Anticancer Res 2001; 21: 25372545. PubMed

42 Hemmati PG et al. Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis. Oncogene 2002; 21: 31493161. Article PubMed

43 Kuo ML et al. Arf induces p53-dependent and -independent antiproliferative genes. Cancer Res 2003; 63: 10461053. PubMed

44 Lu W et al. Expression of p14ARF overcomes tumor resistance to p53. Cancer Res 2002; 62: 13051310. PubMed

45 Saadatmandi N et al. Growth suppression by a p14(ARF) exon 1beta adenovirus in human tumor cell lines of varying p53 and Rb status. Cancer Gene Ther 2002; 9: 830839. Article PubMed

46 Tango Y et al. Adenovirus-mediated p14ARF gene transfer cooperates with Ad5CMV-p53 to induce apoptosis in human cancer cells. Hum Gene Ther 2002; 13: 13731382. Article PubMed

47 Yang CT et al. Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells. J Natl Cancer Inst 2000; 92: 636641. Article PubMed

48 Deng X et al. Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin. Biochem Biophys Res Commun 2002; 296: 792798. Article PubMed

49 Weber HO et al. Human p14(ARF)-mediated cell cycle arrest strictly depends on intact p53 signaling pathways. Oncogene 2002;

21: 32073212. Article PubMed

50 Eymin B et al. Human ARF binds E2F1 and inhibits its transcriptional activity. Oncogene 2001; 20: 10331041. Article PubMed

51 Fatyol K, Szalay AA. The p14ARF tumor suppressor protein facilitates nucleolar sequestration of hypoxia-inducible factor-1alpha (HIF-1alpha ) and inhibits HIF-1-mediated transcription. J Biol Chem 2001; 276: 2842128429. Article PubMed

52 Karayan L et al. Human ARF protein interacts with topoisomerase I and stimulates its activity. Oncogene 2001; 20: 836848. Article PubMed

53 Eymin B et al. p14ARF induces G2 arrest and apoptosis independently of p53 leading to regression of tumours established in nude mice. Oncogene 2003; 22: 18221835. Article PubMed

54 Stahl JM et al. Loss of PTEN promotes tumor development in malignant melanoma. Cancer Res 2003; 63: 28812890. PubMed

55 Fernandez M, Eng C. The expanding role of PTEN in neoplasia: a molecule for all seasons? Clin Cancer Res 2002; 8: 16951698. PubMed

56 Huang H et al. PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression. J Biol Chem 2001; 276: 3883038836. Article PubMed

57 Yuan XJ, Whang YE. PTEN sensitizes prostate cancer cells to death receptor-mediated and drug-induced apoptosis through a FADD-dependent pathway. Oncogene 2002; 21: 319327. PubMed

58 Davies MA et al. Adenoviral-mediated expression of MMACPTEN inhibits proliferation and metastasis of human prostate cancer cells. Clin Cancer Res 2002; 8: 19041914. PubMed

59 Tanaka M, Grossman HB. In vivo gene therapy of human bladder cancer with PTEN suppresses tumor growth, downregulates phosphorylated Akt, and increases sensitivity to doxorubicin. Gene Therapy 2003; 10: 16361642. Article PubMed

60 Huang J, Kontos CD. PTEN modulates vascular endothelial growth factor-mediated signaling and angiogenic effects. J Biol Chem 2002; 277: 1076010766. Article PubMed

61 Wen S et al. PTEN controls tumor-induced angiogenesis. Proc Natl Acad Sci USA 2001; 98: 46224627. Article PubMed

62 Abe T et al. PTEN decreases in vivo vascularization of experimental gliomas in spite of proangiogenic stimuli. Cancer Res 2003; 63: 23002305. PubMed

63 Ekmekcioglu S et al. Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas. Int J Cancer 2001; 94: 5459. Article PubMed

64 Saeki T et al. Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther 2000; 7: 20512057. Article PubMed

65 Mhashilkar AM et al. Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med 2001; 7: 271282. PubMed

66 Saeki T et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene 2002; 21: 45584566. Article PubMed

67 Pataer A et al. Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res 2002; 62: 22392243. PubMed

68 Mhashilkar AM et al. MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells. Mol Ther 2003; 8: 207219. Article PubMed

69 Ramesh R et al. Melanoma differentiation-associated gene 7interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res 2003; 63: 51055113. PubMed

70 Sellar GC et al. OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer. Nat Genet 2003; 34: 337343. Article PubMed

Figures

Figure 1 Rb pathway. In response to a mitogenic stimulus, cyclin Dcdk4 complexes form and sequester p27 and other Waf1Cip1 family members. The cyclin Dcdk4 and cyclin Ecdk2 complexes are then free to phosphorylate Rb. This frees members of the E2F family to transactivate genes necessary for S-phase entry. p16INK4A inhibits cyclin Dcdk4 and thus prevents Rb phosphorylation.

Figure 2 The AktPTEN pathway. Oncogenic and mitogenic stimuli that activate PI3kinase can lead to Akt activation, either directly, via the actions of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4,-bisphosphate (PI(3,4)P2) on the plectrin homology (PH domain), or indirectly, via 3'-phosphoinositide-dependent kinase 1 (PDK1) and PDK2-mediated phosphorylation at positions T308 and S473. Activated Akt can then modulate multiple cellular pathways, leading to the inhibition of apoptosis and stimulation of cell growth. PTEN has intrinsic lipid phosphatase activity that removes the phosphate moeity from the 3' position of PIP3 and PI(3,4)P2, and thus counters the antiapoptotic and growth stimulatory activities of PI3kinase and Akt.

Received 22 September 2003; accepted 19 December 2003; published online 5 February 2004

March 2004, Volume 11, Number 6, Pages 497-503

January (2) 2004, Volume 11, Number 2, Pages 115-125

Table of contents    Previous  Article  Next   PDF

ReviewGene Therapy Progress and Prospects:Noninvasive imaging of gene therapy in living subjects

J J Min1 and S S Gambhir1

1Department of Radiology and Bio-X Program, Stanford University,

USA

Correspondence to: Dr SS Gambhir, Stanford University, James H Clark Center, 318 Campus Drive, East Wing, 1st Floor, Stanford, CA 94305-5427, USA

Abstract

Recent progress in the development of noninvasive imaging technologies should allow molecular imaging to play a major role in the field of gene therapy. These tools have recently been validated in gene therapy models for continuous quantitative monitoring of the location(s), magnitude, and time variation of gene delivery andor expression. This article reviews the use of radionuclide, magnetic resonance, and optical imaging technologies, as they have been used in imaging gene delivery and gene expression for gene therapy applications. The studies published to date lend support that noninvasive imaging tools will help to accelerate preclinical model validation, as well as allow for clinical monitoring of human gene therapy.

Gene Therapy (2004) 11, 115125. doi:10.1038/sj.gt.3302191

Keywords

molecular imaging; radionuclide imaging; magnetic resonance imaging (MRI); optical imaging; positron emission tomography (PET)

In brief

Progress

Diagnostic imaging technologies such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), optical imaging, and magnetic resonance imaging (MRI) have been used to noninvasively monitor transgene expression for gene therapy.

These in vivo molecular imaging technologies have been applied for gene therapy of cancer,

cardiovascular, neurological, musculoskeletal, hepatic and inherited diseases.

Radionuclide imaging (PETSPECT) has been used for imaging the distribution of radiolabeled vector, immune cell trafficking, and assessment of transgene expression using diverse reporter genes and reporter probes.

MRI-based molecular imaging technology provides excellent three-dimensional spatial resolution, but still requires a further improvement in sensitivity.

Bioluminescence imaging using cooled CCD camera shows a minimal background and a very high sensitivity, but lacks detailed tomographic information.

Therapeutic transgene expression can be monitored indirectly using a fusion approach, bicistronic approach, dual-promoter approach, bidirectional transcriptional approach, and two-vector administration approach.

Tumor-restricted gene expression through use of a tissue-specific promoter can be amplified using two-step transcriptional amplification (TSTA) strategies.

Prospects

The explosion in genetic engineering is expected to generate vectors with more robust gene transfer efficiency, bicistronicbidirectional molecular vectors and tissue-specific amplification techniques.

Continued refinement in the chemistry of molecular probe development should give rise to a new generation of molecular imaging probes with greater sensitivity and specificity.

Advances in detector technology and image reconstruction techniques for PET should help to produce a newer generation of imaging instruments with better spatial resolution, sensitivity, and significantly improved throughput time.

New optical technology development including 3-D tomography will expand gene therapy research using small animals, and can play an important role.

Multimodality reporter gene approaches should help gene therapy investigators to more readily move between the various imaging instruments, and should help to accelerate the testing of various preclinical models.

Introduction

Diagnostic imaging technologies have been used to try to monitor transgene expression for gene therapy using MRI, optical imaging, and radionuclide imaging techniques including PET and SPECT.1,2,3,4,5,6,7,8,9,10 Reporter genes with optical signatures (eg, fluorescence and bioluminescence) are low-cost alternatives for real-time analysis of gene expression in small animal models. Fluorescence imaging uses a fluorescent protein such as green fluorescent protein (GFP) that is excited with external illumination, and the emission is subsequently detected.11 GFP-encoding cDNA can easily be included in the myriad of therapeutic vectors and serve as a monitoring tool for gene therapy. Nevertheless, excitation and emission wavelengths in the range of 500 nm (eg, GFP) have limited penetration in mammalian tissues (15 mm). Since mammalian tissues absorb light that is used to excite these fluors, the tissues also fluoresce when excited at these wavelengths. The combination of absorption of specific signal and autofluorescence of tissues can result in poor signal-to-noise.3 Recently, red-shifted mutants of GFP (RFP) have been known to have an advantage over GFP that red light penetrates tissues more efficiently than green.12 Bioluminescent photoproteins such as luciferase have been used as reporter proteins in living animals.2,13,14 Firefly luciferase (FL) catalyzes D-luciferin to produce oxyluciferin in the presence of oxygen, cofactors, Mg+2, and ATP to produce light with peak at 562 nm. Recently, validated renilla luciferase (RL) catalyzes the oxidation of coelenterazine in the presence of oxygen, to generate a flash of blue luminescence with a peak wavelength at 482 nm. The advantage of bioluminescence is the minimal background noise, since luciferase is not a natural constituent of mammalian organisms. Bioluminescence-based approaches currently lack detailed tomographic information, and are limited to relatively small animals.10,15,16 A newer approach to fluorescence imaging of deeper structures uses fluorescence-mediated tomography.17 The subject is exposed in an imaging chamber to continuous wave or pulsed light from

different sources, and detectors arranged in a spatially defined order capture the emitted light. Mathematical processing of this information results in a reconstructed tomographic image. Fluorescence-mediated tomography is still in its infancy, requiring extensive mathematical validation prior to routine implementation. The advantage of MR for the imaging of gene expression is the excellent three-dimensional spatial resolution (tens of m range) at imaging. Owing to the indirect nature of enhancement produced by MR contrast agents, much higher concentrations of injected material, on the order of 10100 M concentrations and higher, are generally necessary to produce sufficient image contrast.2,3,6,7,10 The low sensitivity often entails long imaging times, and consequently slow data acquisition.7 While magnetic resonance spectroscopy (MRS) does not usually produce three-dimensional images, the technique does provide accurate measurement of gene expression in short time frames, and may eventually be harnessed to produce true spatial images, but at a much poorer spatial resolution than MRI. Radionuclide imaging with PET and SPECT has been used to characterize enzyme activity, receptortransporter status, and biodistribution of various radiolabeled substrates (tracers).8 For these reasons, it has made the most significant progress for imaging gene therapy by monitoring gene delivery and identifying therapeutic andor reporter gene expression in living subjects. While sensitivity in PET imaging is high (as little as 10-1110-12 M of tracer can be detected) and the speed of imaging is relatively rapid (min), these techniques lack micrometer spatial resolution (12 mm with micro-PET).3,7,9 An alternative approach to PET is SPECT imaging. While the sensitivity of the single-photon system is intrinsically about one to two orders of magnitude less than PET systems, the required radiopharmaceuticals and imaging systems are more readily available. Further details of the instrumentation available and relative advantages between the various types of imaging instrumentation may be found elsewhere.1,2,18

In the following sections, we will review several imaging technologies for monitoring gene delivery or transgene expression. Much of the current focus of molecular imaging in gene therapy is directed towards oncological applications; however, preliminary studies for cardiovascular and neurological applications have also been reported. All the applications are briefly described in the text and are also summarized in Tables 1 and 2.

Oncology

Radionuclide imaging

Imaging the vector utilized for gene delivery: To assess the efficiency of vector delivery, radionuclide imaging can be used to look at the distribution of the radiolabeled vector itself. The ideal gene therapy paradigm for brain tumors may consist of a combination of intratumoral injection and intra-arterial administration of vectors bearing therapeutic transgenes. In previous studies, herpes simplex virus (HSV) was radiolabeled with lipophilic 111In-oxine complex to be administered to intracerebral glioma-bearing rats. Intracarotid injection of radiolabeled HSV revealed the low efficiency of viral uptake in the tumor (0.10% of the injected dose per gram of tissue) at 1 h. When animals received virus injections stereotactically into the tumor, 71.335.0% of the total dose was found in the tumor at 24 h.19 An alternative labeling approach based on 99mTc-labeled recombinant adenovirus serotype 5 knob (Ad5K) has also been validated.3 Imaging data for the hepatic uptake studies were in agreement with the biodistribution determined by removing and measuring tissues. Recently, we have investigated the potential of labeling adenovirus with 99mTc or 124I to study the viral biodistribution and demonstrated stable labeling with 99mTc using an Isolink carbonyl kit20 and 124I using the standard iodogen method21 without loss of viability or infectivity. This allows imaging viral biodistribution and reporter gene expression.

Imaging of nonviral vector delivery has been studied for direct visualization of the distribution of double-stranded DNA (pCMV-GFP). The generic structure of the probe comprises three elements: (1) a peptide-based chelate that binds the 99mTc; (2) a positively charged linker for binding to DNA phosphodiester backbone; (3) an intercalating psoralen group.22 The formation of a stable complex between the probe and the DNA is achieved by ultraviolet crosslinking of psoralen and DNA. The feasibility of imaging the delivery of plasmid DNA was shown in normal and tumor-bearing animals using gamma camera imaging. The nonviral genetic vector pegylated liposome was also radiolabeled to monitor the gene delivery. A preliminary study demonstrated gamma camera images after intravenous infusion of 111InDTPA-labeled pegylated liposome revealed a high level of accumulation in the head and neck cancer lesions of patients,23 and therefore strongly supported the use of pegylated liposome as a targeting vehicle of therapeutic gene for solid tumors. The imaging of gene delivery in vivo could serve as a general predictor of the ability of the viral or nonviral vectors to reach the tissue(s) of interest. However, mere visualization of exogenous DNA accumulation at a certain site in the body might not correlate with the expression levels of desired gene product.

Imaging cell trafficking: In vivo imaging of cell trafficking has been investigated in many immunological and oncological studies to track the selective recruitment and time of arrival and departure of specific cells. These studies may be useful for investigators using various cell types transfected with gene(s) of

interest. Tracking the migration of cells in living small animals has been performed with radionuclide,24,25,26,27,28,29 MR,30,31 and both fluorescence32 and bioluminescence33 optical imaging. Radionuclide imaging has been used to monitor the trafficking of therapeutic cells in living subjects. For example, gene-modified ovarian cancer cells expressing HSV1-tk (PA1-STK) were radiolabeled with 99mTc and infused into the pleural space of patients with malignant pleural mesothelioma for suicide gene therapy. Radiolabeled PA1-STK cells adhere preferentially to intrapleural mesothelioma deposits, and are retained for at least 24 h in the chest cavity.24 Rat glioma (C6) cells and lymphocytes were radiolabeled using 64Cupyruvaldehyde-bis(N4-methylthiosemicarbazone) (64CuPTSM) and imaged with microPET in living nude mice.25 MicroPET images indicated trafficking of tail-vein-injected C6 cells to the lungs and liver, and transient splenic accumulation of lymphocytes at 3.33 h postinfusion. Reporter gene imaging is also being used to follow the specific localization and expansion of adoptively transferred immune T lymphocytes to the antigen-positive tumor and other sites within the animal.28,29 This approach can be used to assess the effects of immunomodulatory agents intended to potentiate the immune response to cancer, and can also be useful for the study of other cell-mediated immune responses, including autoimmunity.

Imaging therapeutic gene expression: Radionuclide imaging technologies, especially PET and SPECT, can play a significant role in imaging gene expression using diverse reporter genes and reporter probes. A reporter gene can be introduced into the target tissue(s) by various methods including viral and nonviral delivery vectors. If the promoter leads to transcription of the reporter gene, then translation of the imaging reporter gene mRNA leads to a protein product which can interact with the imaging reporter probe (administered in trace amounts for PETSPECT and sometimes referred to as a tracer). This interaction may be based on intracellular enzymatic conversion of the reporter probe with retention of the metabolite(s), or a receptorligand-based interaction. Examples of intracellular reporters include herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its mutant gene (HSV1-sr39tk).34 Note that HSV1-tk or HSV1-sr39tk refers to the genes and HSV1-TK or HSV1-sr39TK refers to the respective enzymes. Substrates that have been studied to date as PET reporter probes for HSV1-TK can be classified into two main categories pyrimidine nucleoside derivatives (eg, 5-iodo-2'-fluoro-2'-deoxy-1- -D-arabinofuranosyluracil (FIAU)) and acycloguanosine derivatives (eg, 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)), and have been studied in terms of sensitivity and specificity.35,36 Examples of reporters on or in the cell surface in the form of receptors include the dopamine 2 receptor (D2R),37 receptors for human type 2 somatostatin receptor (hSSTr2),38 and the sodium iodide symporter (NIS).39,40 Among these reporter genes, the HSV1-tk gene may alter the cellular behavior towards apoptosis by changes in the dNTP pool,41 and receptors may result in second messenger activation such as triggering of signal transduction pathways. For the D2R system, a mutant gene has been studied, which shows uncoupling of signal transduction but preservation of the affinity of receptor for tracer ligand.42

The reporter gene can itself be the therapeutic gene or can be coupled to the therapeutic gene.9 In the former approach, the reporter gene and therapeutic gene are one and the same. For example, anticancer gene therapy using HSV1-tk and ganciclovir (GCV) can be coupled with imaging of the accumulation of radiolabeled probes (18FFHBG or 131124IFIAU).35,36,43 Jacobs et al44 used 124IFIAU PET imaging of humans in a prospective gene-therapy trial of intratumorally infused liposomegene complex (LIPO-HSV1-tk), followed by GCV administration in five recurrent glioblastoma patients. These preliminary findings showed that 124IFIAU PET is feasible and that vector-mediated gene expression may predict a therapeutic effect. Recently, sodiumiodide symporter (NIS), which facilitates the uptake of iodide by thyroid follicular cells, is also being applied in radioiodide gene therapy.39,45 The conventional radioiodide or 99mTcpertechnetate scintigraphy has been used to directly monitor NIS expression.45,46,47,48 NIS has many advantages as an imaging reporter gene that includes wide availability of its substrates, well-understood metabolism, and clearance of these substrates in the body, and no likely interaction with the underlying cellular biochemistry. Since the iodine is not trapped, issue of efflux has to be optimized, but initial studies show significant promise. Further studies are needed with regard to NIS as an imaging reporter gene.

A second approach involves indirect imaging of therapeutic transgene expression using expression of a reporter gene, which is coupled to a therapeutic transgene of choice. This strategy requires proportional and constant co-expression of both the reporter gene and the therapeutic gene over a wide range of transgene expression levels. An advantage of this approach is that it provides for a much wider application of therapeutic transgene imaging, because various imaging reporter genes can be coupled to various therapeutic transgenes, while utilizing the same imaging probe each time. Linking the expression of a therapeutic gene to a reporter gene has been validated using PET through a variety of different molecular constructs. Examples include fusion approaches,49,50,51 bicistronic approaches using internal ribosomal entry site (IRES),52,53,54 dual-promoter approaches,55,56 a bidirectional transcriptional approach,57 and a two-vector administration approach.58 An advantage of the fusion gene approach is that the expression of the linked genes is absolutely coupled (unless the spacer between the two proteins is cleaved). However, the fusion protein does not always yield functional activity for both of the individual proteins andor may not localize in an appropriate subcellular compartment. Although the IRES sequence leads to proper translation of the downstream cistron from a bicistronic vector, translation from the IRES can be cell type specific and the magnitude of expression of the gene placed distal to the IRES is often attenuated.53 This can lead to a lower imaging sensitivity, and methods to improve this approach are currently under investigation.54 Two different genes expressed from distinct promoters within a single vector (dual-promoter approach) may avoid some of the attenuation and tissue-variation problems of an IRES-based approach.55 The potential problem of this approach is that the expression of the two genes may become uncoupled if the two identical promoters have different transcriptional activity based on where the vector integrates into the host genome or if a mutation occurs in one or both promoters that changes transcriptional activity. A bidirectional transcriptional approach utilizes a vector in which the therapeutic and the reporter genes are driven by each minimal CMV promoter induced by tetracycline-responsive element (TRE), transcribing separated mRNA from each gene which would then be translated into separate protein products.57 This system also avoids the attenuation and tissue-variation problems of the IRES-based approach, and may prove to be one of the most robust approaches developed to date. It is

limited by the fact that a fusion protein also needs to be co-expressed, but future vectors should be able to encode for both the fusion protein and the bidirectional transcriptional system on a single vector. Another way to image both the therapeutic and reporter genes can be through administration of two separate vectors, by cloning of the therapeutic and reporter genes in two different vectors, but driven by same promoter. This system may eliminate the need for making a new construct for each therapeutic gene, and has been validated through the expression of two PET reporter genes and showed good correlation.58 However, it is important to realize that the trans effects between two promoters can potentially affect reporter gene expression, and that not all cells may be equally infected with the PET reporter gene vector and therapeutic gene vector.

Tumor-restricted gene expression through tissue-specific transcriptional targeting is an attractive approach for gene therapy. It has been demonstrated that gene expression of highly efficient gene therapy vectors can be targeted to tumors using cell-type or tissue-type specific promoter elements. Approaches have also been developed to image the transcriptional regulation of the PET reporter gene in living animals.27,57,59 For example, transcriptional regulation at the level of induction has been reported in living mice using two PET imaging reporter genes (HSV1-tk and D2R) under the control of a tetracycline-inducible promoter.57 Using PET, correlative expression of both reporters after doxycycline treatment was measured in animals harboring stably transfected tumor. Low levels of imaging reporter gene expression owing to relatively weak tissue-specific promoters were circumvented with VP16 transactivating domains fused to yeast GAL4 DNA-binding domains. This two-step transcriptional amplification (TSTA) system was valuable in demonstrating PSE- or CEA-driven reporter gene expression in vivo using HSV1-sr39tk or HSV1-tk under the control of GAL4-responsive elements.60,61,62,63,64,65 Further studies are necessary to link this system to amplify both therapeutic and reporter gene expression. These approaches hold significant promise for the development of tissue-specific vectors with high levels of gene expression.

The lytic properties of herpes, adeno or Newcastle viruses are also being tailored for the destruction of various tumors. Although these oncolytic viruses are promising agents for treatment of malignancy due to their direct, selective toxicity for tumor cells, it is not easy to document viral replication in living subjects, as serial tissue sampling was required to assess viral titers over time. 124IFIAU PET scanning was capable of distinguishing a half-log difference between viral doses, and was able to document viral proliferation in xenograft tumor infected by oncolytic HSV infection.66,67 This PET data might provide a new direction for evaluating viral infection and proliferation in future clinical trials involving oncolytic viral therapy.

MRI and MRS

In spite of its high spatial resolution (10100 m), MR imaging has only a micromolar sensitivity to paramagnetic contrast agents, so robust signal-amplification strategies are necessary. Relatively large amounts of reporter probe (metals) have to accumulate in cells in order to lead to signal changes that can be imaged in the MR scanner. Targeted MR contrast agents in conjunction with biochemical amplification strategies have been preliminarily studied. Studies highlight the use of the transferrin receptor (Tf-R) as a potential transporter for accumulation of contrast agents, which consist of human holotransferrin covalently conjugated to low-molecular-weight dextrans coating monocrystalline iron oxide nanoparticles (Tf-MION)68,69 or crosslinked iron oxide (Tf-CLIO).70 The transferrin is recognized by the receptor and the entire particle is endocytosed by the cell, bringing in iron, a paramagnetic ion that acts as a contrast agent by affecting the T2 rate. It has been demonstrated that overexpression of the Tf-R in rat gliosarcoma cells in conjunction with the Tf-MION successfully increases the iron content in the cells, such that measurable MRI contrast can be achieved in living mice implanted with tumors. Further studies are under investigation to verify whether the Tf-R can be engineered to coexpress with a therapeutic gene,70 and are necessary to assess the effect of overexpressing Tf-R and increased level of iron on normal cellular function.

Contrast agents that change the magnetic properties at enzymatic hydrolysis have been used recently to image transgene expression. Contrast agents, (1-(2-( -galactopyranosyloxy)propyl)-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane)gadolinium(III) (EgadMe), are based on the framework of a clinical constrast agent, Gd(HP-DO3A), that has been modified with a carbohydrate 'cap' that blocks the access of water to the gadolinium. When access of water to gadolinium is blocked, signal enhancement by the contrast agent is turned 'off'. The cap is attached to the contrast agent through a -galactosidase-cleavable linker. Enzyme cleavage releases the cap and opens water access to the gadolinium ion, turning the contrast agent 'on'.71 Experiments in Xenopus embryo system revealed regions of higher intensity in the MR image correlates with regions expressing -galactosidase, and demonstrated the ability of MRI to detect gene expression in living animals.71 In this report, the contrast agent was introduced to animal systems by microinjection. Such application will require further refinement of the contrast agents in order to be delivered to cells without direct injection.

The ability of MRS to distinguish signals from chemically distinct compounds also offers the potential to measure gene expression. Conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil (5-FU) by yeast cytosine deaminase (yCD) could be observed and quantitated in colorectal tumor xenograft in living subjects, using 19F MRS.72 This study demonstrates the feasibility of using MRS to noninvasively monitor therapeutic transgene expression in tumors.

Optical imaging

A variety of different optical imaging approaches have been used to image gene expression in living

subjects in optically transparent organisms. Using adenovirus encoding GFP as a reporter gene and illumination in a light box by blue light fiber optics, noninvasive, whole-body, real-time fluorescence optical imaging of transgene expression was demonstrated in the major organs of nude mice including the brain and liver.11 GFP also has been used to image the transduction of lentivirus in nondividing hepatocytes in living nude mice.73

Bioluminescence imaging exploits the emission of visible photons at specific wavelengths, on the basis of energy-dependent reactions catalyzed by various luciferases.14,16 The emitted photons can be detected and counted using low-light CCDs or photon-counting cameras. The kinetics of gene expression after vector administration has been examined by injecting lentiviral vector encoding FL.74

To improve the activity and specificity of prostate-targeted gene expression, enhanced promoters were developed by multimerizing key regulatory elements in the prostate-specific antigen (PSA) enhancer and promoter. The resulting PSE-BC construct was incorporated into an adenovirus vector with FL (AdPSE-BC-luc), and applied in the prostate cancer model to identify metastases. Cooled CCD camera imaging localized and illuminated metastases in the lung and spine, and demonstrated the potential use of noninvasive imaging modality in therapeutic and diagnostic strategies for prostate cancer (Figure 1).75 This tissue-specific approach was also applied to the TSTA system, and revealed 20-fold higher levels of expression than the cytomegalovirus enhancer.60,63,65 These approaches were partly validated in a clinically relevant imaging modality such as micro-PET, as mentioned earlier;61,62 however, these approaches are still in need of further studies.

Different kinds of nonviral vectors have been evaluated in the small animal model using optical imaging techniques. The cationic lipid 1,2-dioleoyl-3-trimethyl ammonium-propane (DOTAP):cholesterol DNA liposome complexes76 and transferrin targeted DNApolyethylenimine (PEI) complexes77 were evaluated with FL reporter gene imaging.

Cardiovascular disease

Gene therapy holds much promise as a potential treatment for various cardiovascular diseases. These treatments include the prevention of restenosis after angioplasty, promotion of angiogenesis, and treatment of end-stage heart failure.78

Initial studies using autoradiography detected the uptake of 125IFIAU in rat myocardium transduced with adenoviral-mediated HSV1-tk reporter gene. The authors hypothesized that in vivo cardiac gene imaging is feasible and may eventually be used for the noninvasive monitoring of gene therapy.79 The first demonstration of cardiac reporter gene imaging in living subjects was reported with FL bioluminescence imaging. This study demonstrated the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats.80 The optical study was validated in a clinically relevant microPET (Figure 2). Rat myocardium was transduced with adenovirus carrying HSV1-sr39tk. The presence of 18FFHBG uptake in microPET images was confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay, while utilizing myocardial tissue samples.81 More detailed quantitative microPET studies have also been performed using the same model.82 Further studies are under investigation to construct bicistronic vectors containing both therapeutic (eg, VEGF) and PET reporter genes.83 Recently, we developed novel imaging approaches that allow noninvasive assessment of myocardial response to cell therapy using embryonic cardiomyoblasts expressing HSV1-sr39tk andor FL.84

The location, magnitude, and survival duration of the transplanted cells were monitored noninvasively using PET and bioluminescence optical imaging.

Neurological applications

Both the HSV1-tkHSV1-sr39tk and the D2R PET reporter genes are not optimal for most central nervous system application. All the reporter probes for HSV1-tkHSV1-sr39tk developed to date show very poor penetration across the bloodbrain barrier (BBB). Therefore, imaging with any of these reporter probes

may be useful only if some BBB disruption is present (eg, brain tumors). The D2R reporter gene with 18FFESP can work with brain-specific applications, but only if reporter gene expression is not in the vicinity of normal D2R expression in the striatum.

Recently, in Parkinson's disease research, aromatic-amino-dopadecarboxylase (AADC) gene was delivered to striatum as a therapeutic gene via an adeno-associate viral vector (AAV), and its expression was imaged by 18Ffluoro-m-tyrosine (18FFMT) PET. The AADC tracer 18FFMT is both decarboxylated and stored in the striatal neurons of monkeys providing a method for in vivo visualization of gene expression in the brain.85 For monitoring of stem cells after grafting by a noninvasive imaging technique, embryonic stem cells were labeled by a lipofection procedure with a MRI contrast agent SINEREM consisting of ultrasmall super-paramagnetic iron-oxide particles (USPIO). MRI at 78- m isotropic spatial resolution permitted the observation of the implanted cells in the corpus callosum and their migration to the ventricular walls.86

Miscellaneous diseases

Cellular radiolabeling techniques can be useful to assess the biodistribution of various cells for cellular therapy applications. Hepatocyte transplantation has been shown to provide temporary liver function in acute hepatic failure and various metabolic diseases. 111In-labeled hepatocytes were useful for the short-term noninvasive analysis of the biodistribution of transplanted hepatocytes.87 In another study, myoblasts were radiolabeled with 99mTcbis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt) and injected to assess the biodistribution of these cells in Duchenne-type human muscular dystrophy model.88

Creatine kinase (CK) and arginine kinase (AK) expression was monitored by using 31P MRS in the liver and skeletal muscle.89,90 Walter et al89 have introduced Drosophila melanogaster AK as a reporter gene for MRS detection of gene therapy of muscle diseases. This enzyme phosphorylates arginine, leading to the production of arginine phosphorylate, a unique metabolite that is not otherwise found in mammalian tissues and is readily detected by 31P MRS. CK is expressed primarily in the muscle (MM isoform) and brain (BB isoform), but is absent in the liver, kidney, and pancreas. In a recent study, syngeneic enzyme CK was used as a reporter gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver, the key target for gene therapy applications.90

Future prospects

Molecular imaging strategies associated with gene therapy will likely expand significantly over the next few years as gene therapy continues to evolve. The explosion in genetic engineering is expected to generate more robust gene-transfer vectors, both viral and nonviral. Bicistronicbidirectional vectors that can be easily modified, and tissue-specific amplification techniques, will likely expand. Continued refinements in the chemistry of molecular probe development should give rise to a new generation of probes with greater sensitivity and specificity. Advances in detector technology and image reconstruction techniques for PET should help to produce a newer generation of imaging instruments with better spatial resolution, sensitivity, and significantly improved throughput time. The optical technologies including fluorescence tomography may allow optical strategies to be the method of choice for small animal gene therapy research. Multimodality reporter gene approaches, so that gene therapy investigators may readily move between the various technologies, should help to also test various preclinical models. It will be very important for preclinical imaging strategies to be extended into patient studies where gene therapy is directly or indirectly monitored throughout the use of state-of-art imaging. Ultimately, all of the imaging technologies of gene delivery andor expression will be used as an early measure of successful gene therapy in patients.

References

1 Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2002; 2: 683693. Article PubMed

2 Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 2003; 17: 545580. Article PubMed

3 Bogdanov A, Weissleder R. In vivo imaging of gene delivery and expression. Trends Biotechnol 2002; 20: S11S18. Article PubMed

4 Blasberg R. PET imaging of gene expression. Eur J Cancer 2002; 38: 21372146. Article PubMed

5 Blasberg R. Imaging gene expression and endogenous molecular processes: molecular imaging. J Cereb Blood Flow Metab 2002; 22: 11571164. Article PubMed

6 Wunderbaldinger P, Bogdanov A, Weissleder R. New approaches for imaging in gene therapy. Eur J Radiol 2000; 34: 156165. Article PubMed

7 Weissleder R, Mahmood U. Molecular imaging. Radiology 2001; 219: 316333. PubMed

8 Nichol C, Kim EE. Molecular imaging and gene therapy. J Nucl Med 2001; 42: 13681374. PubMed

9 Gambhir SS et al. Imaging transgene expression with radionuclide imaging technologies. Neoplasia 2000; 2: 118138. PubMed

10 Allport JR, Weissleder R. In vivo imaging of gene and cell therapies. Exp Hematol 2001; 29: 12371246. Article PubMed

11 Yang M et al. Visualizing gene expression by whole-body fluorescence imaging. Proc Natl Acad Sci USA 2000; 97: 1227812282. Article PubMed

12 Jakobs S et al. EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Lett 2000; 479: 131135. Article PubMed

13 Contag CH, Ross BD. It's not just about anatomy: in vivo bioluminescence imaging as an eyepiece into biology. J Magn Reson Imaging 2002; 16: 378387. Article PubMed

14 Bhaumik S, Gambhir SS. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci USA 2002;

99: 377382. Article PubMed

15 Wu JC, Sundaresan G, Iyer M, Gambhir SS. Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. Mol Ther 2001; 4: 297306. Article PubMed

16 Contag CH, Jenkins D, Contag PR, Negrin RS. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2000; 2: 4152. PubMed

17 Ntziachristos V, Tung CH, Bremer C, Weissleder R. Fluorescence molecular tomography resolves protease activity in vivo. Nat Med 2002; 8: 757760. Article PubMed

18 Weissleder R. Scaling down imaging: molecular mapping of cancer in mice. Nat Rev Cancer 2002; 2: 1118. Article PubMed

19 Schellingerhout D, Rainov NG, Breakefield XO, Weissleder R. Quantitation of HSV mass distribution in a rodent brain tumor model. Gene Therapy 2000; 7: 16481655. Article PubMed

20 Murugesan S, Sundaresan G, Gambhir S. In vivo imaging of Tc-99m adenovirus and reporter gene expression in living subjects. Mol Imaging 2003; 2: 288.

21 Sundaresan G, Murugesan S, Gambhir S. MicroPET imaging of I-124 adenovirus biodistribution and optical imaging of reporter gene expression in living subjects. Mol Imaging 2003; 2: 302.

22 Bogdanov Jr A, Tung CH, Bredow S, Weissleder R. DNA binding chelates for nonviral gene delivery imaging. Gene Therapy 2001; 8: 515522. Article PubMed

23 Harrington KJ et al. Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes. Clin Cancer Res 2001; 7: 243254. PubMed

24 Harrison Jr LH et al. Gene-modified PA1-STK cells home to tumor sites in patients with malignant pleural mesothelioma. Ann Thorac Surg 2000; 70: 407411. Article PubMed

25 Adonai N et al. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci USA 2002; 99: 30303035. Article PubMed

26 Le LQ et al. Positron emission tomography imaging analysis of G2A as a negative modifier of lymphoid leukemogenesis initiated by the BCR-ABL oncogene. Cancer Cell 2002; 1: 381391. Article

PubMed

27 Ponomarev V et al. Imaging TCR-dependent NFAT-mediated T-cell activation with positron emission tomography in vivo. Neoplasia 2001; 3: 480488. PubMed

28 Dubey P et al. Quantitative imaging of the T cell antitumor response by positron-emission tomography. Proc Natl Acad Sci USA 2003; 100: 12321237. Article PubMed

29 Koehne G et al. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nat Biotechnol 2003; 21: 405413. Article PubMed

30 Modo M et al. Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging. Neuroimage 2002; 17: 803811. Article PubMed

31 Lewin M et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 2000; 18: 410414. Article PubMed

32 Weninger W, Manjunath N, Von Andrian UH. Migration and differentiation of CD8+ T cells. Immunol Rev 2002; 186: 221233. Article PubMed

33 Hardy J et al. Bioluminescence imaging of lymphocyte trafficking in vivo. Exp Hematol 2001; 29: 13531360. Article PubMed

34 Ray P et al. Monitoring gene therapy with reporter gene imaging. Semin Nucl Med 2001; 31: 312320. PubMed

35 Min JJ, Iyer M, Gambhir SS. Comparison of FHBG and FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection versus stable transfection. Eur J Nucl Med Mol Imaging 2003; 30: 15471560. Article PubMed

36 Tjuvajev JG et al. Comparison of radiolabeled nucleoside probes (FIAU, FHBG, and FHPG) for PET imaging of HSV1-tk gene expression. J Nucl Med 2002; 43: 10721083. PubMed

37 Herschman HR. Micro-PET imaging and small animal models of disease. Curr Opin Immunol 2003; 15: 378384. Article PubMed

38 Zinn KR, Chaudhuri TR. The type 2 human somatostatin receptor as a platform for reporter gene imaging. Eur J Nucl Med Mol Imaging 2002; 29: 388399. Article PubMed

39 Chung JK. Sodium iodide symporter: its role in nuclear medicine.

J Nucl Med 2002; 43: 11881200. PubMed

40 Spitzweg C, Morris JC. The sodium iodide symporter: its pathophysiological and therapeutic implications. Clin Endocrinol (Oxf) 2002; 57: 559574. Article PubMed

41 Haberkorn U, Altmann A, Eisenhut M. Functional genomics and proteomics the role of nuclear medicine. Eur J Nucl Med Mol Imaging 2002; 29: 115132. Article PubMed

42 Liang Q et al. Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction. Gene Therapy 2001; 8: 14901498. Article PubMed

43 Gambhir SS et al. A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography. Proc Natl Acad Sci USA 2000; 97: 27852790. Article PubMed

44 Jacobs A et al. Positron-emission tomography of vector-mediated gene expression in gene therapy for gliomas. Lancet 2001; 358: 727729. Article PubMed

45 Haberkorn U et al. Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose. Gene Therapy 2003; 10: 774780. Article PubMed

46 Cho JY et al. In vivo imaging and radioiodine therapy following sodium iodide symporter gene transfer in animal model of intracerebral gliomas. Gene Therapy 2002; 9: 11391145. Article PubMed

47 Min JJ et al. In vitro and in vivo characteristics of a human colon cancer cell line, SNU-C5N, expressing sodium-iodide symporter. Nucl Med Biol 2002; 29: 537545. Article PubMed

48 Groot-Wassink T et al. Adenovirus biodistribution and noninvasive imaging of gene expression in vivo by positron emission tomography using human sodiumiodide symporter as reporter gene. Hum Gene Ther 2002; 13: 17231735. PubMed

49 Ray P, Wu AM, Gambhir SS. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 2003; 63: 11601165. PubMed

50 Richard JC et al. Imaging pulmonary gene expression with

positron emission tomography (PET). Am J Respir Crit Care Med 2002; 167: 12571263. Article

51 Ray P, Min JJ, Gambhir S. Multimodality imaging of reporter gene expression in single cells and living mice using a novel triple fusion vector. J Nucl Med 2003; 44: 30. PubMed

52 Liang Q et al. Noninvasive, repetitive, quantitative measurement of gene expression from bicistronic message by positron emission tomography, following gene transfer with adenovirus. Mol Ther 2002; 6.

53 Yu Y et al. Quantification of target gene expression by imaging reporter gene expression in living animals. Nat Med 2000; 6: 933937. Article PubMed

54 Wang YL et al. New approaches for linking PET & therapeutic reporter gene expression for imaging gene therapy with increased sensitivity. J Nucl Med 2001; 42: 75.

55 Zinn KR et al. Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice. Radiology 2002; 223: 417425. PubMed

56 Hemminki A et al. In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst 2002; 94: 741749. Article PubMed

57 Sun X et al. Quantitative imaging of gene induction in living animals. Gene Therapy 2001; 8: 15721579. PubMed

58 Yaghoubi SS et al. Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors. Gene Therapy 2001; 8: 10721080. Article PubMed

59 Doubrovin M et al. Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo. Proc Natl Acad Sci USA 2001; 98: 93009305. Article PubMed

60 Iyer M et al. Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci USA 2001; 98: 1459514600. Article PubMed

61 Iyer M et al. Two step transcriptional amplification (TSTA) for enhancing HSV1-sr39tk reporter gene expression using a new single vector feedback strategy. J Nucl Med 2002; 43: 68.

62 Qiao J et al. Tumor-specific transcriptional targeting of suicide

gene therapy. Gene Therapy 2002; 9: 168175. Article PubMed

63 Zhang L et al. Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer. Mol Ther 2002; 5: 223232. Article PubMed

64 Zhang L et al. Interrogating androgen receptor function in recurrent prostate cancer. Cancer Res 2003; 63: 45524560. PubMed

65 Sato M et al. Optimization of adenoviral vectors to direct highly amplified prostate-specific expression for imaging and gene therapy. Mol Ther 2003; 8: 726737. Article PubMed

66 Bennett JJ et al. Positron emission tomography imaging for herpes virus infection: implications for oncolytic viral treatments of cancer. Nat Med 2001; 7: 859863. Article PubMed

67 Jacobs A et al. Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. Cancer Res 2001; 61: 29832995. PubMed

68 Weissleder R et al. In vivo magnetic resonance imaging of transgene expression. Nat Med 2000; 6: 351355. Article PubMed

69 Moore A et al. Human transferrin receptor gene as a marker gene for MR imaging. Radiology 2001; 221: 244250. PubMed

70 Ichikawa T et al. MRI of transgene expression: correlation to therapeutic gene expression. Neoplasia 2002; 4: 523530. PubMed

71 Louie AH et al. In vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 2000; 18: 321325. Article PubMed

72 Stegman LD et al. Diffusion MRI detects early events in the response of a glioma model to the yeast cytosine deaminase gene therapy strategy. Gene Therapy 2000; 7: 10051010. Article PubMed

73 Pfeifer A et al. Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging. Mol Ther 2001; 3: 319322. Article PubMed

74 Tsui LV et al. Production of human clotting Factor IX without toxicity in mice after vascular delivery of a lentiviral vector. Nat Biotechnol 2002; 20: 5357. Article PubMed

75 Adams JY et al. Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and

optical imaging. Nat Med 2002; 8: 891897. Article PubMed

76 Iyer M, Berenji M, Templeton NS, Gambhir SS. Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice. Mol Ther 2002; 6: 555562. Article PubMed

77 Hildebrandt IJ, Iyer M, Wagner E, Gambhir SS. Optical imaging of transferrin targeted PEIDNA complexes in living subjects. Gene Therapy 2003; 10: 758764. Article PubMed

78 Hajjar RJ, del Monte F, Matsui T, Rosenzweig A. Prospects for gene therapy for heart failure. Circ Res 2000; 86: 616621. PubMed

79 Bengel FM et al. Uptake of radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil in cardiac cells after adenoviral transfer of the herpesvirus thymidine kinase gene the cellular basis for cardiac gene imaging. Circulation 2000; 102: 948950. PubMed

80 Wu JC et al. Optical imaging of cardiac reporter gene expression in living rats. Circulation 2002; 105: 16311634. Article PubMed

81 Wu JC et al. Positron emission tomography imaging of cardiac reporter gene expression in living rats. Circulation 2002; 106: 180183. Article PubMed

82 Inubushi MWJ et al. PET reporter gene expression imaging in rat myocardium. Circulation 2002; 107: 326332. Article

83 Chen IY et al. MicroPET imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery. J Nucl Med 2003; 44: 1. PubMed

84 Wu JC et al. Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography. Circulation 2003; 108: 13021305. Article PubMed

85 Bankiewicz KS et al. Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach. Exp Neurol 2000; 164: 214. Article PubMed

86 Hoehn M et al. Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci USA 2002; 99: 1626716272. Article PubMed

87 Bohnen NI et al. Use of indium-111-labeled hepatocytes to determine the biodistribution of transplanted hepatocytes through

portal vein infusion. Clin Nucl Med 2000; 25: 447450. Article PubMed

88 Colombo FR et al. Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy. Nucl Med Biol 2001; 28: 935940. Article PubMed

89 Walter G, Barton ER, Sweeney HL. Noninvasive measurement of gene expression in skeletal muscle. Proc Natl Acad Sci USA 2000; 97: 51515155. Article PubMed

90 Auricchio A, Zhou R, Wilson JM, Glickson JD. In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene. Proc Natl Acad Sci USA 2001; 98: 52055210. Article PubMed

Figures

Figure 1 Visualization of primary and metastatic lesions of advanced human prostate cancer by a targeted gene transfer vector and optical bioluminescence imaging. PSE-BC was incorporated into an adenovirus vector encoding firefly luciferase (AdPSE-BC-luc) to make the prostate-specific gene transfer vector. A total of 1.8 109 infectious units of AdPSE-BC-luc were injected into 7-mm diameter tumors of severe combined immunodeficient (SCID) mice. (a) At 21 days postinjection, not only tumoral (arrowhead) but also low-magnitude extratumoral signals (arrows) were visible (200 RLUmin), emanating from the lower back and chest. The signals in the chest and lower back were found to originate from the lung and spine, respectively, after re-imaging the isolated organs of the mouse. (b) Histological analysis (H&E staining) of the spinal column revealed an elongated lesion embedded in the spinal musculature,

characterized by large pleomorphic nuclei and a high mitotic rate consistent with neoplasia (arrow); reproduced from Adams et al,65 with permission.

Figure 2 PET and optical bioluminescence imaging of cardiac reporter gene expression in living rats. Replication-defective adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 thymidine kinase (AdCMV-HSV1-sr39tk) and replication-defective adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (AdCMV-FL) viruses (1 109 pfu) were injected into the anterolateral wall of the left ventricular myocardium of a SpragueDawley rat. (a) The whole-body microPET image of a rat shows focal cardiac 18FFHBG activity at the site of intramyocardial AdCMV-HSV1-sr39tk injection. Liver 18FFHBG activity is also seen because of systemic adenoviral leakage with transduction of hepatocytes. Control rats injected with AdCMV-FL show no 18FFHBG activity in either of the cardiac of hepatic regions. Significant gut and bladder activities are seen for both the study and control rats, because of the route of 18FFHBG clearance. (b) Tomographic views of cardiac microPET images. The 13NNH3 (gray scale) images of perfusion are superimposed on 18FFHBG images (color scale), demonstrating HSV1-sr39tk reporter gene expression. (c) Bioluminescence image in rat myocardium transfected with AdCMV-FL emits significant bioluminescence due to cardiac firefly luciferase activity.

Tables

Table 1 Application of in vivo molecular imaging in gene therapy for oncology

Table 2 Application of in vivo molecular imaging in gene therapy for

cardiovascular, neurological and miscellaneous diseases

Received 15 January 2003; accepted 16 October 2003

January (2) 2004, Volume 11, Number 2, Pages 115-125

November 2003, Volume 10, Number 24, Pages 1999-2004

Table of contents    Previous  Article  Next   PDF

ReviewGene therapy progress and prospects: gene therapy for severe combined immunodeficiency

H B Gaspar1, S Howe1 and A J Thrasher1

1Molecular Immunology Unit, Institute of Child Health, London, UK

Correspondence to: Dr AJ Thrasher, Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK

Abstract

Severe combined immunodeficiencies have long been targeted as a group of disorders amenable to gene therapy because of their defined molecular biology and pathophysiology, and the prediction that corrected cells would have profound growth and survival advantage. Recently, several clinical studies have shown that conventional gene transfer technology can produce major beneficial therapeutic effects in these patients, but, as for all cellular and

pharmacological treatment approaches, with a finite potential for toxicity.

Gene Therapy (2003) 10, 19992004. doi:10.1038/sj.gt.3302150

Keywords

SCID-X1; ADA; PEG-ADA; LMO-2; insertional mutagenesis

In brief

Progress

Sustained correction of X-severe combined immunodeficiency (SCID) has been observed in clinical trials.

Cells in patients with adenosine deaminase (ADA) deficiency, treated by lymphocyte or stem cell gene therapy, persist and maintain transgene expression for many years.

Withdrawal of PEG-ADA from patients treated by lymphocyte gene therapy for ADA-deficient SCID results in enhanced immunological reconstitution.

Successful gene therapy for ADA-deficient SCID can be achieved in the absence of PEG-ADA and in combination with myelosuppression.

Animal models of RAG-2- and JAK-3-deficient SCID have been corrected using similar strategies.

Insertional mutagenesis has been observed in human studies, reinforcing the need to develop methods for optimization of protocol safety.

Prospects

As a group of well-defined disorders, SCIDs are amenable to treatment by gene therapy, and an extended range will enter clinical study over the next few years.

The durability of immunological reconstitution will determine the effectiveness and the need for repeated administration.

The absolute risk of clinically manifesting mutagenesis using retroviral vectors is at present unknown, and will only be determined by the extended observation of more patients, and by the development of clinically relevant models to test for toxicity in a rigorous way.

The characteristics of retroviral and lentiviral integration in human patients will be determined by mapping integration sites.

Risks of mutagenesis will be reduced by improved design of vectors that restrict promoter activity to relevant cell types and

within the domain of the therapeutic transgene. Development of targeted integration or of stable episomal vector

systems will also enhance safety.

Gene-repair strategies may have particular efficacy in SCID because of the profound growth and survival advantage conferred to corrected cells.

Sustained correction of X-severe combined immunodeficiency (SCID) has been observed in clinical trials

The most severe forms of primary immunodeficiency are known as SCIDs. These are a group of diseases in which T-lymphocyte development is invariably interrupted, and associated with diverse disorders of development and functionality of B lymphocytes, and natural killer (NK) cells.1 X-linked SCID (SCID-X1) accounts for approximately 5060% of all SCIDs, and is caused by mutations in the gene encoding the common cytokine receptor gamma chain ( c). This is a subunit of the cytokine receptor complex for interleukins (IL) 2, 4, 7, 9, 15 and 21.2 In the absence of c signaling, many aspects of immune cell development and function are compromised. The classical immunophenotype of SCID-X1 is the absence of T and NK cells, and persistence of dysfunctional B cells (T-B+NK-SCID). If a genotypically matched family donor is available, bone marrow transplantation is a highly successful procedure with a long-term survival rate of over 90%. The high survival rates are partly due to the fact that the absence of T and NK cells in SCID-X1 patients allows engraftment in the absence of myelosuppressive conditioning. For the majority of individuals, this is not possible, and the survival from mismatched family (usually parental donors) transplants is less good, and is associated with predictable toxicity.

Many incremental advances in gene transfer technology have recently been translated into successful gene therapy for SCID-X1.3 These have included the activation of cells with high concentrations of cytokines (thereby making them susceptible to gamma retrovirus vector-mediated gene transfer), and transduction in containers coated with a recombinant fibronectin fragment (RetroNectin) that is believed to facilitate the colocalization of the virus particle and the target cell.4,5 In the first landmark study, a conventional amphotropic retroviral vector encoding a c cDNA (regulated by Moloney murine leukaemia virus long-terminal repeat sequences) was used to transduce ex vivo autologous CD34+ cells (separated by conventional magnetic bead technology from a bone marrow harvest). The cells were reinfused into the patients in the absence of preconditioning. The results obtained from the first five patients have recently been reported in the scientific literature.6 To date, 10 infants in total have now been treated, with good immunological reconstitution in all but one, in whom the graft appears to have become sequestered in a pathologically enlarged spleen6,7 In nearly all patients, NK cells appeared between 2 and 4 weeks after infusion of cells, followed by new thymic T-lymphocyte emigrants at 1012 weeks. With some variation, the number and distribution of these T cells normalized rapidly (more rapidly than observed following haploidentical transplantation). They also appeared to function normally in terms of proliferative response to mitogens, T-cell receptor (TCR), and specific antigen stimulation, and to have a complex phenotypic and molecular diversity of TCR. Functionality of the humoral system was also restored, maybe not quite as effectively, but to a sufficient degree that discontinuation of immunoglobulin therapy was possible. Persistent long-term marking in myeloid cells (between 0.1 and 1%) suggests that long-lived stem or progenitor cells have been successfully transduced. More recently, we have initiated a similar study for the treatment of SCID-X1. The transduction protocols and vector are very similar although we have used a gibbonapeleukaemia virus (GALV) pseudotype, and conditions that obviate the requirement for foetal calf serum. Although the follow-up period for four children treated in our study is short, all have cleared viral infections, and immunological reconstitution has followed a similar pattern8 (Thrasher et al, manuscript in preparation).

The contribution to the initial burst of thymopoiesis from relatively late T-cell precursors in the original transduced CD34+ cell population versus that from cells earlier in the haematopoietic differentiation hierarchy, which have engrafted in the bone marrow, has not yet been determined. This may have important implications for the durability of immunological reconstitution, and for sustained production of new T cells. These issues may be resolved by longitudinal study of naive T-cell production, and by the isolation of common integration sites between myeloid and lymphoid populations. Ultimately, the longevity of functional reconstitution can only be determined by clinical monitoring, but it should also be feasible to repeat gene therapy on multiple occasions. Unknown, however, are the time frames within which this will be clinically effective, particularly bearing in mind that there may be age-related restrictions to the reinitiation of thymopoiesis in these patients.

Cells in patients with ADA deficiency treated by lymphocyte or stem cell gene therapy persist

and maintain transgene expression for many years

Deficiency of the purine salvage enzyme adenosine deaminase (ADA) accounts for approximately 1020% of all SCIDs. ADA catalyses the deamination of deoxy-adenosine (dAdo) and adenosine to deoxyinosine and inosine, respectively, and the lack of ADA leads to the build of the metabolites deoxyATP (dATP) and dAdo, which have profound effects on lymphocyte development and function through a number of cellular mechanisms. There is variation in the severity of the condition but most ADA patients have very low numbers of T and B lymphocytes. Bone marrow transplantation is highly successful in the genotypically matched setting, but human leucocyte antigen (HLA)-mismatched transplants have poor survival outcomes. An alternative modality of treatment is exogenous enzyme replacement with polyethylene glycol-conjugated bovine ADA, which as regular intramuscular injections can result in the correction of metabolic and immunological abnormalities, albeit only partially in a significant number of cases.9

The first human gene therapy studies were conducted on patients with ADA deficiency in the early 1990s. It is generally agreed that these initial studies were unsuccessful in correcting the immune defect in ADA-SCID. This was in part due to the continued use of PEG-ADA enzyme replacement therapy, which in itself improved the immune function but may also have blunted the survival advantage of gene-modified cells (discussed further below). However, a decade on, more detailed analysis of the patients originally treated does provide important information about the longevity and efficacy of gene transfer.10 In the first human clinical gene therapy study, two patients were treated following repeated gammaretroviral vector-mediated ADA gene transfer into stimulated peripheral blood lymphocytes. Patient 1 still shows over 15% of gene-marked cells in peripheral blood mononuclear cells (PBMCs) and ADA activity in PBMCs remains at 25% of the normal. The level of gene marking (0.1% of PBMCs) and ADA activity (<5% of normal) is considerably less in patient 2, which may reflect the smaller number of gene-transduced cells initially infused, but may also be due to the development of an immune response against the retroviral envelope and lipoprotein components of the foetal calf serum used for culturing the cells. However, these findings clearly demonstrate that human T cells have a lifespan greater than 10 years in the peripheral circulation, and also that transgenes regulated by gammaretroviral sequences continue to express in peripheral T cells and resist in vivo silencing. Molecular analysis of TCR diversity, combined with transgene integration analysis, reveals that within individual V family clones, each cell contains multiple unique integration sites. This suggests that, in the initial phase, cells were repeatedly sampled and transduced.

In a later study, umbilical cord blood CD34+ cells were harvested from antenatally diagnosed ADA-SCID patients, transduced and reinfused in the first week of life. In these patients, little clinical benefit was seen, and the level of gene marking was low (110% of T lymphocytes) in all the three children. Recent clonal integration analyses demonstrate that transgene-containing T lymphocytes are monoclonal or oligoclonal (with 15 different integration sites) more than 8 years after gene therapy, and that single prelymphoid clones contribute between 25 and 100% of genetically corrected lymphocytes.11 Marking in other lineages is consistently less than 1%. Again, the continuation of PEG-ADA throughout these early studies almost certainly compromised the efficient engraftment of transduced cells.

Withdrawal of PEG-ADA from patients treated by lymphocyte gene therapy for ADA-deficient SCID results in enhanced immunological reconstitution

Matched sibling donor transplants for ADA-SCID performed without conditioning result in rapid engraftment and persistence of donor T lymphocytes, strongly suggesting that T lymphocytes expressing ADA have a powerful proliferative and survival advantage. The failure of initial gene therapy studies to demonstrate the proliferation and expansion of gene-modified cells seemed to question this premise, although the continued administration of PEG-ADA in all these patients may have abrogated this advantage. Recently, one patient participating in another study demonstrated convincingly that a survival advantage for gene-transduced cells did exist in the absence of PEG-ADA. In this individual, who had received multiple infusions of autologous transduced peripheral blood lymphocytes (PBLs), and who had reached a plateau of 13% gene-transduced T cells, PEG-ADA-associated immune dysregulatory problems led to a gradual discontinuation of enzyme replacement. As the dose was decreased and finally stopped, the percentage of transduced T cells as assessed by PCR quantification increased, eventually reaching nearly 100% of all T lymphocytes.12 Absolute CD3+ T-cell counts also increased and stabilized at levels higher than prediscontinuation values. T-cell proliferative responses were also restored, and analysis of ADA metabolites showed a rise in intracellular PBL ADA activity.

The blunting effect of PEG-ADA may also be responsible for the disappointing observations made in a more recent study that employed enhanced vectors, and transduction conditions similar to those used for SCID-X1 trials.13 As before, transduced CD34+ cells were returned to the patients without conditioning and patients continued PEG-ADA treatment. ADA gene marking was only seen at low levels (in a range from 0.0001 to 3.6%). On the basis of previous observations, the investigators plan to withdraw PEG-ADA 1 year after treatment.

Successful gene therapy for ADA-deficient SCID can be achieved in the absence of PEG-ADA and in combination with myelosuppression

Results of a new study have clearly demonstrated that ADA-SCID can be successfully treated by gene therapy.14 In this protocol, CD34+ cells were transduced with an amphotropic gammaretroviral vector (originally used in PBL transduction studies) under current optimal conditions. Two important changes were incorporated into the protocol. Firstly, for economic reasons, patients were not commenced on PEG-ADA and, secondly, patients received a mild dose of conditioning (4 mgkg of busulphan as 2 mgkg on two successive days) prior to the return of gene-modified cells. A neutrophil and platelet nadir were seen at approximately 3 weeks, but neither child required blood product support. One patient is now 20 months. Over 2 years, postgene transfer one patient has normal numbers of peripheral T, B and NK cells. This patient has normal immunoglobulin production and is not receiving any prophylactic therapy. There has also been impressive correction of the metabolic defects, with dATP levels falling to 10% of that at diagnosis (comparable to that achieved following successful BMT). The second patient is now 12 months postgene therapy, but was an older child (approximately 2.5 years) at the time of treatment and also received a lower cell dosage. In this patient, recovery has been slower and the T-cell reconstitution at present is suboptimal, but significantly improved from pretransplant levels. There is evidence of some immunoglobulin production, but the patient remains on replacement therapy. A third child has been treated more recently using the same protocol,15 and is showing a recovery similar to that in patient 1. Molecular analysis of the first two patients shows a diverse TCR repertoire and an increase in TCR excision circle (TREC) levels, indicating the successful engraftment of prethymic progenitor populations. Lineage-specific transgene analysis by quantitative PCR shows high level marking in T, NK and B cells, and the persistence of gene-modified granulocytes, monocytes and megakaryocytes at levels between 5 and 20%, again suggesting that multipotent progenitors have engrafted. Somewhat surprising is the level to which marking has persisted in myeloid cells, particularly at the dosage of conditioning employed. This may suggest that a survival advantage is not restricted to lymphoid cells, and that it also extends to myeloid and haematopoietic multipotent progenitor cells.

The results from this study are extremely encouraging. At present, it is difficult to determine whether the success of the procedure is due to the lack of PEG-ADA or the use of nonmyeloablative conditioning, but it is likely that the combination is important. The key to correction of the metabolic abnormalities in ADA-SCID seems to be the delivery of large amounts of ADA enzyme, whether exogenously in the form of PEG-ADA or intracellularly as gene-modified cells. The use of conditioning may facilitate the initial engraftment of a greater number of gene-modified cells. Certainly, if immune function in these patients is sustained, and further patients show a similar safety profile and immune response, this strategy holds great promise for ADA-SCID and potentially other haematopoietic conditions.

Animal models of JAK-3- and RAG-2-deficient SCID have been corrected using similar strategies

The molecular basis of autosomal recessive T-B+NK-SCID is mutation of the receptor tyrosine kinase gene JAK-3. The dependence of c on signalling through JAK-3 is responsible for a clinical and immunological phenotype identical to that of SCID-X1, and the rationale for gene therapy is therefore similar. Correction of a murine model of JAK-3-deficient SCID has been achieved using both myelosuppresssive, and more relevant to clinical studies, conditioning-free protocols.16 Patients with mutations of the recombinase-activating genes RAG-1 and RAG-2 characteristically present with the absence of both B and T cells. Moloney-based gammaretroviral vectors have recently been shown to effectively reconstitute RAG-2-deficient mice, and in the absence of detectable toxicity, even though gene expression was not tightly regulated.17 One way to obviate the toxicity arising from dysregulated gene expression in any condition, and to achieve physiological activity, is to correct genetic mutations by gene repair or homologous recombination. Recently, it has been shown that RAG2-- mutant murine embryonic stem (ES) cells, repaired by standard homologous recombination technology, can be grown in vitro to provide sufficient haematopoietic progenitors for engraftment and correction of RAG-2 mutant mice.18 This is the first example of gene therapy combined with a therapeutic cloning strategy and, clearly, has important implications for future treatment of many genetic disorders.

Insertional mutagenesis has been observed in human studies, reinforcing the need to develop methods for optimization of protocol safety

For retroviruses, which depend on chromosomal integration for the stability of transduction, the most prominent safety concern has been for insertional mutagenicity.19 On the basis of numerous animal studies and over 300 clinical trials in which patients have received retroviral vectors, and from theoretical

considerations, the risk of clinically manifesting insertional mutagenesis has been judged to be low. However, in a recent murine HSC retroviral transduction study, insertion of the vector into the oncogene Evi-1 led to development of myeloid leukaemia.20 This has been followed by the reported development of uncontrolled clonal T-cell proliferation in two patients in the Paris SCID-XI clinical trial (Table 1).7,21 Having initially achieved successful immunological reconstitution, both developed lymphoproliferation approximately 3 years after the gene therapy procedure. In both patients, retroviral vector insertion into or near the LMO-2 proto-oncogene resulted in high-level expression of LMO-2 in the clones, almost certainly as a result of retroviral enhancer-mediated activation of transcription. Activation of LMO-2 is known to participate in human leukaemogenesis by chromosomal translocation, and results in the development of T-cell lymphoproliferation and leukaemia in mice, albeit with a long latency. It is therefore likely that other contributing factors are required for these events to manifest. At present, there is no evidence for the contribution of dysregulated c expression in lymphoid cells, although this remains a possibility and is being studied carefully. Cells with high proliferative potential, such as thymocytes, are also likely to be more susceptible to transformation following an insertional event than quiescent cells, if they acquire additional adverse mutations unrelated to the gene therapy itself. This increased risk cannot yet be quantified. The integration of the vector into LMO-2 in both cases strongly suggests that there is some preference for the survival of these clones or less likely, for integration at this site (Hacein-Bey-Abina et al, submitted for publication). The detailed molecular analysis of insertion events in patients undergoing gene therapy will greatly assist in the delineation of integration points within the genome, but is unlikely to be able to predict potential for mutagenesis unless recurrent hotspots associated with clinical disease become evident.22,23,24 In combination with conventional monitoring of lymphocyte numbers and distributions, longitudinal monitoring of integration sites will provide an important way of monitoring for pathological clonal expansions. The applicability of any novel therapy, including gene therapy, ultimately depends on the balance of risks against those of alternative treatments. The accurate characterization of adverse events, the utilization of protocols to test toxicity in a rigorous way, and the development of methods to minimize risks are therefore essential.

Future prospects for SCID

Recent clinical trials have shown that at least some forms of SCID can be effectively treated by gene therapy. Much, however, can be done to improve the efficiency and safety of current protocols. The design of vectors used for gene delivery is clearly important and modifications may be possible, which will limit the risks of mutagenesis, for example by the incorporation of DNA and RNA insulator sequences in integrating vectors, by the use of self-inactivating vectors or by targeting safe regions in the genome.25,26 Alternative vectors based on lentiviruses or foamy viruses that obviate prolonged ex vivo culture may allow the preservation of larger numbers of multipotential progenitor cells, but at the same time may produce higher numbers of insertion events in each cell.27,28,29,30,31 Methods to minimize the number of integration events per cell, and to limit the number of engrafting clones, for example by more stringent purification of stem cell (or defined target cell) populations, may therefore also be beneficial. Probably, the most straightforward way to improve safety is to dispense with the powerful viral enhancer sequences that can dysregulate gene expression over large chromatin domains. Lentiviral vectors, in particular, provide greater capacity for the incorporation of more complex and physiological regulatory sequences. The relative risk for each type of vector modification needs to be determined in clinically relevant animal-model systems, and the effectiveness of these models to predict side effects in humans will have to be validated. The development of homologous recombination or gene repair to correct mutations, or the construction of mitotically stable extrachromosomal vectors, would obviate many of these problems, but current technologies are inefficient.32 Once again, SCID may be a perfect initial target for this strategy, as even limited efficiency will be sufficient to provide clinical benefit. The future for gene therapy of SCID is exciting, but has been clouded by the occurrence of toxicity. As for all novel therapeutic modalities, increased understanding of mechanisms, and increased sophistication of technology will translate into even more effective and safe application. There is no better paradigm for this process than allogeneic bone marrow transplantation.

References

1 Fischer A. Primary immunodeficiency diseases: an experimental model for molecular medicine. Lancet 2001; 357: 18631869. Article PubMed

2 Leonard WJ. Cytokines and immunodeficiency diseases. Nat Rev Immunol 2001; 3: 200208.

3 Cavazzana-Calvo M et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669672. PubMed

4 Demaison C et al. A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukaemia virus-pseudotyped retroviral vector. Hum Gene Ther 2000; 11: 91100. PubMed

5 Hacein-Bey S et al. Optimization of retroviral gene transfer protocol to maintain the lymphoid potential of progenitor cells. Hum Gene Ther 2001; 12: 291301. PubMed

6 Hacein-Bey-Abina S et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 11851193. Article PubMed

7 Hacein-Bey-Abina S et al.. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 ((gamma) c deficiency). In press.

8 Thrasher AJ et al. Immune Recovery Following Retroviral Mediated Common Gamma Chain Gene Therapy for X-linked Severe Combined Immunodeficiency. American Society of Gene Therapy's Sixth Annual Meeting Executive Summaries, Vol. 36; 2003.

9 Hershfield M. ESID 2002.

10 Muul LM et al. Persistence and expression of the adenosine deaminase gene for twelve years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial. Blood 2002; 101: 25632569.

11 Schmidt M et al. Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nat Med 2003; 9: 463468. Article PubMed

12 Aiuti A et al. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 2002; 8: 423425. Article PubMed

13 Candotti F et al. Corrective gene transfer into bone marrow CD34+ cells for adenosine deaminase (ADA) deficiency: results in

four patients after one year of follow-up. Mol Ther 2003; 7: S448.

14 Aiuti A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 24102413. Article PubMed

15 Aiuti A. Correction of the immune and metabolic defect of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Mol Ther 2003; 7: S448.

16 Bunting KD, Lu T, Kelly PF, Sorrentino BP. Self-selection by genetically modified committed lymphocyte precursors reverses the phenotype of JAK3-deficient mice without myeloablation. Hum Gene Ther 2000; 11: 23532364. Article PubMed

17 Yates F et al. Gene therapy of RAG-2-- mice: sustained correction of the immunodeficiency. Blood 2002; 100: 39423949. PubMed

18 Rideout III WM et al. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 2002; 109: 1727. PubMed

19 Baum C et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003; 101: 20992114. Article PubMed

20 Li Z et al. Murine leukemia induced by retroviral gene marking. Science 2002; 296: 497. Article PubMed

21 Hacein-Bey-Abina S et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348: 255256. Article PubMed

22 Schroder AR et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 2002; 110: 521529. PubMed

23 Laufs S et al. Retroviral vector integration occurs into preferred genomic targets of human bone marrow repopulating cells. Blood 2002; 101: 21912198.

24 Wu X et al. Transcription start regions in the human genome are favored targets for MLV integration. Science 2003; 300: 17491751. Article PubMed

25 Burgess-Beusse B et al. The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci USA 2002; 99 (Suppl 4): 1643316437. Article PubMed

26 Olivares EC et al. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol 2002; 20: 11241128. Article PubMed

27 Follenzi A et al. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217222. Article PubMed

28 Glimm H, Oh IH, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their SG(2)M transit and do not reenter G(0). Blood 2000; 96: 41854193. PubMed

29 Demaison C et al. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency correction of immunodeficiency virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 2002; 13: 803813. Article PubMed

30 Josephson NC et al. Transduction of human NODSCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors. Proc Natl Acad Sci USA 2002; 99: 82958300. Article PubMed

31 Woods NB et al. Lentiviral vector transduction of NODSCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood 2002; 101: 12841289.

32 Van Craenenbroeck K, Vanhoenacker P, Haegeman G. Episomal vectors for gene expression in mammalian cells. Eur J Biochem 2000; 267: 56655678. Article PubMed

Tables

Table 1 Current clinical trials of gene therapy for SCID

November 2003, Volume 10, Number 24, Pages 1999-2004

September 2003, Volume 10, Number 20, Pages 1721-1727

Table of contents    Previous  Article  Next   PDF

ReviewGene therapy progress and prospects: Parkinson's disease

E A Burton1, J C Glorioso2 and D J Fink3,4

1Department of Clinical Neurology, University of Oxford, Radcliffe Infirmary, Oxford, UK

2Department of Molecular Genetics and Biochemistry, University of Pittsburgh, USA

3Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA

4Geriatric Research, Education and Clinical Center (GRECC), Pittsburgh VA Healthcare System, Pittsburgh, PA, USA

Correspondence to: Dr DJ Fink, Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA

Gene Therapy (2003) 10, 17211727. doi:10.1038/sj.gt.3302116

In brief

Progress

Inhibition of apoptosis by gene delivery prevents development of the disease phenotype in animal models

Transgene-mediated expression of glial cell line-derived neurotrophic factor may prevent progression after an initial insult, and may even be restorative in animal models

Combination of antiapoptotic and glial cell line-derived neurotrophic factor (GDNF) gene therapy protects dopaminergic neurons against a toxic insult, more effectively than either intervention alone

Transgene-mediated production of the inhibitory neurotransmitter -amino butyric acid (GABA) in neurons of the subthalamic nucleus ameliorates the behavioral phenotype and may be neuroprotective, in an animal model

Delivery of transgenes encoding enzymes involved in dopamine biosynthesis enhances dopamine production in the striatum

Stem cells may be driven to differentiate into functioning dopaminergic cells by genetic modification

Isolation of genes implicated in rare genetic forms of Parkinson's disease (PD) has allowed generation of new animal models and identification of new candidate targets for intervention

One human gene therapy trial is about to commence in PD The optimal vector remains uncertain

Prospects

Development of presymptomatic diagnostic tests will facilitate neuroprotective studies

Better understanding of the pathogenesis may lead to the development of improved animal models that more closely resemble the human disease

Studies may broaden their scope to include the important nonmotor manifestations of PD

Further characterization of ES and adult stem cell populations will establish whether ex vivo transduction can drive their differentiation into dopaminergic neurons in a therapeutically useful way

Well-designed clinical trials for PD gene therapy may take their lead from cell transplantation trials

PD is an attractive target for central nervous system (CNS) gene therapy for several reasons. First, the pathology in early PD is, to a first approximation, limited to dopaminergic neurons projecting from the substantia nigra pars compacta (SNc) to the caudate aputamenl, so that localized gene delivery is a viable therapeutic strategy. Second, the neurochemical deficits and the functional consequences of dopaminergic cell loss on local basal ganglia circuitry are well characterized; gene transfer can be designed either to improve cell survival, or to modify functional activity in the damaged basal ganglia circuitry (summarized in Figures 1 and 2). Third, PD is common and disabling despite treatment; no current intervention is uniformly accepted as altering the natural history of disease progression; hence, development of novel therapeutics is desirable. A variety of therapeutic transgenes has been delivered in experimental models of PD, using a number of different vectors. In this article, we survey the literature from 2000 to 2003, and briefly review recent progress in the development of gene transfer strategies for treating PD.

Inhibition of apoptosis by gene delivery prevents development of the disease phenotype in animal models

The most frequently studied animal models of PD involve chemical induction of lesions to the SNc of rodents, using the toxins 6-hydroxydopamine (6-OHDA) or 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In each case, the toxic insult leads to a pathogenic cascade, resulting in apoptotic cell death of dopaminergic (DA) neurons. Local expression of apoptotic inhibitors, in the SNc of 6-OHDA- and MPTP-lesioned animals, prevents both the loss of DA neurons and the development of a PD-like phenotype, following the chemical insult. Our previous work showed that expression of bcl-2 from a nonreplicating herpes simplex virus (HSV)-based vector within the SNc protected dopaminergic cells from apoptosis following administration of 6-OHDA, with the resulting preservation of motor function. These findings have now been extended to other experimental paradigms. The human neuronal apoptosis inhibitor protein (NAIP), delivered to the SN of rats by intrastriatal inoculation of a recombinant type 5 adenovirus (Ad) vector,1 protected the animals against 6-OHDA toxicity administered 1 week after the vector, as measured by immunohistochemistry and motor phenotype studies up to 28 days later. Prevention of the apoptotic protease-activating factor-1 (apaf-1)-dependent activation of caspase 9, using a recombinant adenoassociated virus (AAV) vector expressing a dominant-negative apaf-1 derivative,2 protected mice against the effects of intra-peritoneal injections of MPTP administered 2 weeks after vector inoculation. At 1 week following MPTP intoxication, neuronal survival was 75% on the transduced side of the brain compared with 25% on the contralateral control.

These protein inhibitors of apoptosis must be expressed intracellularly in order to block the apoptotic cascade, so that gene transfer is uniquely suited to this approach. However, the pathogenic trigger for PD in humans is unknown, and the role of apoptosis in dopaminergic cell death in naturally occurring PD is controversial. To test the hypothesis that inhibition of apoptosis within the SNc of humans could arrest or slow the progression of PD, it would be necessary to identify and treat patients early in the course of their illness, or to generate better animal models that more directly model the pathogenesis of human PD. Finally, the effects of prolonged expression of antiapoptotic factors in the brain have not been fully explored; some of these proteins are proto-oncogene products, and there might be important issues regarding their safety.

Transgene-mediated expression of glial cell line-derived neurotrophic factor may prevent progression after an initial insult, and may even be restorative in animal models

GDNF was originally isolated by virtue of its trophic effects on dopaminergic cells in culture. It was subsequently demonstrated that GDNF could promote the survival of dopaminergic neurons in the face of a toxic insult in both rat and monkey models of PD, and a putative role for GDNF as a neuroprotective agent in PD was suggested. However, the delivery of potent biologically active peptides with short half-lives to the brain is difficult, and attempts at intraventricular infusion of recombinant GDNF were disappointing. The alternatives include continuous intraparenchymal infusion of recombinant GDNF,3 transplantation of genetically modified cells as production sites for GDNF,4,5,6,7,8 or vector-mediated transfer of the gene encoding GDNF into the CNS parenchyma.

Several different vector systems have been successfully used to effect GDNF gene transfer in experimental models, including lentivirus,9,10,11 adenovirus, adeno-associated virus12,13,14 and herpes simplex virus.15 Various points emerge from these studies, which differ mainly in the details of the experimental paradigms used. First, robust GDNF expression can be seen after gene transfer into the striatum or substantia nigra, and anterograde transport of GDNF to nerve terminals after transduction of the neuronal soma seems to be a property of GDNF rather than the vector system used. Second, GDNF appears to provide trophic support, preventing degeneration of dopaminergic cells and loss of dopaminergic nerve terminals in both the 6-OHDA and MPTP models. This protection correlates both with some behavioral measures of

nigrostriatal integrity and neurochemical assays examining dopamine production. Finally, in many circumstances, the application of GDNF is protective or restorative even after the toxic insult has taken place.

As is the case with antiapoptotic gene therapy for PD, the applicability of the experimental studies to human patients is uncertain, because the etiology and pathogenesis of the human disease are likely to be different from the animal models. However, GDNF appears to provide generic trophic support to dopaminergic neurons in the face of a range of challenges, and a phase I study examining direct intraputaminal infusion of the recombinant protein in patients was recently reported.16 Adverse events were limited to repositioning one infusion catheter and asymptomatic signal changes on MRI that resolved when the concentration of infused GDNF was reduced. Secondary end points in this nonblinded nonrandomized study implied possible clinical benefit and improvement in functional imaging surrogates of dopaminergic terminal integrity. Should the recombinant factor prove efficacious in phase II trials, it is possible that gene delivery will offer advantages for long-term focal treatment.

A recent study has sounded a note of caution for GDNF therapy.17 Careful study of 6-OHDA rats showed that, although pharmacologically induced circling behavior, a marker of dopaminergic neural function, was ameliorated in animals treated with lentivirus-expressing GDNF over periods of up to 9 months, the spontaneous motor behavior was abnormal. This correlated with abnormal axonal sprouting within the pallidum and other brain areas where GDNF expression occurred, and with loss of the tyrosine hydroxlase (TH)-positive phenotype in SNc neurons that were preserved by GDNF treatment. Further characterization of these models, in conjunction with the outcome of clinical trials, will determine whether these concerns relate appropriately to GDNF therapy for PD.

Combination antiapoptotic and GDNF gene therapy protects dopaminergic neurons against a toxic insult, more effectively than either intervention alone

Two recent studies have exploited simultaneous delivery of genes encoding an antiapoptotic factor and GDNF, to enhance the dopaminergic cell survival seen with the corresponding single interventions.

Adenoviral delivery of X-linked inhibitor of apoptosis (XIAP), aimed at preventing apoptosis of SNc neurons in MPTP-treated animals, led to preservation of DA cells but did not prevent loss of striatal DA nerve terminals, resulting in failure of behavioral recovery.18 Combination of adenovirus-expressing XIAP, with another adenovirus-expressing GDNF, however, produced a synergistic effect with functional recovery that was not seen in animals treated with the GDNF-encoding vector alone.

In another study, GDNF was combined with bcl-2 gene delivery using two HSV vectors encoding expression cassettes for each of the factors.15 The 6-OHDA rat model was used, and either intervention (GDNF or Bcl-2) increased cell survival from 25% (control) to 55% (pretreated). However, coadministration of the two vectors increased cell survival to 75%, indicating that the effects of the different modalities were additive.

Transgene-mediated production of the inhibitory neurotransmitter -amino butyric acid (GABA) in neurons of the subthalamic nucleus ameliorates the behavioral phenotype and may be neuroprotective, in an animal model

One functional disturbance found in the basal ganglia of PD patients is the overactivity of neurons within the subthalamic nucleus (STN) that project to the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr). These excitatory neurons serve to increase the firing rate of GPi and SNr neurons that, in turn, inhibit brainstem and thalamic projections to downstream motor pathways, thereby inhibiting the initiation of voluntary movement. Inhibition of over-active STN neurons by stereotactic ablation or deep brain stimulation has been shown to ameliorate motor signs in late-stage PD.

A gene transfer strategy based on this approach has recently been reported.19 Transduction of STN neurons with glutamic acid decarboxylase (GAD), the rate-limiting enzyme for synthesis of the inhibitory neurotransmitter gamma-amino butyric acid (GABA), using an adeno-associated virus vector, resulted in synthesis and activity-dependent release of GABA from STN nerve terminals. Microelectrode studies in control animals showed that stimulation of the STN resulted in excitation of the majority of SNr neurons from which recordings were obtained, consistent with the known glutamatergic neurochemical phenotype of STN neurons. However, stimulation of GAD-transduced STN neurons produced a preponderance of inhibitory responses in the SNr neuron pool, suggesting that expression of GAD and consequent modification of the neurochemical phenotype had altered the physiological properties of the STN-SNr projection. Intriguingly, GAD transduction of the STN appeared to protect SNc dopaminergic neurons from a neurotoxic insult following administration of 6-OHDA. The protective effect seemed dependent on the induction of an inhibitory phenotype in the STN neurons, as destruction of the STN using ibotenic acid did not protect the SNc DA neurons from 6-OHDA. Combining neuroprotection with functional compensation is attractive; a phase I clinical trial has been approved to begin soon (see below).

Delivery of transgenes encoding enzymes involved in dopamine biosynthesis enhances dopamine production in the striatum

Pharmacologic therapy of PD involves correction of the neurochemical deficit by systemic delivery of the dopamine precursor, L-DOPA, or by use of agents that act directly on striatal dopamine receptors. In the first gene therapy study of PD, gene transfer was employed to deliver the rate-limiting enzyme for dopamine formation, tyrosine hydroxylase, to the striatum. This resulted in enhanced dopamine production and observable behavioral benefit in a rodent model.

More recently, it has been demonstrated that simultaneous delivery of multiple genes encoding enzymes that drive DA synthesis, more effectively corrects the DA-deficient phenotype than single-enzyme replacement. Synthesis of DA from tyrosine depends on two reactions, catalyzed by the enzymes tyrosine hydroxylase (TH) and aromatic acid decarboxylase (AADC). The former step is rate limiting, and requires a cofactor that is synthesized by GTP-cyclohydrolase I (GCH1). Various vector systems have been used in recent preclinical studies to deliver different combinations of these enzymes. These include multicistronic lentiviruses simultaneously encoding GCH1, TH and AADC;20 combinations of AAV vectors separately encoding GCH1 and TH21 or GCH1, TH and AADC;22,23 an HSV vector coexpressing AADC and TH.24 In all cases, coexpression of the enzymes and functional recovery of the experimentally lesioned animals was observed.

Long-term dopamine therapy in PD is associated with declining therapeutic efficacy and increasing adverse effects as the disease progresses. While transgene-mediated dopamine expression effectively corrects the motor phenotype in lesioned rodent and primate models, it is unclear at present whether the nonphysiological sustained delivery of dopamine in the striatum by these kinds of approaches will alleviate or exacerbate the problem of adverse effects. Since the therapeutic and toxic doses of dopaminergic agents alter in individual patients over the course of the disease, control over the production of dopamine following gene transfer will be essential before the use of this approach can be contemplated clinically. This might be accomplished using either (i) vectors with inducible enzyme expression, (ii) enzymes with controllable activity, or (iii) a prodrug approach using AADC to activate L-DOPA.

Stem cells may be driven to differentiate into functioning dopaminergic cells by genetic modification

Restoration of the dopaminergic projection from the SNc to the striatum has been a major goal of cell transplantation strategies. One major hurdle for this approach to therapy has been the difficulty in obtaining a suitable source of donor tissue that is both accessible and acceptable. One potential means for achieving this might rely on the use of human stem cell populations, driven to differentiate into dopaminergic neurons by appropriate manipulations. Much effort has been invested in determining which extracellular cues to stem cells are important in directing their differentiation into the desired cell population. It is possible to direct the differentiation of ES cells, for example, into dopaminergic neurons by a series of tissue culture manipulations, with an efficiency of around 15%.25 This can be enhanced to around 50% after transfection of the cells with a transgene encoding Nurr-1,26 an orphan nuclear receptor of the retinoic acid receptor superfamily, which has been implicated in the later stages of dopaminergic neuronal differentiation. It is possible that directed differentiation of dopaminergic neurons from a variety of stem cell sources might depend on ex vivo transduction of stem cell populations to effect genetic modifications that favor adoption of the desired cell fate. Dopaminergic cells formed from ES cells appear to have similar functional and neurochemical properties as native dopaminergic neurons,26 and can rescue an animal model of PD.26,27

Isolation of genes implicated in rare genetic forms of PD has allowed generation of new animal models and identification of new candidate targets for intervention

Although the common form of PD is sporadic and of unknown etiology, rare genetic forms have allowed isolation of genes involved in their pathogenesis, and thus highlighted cellular pathways that may be vulnerable in dopaminergic neurons and form potential targets for molecular intervention in PD. Mutations have been described in the gene encoding -Synuclein,

resulting in autosomal dominant PD. -Synuclein is abundantly expressed in the brain and normally localized at nerve terminals. Aggregates of -Synuclein comprise a major component of the Lewy body, which is the pathological hallmark of the common sporadic form of PD. A second form of familial PD is autosomal recessive, and results from mutations in the gene encoding Parkin, a ubiquitin ligase. Intriguingly, -synuclein is a substrate of Parkin,28 linking the two dissimilar proteins into a common functional pathway. Pathogenic mutations resulting in a PD phenotype have recently been described in two other genes: DJ-1, of unknown function,29 and NR4A2, encoding Nurr-1, a nuclear receptor (see above).30

Elucidation of the pathways involved in genetic forms of PD has provided new animal models of specific SN degeneration, which do not rely on toxicity caused by chemical insults. These may more closely resemble the pathogenesis of human disease. Thus, transgenic mice overexpressing either wild-type31 or mutated forms of human -synuclein32,33,34 develop neuronal inclusions and cell loss. In addition, rat35 and monkey36 models have been developed using virally mediated -synuclein gene transfer. Finally, a transgenic Drosophila model of -synucleinopathy has been described.37 Work in Drosophila may accelerate understanding of the human disease by identifying candidate pathways for disease modification.38

The genetic forms of PD are uncommon, but gene therapy targeting the -synuclein, Parkin or other pathways may also turn out to be an appropriate intervention for idiopathic PD. Using -synuclein transgenic mice, it was shown that overexpression of -synuclein prevented aggregation of -synuclein and the resulting abnormal phenotype.39 In addition, it appears that Parkin is capable of blocking the toxic effects of mutant -synuclein expression and proteasome inhibition in catecholaminergic neurons in culture.40 If deposition of -synuclein, formation of Lewy bodies, and proteasome dysfunction are pivotal events in the pathogenesis of PD, then -synuclein or Parkin gene delivery might be effective measures to disrupt the pathogenic cascade causing neurodegeneration.

One clinical gene therapy trial is about to commence in Parkinson's disease

Despite the wealth of experimental data on preclinical studies of gene therapy for PD, only one gene therapy trial is poised to start recruiting patients.41 The trial is based on GAD gene transfer to the subthalamic nucleus using an adeno-associated virus vector, as detailed in the section above.

In all, 12 patients with asymmetric disease will be selected by standard criteria, to undergo unilateral STN stimulator implantation.41 The trial is a dose-escalation safety study, and as approved by US Food and Drug Administration three cohorts of patients will receive between 1011 and 1012 particles of rAAV-GAD at the time of STN stimulator implantation. The assessors will monitor the patients' clinical state and PET scans. In the worst case, if GAD gene transfer has an unanticipated deleterious effect, then the STN can be either electrically silenced or ablated, both standard treatments for PD, using the stimulator leads without additional surgery.

Although the molecular strategy used in this trial is highly specific to PD, the wider field will view this pioneering study of in vivo gene transfer to the brain to treat neurodegeneration with considerable interest.

The optimal vector remains uncertain

Is there an optimal gene transfer vector with special utility for the development of treatments for PD? Nonviral gene transfer (liposomes or naked plasmids) is in general ineffective for gene transfer to the brain parenchyma, but each of the major viral vectors have demonstrated utility in experimental models of PD. In both earlier and recent studies viral vectors based on Ad1,42 lentivirus (LV),11,20 AAV13,19 and HSV15,24 have all been used to transfer relevant genes to the substantia nigra or striatum of experimental animals. Owing to the differences between studies in the animal model of PD employed, the site and volume of vector inoculation, the transgene and promoter constructs tested, the vector dose and the outcome measures assessed, the published literature does not allow one to make a direct comparison between vectors. The immunogenicity of Ad is likely to exclude that vector from human trials for PD, but each of the remaining vector platforms are likely to come to trial in the next few years. Although AAV and LV result in high-level long-term expression in brain, the results of the human trial using retroviral gene transfer to treat X-linked SCID in which two treated children developed leukemia as a result of insertional mutagenesis,43 coupled with the recent observation that AAV integrates more frequently into active genes than noncoding regions,44 may favor the use of a non-integrating vector such as HSV for these

applications.

Prospects for the next 2 years

The development of readily available presymptomatic diagnostic tests for PD will be necessary to enable the use of neuroprotective strategies to retard the progression of SNc cell loss. Better understanding of the pathogenesis of the common idiopathic form of PD may lead to the development of improved animal models that more closely resemble the etiology and cellular pathophysiology of human PD. This advance might enable identification of further targets for molecular intervention, in addition to providing a necessary resource for studies aimed at tackling the nonmotor features of PD. Depression, cognitive and autonomic dysfunctions are important contributors to morbidity in PD; further research aimed at addressing these components of the illness would be welcome. Further characterization of ES and adult stem cell populations will establish whether ex vivo transduction can drive their differentiation into dopaminergic neurons in a therapeutically useful way. Finally, further clinical trials for PD gene therapy are likely to commence using a variety of strategies. The trial designs may take their lead from recent well-executed clinical trials of cell transplant therapy.45,46 It will be important to measure disability, depression and quality of life in addition to motor outcome, in order to be certain about which aspects of the illness are favorably altered by gene transfer, and whether there is likely to be an overall beneficial effect from these interventions in patients.

References

1 Crocker SJ et al. NAIP protects the nigrostriatal dopamine pathway in an intrastriatal 6-OHDA rat model of Parkinson's disease. Eur J Neurosci 2001; 14: 391400.

2 Mochizuki H et al. An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease. Proc Natl Acad Sci USA 2001; 98: 1091810923. Article PubMed

3 Grondin R et al. Chronic, controlled GDNF infusion promotes structural and functional recovery in advanced parkinsonian monkeys. Brain 2002; 125: 21912201.

4 Date I et al. Grafting of encapsulated genetically modified cells secreting GDNF into the striatum of parkinsonian model rats. Cell Transplant 2001; 10: 397401. PubMed

5 Akerud P, Canals JM, Snyder EY, Arenas E. Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease. J Neurosci 2001; 21: 81088118. PubMed

6 Ostenfeld T et al. Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons. J Neurosci Res 2002; 69: 955965. Article PubMed

7 Park KW, Eglitis MA, Mouradian MM. Protection of nigral neurons by

GDNF-engineered marrow cell transplantation. Neurosci Res 2001; 40: 315323.

8 Shingo T, Date I, Yoshida H, Ohmoto T. Neuroprotective and restorative effects of intrastriatal grafting of encapsulated GDNF-producing cells in a rat model of Parkinson's disease. J Neurosci Res 2002; 69: 946954.

9 Bensadoun JC et al. Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF. Exp Neurol 2000; 164: 1524. Article PubMed

10 Kordower JH et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 2000; 290: 767773. Article PubMed

11 Palfi S et al. Lentivirally delivered glial cell line-derived neurotrophic factor increases the number of striatal dopaminergic neurons in primate models of nigrostriatal degeneration. J Neurosci 2002; 22: 49424954. PubMed

12 Kirik D, Rosenblad C, Bjorklund A, Mandel RJ. Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system. J Neurosci 2000; 20: 46864700. PubMed

13 Wang L et al. Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease. Gene Therapy 2002; 9: 381389. Article

14 McGrath J et al. Adeno-associated viral delivery of GDNF promotes recovery of dopaminergic phenotype following a unilateral 6-hydroxydopamine lesion. Cell Transplant 2002; 11: 215227.

15 Natsume A et al. Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration. Exp Neurol 2001; 169: 231238. Article PubMed

16 Gill SS et al. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 2003; 9: 589595. Article

17 Georgievska B, Kirik D, Bjorklund A. Aberrant sprouting and downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer. Exp Neurol 2002; 177: 461474. Article PubMed

18 Eberhardt O et al. Protection by synergistic effects of adeno-virus-mediated X-chromosome-linked inhibitor of apoptosis and glial cell line-derived neurotrophic factor gene transfer in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. J Neurosci 2000; 20: 91269134. PubMed

19 Luo J et al. Subthalamic GAD gene therapy in a Parkinson's disease rat model. Science 2002; 298: 425429. Article

20 Azzouz M et al. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease. J Neurosci 2002; 22: 1030210312. PubMed

21 Kirik D et al. Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci USA 2002; 99: 47084713.

22 Muramatsu S et al. Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum Gene Ther 2002; 13: 345354. Article PubMed

23 Shen Y et al. Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease. Hum Gene Ther 2000; 11: 15091519. Article PubMed

24 Sun M et al. Correction of a rat model of Parkinson's disease by coexpression of tyrosine hydroxylase and aromatic amino acid decarboxylase from a helper virus-free herpes simplex virus type 1 vector. Hum Gene Ther 2003; 14: 415424.

25 Lee SH et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000; 18: 675679. Article PubMed

26 Kim JH et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 2002; 418: 5056. Article PubMed

27 Bjorklund LM et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 2002; 99: 23442349. Article PubMed

28 Shimura H et al. Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease. Science 2001; 293: 263269. Article PubMed

29 Bonifati V et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 2003; 299: 256259.

30 Le WD et al. Mutations in NR4A2 associated with familial Parkinson disease. Nat Genet 2003; 33: 8589. Article PubMed

31 Masliah E et al. Dopaminergic loss and inclusion body formation in alpha-

synuclein mice: implications for neurodegenerative disorders. Science 2000; 287: 12651269. Article PubMed

32 Lee MK et al. Human alpha-synuclein-harboring familial Parkinson's disease-linked Ala-53 > Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci USA 2002; 99: 89688973. Article PubMed

33 Giasson BI et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron 2002; 34: 521533. PubMed

34 Richfield EK et al. Behavioral and neurochemical effects of wild-type and mutated human alpha-synuclein in transgenic mice. Exp Neurol 2002; 175: 3548. Article PubMed

35 Lo Bianco C et al. alpha-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease. Proc Natl Acad Sci USA 2002; 99: 1081310818. Article PubMed

36 Kirik D et al. Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease. Proc Natl Acad Sci USA 2003; 100: 28842889.

37 Feany MB, Bender WW. A Drosophila model of Parkinson's disease. Nature 2000; 404: 394398. Article PubMed

38 Auluck PK et al. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 2002; 295: 865868. Article PubMed

39 Windisch M et al. Development of a new treatment for Alzheimer's disease and Parkinson's disease using anti-aggregatory beta-synuclein-derived peptides. J Mol Neurosci 2002; 19: 6369.

40 Petrucelli L et al. Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 2002; 36: 10071019. PubMed

41 During MJ, Kaplitt MG, Stern MB, Eidelberg D. Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation. Hum Gene Ther 2001; 12: 15891591. PubMed

42 Xia XG et al. Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson's disease. Proc Natl Acad Sci USA 2001; 98: 1043310438. Article PubMed

43 Check E. Regulators split on gene therapy as patient shows signs of cancer. Nature 2002; 419: 545546. Article PubMed

44 Nakai H et al. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet 2003; 34: 297302. Article PubMed

45 Nakamura T et al. Blinded positron emission tomography study of dopamine cell implantation for Parkinson's disease. Ann Neurol 2001; 50: 181187.

46 Freed CR et al. Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N Engl J Med 2001; 344: 710719. Article PubMed

Figures

Figure 1 The human basal ganglia. A coronal section of a human brain is shown, illustrating the anatomical locations of the basal ganglia. (Photograph of human autopsy specimen kindly provided by Dr Olaf Ansorge, Department of Neuropathology, Radcliffe Infirmary, Oxford).

Figure 2 Gene therapy strategies for PD. The putative events and functional consequences involved in loss of SNc neurons are depicted. The complex pathogenic and pathophysiological cascade provides several candidate targets for molecular intervention, which are labeled with black arrows and white text; some of these are supported by experimental evidence, which is discussed in the text. In addition, alternative strategies to gene delivery, involving functional neurosurgery, cell transplantation and neuropharmacology are shown for contextual comparison. Abbreviations: P, putamen; GPe, external segment of globus pallidus; GPi, internal segment of globus pallidus; STN, subthalamic nucleus; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase; GTPCH1, GTP-cyclohydrolase-1.

September 2003, Volume 10, Number 20, Pages 1721-1727

August 2003, Volume 10, Number 16, Pages 1275-1281

Table of contents    Previous  Article  Next   PDF

ReviewGene therapy progress and prospects: gene therapy of lysosomal storage disorders

S H Cheng1 and A E Smith1

1Genzyme Corporation, 31 New York Avenue, Framingham, MA, USA

Correspondence to: Dr SH Cheng, Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701-9322, USA

Abstract

Despite disappointments with early clinical studies, there is continued interest in the development of gene therapy for the group of metabolic diseases referred to as lysosomal storage disorders (LSDs). The LSDs are monogenic and several small and large, representative animal models of the human diseases are available. Further, the successful reconstitution of only low and unregulated tissue levels of the affected lysosomal enzymes are expected to be sufficient to correct the disease at least in the case of some of the LSDs. For these reasons, they are perceived as good models for the evaluation of different gene delivery vectors and of different strategies for treating chronic genetic diseases by gene transfer. In this review, we will highlight the progress that has been made over the past 2 years in preclinical research for this group of disorders and speculate on future prospects.

Gene Therapy (2003) 10, 12751281. doi:10.1038/sj.gt.3302092

Keywords

lysosomal storage disorders; lysosomal enzymes; metabolic diseases; central nervous system; depot organs

In brief

Progress

Current therapeutic options for lysosomal storage disorders have been shown to be effective but limited

Lysosomal storage disorders are good candidates for therapy by gene transfer

Early clinical studies using ex vivo gene therapy vectors were ineffective

Proof of concept for use of in vivo gene therapy of LSDs has been demonstrated with different vector platforms and in several animal models

AAV serotypes with improved liver transduction activity are a promising vector platform for gene therapy of LSDs

Feasibility of in vivo gene therapy of LSDs with retroviral vectors has been demonstrated

Use of liver-specific promoters minimizes the induction of antibodies to the transgene product

Relative ability of the liver, muscle, and lung to support the secretion of lysosomal enzymes has been evaluated

Encouraging progress has been made in gene therapy of LSDs with CNS disease

Prospects

Candidate in vivo gene transfer vectors such as AAV8 for potential application in the treatment of LSDs will be selected for further characterization

Issues pertaining to safety, readministration of vector and manufacturing will be improved

Performance of the vector platforms in more relevant larger animal models will be addressed

The feasibility and necessity of incorporating gene regulation elements will be evaluated

Candidate lysosomal storage disorders with minimal CNS disease will be targeted for clinical studies

Methods to deliver gene or cell therapies that result in the global rescue of CNS disease will be developed

Current therapeutic options for lysosomal storage disorders have been shown to be effective but limited

Lysosomal storage disorders are a group of more than 40 heritable diseases that are caused by the pronounced deficiency of one or more lysosomal enzymes.1 This loss in enzymatic activity results in the progressive accumulation of undegraded substrate within the lysosomes with resultant engorgement of the organelle. This leads to cellular and tissue damage, subsequent organ dysfunction, and in some diseases to early mortality. Although the incidence of individual LSDs can be quite low, as a group they occur in approximately 1 in 7500 live births, and as such represent one of the more prevalent groups of genetic diseases in humans.

Currently, treatments for these rare disorders are limited to bone marrow transplantation and enzyme replacement therapy.2 The latter has been shown to be effective in the treatment of the non-neuropathic form of Gaucher disease3 and of Fabry disease,4,5 and enzyme replacement therapy is currently being developed for several other LSDs such as mucopolysaccharidosis I (MPS I), II and VI, Pompe disease, and Niemann-Pick B disease. However, because these enzymes generally have short circulating and intracellular half-lives, therapy requires regular, often biweekly, parenteral administrations of relatively large amounts of the relevant enzyme. Repeated bolus administrations of enzyme increase the likelihood of an immune response against the infused proteins, particularly in patients with null mutations, and this could affect the efficacy of subsequent treatments. Furthermore, because systemically administered enzyme is unable to traverse the bloodbrain barrier, enzyme therapy is only effective for those manifestations that do not involve the central nervous system (CNS). Therefore, other approaches, including the use of gene- and cell-based therapies that offer the opportunity for a more prolonged therapeutic effect than can be realized with enzyme replacement therapy, as well as the possibility of treating the CNS, are also being evaluated.

Lysosomal storage disorders are good candidates for therapy by gene transfer

There are several features of LSDs that make them particularly attractive candidates for intervention by gene therapy. For one, they are generally well-characterized single gene disorders. Importantly, it has also been shown that a proportion of many newly synthesized lysosomal enzymes are secreted into systemic circulation. Enzymes secreted in this way are recaptured by adjacent and distant cells, primarily through the cation-independent mannose-6-phosphate receptor, which is present, albeit in different amounts, on the surface of virtually all cells.1 Localized gene transduction of a depot organ such as the liver or muscle could allow for secretion of therapeutic levels of the affected enzymes into circulation. The amount of enzyme required for correction will vary with each disease, but may be only 110% of normal levels, based on the observed enzyme levels in individuals with milder, late-onset disease. In this regard, the potency of the gene transfer vectors necessary for facilitating the production of therapeutic levels of these enzymes may not need to be very high. Although it is unclear whether sustained overexpression of the hydrolases will have untoward consequences, tight regulation of enzyme production levels is unlikely to be necessary in part because the pH optimas for their enzymatic activities are likely to render them inactive in circulation at neutral pH. Moreover, a number of genetically engineered mouse models as well as naturally occurring mouse and large animal models of LSDs are available, which should allow assessment of these predictions.6

Early clinical studies using ex vivo gene therapy vectors were ineffective

Early attempts at gene therapy for subjects with the lysosomal storage diseases Gaucher, Hunter and Hurler were with transplanted retrovirally transduced bone marrow stem cells or mobilized peripheral blood monocytes. However, it was determined that the efficiency of retroviral transduction of these cells was low, and engraftment of the modified stem cells, particularly in the absence of myeloablation, was transient and occurred at a low and ineffective level.7 Nevertheless, with ongoing improvements in vectors and processes for transduction, this approach continues to be of interest for use in LSDs.8,9,10,11

Recent improvements include the development and use of cytokine combinations that induce hematopoietic cell cycling to enhance retroviral transduction,12 pseudotyped virions with envelopes that recognize different and perhaps more abundant receptors in the target cells,13 lentiviral vectors that infect nondividing cells with greater efficiency,14 and methods to enrich for the transduced cells prior to transplantation.10 The adaptation of some of these methods has led to clinical success in treating humans with the genetic disorder severe combined immunodeficiency disease (SCID).15 However, this outcome was due in part to the strong positive selective pressure provided to the corrected lymphoid progenitors upon gene transfer and this is unlikely to occur in corrected cells from LSDs patients. Moreover, two cases of a T-cell leukemia-like disease was recently noted in the treated SCID patients that may have resulted from vector integration. This observation together with the finding that some myeloablation is likely necessary for efficient engraftment of transduced stem cells makes this gene therapy approach less compelling, particularly for those LSDs where enzyme replacement therapy is available.

Proof of concept for use of in vivo gene therapy of LSDs has been demonstrated with different vector platforms and in several animal models

The concept of directly transducing a depot organ to effect the production and secretion of lysosomal enzymes to treat the visceral disease has been demonstrated using a variety of gene delivery systems (Table 1). Understandably, the initial focus of interest has been directed primarily at those LSDs with no or minimal neurological involvement, and for which a viable animal model is available.

Since systemic delivery of recombinant adenoviral vectors results in efficient transduction of the liver and high-level expression and secretion of lysosomal enzymes, a large number of studies have been performed with this vector.16,17 Intravenous delivery of the corresponding recombinant adenoviral vector provided high-level secretion from the liver, and importantly, re-uptake of the lysosomal enzymes by other affected tissues. Depending on the dose of virus used, the levels of enzyme attained in the different tissues varied from 10- to 1000-fold higher than normal levels. In all cases, reconstitution of the enzymes to these levels was sufficient to reduce rapidly the abnormal storage in the lysosomes to normal or near normal levels.

However, these studies also showed that expression of the desired protein was transient, declining to basal levels within several weeks. Moreover, an inflammatory response that included a significant cytotoxic T-lymphocyte response was observed that likely attenuated expression. Hence, while these studies demonstrated the potential of in vivo gene therapy for LSDs, they also highlighted the need for significant improvements in the performance of the adenoviral vectors before clinical studies can usefully be contemplated. The so-called 'gutless', 'PAV', or helper-dependent vectors which are essentially devoid of viral genes, are purportedly less inflammatory, effect less liver toxicity, and support greater longevity of transgene expression. Although these vectors are presently difficult to produce in large quantities and with high purity, their superior properties support their further evaluation in animal models of LSDs.

Synthetic vectors in the form of cationic lipids or polymers have also been considered for in vivo gene therapy of LSDs.18 Since synthetic vectors are nonproteinaceous, a significant advantage is their ability to be readministered following the attenuation of gene expression. However, the therapeutic window of current formulations of synthetic vectors is quite narrow and improvements in their transduction activity as well as their toxicity profile are necessary.19 Another nonviral approach is the use of encapsulated gene-modified cells or organoids. Implantation of nonautologous cells expressing -glucuronidase has been shown to alleviate some of the storage burden in MPS VII mice.20 However, expression was transient and was associated with the induction of antibodies against the enzyme. Limitations associated with the number of cells that can be encapsulated in the current devices, coupled with the potential for dissolution of the membrane biomaterials with ensuing loss of cell viability or escape of the cells need to be further addressed before this can be considered for human application.

AAV serotypes with improved liver transduction activity are a promising vector platform for gene therapy of LSDs

A viral vector that is gaining increasing interest for use in lysosomal storage and other genetic diseases is the adeno-associated viral vector.21 AAV reportedly exhibits low toxicity and supports long-term transgene expression in mice as well as large animals.22,23,24 Although the vector has a limited capacity for inserted sequences, this is not problematic for most of the cDNAs that encode lysosomal enzymes, since they are relatively small. Several distinct AAV serotypes have been isolated, of which AAV2 has been the most studied. Recombinant AAV2 vectors have so far been constructed for the LSDs, Fabry,24,25 Pompe,26 MPS VII,27 MPS I, and MPS IIIB.28 Systemic delivery of recombinant AAV2 encoding -galactosidase A into Fabry mice, or encoding -glucuronidase into neonatal MPS VII mice, resulted in the reconstitution of the respective enzymes in several tissues to 1080% of normal levels. These levels are 100- to 1000-fold lower than those attained using recombinant adenoviral vectors, despite the use of much higher doses of AAV2.

The kinetics of expression were consistent with those reported previously for AAV2 vectors containing other transgenes, with peak expression levels generally attained between 2 and 4 weeks, and these levels persisting for several months post-treatment. Although there was only a modest increase in enzyme levels in some of the tissues, they were sufficient to reduce measurably the amount of the substrates that had accumulated in the different animal models. This observation supports the notion that continuous expression of low levels of enzyme activity is sufficient to reduce the extent of storage in the lysosomes. However, the kinetics for the reduction of the storage materials were slower in the AAV- than in adenoviral-treated animals, with the AAV2-treated animals requiring several more weeks to effect clearance.

When coupled with its positive safety profile, these results suggest that AAV2 vectors have great potential for treating LSDs. However, since AAV2-mediated expression levels were relatively low and close to the threshold for therapeutic efficacy in some of the affected tissues, an improvement in transduction activity would be beneficial. In this regard, other AAV serotypes such as AAV1, and in particular AAV8, have recently been shown to have substantially greater liver transduction activity than AAV2.29 The 10- to 100-fold higher expression levels attained with AAV8 were correlated with a higher number of transduced hepatocytes and greater persistence of vector DNA. Moreover, AAV8 was shown to have a low reactivity to neutralizing antibodies directed to human AAVs. This relative lack of pre-existing immunity to AAV8 coupled with its superior tropism for liver argues that it is a good candidate for further evaluation as a vector for gene therapy of LSDs. However, the induction of a humoral response to the viral proteins following the first administration will present challenges for subsequent readministrations. Although several strategies to overcome this limitation have been reported, none are facile or involve the use of clinically approved agents.

Feasibility of in vivo gene therapy of LSDs with retroviral vectors has been demonstrated

Yet another vector that is under consideration for in vivo gene therapy of LSDs is the oncoretroviral vector. As these vectors have the capacity to integrate into the host genome, they offer the possibility for long-term expression. Both Moloney murine leukemia virus (MLV)-based and lentiviral-based retroviral vectors have been shown to be capable of transducing hepatocytes with sufficient efficiency to facilitate the production and secretion of lysosomal enzymes.30,31 Intravenous delivery to MPS VII mice of either a MLV or lentiviral vector encoding -glucuronidase resulted in expression of enzyme to approximately 1% of normal levels in the liver. Expression was sustained for the duration of the studies (3.55 months) and was associated with a reduction in lysosomal storage. As these vectors preferentially transduce replicating hepatocytes, improved transfection was realized when the animals were pretreated with hepatocyte growth factor. Greater transduction of hepatocytes (220%) could also be attained when the retroviral vectors were administered to neonatal animals as was the case in the recent study in MPS VII dogs.32,33 Clonal expansion of the transduced hepatocytes during liver development resulted in the production of -glucuronidase levels that were within the normal range. These expression levels were sustained in the dogs for several months and were associated with dramatic improvements in the clinical manifestations. Although this intervention is conceivable for humans, implementation would necessarily require an assessment of the risk for insertional mutagenesis, as well as the detection of disease at a very early stage.

Use of liver-specific promoters minimizes the induction of antibodies to the transgene product

Expression of lysosomal hydrolases following viral-mediated gene transfer to immunocompetent mouse models of LSDs is invariably associated with the generation of a robust humoral response against the enzymes. This has the effect of extinguishing transgene expression and thereby limiting the duration of therapy. This problem is likely to be particularly pertinent in LSD subjects that harbor null mutations. The proportion of patients among the different LSDs carrying null mutations is varied but can be as high as 70% as in the case of Hurler syndrome. However, it has been shown that this immune response can be circumvented in both adenovirus- and AAV-treated animals, provided a tissue-restricted promoter was used to direct the transgene expression.34 Systemic injection of either a recombinant adenoviral or AAV vector in which the transgenes were placed under the transcriptional control of liver-restricted promoters reduced the extent of antibodies induced and increased the longevity of transgene expression in immunocompetent mice. The reduced tendency to provoke an immune response is thought to be related to the reduced expression of the transgene in antigen-presenting cells.

In Fabry mice, injection of an AAV vector encoding -galactosidase A under the control of a chimeric human liver-restricted promoter consisting of two copies of the prothrombin enhancer linked to a human serum albumin promoter resulted in undiminished expression for up to 1 year.35 In contrast to Fabry mice treated with a CMV expression cassette, no antibodies to the transgene product were detected in the animals treated with the liver-restricted promoter cassette. The ability of this chimeric promoter to sustain prolonged expression in larger animals such as in a nonhuman primate has also been demonstrated.

Relative ability of the liver, muscle, and lung to support the secretion of lysosomal enzymes has been evaluated

Although early in vivo gene therapy efforts for LSDs have focused primarily on the use of the liver as a depot for the production of lysosomal enzymes, consideration has also been given to skeletal muscle and lung as alternate portals. The liver was selected in part because of the tropism of the adenoviral and AAV vectors for this organ. The rationale for selecting hepatocytes as the target for genetic modification was also supported by the knowledge that they are adept at secreting a variety of proteins. However, parenteral administration of viral vectors, particularly adenoviral vectors, has been shown to be associated with liver toxicity. This characteristic is obviously undesirable, especially for those LSDs where liver function is already compromised. Systemic delivery also carries the potential risk of transfecting the gonads and of subsequent germline alteration.

Using the skeletal muscle as the depot in lieu of the liver circumvents some of these concerns. Using intramuscular injection to produce lysosomal enzymes has been reported for MPS VII,36 Pompe disease,26,37,38 and Fabry disease.25 Although high levels of localized expression of the enzymes could be realized in the muscle, only very low levels of the enzymes were secreted into the circulation. In the case of MPS VII and Pompe disease, these levels were determined to be insufficient to provide therapeutic benefit to affected tissues that were distant from the injected muscle. These findings were consistent with the report by Raben et al showing that the muscle was significantly less efficient than the liver at secreting the lysosomal enzyme -glucosidase for Pompe disease.39 However, these observations did not extend to Fabry disease. Despite attaining only low-level secretion of -galactosidase A, correction of the storage defect was observed not only in the injected muscle but also globally in AAV2-treated Fabry mice. Therefore, it would appear that the selection of muscle as a depot organ might be applicable for use in some but not all LSDs. Perhaps the use of other AAV serotypes, such as AAV1 or AAV7 that reportedly exhibit higher transduction efficiencies in muscle than AAV2, could further improve the utility of this organ for the production of lysosomal enzymes.29

Another organ that is gaining interest as a metabolic factory for the production and secretion of therapeutic proteins into systemic circulation is the lung. Use of the lung as a portal for systemic delivery of proteins offers several advantages. Foremost is the large lumenal surface area of the lung that can be accessed noninvasively by liquid or dry powder aerosols. The lung also has an extensive capillary network that could support the delivery of proteins into the systemic circulation. Presently, a number of metabolic hormones such as insulin and growth hormone are under consideration for systemic delivery by pulmonary inhalation.40 Restricting delivery to the lumen also limits the dissemination and therefore any systemic toxicity that may be associated with the gene transfer vector. Finally, several vector systems, including adenoviral, AAV5 and pseudotyped lentiviral vectors have been shown to transduce airway epithelial cells after pulmonary delivery.41,42

The feasibility of genetically modifying the lung and using this as a portal to administer proteins into the blood has been demonstrated for the lysosomal enzyme -galactosidase A43 and also for erythropoietin (epo) and factor IX.44 Instillation of a recombinant adenoviral vector encoding -galactosidase A into Fabry mice resulted in high level expression in the lung, secretion into the circulation and subsequent uptake of the lysosomal enzyme by the visceral organs. The levels of enzyme detected in the different organs were sufficient to reduce the burden of storage in the affected lysosomes. Although expression in the lung was transient using a recombinant adenoviral vector, expression kinetics were dramatically improved when an AAV5 vector was used for factor IX and epo. The availability of vectors that support high and sustained expression of proteins in the lung and of methods to deliver these in a noninvasive manner to the lumen suggest that the lung may be a viable alternative depot for the production of lysosomal enzymes. As for the gene delivery vectors, the final selection of the depot organ of choice will likely depend on the particulars of the LSDs. A better understanding of the relative abilities of the different organs to confer the necessary post-translational modifications to facilitate effective targeting of the enzymes to the lysosomal compartment may also aid with the selection process.

Encouraging progress has been made in gene therapy of LSDs with CNS disease

To treat LSDs with CNS manifestations requires the development of strategies that not only bypass the physical and bloodbrain barriers but that also support the delivery of enzyme throughout the brain. A further challenge for gene transfer within the CNS is the relatively quiescent state of the resident cells that precludes the use of vectors requiring cell division for transduction. Nevertheless, there have been ample demonstrations of the feasibility of gene therapy with several vector platforms in animal models of LSDs with CNS involvement. Intraventricular or stereotactic injections of recombinant adenoviral, AAV, lentiviral, and herpes simplex viral (HSV) vectors into various cerebral structures can result in transduction of both neuronal and glial cells. Of these, AAV and lentiviral vectors appear to be gaining increasing favor because of their safety profile, and their ability to transduce apparently quiescent cells within the CNS and to confer prolonged expression.

Intracranial injections of recombinant AAV245 or feline immunodeficiency viral46 vectors encoding -glucuronidase into MPS VII mice resulted not only in the correction of the characteristic cellular pathologies but also in improvements in cognitive function. A similar demonstration of protection from disease-associated pathology was also reported for metachromatic leukodystrophy using a lentiviral vector encoding the lysosomal enzyme arylsulfatase A.47 In all cases, although expression of the enzymes was concentrated at the sites of injection, pathology resulting from storage was reduced in most areas of the brain. This suggested that the enzyme was secreted from the transduced cells and taken up by uninfected adjacent cells, resulting in a zone of correction that extended beyond the site of injection. In addition to enzyme diffusion, evidence has also been presented suggesting that the lysosomal enzymes could be further distributed by axonal transport and by cells of the rostral migratory stream.48 Further improvement in the biodistribution of the enzyme in MPS VII-injected mice could also be realized through the incorporation of protein transduction motifs such as that derived from HIV Tat.49 While these results in LSDs mouse models are encouraging, confirmatory studies are required to ascertain whether these observations are translatable to animals with larger brains. In addition, although a few of the studies were performed in adult mice with developed CNS pathology rather than very young animals, it will be important to determine the temporal window for effective interventional therapy.

Summary

Significant progress has been made in studies of gene therapy for lysosomal storage disorders. While the ability to deliver therapy to the CNS is still at a formative stage, the ability to treat the visceral component of the disease, particularly using the recently described gene transfer vectors, appears feasible. Those LSDs that lack or have only moderate CNS disease could therefore be initially considered for therapy using

this approach. Examples of such LSDs include Type I Gaucher disease, Fabry disease, Niemann-Pick B disease, Pompe disease, MPS IS, IHS, IV and VI.

Prospects

Although more validation is required, the reported higher liver transduction activity of AAV8, coupled with its good safety profile and ability to confer long-term expression suggest that this is the vector choice for evaluation in human clinical studies. Studies to confirm that AAV8 transduces human hepatocytes with high efficiency, as seen in mouse models, and that pre-existing neutralizing antibodies to AAV8 are present at a low frequency in the general population are undoubtedly ongoing. However, depending on the dose of vector needed in humans, consistent manufacture of clinical grade AAV vectors at scale may still present a technical challenge. Other issues include the possibility of neutralizing antibodies generated against the newly synthesized enzymes and the ability to readminister the recombinant virus. While the former could be addressed in part through the use of tissue-restricted promoters, the possible incorporation of gene regulation or gene switch technologies may further alleviate this safety concern.50 However, readministration of the recombinant virus without the unwieldy use of additional manipulations remains a significant challenge. Most animal studies would predict that AAV-mediated gene expression is likely to persist for a very significant period in time. It is quite possible that the antibody titer to the virus will drop sufficiently during the intervening period to allow readministration.

There is clearly much optimism for the potential use of gene therapy for LSDs, at least initially, for those with minimal CNS involvement. Over the past few years, investigators have better defined what constitutes an effective and safe gene transfer vector for use in this group of metabolic disorders. If the remaining issues associated with the viral gene delivery vectors can be adequately addressed over the next period, it is likely renewed clinical studies for LSDs will proceed.

References

1 Suzuki K. Lysosomal diseases. In: Graham DI, Lantos PL (eds) Greenfield's Neuropathology. Arnold: London, 2002, pp 653735.

2 Wraith JE. Advances in the treatment of lysosomal storage disease. Dev Med Child Neurol 2001; 43: 639646. PubMed

3 Weinreb NJ et al. Effectiveness of enzyme replacement therapy in 1028 patients with Type I Gaucher disease after 2 to 5 years of treatment: a report from the Gaucher registry. Am J Med 2002; 113: 112119. Article PubMed

4 Eng CM et al. Safety and efficacy of recombinant human -galactosidase A replacement in Fabry disease. N Engl J Med 2001; 345: 916. Article PubMed

5 Schiffman R et al. Enzyme replacement therapy in Fabry disease: a randomised controlled trial. JAMA 2001; 285: 27432749. Article PubMed

6 Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet, AL, Sly WS, Valle D (eds) The Metabolic and Molecular

Basis of Inherited Disease. McGraw-Hill: New York, 2001, pp 34213452.

7 Novelli EM, Barranger JA. Gene therapy for lysosomal storage disorders. Expert Opin Biol Ther 2001; 1: 857867. PubMed

8 Miranda et al. Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of NiemannPick disease. Gene Therapy 2000; 7: 17681776. PubMed

9 Matzner U, Habetha M, Gieselmann V. Retrovirally expressed human arylsulfatase A corrects the metabolic defect of arylsulfatase A-deficient mouse cells. Gene Therapy 2000; 7: 805812. PubMed

10 Qin G et al. Preselective gene therapy for Fabry disease. Proc Natl Acad Sci USA 2001; 98: 34283433. Article PubMed

11 Leimig et al. Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells. Blood 2002; 99: 31693178. PubMed

12 Halene S, Kohn DB. Gene therapy using hematopoietic cells: sisyphus approaches the crest. Hum Gene Ther 2000; 11: 12591267. Article PubMed

13 Horn PA et al. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 2002; 100: 39603967. PubMed

14 Vigna E, Naldini L. Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J Gene Med 2000; 2: 308316. Article PubMed

15 Cavazzana-Calvo M et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669672. PubMed

16 Marshall J et al. Demonstration of feasibility of in vivo gene therapy for Gaucher disease using a chemically induced mouse model. Mol Ther 2002; 6: 179189. PubMed

17 Du H et al. Lysosomal acid lipase deficiency: correction of lipid storage by adenovirus-mediated gene transfer in mice. Hum Gene Ther 2002; 13: 13611372. PubMed

18 Przybylska MJ et al. Correction of the -galactosidase A deficiency in Fabry mice using synthetic gene delivery vectors. Mol

Ther 2002; 5: S343.

19 Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene Therapy 2002; 9: 16471652. PubMed

20 Ross CJD et al. Treatment of a lysosomal storage disease, mucopolysaccharidosis VII, with microencapsulated recombinant cells. Hum Gene Ther 2000; 11: 21172127. PubMed

21 Atthanaspoulos T, Fabb S, Dickson G. Gene therapy vectors based on adeno-associated virus: characteristics and applications to acquired and inherited diseases. Int J Mol Med 2000; 6: 363375. PubMed

22 Zaiss AK et al. Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors. J Virol 2002; 76: 45804590. PubMed

23 Mount JD et al. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy. Blood 2002; 99: 26702676. Article PubMed

24 Jung SC et al. Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice. Proc Natl Acad Sci USA 2001; 98: 26762681. PubMed

25 Takahashi H et al. Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer. Proc Natl Acad Sci USA 2002; 99: 1377713782. Article PubMed

26 Fraites TJ et al. Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors. Mol Ther 2002; 5: 571578. PubMed

27 Daly TM et al. Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer. Gene Therapy 2001; 8: 12911298. PubMed

28 Fu H et al. Neurological correction of lysosomal storage in a mucopolysaccharidosis IIIB mouse model by adeno-associated virus-mediated gene delivery. Mol Ther 2002; 5: 4249. PubMed

29 Gao G et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 1185411859. Article PubMed

30 Gao C, Sands MS, Haskins ME, Ponder KP. Delivery of a retroviral vector expressing human -glucuronidase to the liver and

spleen decreases lysosomal storage in mucopoly-saccharidosis VII mice. Mol Ther 2000; 2: 233244. PubMed

31 Stein CS et al. In vivo treatment of hemophilia A and mucopolysaccharidosis type VII using nonprimate lentiviral vectors. Mol Ther 2001; 3: 850856. Article PubMed

32 Xu L et al. Transduction of hepatocytes after neonatal delivery of a Moloney murine leukemia virus based retroviral vector results in long-term expression of -glucuronidase in mucopolysaccharidosis VII dogs. Mol Ther 2002; 5: 141153. PubMed

33 Ponder KP et al. Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs. Proc Natl Acad Sci USA 2002; 99: 1310213107. PubMed

34 Wang L et al. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol Ther 2000; 1: 154158. Article PubMed

35 Ziegler R et al. Improved adeno-associated viral vectors for gene therapy of Fabry disease. Mol Ther 2002; 5: S91.

36 Daly TM et al. Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer. Gene Therapy 2001; 8: 12911298. PubMed

37 Ding E et al. Efficacy of gene therapy for a prototypical lysosomal storage disease (GSD-II) is critically dependent on vector dose, transgene promoter, and the tissues targeted for vector transduction. Mol Ther 2002; 5: 436446. PubMed

38 Martin-Touaux E et al. Muscle as a putative producer of acid glucosidase for glycogenosis type II gene therapy. Hum Mol Genet 2002; 11: 16371645. PubMed

39 Raben N et al. Conditional tissue-specific expression of the acid -glucosidase (GAA) gene in the GAA knockout mice: implications for therapy. Hum Mol Genet 2001; 10: 20392047. PubMed

40 Agu RU et al. The lung as a route for systemic delivery of therapeutic proteins and peptides. Respir Res 2001; 2: 198209. PubMed

41 Zabner J et al. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol 2000; 74: 38523858. Article PubMed

42 Kobinger GP, Weiner DJ, Yu QC, Wilson JM. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce

airway epithelia in vivo. Nat Biotechnol 2001; 19: 225230. PubMed

43 Li C et al. Adenovirus-transduced lung as a portal for delivering -galactosidase A into systemic circulation for Fabry disease. Mol Ther 2002; 5: 745754. PubMed

44 Auricchio A et al. Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins. J Clin Invest 2002; 110: 499504. PubMed

45 Frisella WA et al. Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis Type VII. Mol Ther 2001; 3: 351358. Article PubMed

46 Brooks AI et al. Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors. Proc Natl Acad Sci USA 2002; 99: 62166221. Article PubMed

47 Consiglio A et al. In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. Nat Med 2001; 7: 310316. Article PubMed

48 Passini MA, Lee EB, Heuer GG, Wolfe JH. Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream. J Neurosci 2002; 22: 64376446. PubMed

49 Xia H, Mao Q, Davidson BL. The HIV Tat protein transduction domain improves the biodistribution of -glucuronidase expressed from recombinant viral vectors. Nat Biotechnol 2001; 19: 640644. Article PubMed

50 Clackson T. Regulated gene expression systems. Gene Therapy 2000; 7: 120125. PubMed

Tables

Table 1 Lysosomal storage disorders

August 2003, Volume 10, Number 16, Pages 1275-1281

July 2003, Volume 10, Number 14, Pages 1135-1141

Table of contents    Previous  Article  Next   PDF

ReviewGene therapy progress and prospects: adenoviral vectors

J A St George1

1Genzyme Corporation, Framingham, MA, USA

Correspondence to: Dr J St George, Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701-9322, USA

Abstract

In September 1999, the perceptions of the use of adenoviral (Ad) vectors for gene therapy were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector succumbed to the toxicity related to vector administration. Appropriately, concerns were raised about continued use of the Ad vector system and, importantly, there were increased efforts to more fully understand the toxicity. Today it is recognized that there is no ideal vector system, and that while Ad vectors are not suitable for all applications, the significant advantages over other vector systems including efficient transduction of a variety of cell types, both quiescent and dividing, make it optimal for certain applications. These include protocols where high levels of short-term expression are sufficient to provide a therapeutic benefit. Potential target applications include therapeutic angiogenesis, administration into immune-privileged sites such as the CNS, or treatments where the

adjuvant effect of adenovirus can be of benefit such as cancer vaccines. Broader applicability of Ad vectors will require resolution of toxicity issues. This review will therefore focus on studies conducted over the last 2 years that have advanced our understanding of the toxicity associated with Ad vectors, studies that have employed methods to reduce toxicity and improvements in Ad vectors themselves that will reduce toxicity by one of several mechanisms. These mechanisms include retargeting vector to the tissue of interest, minimizing or eliminating viral gene expression that is thought to result in loss of transduced cells, or by methods that seek to reduce the vector dose required for therapeutic benefit. An area where there remains significant room for improvement is when readministration of vector is required because transgene expression has decreased to background levels.

Gene Therapy (2003) 10, 11351141. doi:10.1038/sj.gt.3302071

Keywords

adenovirus; targeting; toxicity

In brief

Progress

Application of Ad vectors to select applications such as therapeutic angiogenesis has generated cautious optimism.

An understanding of Ad vector-induced toxicity is continuing to grow and is leading to methods to minimize it's effects.

Refinements in targeting of Ad vectors are improving the therapeutic index.

Advanced generation Ad vectors have reduced toxicity and improved persistence of transgene expression.

Prospects

Methods to reduce toxicity will continue to be advanced and will lead to safer use of Ad vector systems.

The use of targeting methods will be improved. Production methods of fully deleted Ad vectors will continue to

improve.

Further efforts will be focused on readministration challenges.

Ad vectors continue to be tested in clinical trials and have demonstrated a therapeutic effect

Although many have questioned the use of Ad vectors, the number of clinical protocols employing Ad vectors has not decreased. Ad vector use remains around 27% (636 protocols) and the percentage of

patients treated at 18% (3494) (Journal of Gene Medicine Website, www.wiley.co.uk/genmed/clinical/). This proportion is second only to retroviral vectors. The success of an Ad vector in humans was reported recently when the results of a randomized, placebo-controlled, double-blinded phase II trial demonstrated that an Ad vector coding for VEGF gene increased vascularity after percutaneous translumenal angioplasty.1 Currently, there are five phase IIIII or phase III clinical trials where Ad vectors are being tested in either angiogenic or cancer applications.

Ad vector-induced toxicity is complex

As a reflection of the importance of Ad-induced toxicity, the entire January 2002 issue of Human Gene Therapy was devoted to the topic. A call by the National Institutes of Health Recombinant DNA Advisory Committee for a better understanding of the toxicity of Ad vectors included recommendations to develop standards, so that data collected by various laboratories can be compared, to develop a database for the collection and organization of safety and toxicity data and several recommendations to improve the safety of research participants.2 A standard that has been developed includes adenoviral vector reference material that is available to investigators for comparison to their vectors. It is apparent that only with a thorough understanding of all aspects of the toxic interaction between a vector and the host that the full utility of any vector system will be realized. However, the toxicity associated with the use of Ad vectors is extremely complex involving both the innate and adaptive immune responses. It is dose dependent, occurs in phases, is related to route of administration, is dependent on tissue and cell type targeted and varies with species. In addition, the role of the response to a foreign or reporter transgene is often ignored but can complicate interpretation of the findings. Finally, it is important to remember that extrapolations from preclinical studies to clinical studies should take into account that the vast majority of preclinical studies are performed in normal, naïve animals, whereas clinical studies are conducted in a population likely not naïve to adenovirus and that has underlying disease.

The initial response to Ad vectors administered intravascularly occurs within minutes, occurs in the absence of viral gene expression and is attributed to the innate response. Building on earlier studies has shown that, as the vector distributes in the blood, it interacts with cells of the reticuloendothelial system (RES) and induces the release andor production of several proinflammatory cytokines including IL-6, TNF, IL-8, GM-CSF and MIP-2.3,4 Cytokine production appears to occur through the NF B pathway possibly via RafMAP kinase phosphorylation of I B.4,5 Higginbotham et al4 reported that exposure of normal human peripheral blood mononuclear cells to Ad vector resulted in the release of multiple proinflammatory cytokines in vitro. The authors suggested that transcriptional stimulation of these cytokines was the result of cellular binding of virionscapsids. In addition to the mechanism described above, Ad vectors have been shown to activate complement in vitro,6 but the significance of this response has yet to be demonstrated in vivo.

The immediate response peaks around 6 h and the primary cells thought to be responsible are macrophages and dendritic cells that serve as a functional bridge between the innate and acquired immune response. These cells are activated and induced to mature, which results in upregulation of MHC antigens as well as co-stimulatory and adhesion molecules.7 The inadvertent targeting of these antigen-presenting cells leads to a systemic acquired immune response.8 In addition, vector interaction with epithelial cells results in release of C-X-C chemokines, especially IP-10.5 IP-10 is a potent chemoattractant for activated T lymphocytes and pushes the reaction towards a Th-1 type or cytotoxic T-lymphocyte (CTL) response. Thus, the innate response bridges via multiple pathways with the acquired response that follows.

Subsequent responses or possible sequelae to the initial response can be lethal, can occur within days and can be correlated, at least in part, with Ad gene expression. For the most part, this response has been well characterized and is correlated with the infiltration of lymphocytes, the formation of anti-Ad antibodies and the loss of transduced cells. When a lethal response occurs following administration of high doses, systemic changes have been described that include extensive endothelial damage and disseminated intravascular coagulopathy.9 Several studies described a decrease in platelets and an increase in the von Willebrand factor.8,9,10 It is of note, however, that Ad vectors do not directly induce platelet aggregation.11

The January 2002 issue of Human Gene Therapy mentioned above contains two studies describing the responses in nonhuman primates.9,12 Studies in non-human primates may be the most instructive to our understanding of Ad-mediated toxicity as the responses have been shown to mirror those described in patients.10 The findings of studies conducted in primates, including humans, are summarized briefly in Table 1. It is important to note that on balance, other manuscripts in that issue of Human Gene Therapy concluded that in studies using several routes of local administration of low to intermediate doses of Ad vectors were well-tolerated in humans.13,14

In summary, the toxicity associated with the administration of Ad vectors must be fully appreciated and steps taken to reduce it. An additional complication underscoring the need for treatment to reduce toxic

effects, was the demonstration that the physical act of some methods of administration themselves, even in the absence of vector, result in elevated levels of IL-6.15 A straightforward approach to reduce toxic responses has been the pretreatment or coadministration of anti-inflammatory agents. Pretreatment of animal models with steroids such as dexamethasone and budesonide was shown to decrease transcription of chemokines and cytokines, thereby reducing the innate and downstream-acquired response including production of antibodies to Ad vectors.16

Targeting of Ad vectors will improve the therapeutic index

There have been significant efforts in the last 2 years to retarget Ad vectors away from the primary receptor (coxsackieadenovirus receptor CAR) to a tissue or cell-specific receptor. The aims are (i) to restrict transduction to the organ of interest, thereby gaining the greatest benefit with the lowest dose, (ii) to minimize the innate response by limiting vector interaction with the RES and (iii) to potentially avoid the effects of an anti-Ad neutralizing antibody response.17,18,19 Another rationale for targeting Ad vectors is that CAR is present in low levels in some cell types that are potential targets for gene transfer.

The most straightforward method of detargeting of CAR binding is to pseudotype human serotypes with capsids from other species20,21,22 or to simply employ Ad vectors from nonhuman serotypes.23 This approach has the added benefit of avoiding the neutralization of input vector by preexisting antibodies to human adenoviruses. Other targeting approaches are grouped into two categories; those that modify the viral capsid through genetic alteration especially of fiber and fiber knob DNA and those that employ two components where one binds to the Ad vector and is linked to another that will target the complex to a specific receptor. Genetic retargeting of Ad vectors involves the ablation of the normal tropism by mutating or deleting the CAR-binding sequences and incorporating foreign sequences into the loops within the knob of the fiber shaft redirecting the vector to specific receptors on selected cell types. The feasibility of this approach was demonstrated with the identification of a conserved receptor-binding region on the side of three divergent CAR-binding knobs. However, the complexity of the fiber knob is such that modifications may destabilize the fiber with failure to trimerize, rendering the vector nonfunctional.24 A modified approach therefore incorporated the deletion of the entire knob domain of the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization function for the knobless fiber and specific binding to the target cell.25 These approaches have been tested extensively and demonstrated efficient transduction (up to a three log increase)26 of the desired targets including endothelial27 and smooth muscle cells,22 brain microcapillary bed,28 synovial cells29 and tumor cells.30 A Phase I clinical trial is underway in which an Ad vector genetically modified to target integrins via the Arg-Gly-Asp (RGD) peptide motif is being tested in ovarian cancer and recurrent cancer of the oral cavity.26

Ablation of native tropism requires the development of additional packaging cell lines and the size of the modifications to the fiber knob is restricted to about 30 amino acids. Even with these limitations and added complexities, the advantages of the single component system are that vector production and qualification for clinical use are more straightforward than the challenges of characterizing the heterogeneous mixture of vector, targeting ligand and vector-binding ligand of a two-component system. Advantages of the two-component system are that many receptors can be targeted and there is no need to genetically engineer the vector. Acommonly used example of the two-component method employs bispecific antibodies,31 one to the fiber and the other to a cell receptor expressed for example on tumor cells32 or cell surface antigen that is upregulated in angiogenic areas of tumors.33 This approach has been used frequently to demonstrate proof of concept of a given targeting ligand.

In addition to the viral particle targeting methods described above, the use of tissue-'specific' promoters can limit transcription to the tissue of interest.34 While this approach does not avoid vector interaction with the RES, it has provided an unexpected benefit where in the case of muscle or liver-specific expression, there was no detectable antibody response to the foreign transgene.35 Transgene expression in these instances was also prolonged.

These are but a fraction of the studies that have been published recently in the area of targeting of Ad vectors and it is clear that this intense effort will advance the field. It will be extremely important to evaluate the modified vectors comprehensively in vivo, as exemplified in the recent report from Smith et al,36 who demonstrated that it is not sufficient to simply mutate the CAR-binding sites in fiber knob to redirect transduction in vivo.36 These investigators mutated CAR binding in Ad5 fiber only to discover that upon injection into mice there was increased transduction in the liver with the modified vector. This observation may be related to CAR-independent transduction via heparin sulfate glycosoaminoglycans37 and may call into question the currently accepted cell entry pathway.

Advanced generations of Ad vectors provide increased persistence with reduced toxicity

Many applications of gene therapy will require lifelong transgene expression, but most current Ad vectors

result in an expression that is transient. Transgene expression is complex with at least three factors that can impact persistence including the expression cassette itself, the immune response to the vector andor the transgene product and turnover of the transduced cell. The importance of the expression cassette was demonstrated by De Geest et al,38 who reported that gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA and four apo E enhancers, all of human origin, resulted in human apo A-I expression above 20 mgdl for up to 6 months in the absence of significant hepatotoxicity.38 Another study concluded that 'strong promoters are the key to highly efficient, noninflammatory and noncytotoxic adenoviral-mediated transgene delivery into the brain in vivo'.39 These investigators demonstrated that by using a very strong promoter the required dose to achieve sufficient transduction could be reduced 100-fold, thereby reducing the toxicity. However, an often-ignored factor is what impact such a strong expression will have on normal cellular function, and this will likely vary between cell types.

It is widely held that transgene expression will decrease to approximately background levels within 3 weeks following administration of first-generation vectors. However, there are examples of first-generation vectors that provide prolonged expression.40 In addition, partially deleted vectors have resulted in persistent expression.41 It is therefore unclear how much of the viral genome must be deleted to achieve the desired length of expression.42 There may, in fact, be a benefit in retaining some viral genes as they function to modulate the cellular immune response.43 However, studies characterizing the function of the E4 gene products led the investigators to conclude that it is 'prudent' to remove all of the E4 region.44 It is important to remember that with each deletion of a viral gene, a cell line must be created to complement that gene45 and this can create production issues.

Helper-dependent, fully deleted Ad vectors offer great potential

With the recognition that viral gene expression even at low levels can lead to loss of transgene expression, a significant effort is in progress to develop Ad vectors that do not contain any viral genes. These are variously referred to as helper-dependent, high-capacity, gutted or gutless vectors, fully deleted and pseudo-adenovirus (PAV). Although these vectors do not avoid the innate response, many studies have demonstrated reduced toxicity with prolonged expression (for recent review see Kochanek et al46). The helper-dependent (HD) Ad vectors are produced with a helper Ad that provides the necessary viral functions, but cannot be packaged because the packaging signal is excised. This excision is typically mediated by a bacterial phage P1 Cre recombinase that recognizes loxP sites that flank the region essential for packaging. Another approach has been to employ a yeast recombinase (FLPe), where the loxP sites are replaced with FLPe recombinant targets (FRT).47,48 Umana et al47 report greater efficiency with the FLPe recombinase and claim that this should allow large-scale production of HD vectors using column chromatography-based virus purification. A challenge of the HD vector system is that the vector preparations are contaminated with low levels of helper Ad virus. Since the intent is to move away from a vector with any viral genes, there are concerns of what impact the contaminating virus will have on the toxicity and persistence of expression. One study attempted to address this by adding back increasing levels of helper virus and did not detect a decrease in persistence with levels up to 10%.49 A novel system for the production of HD vectors that does not require helper Ad, and is therefore not contaminated with helper virus, has been described.50 The adenoviral genes are delivered into producer cells by a baculovirusadenovirus hybrid, Bac-B4, carrying a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although this resulted in preparations of vectors that were free of contaminating Ad helper, scaling-up was prevented by the eventual emergence of replication-competent adenovirus (RCA). Further optimization of HD vector production that provides increased yields, minimal Ad helper, scalability and minimal levels of RCA has recently been described.51

HD vectors have been used in a variety of applications with an almost uniform improvement in persistence of transgene expression. Several of these were recently reviewed by Kochanek.46 For example, lifetime correction of hyperlipidemia was demonstrated in a mouse model.52 In this study, expression was detected for 2.5 years. In a mouse model of hemophilia, the defect was corrected for greater than 9 months with the expression of factor VIII.49 Along with the advantages of the increased persistence of transgene expression, HD vectors have a greater capacity of up to 36 kb. Although HD vectors have demonstrated advantages, they remain difficult to produce and purify in clinically relevant quantities and it is clear that advances in this area are still required before products can be introduced into the clinic.

Readministration with Ad vectors remains a challenge

Applications in which lifelong expression is necessary will require readministration of vector following the

eventual loss of therapeutic transgene expression. Without intervention or masking of the vector, the neutralizing antibody response to a previous exposure to Ad either through natural infection or administration of vector can preclude or significantly reduce effective readministration. However, several studies have documented effective readministration in certain limited applications. For example, an initial intramuscular administration of low-dose Ad vector producing low but detectable levels of transgene expression did not preclude readministration into the muscle, where systemic readministration was not effective.53 In a phase III trial for recurrent ovarian cancer where intraperitoneal readministration was used, transgene expression was measurable in 17 of 20 samples obtained after two or three cycles.54 Thus, it should be recognized that the ability to effectively repeat administer Ad vectors is dose-dependent and site-specific.

Since effective intravascular readministration in the absence of some intervention is unlikely, various approaches are being tested to circumvent the neutralizing response. A popular and straightforward approach that has been tested involves a serotype switch such that the second administration would not encounter a neutralizing response.55 There are, however, a finite number of serotypes and this approach would represent a complex set of clinical products. An intriguing approach attempts to physically remove the neutralizing antibodies. Plasmapheresis is a clinical procedure commonly used to reduce the antibody levels in patients with some autoimmune conditions. A method similar to this clinical practice was used to remove anti-Ad antibodies from serum by affinity chromatography and the resulting eluate was tested both in vivo and in vitro for neutralization capacity.56 These authors reported that depletion of antiadenoviral antibodies restores transduction in vivo during systemic Ad gene therapy in hosts previously exposed to adenovirus. Although this approach shows promise, it remains to be determined to what level the antibodies must be reduced and whether this is feasible in humans. Another approach has involved the masking of vectors to both reduce the response to the initial Ad treatment and evade the neutralizing response on subsequent exposures. Studies have employed a variety of molecules including polyethylene glycol polymers,57 multivalent hydrophilic polymers that can incorporate targeting ligands in addition to masking the vector58 and bilamellar cationic liposomes.59 These methods have potential, but remain to be fully characterized in terms of toxicity.

Immune intervention or modulation has also been used to avoid or minimize the neutralizing immune response. This approach seeks to minimize the response to the first Ad vector exposure, thereby permitting subsequent administrations. A straightforward approach involves pretreatment or coadministration of anti-inflammatory drugs such as steroids.16 A more complex method involves the administration of monoclonal antibodies directed against costimulatory signalling molecules that are required for the development of a fully mature immune response. One such approach employed antibodies directed against CD40, CD80B7.1 and CD86B7.2.60 This treatment reportedly fully abrogated the immune response against the Ad vector such that readministration was as effective as that in naïve animals. In addition, these authors claimed that the animals were capable of mounting a normal response to Ad. Another complex approach incorporates genes into the vector that upon expression will attenuate the immune response. This method was tested using two genes that block costimulatory signals that are required for an optimal immune response.61,62 These authors report that the immunomodulatory genes CTLA4Ig and CD40Ig included in one HD vector and coadministered with an Ad vector coding for a reporter gene resulted in higher levels of transgene expression, lower levels of anti-Ad antibody and more prolonged transgene expression, when compared to controls. However, prolonged expression of these immunomodulatory genes compromised the host immune response. Other investigators have incorporated the immune modulatory gene CTLA4Ig into the vector containing the transgene of interest and claimed that this vector was more effective in suppression of the immune response than the two-vector system.63 They speculated that high levels of local expression may be required to completely block both humoral and cellular responses.

Summary

Although the utility of Ad vectors for use in clinics has been seriously challenged in the last 2 years, they remain viable, if not preferred, candidates for some gene therapy applications. With a better understanding of the toxic response to Ad vectors, investigators have begun to utilize a variety of methods to attenuate or avoid this potentially lethal response. Future Ad vectors incorporating features outlined in this review would be fully deleted nonhuman Ads, modified to target away from the RES in favor of the organ of interest and carry, in addition to the gene of interest under control of a tissue-specific promoter, controllable immunomodulatory gene(s) that would attenuate the immune response.

References

1 Makinen K et al. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002; 6: 127133. Article PubMed

2 Assessment of adenoviral vector safety and toxicity: report of the National Institutes of Health Recombinant DNA Advisory Committee. Hum Gene Ther 2002; 13: 313.

3 Zhang Y et al. Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages. Mol Ther 2001; 3: 697707. Article PubMed

4 Higginbotham JN, Seth P, Blaese RM, Ramsey WJ. The release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid. Hum Gene Ther 2002; 13: 129141.

5 Borgland SL et al. Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB. J Virol 2000; 74: 39413947. PubMed

6 Cichon G et al. Complement activation by recombinant adenoviruses. Gene Therapy 2001; 8: 17941800.

7 Morelli AE et al. Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway. J Virol 2000; 74: 96179628. Article PubMed

8 Schnell MA et al. Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors. Mol Ther 2001; 3(Part 1): 708722. Article PubMed

9 Morral N et al. Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons. Hum Gene Ther 2002; 13: 143154. Article PubMed

10 Raper SE et al. A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency. Hum Gene Ther 2002; 13: 163175. Article

11 Eggerman TL, Mondoro TH, Lozier JN, Vostal JG. Adenoviral vectors do not induce, inhibit, or potentiate human platelet

aggregation. Hum Gene Ther 2002; 13: 125128.

12 Lozier JN et al. Toxicity of a first-generation adenoviral vector in rhesus macaques. Hum Gene Ther 2002; 13: 113124. Article PubMed

13 Crystal RG et al. Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions. Hum Gene Ther 2002; 13: 65100.

14 Harvey BG et al. Safety of local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions. Hum Gene Ther 2002; 13: 1563.

15 Ben Gary H et al. Systemic interleukin-6 responses following administration of adenovirus gene transfer vectors to humans by different routes. Mol Ther 2002; 6: 287297.

16 Kolb M et al. Budesonide enhances repeated gene transfer and expression in the lung with adenoviral vectors. Am J Respir Crit Care Med 2001; 164: 866872. PubMed

17 Nemerow GR. Adenoviral vectors new insights. Trends Microbiol 2000; 8: 391394.

18 Wickham TJ. Ligand-directed targeting of genes to the site of disease. Nat Med 2003; 9: 135139. Article PubMed

19 Krasnykh VN, Douglas JT, van Beusechem VW. Genetic targeting of adenoviral vectors. Mol Ther 2000; 1(Part 1): 391405. Article PubMed

20 Ostapchuk P, Hearing P. Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E. J Virol 2001; 75: 4551.

21 Havenga MJ et al. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J Virol 2002; 76: 46124620. Article

22 Su EJ et al. A genetically modified adenoviral vector exhibits enhanced gene transfer of human smooth muscle cells. J Vasc Res 2001; 38: 471478.

23 Loser P et al. Ovine adenovirus vectors mediate efficient gene transfer to skeletal muscle. Gene Therapy 2000; 7: 14911498.

24 Magnusson MK, Hong SS, Boulanger P, Lindholm L. Genetic

retargeting of adenovirus: novel strategy employing 'deknobbing' of the fiber. J Virol 2001; 75: 72807289.

25 van Beusechem VW et al. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer. Gene Therapy 2000; 7: 19401946.

26 Barnett BG, Crews CJ, Douglas JT. Targeted adenoviral vectors. Biochim Biophys Acta 2002; 1575: 114. PubMed

27 Nicklin SA et al. Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus. Mol Ther 2001; 4: 534542. Article

28 Xia H, Anderson B, Mao Q, Davidson BL. Recombinant human adenovirus: targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium. J Virol 2000; 74: 1135911366. Article PubMed

29 Bakker AC et al. A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis. Gene Therapy 2001; 8: 17851793. Article

30 Biermann V et al. Targeting of high-capacity adenoviral vectors. Hum Gene Ther 2001; 12: 17571769.

31 Levy RJ et al. Localized adenovirus gene delivery using antiviral IgG complexation. Gene Therapy 2001; 8: 659667.

32 Israel BF et al. Enhancement of adenovirus vector entry into CD70-positive B-cell lines by using a bispecific CD70-adenovirus fiber antibody. J Virol 2001; 75: 52155221.

33 Nettelbeck DM et al. Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105). Mol Ther 2001; 3: 882891. Article PubMed

34 Barnett BG, Tillman BW, Curiel DT, Douglas JT. Dual targeting of adenoviral vectors at the levels of transduction and transcription enhances the specificity of gene expression in cancer cells. Mol Ther 2002; 6: 377385.

35 Hartigan-O'Connor D et al. Immune evasion by muscle-specific gene expression in dystrophic muscle. Mol Ther 2001; 4: 525533. Article PubMed

36 Smith T et al. In vivo hepatic adenoviral gene delivery occurs independently of the coxsackievirusadenovirus receptor. Mol Ther

2002; 5: 770779.

37 Dechecchi MC et al. Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5. J Virol 2001; 75: 87728780. Article PubMed

38 De Geest B et al. Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer. Hum Gene Ther 2000; 11: 101112. Article PubMed

39 Gerdes CA, Castro MG, Lowenstein PR. Strong promoters are the key to highly efficient, noninflammatory and noncytotoxic adenoviral-mediated transgene delivery into the brain in vivo. Mol Ther 2000; 2: 330338. Article PubMed

40 Ehrhardt A, Kay MA. A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood 2002; 99: 39233930. Article PubMed

41 Ding EY et al. Long-term efficacy after E1-, polymerase- adenovirus-mediated transfer of human acid-alpha-glucosidase gene into glycogen storage disease type II knockout mice. Hum Gene Ther 2001; 12: 955965.

42 Andrews JL et al. Generation and characterization of E1E2aE3E4-deficient adenoviral vectors encoding human factor VIII. Mol Ther 2001; 3: 329336. Article PubMed

43 Braithwaite AW, Russell IA. Induction of cell death by adenoviruses. Apoptosis 2001; 6: 359370.

44 Tauber B, Dobner T. Molecular regulation and biological function of adenovirus early genes: the E4 ORFs. Gene 2001; 278: 123.

45 Zhou H, Beaudet AL. A new vector system with inducible E2a cell line for production of higher titer and safer adenoviral vectors. Virology 2000; 275: 348357.

46 Kochanek S, Schiedner G, Volpers C. High-capacity 'gutless' adenoviral vectors. Curr Opin Mol Ther 2001; 3: 454463.

47 Umana P et al. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat Biotechnol 2001; 19: 582585.

48 Ng P et al. Development of a FLPfrt system for generating helper-dependent adenoviral vectors. Mol Ther 2001; 3(Part 1):

809815. Article PubMed

49 Reddy PS et al. Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector. Mol Ther 2002; 5: 6373.

50 Cheshenko N, Krougliak N, Eisensmith RC, Krougliak VA. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Therapy 2001; 8: 846854.

51 Sakhuja K et al. Optimization of the generation and propagation of gutless adenoviral vectors. Hum Gene Ther 2003; 14: 243254.

52 Kim IH et al. Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector. Proc Natl Acad Sci USA 2001; 98: 1328213287. Article PubMed

53 Chen P, Kovesdi I, Bruder JT. Effective repeat administration with adenovirus vectors to the muscle. Gene Therapy 2000; 7: 587595. PubMed

54 Buller RE et al. A phase III trial of rAdp53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Ther 2002; 9: 553566.

55 Moffatt S, Hays J, HogenEsch H, Mittal SK. Circumvention of vector-specific neutralizing antibody response by alternating use of human and non-human adenoviruses: implications in gene therapy. Virology 2000; 272: 159167. Article

56 Rahman A et al. Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy. Mol Ther 2001; 3(Part 1): 768778.

57 Croyle MA, Chirmule N, Zhang Y, Wilson JM. 'Stealth' adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 2001; 75: 47924801. Article PubMed

58 Fisher KD et al. Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies. Gene Therapy 2001; 8: 341348.

59 Yotnda P et al. Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses. Mol Ther 2002; 5: 233241. Article PubMed

60 Ziller C, Stoeckel F, Boon L, Haegel-Kronenberger H.

Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector. Gene Therapy 2002; 9: 537546.

61 Jiang Z, Feingold E, Kochanek S, Clemens PR. Systemic delivery of a high-capacity adenoviral vector expressing mouse CTLA4Ig improves skeletal muscle gene therapy. Mol Ther 2002; 6: 369376.

62 Jiang ZL et al. Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle. Mol Ther 2001; 3: 892900. Article

63 Thummala NR et al. A non-immunogenic adenoviral vector, coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term, repeatable transgene expression in the Gunn rat model of CriglerNajjar syndrome. Gene Therapy 2002; 9: 981990.

Tables

Table 1 Systemic Ad vector administration in primates

June 2003, Volume 10, Number 12, Pages 999-1003

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ReviewGene therapy Progress and Prospects: Gene therapy for the hemophilias

Christopher E Walsh MD, Ph,D1

1Mt Sinai School of Medicine, One Gustave Levy Pl, New York City, NY, USA

Correspondence to: Dr CE Walsh, Mt Sinai School of Medicine, One Gustave Levy Pl, Rm 24-42C Annenberg Bldg, New York City, NY 10029, USA

Abstract

Recent gene transfer trials for hemophilia A and B, bleeding disorders lacking either functional factor VIII or IX, respectively, have produced tantalizing results, suggesting that the potential to correct these bleeding disorders at a molecular level may be at hand. Genetic correction of the hemophilias represents a model system to develop a basic understanding of how gene therapy will be achieved. The goals for hemophilia gene transfer require the long-term therapeutic production of the coagulant protein without stimulating an immune response to the transgene product or the vector. Based on a scientific understanding of the molecular and cellular defects, leading to the bleeding phenotype, impressive strides have been made in the last 2 years.

Gene Therapy (2003) 10, 9991003. doi:10.1038/sj.gt.3302024

Keywords

hemophilia, gene transfer, gene repair, stem cells, viral vectors

In brief

Progress

New AAV serotypes and lentiviral vectors have recently been studied for the production of factor VIII or IX.

Organs other than the liver can be used as 'factories' for factor VIII and IX production.

The immune response to endogenous factor synthesis and viral vectors is a potential problem in hemophilia gene transfer.

Hemophilia clinical trials using either retrovirus, AAV or ex vivo transfected fibroblasts have been carried out or are ongoing.

Prospects

Development of improved lentiviral, gutless adenovirus and alternate AAV serotype vectors is in progress

Gene repair, RNA repair (Trans-splicing) offer opportunities for the treatment of hemophila.

Gene-modified circulating endothelial progenitor may prove useful in the future.

Gene-modified stem cell therapy may become available.

Introduction

Current treatment for hemophila-related bleeding episodes utilizes intravenous infusion of purified and recombinant factor protein, which is effective, yet transient because of the short half-life of the proteins. This treatment is expensive, restricts the prophylactic use of factors and can lead to crippling joint disease and susceptibility to infectious agents. Effective hemophilia gene transfer requires that a sustained, long-term (years) production of coagulation factor at therapeutic levels be generated. Thus, the method of gene delivery must be safe, and the risk of immune response to potential neoantigens must be minimal. Given recent scientifictechnical developments, genetic correction of hemophilic patients is now viewed as an achievable goal.

The factor VIII and IX gene and protein products have been extensively studied.1,2 Many tissues and cell types (skeletal muscle, liver, spleen and skin) are capable of producing and expressing fully modified and functional factor IX protein. A therapeutic factor level is considered to be 2% of normal levels. This is sufficient to convert a severely affected patient with frequent spontaneous bleeding episodes (patients with <1% factor level) to a moderate or mildly affected level. Coagulation assays are standardized and animal models (knockout mice and hemophilic canines) that mimic the human phenotype are available for testing. A variety of gene transfer approaches are currently being tested both in the laboratory and in the clinic. Results of two clinical trials, using nonviral and viral-based gene transfer approaches, suggest that

despite low factor levels, patients required less factor infusion and reported fewer bleeding episodes. Although neither trial included a placebo arm, these results enforce clinical observations that low levels of factor dramatically reduce spontaneous bleeding. Despite the current excitement, results point to the need for improved vectors. Here, we will review the recent advances over the past 2 years in this field that mirrors the advances in the field of gene transfer in general.

New adeno-associated virus (AAV) serotypes and lentiviral vectors have recently been studied for the production of factor VIII or IX

In general, viral vectors exhibit long-term gene expression (years), whereas nonviral methods produce transient (weeks to months) factor expression. Hemostatic levels of factors VIII and IX were reached with first- and second-generation adenovirus vectors. Unfortunately, the exuberant cell-mediated immune response engendered by this vector leads to inflammatory response directed at transduced cells with the attendant loss of protein expression. Newer gutless adenovirus with a minimum of endogenous adenoviral genes may limit the immune response, but as a consequence gene expression is drastically reduced. AAV has recently come to the fore based on its now relative ease in preparation, ability to infect both dividing and nondividing cells,3 and although it engenders a humoral immune response, it does not stimulate a cytotoxic lymphocyte response. Using this vector, therapeutic levels of factors IX and VIII were demonstrated in knockout mouse and hemophilic canines. Eight serotypes of AAV, have been isolated and cloned (AAV1-8). Of these AAV, type 2 was the first cloned and most extensively studied. Surprisingly, other serotypes yield factor IX at levels two logs greater than AAV-2 following skeletal muscle injection into mice4 and produce sustained supratherapeutic factor levels leading to complete loss of the bleeding diathesis.5 Such levels are achieved as a result of more efficient effective skeletal muscle gene transfer. Here, a linear relation exists between input vector and factor expression. A unique side benefit is lack of an immune inhibitor response presumably because of continuous production of factor as the major determinant for inducing tolerance. This result is reminiscent of immune tolerance strategies currently used in the clinic, performed by repeated infusion of factor.

In particular, AAV1 produces robust transgene expression in muscle, but the exact mechanism is unclear. Data using AAV1EGFP suggested that the number of transduced muscle fibers infected increases significantly with AAV1 compared to AAV2 (see Figure 1). A linear relation between circulating levels of canine factor IX protein and AAV1 dose suggests that this result is most likely because of the number of myocytes productively infected. One working hypothesis explaining the serotype transduction differences lies in the level of virus specificity for binding cell receptors. At present, the receptor for AAV type 1 has not been identified but appears distinct from type 2, that is, not inhibited by heparin.

AAV serotypes 7 and 8 recently isolated from primates infected with high-dose adenovirus 6 are not neutralized by heterologous antisera raised to the other serotypes. Recombinant serotypes 7 and 8 vector particles carrying the alpha-1-antitrypsin cDNA were compared for transducing effectiveness in mice. AAV7 was equivalent to AAV1 in efficient expression in skeletal muscle, whereas AAV8 expressed at a 10 to 100-fold greater rate in liver-directed expression than all other serotypes. These data confirm that relatively small differences in the capsid structure produce striking differences in transgene expression in a wide variety of tissues.

Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. One study used human immunodeficiency virus (HIV)-based lentiviral vectors containing human factors VIII or IX cDNA expression for portal vein injection into C57Bl6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 5060 ngml. Partial hepatectomy resulted in a 4- to 6-fold increase in serum hFIX of up to 350 ngml compared with the nonhepatectomized animals. The expression of plasma hFVIII reached 30 ngml (15% of normal), but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies.7

Owing to the potential safety issues using an HIV-based vector, an alternate approach is to utilize plasmid-based approaches that carry genetic elements that promote integration. Early attempts using such a system have provided encouraging results.8

Organs other than the liver can be used as sources for factor VIII and IX production

The liver is the principal organ synthesizing the coagulation factors. However, other organs can synthesize factors VIII and factor IX. Factor IX can be expressed from skeletal muscle, fibroblasts, kerintinocytes, intestinal mucosa, cells lining the amniotic cavity and marrow stroma.9 Factor VIII transgene expressing circulating endothelial cells are capable of secreting high levels of factor VIII for a sustained period in

animal models.10 Circulating endothelial cells obtained from peripheral blood are expanded ex vivo and then genetically modified to express a gene of interest. The biochemical elements necessary for high-level factor expression including the endogenous coexpression of factor VIII and vWF may explain the high factor levels observed in vivo. The regulation of infused endothelial precursor cell growth kinetics and half-life of fully differentiated endothelium remains to be determined. Ex vivo gene transfer of hematopoietic progenitor cells and marrow stroma are also capable of factor VIII secretion in vivo.11

The immune response to endogenous factor synthesis and viral vectors is a potential problem in hemophilia gene transfer

The antibody responses to exogenous factor replacement, termed inhibitors, effect nearly 20% of factor VIII patients and 3% of factor IX patients. Inhibitory antibodies that bind to the particular regions of the factor molecule inactivate by changing factor protein conformation.2 In general, the immune response may in part be related to the type of mutation. For example, a large deletion in the factor VIII gene and complete loss of protein typically leads to a greater incidence of inhibitor formation. Bleeding episodes of patients with inhibitors are difficult to manage, relying on activated bypass factors and recombinant factor VIIa.12 Will a constant source of factor engender high titer inhibitory antibodies negating any positive benefit and with consequences of worsening bleeding? The clinical trials described below carefully screen for noninhibitor patients or patients with frequent infusions where the chances of inhibitor are reduced. CD4+ subset activation in humans and Th-1 and Th-2 lymphocytes in mice suggests that both MHC class I and II mechanisms are involved. Involvement of both central (marrow, thymus) and peripheral (lymph nodes, Peyers patch) tolerance mechanisms to factor VIII is described but poorly understood.13,14,15 In addition to the immune response to the transgene factor proteins, immune response to viral vectors is well described for adenovirus and AAV and thus prevent readministration of vector.

Hemophilia clinical trials using either retrovirus, AAV or ex vivo transfected fibroblasts have been carried out or are ongoing

Within the past 2 years, five gene transfer trials were approved (three for hemophilia A, two for hemophilia B) in the US. Biotech firms that developed vectors specifically for factors VIII and IX sponsor all five trials. In a phase I dose escalation study, 13 subjects with hemophilia A received by peripheral intravenous infusion an amphotropic retroviral vector carrying a B-domain deleted human factor VIII gene. Infusions were administered to patients with HIV and HCV infections and were well-tolerated. Factor VIII was measured and no subject had sustained repeated FVIII levels >1% of normal levels. Patients were treated with vector ranging from 3 107 to 9 108 vpkg. Overall, there was no significant change in bleeding frequency. And there was no correlation between vector dose and time to FVIII activity response. This clinical outcome is consistent with the limited capability of retroviral integration into nondividing liver cells and the lack of a liver-specific promoter in the retroviral vector used.

A trial using an AAV2 vector carrying the human factor IX cDNA injected intramuscularly was carried out in eight patients in a dose escalation study.16,17 One patient receiving the lowest dose (2 1011 vpkg) was reported to maintain factor levels at 12% and reported a 50% reduction in factor usage and bleeding episodes for a period up to 40 months postinjection. No evidence of inhibitor was reported despite preclinical data in dogs of a transient inhibitory response. Virus dissemination was transiently detectable in all body fluids, excluding semen. At higher doses, no significant plasma factor levels were reported. However, reduced frequency of factor usage was one end point signifying treatment effectiveness. Molecular analysis of virus dissemination was detectable in all body fluids (saliva, blood and urine) but not in germ cells. All patients had low (1:1001000) preinjection anti-AAV2 neutralizing titers that increased after vector administration. An increase in the number of injection sites from 10 to 90 produced no significant increase in factor level. High titer neutralizing antibodies to AAV developed in all patients at levels sufficient to preclude readministration of vector. Muscle biopsy confirmed previous observations in animals that slow twitch muscle fibers expressed factor IX.

A dose-escalation study based on AAV2 vectors carrying human factor IX cassettes delivered via the hepatic artery has begun. Two patients received the lowest dose of virus (2 1011kg) via intra-arterial delivery without adverse effects. However, the trial was temporarily halted because of detection of the

transgene in seminal fluid. Trial resumption was based on data that germ cells were not infected with the virus. No detectable levels of factor FIX were observed above background in the two patients receiving the lowest dose of vector. Two patients received moderate doses of vector (1 1012kg). One patient developed FIX levels up to 1012% within 23 weeks after injection; however, factor levels subsequently dropped coincident with an elevation of the liver transaminases. No data were presented on the second patient. Whether this represents toxicity of the vector at this dose remains to be determined. It is interesting to note that based on doseresponse experiments in hemophilic dogs, this moderate dose of virus produced 414% of canine FIX for 12 years without significant toxicity.

A nonviral approach used a factor VIII plasmid electroporated into autologous skin fibroblasts: cells were expanded in vitro and 100 or 400 million cells injected into the greater omentum.18 Levels of factor VIII above pretreatment levels were measured in four of six patients with either a concommitant reduction in the use of recombinant factor VIII or decreased number of spontaneous bleeding episodes. However, factor VIII decreased to pretreatment levels in all the patients after 12 months. The explanation for the decline in factor expression may have been because of gene silencing, immunological clearance or senescence of the fibroblasts after reimplantation.

What do these clinical results tell us? No significant toxicity in any of the patients was reported. Furthermore, the factor levels predicted from animal models were not observed in patients. Thus, although testing of new factor proteins in hemophilic animals is traditionally used because of similar pharmacokinetic profiles seen in man, such extrapolation using gene transfer vectors may not be clear-cut. A review of the preclinical data also suggests that animal studies may not be predictive of the clinical outcome. For example, vector dosing based on a vector particle-to-weight ratio produced discrepant results when comparing equivalent AAV vector dosing in mice and hemophilic dogs. Whereas experiments in hemophilic mice are dose-dependent and can produce supraphysiologic levels of factor IX (300% of normal), equivalent doses in hemophilic dogs produce factor IX at levels around 5% of normal and do not appear to be dose-dependent.19 Recent data testing AAV2human factor IX vectors in non-human primates produced 410% factor IX,20 similar to data generated in hemophilic dogs,21 for a period of 1 year. These outcomes reflect species differences in terms of the rate of cell infectivity, gene expression, protein modification and processing. However, testing in different animal models serves to confirm the validity of each new approach.

Future prospects

RNA repair (Trans-splicing) offers opportunities for the treatment of hemophila

A novel approach for genetic correction involves the use of premessenger RNA (pre-m-RNA) repair. RNA trans-splicing utilizes endogenous splicing mechanisms to correct a portion of the defective RNA. A pre-mRNA is designed to base pair with a pre-mRNA transcribed from the defective gene. The pre-mRNA also contains all the requisite splicing signals that allows two independent mRNAs to splice together, resulting in a correct copy of mRNA that is translated into a normal protein.22 The advantage of this system is that large genes, unable to be packaged into viral vectors, or genes that contain large regulatory elements could be corrected by using the smaller spliced sequences. We developed such a system for factor VIII correction . Using the factor VIII exon 16 knockout mice as a model to test trans-splicing, we demonstrated that by injecting plasmid or AAV carrying pre-mRNA encoding for exons 1626 around 26% of factor VIII, that prevented bleeding challenge, was generated for 35 days and 34 months, respectively (Chao et al, submitted). As trans-splicing efficiency improves, this may be useful for the treatment of autosomal dominant disorders involving other coagulation and thrombotic defects.

Gene-modified circulating endothelial progenitor may prove useful in the future

The use of blood outgrowth endothelial cells (BOEC) as a source of cells synthesizing factor VIII has been described.10 These circulating endothelial progenitor cells are isolated from peripheral blood, expanded in culture, and modified genetically to carry the normal FVIII gene. A significant advantage includes the expansion of BOEC clones that synthesize vWF, the carrier protein necessary for FVIII stability in plasma. Major questions of BOEC use include the half-life of these cells in vivo, and potential for uncontrolled growth following transplantation.

Gene-modified stem cell therapy may become available

Recent reports on the plasticity of stem cells derived from adult tissue forming liver, brain, muscle, skin,

fat have generated enormous interest on their use for the genetic correction of hemophilia. A multipotential subset of mesenchymal stem (MAPC) cells derived from marrow stroma can be induced to differentiate into cell types with neuroectoderm, endoderm and mesoderm characteristics.23 When MAPCs are injected into irradiated animals, they differentiate into hematopoietic lineages as well as epithelium of the liver, gut and lung. Potentially MAPCs could be genetically modified to synthesize coagulation factors before re-transplantation. Advantages include ex vivo expansion and gene modification with selected clones producing high levels of factor. Autologous stem cells derived from each patient would avoid transplantation rejection and immunosuppression. Current disadvantages include the long lead time (months) required to generate the number of cells for transplantation and the ability to control the differentiated fates of the transplanted multipotential cells.

Summary

A body of data suggests that genetic correction of the hemophilias is feasible. The subjective reporting by patients of decreased bleeding episodes and an apparent self-declared reduction in bleeding episodes at nominal levels of factor strongly hint that reasonable factor levels if reached will achieve a major breakthrough in the treatment of hemophilia. Hemophilia gene transfer represents the combination of vector delivery systems, animal models and clinical studies designed to answer specific questions. Not only will these studies benefit hemophilic patients, but should also instruct others in the field as well. Hopefully, this work will represent a milestone in the use of genetics for treatment of human ailments.

References

1 Kaufman R, Antonarakis S. Structure, biology, and genetics of factor VIII, In: Hoffman R, Benz EJ, Shattil S, Furie B, Cohen H, Silberstein L, McGlave P (eds). Hematology: Basic Principles and Practice, Vol. VIII-108, 3rd edn. Church Livingstone: New York, 2000, pp 18501868.

2 Lillicrap D. Hemophilia treatment. Gene therapy, factor VIII antibodies and immune tolerance: hopes and concerns. Haematologica 85 (Suppl 10): 2000.

3 Monahan P, Samulski R. AAV vectors: is clinical success on the horizon? Gene Therapy 2000;7:2430. Article PubMed

4 Chao H et al. Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors. Mol Ther 2000;2:619623. Article PubMed

5 Chao H et al. Sustained and complete phenotype correction of hemophilia b mice following intramuscular injection of aav1 serotype vectors. Mol Ther 2001;4:217222. Article

6 Gao G et al. Novel adeno-associated viruses from rhesus monkeys

as vectors for human gene therapy. Proc Natl Acad Sci USA 2002;99:1185411859. Article PubMed

7 Park F, Ohashi K, Kay M. Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 2000;96:11731176. PubMed

8 Yant S et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 2000;25:3541. Article PubMed

9 Krebsbach P, Zhang K, Malik A, Kurachi K. Bone marrow stromal cells as a genetic platformfor systemic delivery of therapeutic proteins in vivo: human factor IX model. J Gene Med 2003;5:1117. Article

10 Lin Y et al. Use of blood outgrowth endothelial cells for gene therapy for hemophilia A. Blood 2002;99:457462. Article

11 Chuah M et al. Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum Gene Ther 2000;11:729738. Article PubMed

12 Poon M. Use of recombinant factor VIIa in hereditary bleeding disorders. Curr Opin Hematol 2001;8:312318. Article

13 Chao H, Walsh C. Induction of tolerance to human factor VIII in mice. Blood 2001;97:33113312. Article

14 Qian J, Collins M, Sharpe A, Hoyer L. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000;95:13241329.

15 Brown B, Lillicrap D. Dangerous liaisons: the role of 'danger' signals in the immune response to gene therapy. Blood 2002;100:11331140. Article

16 Kay M et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000;24:257261. Article PubMed

17 Manno C et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood, Prepublished online Dec. 19, 2002.

18 Roth D et al. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A. N Engl J Med

2001;344:17351742. Article PubMed

19 Wang L et al. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol Ther 2000;1:154158. Article PubMed

20 Nathwani A et al. Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques. Blood 2002;100:16621669. Article

21 Mount J et al. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy. Blood 2002;99:26702676. Article

22 Puttaraju M et al. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol Ther 2001;4:105114. Article PubMed

23 Jiang Y et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:4149. Article PubMed

Figures

Figure 1 AAV serotype transduction of skeletal muscle. Fluorecence of skeletal muscle samples following injection of equivalent doses of serotype AAV1 and two vectors carrying an EGFP expression cassette. A uniform fluorescence pattern is observed with AAV1 compared to the patchy appearance of AAV2.

April 2003, Volume 10, Number 8, Pages 605-611

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ReviewGene Therapy Progress and Prospects: Gene therapy in organ transplantation

J Bagley1 and J Iacomini1

1Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, MGH-East, Boston, MA 02129, USA

Correspondence to: Dr J Iacomini, Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, MGH-East, Building 149, 13th Street, Boston, MA 02129, USA

Abstract

One major complication facing organ transplant recipients is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with an increased incidence of malignancy and susceptibility to opportunistic infections. Gene therapy has the potential to eliminate problems associated with immunosuppression by allowing the production of immunomodulatory proteins in the donor grafts resulting in local rather than systemic immunosuppression. Alternatively, gene therapy approaches could eliminate the requirement for general immunosuppression by allowing the induction of donor-specific tolerance. Gene therapy interventions may also be able to prevent graft damage owing to nonimmune-mediated graft loss or injury and prevent chronic rejection. This review will focus on recent progress in preventing transplant rejection by gene therapy.

Gene Therapy (2003) 10, 605611. doi:10.1038/sj.gt.3302020

Keywords

transplantation; gene transfer; tolerance

In brief

Progress

Gene therapy-mediated CD28B7 costimulatory blockade can prolong graft survival, but results in nonspecific immunosuppression.

Gene therapy-mediated CD40CD154 costimulatory blockade can prevent acute allograft rejection, but does not prevent chronic alloreactivity.

Hyporesponsiveness to virally encoded MHC genes has been induced in both rodent and large animal models.

Induction of stable long-term T-cell tolerance to MHC class I antigens through central deletion of alloreactive T cells following induction of molecular chimerism.

Evidence for induction of regulatory cells that can prevent allograft rejection in gene therapy models.

Induction of B-cell tolerance by molecular chimerism.

Induction of tolerance in sensitized recipients.

Gene therapy-mediated cytokine production, or immune deviation, prolongs graft survival, but does not prevent rejection.

Transduction of dendritic cells with genes encoding immunomodulatory proteins can moderately prolong graft survival.

Improved graft survival through antiapoptotic and antiproliferative gene therapy.

Prospects

Better understanding of activation of alloreactive cells will allow for the development of more targeted gene therapy aimed at preventing this activation.

Increased understanding of costimulatory pathways will reveal additional targets for gene therapy interventions.

Elucidation of the nature of regulatory cells may lead to gene therapy approaches designed to efficiently generate these populations.

Induction of tolerance through molecular chimerism will be tested in large animal models.

Development of improved vectors for transduction of terminally differentiated tissue will improve efficacy of gene therapy approaches that depend on intracellular protein expression.

Gene therapy-mediated CD28B7 costimulatory blockade can prolong graft survival, but results in nonspecific immunosuppression

The major effectors of transplant rejection are host CD4 and CD8 T cells. In order for T cells to become activated and participate in transplant rejection, they must first receive a signal through the T-cell receptor (TCR), which occurs after either direct or indirect recognition of alloantigens on the surface of antigen-presenting cells (APCs). However, in order for T cells to become fully activated and acquire effector function, such as the ability to produce cytokines, they must also receive signals from the interaction of costimulatory molecules expressed on their surface with ligands expressed on APCs. Costimulatory molecule interactions can result in T-cell activation or inhibition. For example, following TCR ligation, the interaction of the costimulatory molecule CD28 expressed on T cells with CD80 and CD86 (B7.1 and B7.2, respectively) expressed on APCs results in T-cell activation (reviewed in Sharpe and Freeman1). In contrast, signaling through cytolytic T-lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells following ligation by CD80 and CD86 downregulates immune responses.

T cell costimulatory pathways are central to T-cell activation, and therefore several groups have tried to block or manipulate these pathways using gene therapy approaches in order to prevent T-cell responses. Blocking the interaction of CD28 with CD80 and CD86 using an immunoglobulin fusion protein containing the extracellular portion of CTLA-4 (CTLA-4Ig) can result in immunosuppression in vivo, and prevent transplant rejection. To overcome systemic immunosuppression as a consequence of CTLA-4Ig administration, groups have tried to prevent transplant rejection by expressing CTLA-4Ig locally within transplanted tissues or organs. Expression of the gene encoding CTLA-4Ig in donor organs has generally resulted in the prolongation of transplant survival, but has not permitted permanent acceptance (reviewed in Guillot et al2). Recently, indefinite graft survival was achieved in a cardiac transplantation model.3 However, in this model, it is likely that the transplanted organs themselves may have participated in the maintenance of hyporesponsiveness that was initially induced by transient expression of CTLA-4Ig. Indeed, cardiac allografts themselves can be tolerogenic. In experimental systems involving transplantation of tissues expressing CTLA-4Ig which are not thought to be tolerogenic, such as skin,4 hepatocytes,5 corneal grafts,6 and fetal cardiomyocytes,7 only modest graft prolongation was achieved. In these studies, graft rejection was associated with a loss or decrease in gene expression.

Analysis of rat cardiac allografts expressing adenovirus encoded CTLA-4Ig revealed that while permanent acceptance of the genetically modified transplants could be achieved in allogeneic hosts, acceptance was associated with nonspecific inhibition of T-cell responses to unrelated third-party antigens.3 Responses to third-party antigens were diminished immediately after transplantation, and hyporesponsiveness was apparent even after a five-fold reduction in serum levels of CTLA-4Ig was observed 120 days after transplantation. These data suggest that intragraft expression of CTLA-4Ig can result in systemic immunosuppression, and therefore long-term expression of this gene product may be detrimental to host immunity. In an attempt to overcome the long-term immunosuppressive effects of CTLA-4Ig expression, adenovirus vectors carrying the gene encoding CTLA-4Ig flanked by two loxP sequences were developed.8 Following intravenous injection of adenoviruses carrying this novel CTLA-4Ig gene construct, subsequent administration of adenoviruses carrying the gene encoding Cre recombinase permitted excision of the CTLA-4Ig gene, terminating expression in vivo and vitro. Pancreatic islets transplanted into the liver of mice receiving loxP-flanked adenovirus encoded CTLA-4Ig remained functional 40 days after serum CTLA-4Ig was no longer detectable following Cre-mediated excision of the CTLA-4Ig gene.8 However, long-term islet function after cessation of CTLA-4Ig expression was not examined, so it is not possible to determine if long-term survival occurred. Skin allograft survival was also prolonged, however, all skin grafts were eventually rejected. In addition, Cre-mediated deletion of CTLA-4Ig was associated with the restoration of responses to adenovirus, suggesting that long-term hyporesponsiveness was averted using this approach, effectively allowing one to control the duration of immunosuppression.

Gene therapy-mediated CD40CD154 (CD40 ligand) costimulatory blockade can prevent acute allograft

rejection, but does not prevent chronic alloreactivity

In addition to the CD28-CD8086 costimulatory pathway discussed above, the interaction of CD40 ligand (CD154) expressed on T cells with CD40 on APCs has also been shown to be an important component in the initiation and maintenance of T-cell responses. Gene therapy approaches have been developed in which an adenovirus encoded CD40-Ig fusion protein is used to block intragraft CD40-CD154 interactions in order to prevent graft rejection. Adenoviral-mediated transfer of the gene encoding CD40-Ig into rat livers resulted in long-term survival following transplantation into allogeneic recipients.9 Immunocompetence was tested by analyzing skin allograft survival 120 days after liver transplanta-tion, long after CD40-Ig production had ceased. Therefore, it is unclear whether recipients were able to respond to third-party antigens at earlier time points while CD40-Ig was expressed in the transplanted livers. Expression of CD40-Ig in rat cardiac transplants has also been shown to delay acute rejection, although, at early time points after transplantation, recipients of gene-modified hearts exhibited nonspecific immunosuppression.10 At later time points, nonspecific immuno-suppression abated and responses to third-party alloantigens were restored. However, when immune responses to third-party antigens were restored, antidonor T-cell activity returned in these animals, and was associated with chronic rejection.10 The development of chronic rejection suggests that intragraft expression of CD40-Ig needs to be complemented by other therapeutic strategies to obtain long-term transplant survival.

Several other molecules involved in T-cell costimulation have recently been identified, but the function of many newly discovered costimulatory pathways has not been fully elucidated.1,11 However, it is becoming apparent that other costimulatory pathways, such as the inducible costimulator (ICOS)-B7 related protein-1 (B7RP-1) pathway, are important for the activation of effector T cells.12 Indeed, it has been shown that an ICOS-Ig recombinant protein used in conjunction with the CD40CD154 blockade may prevent chronic rejection of cardiac allografts.13 While no gene transfer approach targeting ICOS-B7RP-1 interactions has been described to date, inhibiting this costimulatory pathway may represent an interesting approach for preventing chronic graft rejection.

Hyporesponsiveness to virally encoded MHC genes has been induced in both rodent and large animal models

It has been known for many years that a state of mixed hostdonor hematopoietic cellular chimerism, induced by allogeneic bone marrow transplantation, leads to long-term stable donor-specific tolerance (reviewed in Sykes14). Building on the concept of mixed chimerism, it has been suggested that a state of molecular chimerism involving the transfer of genes encoding allogeneic donor-type MHC proteins, or other antigens, into autologous hematopoietic stem cells may also result in tolerance.15 The induction of molecular chimerism through genetic modification of autologous hematopoietic stem cells has the potential to induce donor-specific tolerance without the complications associated with allogeneic bone marrow transplants, such as graft-versus-host disease. Expression of retrovirally transduced allogeneic donor-type MHC genes in bone marrow-derived cells has been shown to be sufficient to induce donor-specific hyporesponsiveness to the introduced gene product, allowing for prolonged survival of cardiac and skin allografts without affecting rejection of third-party control grafts.15 Hyporesponsiveness to allogeneic renal transplants has also been induced in pigs following induction of molecular chimerism, although it is not clear to what extent the transplanted organ itself contributed to establishing allograft acceptance.16 The induction of hyporesponsiveness to marker genes expressed in bone marrow-derived cells has also been achieved in rhesus macaques.17 Thus, the induction of molecular chimerism has been shown to be capable of inducing hyporesponsiveness in multiple animal models.

Induction of stable long-term T-cell tolerance to MHC class I antigens through central deletion of alloreactive T cells following induction of molecular chimerism

The ability to induce hyporesponsiveness through molecular chimerism has been well established; however, it has also been shown that this hyporesponsiveness can be abrogated by providing T-cell help (reviewed in Bagley et al15). In order for molecular chimerism to become clinically relevant, it is important to demonstrate that stable long-term T-cell tolerance can be achieved using this approach. Recently, it has been shown that efficient expression of an allogeneic MHC class I gene in bone marrow-derived cells is sufficient to allow for permanent survival of MHC class I disparate skin grafts without affecting rejection of third-party grafts.18 Cytotoxic T cells capable of lysing donor-type targets remained undetectable in vitro even after rigorous antigen challenge. These results are in contrast to previous studies in which T-cell hyporesponsiveness induced by genetic engineering of bone marrow could be broken by provision of T-cell help.

These results suggest that donor-specific tolerance can be established by inducing molecular chimerism.

The mechanism by which induction of molecular chimerism induces CD8 T-cell tolerance has been recently elucidated using a T-cell receptor transgenic mouse model. In this system, H-2Kb-specific transgenic CD8 T cells were observed to undergo negative selection in the thymus upon encountering bone marrow-derived cells expressing the transduced MHC class I gene.19 Expression of the transduced antigen on T cells appears to participate in trafficking alloantigen to the thymus to facilitate negative selection, as suggested by a study that demonstrated that in the absence of transduced MHC class I expression on T and B cells, mice failed to become tolerant.20 Since central deletion of alloreactive T cells is the most stable form of tolerance, the proof that deletion occurs in molecular chimeras is encouraging. Collectively, these data strongly suggest that gene therapy can be used to permanently reshape the T-cell repertoire.

Evidence for induction of regulatory cells that can prevent allograft rejection in gene therapy models

In recent years, a significant amount of evidence suggests that subpopulations of CD4 T cells can suppress allograft rejection. These regulatory cells can be induced using a variety of approaches including administration of nondepleting anti-CD4 antibodies and exposure to a tolerizing antigen in the form of donor-specific transfusions.21,22,23 Often regulatory cells are able to induce tolerance to both the tolerizing antigen and third-party antigens, as long as they are both expressed on the same graft, suggesting that inhibition occurs locally.21,22 Recently, it has been shown that treatment of immunocompetent mice with nondepleting anti-T-cell antibodies together with syngeneic bone marrow infected with adenoviruses carrying an allogeneic MHC class I gene can lead to the acceptance of fully-allogeneic cardiac transplants which share the same MHC class I antigen carried by the adenovirus construct.24 This approach essentially mimics results obtained using donor-specific transfusion, coupled with the use of nondepleting anti-T-cell antibodies to induce tolerance. Although the mechanism by which acceptance was achieved is unknown, transplant acceptance was observed even though gene expression was very short lived in vivo. These experiments suggest that the use of gene therapy-modified bone marrow may be capable of inducing regulatory T cells that can inhibit the responses to the transduced gene product as well as additional transplantation antigens when combined with other therapies. This is a potentially important extension of the use of molecular chimerism. However, it is not clear whether similar approaches would work in models in which the organ itself does not participate in the maintenance of tolerance, or prevent chronic rejection.

Induction of B-cell tolerance by molecular chimerism

In addition to inducing T-cell tolerance, molecular chimerism has been shown to be capable of inducing B-cell tolerance. Natural antibodies specific for the carbohydrate epitope Gal 1-3Gal 1-4GlcNac-R ( Gal) are the main mediators of hyperacute xenograft rejection in pig to primate xenotransplantation. Mutant mice, which lack (1,3)galactosyltransferase ( GT), the enzyme that synthesizes the Gal epitope, produce Gal-specific natural antibodies, as do humans. Expression of porcine GT in bone marrow-derived cells of GT knockout mice has been shown to prevent the production Gal-reactive natural antibodies, resulting in stable long-term tolerance to Gal even after rigorous antigen challenge.15,25,26 Importantly, tolerance remained intact even after mice received cardiac transplants from wild-type mice that expressed Gal, and Gal antibody-mediated rejection was prevented in molecular chimeras.27 Analysis of B cells from mice reconstituted with GT-transduced bone marrow revealed that B cells which produce Gal-specific antibodies were eliminated from the immunological repertoire following gene therapy. Collectively, these data suggest that gene therapy may be used to induce both B- and T-cell tolerance through the establishment of molecular chimerism.

Induction of tolerance in sensitized recipients

The extent to which pre-existing host immune responses can affect the induction of molecular chimerism and establishment of donor-specific tolerance is an important issue. Many patients have been presensitized to organ allografts by blood transfusions or previous transplants, and all humans have natural antibodies to Gal epitopes on xenogeneic organs. To determine the extent to which a preformed immune response was a barrier to the induction of molecular chimerism, GT knockout mice were immunized with Gal-expressing pig cells. Since Gal is a T-dependent antigen28 this immunization resulted in T-cell priming and increased titers of anti-Gal antibodies. Despite high titers of serum Gal-specific antibodies in immunized hosts, molecular chimerism could be established by increasing the dose of transduced bone marrow used for reconstitution of lethally irradiated recipient.29 Once molecular chimerism was established in sensitized hosts, tolerance to Gal resulted and production of Gal-specific antibodies ceased. These data demonstrated that the induction of molecular chimerism could be used to reshape the pre-existing B-cell repertoire in appropriately conditioned sensitized hosts.

Gene therapy-mediated cytokine production, or immune deviation, prolongs graft survival, but does not prevent rejection

Acute rejection events in immunosuppressed patients correlate with intragraft production of T helper type 1 (Th1) cytokines, such as IFN- , while the lack of acute rejection is associated with production of T helper type 2 cytokines (Th2), such as IL-10. It has therefore been hypothesized that production of Th2 cytokines might act to downregulate the immune response to organ allografts. Thus, the concept of immune deviation was proposed as a way to prevent organ allograft rejection by fostering a Th2-type response, rather than Th1. Since systemic administration of cytokines often results in unacceptable side effects, gene therapy approaches were developed to test the hypothesis that modifying the local graft environment to promote Th2 rather than Th1 responses would result in prolonged transplant survival. The ability of several Th2-type cytokines to prolong allograft graft survival when expressed locally has been assessed both with and without additional immunosuppression. In general, prolongation of graft survival has been observed using this approach. However, expression of cytokine genes within donor tissues did not lead to permanent graft acceptance. More recently, it has been shown that localized liposome-mediated IL-10 gene transfer into rabbit cardiac transplants could induce alloreactive T-cell apoptosis and prolong cardiac allograft survival.30 In addition, transfer of the gene encoding viral IL-10 delayed graft rejection in a rat model.31 However, in both these studies, prolongation was extremely modest, and it is therefore unclear what kind of clinical significance can be attached to such results in model systems that are relatively sensitive to tolerance induction.

Transduction of dendritic cells with genes encoding immunomodulatory proteins can moderately prolong graft survival

An increased understanding of the role of dendritic cells in T-cell activation has led to an interest in modification of dendritic cells to induce tolerance. While mature dendritic cells express high levels of costimulatory molecules and induce strong T-cell responses, there is some evidence that dendritic cells expressing low levels of costimulatory molecules can induce anergy in T-cells.32 Attempts have been made to modify graft rejection by genetically modifying dendritic cells to express cytokines associated with delayed graft rejection. Retroviral delivery of TGF- into myeloid dendritic cells has been shown to decrease their ability to stimulate alloreactive cells, resulting in moderately prolonged graft survival of cardiac allografts in mice.33 Human myeloid dendritic cells transduced with the gene encoding IL-10 prolonged human skin allograft survival in a humanized NOD-scid chimeric model.34 However, other studies have indicated that adenovirus transduction of the IL-10 gene into murine myeloid dendritic cells can enhance alloreactive responses to cardiac grafts.35 Overall, the results obtained with cytokine-gene-transduced dendritic cells were similar to those obtained with transduced organs. Both resulted in only a modest increase in transplant survival. Similar results have been observed in models where dendritic cells were transduced with genes encoding CTLA-4Ig.36

Dendritic cells have also been genetically engineered to express Fas ligand (CD95L).37,38,39 Engagement of Fas (CD95) on the surface of T cells by CD95L leads to the induction of apoptosis in activated T cells. Apoptosis induced by CD95CD95L interactions is thought to play a role in the establishment of immunoprivileged sites such as the eye and testis, and may

be involved in the killing of CD4 T cells. Dendritic cells genetically engineered to express CD95L on their surface are able to inhibit alloreactive T-cell proliferation in vitro, and cause a slight prolongation of cardiac graft survival when administered in vivo.38 However, as discussed above, since mouse cardiac transplants are relatively sensitive to tolerance induction, it is unclear how significant these results will be in more rigorous transplantation models.

Improved graft survival through antiapoptotic and antiproliferative gene therapy

In addition to host immunity, other nonimmunological factors play a role in transplant survival. Therefore, approaches have been developed to protect donor organ or tissue grafts at the time of transplantation from damage owing to nonimmune-mediated inflammation and ischemiareperfusion injury. The use of gene transfer to prevent damage that occurs at the time of transplant has the advantage that it does not require long-term gene expression, making these therapies potentially clinically relevant.

A significant proportion of cellular transplants such as islets or hepatocytes are lost due to anoxiaischemia reperfusion injuries at the time of transplantation that trigger the generation of reactive oxygen species leading to cellular death and localized inflammation. Endogenous scavenger systems can eliminate toxic radicals. However, the components of these systems are usually degraded rapidly when given exogenously. The expression of scavenging factors locally through genetic engineering of transplants would therefore be one way to protect organs and tissues. In a rat liver transplantation model, introduction of the gene encoding copperzinc superoxide dismutase through adenoviral transduction allowed for the survival of 100% of recipients, whereas only 25% of mock-treated controls survived. Expression of copperzinc superoxide dismutase in transplanted livers also significantly reduced necrosis within the transplant.40,41 These data suggest that expression of copperzinc superoxide dismutase decreases injury resulting from the generation of reactive oxygen species during transplantation. Similarly, gene therapy approaches using other cytoprotective genes such as hemeoxygenase-142,43 and catalase44 have also been shown to protect organs against the effects of ischemiareperfusion. Expression of hemeoxygenase-1 in rat liver transplants following adenovirus-mediated gene transfer resulted in an increased survival of recipients from 50% in controls to 80% in those receiving hemeoxygenase-1-transduced livers. Gene transfer in these studies was performed from 4 to 24 h in advance of transplant, which may be compatible with clinical application of these techniques.

The overexpression of antiapoptotic genes in transplanted tissue may also protect the graft from nonimmune as well as immune-mediated injury. Bcl-2, an antiapoptotic cell survival factor, blocks the release of cytochrome 3 from the mitochondria and subsequent activation of caspases, proteases involved in cell death. The overexpression of bcl-2 in macaque pancreatic islets was able to enhance insulin production after transplant into diabetic SCID mice45 and protect porcine islets after exposure to rhesus monkey serum.46 In addition, the expression of bcl-2 protected human endothelial cells from CTL activity.47 However, since the function of bcl-2 is intracellular, the effectiveness of this approach is dependent on a high transduction efficiency that will be more difficult to achieve in the terminally differentiated cells of vascularized organs.

Prospects

In the coming years, in order for gene therapy to be used to prevent transplant rejection clinically, it will be crucial to gain a better understanding of how alloreactive T cells are activated and regulated, so that rational gene therapy interventions can be designed to prevent transplant rejection (Figure 1). Blockade of yet to be defined costimulatory pathways needs to be evaluated in order to determine if targeting these pathways using gene therapy approaches will exhibit utility for clinical transplants. In addition, a greater understanding of the mechanisms by which regulatory T cells are induced needs to be achieved in order to design strategies to exploit these cells to achieve transplant acceptance. Tolerance induction remains a major goal in the field, and if it can be achieved clinically may represent the most attractive approach, completely overcoming the need for immunosuppression. Results obtained in molecular chimeras have demonstrated that gene therapy approaches can be used to induce tolerance. Coupling the induction of molecular chimerism to, for instance, induction of regulatory cells may allow for survival of organs mismatched at major and minor histocom-patibility loci, extending the clinical utility of this approach. However, the ability of molecular chimerism to induce tolerance must also be assessed in preclinical non-human primate models to determine if similar approaches could eventually be used in humans. In addition, methods must be developed to allow the induction of molecular

chimerism using clinically acceptable host conditioning. Lastly, as has been true for many years, the development of more efficient vectors for gene transfer continues to be of great importance, allowing for more efficient transduction and improved gene expression.

Acknowledgements

We thank Drs David KC Cooper and Yong Guang Yang for critical review of the manuscript, and members of the Iacomini Laboratory for helpful suggestions and comments. This work was upported by NIH grants RO1 AI43619 and RO1 AI44268 to JI. JB is supported by NIH Training grant T32 AI07529, and in part by a grant from the Children's A-T Project.

References

1 Sharpe AH and Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2: 116126. Article PubMed

2 Guillot C et al. Gene therapy in transplantation in the year 2000: moving towards clinical applications? Gene Therapy 2000; 7: 1419.

3 Guillot C et al. Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression. J Immunol 2000; 164: 52585268. PubMed

4 Malm H et al. CTLA4ig induces long-term graft survival of allogeneic skin grafts and totally inhibits T-cell proliferation in LFA-1-deficient mice. Transplantation 2002; 73: 293297.

5 Reddy B et al. The effect of CD28B7 blockade on alloreactive T and B cells after liver cell transplantation. Transplantation 2001; 71: 801811.

6 Comer RM et al. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci 2002; 43: 10951103.

7 Li TS et al. Prolonged survival of xenograft fetal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression. Transplantation 2001; 72: 19831985.

8 Takehara M et al. Long-term acceptance of allografts by in vivo gene transfer of regulatable adenovirus vector containing CTLA4IgG and loxP. Hum

Gene Ther 2001; 12: 415426.

9 Nomura M et al. Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation. Transplantation 2002; 73: 14031410.

10 Guillot C et al. Prolonged blockade of CD40CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection. J Immunol 2002; 168: 16001609.

11 Greenwald RJ et al. CTLA-4 regulates induction of anergy in vivo. Immunity 2001; 14: 145155. PubMed

12 Coyle AJ, Gutierrez-Ramos JC. The expanding B7 superfamily: increasing complexity in costimulatory signals regulating T cell function. Nat Immunol 2001; 2: 203209. Article PubMed

13 Ozkaynak E et al. Importance of ICOS-B7RP-1 costimulation in acute and chronic allograft rejection. Nat Immunol 2001; 2: 591596. Article PubMed

14 Sykes M. Mixed chimerism and transplant tolerance. Immunity 2001; 14: 417424. Article PubMed

15 Bagley J et al. Establishing immunological tolerance through the induction of molecular chimerism. Front Biosci 2002; 7: 13311337.

16 Sonntag KC et al. Tolerance to solid organ transplants through transfer of MHC class II genes. J Clin Invest 2001; 107: 6571. PubMed

17 Heim DA et al. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model. Mol Therapy 2000; 1: 533544.

18 Bagley J et al. Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy. Blood 2002; 99: 43944399.

19 Kang ES, Iacomini J. Induction of central deletional T cell tolerance by gene therapy. J Immunol 2002; 169: 19301935.

20 Tian C, Bagley J, Iacomini J. Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy. J Immunol 2002; 169: 3776.

21 Zelenika D et al. The role of CD4+ T-cellsubsets in determining transplantation rejection or tolerance. Immunol Rev 2001; 182: 164179. Article PubMed

22 Waldmann H, Cobbold S. Regulating the immune response to transplants. A role for CD4+ regulatory cells? Immunity 2001; 14: 399406.

Article PubMed

23 Graca L et al. Both CD4(+)CD25(+) and CD4(+)CD25(-) regulatory cells mediate dominant transplantation tolerance. J Immunol 2002; 168: 55585565. PubMed

24 Fry JW, Morris PJ, Wood KJ. Adenoviral transfer of a single donor-specific MHC class I gene to recipient bone marrow cells can induce specific immunological unresponsiveness in vivo. Gene Therapy 2002; 9: 220226.

25 Bracy JL, Iacomini J. Induction of B cell tolerance by retroviral gene therapy. Blood 2000; 96: 30083015.

26 Iacomini J, Bracy JL. Inducing tolerance by molecular chimerism. Graft 2001; 4: 102104.

27 Bracy JL et al. Induction of molecular chimerism by gene therapy prevents antibody mediated heart transplant rejection. Gene Therapy 2001; 8: 17381744.

28 Cretin N et al. The role of T cell help in the production of antibodies specific for Gal alpha 13Gal. J Immunol 2002; 168: 14791483.

29 Bracy J, Iacomini J. Engraftment of genetically modified bone marrow cells in sensitized hosts. Mol Ther 2002; 6: 252.

30 Oshima K et al. Localized interleukin-10 gene transfer induces apoptosis of alloreactive T cells via FASFASL pathway, improves function, and prolongs survival of cardiac allograft. Transplantation 2002; 73: 10191026.

31 David A et al. Interleukin-10 produced by recombinant adenovirus prolongs survival of cardiac allografts in rats. Gene Therapy 2000; 7: 505510.

32 Hawiger D et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001; 194: 769779. Article PubMed

33 Takayama T et al. Retroviral delivery of transforming growth factor-beta1 to myeloid dendritic cells: inhibition of T-cell priming ability and influence on allograft survival. Transplantation 2002; 74: 112119.

34 Coates PT et al. Human myeloid dendritic cells transduced with an adenoviral interleukin-10 gene construct inhibit human skin graft rejection in humanized NOD-scid chimeric mice. Gene Therapy 2001; 8: 12241233.

35 Lee WC et al. Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin-10 on organ allograft and tumour rejection. Immunology 2000; 101: 233241.

36 O'Rourke RW et al. A dendritic cell line genetically modified to express

CTLA4-IG as a means to prolong islet allograft survival. Transplantation 2000; 69: 14401446.

37 Matsue H et al. Immunosuppressive properties of CD95L-transduced "killer" hybrids created by fusing donor- and recipient-derived dendritic cells. Blood 2001; 98: 34653472.

38 Min WP et al. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. J Immunol 2000; 164: 161167. PubMed

39 Min W et al. Fas ligand-transfected dendritic cells induce apoptosis of antigen-specific T cells. Transplant Proc 2001; 33: 234.

40 Lehmann TG et al. Delivery of CuZn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat. Transplantation 2000; 69: 10511057. PubMed

41 Lehmann TG et al. Gene delivery of CuZn-superoxide dismutase improves graft function after transplantation of fatty livers in the rat. Hepatology 2000; 32: 12551264.

42 Kato H et al. Heme oxygenase-1 overexpression protects rat livers from ischemiareperfusion injury with extended cold preservation. Am J Transplant 2001; 1: 121128.

43 Coito AJ et al. Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemiareperfusion injury. Transplantation 2002; 74: 96102.

44 Zhu HL et al. Blocking free radical production via adenoviral gene transfer decreases cardiac ischemiareperfusion injury. Mol Ther 2000; 2: 470475.

45 Contreras JL et al. Cytoprotection of pancreatic islets before and early after transplantation using gene therapy. Kidney Int 2002; 61(Suppl 1): 7984.

46 Contreras JL et al. Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement. Surgery 2001; 130: 166174. PubMed

47 Zheng L et al. Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses. J Immunol 2000; 164: 46654671. PubMed

Figures

Figure 1 Preventing transplant rejection by gene therapy. Gene therapy-based strategies can be used to prevent activation of alloreactive host T cells in peripheral lymphoid tissue, such as the lymph node (shown), by blocking costimulatory molecule interactions between T cells and APCs (a). Such interventions leave T cells in a non-responsive state, and can also result in anergy. Reconstitution of conditioned hosts with autologous hematopoietic stem cells engineered to express donor type transplantation antigens using retroviruses allows for expression of donor type antigen on bone marrow-derived cells (b). Expression of retrovirally transduced antigen on bone marrow-derived cells within the recipient thymus mediates negative selection of newly formed alloreactive T cells (c). It is unclear whether expression in the thymus can also lead to the generation of regulatory T cells capable of controlling allograft rejection. Direct modification of the donor graft itself (d) using a wide variety of gene delivery systems prior to transplantation offers the possibility of reducing graft damage owing to ischemiareperfusion injury. This approach has also been proposed as a method to allow the destruction of alloreactive T cells that reach the graft.

Received 24 September 2002; accepted 30 January 2003

April 2003, Volume 10, Number 8, Pages 605-611

March 2003, Volume 10, Number 6, Pages 453-458

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ReviewProgress and prospects: naked DNA gene transfer and therapy

H Herweijer1 and J A Wolff1

1Mirus Corporation and University of Wisconsin-Madison Madison, WI, USA

Correspondence to: Dr J Wolff, University of Wisconsin-Madison, Department of Pediatrics, Waisman Center, 1500 Highland Avenue, Madison, WI 53705, USA

Abstract

Increases in efficiency have made naked DNA gene transfer a viable method for gene therapy. Intravascular delivery results in effective gene delivery to liver and muscle, and provides in vivo transfection methods for basic and applied gene therapy and antisense strategies with oligonucleotides and small interfering RNA (siRNA). Delivery via the tail vein in rodents provides an especially simple and effective means for in vivo gene transfer. Electroporation methods significantly enhance direct injection of naked DNA for genetic immunization. The availability of plasmid DNA expression vectors that enable sustained high level expression, allows for the development of gene therapies based on the delivery of naked plasmid DNA.

Gene Therapy (2003) 10, 453458. doi:10.1038/sj.gt.3301983

Keywords

gene transfer; naked DNA; nonviral; intravascular; hydrodynamic

In brief

Progress

Injection of naked DNA in muscle results in transfection of cells in vivo. Electroporation enhances uptake of injected plasmid DNA into muscle and skin.

Intravascular delivery of plasmid DNA results in a very effective gene transfer of hepatocytes.

Tail vein pDNA delivery is a simple and effective method for transfecting liver cells in mice and rats.

Effective transfection of skeletal muscle cells in mice, rats, dogs, and monkeys via intravascular delivery of plasmid DNA.

Novel pDNA expression vectors allow for high-level sustained expression.

The mechanism of intravascular naked DNA delivery is thought to involve active cellular pDNA uptake.

Naked DNA delivery has entered clinical studies for peripheral arterial occlusion disease (PAOD).

Small interfering RNA (siRNA) can be delivered very efficiently using intravascular gene transfer methods and results in potent knock out of the target gene expression.

Prospects

Nonviral gene transfer will become more important as better delivery methods become available.

Clinical trials for genetic diseases (eg, Duchenne muscular dystrophy, ischemia, hemophilia) will be initiated in the next few years.

Tail vein injections in rodents will become a widely used technique for rapidly testing expression vectors and gene therapy approaches.

RNA interference will become a major element in the gene therapy field.

Injection of naked DNA in muscle results in transfection of cells in vivo

Gene therapy would be a lot simpler if we could forget about recombinant viruses or complexing DNA with polymers or lipids. It has been known for years that naked DNA can be delivered to cells in vivo and result in gene expression. However, the efficiency of gene transfer into skeletal or cardiac muscle is relatively low and variable. Attempts to increase pDNA uptake, for instance by inducing muscle regeneration, have not increased efficiency to a level that would allow for clinical use in gene therapy protocols. As a result, naked DNA gene transfer has been mainly used for genetic immunization studies. In recent years, work in several laboratories has shown that naked plasmid DNA (pDNA) can be delivered efficiently to cells in vivo either via electroporation, or by intravascular delivery, and has great prospects for basic research and gene therapy.

Electroporation enhances uptake of injected plasmid DNA into muscle and skin

The last few years has seen a marked increase in the number of studies employing intramuscular or intradermal injection of naked DNA followed by electroporation. Technical improvements in electroporation equipment made in recent years, as well as better methodology following years of experimentation, have enabled increases both in gene transfer efficiency and safety.1 A very large number of publications in the last few years have demonstrated gene transfer to a variety of different cell types in

vivo. Expression levels in muscle are at least 10-fold higher compared to injection of pDNA without electroporation, but are accompanied by elevation of serum creatine kinase levels.2 It is not clear whether these increases in transgene expression (especially of secreted proteins) are due to enhanced gene transfer into myofibers, or to simultaneous transfer into different cell types (eg, endothelial cells). Expression levels are considered sufficient to warrant further investigation of this method for gene therapy, for instance for chronic anemia3 or muscular dystrophies.4 Yet, expression levels are not as high as those achieved following intravascular delivery,5 (see below). Injection of naked DNA in conjunction with electroporation into skeletal muscle or skin should enhance the efficacy of genetic immunization procedures.6

Intravascular delivery of plasmid DNA results in very effective gene transfer of hepatocytes

Intravascular delivery of genes is attractive because it avoids the necessity for multiple intraparenchymal injections into the target tissue. The gene is disseminated throughout the tissue since the vascular system accesses every cell. Vascular delivery could be systemic or regional in which injections are into specific vessels that supply a target tissue.7 The intravascular delivery of adenoviruses or cationic lipidDNA complexes in adult animals mostly results in expression in vascular-accessible cells such as endothelial cells or hepatocytes reached via the sinusoid fenestrae. Efficient transgene expression in hepatocytes throughout the liver can be obtained following delivery of naked pDNA via the portal vein, the hepatic vein, or the bile duct in mice and rats. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels, although it was later shown that a hyperosmotic solution is not absolutely necessary.

Tail vein pDNA delivery is a simple and effective method for transfecting liver cells in mice and rats

A major advance in the intravascular delivery of pDNA was the recent development of the tail vein delivery procedure in the Wolff and Liu laboratories. As a logical extension to rapid delivery of a relatively large volume into a vessel leading into or from the liver, the tail vein itself can be employed. The tail vein drains into the vena cava. Delivery of a large bolus presumably results in a liquid volume in the vena cava that is too large for the heart to handle rapidly. The fluids back up and end up (predominantly) in the liver, resulting in gene transfer. This explains the rather delicate sensitivity of this method of gene delivery upon delivery volume and speed. Several groups have found that the optimal volume is around 10% of the body weight of a mouse or rat.7,8 The delivery time should be between 5 and 7 s in mouse; 1520 s in rat, these times being the fastest rates that can be practically achieved by skilled operators.

Tail vein or hydrodynamic delivery results in very high levels of gene transfer. Typically, 1015% of the hepatocytes are transfected in mouse liver following injection of 10 μg pDNA, but levels up to 40% have been reported. We have measured high levels of reporter genes (about 2 μg luciferase, 50 μg secreted alkaline phosphatase) and supranormal levels of therapeutic genes (55 μgml human factor IX, 1.6 μgml human factor VIII, 915 ngml rhesus erythropoietin) one day after gene delivery. Transgene expression is also found in heart, spleen, and kidneys, at levels about 100-fold lower than in liver. While the procedure seem harsh, nearly all animals survive (99%) and show no ill effects. Liver enzymes are transiently elevated. For instance, we observed serum alanine aminotransaminase (ALT) levels around 1000 Uml 24 h after injection in mice. The ALT levels diminished to normal over the next few days. Liver histology shows minimal damage that resolves within a week, which is similar to the data we observed for intravascular delivery into liver vessels.9

The tail vein method has been adopted remarkably quickly in the gene therapy field for basic research and gene therapy evaluation. At the recent meeting of the American Society of Gene Therapy (June 59, 2002), 22 abstracts were presented employing this novel technique. Owing to its simplicity and reproducibility, it allows for the rapid testing of novel expression vectors (whether or not the expression cassette will eventually be used in a viral or a nonviral vector). We, and others, have used tail vein injections to evaluate the expression cassettes capable of driving sustained high level expression in the liver.9,10,11,12,13,14 As it is easy to regulate the level of gene expression by adjusting the amount of plasmid DNA, it is now possible to test accurately the level of transgene expression required for achieving a physiological effect in a disease model (eg, the level of phenylalanine hydroxylase (PAH) expression in PAH-deficient phenylketonurea (PKU) mice; Cary Harding, personal communication).

As the liver is the organ that is predominantly transfected, testing gene therapy approaches may be limited to those diseases where liver transgene expression is appropriate. However, the liver may be used as an ectopic expression site for secreted proteins (eg, erythropoietin), or in a situation where hepatocytes can take over the function of other cells (eg, as in clearing of metabolites). An often asked

question is how this technique can be translated to the human situation. Since the human tail is on the short side, other entry points need to be found. We have extensively investigated intravascular delivery to the liver in large animals (unpublished data). It appears that similar efficiencies can be obtained in monkeys as in mice by injecting pDNA into the afferent or efferent vessel of the liver. Such injections can be done via catheters in humans, making this a relatively simple procedure. Eastman et al15 recently presented data in rabbits, reporting high expression levels following delivery of pDNA via balloon occlusion catheters introduced into the portal vein. Expression levels were somewhat lower than in mice following tail vein injections; toxicity measurements showed a similar transient rise in liver enzymes. These studies demonstrate the feasibility of intravascular delivery to the liver using catheters, and are a step in the direction of human clinical trials. Intravascular delivery to other organs is also being explored (eg, Zhang et al,7 and Maruyama et al16).

Effective transfection of skeletal muscle cells in mice, rats, dogs, and monkeys via intravascular delivery of plasmid DNA

The intravascular delivery of naked pDNA to muscle cells is also attractive particularly since many muscle groups would have to be targeted for intrinsic muscle disorders such as Duchenne muscular dystrophy. An intravascular approach would avoid the limited distribution of pDNA through the interstitial space following intramuscular injection. Muscle has a high density of capillaries that are in close contact with the myofibers. Delivery of pDNA to muscle via capillaries puts the pDNA into direct contact with every myofiber and substantially decreases the interstitial space the pDNA has to traverse in order to access a myofiber. However, the endothelium in muscle capillaries is of the continuous, nonfenestrated type and has low solute permeability, especially to large macromolecules. Nonetheless, rapid delivery of relatively large volumes of pDNA solutions (10 ml injected into rat iliac artery) resulted in very efficient gene transfer into myofibers. With the best injection condition, up to 50% of myofibers expressed β-galactosidase in many areas of the muscles. Experiments have successfully been performed in mice, although these are technically difficult to do. Expression levels and percentage of transfected cells do vary significantly from mouse to mouse, yet up to 20% of transfected myofibers have been observed regularly.

Studies in larger animals have now demonstrated the clinical relevance of this method of gene delivery. Intravascular delivery to limb skeletal muscle has successfully been performed in rabbits, dogs, and rhesus monkeys (Zhang et al17; unpublished results). Several alternative methods for delivering the pDNA solution and blocking limb blood flow have been evaluated. Delivery via a catheter to regional target muscle groups, in combination with blocking blood flow with a tourniquet or blood pressure cuff, is very effective in larger animals. In rhesus monkeys, transfection efficiencies of 40% have been observed,17 a number that is considered sufficient for treatment of muscle defects such as Duchenne muscular dystrophy. A transient increase in serum creatine kinase levels was measured, which resolved within a few days. The short time required for occluding blood flow to skeletal muscle should be well tolerated in a human clinical setting since ischemia can be tolerated by muscles for 23 h. In fact, a common anesthetic procedure for distal limb surgery (eg, carpal tunnel repair) involves the placement of a tourniquet to block both venous and arterial blood flow and the intravenous administration of a local anesthetic (eg, lidocaine) distal to the tourniquet. Surgery in humans can be performed for a couple of hours using these anesthetic procedure. Similarly, histologic analyses of the rat, dog, and rhesus muscles in our experiments indicated that the ischemia did not cause myofiber damage. Besides gene therapy for muscle defects, it appears worthwhile to evaluate this method for delivery of angiogenic genes for the treatment of ischemia. Delivery is not limited to skeletal muscle, but can also target cardiac muscle (retrograde delivery via standard angioplasty catheters) and can thus potentially be used for treating heart ischemia.18

Novel pDNA expression vectors allow for high level sustained expression

One of the problem areas in gene therapy is the sustained expression of transgenes at high levels. We have recently analyzed five explanations for the rapid loss of expression observed after intravascular pDNA gene delivery to the liver.9 While these analyses were performed following portal vein delivery, the conclusions appear valid for other intravascular delivery routes to the liver. First, the injection procedure or the presence of intracellular pDNA may induce cell death causing loss of the vector. As described above, it is clear that the injection procedure (with pDNA or with vehicle alone) does induce damage. But, all pathological reactions observed were transient and liver morphology was restored at day 4 after manipulation.

Second, intravascular delivery of pDNA may result in increased cell cycling causing the loss of the nonintegrated pDNA. While an increase in the number of cycling cells was found (BrdU labeling studies),

peaking at 2 days at 11.5%, cycling appears to be insufficient to fully account for the 5000-fold drop in expression observed for CMV promoter-driven luciferase expression (day 1 to day 7). Third, pDNA may be lost independent of cell replication. Southern blotting experiments allowed us to determine the time course of pDNA loss. pDNA is rapidly lost in the first 24 h, likely reflecting the loss of extracellular pDNA or pDNA associated with Kupffer cells.19 The decline in pDNA levels from day 1 to day 7 was 1040 fold, much less than the loss of reporter gene expression in the same period (5000 fold).

Fourth, the promoter driving reporter gene expression may be inactivated, thus resulting in the loss of reporter gene expression. Comparison of viral- and tissue-specific promoters revealed that (1) viral promoters generally express at very high levels 1 day after injection; (2) viral promoter-driven reporter gene expression falls precipitously after day 1; (3) the liver-specific albumin promoter expresses at much lower levels on day 1, but expresses at a similar level on day 7. We therefore hypothesized that the enormous drop in expression measured for the viral promoters is the result of promoter inactivation. Fifth, the expressed proteins may induce an antigen-specific immune response, resulting in a further loss of expression after 23 weeks.

Our observations indicate that loss of transgene expression following pDNA gene transfer to the liver occurs in two phases. There is a rapid loss of expression in the first few days, followed by a slower decrease after about 1 week. The loss of expression in this latter phase appears to be the result of an antigen-specific immune response in normal, immunocompetent mice, as expression is much prolonged in immunocompromised mice. The early phase of expression loss appears multifactorial. The delivery procedure does result in liver damage, as evidenced by histological observation and an increase in hepatocyte cell cycling. It is therefore likely that part of the transfected cells are destroyed, or nonintegrated plasmid DNA is lost during cell cycling. Overall, the loss of pDNA during this phase is not nearly as great as the loss of expression. Relatively sustained expression driven by the liver-specific albumin promoter (albeit at low levels) supports this hypothesis. This has recently been confirmed more conclusively by Miao et al, who described long-term expression of human factor IX in mice following tail vein injection of a plasmid DNA vector employing an α1-antitrypsin promoter in conjunction with a hepatic control region.10,11 Similar expression vectors with liver-specific promoters and transcriptional regulatory elements developed in our lab have corroborated these results.

It is now well established that certain sequences in bacterial DNA stimulate the immune system.20 This appears to be based on the absence of CpG methylation in bacterial DNA, whereas in mammalian DNA most CpG sequences are methylated. By inclusion of these sequences in genetic vaccines, an enhanced immune response can be induced that is skewed to Th-1.21 If the objective is long-term expression, minimizing the CpG content of the pDNA vector is beneficial.13 Combination of liver-optimized expression cassettes in CpG-minimized vectors has made sustained, high-level transgene expression in the liver following naked pDNA delivery a reality.

The mechanism of intravascular naked DNA delivery is thought to involve active cellular pDNA uptake

The combined intraparenchymal and emerging intravascular data indicates that the uptake and expression of naked DNA is a general property of animal cells within a tissue architecture. It is common to cells of all three lineages: endoderm (eg, hepatocytes), mesoderm (eg, muscle), and ectoderm (eg, skin). This property is typically lost when the cells are removed and maintained in culture. Tissue disruption and cell isolation may modify the cell so that it can no longer take up naked DNA.

The intravascular injection conditions presumably enhance DNA transfer to hepatocytes by opening transiently the hepatic endothelial barrier. Under normal conditions, the 0.1 μm size of the fenestrae would prevent the exit from the sinusoids of plasmid DNA, has a gyration radius of 0.1 μm. Raising the intraportal pressure may transiently enlarge their size and thereby increase the extravasation of the pDNA complexes. In fact, results using fluorescent-labeled DNA showed that the increased pressure was required for movement of the DNA out of the sinusoids and to the hepatocytes. Several observations suggest that the mechanism of pDNA uptake may involve native cellular uptake processes. pDNA uptake was time-dependent, suggesting that it is due in part to a cellular process.19 A receptor-mediated process is suggested by the inhibition of expression (from intramuscularly injected pDNA) by excess salmon sperm DNA, dextran sulfate,19 or heparin.22 These cellular processes may be aided or initiated by the rapid injection of the large volumes.

After traversing the plasma membrane, the pDNA must enter the nucleus since it is highly unlikely that plasmid DNA containing RNA polymerase II promoters could be expressed anywhere else. Although it is often assumed that DNA enters the nucleus from the cytoplasm little is known about the actual nuclear uptake process despite many advances toward an understanding of protein and RNA nuclear transport.

Our preliminary model of DNA nuclear uptake is as follows. After cytoplasmic delivery, the small amount of DNA that avoids binding to or sequestration by cytoplasmic elements enters the intact nucleus through the nuclear pore. The relatively rare entry of DNA into the nucleus (in comparison with karyophilic proteins) could be explained by its rapid and substantial cytoplasmic sequestration and its low rate of transport through the nuclear pore. This understanding of DNA nuclear transport provides a basis for future efforts to increase the efficiency of this process and is consistent with efforts to increase the amount of DNA delivered to the cytoplasm. For example, we have observed increased nuclear entry in digitonin-permeabilized cells of pDNA containing covalently attached SV40 T antigen nuclear localizing signal.

Naked DNA delivery has entered clinical studies for peripheral arterial occlusion disease (PAOD)

Several studies have been initiated for the treatment of limb ischemia or PAOD, using direct injection of pDNA into skeletal muscle as the gene transfer method. While notoriously difficult to evaluate, a benefit was noted in several early-phase clinical studies. Simovic et al23 performed a dose-escalation study in 29 patients with critical leg ischemia. After intramuscular injection of naked pDNA expressing the human vascular endothelial growth factor (VEGF) driven by a CMV promoter, patients had significant clinical improvements in examination scores and vascular ankle-brachial index in the treated limb.23 This was accompanied with improvements in electrophysiologic measures 6 months after gene transfer. Similar results were reported by Comerota et al 24 in a trial involving FGF-1 gene transfer into 51 patients with lower leg ischemia.24 A significant decrease in reported pain and ulcer size was observed, as well as an improved transcutaneous oxygen pressure and ankle-brachial index. No increase in FGF-1 serum levels was measured, suggesting that localized expression may be effective. While clearly encouraging, the gene therapy clinical trials to date have been predominantly small and noncontrolled (open label). Initial results show encouraging improvements, although absolute increases in study parameters remain relatively small.

Several questions are in need of answering prior to entering large-scale clinical trials: (1) Which angiogenic gene, or cocktail of genes, should be used? VEGF and FGF-1 appeared to work in these early trials, but are likely not optimal factors. The basic biology of angiogenesis needs to be developed more fully to provide a better understanding of this complicated process, allowing for more rational choices of therapeutic genes. (2) What naked DNA gene transfer method is optimal, direct injection (with or without electroporation) resulting in localized expression, or intravascular delivery resulting in widespread gene delivery and expression throughout the limb? This can likely be studies in an animal model, although no current model closely mimics human disease. (3) What duration of expression is desired? A benefit of naked DNA gene transfer into skeletal muscle is that generally long-term expression is obtained: however, that may not be optimal for the expression of angiogenic factors for the treatment of PAOD. While one could use regulated expression vectors, these introduce recombinant transcription factors that may lead to immune rejection and other problems. Different expression vectors therefore may need to be developed. Also, do we need to use optimized expression vectors (eg, CG-less) to avoid activating the host immune system. (4) What criteria for successful gene therapy are used? Subjective measures (eg, leg pain) are notoriously inaccurate. Measurement of increased blood flow appears a much better choice, but may underestimate benefit of therapy. (5) When can new gene therapy approaches be tested in clinical trials? Beyond basic safety testing, it may be difficult to assess effectiveness in animal models. Patients with severe limb ischemia (near amputation) may benefit from gene therapy, even while not all details (eg, optimal gene, optimal delivery method) are known. Based on recent history, it appears prudent to approach clinical trials with great care.

Small interfering RNA (siRNA) can be delivered very efficiently using intravascular gene transfer methods and results in potent knockout of target gene expression

RNA interference (RNAi) has been demonstrated to be highly effective for gene knockdown in a number of experimental organisms, as well as in mouse oocytes and preimplantation embryos. RNAi is mediated by double-stranded RNA (dsRNA), where mRNAs with sequence identity to the double-stranded RNA are degraded rapidly. It has been demonstrated using cultured mammalian cells that RNAi can be accomplished by delivering short interfering RNAs (siRNAs, dsRNA of 2125 bp length), which circumvents the induction of an interferon response normally associated with the delivery of longer dsRNA. We and others have determined that siRNAs can be delivered to tissues in vivo by intravascular delivery.25,26

Expression of reporter genes (luciferase from codelivered pDNA or GFP in transgenic mice) and endogenous genes can be downregulated by 90% in a majority of liver cells following one injection with an appropriate siRNA. We have also observed efficient reporter gene downregulation in heart, lung, spleen, and other tissues following tail vein delivery in mice. It is likely that several hurdles, associated with gene delivery, are easier to overcome for siRNA delivery. siRNAs are much smaller than pDNA, and therefore vascular extravasation and cellular uptake may be easier. siRNAs exert their effect in the cytoplasm; therefore, the slow step of nuclear entry is avoided. It is not clear yet how long the RNAi effect lasts following delivery of a single dose of siRNA. Nonetheless, it appears that RNAi is becoming a very important extension of what we should call now the naked nucleic acid transfer field.

Summary

The direct intramuscular injection of naked DNA is in common use for genetic immunization and an HIV vaccine clinical trial is in progress. The efficiency of gene expression following the intravascular delivery of naked DNA is high in muscle and liver, even approaching what can be achieved with viral vectors. High expression is also possible in larger animals including primates and there are potential applications of the technique for treating human disorders. Given that tail vein injections of naked DNA under certain conditions lead to high levels of foreign gene expression in hepatocytes in rodents and is convenient, the technique is gaining increasing favor for studying gene expression in vivo. The technique also enables delivery of siRNA to the liver and other organs in mice and should be useful for attenuating gene expression.

References

1 Somiari S et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000; 2: 178187. Article PubMed

2 Hartikka J et al. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188. Mol Ther 2001; 4: 407415.

3 Terada Y et al. Efficient and ligand-dependent regulated erythropoietin production by naked DNA injection and in vivo electroporation. Am J Kidney Dis 2001; 38: S50S53.

4 Vilquin JT et al. Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies. Gene Ther 2001; 8: 10971107. Article

5 Jiang J, Yamato E, Miyazaki J. Intravenous delivery of naked plasmid DNA for in vivo cytokine expression. Biochem Biophys Res Commun 2001; 289: 10881092.

6 Drabick JJ, Glasspool-Malone J, King A, Malone RW. Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization. Mol Ther 2001; 3: 249255. Article

7 Zhang G et al. Surgical procedures for intravascular delivery of plasmid DNA to organs. Methods Enzymol 2002; 346: 125133.

8 Maruyama H et al. High-level expression of naked DNA delivered to rat liver via tail vein injection. J Gene Med 2002; 4: 333341.

9 Herweijer H et al. Time course of gene expression after plasmid DNA gene transfer to the liver. J Gene Med 2001; 3: 280291.

10 Miao CH et al. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 2000; 1: 522532. Article PubMed

11 Miao CH, Thompson AR, Loeb K, Ye X. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther 2001; 3: 947957.

12 Yew NS et al. High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther 2001; 4: 7582. PubMed

13 Yew NS et al. CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 2002; 5: 731738.

14 Chen ZY et al. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403410. Article PubMed

15 Eastman SJ et al. Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA. Hum Gene Ther 2002; 13: 20652077.

16 Maruyama H et al. Kidney-targeted naked DNA transfer by retrograde renal vein injection in rats. Hum Gene Ther 2002; 13: 455468.

17 Zhang G et al. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001; 12: 427438. Article PubMed

18 Herweijer H et al. Retrograde coronary venous delivery of naked plasmid DNA. Mol Ther 2000; 1: S202.

19 Budker V et al. Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process Review. J Gene Med 2000; 2: 7688. Article PubMed

20 Krieg AM. Now I know my CpGs. Trends Microbiol 2001; 9: 249252. Article PubMed

21 Krieg AM, Davis HL. Enhancing vaccines with immune

stimulatory CpG DNA. Curr Opin Mol Ther 2001; 3: 1524.

22 Satkauskas S, Bureau MF, Mahfoudi A, Mir LM. Slow accumulation of plasmid in muscle cells: supporting evidence for a mechanism of DNA uptake by receptor-mediated endocytosis. Mol Ther 2001; 4: 317323.

23 Simovic D et al. Improvement in chronic ischemic neuropathy after intramuscular phVEGF165 gene transfer in patients with critical limb ischemia. Arch Neurol 2001; 58: 761768. PubMed

24 Comerota AJ et al. Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment of end-stage unreconstructible lower extremity ischemia: preliminary results of a phase I trial. J Vasc Surg 2002; 35: 930936.

25 Lewis DL et al. Efficient delivery of siRNA and inhibition of gene expression in post-natal mice. Nat Genet 2002; 32: 107108. Article PubMed

26 McCaffrey AP et al. Gene expression: RNA interference in adult mice. Nature 2002; 418: 3839. Article PubMed

Received 22 July 2002; accepted 13 December 2002

March 2003, Volume 10, Number 6, Pages 453-458

February 2003, Volume 10, Number 4, Pages 285-291

Table of contents    Previous  Article  Next   PDF

ReviewGene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia

T A Khan1, F W Sellke1 and R J Laham2

1Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

2Division of Cardiology and Angiogenesis Research Center, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

Correspondence to: Dr RJ Laham, Angiogenesis Research Center, Beth Israel Deaconess Medical Center, 330 Brookline Ave., SL-423, Boston, MA 02215, USA

Abstract

After extensive investigation in preclinical studies and recent clinical trials, gene therapy has been established as a potential method to induce therapeutic angiogenesis in ischemic myocardial and limb disease. Advancements in viral and nonviral vector technology including cell-based gene transfer will continue to improve transgene transmission and expression efficiency. An alternative strategy to the use of transgenes encoding angiogenic growth factors is therapy based on transcription factors such as hypoxia-inducible factor-1 (HIF-1 ) that regulate the expression of multiple angiogenic genes. Further understanding of the underlying biology of neovascularization is needed to determine the ability of growth factors to induce functionally significant angiogenesis in patients with atherosclerotic disease and associated comorbid conditions including endothelial dysfunction, which may inhibit blood vessel growth. The safety and tolerability of therapeutic angiogenesis by gene transfer has been demonstrated in phase I clinical trials. However, limited evidence of efficacy resulted from early phase II studies of angiogenic gene therapy for ischemic myocardial and limb disease. The utility of therapeutic angiogenesis by gene transfer as a treatment option for ischemic cardiovascular disease will be determined by adequately powered, randomized, placebo-controlled phase II and III clinical trials.

Gene Therapy (2003) 10, 285291. doi:10.1038/sj.gt.3301969

Keywords

angiogenesis; gene transfer; ischemic heart disease; coronary artery disease; peripheral vascular disease; vascular endothelial growth factor; fibroblast growth factor

In brief

Progress

Improvements in adenoviral vector and plasmid DNA technology have improved transmission and expression efficiency in myocardial and skeletal muscle tissue.

Newer techniques of gene transfer including AAV vectors and liposome complexes have been shown to be effective in preclinical studies of angiogenesis for myocardial and vascular disease.

Studies in animal models of therapy based on HIF-1 have provided evidence of angiogenesis using an approach of transcriptional regulation.

Preclinical studies of angiogenesis have identified disease processes that may contribute to attenuated angiogenesis such as endothelial dysfunction.

Phase I and II clinical trials have demonstrated the safety and suggested the efficacy of gene transfer in therapeutic angiogenesis for CAD and PVD.

Minimally invasive catheter-based and surgical techniques of delivery have been effective in clinical trials.

Prospects

Further improvements in viral technology will increase transmission efficiency and reduce toxicity because of inflammatory and immune responses.

The advancement of nonviral vector technology will allow for efficient gene transfer while avoiding the safety concerns associated with viral vectors.

Refinements in cell-based gene transfer to allow multigene and sequential gene expression in addition to regulatable expression with organ and tissue specificity will be needed.

Gene transfer using transcription factors that regulate the expression of multiple angiogenic genes may be preferable for the induction of angiogenesis.

Current investigations of the molecular mechanisms of disease states that have been shown to inhibit angiogenesis will provide vital information to develop an effective therapeutic strategy of neovascularization.

The method of delivery of angiogenic agents and timing of treatment will become essential factors in the strategy of therapeutic angiogenesis.

Phase II and III clinical trials will be critical in determining the utility of angiogenic gene therapy in the treatment of ischemic limb and heart disease.

Gene therapy is a potential method for therapeutic angiogenesis

Therapeutic angiogenesis has emerged as a promising investigational strategy for the treatment of patients with ischemic limb and heart disease. After over a decade of preclinical studies and recent clinical trials, gene therapy has been established as a potential method to induce therapeutic angiogenesis in patients with ischemic cardiovascular disease. Therapeutic angiogenesis using gene therapy vectors will be the subject of this review with emphasis on recent advancements and future directions in the treatment of ischemic limb and myocardial disease.

Neovascularization involves a complex series of events that likely include the coordinated action of several cytokines to produce new conduits of blood flow. Vasculogenesis, angiogenesis, and arteriogenesis are the three processes that may contribute to the growth of blood vessels.1 Vasculogenesis is the formation of new vessels from pluripotent stem cells as seen in embryonic development. Increasing evidence suggests that vasculogenesis also occurs in the adult as seen in the mobilization of endothelial progenitor cells from bone marrow and the incorporation of these cells into foci of neovascularization. Angiogenesis describes capillary growth from enlarged venules that sprout capillary buds, become divided by periendothelial cells (intussusception), or are separated by transendothelial cell bridges (bridging) to form capillaries. The process involves vasodilation and increased permeability to allow extravasation of proteins to form an extracellular matrix, endothelial cell proliferation and migration, and vessel formation. Endothelial cell differentiation follows in response to the local tissue environment.2 Angiogenesis is the manner by which capillaries proliferate in healing wounds and along the border of myocardial infarctions. Arteriogenesis is the process that produces arteries possessing a fully developed tunica media resulting in true collateral arteries. Smooth muscle cells may differentiate from various cell types including endothelial cells and bone marrow precursors. Arteriogenesis involves smooth muscle cell growth and proliferation, migration, and differentiation to a contractile phenotype.2 An example of arteriogenesis is the development of angiographically visible collaterals in patients with advanced obstructive atherosclerotic disease (Figure 1).

In patients with coronary artery disease (CAD) and peripheral vascular disease (PVD), progressive occlusion of arteries often leads to the development of collateral vessels that supply the ischemic tissue. However, this natural compensatory process of neovascularization is often not sufficient as evidenced by the large number of revascularization procedures performed annually. The lack of an adequate angiogenic response in part may be related to reduced production of angiogenic factors. Therapeutic angiogenesis describes the method of improving blood flow to ischemic tissue by the induction of neovascularization by angiogenic agents administered as recombinant protein or by gene transfer. The administration of recombinant protein or the genes that encode these proteins both have been used as techniques of angiogenic therapy in preclinical and clinical trials. Advantages of gene transfer include persistent expression of the angiogenic factor providing prolonged, local exposure, potential for single-dose regimens, and cell-specific angiogenic therapy. However, low efficiency of gene expression and immune deactivation of the foreign material are limiting factors. Furthermore, induction of an inflammatory response, nonspecific gene transfer to other cell types, and lack of regulation of gene expression and the resulting uncontrolled level of growth factor are additional risks to the patient.3,4 Protein formulations provide predictable pharmacokinetics and tissue therapeutic levels that allow for controlled dosing at the time of growth factor administration. In using protein-based angiogenic therapy, the administration of viral vectors and foreign genetic material is avoided. The short half-life of angiogenic proteins limits the duration of exposure and presents a possible need for additional doses. However, slow-release delivery systems may circumvent this issue and repeat dosing may be more effective because of the relative lack of significant inflammatory and immune responses to protein therapy.

As important as the type of angiogenic therapy is the delivery of the angiogenic agent. Intravascular delivery may lead to nonspecific and systemic exposure, while intramyocardial delivery techniques can allow for a local and sustained angiogenic effect.5 In addition, therapy based on single growth factor agents may not be adequate to induce functionally significant angiogenesis in humans because of the complexity of the angiogenic process, particularly in the context of advanced atherosclerotic disease and associated comorbid conditions. Multi-agent therapy may be necessary to achieve angiogenesis and provide significant improvements in myocardial perfusion and function as well as clinical outcome in patients.

Viral and nonviral gene transfer agents have been successfully studied

Among the current clinical trials of therapeutic angiogenesis for CAD and PVD, adenoviruses and plasmids are the vectors most often studied likely because of the ease of production, reasonable transfection efficiency, and expression in nonproliferating cells. First-generation E1-deleted adenoviral vectors were limited in vascular gene transfer because of endothelial injury and the inflammatory response. The development of attenuated adenoviral vectors with further deleted elements of the viral genome has produced vectors that result in increased transgene expression and reduced inflammation in cardiovascular gene transfer. Deletions of the E1 and E4 regions to produce a second-generation adenoviral vector have been shown to result in improved transgene expression with reduced inflammatory response and preserved endothelium-dependent relaxation. However, lack of prolonged transgene expression at 28 days may have been representative of residual, although substantially less, inflammation induced by the second-generation adenoviral vector because of low-level, late gene expression.6 Fully deleted adenoviral vectors may potentially eliminate this late expression and any associated inflammatory response. This technology may improve transgene expression despite the presence of antiadenoviral neutralizing antibodies.7 Production of fully deleted vectors is made possible by a helper virus that provides viral proteins required for replication and packaging. A study of fully deleted adenoviral vectors, also known as gutless or helper-dependent, carrying a marker transgene, erythropoietin, demonstrated that intramuscular delivery resulted in efficient and prolonged expression in both immunocompetent mice and those immunized against the adenovirus serotype.7 The results of the study are clinically important

considering the significant proportion of the population with pre-existing immunity to adenovirus. In addition, recombinant adeno-associated viruses (AAV) are potential vectors for therapeutic angiogenesis. Advantages of the AAV vectors for gene transfer include the transduction of nonproliferating cells, lasting transgene expression, and reduced inflammatory response, while limitations involve difficulty with production and a small packaging capacity.

Nonviral methods of gene transfer studied in clinical trials include plasmid DNA and liposomal complexes. The use of nonviral techniques avoids the concerns over the toxicity associated with viral vectors. While the inflammatory response to adenoviral vectors is well described, both plasmid DNA and liposomal complexes also potentially induce inflammation.8,9 Despite the ease of production and scale-up of plasmid and liposomal complexes, low transmission efficiency and transgene expression are limiting. Transmission efficiency of plasmid DNA may be improved by the use of ultrasound. Ultrasound exposure with microbubble echocontrast agents increase transgene expression significantly after naked DNA transfection by cell membrane permeabilization. This technique of membrane permeabilization, or acoustic cavitation, with microbubble echocontrast was reported to increase transgene expression by approximately 300-fold through the creation of transient small holes in the cell surface membrane through which naked DNA is rapidly translocated.10 In a study of plasmid DNA transmission efficiency, luciferase plasmid transfection using ultrasound with microbubble echocontrast was increased approximately 10-fold compared to plasmid alone in cultured human skeletal muscle. In the same report, gene transfer of a hepatocyte growth factor (HGF) plasmid in a rabbit model of hindlimb ischemia produced increased angiographic score and capillary density in animals transfected using ultrasound with microbubble echocontrast versus transfection with plasmid alone.11 The use of ultrasound with microbubble contrast also has been demonstrated to increase the transfection efficiency of plasmid DNA in human aortic endothelial and vascular smooth muscle cells without apparent toxicity.12 This technique has the potential to improve the efficiency of plasmid DNA transfection in human myocardial tissue as well. The transmission efficiency of liposomes may be enhanced by improvements in cationic polymers.13 Liposomes have been shown to be effective in the transfer of growth factors in animal models of angiogenesis. In a rabbit ischemic hind limb model, vascular endothelial growth factor (VEGF) gene transfer by cationic liposome resulted in neovascularization and improved blood flow in the ischemic limb.14 Liposome carriers also have been demonstrated to be effective in angiogenesis based on HGF. Transfer of HGF with the hemagglutinating virus of Japan (HVJ)liposome method has been shown to induce angiogenesis in normal and infarcted myocardium.15

Cell-based gene transfer is a novel strategy that utilizes autologous cells as vectors after in vitro transfection with a transgene of interest.16 Such a system is able to circumvent the inflammatory response by using autologous cells and achieves prolonged expression by stable transfection using various measures including electroporation and in vitro retroviral or lentiviral transfection. In addition, complex constructs can be synthesized that would allow stable, regulatable expression and multiple transgene expression. Recent investigations have focused on gene transfer by cellular transplantation to induce neovascularization in ischemic tissue using skeletal myoblasts and angioblasts.17,18,19

VEGF and FGF are the most widely studied growth factors

Numerous growth factors and transcription factors have been associated with physiologic and pathologic angiogenesis. Among the transgenes under investigation in clinical trials for CAD and PVD, genes that encode growth factors predominate. VEGF and fibroblast growth factor (FGF) are the agents most widely studied in clinical trials, specifically the 121 and 165 amino-acid isoforms of VEGF1, VEGF2, FGF1, and FGF4. Gene transfer of VEGF and FGF has been shown to induce functionally significant angiogenesis in numerous preclinical studies of angiogenic therapy for ischemic heart disease20 and peripheral arterial disease.21 Another strategy under investigation in clinical trials is a therapy based on the transcription factor hypoxia-inducible factor-1 (HIF-1 ). The expression of many angiogenesis-related genes, including VEGF and the VEGF receptor FLT-1, is regulated by HIF-1 . A hybrid, constitutively active form of HIF-1 , has been synthesized from the DNA-binding and dimerization domains of the HIF-1 subunit and the transactivation domain of the VP16 protein of the herpes simplex virus. Angiogenic gene transfer of this hybrid form has been reported in preclinical studies as described below. Another potential factor that regulates angiogenesis is the peptide PR-39. This peptide increases the cellular levels of HIF-1 by inhibiting its degradation in the ubiquitinproteasome complex. PR39 has been shown to increase the expression of VEGF, the VEGF receptors KDR and FLT-1, and the FGF receptor 1.22 Concerns over a nonspecific action of PR39 related to charge are being investigated.

Preclinical and clinical trials in PVD have demonstrated safety and suggested efficacy

Angiogenic responses to growth factor gene transfer using plasmid and adenoviral vectors have been well documented in animal models of chronic limb ischemia. VEGF gene transfer techniques using nonviral and adenoviral vectors have been the more common methods in preclinical studies. Recently, angiogenesis has been induced in animal models of limb ischemic using AAV vector-mediated therapy with VEGF. In rat hind limb models of ischemia, VEGF-based therapy via AAV vectors produced increased blood flow and capillary growth in treated, ischemic limbs compared to controls.23,24 HGF also has emerged as a potential agent in therapeutic angiogenesis. In a rabbit ischemic hind limb model, intramuscular injection of human HGF plasmid resulted in enhanced collateral development by angiography and increased blood flow and blood pressure in the ischemic limb.25 Furthermore, evidence has recently been published that suggests FGF-2 contributes to the regulation of HGF expression.26

Recent results of clinical trials of therapeutic angiogenesis for peripheral arterial disease have provided further information on the clinical response to angiogenic gene therapy including measures of neovascularization as well as known side effects such as edema formation. Lower extremity edema was evaluated in 90 patients that were treated with VEGF165 by intra-arterial or intramuscular gene transfer. Edema was observed in 34% of patients, being more common in those patients with rest pain and ischemic ulcers as compared to those with claudication only. The increased vascular permeability was an effect attributed to the VEGF therapy.27 Recently, the results of a phase II clinical trial of VEGF-1 gene therapy for chronic limb ischemia were published. In the randomized, placebo-controlled, double-blinded study of catheter-based VEGF-1 gene therapy after percutaneous transluminal angioplasty (PTA), patients in the treatment groups received intra-arterial VEGF-1 by adenoviral vector or liposomeplasmid carrier while those in the control group received crystalloid solution. Digital subtraction angiography (DSA) revealed increased vascularity in both treated groups. Both the VEGF-adenoviral and VEGF-liposomeplasmid groups showed increased vascularity distal to the site of gene transfer. In addition, the VEGF-adenoviral group demonstrated significantly increased vascularity in the clinically most severe region of ischemia. However, the ratio of lower extremity compared to upper extremity blood pressure, or ankle-brachial index (ABI), was not significantly different between treated and control groups. In addition, antiadenoviral antibodies increased in 11 of 18 patients administered VEGF-1 by adenoviral vector.28

Phase III clinical trials in patients with myocardial ischemia have been completed

Several preclinical studies have demonstrated efficacy of angiogenic gene therapy for myocardial ischemia. FGF- and VEGF-based protocols have been the most widely studied. A recent report of VEGF121 gene transfer by an adenoviral vector in a porcine model of myocardial ischemia demonstrated that intramyocardial delivery resulted in transient, focal VEGF expression in the target, ischemic area of the myocardium. The localized VEGF expression was 10-fold greater than in intracoronary delivery and produced regional improvement in myocardial blood flow.29 Other studies of late have reported alternative strategies producing favorable results in animal models of myocardial angiogenesis. In a mouse model of myocardial ischemia, an AAV-VEGF vector injected around ischemic myocardium resulted in neovascularization without evidence of inflammation.30 Gene transfer of the HIF-1 VP16 hybrid in a rabbit model of hind limb ischemia was associated with increased regional blood flow and capillary density.31 In a rat model of acute myocardial ischemia, intramyocardial delivery of the HIF-1 VP16 hybrid plasmid was able to reduce the size of myocardial infarction and increase capillary density in the border zone of the infarct area. The induction of angiogenesis was similar to the neovascularization that resulted from VEGF therapy by plasmid gene transfer in the same model.32 In a study of transgenic mice with porcine PR39 cDNA, angiogenesis was induced in the PR39 mice compared to age-matched controls as seen by increased CD-31-stained vascular structures.22 These results suggest that agents that produce a multifactorial angiogenic response such as HIF-1 and PR39 are effective in neovascularization and may represent a more effective strategy in therapeutic angiogenesis.

Results of several clinical trials of gene transfer for therapeutic angiogenesis have been reported. These trials include uncontrolled, open label designs primarily investigating safety and feasibility. The results of these studies should be interpreted with caution considering the significant placebo effect observed in patients with CAD. In a study of 129 patients enrolled in control groups of phase I and II clinical trials of therapeutic angiogenesis and laser myocardial revascularization, the mean CCS angina class was 3.00.5 at baseline and 2.10.6 at 6 months (P<0.001), with 24.8% of patients improved by two or more angina classes. Mean follow-up was 306 months and at last follow-up, mean CCS angina class was 2.30.8 (P<0.001). The results of this study underscore the significance of the placebo effect in this patient population of severe CAD.33

Two studies of surgical angiogenic therapy using gene transfer have been published recently. A phase I

study evaluated the surgical delivery of VEGF165 plasmid DNA through a mini-thoracotomy in seven patients. As previously reported, both nitroglycerin intake and CCS angina class were significantly reduced, while improved myocardial perfusion was suggested by single-photon emission computed tomography (SPECT) and coronary angiography.34 A study of intramyocardial gene transfer through a limited thoracotomy was initiated using VEGF-2 plasmid DNA in patients with chronic stable angina. An initial report on 11 patients revealed reductions in angina episodes and nitroglycerin use as well as improvement in exercise tolerance testing.35

Two trials of catheter-based myocardial gene transfer of plasmid DNA encoding for VEGF-2 have been reported of late. An initial randomized, single-blind, placebo-controlled study investigated the safety and feasibility of VEGF-2-based therapy in six patients. Patients that received VEGF-2 plasmid DNA experienced reduced angina compared to control patients at 90 days of follow-up. Reduction in ischemia and improvement in myocardial perfusion were suggested by electromechanical mapping and SPECT imaging, respectively.36 A multicenter, randomized, double-blind, placebo-controlled phase III clinical trial followed. VEGF-2 plasmid DNA was administered as part of a dose-escalating protocol to patients with Canadian Cardiovascular Society (CCS) class III or IV angina. The catheter-based delivery to the endocardial surface of the left ventricle resulted in no hemodynamic alterations, sustained ventricular arrhythmias, or electrocardiographic evidence of infarction. A recent interim report after enrollment of 19 patients described a significant reduction in CCS angina class in the treated group compared to controls.37 Although these results as a whole suggest the therapeutic efficacy of angiogenesis by gene transfer, a study of intramyocardial gene transfer of plasmid DNA encoding VEGF-A and VEGF-B as an adjunct to CABG in 24 patients was published recently that provided only modest evidence of improved perfusion.38

The angiogenic gene therapy (AGENT) study was the first randomized, double-blind, placebo-controlled trial of therapeutic angiogenesis by gene transfer for myocardial ischemia. FGF-4 carried by an adenoviral vector was given intracoronary to 79 patients with chronic stable angina randomized in a 1:3 ratio to produce 19 in the control group and 60 in the treatment group. Overall, FGF-4 therapy using an adenoviral vector was well tolerated without significant safety concerns. No significant difference was observed in stress-induced wall motion by echocardiography between groups. The results of the primary end point, exercise treadmill testing (ETT), after 4 and 12 weeks of follow-up demonstrated a nonsignificant trend towards improvement in the FGF-4-treated group. Subgroup analysis revealed a significant improvement in those patients with baseline ETT of less than 10 min.39

Patient selection and end-point evaluation are important factors in clinical trial design

Therapeutic angiogenesis has provided a new treatment strategy for patients with end-stage CAD and PVD. Currently, results of adequately powered, randomized, double-blind, placebo-controlled trials are lacking. For enrollment in clinical trials, randomization should include baseline angiogenic response manifest as collateralization. Patients selected for trials of therapeutic angiogenesis often previously have had multiple percutaneous and surgical revascularization attempts. These individuals may possess resistance to stimulation of neovascularization, considering they likely suffer from failure of natural angiogenic responses. Thus, patients enrolled in current trials may represent the group least likely to respond. Ideal candidates are those with ischemic but viable myocardium and diffuse multivessel disease. Exclusion criteria generally include a history of malignancy or proliferative retinopathy because of concerns of pathogical angiogenesis. Patients with abnormal baseline renal function and proteinuria are excluded from trials of FGF therapy because of the risk of renal toxicity.

End points of cardiac morbidity and mortality including myocardial infarction and death may provide objective measures of outcome. However, the low frequency of these events in clinical trials of treatment of myocardial disease indicate that a prohibitively large study population may be required to show significant reductions in these outcomes. Limb salvage from amputation may be used in a patient population with advanced peripheral vascular disease. Other end points in cardiac trials include exercise tolerance testing and measures of myocardial perfusion using single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI), while end points in peripheral vascular trials may include vascularity by DSA or magnetic resonance angiography, lower extremity blood pressure by ABI, and healing of ischemic ulcers. The response to angiogenic gene therapy for limb ischemia may be assessed through improvements in claudication and rest pain. Relief of myocardial ischemic symptoms and improvements in quality of life may be determined by methods such as the Seattle Angina Questionnaire or the Canadian Cardiovascular Society angina classification. However, the placebo effect has been shown to be very powerful in studies of patients with severe CAD and PVD.

Prospects

Recent advances in gene transfer have allowed therapeutic angiogenesis to become a potential treatment for CAD and PVD.

Continued development of viral and nonviral vector technology will improve transgene transmission and expression efficiency. Refinements in cell-based therapy may provide for regulatable and multiagent therapy. The attenuated angiogenic response in patients enrolled in clinical trials may be related to comorbid pathophysiology that is associated with CAD and PVD. Endothelial dysfunction is associated with atherosclerotic disease, and may play a role in a reduced angiogenic response. We investigated the effect of endothelial dysfunction secondary to hypercholesterolemia on therapeutic angiogenesis by perivascular delivery of FGF-2 through a mini-thoracotomy.40 In our pig ameroid constrictor model of chronic myocardial ischemia, hypercholesterolemic animals showed significant endothelial dysfunction and impaired angiogenesis manifest as decreased perfusion compared to the control, normal diet group. Thus, endothelial dysfunction may represent one of many factors that prevent a significant angiogenic response in patients with CAD and PVD. The future development of treatment options for endothelial dysfunction may allow for an improved angiogenic response to growth factor therapy in humans with atherosclerotic occlusive disease.

Improvements in delivery modalities with increased local distribution and retention and reduced systemic circulation are needed. Intramyocardial and intramuscular delivery techniques currently under investigation in phase I and II clinical trials may provide evidence of efficacy that was lacking in studies using intravascular routes of administration. Delivery optimization studies also should be conducted for specific agents prior to preclinical and clinical investigation. Multiagent therapy may be needed to achieve the complex process of angiogenesis in humans. The natural angiogenic response is likely a result of the actions of multiple growth factors at various points in time. The sequential or concomitant administration of the agents may be an important factor as well. In addition, a synergistic mechanism of action between growth factors in angiogenesis has been suggested. Furthermore, it has become clear that the timing of growth factor therapy may be critical in inducing an angiogenic response. A recent study of VEGF-based angiogenesis has demonstrated that a critical duration of growth factor exposure is required to prevent regression of the newly formed vasculature.41 Thus, therapeutic angiogenesis using methods with prolonged presence of growth factors over a few weeks may be necessary, as suggested by the promising results of the clinical trial using surgically implanted heparin-alginate microcapsules that release FGF-2 over a course of 34 weeks.42

Conclusions

Therapeutic angiogenesis is a promising therapy for patients with CAD and PVD not amenable to current revascularization techniques. Clinical trials of gene transfer for therapeutic angiogenesis in the treatment of ischemic limb and heart disease have been limited to predominantly uncontrolled phase I studies that have demonstrated safety and preliminary reports of phase II trials that have provided modest evidence of clinical efficacy. As further investigation of gene transfer proceeds, monitoring of potential toxicities must be continued to maximize the benefit and minimize the risk of angiogenic therapy. Overall, the role of therapeutic angiogenesis by gene transfer as a potential treatment option for ischemic limb and heart disease will be determined by adequately powered, randomized, placebo-controlled phase II and III clinical trials.

Acknowledgements

Supported in part by NIH Grants MO1-RR01032 and HL63609 (RJL), and HL46716 and HL69024 (FWS). Dr Khan is supported by an NIH Individual National Research Service Award, HL69651-01.

References

1 Laham R, Simons M. Growth factor therapy in ischemic heart disease. In: Rubanyi G (ed). Angiogenesis in Health and Disease. Marcel Decker: New York, 2000, pp 451475.

2 Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000; 6: 389395. Article PubMed

3 Laham RJ, Simons M, Sellke F. Gene transfer for angiogenesis in coronary artery disease. Annu Rev Med 2001; 52: 485502. Article

4 Laham RJ, Mannam A, Post MJ, Sellke F. Gene transfer to induce angiogenesis in myocardial and limb ischaemia. Expert Opin Biol Ther 2001; 1: 985994.

5 Simons M et al.. Clinical trials in coronary angiogenesis: issues, problems, consensus: an expert panel summary. Circulation 2000; 102: E73E86. PubMed

6 Qian HS et al.. Improved adenoviral vector for vascular gene therapy: beneficial effects on vascular function and inflammation. Circ Res 2001; 88: 911917.

7 Maione D et al.. An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus. Proc Natl Acad Sci USA 2001; 98: 59865991. Article

8 Hofman CR et al.. Efficient in vivo gene transfer by PCR amplified fragment with reduced inflammatory activity. Gene Therapy 2001; 8: 7174. Article

9 Norman J et al.. Liposome-mediated, nonviral gene transfer induces a systemic inflammatory response which can exacerbate pre-existing inflammation. Gene Therapy 2000; 7: 14251430. Article

10 Lawrie A et al.. Microbubble-enhanced ultrasound for vascular

gene delivery. Gene Therapy 2000; 7: 20232027. Article PubMed

11 Taniyama Y et al.. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Therapy 2002; 9: 372380. Article

12 Taniyama Y et al.. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002; 105: 12331239. Article

13 Simberg D et al.. Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity. J Biol Chem 2001; 276: 4745347459. Article

14 Gowdak LH et al.. Induction of angiogenesis by cationic lipid-mediated VEGF165 gene transfer in the rabbit ischemic hindlimb model. J Vasc Surg 2000; 32: 343352. Article

15 Aoki M et al.. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets. Gene Therapy 2000; 7: 417427. Article PubMed

16 Powell C, Shansky J, Del Tatto M, Vandenburgh HH. Bioartificial muscles in gene therapy. Methods Mol Med 2002; 69: 219231.

17 Lu Y et al.. Recombinant vascular endothelial growth factor secreted from tissue-engineered bioartificial muscles promotes localized angiogenesis. Circulation 2001; 104: 594599.

18 Suzuki K et al.. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 2001; 104: I207I212.

19 Iwaguro H et al.. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002; 105: 732738. Article

20 Khan TA, Sellke FW, Laham RJ. Therapeutic angiogenesis for coronary artery disease. Curr Treat Options Cardiovasc Med 2002; 4: 6574.

21 Faries PL, Pomposelli Jr FB, Quist WC, LoGerfo FW. Assessing the role of gene therapy in the treatment of vascular disease. Ann Vasc Surg 2000; 14: 181188. Article

22 Li J et al.. PR39, a peptide regulator of angiogenesis. Nat Med 2000; 6: 4955. Article PubMed

23 Byun J et al.. Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol 2001; 33: 295305. Article PubMed

24 Shimpo M et al.. AAV-mediated VEGF gene transfer into skeletal muscle stimulates angiogenesis and improves blood flow in a rat hindlimb ischemia model. Cardiovasc Res 2002; 53: 9931001. Article

25 Taniyama Y et al.. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease. Gene Therapy 2001; 8: 181189. Article

26 Onimaru M et al.. Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs. Circ Res 2002; 91: 923930. Article

27 Baumgartner I et al.. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor. Ann Intern Med 2000; 132: 880884. PubMed

28 Makinen K et al.. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002; 6: 127133. Article

29 Lee LY et al.. Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121. Ann Thorac Surg 2000; 69: 1423; discussion 2314. Article PubMed

30 Su H, Lu R, Kan YW. Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart. Proc Natl Acad Sci USA 2000; 97: 1380113806. Article PubMed

31 Vincent KA et al.. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alphaVP16 hybrid transcription factor. Circulation 2000; 102: 22552261. PubMed

32 Shyu KG et al.. Intramyocardial injection of naked DNA encoding HIF-1alphaVP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat. Cardiovasc Res 2002; 54: 576583. Article

33 Laham RJ et al.. Longevity of the placebo effect in patients enrolled in angiogenesis and laser myocardial revascularization trials. J Am Coll Cardiol 2002; 39: 11A.

34 Sarkar N et al.. Effects of intramyocardial injection of phVEGF-A165 as sole therapy in patients with refractory coronary artery disease 12-month follow-up: angiogenic gene therapy. J Intern Med 2001; 250: 373381. Article

35 Vale PR et al.. Effective gene transfer of phVEGF-2 for therapeutic angiogenesis in chronic myocardial ischemia as assessed by NOGATM left ventricular electromechanical mapping. Circulation 2000; 102: II689.

36 Vale PR et al.. Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia. Circulation 2001; 103: 21382143. PubMed

37 Losordo DW et al.. Phase 12 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation 2002; 105: 20122018. Article

38 Huwer H et al.. Simultaneous surgical revascularization and angiogenic gene therapy in diffuse coronary artery disease. Eur J Cardiothorac Surg 2001; 20: 11281134. Article

39 Grines CL et al.. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation 2002; 105: 12911297. Article

40 Ruel M et al.. Inhibited angiogenic response to surgical FGF-2 protein therapy in a swine model of endothelial dysfunction. Circulation (in press).

41 Dor Y et al.. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 2002; 21: 19391947. Article PubMed

42 Ruel M et al.. Long-term effects of surgical angiogenic therapy with fibroblast growth factor 2 protein. J Thorac Cardiovasc Surg 2002; 124: 2834. Article

Figures

Figure 1 Arteriogenesis in an animal model of myocardial ischemia. Batson casts were performed on explanted hearts following left circumflex coronary artery ameroid constrictor placement. After myocardial digestion, vessels were visualized (blue for occluded left circumflex, red for left anterior descending (LAD), and white for right coronary arteries). Arrows point to epicardial collateral vessels going from the LAD and right coronary arteries to the ischemic left circumflex territory.

Received 10 September 2002; accepted 3 December 2002

January 2003, Volume 10, Number 2, Pages 95-99

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ReviewGene Therapy Progress and Prospects: Alpha-1 antitrypsin

A A Stecenko and K L Brigham

Center for Translational Research in the Lung, McKelvey Center for Lung Transplantation and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

Correspondence to: KL Brigham, Department of Medicine, Emory University School of Medicine, Whitehead Biomedical Research Building, 615 Michael Street, Suite 205, Atlanta, GA 30322, USA

Abstract

Over the 2 years covered here, there has been one clinical study in which a normal alpha-1 antitrypsin (AAT) gene was delivered to the nasal epithelium of AAT-deficient subjects using plasmidliposome complexes; a second study using an adeno-associated vector should begin soon. Although progress in clinical studies has been slow, advances in both viral and nonviral vector designs show considerable promise. Strategies that combine liposome technology with imaginative vector design may permit long-term expression of a normal transgene that is sufficient to achieve therapeutic serum AAT concentrations. While reproducing the normal physiology by targeting normal AAT gene expression to the liver is logical, local expression in lung cells may be less demanding of the technology and offers therapeutic benefits that are produced neither by AAT protein therapy nor by AAT gene therapy targeted to the liver. Developing technologies may permit direct correction of the mutant AAT gene using innovative approaches to in vivo gene repair.

Gene Therapy (2003) 10, 9599. doi:10.1038/sj.gt.3301947

Keywords

alpha-1 antitrypsin; organ-targeted gene therapy; gene repair; emphysema

In brief

Progress

Human gene therapy trials: cautious optimism Strategies for reducing the cellular immune response and eliminating need for receptors may improve

utility of adenoviral vectors

Adeno-associated viral vectors have low toxicity and effectively deliver an AAT transgene in animals

Nonviral vectors can deliver a functioning AAT gene in vivo and can be designed to prolong transgene expression

Prospects

Lung-directed AAT gene transfer may be therapeutic without 'therapeutic' protein levels in body fluids: applications beyond AAT deficiency

RNADNA oligonucleotide (RDA)-directed repair of the defective AAT gene

Extension of AAT gene therapy to treat acquired emphysema

Expanded use of AAT gene therapy as 'antiprotease therapy' (antiviral, anti-inflammatory)

Human gene therapy trials: cautious optimism

Alpha-1 antitrypsin (AAT) is the most prominent endogenous serine proteinase inhibitor (SERPIN) in humans. An inherited AAT deficiency is second in prevalence only to cystic fibrosis among inherited diseases of the lungs. The genetic abnormality is thoroughly described, current therapy is suboptimal and multiple vectors capable of delivering the normal AAT gene in vivo have been developed. In addition, unlike the cystic fibrosis transmembrane conductance regulator, AAT is a secreted protein so that gene therapy should be less demanding of the technology. However, in contrast to the situation with cystic fibrosis, there have been few clinical trials of gene therapy with AAT. There are currently no active AAT gene therapy trials in progress.

A general surrogate criterion for efficacy of AAT gene therapy has been achieving 'therapeutic' concentrations (that is approaching normal) in circulating blood or (receiving more emphasis) in bronchoalveolar lavage fluid. If the goal is to reproduce normal physiology, production and secretion of sufficient amounts of normal AAT protein by the liver to mimic the normal condition, those criteria are appropriate. The criteria are based primarily on the rationale used to approve AAT protein for clinical use as an orphan drug without proven effect on the course of the disease. Although some of the more recent technologies are approaching levels of gene delivery and expression sufficient to meet these criteria, recent evidence suggests that if the AAT gene is expressed in the lungs (ie at the site of the disease for which the therapy is intended), the level of AAT transgene expression required may be less than previously thought.

There are two clinical trials in humans either imminent or nearly so that propose delivery of a normal AAT transgene to the respiratory tract of humans. One proposed study is based on animal studies indicating that the normal AAT gene delivered in an adeno-associated viral vector (AAV-AAT) can produce prolonged 'therapeutic' serum concentrations of the normal protein (see discussion of the animal studies below).1 This proposal would inject AAV-AAT intramuscularly with the goal of achieving normal blood AAT concentrations.2 Studies would be done in 812 relatively healthy subjects with the common PiZZ phenotype AAT deficiency. According to the information on the Alpha-1 web site,2 these studies are nearing approval by the appropriate regulatory agencies and should begin soon.

The one reported study in human subjects used an unmodified cationic liposome (DOTMA:DOPE) and a plasmid vector containing the normal human AAT gene driven by a CMV promoter.3 In this study, the lipoplex was instilled into one nostril of five subjects with ZZ phenotype AAT deficiency, with the contralateral nostril serving as a control. AAT concentrations were measured in serial samples of nasal lavage fluid. As shown in Figure 1, AAT concentrations increased significantly in the transfected nostril, peaking on day 5 after transfection and returning to baseline by day 14. The mean peak AAT concentration in the transfected nostril in these five subjects was about one third the normal level (Figure 2). Those studies demonstrate that lipoplex technology can deliver a normal AAT gene to human respiratory epithelium in vivo, but delivery to the entire lung presents other challenges.

A new proposal is to conduct a similar investigation in subjects with cystic fibrosis where lung destruction may be

partly attributable to the presence of large amounts of protease, but to extend the study to determine the effects of repeated dosing of the lipoplex. Two subjects have completed this protocol, but the study is now on clinical hold by the FDA awaiting production of new plasmid and liposome reagents.

Lung-directed AAT gene transfer may be therapeutic without 'therapeutic' protein levels in body fluids: applications beyond AAT deficiency

Proteolytic events are ubiquitous in both physiologic and pathologic processes. AAT is normally produced primarily in the liver, reaching the lung via the circulation. Since AAT is a relatively large hydrophilic protein, it is excluded from intracellular and other cryptic spaces where pathologic proteolytic events occur. If lung cells are made to produce AAT, the antiproteolytic activity might locate inside cells where it is produced as well as in interstitial and intercellular spaces not usually available to circulating AAT. Thus, locally expressed AAT might have potentially therapeutic activities similar to those of small molecule antiproteases.

Figure 2 summarizes data from nasal lavage fluid in studies on AAT-deficient subjects who had a single nostril transfected with a normal human AAT gene delivered as a lipoplex.3 Shown are measurements of AAT and of interleukin-8, a proinflammatory cytokine measured in these studies as an indicator of inflammation. When on no therapy, AAT concentrations in nasal lavage fluid were very low as expected, and IL-8 concentrations were significantly elevated compared to normal, indicating some level of continuing inflammation. While these subjects were receiving intravenous AAT protein therapy, nasal lavage fluid concentrations of AAT were in the normal range, but IL-8 levels remained elevated. In contrast, when nasal epithelium was expressing the normal AAT transgene, nasal lavage fluid AAT concentrations were only about one-third the normal mean, but IL-8 concentrations were normal. These findings suggest that if the normal AAT gene is expressed locally, it may not be necessary to achieve 'therapeutic' AAT concentrations in plasma or bronchoalveolar lavage fluid in order to have a therapeutic effect. In addition, the anti-inflammatory effect of local expression may expand the disease targets for this therapy to include a broad range of chronic lung diseases in which inflammation is a major part of the pathophysiology.

Strategies for reducing the cellular immune response and eliminating need for receptors may improve utility of adenoviral vectors

Replication-deficient adenoviral vectors, originally promising as vectors for delivering therapeutic transgenes to the lungs, have been disappointing for several reasons. Human respiratory epithelium is not rich in the requisite specific cell receptors, so that adenoviral vectors are inefficient at delivering transgenes to the lungs of humans.4 In addition, even replication-deficient adenoviruses cause an acute inflammatory response and immunity is common. Several strategies have been used in attempts to improve efficiency and decrease toxicity of these vectors.

One strategy to overcome pre-existing immunity is to employ species-specific adenoviral vectors in a heterologous species. This approach should diminish the issue of pre-existing immunity. An ovine adenoviral vector was used to deliver an AAT transgene to mice by intramuscular injection.5 Moderate doses of the vector resulted in high serum AAT levels, and expression of the gene was limited to the site of injection, but expression was transient and viral DNA was rapidly cleared and was accompanied by a cellular immune response. Viral doses low enough to minimize the immune response still produced potentially therapeutic serum AAT levels (>100 ngml) and a second intramuscular injection produced serum AAT levels similar to the first. These studies address some of the problems limiting the clinical potential of adenoviral vectors, but requirements for long-term repeated dosing with unknown efficacy and safety are limitations to clinical application.

Although the bulk of work with liposomes as a technology for delivering genes in vivo has been with lipoplex formulations (plasmid-cationic liposome complexes), a recent report uses liposomes as an escort for an adenoviral vector. Yotdna et al6 encapsulated adenoviral vectors into bilamellar DOTAP:chol liposomes with the hypothesis that the lipid-coated virus could enter cells independent of specific receptors and would also be unaffected by virus-specific antibodies. Adenoviral vectors expressing either the reporter gene, -galactosidase, or the human alpha-1 antitrypsin gene were coated with liposomes and their transfection efficiency in cultured cells with and without adenoviral receptors was compared with uncoated vector. The liposomeviral complexes infected cells independent of receptors. The complexes also protected the virus from neutralization by human antisera. In mice, intravenous delivery of viralliposome complexes expressing the AAT gene produced detectible serum AAT levels for up to 30 days (uncoated vector expressed for only about a week). The coated vector also expressed more exuberantly after a second dose delivered a month after the first. The acute inflammatory response (as measured by serum levels of IL-6) was significantly less in animals given the liposomeviral complex than those given the

uncoated viral vector. More advanced formulations employing combinations of liposome and viral technologies for gene delivery may eventually be clinically useful.

Adeno-associated viral vectors have low toxicity and effectively deliver an AAT transgene in animals

As it becomes obvious that adenoviral vectors are less than optimal, other viral vectors are being explored. Adeno-associated virus (AAV) vectors have the advantage of apparent high in vivo transgene expression and low toxicity. A single injection of an AAV vector expressing the AAT gene into the liver can achieve potentially therapeutic serum AAT levels that persist for at least several weeks.7

However, in order to achieve potentially therapeutic serum AAT levels with intramuscular injection, it is necessary to give fairly high doses of vector. A more efficient system is desirable both to minimize toxic potential and because production of AAV vector is complicated and expensive. In addition, AAT deficiency causes liver as well as lung disease. The liver disease results from aberrant intracellular trafficking of the misfolded mutant AAT protein. So, although the lung disease might be prevented by achieving sufficient extracellular AAT concentrations, correcting the liver disease will likely require expression of a normal AAT transgene in the majority of hepatic cells since suppression of synthesis of the abnormal protein by expressing the normal gene would only occur in cells expressing the normal transgene.

The group that undertook the original studies with intramuscular injection of an AAT-containing AAV vector now report studies in which such vectors were injected into the portal vein of adult mice.7 They compared results to those with intravenous injection. They injected mice intravenously with an AAV construct containing the AAT gene driven by a CMV- actin hybrid promoter at varying doses and followed serum AAT protein concentrations. There was a dose-related elevation in serum AAT with potentially therapeutic levels sustained for up to 50 weeks at the higher viral doses. Southern blot analysis of liver tissue indicated that the vector was predominantly episomal at an average density of 25 copies per cell. However, immunohistochemical analysis of liver tissue using an antibody specific for the human protein showed a patchy distribution of the protein and a relatively small fraction of cells (6.4%) containing the protein. There was no evidence of toxicity.

AAV vectors appear to have some distinct advantages over adenoviral vectors. The lack of an acute inflammatory response, even on direct injection into the liver, and the prolonged expression of the normal AAT gene at levels sufficient to produce potentially therapeutic serum AAT concentrations are encouraging for possible treatment of the lung disease in AAT-deficient subjects. However, the efficacy of intraportal delivery of the normal AAT gene in an AAV vector for treating the liver disease of AAT deficiency will likely require strategies to increase the fraction of hepatocytes expressing the transgene.

Nonviral vectors can deliver a functioning AAT gene in vivo and can be designed to prolong transgene expression

Nonviral vectors for gene therapy are limited by low efficiency of transgene expression and short duration of expression. They continue to be attractive, however, because plasmids are not human pathogens in any setting and therefore carry less potential for harm than most viral vectors. Recent studies attempt to address these issues.

There are several reasons for the short duration of expression of transgenes delivered to mammals in plasmid vectors. One reason is that plasmids do not replicate in mammalian cells so that host cell replication obligatorily dilutes the vector. However, plasmids can be constructed to include eukaryotic replication initiation sites that render them capable of replicating in cycle with replication of the host cell. Stoll et al8 constructed a plasmid expression vector that contained the AAT gene and incorporated eucaryotic replication initiation sequences from EpsteinBarr virus, EBNA1 and its family of binding sites. After intravenous injection using the interesting technique of 'hydrodynamic injection' of the naked plasmid into mice, they found greater than 300 gml of AAT in serum, and increased serum AAT concentrations were maintained for greater than 9 months after a single administration of the vector. Although it is well established that these vectors containing EBV eukaryotic replication initiation sequences undergo extrachromosomal replication in dividing cells in culture, the fact that essentially nondividing liver cells in vivo retain the expressing vector for long period of time is somewhat unexpected. The favorable safety profile of plasmid vectors over viral vectors makes these observations interesting as the technology for clinical application develops.

Intravenous injection of linear DNA encoding the AAT gene driven by an RSV promoter into mice is reported to achieve 10100 fold higher serum AAT concentrations than similar administration of circular DNA with expression persisting for at least 9 months.9 The administered DNA localized to the liver and rapidly formed large unintegrated concatemers. This physical form of the DNA may contribute to more exuberant and prolonged in vivo expression than seen with circular vectors. However, those results were obtained after rapid intravenous injection of 40 g of DNA in 2 ml saline. Assuming a mouse of approximately 25 g, similar administration to a 50 kg human would require rapid intravenous injection of 80 mg of DNA in 4 l of saline, so that the clinical applicability of this approach

is questionable at present.

The clinical potential of cationic liposomeplasmid complexes (lipoplexes) remains interesting because of their apparent safety, but their limitations are similar to those of other nonviral technologies. Dasi et al10 covalently coupled an asialoglycoprotein (asialofetuin) ligand for a unique hepatocyte receptor to either cationic or anionic liposomes and administered lipoplexes containing these modified liposomes and a plasmid vector containing the AAT gene intravenously to mice. They produced serum AAT levels of 50300 ngml that persisted for up to 12 months with DNA doses of 200 ngmouse. The asialoglycoprotein conjugate conferred considerably greater efficacy than unmodified liposomes, either cationic or anionic.

RNADNA oligonucleotide (RDA)-directed repair of the defective AAT gene

Although not uniformly accepted as a valid approach to therapy, a newly reported technology under development may permit site-specific correction of a single base carried in a chromosome and therefore, in theory, provide a basis for correcting the most common genetic mutations leading to AAT deficiency.11 This technology (designated Genoplasty (Valigen, Lawrenceville, NJ, USA)) uses RNADNA chimeric molecules with short targeting sequences that are completely homologous with the region of interest except for the single base to be corrected. These regions are thought to be recognized and corrected by the cell's endogenous DNA repair systems. In vivo feasibility of this technology has apparently been reported for some genes in mice, but application to AAT deficiency will require confirmation of the concept and developing efficient systems for delivering the chimeric molecules to the liver that is efficient enough to correct the requisite number of hepatocytes.

Conclusions

AAT deficiency should be a good target for gene therapy since it is a single gene defect, the molecular biology is well-understood and the gene encodes a secreted protein. The possibility that expressing the normal gene locally in lung cells may preclude the necessity for normal circulating levels of the protein may make gene therapy more practical. Developing vectors and technologies encourage cautious optimism that toxicity can be reduced and efficacy increased.

References

1 Flotte TR. Recombinant adeno-associated virus gene therapy for cystic fibrosis and alpha1-antitrypsin deficiency. Chest 2002; 121: 98S102S.

2 http:www.alphaone.orgresearchclinical_trialsGene_Therapy_Trials.htm. 2002.

3 Brigham KL et al. Transfection of nasal mucosa with a normal alpha-1 antitrypsin (AAT) gene in AAT deficient subjects: comparison with protein therapy. Human Gene Therapy 2000; 11: 10231032.

4 Albelda SM, Wiewrodt R, Zuckerman JB. Gene therapy for lung disease: hype or hope? Ann Int Med 2000; 132: 649660.

5 Loser P, Hillgenberg M, Arnold W, Both GW, Hofmann C. Ovine adenovirus vectors mediate efficient gene transfer to skeletal muscle. Gene Ther 2000; 7: 14911498.

6 Yotdna P et al. Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses. Mol Ther 2002; 5: 233241. Article PubMed

7 Song JE et al. Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Ther 2001; 8: 12991306. Article

8 Stoll SM et al. Epstein-Barr virushuman vector provides high-level long-term expression of 1-antitrypsin in mice. Mol Ther 2001; 4: 122129. Article PubMed

9 Chen Z-Y et al. Linesa Dnas concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403410. Article PubMed

10 Dasi F, Benet M, Crespo A, Alino SF. Asialofetuin liposome-mediated human 1- antitrypsin gene transfer in vivo results in stationary long-term gene expression. J Mol Med 2001; 79: 205212.

11 Metz R et al. Mode of action of RNADNA oligonucleotides: progress in the development of gene repair as a therapy for alpha1-antitrypsin deficiency. Chest 2002; 121: 91S97S.

Figures

Figure 1 Concentrations of transgene-derived normal alpha-1 antitrypsin protein in nasal lavage fluid from subjects with PiZZ AAT deficiency. Time course of responses to intranasal lipoplex delivery of a normal AAT gene with the untreated contralateral nostril serving as control. Data are normalized to total protein concentration. (Reprinted with permission from reference 3.)

Figure 2 AAT and IL-8 concentrations in nasal lavage fluid from PiZZ AAT-deficient subjects when on no therapy, while receiving treatment with intravenous AAT protein at weekly intervals and at day 5 (time of peak transgene expression) after lipoplex intranasal transfection with the normal AAT gene. (Reprinted with permission from reference 3.)

January 2003, Volume 10, Number 2, Pages 95-99

December 2002, Volume 9, Number 24, Pages 1647-1652

Table of contents    Previous  Article  Next   [PDF]

ReviewGene Therapy Progress and Prospects: Nonviral vectors

T Niidome and L Huang

Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA

Correspondence to: L Huang, Center for Pharmacogenetics, School of Pharmacy, 633 Salk Hall, University of Pittsburgh, Pittsburgh, PA 15213, USA

Abstract

The success of gene therapy is largely dependent on the development of the gene delivery vector. Recently, gene transfection into target cells using naked DNA, which is a simple and safe approach, has been improved by combining several physical techniques, for example, electroporation, gene gun, ultrasound and hydrodynamic pressure. Chemical approaches have been utilized to improve the efficiency and cell specificity of gene transfer. Novel gene carrier molecules, which facilitate DNA escape from the endosome into the cytosol, have been developed. Several functional polymers, which enable controlled release of DNA in response to an environmental change, have also been reported. Plasmids with reduced number of CpG motifs, the use of PCR fragments and the sequential injection method have been established for the reduction of immune response triggered by plasmid DNA. Construction of a long-lasting gene expression system is also an important theme for nonviral gene therapy. To date, tissue-specific expression, self-replicating and integrating plasmid systems have been reported. Improvement of delivery methods together with intelligent design of the DNA itself has brought about large degrees of enhancement in the efficiency, specificity and temporal control of nonviral vectors.

Gene Therapy (2002) 9, 16471652. doi:10.1038/sj.gt.3301923

Keywords

non-viral vector; gene therapy; cationic lipid; cationic polymer; naked DNA

In brief

Progress

Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene expression in vivo

Gene delivery using a chemical carrier: to establish functional gene delivery in vivo

Nonviral vector modifications with peptides to increase intracellular gene delivery

Reduction of immune responses by modifying the administration protocol or the composition of the DNA

Design of tissue-specific, self-replicating and integrating plasmid expression systems to facilitate long-lasting gene expression

Prospects

Physical techniques for gene delivery into cells such as electroporation, with and without adjuvants, will be significantly optimized

Knowledge of the interaction of naked DNA with serum components and cell surface receptors will continue to accumulate. Immune responses originating from CpG motifs and nonviral gene carriers will diminish

The structure of gene carriers will be further optimized and tailored for specific uses such as systemic administration, local injection or organ-specific delivery

Novel ligands for targeted delivery of DNA will be found

Translocation mechanisms for plasmid DNA within the cell will be identified these may provide novel strategies for efficient delivery

More tissue-specific, site-specific integrating or self-replicating plasmid vectors are likely to appear

Introduction

The development of gene carriers for effectively delivering genes into cells has attracted a great deal of attention in recent years. Nonviral vectors should circumvent some of the problems occurring with viral vectors such as endogeneous virus recombination, oncogenic effects and unexpected immune response. Further, nonviral vectors have advantages in terms of simplicity of use, ease of large-scale production and lack of specific immune response. These techniques are categorized into two general groups: (1) naked DNA delivery by a physical method, such as electroporation and gene gun and (2) delivery mediated by a chemical carrier such as cationic polymer and lipid. In this review, we focus on the progress made over the last two years and discuss techniques in these two categories.

Naked DNA delivery by physical method: to overcome safety issue and to realize efficient gene expression in vivo

Many mechanical techniques are included in this section. The simplest way for administration of DNA is direct injection of naked plasmid DNA into the tissue or systemic injection from a vessel. Use of naked DNA without any carrier molecule is also the safest method. Little attention needs to be paid on issues of complex formation and its safety assessment. So far, site of the direct injection includes skeletal muscle, liver, thyroid, heart muscle, urological organs, skin and tumor.1 Systemic injection is also a convenient route for gene administration. However, owing to rapid degradation by nucleases in the serum and clearance by the mononuclear phagocyte system, the expression level and the area after injection of naked DNA are generally limited. Various physical manipulations have been used to improve the efficiency. Electroporation, bio-ballistic (gene gun), ultrasound, hydrodynamics (high pressure) injection and others have been established (Figure 1).2

Electroporation, the application of controlled electric fields to facilitate cell permeabilization, is used for enhancement of gene uptake into cells after injection of naked DNA.3 In addition, electroporation can achieve long-lasting expression and can be used in various tissues. Skin is one of the ideal targets because of the ease of administration. Drabick et al4 established cutaneous transfection method for the purpose of DNA vaccination. To optimize the condition of electroporation, factors such as dose of DNA, electrode shape and number, electrical field strength and duration have been optimized for expression of hepatitis B surface antigen,4 erythropoietin5 and IL-12.6 High ionic strength in the injection medium is also favorable for gene expression in the skin.7 Muscle is also a good candidate for electroporation. Most of reports published recently relate to immunological applications. For DNA vaccination, potent immune responses against hepatitis B surface antigen and HIV gag protein were obtained by electroporation of muscle after intramuscular injection of naked plasmid DNA.8 Therapeutic effect of cytokines, such as IL-129 and IFN- ,10 for inhibition of tumor growth located at a distant site has been demonstrated. IL-12 was also employed for electroporation after intratumor injection.11 Our laboratory recently reported the use of a syringe electrode, with which same transfection efficiency could be achieved by using much lower electric field strength than that of conventional electrode. Tissue damage by the electric field is thus minimized.12 Electrically mediated DNA delivery to hepatocellular carcinoma in the liver was reported by Heller et al.13 All of the electroporation protocols employ local injection of the plasmid DNA. However, our group recently demonstrated efficient gene transfer to the liver by electroporation following tail-vein injection of naked DNA.14 Comparing with local injection of DNA to the liver, systemic injection has the advantage of delivering genes more evenly to the liver.

Gene gun can achieve direct gene delivery into tissues or cells. Shooting gold particles coated with DNA allows direct penetration through the cell membrane into the cytoplasm and even the nucleus, bypassing the endosomal compartment. Majority of the efforts reported in the last 2 years are to introduce genes for antigen or cytokines such as IL-12 into skin15,16 or liver17,18,19 for vaccination and immunotherapy, respectively. However, a disadvantage of this method is the shallow penetration of DNA into the tissue.

Ultrasound can increase the permeability of cell membrane to macromolecules such as plasmid DNA. Indeed, enhancement of gene expression was observed by irradiating ultrasonic wave to the tissue after injection of DNA.20,21 Since ultrasound application is flexible and safe, its use in gene delivery has a great advantage in clinical use. Recently, it was reported that combination of microbubble with ultrasound could further increase the gene expression level. Microbubbles, or ultrasound contrast agents, lower the threshold for cavitation by ultrasound energy. In most cases, perfluoropropane-filled albumin microbubbles or Optison (Mallinckrodt, San Diego, USA) were used as microbubbles. It was modified with plasmid DNA before injection, followed by irradiation of ultrasound. At present, this technique is used for gene delivery to vascular cells,22,23,24,25,26 muscle26,27 and fetal mouse.28

Hydrodynamic injection, a rapid injection of a large volume of naked DNA solution (eg 5 g plasmid DNA injected in 58 s in 1.6 ml saline solution for a 20 g mouse) via the tail vein, can induce potent gene transfer in internal organs, especially the liver. Budker et al hypothesized that naked plasmid DNA is taken up by receptor-mediated pathway by hepatocytes.29 Certain DNA receptors have been found in various tissues;30 however, their function has not been elucidated. It has been proposed that the injected DNA solution accumulates mainly in the liver because of its flexible structure, which can accommodate large volume of solution, and the hydrostatic pressure forces DNA into the liver cells before it is mixed with blood. Furthermore, breaking of the endothelial barrier by the pressure has been proposed as the major mechanism responsible for the highly efficient expression in the liver. Recently, our group reported that external massage of the abdomen after small-volume injection of DNA via the tail vein can enhance gene expression in the liver.31 The observation suggests that mechanical stretching of the endothelial barrier may affect uptake of DNA into the hepatocytes. This pressure-mediated transfection method can be applicable to other tissues. Wolff's group showed that large-volume injection with high speed via the portal vein of liver or the artery of limb muscles achieved high gene expression in the respective organ.29,32

Our group has demonstrated that significant gene expression can be achieved in the liver by transiently restricting blood flow through the liver immediately following peripheral intravenous injection of naked DNA.33 Occlusion of blood flow either at vena cava or at hepatic artery and portal vein increased the expression level in the liver. Presumably, the injected DNA is internalized into the hepatic cells by receptor-mediated mechanism as proposed by Budker et al29 or via a nonreceptor-mediated pathway. However, the binding of DNA to the surface of hepatic cells might be so weak that DNA could be easily dissociated and washed away by the blood flow in the normal physiological condition. Only when the blood flow is transiently stopped, the DNA can stably bind with the receptor and be internalized into cells. A similar uptake of DNA by the diaphragm muscle cells was achieved by a brief occlusion of the blood flow through the diaphragm immediately after peripheral intravenous injection of DNA.34

Gene delivery using a chemical carrier: to establish functional gene delivery in vivo

Novel carriers to achieve high-level gene expression and functional delivery have been designed. Gene carriers can be categorized into several groups: (1) those forming condensed complexes with the DNA to protect the DNA from nucleases and other blood components; (2) those designed to target delivery to specific cell types; (3) those designed to increase delivery of DNA to the cytosol or nucleus; (4) those designed to dissociate from DNA in the cytosol and (5) those designed to release DNA in the tissue to achieve a continuous or controlled expression. Lipids and polymers are mainly used for gene delivery.

Lipid-mediated gene delivery

Liposome-based gene delivery, first reported by Felgner in 1987, is still one of the major techniques for gene delivery into cells. In 1990s, a large number of cationic lipids, such as quaternary ammonium detergents, cationic derivatives of cholesterol and diacylglycerol, and lipid derivatives of polyamines, were reported. However, the development of novel types of lipid molecules appears to be saturated, and most of the efforts have shifted to improving efficacy by the modification listed above, as well as to specific in vivo applications. We will highlight a number of new concepts that have appeared in the last 2 years.

The reductionoxidation (redox) sensitive character of thiol groups has been exploited to control DNAlipid complex formation. Dauty et al35 reported a dimerizable cationic detergent, which contains free thiol, amine and alkyl groups. This alkylated ornithinyl cysteine derivative forms a complex with plasmid DNA. Subsequent oxidation of the thiol groups to disulfides converts the complex into stable nanometric particles. The particle is made of a single molecule of condensed plasmid DNA with a uniform diameter of less than 40 nm and showed reasonable transfection activity in vitro. Practical advantages include the small size for in vivo gene delivery (improved particle diffusion) and that the disulfide bonds should be reduced to thiols in the cytosol because of the reductive environment provided by intracellular glutathione, thus resulting in DNA release.

Peptide-mediated gene delivery

Redox-sensitive thiols have also been incorporated into peptide gene carriers. McKenzie et al36 developed peptides containing a cysteine residue and a continuous sequence of lysine residues, for example, Cys-Trp-Lys18.36 This peptide can also condense plasmid DNA, and the thiol group is spontaneously oxidized, resulting in a highly stable complex with potent transfection activity in vitro. Cross-linking the peptide caused elevated gene expression, without increasing DNA uptake by the cells, suggesting that intracellular release of the DNA triggered by disulfide bond reduction played a key role. Furthermore, Park et al37 have also synthesized sulfhydryl cross-linking poly(ethylene glycol)-peptides (for stealth activity) and glycopeptides for targeted delivery of genes in vivo.

Polymer-mediated gene delivery

Wightman et al38 systematically compared the ability of branched and linear PEIDNA complexes to transfect cells in vitro and in vivo at various aminephosphate ratios and salt concentrations. They showed that salt-free DNA complexes of linear PEI (22 kDa), which showed high transfection efficiency in the lung, were small, but subsequently aggregated when salt was added. In contrast, DNA complex of branched PEI (25 kDa), which showed low transfection efficiency in most of the organs, remained small even after salt was added. The greater efficiency of linear PEI in vivo might be because of a dynamic structure change of the complex under high salt concentrations as found in blood. Understanding of the interaction between linear PEI and DNA could help in designing future vectors.

Biodegradable polymers are known for their low toxicity and high biocompatibility. Recently, a biodegradable polymer, poly -(4-aminobutyl)-L-glycolic acid (PAGA), a derivative of poly-L-lysine, in which the ester link is substituted with amide, was designed by Kim's group.39 This biodegradable and water-soluble polymer condenses DNA and subsequently releases DNA upon hydrolysis of the polymer. The complex showed higher in vitro gene transfection efficiency with lower cytotoxicity than poly-L-lysine. Significant expression of murine IL-10 was observed in the serum after tail-vein injection of PAGADNA complexes, and the systemic administration of murine IL-10 gene with PAGA into NOD mice markedly reduced insulitis.40 The murine IL-12

gene was also injected with PAGA into subcutaneous tumors in BALBc mice. Significant level of the protein expression and reduction of tumor growth was observed.41 Recently, other types of biodegradable polymers were reported by Kim's and Leong's groups, who have synthesized cationic copolymers derived from PEI and polyethylene glycol (PEG)42 and cationic polyphosphoester,43 respectively.

Thermosensitive polymers can control the release of encapsulated DNA in response to temperature changes that lead to swelling or de-swelling of the hydrated polymer. Kurisawa et al44 synthesized a thermosensitive copolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA), and investigated its thermosensitive character and transfection efficiency at different incubation temperatures.44 A polymer containing 8 mol% DMAEMA and 11 mol% BMA had a low critical solution temperature of 21°C and complex formationdissociation was modulated by temperature alteration. Transfection efficiency in vitro also depended on the incubation temperature. Kim's group have developed the biodegradable and thermosensitive polymer, PEGpoly(D,L-lactic acid-co-glycolic acid) (PLGA)PEG triblock co-polymer. This nonionic, hydrophilic polymer shows temperature-dependent solution-to-gel transitions45,46 and can be loaded with plasmid DNA in aqueous phase at 420°C. At above 303°C (eg, at the body temperature), the solution-to-gel transition occurs. It is conceivable that DNA could be formulated and injected in the polymer solution at room temperature, and slowly released from the hydrogel for prolonged transfection at the injection site.

PEG-PLL block contains a hydrophilic part consisting of PEG and a DNA-binding moiety consisting of PLL and forms self-assembling particles with DNA in a core-shell structure with electrostatic interaction as the main driving force. These polyion complex micelles are water-soluble and nuclease-resistant nanoparticles, suitable for in vivo gene delivery. Thus, DNA in the complex remained intact in the blood stream for 30 min, although gene expression after injection via the tail vein of mice was only seen in the liver.47

Nonviral vector modifications with peptides to increase intracellular gene delivery

Many anionic pH-sensitive peptides48 and cationic fusogenic peptides49 show an enhancing effect on gene expression mediated by cationic liposome and PEI, respectively. These peptides show membrane disrupting activities in weakly acidic condition, which is similar to that in the endosome compartment, and could enhance the translocation of the DNA to cytosol. Rittner et al50 reported that a bifunctional peptide with both DNA-binding and membrane-disrupting activities showed significant gene expression in the lung after tail-vein injection.50

Inefficient entry of DNA into the nucleus is a major limiting step in the development of nonviral gene delivery system. The problem is particularly serious in nondividing cells, where entry into the nucleus is thought to occur only through the nuclear pore complex. To achieve active transport to the nucleus, nucleus localizing signal (NLS) peptides have been widely used. Recent effort has been summarized in excellent reviews.51,52,53 In most cases, NLS is conjugated with a gene carrier such as PEI, or with DNA directly.

Reduction of immune responses by modifying the administration protocol or the composition of the DNA

Although it is well known that nonviral gene delivery produces a less severe immune responses than virus-mediated delivery, problems still remain. The DNAgene complex is recognized by macrophages, dendritic and other immune cells. For cationic liposomes, toxicity relates to the rapid induction of proinflammatory cytokines such as TNF- , IL-6, IL-12 and IFN- .54 This response stems from the stimulation of the immune cells by the unmethylated CpG motifs in the plasmid DNA. Various approaches have been taken to reduce this inflammatory toxicity, including elimination of CpG motifs in the plasmid DNA,55 use of PCR fragments with reduced numbers of CpG motifs56 and active targeting of the DNA to the endothelium, which minimizes interaction with immune cells.57 Furthermore, sequential injection of cationic liposomes followed by naked plasmid DNA, first reported by Liu's group,14 reduces the inflammatory response.58 Thus, when plasmid DNA was injected into the tail vein of mice 25 min after the injection of cationic liposome, 5080% lower levels of

proinflammatory cytokines (compared to lipoplexes) were observed, without affecting gene expression level in the lung.

Design of tissue-specific, self-replicating and integrating plasmid expression systems to facilitate long-lasting gene expression

Producing sustained gene expression is also an important goal for nonviral gene therapy. Tissue-specific expression systems can produce stable expression by reducing the probability of inducing an immune res-ponse to the transgene. Thus, Kay's group constructed a plasmid DNA containing the apolipoprotein E locus control region, 1-antitrypsin promoter, human factor IX minigene sequence including a portion of the first intron, 3'-untranslated region, and the bovine growth hormone polyadenylation signal.59 When the plasmid DNA was delivered to mouse liver by hydrodynamic injection, it produced not only increased gene expression of factor IX (in the therapeutic range), but also maintained these levels for at least 10 months. Furthermore, a linear DNA expression cassette originating from this plasmid showed 10- to 100-fold higher expression than the closed circular DNA for a period of 9 months.60

The EpsteinBarr virus (EBV)-based plasmid vector is known to self-replicate in cells. It carries two genetic elements from EBV, the EBV nuclear antigen 1 (EBNA1) gene and the oriP element. The EBNA1 protein binds to oriP, and facilitates the replication of the plasmid in synchrony with chromosomal DNA. Furthermore, the EBNA1 also facilitates nuclear localization of the plasmid DNA. This approach has been used for tumour suicide therapy61 (coupled to a polyamidoamine dendrimer), long-term expression of the 2-adrenergic receptor in cardiomyocytes,62 and efficient and long-lasting luciferase expression in murine liver after hydrodynamic injection.63 Stoll et al64 have also reported high-level and long-lasting expression of the 1-antitrypsin gene in mouse liver using the hydrodynamic injection protocol.

Controllable integration of plasmid DNA into the genome of mammalian cells would also provide long-lasting gene expression. Reconstitution of an ancient transposon, Sleeping Beauty, from sequence alignment of nonfunctional remnants of members in the Tc1mariner superfamily of transposons within the genomes of salmonids, provided the first functional transposon for use in vertebrate species.65 Sleeping Beauty has been used to accomplish stable chromosomal integration of functioning genes in somatic cells of adult mice.66 In addition, several phage integrases and their corresponding recognition elements, which can mediate integration into mammalian chromosomes, were reported by Calos's group.67,68,69,70 Although the integration efficiency of integration system is still low, this technology may one day enable site-specific and high-efficiency integration into the host chromosome without the potential for mutagenesis.

Summary

To establish efficient and safe gene delivery in vivo, a number of new techniques and concepts have been introduced in the last 2 years, with improvements in targeted or controlled delivery of genes. However, we are still far from the perfect gene carrier suitable for clinical use. We have come a long way in understanding the cellular barriers which prevent proper delivery of DNA, but still relatively ignorant about factors controlling the stability, pharmacokinetics and biodistribution of nonviral vectors. Much of the above effort has been carried out in rodents and whether the new improvements are applicable to larger animals remains to be seen. We are still far from the perfect gene carrier suitable for clinical use, and much more work is still ahead of us.

References

1 Nishikawa M, Huang L. Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 2001; 12: 861-870. Article MEDLINE

2 Li S, Ma Z. Nonviral gene therapy. Curr Gene Ther 2001; 1: 201-226.

3 Somiari S et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000; 2: 178-187. Article MEDLINE

4 Drabick JJ et al. Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization. Mol Ther 2001; 3: 249-255. Article

5 Maruyama H et al. Skin-targeted gene transfer using in vivo electroporation. Gene Ther 2001; 8: 1808-1812. Article

6 Dujardin N, Van Der Smissen P, Preat V. Topical gene transfer into rat skin using electroporation. Pharm Res 2001; 18: 61-66. Article

7 Chesnoy S, Huang L. Enhanced cutaneous gene delivery following intradermal injection of naked DNA in a high ionic strength solution. Mol Ther 2002; 5: 57-62. Article

8 Widera G et al. DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol 2000; 164: 4635-4640. MEDLINE

9 Hanna E et al. Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at distant site. Cancer Gene Ther 2001; 8: 151-157. Article MEDLINE

10 Li S et al. Intramuscular electroporation delivery of IFN- gene therapy for inhibition of tumor growth located at a distant site. Gene Ther 2001; 8: 400-407. Article

11 Tamura T et al. Intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer. Hum Gene Ther 2001; 12: 1265-1276. Article

12 Liu F, Huang L. A syringe electrode device for simultaneous injection of DNA and electrotransfer. Mol Ther 2002; 5: 323-328. Article

13 Heller L et al. Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo. Gene Ther 2000; 7: 826-829. Article MEDLINE

14 Liu F, Hunag L. Electric gene transfer to the liver following

systemic administration of plasmid DNA. Gene Ther 1998; 8: 1531-1537.

15 Lin MT, Pulkkinen L, Uitto J, Yoon K. The gene gun: current application in cutaneous gene therapy. Int J Dermatol 2000; 39: 161-170. Article

16 Davidson JM, Krieg T, Eming SA. Particle-mediated gene therapy of wounds. Wound Repair Regen 2000; 8: 452-459. Article

17 Muangmoonchai R et al. Transfection of liver in vivo by biolistic particle delivery: its use in the investigation of cytochrome P450 gene regulation. Mol Biotechnol 2002; 20: 145-151.

18 Kuriyama S et al. Particle-mediated gene transfer into murine livers using a newly developed gene gun. Gene Ther 2000; 7: 1132-1136. Article

19 Yoshida S et al. Direct immunization of malaria DNA vaccine into the liver by gene gun protects against lethal challenge of Plasmodium berghei sporozoite. Biochem Biophys Res Commun 2000; 271: 107-115. Article

20 Newman CM, Lawrie A, Brisken AF, Cumberland DC. Ultrasound gene therapy: on the road from concept to reality. Echocardiography 2001; 18: 339-347. MEDLINE

21 Amabile PG et al. High-efficiency endovascular gene delivery via therapeutic ultrasound. J Am Coll Cardiol 2001; 37: 1975-1980. Article

22 Taniyama Y et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002; 105: 1233-1239. Article

23 Teupe C et al. Vascular gene transfer of phosphomimetic endothelial nitric oxide synthase (S1177D) using ultrasound-enhanced destruction of plasmid-loaded microbubbles improves vasoreactivity. Circulation 2002; 105: 1104-1109. Article

24 Unger EC, Hersh E, Vannan M, McCreery T. Gene delivery using ultrasound contrast agents. Echocardiography 2001; 18: 355-361.

25 Lawrie A et al. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 2000; 7: 2023-2027. Article

26 Song J et al. Influence of injection site, microvascular pressure and ultrasound variables on microbubble-mediated delivery of microspheres to muscle. J Am Coll Cardiol 2002; 39: 726-731. Article

27 Shohet RV et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation

2000; 101: 2554-2556.

28 Endoh M et al. Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound. Mol Ther 2002; 5; 501-508.

29 Budker V et al. Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process. J Gene Med 2000; 2: 76-88. Article MEDLINE

30 Siess DC et al. A human gene coding for a membrane-associ-ated nucleic acid-binding protein. J Biol Chem 2000; 275: 33655-33662. Article

31 Liu F, Huang L. Noninvasive gene delivery to the liver by mechanical massage. Hepatology 2002; 35: 1314-1319. Article

32 Zhang G et al. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 2001; 12: 427-438. Article MEDLINE

33 Liu F, Huang L. Improving plasmid DNA-mediated liver gene transfer by prolonging its retention in the hepatic vasculature. J Gene Med 2001; 3: 569-576. Article

34 Liu F, Nishikawa M, Clemens PR, Huang L. Transfer of full-length Dmd to the diaphragm muscle of Dmd(mdxmdx) mice through systemic administration of plasmid DNA. Mol Ther 2001; 4: 45-51. Article

35 Dauty E, Remy JS, Blessing T, Behr JP. Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in culture. J Am Chem Soc 2001; 123: 9227-9234. Article MEDLINE

36 McKenzie DL, Kwok KY, Rice KG. A potent new class of reductively activated peptide gene delivery agents. J Biol Chem 2000; 275: 9970-9977. Article MEDLINE

37 Park Y, Kwok KY, Boukarim C, Rice KG. Synthesis of sulfhydryl cross-linking poly(ethylene glycol)-peptides and glyco-peptides as carriers for gene delivery. Bioconjug Chem 2002; 13: 232-239. Article

38 Wightman L et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 2001; 3: 362-372. Article MEDLINE

39 Lim YB et al. Biodegradable polyester, poly -(4-aminobutyl)-L-glycolic acid, as a non-toxic gene carrier. Pharm Res 2000; 17: 811-816. Article MEDLINE

40 Koh JJ et al. Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice. Gene Ther 2000; 7: 2099-2104. Article MEDLINE

41 Maheshwari A et al. Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment. Mol Ther 2000; 2: 121-130. Article MEDLINE

42 Ahn CH, Chae SY, Bae YH, Kim SW. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release 2002; 80: 273-282. Article

43 Wang J, Mao HQ, Leong KW. A novel biodegradable gene carrier based on polyphosphoester. J Am Chem Soc 2001; 123: 9480-9581. Article MEDLINE

44 Kurisawa M, Yokoyama M, Okano T. Gene expression control by temperature with thermo-responsive polymeric gene carriers. J Control Release 2000; 69: 127-137. Article

45 Jeong B, Kim SW, Bae YH. Thermosensitive sol-gel reversible hydrogels. Adv Drug Deliv Rev 2002; 54: 37-51. Article

46 Jeong B, Bae YH, Kim SW. Drug release from biodegradable injectable thermosensitive hydrogel of PEGPLGAPEG triblock copolymers. J Control Release 2000; 63: 155-163. Article

47 Harada-Shiba M et al. Polyion complex micelles as vectors in gene therapy pharmacokinetics and in vivo gene transfer. Gene Ther 2002; 9: 407-414. Article

48 Turk MJ, Reddy JA, Chmielewski JA, Low PS. Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs. Biochim Biophys Acta 2002; 1559: 56-68. Article MEDLINE

49 Lee H, Jeong JH, Park TG. A new gene delivery formulation of polyethylenimineDNA complexes coated with PEG conjugated fusogenic peptide. J Control Release 2001; 76: 183-192. Article

50 Rittner K et al. New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo. Mol Ther 2002; 5: 104-114. Article

51 Bremner KH, Seymour LW, Pouton CW. Harnessing nuclear localization pathways for transgene delivery. Curr Opin Mol Ther 2001; 3: 170-177.

52 Tachibana R, Harashima H, Shinohara Y, Kiwada H. Quantitative studies on the nuclear transport of plasmid DNA and gene

expression employing nonviral vectors. Adv Drug Deliv Rev 2001; 52: 219-226. Article

53 Cartier R, Reszka R. Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Ther 2002; 9: 157-167. Article

54 Tousignant JD et al. Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice. Hum Gene Ther 2000; 11: 2493-2513. Article

55 Yew NS et al. Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs. Mol Ther 2000; 1: 255-262. Article MEDLINE

56 Hofman CR et al. Efficient in vivo gene transfer by PCR amplified fragment with reduced inflammatory activity. Gene Ther 2001; 8: 71-74. Article

57 Li S et al. Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody. Am J Physiol Lung Cell Mol Physiol 2000; 278L: 504-511.

58 Tan Y et al. Sequential injection of cationic liposome and plasmid DNA effectively transfects the lung with minimal inflammatory toxicity. Mol Ther 2001; 3: 673-682. Article MEDLINE

59 Miao CH et al. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 2000; 1: 522-532. Article MEDLINE

60 Chen ZY et al. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403-410. Article MEDLINE

61 Maruyama-Tabata H et al. Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer. Gene Ther 2000; 7: 53-60. Article

62 Tomiyasu K et al. Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters. Gene Ther 2000; 7: 2087-2093. Article MEDLINE

63 Cui FD et al. Highly efficient gene transfer into murine liver achieved by intravenous administration of naked EpsteinBarr virus (EBV)-based plasmid vectors. Gene Ther 2001; 8: 1508-1513. Article

64 Stoll SM et al. EpsteinBarr virushuman vector provides high-level, long-term expression of 1-antitrypsin in mice. Mol Ther 2001; 4:

122-129. Article MEDLINE

65 Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997; 91: 501-510. MEDLINE

66 Yant SR et al. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 2000; 25: 35-41. Article MEDLINE

67 Thyagarajan B et al. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol Cell Biol 2001; 21: 3926-3934. Article MEDLINE

68 Olivares EC, Hollis RP, Calos MP. Phage R4 integrase mediates site-specific integration in human cells. Gene 2001; 278: 167-176. Article

69 Sclimenti CR, Thyagarajan B, Calos MP. Directed evolution of a recombinase for improved genomic integration at a native human sequence. Nucleic Acids Res 2001; 29: 5044-5051. Article MEDLINE

70 Stoll SM, Ginsburg DS, Calos MP. Phage TP901-1 site-specific integrase functions in human cells. J Bacteriol 2002; 184: 3657-3663. Article

Figures

Figure 1 Overview of nonviral gene delivery technologies. Different injection routes of naked DNA and enhancement strategies are outlined.

November 2002, Volume 9, Number 22, Pages 1487-1491

Table of contents    Previous  Article  Next   [PDF]

Progress And ProspectsPost-intervention vessel remodeling

J Rutanen1, H Puhakka1 and S Ylä-Herttuala1,2,3

1AI Virtanen Institute, University of Kuopio, Kuopio, Finland

2Department of Medicine, University of Kuopio, Kuopio, Finland

3Gene Therapy Unit, University of Kuopio, Kuopio, Finland

Correspondence to: S Ylä-Herttuala, Department of Molecular Medicine, AI Virtanen Institute, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland

Abstract

By-pass surgery and percutaneous transluminal (coronary) angioplasty, PT(C)A, are standard techniques for the treatment of vascular occlusions. Their usefulness is limited by by-pass graft failure and restenosis occuring after the procedures. Twenty percent of patients treated with PTCA/PTA need a new revascularization procedure within 6 months, despite a successful procedure. Stents are used to prevent restenosis in selected lesions, but in-stent restenosis also remains an important clinical problem. In this review we discuss progress of gene therapy for the treatment of post-PT(C)A restenosis, in-stent restenosis and by-pass graft stenosis over the last 2 years (20002002).

Gene Therapy 2002 9, 14871491. doi:10.1038/sj.gt.3301866

Keywords

artery; stenosis; intimal hyperplasia; vein graft; neointima

Progress

Low gene transfer efficiency in blood vessels is still a significant problem Identification of new treatment genes has provided more opportunities for therapy

Combination therapy may be more efficient than single gene treatment and first evidence of prolonged therapeutic effect has been achieved

Inhibition of vein graft stenosis using gene therapy has been successful in animal models

Clinical trials have indicated cautious optimism only in vein graft stenosis

Prospects

Low gene transfer efficiency in blood vessels is still a significant problem Identification of new treatment genes has provided more opportunities for therapy

Combination therapy may be more efficient than single gene treatment and first evidence of prolonged therapeutic effect has been achieved

Inhibition of vein graft stenosis using gene therapy has been successful in animal models

Clinical trials have indicated cautious optimism only in vein graft stenosis

Identification of new treatment genes should continue

Improved gene delivery methods to blood vessels need to be developed

Regulated, targeted vectors, gene cocktails and combination therapies need to be studied

Randomized, blinded, controlled phase II/III studies are needed to establish safety and efficacy of gene therapy

Other novel methods, such as arterial grafting and drug eluting stents may overcome some problems related to post-intervention vessel occlusion

Low gene transfer efficiency in blood vessels is still a significant problem

Various types of catheters are available for gene transfer into the vessel wall.1,2 Unfortunately, even with powerful viral vectors gene transfer efficiency through human atherosclerotic lesions and lipid-rich atheroma is 5%, and results in a potentially harmful biodistribution of the vector.3 Therefore, there is great interest in developing better strategies for efficient, targeted gene delivery into the vessel wall. Accordingly several biologic targeting systems have recently been introduced.4,5,6,7,8,9,10 In spite of relatively low transfection efficiency adenoviruses are still the most widely used viral vectors for vascular applications since they can transfect both proliferating and non-proliferating cells.2,11 However, adenoviruses also transfect many unwanted organs and peripheral blood monocytes.3 Therefore, novel methods to target adenoviruses to the vessel wall have been developed.6,9,10,12,13 These include matrix metalloproteinase-2 and -9 (MMP-2 and -9) targeted tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) encoding adenoviruses,10 integrin targeted human interleukin-2 encoding adenoviruses14 and endothelial cell targeted adenoviruses.9 However, longer expression times are probably needed for better

clinical efficacy. Thus, adeno-associated viruses (AAV) have been successfully used to transduce rabbit jugular veins with expression lasting beyond 30 days,15 and a method to target AAV gene therapy to vascular endothelial cells has been described.8 Furthermore, modified retrovirus, Semliki Forest virus, Baculovirus and herpes simplex virus vectors have been used for vascular gene transfer in vivo, but these vectors have not yet solved all problems related to vascular gene transfer.7,16,17,18 The most widely used non-viral vector for vascular gene therapy is plasmid DNA with or without carrier molecules. Due to the safety aspects and relatively easy processibility, there is a great interest in the development of targeted non-viral gene delivery methods. However, relatively low tranfection efficiency has limited their use in vascular applications. It should be pointed out that different treatments strategies require different gene transfer efficiencies, eg with secreted compounds, such as growth factors or NO, even a relatively inefficient vector may be useful, whereas antisense or decoy constructs for cell cycle mediators need to be delivered in the majority of vascular cells before a therapeutic effect can be expected.

Therapeutic ultrasound has also proven efficient in augmenting intravascular gene delivery.19 For the treatment of in-stent restenosis a good method to deliver vectors locally to the target area is the stent itself. Klugherz et al succeeded in transducing porcine coronary arteries with a plasmid DNA eluting polymer-coated stent.20 Also, they reported approximately 6% transfection efficiency using a stent-based antibody-tethered adenoviral gene transfer system.21 An ex vivo approach using genetically engineered cells attached to the stent surface could be another possibility to achieve long-term transgene expression. Panetta et al have demonstrated GFP expression 1 month after implantation of a stent carrying engineered autologous smooth muscle cells (SMCs) into porcine coronary artery.22 However, these approaches still suffer from fast wash-out of the vectors from the stent coating and detachment of the seeded cells from the stent struts.

Identification of new treatment genes has provided more opportunities for therapy

Smooth muscle cell migration and proliferation are key factors in the development of restenosis, in-stent restenosis and vein graft stenosis23 and most gene therapy strategies are directed towards these targets (Table 1). Early markers of SMC activation, such as oncogenes are detectable shortly after arterial injury. Antisense oligonucleotides directed against oncogenes and cell cycle regulators have been used to decrease neointimal thickening in animal models. Promising results have been obtained with antisense oligonucleotides against NF B,24 E2F25 and c-myc.26 Also, adenoviral Gax (growth arrest homeobox) gene transfer has been shown to reduce neointimal hyperplasia in stented rabbit iliac arteries.27 The platelet-derived growth factor (PDGF) gene family is one of the most potent chemoattractants of vascular SMC. PDGFs mediate neointimal growth after vascular injury and this can be prevented by inhibition of PDGF expression with antibodies against PDGF or its receptors.28 However, clinical use of antibodies carries the risk of immunoreactivity and thus gene-based approaches against PDGFs have been introduced.29 MMPs are also important effectors in the migration of SMCs and can be inhibited by overexpressing tissue inhibitors of metalloproteinases (TIMPs). Gene transfer of TIMP-1 has been shown to reduce neointimal growth in rat and rabbit denudation models.10

Since intravascular manipulation causes damage to endothelium, it is hypothesized that rapid re-endothelialization of arterial wall after balloon dilatation should reduce restenosis. Members of the vascular endothelial growth factor (VEGF) family induce endothelial cell proliferation and migration. Hiltunen et al demonstrated that both VEGF-A and VEGF-C gene therapy reduced restenosis after arterial injury.30 Also, gene therapy with VEGF-D attenuates neointimal growth in rabbits.31 In addition to re-endothelization, the effects of VEGFs may be at least partially due to increased nitric oxide (NO) production which inhibits SMC migration and proliferation. Hepatocyte growth factor (HGF) is another gene that is reported to reduce neointimal hyperplasia through re-endothelization and increased NO production.32 Aiming at increased NO production Janssens et al have previously shown that eNOS gene transfer reduced neointima formation in a rat model and Varenne et al noticed the same effect with eNOS gene transfer in a pig model. Also, van der Leyen et al have achieved prevention of restenosis with iNOS gene transfer. Further, extracellular superoxide dismutase (EC-SOD) gene transfer has been shown to reduce neointima formation in a rabbit model.33 Platelet-activating factor (PAF)-like phospholipids are characteristic of proinflammatory conditions. These phospholipids are inactivated by PAF-acetylhydrolase (PAF-AH). Quarck et al showed that PAF-AH prevents neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice.34 It has also recently been demonstrated that a therapeutic effect on intimal hyperplasia can be achieved by inhibiting tissue factor (TF) with TF pathway inhibitor (TFPI).35,36,37 Genes that have an effect on various pathological processes in the vessel wall,

such as VEGFs, HGF or E2F decoy may offer the most promising tools for gene therapy against post-intervention vessel occlusion. On the other hand, similar effects may be achieved with combination therapy.

Combination therapy may be more efficient than single gene treatment and first evidence of prolonged therapeutic effect has been achieved

The majority of gene therapy studies for restenosis, in-stent restenosis and vein graft stenosis have evaluated only single genes. Considering the complex pathophysiology of these events it is possible that combination therapy or 'gene cocktails' may provide a better treatment effect. Thus, utilization of different mechanisms which are known to reduce restenosis may lead to an additive treatment effect. So far, there have been only a few reports on combination therapy and its effects on these remodeling processes. Puhakka et al studied peptide re-targeted TIMP-1 plus VEGF-C combination gene therapy in a rabbit model and found a prolonged treatment effect.38 Leppänen et al have shown that combination of VEGF-C gene transfer and treatment with PDGF receptor kinase inhibitor STI571 leads to a long-term reduction in neointima formation in a balloon-denuded rabbit aorta.39 These studies have shown prolonged therapeutic effect compared with single treatment with VEGF-C or STI571.28,30 As another example of combination therapy Atsuchi et al reduced neointima formation in a rabbit model by brief irrigation with tissue factor pathway inhibitor (TFPI) combined with adenovirus-mediated local TFPI gene transfer.37

Inhibition of vein graft stenosis using gene therapy has been successful in animal models

Surgical approach provides an opportunity to use ex vivo approach, which allows longer contact time with vector and may result in a better gene transfer efficiency. After surgical revascularization vein grafts are exposed to high blood pressure, which causes endothelial cell damage, platelet and leukocyte adhesion, thrombosis, matrix destruction and SMC proliferation. Although neointimal hyperplasia caused by SMC proliferation and migration may not be the most prominent factors producing significant stenosis, it exposes veins to the later development of graft atheroma. Vein graft stenosis has similar features to neointimal hyperplasia in arteries, and thus the same therapeutic approaches may apply. Suppressing SMC migration and proliferation by inhibiting MMPs with TIMP-1, TIMP-2 or TIMP-340 has been shown to inhibit neointima formation. TIMP-2 reduces neointima formation in human saphenous veins and TIMP-3 decreased stenosis in human and porcine veins.40 Also, long-term stabilization of the vein grafts with ex

vivo pressure-mediated E2F decoy oligonucleotide gene transfer has been achieved.41 Endothelial damage predisposes vein grafts to thrombus formation and stimulates SMC migration to intima. Thus, re-endothelization of the graft should be useful. Ohno et al demonstrated accelerated re-endothelization with suppressed thrombogenesis and neointimal hyperplasia using gene transfer of C-type natriuretic peptide (CNP).42 An emerging new area of gene therapy is prevention of stenosis in dialysis access grafts and arterio-venous loops.

Clinical trials have indicated cautious optimism only in vein graft stenosis

In spite of promising results achieved in preclinical animal models, only a few clinical trials have been started (Table 2). To date, there are only two successful reports on the prevention of neointimal hyperplasia in clinical trials. Previously, Mann et al reduced bypass vein graft failure rate with E2F antisense decoy by using an ex vivo gene transfer method and Grube et al have now reported significant efficacy of the same construct in coronary vein grafts.43 Laitinen et al demonstrated the safety and

feasibility of intravascular catheter-mediated VEGF-A plasmid/liposome gene transfer to human coronaries in conjunction with PTCA in a randomized, controlled phase I study, but no efficacy was detected in control angiography.1 In a randomized, controlled phase II study of catheter-mediated VEGF-A gene transfer with plasmid/liposome or adenovirus to infrainguinal arteries after PTA, increased vessel formation was seen in the VEGF treatment groups in the follow-up angiography 3 months after the gene transfer, but no effect was found on restenosis.44 The first anti-sense-based clinical study for in-stent restenosis was recently published by Kutryk et al.45 They used antisense oligonucleotides against c-myc to inhibit cell proliferation in stented lesions. Although the pre-clinical results in preventing intimal hyperplasia with this cell cycle regulator were very promising, the study did not show any therapeutic effect measured by intravascular ultrasound and angiography.45 This may be explained by compromised efficacy of intravascular delivery compared with ex vivo approach used for vein graft stenosis. Thus, vascular gene therapy still faces the same problem as any pharmacological therapy: how to transfer positive pre-clinical results to clinically successful therapy.

Summary

Gene therapy offers an alternative approach for the treatment of vessel remodelling. First clinical trials have established the safety of gene therapy for the treatment of these remodeling processes and some positive clinical results have been reported for vein graft stenosis. Currently several genes and proteins have shown promising results in pre-clinical studies. However, randomized, double-blinded, placebo-controlled phase II/III studies are needed to determine usefulness of gene therapy for these diseases.

Acknowledgements

This study was supported by grants from Finnish Academy and Sigrid Juselius Foundation. We thank Ms Marja Poikolainen for preparing the manuscript.

References

1 Laitinen Met al. . Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. Hum Gene Ther 2000; 11: 263-270. Article MEDLINE

2 Hiltunen MO, Turunen MP, Ylä-Herttuala S. Gene therapy methods in cardiovascular diseases. Meth Enzymol 2002; 346: 311-320.

3 Hiltunen MOet al. . Biodistribution of adenoviral vector to

nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. FASEB J 2000; 14: 2230-2236. Article MEDLINE

4 Nicklin SAet al. . Selective targeting of gene transfer to vascular endothelial cells by use of peptides isolated by phage display. Circulation 2000; 102: 231-237. MEDLINE

5 Hall FLet al. . Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor. Hum Gene Ther 2000; 11: 983-993. Article MEDLINE

6 Ribault Set al. . Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res 2001; 88: 468-475.

7 Gordon EMet al. . Lesion-targeted injectable vectors for vascular restenosis. Hum Gene Ther 2001; 12: 1277-1287.

8 Nicklin SAet al. . Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells. Mol Ther 2001; 4: 174-181.

9 Nicklin SAet al. . Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus. Mol Ther 2001; 4: 534-542.

10 Nicklin SAet al. . Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus. Turunen MP et al. Peptide re-targeted adenovirus encoding tissue inhibitor of metalloproteinase-1 decreases restenosis after intravascular gene transfer. Mol Ther 2002 (in press).

11 Ylä-Herttuala S, Martin JF. Cardiovascular gene therapy. Lancet 2000; 355: 213-222. Article MEDLINE

12 Kibbe MRet al. . Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors. Arch Surg 2000; 135: 191-197.

13 Hay CMet al. . Enhanced gene transfer to rabbit jugular veins by an adenovirus containing a cyclic RGD motif in the HI loop of the fiber knob. J Vasc Res 2001; 38: 315-323.

14 Mizuguchi Het al. . CAR- or alhav integrin-binding ablated adenovirus vectors, but not fiber-modified vectors containing RGD peptide, do not change the systemic gene transfer properties in

mice. Gene Therapy 2002; 9: 769-776.

15 Eslami MHet al. . Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors. J Vasc Surg 2000; 31: 1149-1159. Article MEDLINE

16 Skelly CLet al. . Prevention of restenosis by a herpes simplex virus mutant capable of controlled long-term expression in vascular tissue in vivo. Gene Therapy 2001; 8: 1840-1846.

17 Roks AJet al. . Recombinant Semliki Forest virus as a vector system for fast and selective in vivo gene delivery into balloon-injured rat aorta. Gene Therapy 2002; 9: 95-101.

18 Airenne KJet al. . Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery. Gene Therapy 2000; 7: 1499-1504.

19 Amabile PGet al. . High-efficiency endovascular gene delivery via therapeutic ultrasound. J Am Coll Cardiol 2001; 37: 1975-1980.

20 Klugherz BDet al. . Gene delivery from a DNA controlled-release stent in porcine coronary arteries. Nat Biotechnol 2000; 18: 1181-1184. Article MEDLINE

21 Klugherz BDet al. . Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum Gene Ther 2002; 13: 443-454.

22 Panetta CJet al. . A tissue-engineered stent for cell-based vascular gene transfer. Hum Gene Ther 2002; 13: 433-441.

23 Brasen JHet al. . Angiogenesis, vascular endothelial growth factor and platelet-derived growth factor-BB expression, iron deposition, and oxidation-specific epitopes in stented human coronary arteries. Arterioscler Thromb Vasc Biol 2001; 21: 1720-1726.

24 Yoshimura Set al. . Inhibition of intimal hyperplasia after balloon injury in rat carotid artery model using cis-element 'decoy' of nuclear factor-kappaB binding site as a novel molecular strategy. Gene Therapy 2001; 8: 1635-1642.

25 Nakamura Tet al. . Molecular strategy using cis-element 'decoy' of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model. Gene Therapy 2002; 9: 488-494.

26 Kipshidze NNet al. . Intramural coronary delivery of advanced antisense oligonucleotides reduces neointimal formation in the porcine stent restenosis model. J Am Coll Cardiol 2002; 39: 1686-1691.

27 Maillard Let al. . Effect of percutaneous adenovirus-mediated Gax gene delivery to the arterial wall in double-injured atheromatous stented rabbit iliac arteries. Gene Therapy 2000; 7: 1353-1361.

28 Leppanen Oet al. . Intimal hyperplasia recurs after removal of PDGF-AB and -BB inhibition in the rat carotid artery injury model. Arterioscler Thromb Vasc Biol 2000; 20: E89-E95. MEDLINE

29 Ding Het al. . Adenovirus-mediated expression of a truncated PDGFbeta receptor inhibits thrombosis and neointima formation in an avian arterial injury model. Thromb Haemost 2001; 86: 914-922.

30 Hiltunen MOet al. . Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta. Circulation 2000; 102: 2262-2268. MEDLINE

31 Rutanen Jet al. . EGD-D is abundantly expressed in human arteries and gene transfer of VEGF-D N C reduces neointima formation in balloon-denuded rabbit aorta. Mol Ther 2002; 5: S354-S355.

32 Hayashi Ket al. . In vivo transfer of human hepatocyte growth factor gene accelerates re-endothelialization and inhibits neointimal formation after balloon injury in rat model. Gene Therapy 2000; 7: 1664-1671. Article MEDLINE

33 Hayashi Ket al. . In vivo transfer of human hepatocyte growth factor gene accelerates re-endothelialization and inhibits neointimal formation after balloon injury in rat model. Laukkanen MO. Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon denuded rabbit aorta. Circulation 2002 (in press).

34 Quarck Ret al. . Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice. Circulation 2001; 103: 2495-2500.

35 Zoldhelyi Pet al. . Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries. Proc Natl Acad Sci USA 2001; 98: 4078-4083.

36 Golino Pet al. . Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries. J Am Coll Cardiol 2001; 38: 569-576.

37 Atsuchi Net al. . Combination of a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI gene transfer additively reduces neointima formation in balloon-

injured rabbit carotid arteries. Circulation 2001; 103: 570-575.

38 Puhakka HPet al. . eptide re-targeted adenoviral combination gene therapy coding TIMP-1 and VEGF-C has a long term effect on reducing restenosis. Mol Ther 2002; 5: S163.

39 Leppänen Oet al. . Combination of VEGF-C gene transfer and treatment with the PDGF receptor kinase inhibitor STI571 leads to persistent reduction in neointima formation in balloon-denuded rabbit aorta. J Am Coll Cardiol 2002; 39: 872.

40 George SJet al. . Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation 2000; 101: 296-304. MEDLINE

41 Ehsan Aet al. . Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg 2001; 121: 714-722. Article MEDLINE

42 Ohno Net al. . Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation 2002; 105: 1623-1626.

43 Ohno Net al. . Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Grube, E. E2F Decoy-gene therapy in bypass grafts. American Heart Association 2001 Meeting (Plenary Session: Late Breaking Clinical Trials). 2001.

44 Makinen Ket al. . Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002; 6: 127-133.

45 Kutryk MJet al. . Local intracoronary administration of antisense oligonucleotide against c-myc for the prevention of in-stent restenosis. Results of the randomized investigation by the thoraxcenter of antisense dna using local delivery and ivus after coronary stenting (ITALICS) trial. J Am Coll Cardiol 2002; 39: 281-287. Article MEDLINE

46 Tsui LVet al. . p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion. Circ Res 2001; 89: 323-328.

47 Wu CHet al. . Inhibition of neointimal formation in porcine

coronary artery by a Ras mutant. J Surg Res 2001; 99: 100-106.

48 Condorelli Get al. . Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice. FASEB J 2001; 15: 2162-2170.

49 Ascher Eet al. . Effect of p53 gene therapy combined with CTLA4Ig selective immunosuppression on prolonged neointima formation reduction in a rat model. Ann Vasc Surg 2000; 14: 385-392. Article MEDLINE

50 Luo Zet al. . Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35. Hum Gene Ther 2001; 12: 2191-2202.

51 Larson RAet al. . Adenoviral-mediated uteroglobin gene transfer inhibits neointimal hyperplasia after balloon injury in the rat carotid artery. J Vasc Surg 2000; 32: 1111-1117.

52 Yamamoto Ket al. . Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease. Circulation 2000; 102: 1308-1314.

53 Eto Yet al. . Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol Heart Circ Physiol 2000; 278: H1744-H1750. MEDLINE

54 Varenne Oet al. . Percutaneous gene therapy using recombinant adenoviruses encoding human herpes simplex virus thymidine kinase, human PAI-1, and human NOS3 in balloon-injured porcine coronary arteries. Hum Gene Ther 2000; 11: 1329-1339.

55 Lamfers MLet al. . In vivo suppression of restenosis in balloon-injured rat carotid artery by adenovirus-mediated gene transfer of the cell surface-directed plasmin inhibitor ATF.BPTI. Gene Therapy 2001; 8: 534-541.

56 Izumi Yet al. . Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in balloon-injured rat artery. Circ Res 2001; 88: 1120-1126.

Tables

Table1 Gene therapy approaches to reduce restenosis or vein graft

stenosis

Table2 Clinical gene therapy trials to prevent restenosis and vein graft stenosis

Received 2 July 2002; accepted 24 July 2002

October 2002, Volume 9, Number 20, Pages 1344-1350

Table of contents    Previous  Article  Next   [PDF]

ReviewGene Therapy Progress and Prospects: Cystic fibrosis

U Griesenbach, S Ferrari, D M Geddes and E W F W Alton

Department of Gene Therapy, National Heart and Lung Institute, Imperial College, Faculty of Medicine, London, UK

Correspondence to: U Griesenbach, Department of Gene Therapy, Faculty of Medicine, National Heart and Lung Institute, Imperial College, London, SW3 6LR, UK

Abstract

Since the cloning of the cystic fibrosis gene (CFTR) in 1989, 18 clinical trials have been carried out, including five in the 2 years reviewed here. Most trials demonstrated proof-of-principle for gene transfer to the airway. However, gene transfer efficiency with each of the three gene transfer agents (adenovirus (Ad), adeno-associated virus 2 (AAV2) and cationic liposomes) was low, and most likely insufficient to achieve clinical benefit. Here, we will review the clinical and pre-clinical progress for the last 2 years (20002001) and briefly speculate on future prospects for the next 2 in CF gene therapy.

Gene Therapy 2002 9, 13441350. doi:10.1038/sj.gt.3301791

Keywords

cystic fibrosis; gene therapy; airway gene transfer

In brief

Progress

Five clinical trials for CF have been carried out between 2000 and 2001 Encouraging progress has been made to overcome some of the extracellular barriers to airway

gene transfer, such as mucus and the glycocalyx

Important intracellular barriers, such as cytoplasmic nucleases and the nuclear membrane have been identified and first attempts been made to overcome these barriers

Viruses that recognise receptors on the apical surface of airway epithelial cells have been identified

Targeted receptor-mediated endocytosis of synthetic vectors has increased transfection efficiency of airway epithelial cells

Repeated administration of viral vectors, but not non-viral vectors, remains a significant problem

Expression cassettes have been improved to enable prolonged transgene expression

Intravenous and in utero gene delivery have been evaluated

Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced for CF

Prospects

Five clinical trials for CF have been carried out between 2000 and 2001 Encouraging progress has been made to overcome some of the extracellular barriers to airway

gene transfer, such as mucus and the glycocalyx

Important intracellular barriers, such as cytoplasmic nucleases and the nuclear membrane have been identified and first attempts been made to overcome these barriers

Viruses that recognise receptors on the apical surface of airway epithelial cells have been identified

Targeted receptor-mediated endocytosis of synthetic vectors has increased transfection efficiency of airway epithelial cells

Repeated administration of viral vectors, but not non-viral vectors, remains a significant problem

Expression cassettes have been improved to enable prolonged transgene expression

Intravenous and in utero gene delivery have been evaluated

Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced for CF

Integrating viral and non-viral GTAs will prolong gene expression in the airways and expression will be regulated through on/off cassettes

Repeated administration of viral vectors will be improved

Physical methods of gene delivery will be developed and will overcome some of the barriers to topical gene transfer

Better regulatory elements will be developed

Stem cell gene therapy will advance

Better animal models will begin to be developed

New imaging techniques to monitor the success and effect of gene transfer and more clinically relevant endpoint assay will be developed

Five clinical trials for CF have been carried out between 2000 and 2002

Three out of the five trials used liposome-mediated gene transfer strategies, one trial used Ad2 and one AAV2. Noone et al transfected the nasal epithelium of CF patients and was able to detect vector-specific DNA up to 10 days after gene transfer, but not mRNA or functional correction of the CFTR defect.1 The discrepancy between this trial and previously published nose trials that were able to detect CFTR mRNA and partial correction of the CFTR defect, is likely due to the lower transfection efficiency of the cationic lipid used. In a dose-escalating safety trial, liposome DNA complexes were aerosolised into the lungs of CF subjects, half of which, had a transient fever, muscle and joint pain shortly after liposome/DNA administration.2 This was attributed to an immunological response against the liposome/DNA complexes; similar results were previously reported in a lung trial, using the same liposome/DNA formulation. Interestingly, Hyde et al demonstrated for the first time, that liposome/DNA complexes could be successfully re-administered to the nose of CF patients. Each subject received three doses, administered 4 weeks apart and samples were analysed 4 days after each treatment. Six out of 10 treated subjects were positive for CFTR gene transfer after each dose.3

Perricone et al reported the results of a phase I clinical trial, in which recombinant adenovirus (Ad2/CFTR) was administered through bronchoscopic instillation or aerosolisation to the lungs of CF patients. In contrast to previous studies, the authors carefully determined that inefficient gene expression was due to the very low transduction efficiency of Ad in human airways. In addition, the vast majority of transfected cells (97%), did not appear to be epithelial cells.4 The authors therefore concluded that further improvements in Ad vector design are urgently required. An AAV2 vector was nebulised into the lung of CF subjects to assess delivery and safety. AAV2 administration to the lung appeared to be safe and vector genome was detected, at the highest dose, up to 30 days after administration. However, vector-specific mRNA could not be detected and therefore evidence for gene transfer was not provided.5

In summary, these new studies consolidate the view that proof-of-principle of gene transfer can be demonstrated in some, but not all studies, but that gene transfer efficacy is currently insufficient to warrant phase II/III trials. Thus, significant improvements in all aspects of gene transfer need to be made.

Encouraging progress has been made to overcome some of the extracellular barriers to airway gene transfer, such as mucus and the glycocalyx

A major function of the airway epithelium is to prevent uptake of foreign materials, including gene transfer agents (GTAs). For this purpose several very effective extracellular barriers, such as mucus, the gycocalyx, tight junctions and mucociliary clearance have evolved (Figure 1). Mucus, for example, reduces the transfection efficiency of most viral and non-viral gene transfer agents. However, transfection efficiency could be increased through pre-treatment with mucolytics or the antichiolinergic drug glycopyrolate in vitro and in vivo.6 In addition to mucus, sputum and bronchoalveolar lavage fluid recovered from CF patients have been shown to inhibit liposome,7 adenovirus8 and AAV-mediated gene transfer efficiency.9 Although recombinant DNase reduced sputum viscoelasticity and improved nanosphere migration in vitro,10 the effect of DNase on gene transfer in vivo is unclear. To avoid the confounding effect of sputum in vivo, gene transfer should ideally be studied in CF children, before their lungs become filled with secretions. Indirect evidence has indicated, that the glycocalyx is also a barrier to gene transfer and that neuroaminidase enhances Ad transfection of polarised cells in vitro.11

Receptors for Ad and AAV2 are located on the basolateral membrane of human airway epithelial cells and uptake of non-viral gene transfer agents also appears to be enhanced on the basolateral site, due to the presence of heparan receptors and a higher endocytosis rate. The effect of several tight junction 'openers', including EGTA,12,13 anti-E-cadherin antibody,14 sodium caprate,15 a blend of sucrose, mannitol and Pluronic F6816 or perfluorochemical17,18 on gene transfer has been studied in vitro and in vivo. Although a 10- to 50-fold increase in virus- and non-virus-mediated gene transfer was generally seen in vitro, it is unlikely that the opening of tight junctions, even if transient, will be clinically applicable, given that the airways of most CF patients are heavily colonised with bacteria.

In summary, although encouraging progress has been made partially to overcome some extracellular barriers, it is unlikely that these strategies on their own will significantly improve gene transfer in the human airways. However, they may prove beneficial in combination with other strategies (see below).

Targeted receptor-mediated endocytosis of synthetic vectors has increased the transfection efficiency of airway epithelial cells

Liposomes and other synthetic vectors are also unable to transfect airway epithelial cells efficiently via the apical membrane. Targeted receptor-mediated endocytosis may increase gene transfer and proof-of-principle for this approach has recently been provided by Ziady et al, who have shown that targeting of the serpin enzyme complex receptor (sec-R) increases gene transfer to the nasal epithelium of mice.19 The sec-R ligand was linked to the CFTR plasmid via a poly-L-lysine bridge, and this formulation partially corrected the CFTR defect (chloride efflux) in the nose of CFTR knockout mice. Most remarkably, it was demonstrated for the first time in vivo that CF-related secondary defects (nitric oxide synthase-2 and sodium hyper-absorption) were partially correctable through gene therapy as well.19 Targeting of sec-R has been successful in increasing airway gene transfer and several groups are currently trying to identify new peptides that bind to airway epithelial cells and promote targeted receptor-mediated endocytosis, using phage display libraries.20

Viruses that recognise receptors on the apical surface of airway epithelial cells have been identified

As mentioned above, the density of receptors for Ad and AAV2 on the apical membrane of human airway epithelial cells is low, which explains in part the inefficient transfection efficiency. Multiple approaches have been studied to overcome this problem. Firstly, several new recombinant viruses have been identified, that appear to be able to enter epithelial cells via the apical membrane efficiently. In addition to respiratory syncytial virus (RSV),21 Sendai virus (SeV)22 has recently been shown to transfect bronchial epithelial cells efficiently in vivo. Two days after infection with SeV 80% of airway epithelial cells expressed the -galactosidase transgene (Figure 2a). SeV uses binding to cholesterol and sialic acid as receptors, which are both present at the apical membrane of airway epithelial cells. In addition, SeV is not greatly inhibited by mucus. AAV entry into airway epithelial cells appears to be serotype-specific and AAV5 has been shown to transfect airway epithelial cells five times more efficiently than AAV2.23,24 AAV in general is a very promising gene transfer agent. Not only does the virus maintain prolonged transgene expression due to integration or concatemerisation of the vector genome, but it is also thought that the virus might be less immunogenic, due to the fact that it does not appear to transduce antigen-presenting dendritic cells. Some reports have suggested that AAV could be repeatedly administered to the lung. However, this possibility has recently been ruled out unless different serotypes or some form of immunosuppression are used.25 An important limitation of AAV as gene transfer vector for CF has been its limited packaging capacity. However, it has recently been demonstrated that this problem can be overcome by using trans-splicing or overlapping vectors26 or by shortening the CFTR cDNA.27

Secondly, proof-of-principle has been established that adeno and AAV vectors can be retargeted to apical surface receptors, such as the bradykinin,28 the urokinase plasminigen29 and the P2Y2-purinoreceptor30 in vitro. However, it remains to be established if these approaches will increase transfection efficiency in

vivo.

A third strategy involves systematic pseudotyping of enveloped virus with different envelope glycoproteins. Glycoproteins from viruses that naturally infect the airway epithelial cells, such as human coronavirus 229E,31 influenza A strains32 and members of the Filovirus family, such as Marburg or Ebola virus, are good candidates. Pseudotyping of lentiviral vectors such as the human, feline or equine immunodeficiency virus is a particularly attractive approach. Lentiviral vectors have the ability to integrate into the genome of dividing and non-dividing, differentiated cells. Expression is therefore prolonged when compared with episomally maintained vectors and may last forever, if integration into a stem cell occurred. Kobinger et al have recently demonstrated that an Ebola-pseudotyped HIV vector efficiently transfects airway epithelial cells in vivo.33 Most interestingly, expression was low 7 days after administration, but strong expression was visible in airway epithelium and submucosal gland cells 28 days after transfection (30% tracheal epithelium expressed -galactosidase). This persisted at least until day 63 (24% of the tracheal epithelium expressed a -galactosidase reporter gene) Figure 2b. Currently large-scale, high titre production of pseudotyped lentiviral vectors is technically difficult and a critical limiting factor.

The identification of viruses that are capable of efficiently transfecting the airway epithelium via the apical membrane has been one of the most exciting findings in the last 2 years. Although big problems such as repeated administration still exist, proof-of-principle studies, demonstrating that the primary CFTR defect can be consistently corrected in humans, should now become possible.

Repeated administration of viral vectors, but not non-viral vectors, remains a significant problem

Transgene expression will remain transient, unless lung repopulating stem cells can be targeted with a stably maintained vector. The treatment of CF with gene therapy will therefore require repeated administrations of gene transfer agents, which is a particular problem for viruses. Strategies such as administration of immunosuppressants and corticosteroids, and treatments aimed at transiently blocking CD4+ T cells34 have been evaluated for repeated administration of adenovirus. However, the success of these strategies has been limited. In general, repeated administration was possible a few times, but ultimately lead to reduced and finally absent transgene expression. The only exception so far is a report by Kolb et al, who demonstrated that administration of the steroid budesonide enabled adenovirus to be re-administered at least five times without loss of transfection efficiency.35 Importantly, treatment with an anti-CD40 ligand monoclonal antibody did not prevent a virus specific antibody response in non-human primates.34

Another and perhaps more promising approach is based on generating a 'stealth virus', which is invisible to the immune system, by coating the virus capsid with polyethylene glycol (PEG). PEGylation of the virus capsid reduced cytotoxic T cells and antibody production and significantly prolonged transgene expression from 4 to 42 days. Repeated administration of the adenovirus modified with the same PEG was not successful, however, when different PEG formulations were used, significant transgene expression was detected after repeat administration.36

Although, it has been demonstrated that repeated administration of liposome/DNA complexes to the nose of CF patients is possible,3 there have been concerns raised regarding the inflammatory components of bacterial DNA.2 The abundance of unmethylated CpG motifs in the bacterial plasmid DNA may at least in part be responsible for the inflammatory response. Several strategies are currently being explored to decrease these unwanted properties, such as (1) methylation of CpG sequences; (2) reduction of the CpG frequency by eliminating non-essential regions or by site-directed mutagenesis; and (3) the use of specific inhibitors of the CpG signalling pathway, such as chloroquine or quinacrine.37

In summary, progress in enabling repeated administration of viruses has been rather slow and concerns regarding viral and non-viral GTA-induced inflammation have been raised. A more detailed understanding of the immune responses against viral and non-viral GTAs is therefore crucial for further improvements in gene transfer.

Important intracellular barriers including cytoplasmic nucleases and the nuclear membrane have been identified and first attempts been made to overcome these barriers

Intracellular barriers limiting transgene expression have also been identified. Endosomal and cytoplasmic degradation are a particular problem for most non-viral gene transfer agents. In the cytoplasm, Ca2+-sensitive cytosolic nucleases restrict the half-life of plasmid DNA to 5090 min.38 Interestingly, even recombinant viruses are inhibited by intracellular degradation. In polarised cells, the AAV capsid is ubiquinated and subsequently enters the ubiquitin proteasome-dependent degradation pathway.39,40 Thus, proteasome inhibitors augmented AAV2-mediated gene transfer in the mouse lung from undetectable levels to 10% of airway epithelial cells transfected.39

In airway epithelial cells the nuclear membrane is an important barrier for non-viral gene transfer.41 It is currently uncertain if strategies, such as the addition of nuclear localisation signals42 or lactosylated poly-L-lysine,43 which increased nuclear transfection in other cell types, will be beneficial in these.

Although, it has been demonstrated that AAV is degraded by proteasomes, non-viral GTAs are probably more affected by intracellular degradation and nuclear entry than viruses. Thus, new strategies are urgently required to overcome the intracellular barriers. It is also important to note, that these barriers are likely to be cell-type specific and therefore studies need to be carried out in the 'relevant' cell type.

Expression cassettes have been improved to enable prolonged trangene expression

Current non-integrating viral and non-viral GTAs are unable to maintain sustained expression. Immuno-suppressants have been reported to prolong transgene expression,44 due to their ability to reduce the T cell-mediated response. In addition, Scaria et al showed that incorporation of the adenoviral gene ICP47 into an adenoviral vector, which reduced MHC class I antigen presentation in CD8+ T cells, prolonged expression in primate lung up to 21 days.45

The strong CMV promoter has traditionally been used in most GTAs. However, more recently evidence has been provided that weaker eukaryotic or hybrid promoters, such as the polyubiquitin C promoter,46 the elongation factor 1 promoter46 and the CMV-Ubiquitin B hybrid promoter47 enable prolonged transgene expression. In addition, there is growing evidence that genomic sequences, either within or flanking the gene, might be essential to provide in vivo long-term expression.48 Alternative strategies to prolong transgene expression include the development of artificial chromosomes and other self-replicating systems. Huertas et al have developed a circular yeast artificial chromosome (YAC) carrying the human CFTR sequence and the oriP and EBNA-1 genes from EpsteinBarr (EBV) virus.49 However, it remains to be established if these large constructs are able to transfect airway epithelial cells in vivo.

Intravenous and in utero gene delivery routes have been evaluated

As noted above topical gene transfer to the lung is severely affected by many extra-cellular barriers. In an attempt to overcome these barriers GTAs or oligonucleotides (ODN) have been administered intravenously (i.v.). It is likely that i.v. administered GTA have to reach the bronchial circulation and avoid lodging in the pulmonary circulation to reach relevant target cells for CF therapy. Fox et al have recently demonstrated that 'naked' ODN are able to leave the bronchial circulation and transfect the cytoplasm, but not the nuclei of airway epithelial cells.50 In addition, Kohler et al have shown that i.v. injection of lipid-complexed

-galactosidase plasmids lead to expression in bronchial epithelium and tracheal submucosal glands of

mice. However, this was only achieved with some, but not all of the cationic lipids tested.51 Importantly, in the past it has sometimes been difficult to discriminate recombinant from endogenous -galactosidase expression and it is therefore important for these potentially exciting findings to be expanded upon.

The possibility for in utero gene therapy for CF has also been investigated. Injection of GTAs into the amniotic fluid, would provide contact with most relevant target sites for CF (pulmonary, gastrointestinal and sinus epithelium). Injection of adenovirus, retrovirus and more recently AAV52 resulted in reporter gene expression in both pulmonary and gastrointestinal epithelium with persistence of transgene expression in the lung ranging from 14 to 30 days after infection. The possibility that in utero gene therapy would tolerise recipients to viral GTAs, has recently been ruled out.53 Larson et al have postulated a role for CFTR during development and provided preliminary evidence that transient expression of CFTR cDNA in utero alleviates the intestinal defect in the CF knockout mouse long term.54,55 These controversial findings, if replicated, may have far reaching implications for traditional gene therapy approaches, as well as other areas of CF research.

Genomic gene repair, mRNA trans-splicing and antisense approaches have been introduced for CF

Gene repair of the endogenous CFTR gene has two major advantages over traditional gene therapy. If successful, gene repair will ensure gene expression for the lifetime of the cells and appropriate control of gene expression will be guaranteed because the endogenous CFTR promoter is utilised. Preliminary results indicated that the genomic CFTR locus could be modified in primary rat hepatocytes using chimeraplasts (DNA/RNA hybrid oligonucleotides).56 Hepatocytes have previously been shown to be easily amenable for gene repair strategies, most likely due to efficient uptake of repair molecules into the nucleus. In addition, a similar approach using small fragment homologous recombination (SFHR) was able to reintroduce the wild-type CFTR sequence into the lungs of CF knockout mice, albeit at very low frequency.57 However, the specificity of targeted gene repair is currently unknown.

Down-regulation of gene expression through antisense molecules may be of therapeutic benefit in CF. Lambert et al showed that antisense inhibition of the B cell antigen receptor-associated protein (BAP) 31 increased expression of both wild-type CFTR and mutant CFTR and partially restored CFTR chloride channel function.58 The exact function of BAP31 is unclear, although the authors speculated that the protein may be involved in retaining mutant CFTR in the ER. Several other chaperone proteins, mucins or the epithelial sodium channel (ENaC), which is up-regulated in CF may be suitable candidates for antisense strategies.

Spliceosome-mediated trans-splicing (SMaRT) has recently been introduced as a means to generate wild-type CFTR mRNA in CF xenograft models. Cells were transfected with very high titres of adenovirus that produced so-called pre-therapeutic wild-type CFTR mRNA molecules (PTMs), which are designed to promote trans-splicing with the endogenous CFTR mRNA and 22% of wild-type CFTR function could be restored.59 Similar to gene repair, SMaRT ensures cell-type specific expression of wild-type CFTR mRNA, however efficiency and specificity of the reaction require further improvements, before clinical trials can be considered.

Prospects

Together with traditional GTAs, new approaches are being developed in order to increase gene transfer efficiency to the airway epithelium. Gersting et al60 recently reported that application of a magnetic field to human primary airway epithelial cells transfected with plasmid DNA mixed with superparamagnetic nanoparticles (magnetofection) resulted in more than 100-fold increase in gene transfer. Stem cell-based gene therapy approaches are also being investigated. Two strategies can be envisaged: either targeting lung-resident stem cells with integrating vectors or engineering bone marrow/embryonic cells ex vivo and inducing engraftment into the airway epithelium. Thus, bone marrow-derived stem cells have recently been observed to diferentiate into airway epithelial cells in the lung.61 Prolonged and sustained CFTR

expression may not be beneficial, especially if integrating vectors are used. To this end, epithelium-specific regulated cassettes are being developed.62 Each of the vectors and technologies will have to be tested in relevant in vivo models. Ideally, in contrast to CF mice, these would reproduce the lung pathology seen in CF patients. For this reason efforts are being made to develop a CF ferret63 and a CF sheep,64 their lung biology being more similar to humans than the mouse. Finally new imaging techniques to monitor the efficiency of gene transfer in living animals/patients will also have to be developed. In addition to positron emission tomography (PET) and magnetic resonance imaging (MRI)-based strategies, laser-induced fluorescence bronchoscopy65 has recently been developed as a way to detect gene expression in a non-invasive way in human airways.

Conclusion

Although topical gene transfer to the airways for CF gene therapy is more challenging than originally thought, significant progress has been made in pre-clinical research over the last 2 years to overcome some of the hurdles. Most importantly, the identification of more effective viral and non-viral gene transfer agents for airway gene delivery, development of new delivery routes and most recently alternative strategies are keeping the development of CF gene therapy well on track.

References

1 Noone PGet al. . Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis. Mol Ther 2000; 1: 105-114. MEDLINE

2 Ruiz FEet al. . A clinical inflammatory syndrome attributable to aerosolised lipid-DNA administration in cystic fibrosis. Hum Gene Ther 2001; 12: 751-761. MEDLINE

3 Hyde SCet al. . Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Therapy 2000; 7: 1156-1165. MEDLINE

4 Perricone MAet al. . Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium. Hum Gene Ther 2001; 12: 1383-1394. Article MEDLINE

5 Aitken MLet al. . A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Hum Gene Ther 2001; 12: 1907-1916. MEDLINE

6 Ferrari Set al. . Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium. Gene Therapy 2001; 8: 1380-1386. MEDLINE

7 Sanders NN, Van Rompaey E, De Smedt SC, Demeester J. Structural alterations of gene complexes by cystic fibrosis sputum. Am J Respir Crit Care Med 2001; 164: 486-493. MEDLINE

8 Perricone MAet al. . Inhibitory effect of cystic fibrosis sputum on

adenovirus-mediated gene transfer in cultured epithelial cells. Hum Gene Ther 2000; 11: 1997-2008. MEDLINE

9 Virella-Lowell Iet al. . Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients. Gene Therapy 2000; 7: 1783-1789. MEDLINE

10 Sanders NNet al. . Cystic fibrosis sputum: a barrier to the transport of nanospheres. Am J Respir Crit. Care Med 2000; 162: 1905-1911.

11 Pickles RJet al. . Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer. J Virol 2000; 74: 6050-6057. MEDLINE

12 Wang Get al. . Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo. Am J Respir Cell Mol Biol 2000; 22: 129-138. MEDLINE

13 Chu Qet al. . EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo. Hum Gene Ther 2001; 12: 455-467. MEDLINE

14 Man Yet al. . Loss of epithelial integrity resulting from E-cadherin dysfunction predisposes airway epithelial cells to adenoviral infection. Am J Respir Cell Mol Biol 2000; 23: 610-617. MEDLINE

15 Coyne CB, Kelly MM, Boucher RC, Johnson LG. Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am J Respir Cell Mol Biol 2000; 23: 602-609. MEDLINE

16 Croyle MA, Cheng X, Sandhu A, Wilson JM. Development of novel formulations that enhance adenoviral-mediated gene expression in the lung in vitro and in vivo. Mol Ther 2001; 4: 22-28. MEDLINE

17 Weiss DJet al. . Perfluorochemical liquid enhances adeno-associated virus-mediated transgene expression in lung. Mol Ther 2000; 2: 624-630. MEDLINE

18 Das A, Niven R. Use of perfluorocarbon (Fluorinert) to enhance reporter gene expression following intratracheal instillation into the lungs of Balb/c mice: implications for nebulized delivery of plasmids. J Pharm Sci 2001; 90: 1336-1344. MEDLINE

19 Ziady AGet al. . Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme

complex receptor. Mol Ther 2002; 5: 413-419. MEDLINE

20 Jost PJet al. . A novel peptide, THALWHT, for the targeting of human airway epithelia. FEBS Lett 2001; 489: 263-269. MEDLINE

21 Zhang L, Collins PL, Boucher RC, Pickles RJ. Respiratory syncytial virus (RSV) infects ciliated cells of airway epithelium via the lumenal membrane. Pediat Pulmonol 2001; 244.

22 Yonemitsu Yet al. . Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat Biotechnol 2000; 18: 970-973. Article MEDLINE

23 Walters RWet al. . Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem 2001; 276: 20610-20616. MEDLINE

24 Zabner Jet al. . Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol 2000; 74: 3852-3858. Article MEDLINE

25 Halbert CLet al. . Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J Virol 2000; 74: 1524-1532. Article MEDLINE

26 Duan D, Yue Y, Engelhardt JF. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol Ther 2001; 4: 383-391. Article MEDLINE

27 Ostedgaard LSet al. . CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo. Proc Natl Acad Sci USA 2002; 99: 3093-3098. MEDLINE

28 Pickles RJ, Hirsh AJ, Gerard RJ, Boucher RC. Retargeting Ad5 fibers to bradykinin receptors expressed on the lumenal surface of human airway epithelium. Pediat Pulmonol 2001; 22: 247.

29 Drapkin PTet al. . Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia. J Clin Invest 2000; 105: 589-596. MEDLINE

30 Kreda SM, Pickles RJ, Lazarowski ER, Boucher RC. G-protein-coupled receptors as targets for gene transfer vectors using natural small-molecules ligands. Nat Biotechnol 2000; 18: 635-640. MEDLINE

31 Wang Get al. . Human coronavirus 229E infects polarized airway epithelia from the apical surface. J Virol 2000; 74: 9234-9239.

MEDLINE

32 Slepushkin VAet al. . Infection of human airway epithelia with H1N1, H2N2 and H3N2 influenza A virus strains. Mol Ther 2001; 3: 395-402. Article MEDLINE

33 Kobinger GP, Weiner DJ, Yu QC, Wilson JM. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 2001; 19: 225-230. MEDLINE

34 Chirmule Net al. . Readministration of adenovirus vector in nonhuman primate lungs by blockade of CD40-CD40 ligand interactions. J Virol 2000; 74: 3345-3352. MEDLINE

35 Kolb Met al. . Budesonide enhances repeated gene transfer and expression in the lung with adenoviral vectors. Am J Respir Crit Care Med 2001; 164: 866-872. MEDLINE

36 Croyle MA, Chirmule N, Zhang Y, Wilson JM. 'Stealth' adenovirus blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 2001; 75: 4792-4801. MEDLINE

37 Yew NSet al. . Reduced inflammatory responses to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs. Mol Ther 2000; 1: 255-262. MEDLINE

38 Pollard Het al. . Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids. J Gene Med 2001; 3: 153-164. MEDLINE

39 Duan Det al. . Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest 2000; 105: 1573-1587. MEDLINE

40 Yan Zet al. . Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. J Virol 2002; 76: 2043-2053. MEDLINE

41 Yan Zet al. . Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. Griesenbach U et al. Cytoplasmic deposition of NF B decoy oligonucleotides is insufficient to inhibit bleomycin induced pulmonary inflammation. Gene Therapy 2002 (in press).

42 Cartier R, Reszka R. Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems. Gene Therapy 2002; 9: 157-167. MEDLINE

43 Klink DT, Chao S, Glick MC, Scanlin TF. Nuclear translocation

of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells. Mol Ther 2001; 3: 831-841. Article MEDLINE

44 Cassivi SDet al. . Transplant immunosuppression increases and prolongs transgene expression following adenoviral-mediated transfection of rat lungs. J Heart Lung Transplant 2000; 19: 984-994. MEDLINE

45 Scaria Aet al. . Adenoviral vector expressing ICP47 inhibits adenovirus-specific cytotoxic T lymphocytes in nonhuman primates. Mol Ther 2000; 2: 505-514. MEDLINE

46 Gill DRet al. . Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1 promoter. Gene Therapy 2001; 8: 1539-1546. MEDLINE

47 Yew NSet al. . High and sustained transgene expression in vivo from plasmid vector containing a hybrid ubiquitin promoter. Mol Ther 2001; 4: 75-82. MEDLINE

48 Stoll SMet al. . Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice. Mol Ther 2001; 4: 122-129. MEDLINE

49 Huertas D, Howe S, McGuigan A, Huxley C. Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells. Hum Mol Genet 2000; 9: 617-629. MEDLINE

50 Fox Eet al. . Towards nucleic acid transfer to the airway epithelium via the systemic route. Mol Ther 2001; 3: S197.

51 Koehler DRet al. . Targeting transgene expression for cystic fibrosis gene therapy. Mol Ther 2001; 4: 58-65. MEDLINE

52 Boyle MPet al. . In utero AAV-mediated gene transfer to rabbit pulmonary epithelium. Mol Ther 2001; 4: 115-121. MEDLINE

53 Lipshutz GS, Flebbe-Rehwaldt L, Gaensler KML. Re-expression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration. Mol Ther 2000; 2: 374-380. MEDLINE

54 Larson JEet al. . Gene transfer into the fetal primate: evidence for the secretion of transgene product. Mol Ther 2000; 2: 631-639. Article MEDLINE

55 Larson JEet al. . CFTR modulates lung secretory cell proliferation and differentiation. Am J Physiol Lung Cell Mol Physiol 2000; 279: L333-L341. MEDLINE

56 Griesenbach Uet al. . Conversion of wild-type CFTR to the G551D mutation in primary rat hepatocytes using RNA/DNA oligonucleotides. Pediat Pulmonol 2001; 252.

57 Goncz KKet al. . Expression of (F508 CFTR in normal lung after site-specific modification of CFTR sequences by SFHR. Gene Therapy 2001; 8: 961-965. MEDLINE

58 Lambert Get al. . Control of cystic fibrosis transmembrane conductance regulator expression by BAP31. J Biol Chem 2001; 276: 20340-20345. MEDLINE

59 Liu Xet al. . Partial correction of endogenous (F508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat Biotechnol 2002; 20: 47-52. Article MEDLINE

60 Liu Xet al. . Partial correction of endogenous (F508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Gersting S et al. Magnetofection of permanent and primary human airway epithelial cells. Mol Ther 2001; 3: Abstr. 1017.

61 Krause DSet al. . Mutli-organ, multi-lineage engraftment by a single bone marrow-derived sem cell. Cell 2001; 105: 369-377. MEDLINE

62 Ye Let al. . Regulated expression of the human CFTR gene in epithelial cells. Mol Ther 2001; 3: 723-733. MEDLINE

63 Ye Let al. . Regulated expression of the human CFTR gene in epithelial cells. Jiang Q, Li Z, Zhang Y, Engelhardt JF. Development of a ferret model of the cystic fibrosis. Pediat Pulmonol 2000; 20 (Suppl.): Abstr. 164.

64 Mouchel Net al. . The sheep genome contributes to localization of control elements in a human gene with complex regulatory mechanisms. Genomics 2001; 76: 9-13. MEDLINE

65 Mouchel Net al. . The sheep genome contributes to localization of control elements in a human gene with complex regulatory mechanisms. Rooney C et al. Laser induced fluorescence bronchoscopy for the detection of gene transfer. Mol Ther 2001; 3: Abstr. 1152.

Figures

Figure 1 Scanning electron micrograph of human airway epithelium showing fields of cilia covered at their tips by flakes of mucus. In hypersecretory disease the mucus usually forms a continuous sheet or 'blanket' overlying the cilia (supplied by courtesy of Professor Peter Jeffery, Imperial College, London, UK).

Figure 2 Viral gene transfer to the lung. (a) Recombinant Sendai virus (SeV) mediated -galactosidase expression 48 h after gene transfer.48 (b) Filovirus pseudotyped lentivirus-mediated -galactosidase expression 63 days after gene transfer (with permission from Ref. 33)..

October 2002, Volume 9, Number 20, Pages 1344-1350