A new method of gene delivery has beendeveloped that uses compacted DNAnanoparticles to penetrate the nuclearmembrane. Researchers have demon-strated the efficacy of the technique in amouse model of cystic fibrosis (CF), andPhase I/II clinical trials have now begun.

A way into the nucleusThe most effective gene therapy methodto date has been to use a modifiedadenovirus as the vector for deliveringgenetic material to the cell nucleus.However, viruses can provoke severe reactions and can not be administeredrepeatedly because of the specific im-mune response they provoke. An alter-native method is to encase the DNA in liposomes that can readily pass throughthe outer membranes of cells. The mainproblem with this technique is that theliposomes are usually too big (~100 nm)to pass through pores in the nuclearmembrane. This is only possible duringcell division, when the nuclear membranebreaks down enough to enable the DNAto pass through. This leads to inefficientuptake and is not practical for slow-dividing cells, such as those lining thelungs. These drawbacks have stimulatedthe search for alternative methods ofgene delivery.

One such alternative is being investi-gated by researchers from Case WesternReserve University (http://www.cwru.edu/)and Copernicus Therapeutics (http://www.cgsys.com). In their technique, asingle DNA molecule is wrapped in acoat of positively charged peptides. Theresulting particle has a diameter of only20–25 nm, small enough to pass througha nuclear pore. Mark Cooper of Copernicuspresented data at the American Society ofGene Therapy meeting (30 May–3 June2001; Seattle, WA,USA) [1]. He defined

the size constraints that permit compactedDNA to be effective: ‘Our compactedDNA nanoparticles robustly transfectpost-mitotic human cells, achieving upto a 6900-fold enhancement of gene expression compared to naked DNA.’

The need for a new type of CFtherapyCF is one of the most common geneticdiseases, affecting ~30,000 people in theUSA alone (http://www.cff.org). A reces-sive genetic mutation causes deficienciesin the transport of salt across the mem-branes of secretory cells. This, in turn,causes the accumulation of a thick, stickymucus in the respiratory and digestivetracts and in the reproductive system,which leads to recurrent lung infections,pulmonary damage and difficulties inobtaining nutrients from food. There isno cure for CF and, until recently, fewsufferers survived childhood; however,with modern supportive therapies andcareful disease management those affected now often live into their 30s.Gene therapy offers the best hope forcuring the disease [2]. However, gettingforeign material into the lung lining isparticularly challenging, especially whenthese tissues are coated in thick mucus.

Studies in a mouse model of CFPam Davis and co-workers from CaseWestern applied the nanoparticle tech-nique to mice with CF [3]. Davis explained how the peptide casing, apolylysine chain of >250 residues, resultsin such a small particle size: ‘The nega-tive charges on the DNA phosphategroups interact with the positive chargeson the lysines, allowing the atoms topack closely together, indeed, down toless than twice the minimum theoreticalvolume based on the size of the atoms

alone.’ A 17-amino-acid ligand for theserpin-enzyme complex receptor wasalso conjugated to the particle. This re-ceptor is present on the apical mem-brane of certain cells and is adapted forthe uptake of large molecules into thecell. Attachment of the ligand to thenanoparticle thus facilitated the efficienttargeted entry of the particle, whichcould then proceed to the nucleus.

The gene therapy was successful, withthe replacement CF gene being ex-pressed in the cells of the nasal lining [3].This partially restored function with littleimmune reaction. However, the airwaystructure of mice is different to that ofhumans, so this is no guarantee that that technique would make an effectivetherapeutic. Davis commented: ‘Basedon the data in mice, we expect that thisreagent will be quite benign, but amouse is not a man.’

Clinical trialsTo address this issue, trials have nowbegun on 12 people with CF. The trialuses a slightly different form of nanopar-ticle (Fig. 1). A much shorter polylysinechain is attached via cysteine to poly-ethylene glycol (PEG). This stabilizes theparticle under normal physiological con-ditions making the nanoparticles moresuitable for clinical studies. According toCooper, it is still unclear how the PEG-conjugated particles enter the cell butthey appear to have equal efficacy tothose used by Davis, as shown in numer-ous animal models.

The current trial is a Phase I/II double-blind, placebo-controlled study in whichsaline (placebo) or compacted DNA ofthe CFTR gene in saline is applied to thenostrils of CF subjects. The Phase I trialwill last 5–6 months and will be followedby a Phase II study in which compacted

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Gene therapy trials for cystic fibrosisMatt Brown, matt.brown@elsevier.com

DNA will be administered by a broncho-scope to an isolated bronchial segment.

Subsequent trials will administer aerosolsof compacted DNA to the whole lung.

Terry Flotte, a researcher of CF genetherapy from the University of Florida(http://www.ufl.edu/), was enthusiasticabout the work: ‘It is essential that thesesorts of therapies continue to be testedin patients because, in the final analysis,there is no other way to move forwardtoward better therapies for this disease’,he commented.

Davis is optimistic about the applica-bility of the technique to a wide varietyof other diseases, including gene therapyto produce blood-clotting factors andsurfactant proteins and as a timed response to afflictions such as severeasthma attacks. She also commented onthe possible use of the technique as a

protection from the side-effects of othertherapies: ‘It may be possible to protectthe lung against known, expected tox-icity from radiation therapy or chemo-therapy drugs by delivering a protectivegene at the right time. This might allow fully effective doses of radiation or chemotherapy to be administered,which might otherwise not be tolerable.’

References1 Liu, G. et al. (2001) Nanoparticles of

compacted DNA transfect post-mitotic cells.Mol. Ther. 3, S14

2 Flotte, T.R. (1999) Gene therapy for cysticfibrosis. Curr. Opin. Mol. Ther. 1, 510–516

3 Ziady, A-G. et al. (2002) Functional evidenceof CFTR gene transfer in nasal epithelium ofcystic fibrosis mice in vivo following luminalapplication of DNA complexes targeted tothe serpin-enzyme complex receptor. Mol. Ther. 5, 413–419

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Figure 1. DNA nanoparticles ofCopernicus Therapeutics. Electronmicrograph of polyethylene glycol(PEG)-stabilized compacted DNAparticles (amplification 3 × 104). Figurecourtesy of Copernicus Therapeutics(http://www.cgsys.com).

An inhibitor of cyclin-dependent kinase(CDK) has been well tolerated in Phase Iaclinical trials and is due to enter Phase Ibtrials for glomerulonephritis (GN), a group of inflammatory kidney diseases,in autumn 2002. Details of this were announced by Spiros Rombotis, ChiefExecutive of Cyclacel (http://www.cyclacel.com), at the BioEquity Europe2002 meeting in Zurich, Switzerland(14–15 May 2002).

GlomerulonephritisGlomerulonephritis is a group of kidneydiseases caused by inflammation and cell proliferation that result in gradual,progressive destruction of internal kid-ney structures (glomeruli). Although rare,GN is the third most common cause of

end-stage renal disease (ESRD). This groupof diseases causes 20–50% of all renalfailures that necessitate kidney dialysis or transplantation. GN also develops inpeople with certain cancers, liver cirrhosisand some infectious diseases.

Current treatments include high-dosagesteroid and cytotoxic drug therapy;however, these are often ineffective andhave a high risk of toxic side effects. Newtherapies directed at specific cytokinesand growth factors are under develop-ment, as are less toxic forms of immuno-therapy [1].

In the developing world, ∼ 700,000people are affected by GN, and in theUSA 330,000 people are on dialysis forESRD. US$17 billion is spent annually inthe USA alone on ESRD.

CYC202The lead drug from Cyclacel, CYC202, isa small-molecule inhibitor of CDK, whichregulates the proliferation of cells. CYC202is a tri-substituted purine and is a highlyspecific inhibitor of CDK2/cyclinE ac-tivity, inducing selective apoptosis in cancer cells. During glomerulogenesis,visceral glomerular epithelial cells exitthe cell cycle and become terminally differentiated. Cell proliferation is underthe control of cell-cycle regulatory proteins, such as p21 [2].

The use of CDK inhibitors in the treat-ment of inflammatory disease of the kidney is to suppress abnormal prolifera-tion of non-cancerous kidney cells that destroy renal function. Cancer treatmentusing CDK inhibitors emulates the activity

CDK inhibitor shows promise forinflammatory kidney diseaseJoanne Clough, joanne.clough@elsevier.com