Gene therapy approaches for cystic fibrosis

Biologicds

f 1995 I 23, 2 1-25

Gene Therapy

Approaches

for Cystic Fibrosis

Charles Coutelle St. Mary’s Hospital Medical School, Department of Biochemistry

Introduction Cystic fibrosis (CF) is a severe autosomal recessive genetic disease with an incidence of about 1 affected child among 2000 newborns. In the U.K., there are about 6000 children and 1500 adults affected by this condition. Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene are the molecular basis of CF. The cystic fibrosis transmembrane conductance regulator protein coded by this gene is an integral membrane protein. Its main physiological role is that of a CAMP-regulated chloride channel.’ CFTR mutations which cause CF lead to a disturbance of the ion and water transport across the luminal surface of the secretory epithelia of the gut, pancreas, lung, biliary ducts and sperm ducts and are responsible for the dehydrated mucus characteristic of cystic fibrosis.” The dehydrated mucus supports repeated infection which leads to chronic inflammation of the airways and gut lumen. To date treatment of CF is symptomatic, including daily physiotherapy and antibiotics directed against respiratory infections, together with pancreatic enzyme supplementation and intensive dietary support. More recently DNase and a,-antitrypsin have been on trial as more specific therapies, but these are still directed at symptoms rather than the underlying defect. Somatic gene therapy for CF aims at the introduction of a normal gene sequence into cells of the affected tissues. This should lead to synthesis of the normal protein, which should correct the ion transport defect and prevent the secondary organ damage due to cellular malfunction. All present strategies for gene therapy of CF are oriented towards complementation of the two mutated alleles by addition of the normal gene sequence and not to gene replacement. Both the cDNA and the genomic DNA for CFTR have been isolated, and it is known that heterozygosity for the mutated gene presents no pathology. CFTR is effective when expressed at very low levels in normal lung cells3 It also does not seem to need sophisticated gene regulation, since neither over1045-1056/95/010021+05

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and Molecular Genetics,

London, U.K.

expression in cell cultureG6 nor permanent synthesis of the human protein in transgenic mice7 causes any cellular toxicity or organ impairment. Experiments with CF cells in culture have shown that the ion transport defect caused by the mutated CFTR gene can be corrected by introduction of a single copy of the normal CFTR coding sequence into the affected cells. It has been shown that normal CFTR expression by transfection with a retroviral CFTR cDNA construct of less than 10% of CF-cells grown in coculture with uncorrected CF cells is sufficient to generate chloride transport in the entire monolayer similar to that observed in an epithelial sheath consisting of 100% corrected cells.8 This is probably due to ionic coupling of adjacent cells through gap junctions.g There are several mouse models for CF where the murine CFTR gene has been mutated, and which are appropriate models for testing somatic gene therapy. CF has therefore many features making it a good choice for gene therapy approaches. Alton and collaborators’0 have developed a noninvasive, sensitive and reliable in uivo method to detect the ion transport defect in CF patients by measuring the electrical potential difference in the epithelial cells lining the nose. This has become a favourite procedure to monitor the effect of gene therapy trials in patients, for use as a surrogate end point during clinical trials. Strategies fibrosis

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The lung, and in particular its bronchi and bronchioli, is the organ most efforts presently focus upon. The probability that only a relatively low level of expression in a limited number of cells is required to restore normal ion transport, and the possibility of vector delivery by inhalation to the appropriate small airways, makes a good case for in vivo gene application by nebulisation to the lung epithelia. Present approaches towards in vivo somatic gene therapy for CF are concentrating on gene delivery by adenovirus vectors and liposomes. @ 1995 The

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C. Coutelle

Adenovirus vectors Most groups using a viral strategy for gene therapy of CF are presently investigating the potential of adenovirus-vectors. Adenovirus naturally infects lung epithelia. It has a genome size of about 36 kb, can infect nondividing cells and expresses large amounts of gene product. The adenoviral vectors constructed for gene therapy are derivatives of the wild type adenovirus types 2 and/or 5. Both belong to the subgroup C of this virus for which no oncogenic potential has been observed in any species investigated, including man.11*12 For use as gene therapy vectors, the El gene region, and in most cases the E3 gene region as well, have been deleted. Deletion of the El gene functions makes the virus replication deficient, and such viral vectors have to be packaged by growth in the human embryonic kidney helper cell-line 293, which expresses the El function constitutively. I3 Adenoviral vectors of this type can package about 38 kb DNA containing an insert of up to about 75 kb. Adenovirus vectors can be produced at high titres and show a very high level of infectivity, reaching almost 100% in cell culture and in uiuo. Two factors contribute to this high efficiency: receptor mediated cell attachment and entry via the fibre protein and. the integrin binding domain of the penton base; and the ability to escape the lysosome degradation pathway into which most endosomally introduced substances are directed. All major cell types of the airway epithelia of rodents14 and primates (Pavirani, personal communication) are infected by adenovirus recombinant vectors carrying the Pgalactosidase marker gene. Human CFTR and or1-antitrypsin cDNA have also been cloned into the deleted El gene region of the adenovirus genome and were successfully expressed in the lung epithelium of cotton rats.‘j,16 Expression of human CFTR protein was detected for about 2 weeks and CFTR mRNA transcripts for up to 6 weeks.15 However, since the adenovirus usually does not integrate into the genome of lung epithelial stem cells, it will eventually be lost due to degradation or the regular replacement of epithelial cells. Repeated re-infection will, therefore, be necessary, which may cause immune reactions against this treatment. The viral proteins are known to induce inflammatory reactions independent of viral replication. This could pose particular clinical difficulties in the context of CF, as reduced resistance to infection and chronic inflammation are general problems in the case of CF patients.

Several different adenovirus/CFTR cDNA constructs have been prepared, with different viral sequences and promoters governing CFTR expression. The CFTR-cDNA recombinant, Ad CFTR, is based on the adenovirus type 5 genome, with extensive deletions in the El and E3 gene regions. The entire 4.5 kb protein coding region of human CFTR is expressed under regulatory control of the adenovirus type 2 C major late promotor and its three tripartite leader sequences at the 5’ end, and the SV40 polyadenylation site at the 3’ end.‘” A similar CFTR expression vector based on adenovirus type 5, Ad.CBCFTR, uses the chicken pactin gene promotor.” The CFTR-cDNA construct Ad2/CFTR-1 in which the E3 gene has been retained is expressed under control of adenovirus 2 transcription elements.lR All three adenovirus/CFTR-constructs are presently being tested in the USA in phase 1 clinical trials. Cationic liposome DNA complexes Cationic liposome DNA complexes’g have been used successfully in nonviral approaches for the introduction of DNA into cells in vitro and in viuo. Various expressing plasmids and even DNA of 650 kb yeast artificial chromosomes (YACS)~~ have been transferred into cells by this method. Since these DNA-lipid complexes have no replicative capability most of the health and safety concerns associated with virus vectors do not apply. Furthermore, liposome formulations containing hydrolizable bonds have been synthesised which reduce general cytotoxicity. Such liposomes are naturally degraded after fusion with the cell membrane and delivery of the complexed DNA. One of these preparations composed of dioleoyl phosphatidylethanolamin/3mN-(N’-N’dimethyl-aminoethane-carbamoyll (DCCholDOPEyZ1 shows efficient in vitro transfection and low cytotoxicity.22 This preparation caused no acute or toxic systemic reactions when given intravenously to mice at a dose of 0.42 nmolz2 and no autoimmune reaction or transfer of DNA into gonadal cells was observed after repeated intravenous, intraperitoneal or subcutaneous injection of plasmid DNA-DC-Chol/DOPE injection in mice. 23 DC-CholDOPE has been applied in human trials for cancer treatment.22.24-“” Instillation2i~2s and nebulisation2g*“0 have been successfully used to introduce different liposome plasmid DNA complexes in uiuo into the airways of mice. Marker genes coding for luciferase, pgalactosidaseZa or CAT,2g as well as human CFTR cDNA,~~ have been expressed in uiuo in the lung of wild type mice after delivery by DOTMADOPE. Lipofectin27 and DOPE/DC-CO~“~ were used to deliver and

Gene

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approaches

express human CFTR cDNA plasmids in uivo in the lung of two different transgenic CF mouse strains by intratracheal instillation and nebulisation respectively. Successful correction of the CAMP-dependent chloride transport defect in the airways of these animals was detected in vitro and in viva by electrophysiological measurements.“0,“7 Phase 1 gene therapy fibrosis

clinical

trials of cystic

Based on these extensive investigations in cell culture, tissue samples, and normal and CF experimental animals, phase 1 clinical trials for somatic gene therapy of the pulmonary manifestations of cystic fibrosis have now started. The main objective of these trials is to obtain information on safety and toxicity of the constructs. Several trials using adenovirus vectors carrying CFTR cDNA sequences have been approved by the Recombinant Advisory Committee (RAC) and the Food and Drug Administration (FDA) in the USA. The use of FDAapproved liposome preparation (DC-ChoVDope) for CFTR-cDNA delivery has been approved by the U.K. Committee on the Ethics of Gene Therapy and the U.K. Medicines Control Agency (MCA). Neither the adenovirus nor the liposome trials intend to provide definitive treatment at this point, even to the very small number of adult volunteer patients involved. They aim at investigating efficacy, safety and dose response of the different vector and delivery systems by measuring the correction of the ion transport defect in duo, as a surrogate end point, on a very small area of the airway epithelia of the patients. In the Iowa/Genzyme adenovirus trial, and the Royal BromptonSt Mary’sEdinburgh liposome trial, the effect of gene administration is being monitored by measuring the changes in the electrical potential difference on the surface of nasal epithelial cells. This non-invasive procedure allows the time course of gene expression and its persistence after application to be followed. The electrophysiological investigations will be supported by tests for expression of normal CFTRcDNA in epithelial brushings and biopsies. Studies of the epithelia lining of the nose have the additional advantage of permitting an initial measuring period in which the baseline electrical potential differences for each CF patient can be established. The first published adenovirus-CFTR trial, performed by the teams of Welsh and Smith (Iowa/Genzyme),31 used increasing doses of virus from 2 X lo6 to 6 X lo7 infectious units per cells, which were delivered to the nasal epithelia of three

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CF patients. All three showed a decrease of the baseline electrical potential difference and increase in the response to antagonists. The correction of the potential differences lasted for about 3 weeks, while CAMP stimulation was only detectable for about 10 days. No adenovirus-related pathology was reported for this initial trial. In a similar so far unpublished investigation by Crystal’s group (N.I.H.) nasal delivery was followed one day later by bronchoscopic application into a large bronchus. This trial had to be suspended temporarily following an inflammatory reaction of one of the patients. It is not clear whether this reaction was idiosyncratic, or represented a dose which would cause difficulties in other patients. Further adenovirus trials conducted by Wilson (Philadelphia), Boucher (North Carolina) and Wilmolt & Whitsett (Cincinnati) have started recently. The first U.K. liposome gene therapy trial was performed as a collaborative study between the CF gene therapy groups at the Royal Brompton Hospital/National Heart and Lung Institute, the Department of Biochemistry and Molecular Genetics of St. Mary’s Hospital Medical School/Imperial College and the MRC Human Genetics Unit in Edinburgh. The trial was conducted double blind, and recruited 15 adult CF patients. The placebo group received liposome alone (three 500 pg, three 1500 pg); the active group was given increasing doses of the CFTR cDNA/hposome complex; ratio DNA to liposome 1:5; DNA 10, 100 and 300 pg per nostril. Nasal electrical potential differences were read from both nostrils on days - 14, -7,O, 1,3,4,5, 7, 11, 18 and 28. Nasal brushings and biopsies were taken on day 4 from one nostril and were analysed for signs of toxicity or inflammation using histology, and for plasmid DNA and CFTR-mRNA by PCR and RT-PCR. The data of this trial are published elsewhere.32 All protocols of these trials ensure monitoring for any signs of inflammation, shedding into the environment or other adverse effects. Provided these phase 1 trials give positive results, protocols for repeated application, and eventually application to the lower airways will follow, and could lead to attempts to treat CF pulmonary disease by gene therapy approaches within the next five to ten years. Ethical and legal considerations All present strategies for gene therapy of CF are oriented solely towards somatic gene supplementation. This aims at the correction of the functional defect in a range of somatic cells expressing the affected phe-

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notype in a particular patient. It is generally agreed that somatic gene therapy does not create new ethical problems.33 However, since it is an experimental treatment, or research towards treatment, ethical approval for individual protocols has to be obtained. For the proposed phase I trials for CF gene therapy approval was given for the adenovirus-vector approach by the NIH Recombinant Advisory Committee (RAC) on the basis of extensive written documentation and a public hearing. The documents submitted to the NIH RAC by the different U.S. groups describe the disease, its molecular and cellular basis, its current therapy and prognosis underlining the relevance of gene therapy for CF. They describe the structure and biology of adenovirus and the rationale behind construction of the proposed CFTR cDNA recombinants. Experimental data of in vitro tests and in vivo experiments in rodents and primates supporting the efficacy and safety of these constructs and a detailed description of the production and quality control of the recombinant vectors are provided. Various safety aspects raised by the proposed use of adenovirus recombinants are commented on; in particular in relation to possible replicative, recombinatory and oncogenic potentials of adenovirus, its ability to integrate into the host genome, the possibility that it may infect germ cells or be shed into the environment and its potential to provoke adverse reactions by the host. The protocols for the proposed phase I trials are described in detail, giving an exact account of the procedures to be followed and defining the criteria for patient selection and for assessment of the results with respect to safety and efficacy. Special consideration is given to confidentiality and informed consent, follow up of the patients and the reporting of adverse effects. Our protocol for a liposome based phase I clinical trial for CF was approved by the Committee on the Ethics of Gene Therapy following clarification of queries relating to patient information and consent, patient selection, information to participating staff, the assessment of safety and efficacy and the preparation of DNA. DNA for the trial was prepared at the MRC Human Genome Mapping Project Resource Centre, at Northwick Park Hospital, according to a detailed protocol agreed by the Medicines Control Agency. The liposomes DC-CholDOPE were prepared using FDA approved procedures by Drs Huang and Gao (University of Pittsburgh). Our experiences concerning the regulatory procedures and production of resources have been published elsewhere.34

Acknowledgements I wish to thank my colleagues in the CF-Gene Therapy Group at St. Mary’s Hospital Medical School and at the Royal Brompton Hospital/National Heart and Lung Institute and in particular Bob Williamson, Natasha Caplen, Duncan Geddes, Eric Alton and Peter Middelton for helpful discussions and suggestions. References 1. Bear CE, Li C, Kartner N et al. Purification and functional reconstitution of the cystic fibrosis transmembrane regulator (CFTR). Cell 1992; 68: 809-818. 2. Boucher RC, Knowles MR, Cantley L et al. Na transport in cystic fibrosis respiratory epithelia. J Clin Invest 1986; 78: 1245-1252. 3. Trapnell BC, Chu C-S, Paakko PK et al. Expression of the cystic fibrosis transmembrane regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis. Proc Nat1 Acad Sci USA 1991; 88: 6565-6569. 4. Drumm ML, Wilkinson DJ, Smit LS et al. Chloride conductance expressed by AF508 and other mutant CFTRs in Xenopus oocytes. Science 1991; 254: 1797-1799. 5. Gregory RJ, Cheng SH, Rich DP et al. Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 1990; 347: 382-386. 6. Rich DP, Anderson MP, Gregory RJ et al. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 1990; 347: 358-363. 7. Whitsett JA, Dey CR, Stripp BR et al. Human cystic regulator fibrosis transmembrane conductance directed to respiratory epithelial cells of transgenic mice. Nature Genet 1992; 2: 13-20. 8. Johnson LG, Olsen JC, Sarkadi B et al. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nature Genet 1992; 2: 21-25. 9. Gilula NB, Reeves OR, Steinbach A. Metabolic coupling, ionic coupling and cell contacts. Nature 1972; 235: 262-265. 10. Alton EWFW, Hay JG, Munro C et al. Measurement of nasal potential difference in adult cystic fibrosis, Young’s syndrome, bronchiectasis. Thorax 1987; 42: 815-817. 11. Horowitz MS. In: Fields BN & Knipe DM, eds. Virology, Raven Press, Ltd, New York, 1990: 1723-1740. 12. Strauss S. In: Ginsberg HS ed. Adenoviruses, Plenum Press, New York, London, 1984: 451-496. 13. Graham FL, Smiley J, Russell WE et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977,36: 59-72. 14. Mastrangeli A, Dane1 C, Rosenfeld MA et al. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated transfer. J Clin Invest 1993; 91: 225-234.

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K, Trapnell BC et al. In 15. Rosenfeld MA, Yoshimura vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 1992; 68: 143-155. 16. Rosenfeld MA, Siegfied W, Yoshimura K et al. Adenovirus-mediated transfer of a recombinant a,antitrypsin gene to the lung epithelium in uiuo. Science 1991, 252: 431-434. for 17. Yang Y, Raper SE, Cohn JA et al. An approach treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc Nat1 Acad Sci USA 1993; 90: 4601-4605. 18. Zabner J, Petersen DM, Puga AP et al. Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nature Genet 1994; 6: 75-83. 19. Gershon H, Ghirlando R, Guttman SB et al. Mode of formation and structural features of DNA-cationic liposome complexes used for transfection. Biochemistry 1993; 32: 7143-7151. 20. Lamb BT, Sisioda SS, Lawler AM et al. Introduction and expression of the 400 kilobase precursor amyloid protein gene in transgenic mice. Nature Genet 1993; 5: 22-29. 21. Gao X, Huang L. A novel cationic liposome for efficient transfection of mammalian cells. Biochem Biophys Res Commun 1991; 179: 280-285. 22. Stewart MJ, Plautz GE, Del Bouno Let al. Gene transfer in vivo with DNA-liposome complexes: Safety and acute toxicity in mice. Hum Gene Ther 1992; 3: 267-276. 23. Nabel EG, Gordon D, Yang Z-Y et al. Gene transfer in vivo with DNA-liposome complexes: of lack of autoimmunity and gonadal localization. Hum Gene Ther 1992; 3: 649-656. 24. Miller AD. Human gene therapy comes of age. Nature 1992; 357: 455-460.

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25. Nabel GJ, Chang A, Nabel EG et al. Clinical protocol; Immunotherapy of malignancy by in uiuo gene transfer into tumors. Hum Gene Ther 1992; 3: 399410. 26. Nabel GJ, Nabel EG, Yang Z et al. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biological activity and lack of toxicity in humans. Proc Nat Acad Sci USA 1993; 90: 11307-11311. 27. Hyde SC, Gill DR, Higgins CF et al. Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 1993; 362: 250-255. 28. Yoshimura K, Rosenfeld MA, Nakamura H et al. Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in uiuo intratracheal plasmid-mediated gene transfer. Nucleic Acids Res 1992; 20: 3233-3240. 29. Stribling R, Brunette E, Liggitt D et al. Aerosol gene delivery in vivo. Proc Nat1 Acad Sci USA 1992; 89: 11277-11281. 30. Alton EWFW, Middleton PG, Caplen NJ et al. Noninvasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nature Genet 1993; 5: 135-142. 31. Zabner J, Couture LA, Gregory RJ et al. Adenovirusmediated gene transfer transiently corrects the transport defect in nasal epithelia of patients with cystic fibrosis. Cell 1993; 75: 207-216. NJ, Alton EWFW, Middelton PG et al. 32. Caplen Liposome mediated CFTR gene transfer to the nasal epithelia of patients with cystic fibrosis. Nature Medicine 1995; 1: 39-46. 33. Clothier CM. Report of the Committee on the Ethics of Gene Therapy. 1992, HMSO. 34. Caplen NJ, Gao X, Hayes Pet al. Gene therapy for cystic fibrosis in human subjects by liposome mediated DNA transfer: regulatory process and production of resources. Gene Therapy 1994; 1: 139-147.