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Genetic Therapies for Cystic Fibrosis Lung Disease

Patrick L. Sinn1, Reshma Anthony1, Paul B. McCray, Jr. 1*

1Program in Gene Therapy, Department of Pediatrics, Carver College of Medicine, The

University of Iowa, Iowa City, Iowa 52242

*Corresponding Author

Department of Pediatrics

200 Hawkins Drive

The University of Iowa College of Medicine

The University of Iowa

Iowa City, IA 52242

Tel: (319) 356-4866

Fax: (319) 356-7171

E‐mail: paul‐[email protected]

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Abstract

The aim of gene therapy for cystic fibrosis (CF) lung disease is to efficiently and safely express

the cystic fibrosis transmembrane conductance regulator (CFTR) in the appropriate pulmonary

cell types. While CF patients experience multi-organ disease, the chronic bacterial lung

infections and associated inflammation are the primary cause of shortened life expectancy. Gene

transfer-based therapeutic approaches are feasible, in part, because the airway epithelium is

directly accessible by aerosol delivery or instillation. Improvements in standard delivery vectors

and the development of novel vectors, as well as emerging technologies and new animal models,

are propelling exciting new research forward. Here we review recent developments that are

advancing this field of investigation.

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INTRODUCTION

Since the discovery of the CFTR gene in 1989, cystic fibrosis (CF) has been in the sights of

scientists hoping to prevent or delay the onset and progression of lung disease through the use of

gene transfer. While loss of CFTR function adversely affects multiple cells and tissues,

progressive lung disease accounts for the majority of the morbidity and mortality. For this

reason, most effort in the field has focused on gene transfer to the airways. CFTR is expressed in

multiple epithelial cell types in the surface and submucosal glands of the conducting airways

where its mRNA is expressed in low abundance. The gene product is an apical membrane anion

channel that is regulated by nucleotides and phosphorylation (1-3). Loss of CFTR function likely

alters the volume and composition of airway secretions, but key details of the molecular

pathogenesis of CF lung disease remain the subject of intense study.

Complementation of this autosomal recessive disease by the delivery of a CFTR cDNA to the

airway epithelium with a viral or non-viral vector holds appeal, as the envisioned target cells are

accessible via direct instillation or aerosol delivery approaches. Furthermore, early studies

indicated that complementation of as few as 6-10% of CF epithelia generated wild type levels of

chloride transport in vitro (4). However, since the completion of the first human gene therapy

trial in 1993, the achievement of this goal has proved challenging.

There is controversy regarding which cells to target for CF gene therapy. Arguments can be

made in support of correcting cells of the surface epithelium, the submucosal glands, or both (5-

8). A heterogeneous population of cell types express CFTR in the airways including ciliated

cells within the surface epithelium and a subpopulation of cells in submucosal gland ducts and



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acini. There appear to be several epithelial cell types in the lung that provide progenitor

functions, providing the possibility of long term correction if such cells can be targeted with

integrating vectors (9). These cells may represent a pluripotent population or serve as progenitors

for a specific lineage. Experiments from several species and model systems identify potential

progenitor populations, including: basal cells (10, 11) and non-ciliated columnar cells of the

airways (10, 12, 13), submucosal gland epithelia (14-16), Clara cells (17, 18) and alveolar type II

cells in the distal lung (19, 20). Studies using integrating vectors (Moloney murine leukemia

virus (MLV)- and lentivirus-based) suggest that if cells with progenitor capacity are targeted in

vitro and in vivo, long-term expression can be attained (21-25).

RECENT VECTOR DEVELOPMENTS

Enveloped Viral Vectors

Current lentiviral vector technology has made considerable progress toward the aims of

efficiently, safely, and persistently expressing CFTR in the appropriate pulmonary cell types.

Studies are beginning to examine the consequences of repeated administration of lentiviral-based

vectors in the airways. Sinn and colleagues repeatedly administered (7 doses, 1 dose/week) a

baculovirus envelope (GP64) pseudotyped feline immunodeficiency virus (FIV)-based lentiviral

vector to the nasal epithelia of mice (26) and observed dose-dependant increases in reporter gene

expression that persisted for the 80 week duration of the experiment (Figure 1). The observed

innate or adaptive immune responses to the vector or vector encoded transgenes were minimal

and failed to curtail reporter or therapeutic gene expression. In contrast, Limberis and coworkers

reported that gene transfer with a VSV-G pseudotyped HIV vector resulted in activation of

transgene–specific T cells in mice (27). Transduction by VSV-G pseudotyped HIV vectors can



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be further improved by formulations including magnetofectins (28), polyethylenimine (29), or

lysophosphatidylcholine (30, 31). Such methods may prove vital for achieving transduction

efficiencies sufficient to correct the chloride transport defect in vivo. Careful preclinical studies

in large animal models will be needed to further assess the safety and efficacy of the different

lentiviral vector platforms under investigation for pulmonary gene transfer.

Fetal and neonatal re-administration of a GP64-pseudotyed HIV vector was also investigated in

mouse lung (32). Buckley et al. compared a single fetal intra-amniotic administration, 3

administrations (1 fetal/2 neonatal), and 2 neonatal administrations. The levels of macrophage

transduction increased with neonatal re-administration. The authors speculated that following the

initial dose of lentiviral vector, macrophages are recruited to the pulmonary lumen and are

subsequently transduced by the second and third doses. The authors further concluded that intra-

amniotic administration of GP64-pseudotyped HIV was the most efficient mode of delivery for

achieving airway epithelial cell transduction in the mouse model.

Mitoma and colleagues described a simian immunodeficiency virus (SIV)-based lentiviral vector

pseudotyped with the Sendai virus envelope proteins, hemaglutinin-neuraminidase (HN) and

fusion (F) protein (33). F/HN-pseudotyped SIV vector transduced nasal epithelial cells, resulting

in sustained transgene expression for the duration of the experiment (8-12 months) in vivo.

Similar to studies with GP64-pseudotyped FIV (26), re-administration was feasible with F/HN-

pseudotyped SIV, where transgene expression remained stable after 3 vector doses. In addition,

F/HN-pseudotyped SIV conferred functional CFTR expression in vitro as determined by iodide

efflux assay.



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Paramyxovirus family members with known airway tropism are currently being explored as

potential CFTR delivery vehicles for the treatment of CF lung disease using reverse genetics

systems. Kwilas and colleagues recently demonstrated that a respiratory syncytial virus (RSV)-

based vector could deliver CFTR and correct the chloride transport defect in primary cultures of

human CF airway epithelia (34). In addition, a human parainfluenza virus (PIV)-based vector

mediated detectable but transient expression of GFP and a-fetoprotein in rhesus macaques (35).

Both the RSV and PIV-based vectors are replication competent; however, these studies may lead

to replication attenuated vectors that are further engineered to reduce the expression of cytotoxic

and/or immunogenic proteins. If such engineering is feasible, it could improve the duration of

gene expression, address the obstacle of pre-existing immunity, allow for repeat administration,

and make these vectors suitable for clinical studies.

Encapsidated Viral Vectors

Helper dependent-adenoviral (HD-Ad) vectors do not express viral-coding sequences and elicit

reduced cell-mediated immune responses, as compared to earlier generations of Ad vectors.

However, HD-Ad capsid proteins remain targets for neutralizing antibodies and may trigger

cytokine responses from innate immune effector cells. Recently, Cao and colleagues

demonstrated that transient immunosuppression significantly enhanced the efficiency of

transgene expression and facilitated readministration of HD-Ad vectors to mouse lungs (36). In

addition to immunosuppression, serotype switching is a proposed technique to allow for redosing

of CFTR expressing HD-Ad vectors in vivo (37). Granio and colleagues delivered an Ad vector

expressing GFP tagged CFTR to primary cultures of human CF airway epithelia. They observed



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that swapping Ad5 fiber with serotype 35 fiber conferred more effective apical transduction and

correction of the Cl- transport defect (38). These data suggest that Ad vectors such as Ad5 that

use the coxackie and adenovirus receptor (CAR) are less effective at transducing the apical

surface of airway epithelial cells than CAR-independent vector serotypes such as Ad35. Taken

together, these studies outline strategies for using HD-Ad, immunosuppression, serotype

switching, and optimal fiber selection to improve the safety and long-term efficacy of adenovirus

for gene transfer to the airways.

Recombinant adeno-associated virus (AAV) has been used for pulmonary gene transfer in

several preclinical and clinical trials. Flotte and colleagues demonstrated that AAV1 offered

advantages over AAV5 in the chimpanzee airways, both in terms of gene transfer efficiency and

reduced immunogenicity (39). Importantly, this observation was validated by studies in well-

differentiated human airway epithelia, suggesting that the dual reporter virus co-infection

approach can help predict efficacy of AAV vectors in vivo. Progress has also been made in

engineering minimal CFTR expression cassettes that can be accommodated by the AAV vector

(40).

Additional strategies to improve the efficiency of AAV transduction to airway epithelia include

using different capsid serotypes or capsid mutants with a greater affinity for airway epithelial

cells (41). As discussed below, other novel AAV capsid variants have resulted from directed

evolution and sequence shuffling (42-44). Although standard triple transfection methodology

remains an option, new developments in baculovirus-based methods are better suited to meet

AAV production requirements (45-47). In conclusion, improvements in AAV engineering,

capsid serotype design, and production methods have made AAV an attractive vector choice for



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delivering CFTR to the airways. Studies of efficacy and vector readministration in large animal

models will help guide vector development.

Non-Viral Vectors

Considerable progress has been made toward developing non-viral vectors for gene transfer to

the lung. Typically, non-viral vectors fall into two categories: 1) non-integrating, such as

plasmids (48), nanoparticles (49), and mini-circles (50), or 2) integrating, such as transposons

(51) and phage phiC31 (52, 53). Both integrating and non-integrating non-viral vectors face

many of the same delivery and transduction obstacles in vivo. Optimizing the delivery efficiency

of DNA-based vectors to the in vivo airways remains a focus of the field (54, 55). Doxorubicin

(56), carboxymethylcellulose (57), and chitosan (58) improve plasmid-based gene transfer and

expression in the airways. As an alternative, some groups are investigating hybrid vector systems

combining features of adenovirus (59) or lentivirus with transposon-based vectors to improve

delivery (60).

RECENT DEVELOPMENT OF CLINICAL STUDIES FOR CF GENE THERAPY

Since 1993, approximately 25 phase I/II trials using either viral or non-viral vectors for CF have

been conducted (61). A currently ongoing clinical trial in the field was initiated by the UK CF

Gene Therapy Consortium, funded by the Cystic Fibrosis Trust, using an aerosolized non-viral

gene transfer agent (62). CF patients are receiving a single dose of a plasmid carrying the CFTR

cDNA that is complexed to the cationic lipid GL67. The plasmid, termed pGM169, is devoid of

putative pro-inflammatory sequences (CpG islands) and gene expression is regulated by a hybrid

elongation factor-1a promoter. When complexed to the cationic lipid GL67A, pGM169 led to >4



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week expression in mouse models upon single dosing (48). The initial single dose clinical trial

will assess safety and duration of expression in patients and will guide a planned (approximately

100 patients) multi-dose placebo controlled trial. The planned trial, due to start in autumn 2011

(personal communication, Uta Griesenbach), will determine if repeated non-viral CFTR gene

transfer (12 doses over 12 months) improves CF lung disease (61).

NEW TECHNOLOGIES

Directed evolution of viral vectors

As vehicles for gene therapy applications, all viral vectors have potential weaknesses, such as

immunogenicity, tropism, transient transgene expression, and production to high titers. The

success in exploiting viral vectors will depend on the ability to overcome these limitations.

Directed evolution of viruses is a method for generating new or improved viral protein properties

using selection-based approaches. AAV and retroviruses have been the subject of combinatorial

engineering approaches in the past decade. AAV has been attractive due to its safety profile, low

immunogenicity, and ability to transduce both dividing and non-dividing cells. The AAV capsid

determines infectivity and cell tropism (63) and is therefore the target of modification by directed

evolution (42) or phage panning (64). The breadth of naturally occurring AAV serotypes

suggests that the capsid is tolerant to changes (65). Directed evolution of the AAV capsid by

PCR-based mutagenesis combined with high throughput in vitro recombination generated a

library of chimeric cap genes with components from two diverse serotypes. These serotypes,

AAV2 and AAV5, use distinct receptors, heparan sulphate and sialic acid, respectively (42).

Further selection of this library for improved transduction of human airway cells in culture

identified a novel AAV variant, AAV2.5T, a chimera between AAV2 (aa1–128) and AAV5



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(aa129–725) with a single point mutation (A581T) that exhibited enhanced binding to the apical

surface of airway epithelia and improved gene transfer (42). Furthermore, AAV2.5T could

efficiently express the CFTR cDNA in human airway cells in culture and correct the Cl- transport

defect in human CF epithelia (42).

Gamma retrovirus vectors are also efficient gene delivery tools but insertional mutagenesis and

potential oncogenesis due to preferential integration at transcriptional start sites (TSS) are

limitations for their clinical use (66-69). Lim et al. recently showed that the random insertion of

engineered zinc finger domains throughout the MLV Gag-Pol region and selection of viable

variants resulted in a shifting of the integration preferences of these vectors (70). Furthermore,

these modified integration patterns did not favor TSS. This approach could be extended to

lentiviruses and may serve as a powerful method to improve the safety profile of retroviruses as

gene transfer vectors for clinical use.

Gene repair and gene addition

A technique known as ‘genome editing’ enables efficient and precise modification of a target

sequence in a genome by introduction of a double stranded break (DSB) followed by

modification of the locus during subsequent DSB repair by homologous recombination. The

DSB is induced by a zinc finger nuclease (ZFN) (71-74), a specifically engineered endonuclease

designed to cleave a chosen target in the genome. The ZFN consists of two components, the zinc

finger protein (ZFP) and the non-specific cleavage domain of Fok1 endonuclease. The ZFP

binds to the target sequence and contains a tandem array of Cys2-His2 fingers (75) each

recognizing 3 bp of DNA. The arrays generally contain three or four fingers that bind a 9 or 12



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bp target, respectively. The Fok1 domains must dimerize to cleave the DNA (76), consequently

the specificity of the recognition site is doubled from 9 to 18 bp for a 3 finger ZFN.

Homology-based genome editing can be exploited for correction of mutated genes responsible

for monogenic disorders (Figure 2). ZFN-based genome editing requires delivery of a donor

DNA repair template along with the target specific ZFN pair. Methods for generating ZFN pairs

targeting specific genomic loci are becoming widely available and include modular design

approaches (77-79) and the selection-based oligomerized pool engineering (OPEN) strategy (80).

ZFNs designed using OPEN technology have been shown to bind the genomic DNA encoding

CFTR (78) and create double strand breaks near the F508 mutation in exon 10. Provision of a

wild-type donor DNA template with a non-integrating vector, such as integrase-deficient

lentiviral vector (81), can facilitate repair of this mutation by homologous recombination.

Other repair strategies using homing endonucleases (82-84) or transcription activator-like

effector (TALE) nucleases (85) provide alternative mechanisms for creating DSBs in genomic

DNA and allowing for gene repair by co-delivery of a homology repair template. A potential

advantage of each of these gene repair approaches is that correction of CFTR in progenitor cell

types could preserve the native regulatory elements and allow for correction in subsequent

daughter cells. Reagent development and delivery to airway epithelia will be important for this

field to advance.

An alternative to the gene repair approach is termed ‘targeted gene addition’ (Figure 2). Here,

ZFNs may be used to create DSBs at potential ‘safe harbor’ loci such as AAVS1 (86), CCR5



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(87), or the mouse Rosa26 locus (88). In this approach, the entire therapeutic transgene with

flanking homology arms would be inserted into the safe harbor loci by homologous

recombination. Modification of such a locus is reasoned much less likely to perturb the

expression of neighboring genes or disrupt the function of other genetic elements (89).

Applications of RNA interference to treat CF

The recent explosion of knowledge in the field of small interfering RNAs has led to applications

of direct relevance to CF. First, RNAi has been used as a tool to identify gene products that

contribute to steps in wild type and mutant CFTR biogenesis including ER and Golgi trafficking,

residence time in the cell membrane, and its removal by proteosomal degradation (90-92). This

has raised the possibility that RNAi-based strategies might be developed to increase the

expression of F508 CFTR, to rescue F508 CFTR from proteosomal degradation, or enhance

its residence time in the cell membrane. Any of these approaches might provide sufficient

residual CFTR function to be therapeutically relevant. Similarly, targeting other cellular

pathways, such as those involved in inflammation, might offer a means to ameliorate disease

symptoms and progression. A significant hurdle for translational studies in this area is

identifying the methods to efficiently deliver RNAi to well-differentiated airway epithelia.

Another area of investigation relevant to the field is the identification of the microRNA

repertoire in airway epithelia and other CFTR expressing cells, as well as their respective target

gene products. Knowledge in this area may identify new targets for therapeutic manipulation.



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Lung tissue engineering

Lung transplantation is currently the only definitive treatment for end stage CF lung disease. The

supply of donor lungs is limited and transplantation achieves only a 10 to 20% survival at 10

years (93). Recently, two groups independently used similar tissue engineering strategies to

develop an autologous bioartificial lung that may begin to help to overcome the limited

availability of donor tissues (94, 95). The bioartificial lungs were created by first generating a

whole lung scaffold by perfusion and decellularization of the adult rat lung, followed by

reseeding of the endothelial and epithelial surfaces of the scaffold with new cells. Evidence for

gas exchange within the resulting grafts was demonstrated. With the further development of this

technology, one could envision the ex vivo correction of patient derived cells, followed by lung

tissue engineering, and transplantation. Although these initial results are very exciting, several

steps need to be further optimized before long-term tissue-engineered lung function can be

translated to the clinic (96).

NEW ANIMAL MODELS OF CF DISEASE

A significant bottleneck for the development of new CF therapeutics has been the lack of animal

models that recapitulate key features of lung and other organ disease pathogenesis. The mouse

models with CFTR null alleles and specific disease mutations available since the early 1990s

have contributed greatly to disease understanding but fail to develop spontaneous lung disease

similar to humans with CF. Recently, two groups used somatic cell targeting of the CFTR gene

with AAV vectors, followed by nuclear transfer and cloning to develop novel models in pigs (97,

98) and ferrets (99, 100). These new animal models recapitulate key features of CF disease (98,

100). At birth, the lungs of CFTR null pigs are free of inflammation but manifest a bacterial host



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defense defect without the secondary consequences of infection (98, 101). CF pigs spontaneously

develop a lung disease phenotype mirroring key features of human CF lung disease in the first

months of life including infection with bacteria, airway remodeling, and mucus hypersecretion

(Figure 3). CFTR null ferrets also develop multiorgan system disease and neonatal animals

manifest a pulmonary host defense defect in the airways associated with colonization by bacteria

(100). There is also early evidence that adult CF ferrets develop a lung disease phenotype with

similarities to human CF, including bacterial colonization (personal communication, John

Engelhardt). These phenotypic features make these new models very attractive for gene therapy

studies. They offer the unique opportunity to test gene therapy interventions prior to the onset of

lung disease and monitor the outcomes for prevention-based treatment strategies.

CONCLUSIONS

This is an unprecedented time in the development of new therapies for CF. The near universal

availability of newborn screening for CF in developed nations has made early diagnosis

commonplace, allowing the potential for treatment of healthy lungs before the onset of chronic

lung disease. However, this opportunity comes at a time where there is a dearth of sensitive and

specific markers of early disease that can be used to assess lung disease onset and monitor

responses to therapy. Additional work is needed to develop new specific and sensitive measures

of the early stages of lung disease suitable for monitoring the response to therapies for use in

infants and young children. Parallel developments in improved gene transfer tools should further

aid the field and lead to new clinical trials.



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ACKNOWLEDGEMENTS

We thank John Engelhardt, and Uta Griesenbach for their helpful discussions, as well as David

Meyerholz for providing the images presented in Figure 3. This work was supported by NIH

grants: R01 HL-075363 (P.B.M.), PO1 HL-51670 (P.B.M.), R21 HL-91808 (P.B.M.), the Cystic

Fibrosis Foundation (P.L.S.), and the Roy J. Carver Charitable Trust (P.B.M.). We also

acknowledge the support of the In Vitro Models and Cell Culture Core and Cell Morphology

Cores, partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-

54759) and the Cystic Fibrosis Foundation.

CONFLICT OF INTEREST STATEMENT

None declared.



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FIGURE LEGENDS

Figure 1. Repeat administration of lentiviral vector results in persistent transgene expression in

the nasal airways. Seven doses of GP64 pseudotyped FIV expressing luciferase were delivered at

1 week intervals for 7 consecutive weeks to mice via nasal instillation (arrows). At the indicated

time points, light release was captured using bioluminescent imaging and data quantified with

living image software. Stable luciferase expression was documented for 80 weeks. Data are

expressed as mean ± standard error. n = 5.

Figure 2. Schematic of targeted gene correction and gene addition strategies. A) Expressed ZFN

pairs bind to and cleave the target genomic locus near the disease causing mutation. ZFNs are

composed of chimeric zinc fingers (blue) and Fok1 endonuclease domains (red). Introduction of

a double stranded break (DSB) promotes homologous recombination, using the repair template

donor. B) For targeted gene addition, the ZFN pair cleaves a predetermined “safe harbor” locus.

The expression cassette with a therapeutic gene (green) is flanked by homology arms to the safe

harbor locus.

Figure 3. Pigs with CFTR mutations develop pulmonary disease with similarity to humans with

CF. In this example from a pig of 5.5 months of age, key pathogenic features observed include

mucus accumulation in the airways, airway obstruction with purulent material containing

neutrophils and bacteria (right panel, black arrows), and airway remodeling. The bacterial

cultures from this animal were positive for Bordetella bronchiseptica. The wild-type pig is an

age matched control. Hemotoxylin & eosin stain. Both images are 100X magnification.



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0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

7 doses

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Figure 1



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ZFN cleavage

* disease mutation

Repair template

Corrected gene

Homologous recombination

A) Targeted correction

Figure 2

B) Gene addition

ZFN cleavage

cDNA of wild-type gene

Homology arm

Homology arm

Wild-type gene added to ‘safe harbor’ locus by homologous recombination

ZFN



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Wild-type pig CF pig

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