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IS DNA DESTINY?
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CYSTIC FIBROSIS
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IS DNA DESTINY? A Cure for Cystic Fibrosis Cynthia B. Robinson, MD
CYSTIC FIBROSIS GENE THERAPY UPDATE
Time, for cystic fibrosis (CF) patients, has been forever divided into the dark years "before" and the bright promise of "after." The singular event that cleaved time in two was the discovery of the gene responsible for CF 28 Before the discovery of the gene, in 1989.27, only the natural history and the genetic nature of the disease were well characterized. After the discovery of the gene, the basic defect became understood and curing the disease became a tangible possibility.', 29 The resultant effort in gene therapy for CF has produced important new insights into lung biology that benefit the pulmonary community at large. This article updates the status of gene therapy for CF, highlights the remaining hurdles, and provides insights into lung biol-
ogy. GENERAL CONSIDERATIONS
For many reasons, CF remains an attractive target for gene therapy. CF is an autosomal recessive single gene defect. This means that
to correct the defect, one does not need to "silence" the existing genes and that provision of a single copy of the normal gene is sufficient. Furthermore, there must be only one gene principally responsible for the disease. Supplying a single normal copy of the gene to all cells of the lung would be a daunting task, but in vitro work has shown that only 7% to 10% of cells need to be corrected to overcome some of the ion transport abnormalities.ls Although the ciliated epithelial cells express cystic fibrosis transmembrane conductance regulator (CFTR), abundant expression is found in submucosal glands of the large airway^.^ It is not known which cell type is most important to target for gene therapy, but aerosol delivery of drug to the epithelium is more feasible. In all phase I trials, the principal outcome measure is safety assessment. Incorporation of pharmacodynamic endpoints, however, is desirable, even in phase I trials. A variety of techniques have been employed to demonstrate gene expression. The most basic demonstration of gene expression is to show evidence of transgene DNA by DNA amplification via polymerase chain reaction (PCR)
From the Department of Clinical Pharmacology and Adult Cystic Fibrosis Program, University of Pennsylvania; and SmithKline Beecham Pharmaceuticals, Presbyterian Hospital, University of Pennsylvania Health System, Philadelphia, Pennsylvania
CLINICS IN CHEST MEDICINE
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in harvested cells. DNA PCR cannot distinguish between exogenously administered DNA (i.e., the dose of DNA) from DNA that has been successfully incorporated into cells, however. A higher level of proof of successful transfection is reverse transcription PCR (RTPCR) from CFTR messenger RNA. In this situation, the only source of CFTR RNA is from the successfully incorporated transgene. In situ hybridization using the radiolabeled DNA (or RNA) antisense strand also requires uptake by the cells of the exogenously provided transgene. Demonstration of protein expression by immunohistochemistry is probably the strongest level of proof of successful gene transfer. Immunohistochemistry, however, is far less sensitive than any of the other aforementioned molecular biology techniques. The second pharmacodynamic endpoint used in several phase I trials for gene therapy in CF has been demonstration of CFTR function. Two distinct parameters of CFTR function have been described in respiratory epithelium-a baseline hyperpolarization caused by excessive sodium absorption and a lack of depolarization (by chloride secretion) in response to CAMP agonists.19 Restoration of the electrical potentials toward the normal response is becoming accepted as proof of concept. It is not clear, however, which electrical potential is the most important to correct-the baseline sodium hyperabsorption or the chloride secretory response to cyclic adenosine monophosphate (CAMP).Another ex vivo method of demonstrating intact CFTR function is to load harvested cells with a fluorescent dye-SPQ (6-methoxy-N-[3sulfopropyl]quinolinium).16~ 35 SPQ's signal is quenched in the proximity of anions like chloride. When chloride secretion is stimulated by CAMP, the SPQ signal is dequenched and increasing fluorescence is measured. Extent of CFTR correction can be ascertained from the magnitude of the change in fluorescence and the number of cells exhibiting this change. The newest measure of protein function is whether defective CF airway defensin function has been restored as a consequence of gene therapy. Airway defensins are primitive antimicrobial peptides that work against a variety of organisms. In high salt environ-
ments, however, the defensins are inoperative, suggesting that the CF airway milieu is hypert~nic.'~, 34 Although numerous measurements have been attempted, it is not clear whether CF airway surface fluid is hypertonic. The ionic content of airway surface fluid is susceptible to changes during measurement. Furthermore, it is likely that the ionic content of glandular secretions is different from the pericellular water layer, confusing the analysis. The ability of CF cells to resist bacterial colonization may be another demonstration of effective CFTR protein function. The clinical trials to date for CF gene therapy have addressed some difficult problems in drug development. At a minimum, these trials have sought to profile the safety and the presence of gene expression. ADENOVIRAL VECTORS
Promises Most of the early experience with gene therapy for CF has been with adenovirus vectors. Adenovirus has a natural tropism for respiratory epithelium and can be grown to high titers in permissive cell lines5,15, 26 Use of adenoviral vectors has been made possible by the ability to modify specific portions of the viral genome that govern viral replication and immune escape while preserving the ability to make intact virions. In the place of the deleted viral genes, an expression cassette comprising the transgene (CFTR) and its promoter is inserted into the viral backbone by recombination. The replication permissive cell lines package the intact virion with the new transgene. Clinical Trials
The first trial (National Institutes of Health/Cornell University) was initiated in April of 1993 and a total of eight CF patients were treated with an adenovirus 5 El, E3deleted vector delivered bronchoscopically into their lungs.8 To date, approximately 33 patients have been treated with adenoviral
IS DNA DESTINY?
vectors.1°An aerosol trial has been completed, as has a repeat-dosing regimen.4 Both the nose and the lung have been dosed. Details of the completed trials are shown in Table 1. In general, adenoviral vectors can achieve scant, patchy gene expression, as shown by RT-PCR, immunohistochemistry, and in situ hybridization in the nose and the lung. A modest dose-proportionality response in the number of patients exhibiting expression is seen, but no dose-response relationship exists for the number of cells transduced. Duration of transgene expression has not been formally evaluated in patients because of the difficulty in obtaining samples and sampling error. Functional correction has been difficult to show. The results from the large, placebocontrolled, dose-rising study from the University of North Carolina showed a trend toward normalization of the baseline hyperpolarization when comparing the highest dose cohort with the lowest dose cohort.19 The Cornell trial also assessed the electrical potentials of nasal epithelium following a single administration of an adenoviral vector containing CFTR to one nares. In this unblinded study, baseline potential difference, amiloride response, and response to both low chloride and cAMP agonist were statistically different from the patients' untreated side ( P
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= 0.01, P = 0.02, P = 0.05, P = 0.05, respectively).l4 Overall, the safety profile of adenoviral vectors is quite good. In the first trial, at a total dose of 2 X lo9 pfu (plaque forming unit), a marked inflammatory response occurred, with fever, hypotension, and pulmonary infiltrates.8 This syndrome was attributed to cytokine release.23 The cytokine release syndrome was attributed, in part, to the high volume of instillate (20 mL) used. High volumes of instillates promote alveolarization of airway materials, including bacteria. High fevers after bronchoalveolar lavage are quite common, even in non-CF individuals. Both the humoral and cytotoxic responses to adenoviral vectors are significant barriers to successful gene transfer. Elaboration of neutralizing antibodies to adenoviral coat proteins, particularly in the pulmonary parenchyma, would prevent adhesion and subsequent internalization of the vector. To date, modest increases in serum-neutralizing antibodies have been reported in some individuals after bronchoscopic instillation but not in patients who received aerosol delivery or nasal instillation (J Wilson, personal communication, 1997).4, Host defense against de novo viral infection involves antigen processing and presen-
Table 1. COMPLETED CLINICAL TRIALS OF ADENOVIRAL VECTORS Vector
Lung
2
x lo9 pfu'
Nose Lung
2 2
x x
Ad5 AEl, A E3
Nose
2
x 1Olopfu
Ad2 AEl, AE4, E3 Ad5 AEl, AE3
Nose
5
Nose Lung (aerosol)
4 x lo8 pfu 5.4 x 108 pfu
National Institutes of Health/Cornell
Ad5 AEl, AE3
University of Pennsylvania
Ad5 AEl, AE3
University of North Carolina University of Iowa
pfu 10' pfu
Expression
+ in situ in 1 of 3 + basal PD, amil, cAMP + in situ in 1 of 8 +
RT-PCR in 5 of 12 in situ basal mean PD + / - cyclic adenosine monophosphate
Reference Crystal RG7 Hay J14 Wilson JM personal communication, 1997 Knowles MR19
-
+
Lyon Transgene S.A.
Highest Dose
Site
x lo7 pfu
+
RT-PCR in 4 of 6
+ immunohisto in 6 of 6 - RT-PCR in 6 of 6 + immuno in 2 of 6
Zabner J42 Bellon G4
'pfu = Plaque-forming units; PD = Potential difference. For the RT-PCR and immunohistochemistry expression data, any positive data at any time point are included. For nasal potential data, only statistically significant results are presented.
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tation via major histocompatibility (MHC) class I to T cells (CD8 ). Virally infected cells display parts of these foreign epitopes via MHC class I, a process that involves viral protein synthesis. Once these antigens are presented to stimulated T cells, infected cells are targeted for destruction by a cytotoxic T lymphocyte (CTL) response supported by yinterferon (IFN) elaboration. In animals, CTL response has been demonstrated against both the vector and the t r a n ~ g e n e .It~is ~ ,too ~ ~early to determine whether a CTL response has been generated against CFTR in the patients. Cytokine profiles have been highly variable and unpredictive of toxicity (J Wilson, personal communication, 1997). None of the studies has been powered sufficiently to examine effects on pulmonary function tests but no substantial declines in spirometry have been observed. At very high doses in the nose, unilateral nasal congestion, earache, and odynophagia were ob~erved.'~
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Hurdles to Development
The principal hurdles for development of adenoviral vectors are immunologic responses to viral and transgene proteins and limitations on infectability of differentiated respiratory epithelium. The viral protein "coat" contains receptor ligands for specific respiratory cell surface markers. Integrins-specifically the aVP5 subtype-appear to mediate viral attachment to specific respiratory cell types.12 This integrin is displayed on the lower airway surface epithelium but not in the nasal epithelium, explaining the difficulty in transducing the nasal epithelium. In vitro studies and animal work have repeatedly shown that it is relatively difficult to transduce ciliated airway epithelial cells and quite easy to transduce the basal cells after Adenoviral receptor expression may be greater on basal cells than on ciliated epithelial cells. The viral protein coat presents receptor ligands for viral penetration into infectable cells and provides antigen to the host immune system. Indeed, approximately 97% of all humans over 3 years of age have antibod-
ies against these adenoviral coat proteins. If these antibodies are neutralizing, then reinfection of the host cells will be difficult, if not impossible, because attachment will be prevented. Readministration (i.e., repeat dosing) of the same adenoviral serotype therefore may not be possible. Because the immunodominant epitopes are the viral coat proteins (late gene products), readministration of transgene using different adenoviral serotypes has been in~estigated.~, 22 The host response to viral infections is mediated principally by CTL-mediated destruction of cells that exhibit processed viral antigen. CTL-mediated attack on virally infected, transgene-bearing cells limits the duration of expression of the t r a n ~ g e n e 40 . ~Initiation ~, of a CTL response requires participation of CD8 + lymphocytes, antigen-presenting cells, and costimulatory molecules, a process supported by y-IFN. By manipulating antigen presentation (or y-IFN elaboration), it should be possible to decrease CTL attack and thereby decrease inflammation and prolong transgene expression. One approach to manipulating antigen presentation is to modify the viral genes that promote viral "escape" from antigen presentation. Recent research has demonstrated the importance of adenoviral protein E4 in modulating antigen pre~entation.~, 38 Another approach to diminishing CTL attack on virally infected cells is to interfere with costimulatory molecule presentation (CTLa B7 for example). Recent animal work has validated this approach to reducing CTL response to both the transgene and the viral proteins by reducing costimulatory molecular i n t e r a ~ t i o nTransient .~~ immunosuppression either by vectoring or by medicating may be important to the success of using adenoviral vectors. When the virion infects cells, viral proteins help direct the synthesis of the new transgene and viral proteins. The viral DNA remains episomally, however, and is lost when the cell replicates. For rapidly dividing cells, rapid loss of transgene therefore would be expected, also limiting transgene expression. The ongoing airway inflammation may make CF airways easier to transduce but the rapid turnover may limit the duration of transgene expression (Table ,).,I, 36
IS DNA DESTINY?
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Table 2. COMPARISON BETWEEN VECTORS CURRENTLY IN CLINICAL TRIALS FOR GENE THERAPY IN CYSTIC FIBROSIS ~
Vector
~~~
Advantages
Adenovirus
High titers, genome characterized, has internal markers
Adenoassociated virus
Host genome integration
Liposomes
No immunologic response
ADENOASSOCIATED VIRUS Promises
One approach to improving duration of transgene expression is to use adenoassociated virus (AAV) as a delivery vector. AAV is a much simpler and smaller virus that requires assistance from adenovirus to infect cells.31AAV has signal sequences that direct integration into a specific place in the host chromosomes. When the host cell replicates, the integrated transgene will also replicate and not be lost. Preclinical work on AAV (at doses up to 5 x 1O1O particles) in neonatal rabbits has shown RT-PCR expression of human CFTR at 3 weeks but not at 6 months after inoculation.ll,30
Clinical Trials
A human phase I trial was begun in 1996 and a total of 19 patients have been treated (T Flotte, personal communication, 1997). Both the nose and the lung were dosed. The contralateral nares served as a vehicle control. An interim analysis after 13 patients showed scant evidence of gene transfer by either potential difference or by DNA PCR and six additional patients were dosed at a higher level (up to lo6 infectious units or approximately lo8 to lo9 particles). No safety concerns arose during the trial. A complementary trial using AAV in the maxillary sinus in 12 patients was completed
Disadvantages
Clinical Implications
Poor transfection of differentiated cells, cytotoxic T lymphocyte and antibody response No markers, difficult purification to make sufficient quantity, low efficiency of transfection
May not be able to give repeat doses, may not be able to transfect correct cells No control over integration poses question of risk of oncogenesis, unable to compare results because of marker absence Impractical to make sufficient quantity
lneff icient, formulation inconsistencies
recently.37An unusual feature of this trial was performance of sinus potential difference, modeled after the technique of nasal potential difference. At doses over 5 X lo4 replication units, evidence of gene transfer was seen by DNA PCR. At lo5 replication units, a trend toward hyperpolarization at baseline was seen in the treated side. Sinus potential difference is a new technique that has not been validated in normal subjects or in disease controls, however, so it is difficult to interpret the physiologic data. Hurdles to Development
Because of its small size, insertion of very large therapeutic gene cassettes (such as CFTR) can decrease efficient packaging by permissive cell lines. To improve packaging of CFTR, the signal sequences that govern site-specific integration have been deleted. When those signal sequences are deleted, random insertion into host DNA can occur. Random integration may pose a concern about carcinogenicity of an AAV vector. Because the AAV backbone is so small, the incorporation of transgene-specific markers suitable for RTPCR amplification has been difficult. It therefore is impossible to compare the relative efficiencies of transduction of adenoviral vectors and AAV vectors using RT-PCR. Growth of AAV in permissive cell lines requires coinfection with adenovirus. Subsequent purification of large titers of AAV from adenovirus has been difficult. It is not clear how the
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dose of AAV should be expressed or how to compare the doses between AAV and adenovirus. For AAV vectors to succeed as delivery vehicles, control over integration sites and improvements in production need to be achieved.
CATIONIC LIPOSOMES Promises
More recently, cationic liposomes complexed with DNA have undergone testing for efficiency and safety of gene transfer. Cationic liposomes are highly positively charged lipophilic complexes that associate with the highly negatively charged DNA. By neutralizing the charge on DNA, more efficient penetration into cells would be predicted. The chief advantage to this nonviral vector system is the absence of an immunologic response to vector and transgene. Preclinical work in CF mutant and transgenic mice demonstrated that cationic liposomes could correct the cAMP defective chloride secretory defect in vivo without convincing evidence of histopathology2,l7 Clinical Trials
The first trial conducted in the United Kingdom evaluated the effects on nasal epithelium of a single administration of DCcholesterol/DOPE and CFTR cDNA (highest dose of DNA was 300 mg) in nine CF patients and six CF patients treated with DCcholesterol/DOPE alone.6* 25 No abnormal histopathology was observed. Nasal potential difference was measured serially and a 20% improvement in the low chloride response was observed at approximately the third day. No changes in basal potential difference or cAMP response were noted in this trial. The majority of patients had molecular evidence of gene transfer by either DNA PCR or RTPCR. Improvements in vectoring design have been incorporated into another trial, in which repeat nasal dosing was administered at monthly intervals for 3 months in 12 patients.
Multiple endpoints were sampled after each dose. DNA PCR was positive after the third dose in eight of the patients. In contrast, all samples were below sensitivity for in situ hybridization. By immunohistochemistry, 2% to 5% of ciliated cells were positive in six of nine analyzed patients. Interestingly, all of the positive immunohistochemistry samples were also positive for SPQ fluorescence. A positive SPQ fluorescence was scored as peak fluorescence of 33% of cells at lox magnification after examining between 50 and 200 ciliated cells. To treat a small area of the nose required 400 p,g of total DNA and 2.4 FM dose of cationic liposomes.”, 33 An aerosol trial using 42 mg of DNA and 229 mg of liposomes in 16 mL has been initiated.
Hurdles to Development
It is not clear how practical DNA-lipid complexes will be for commercial production to treat the lung. The pulmonary toxicity and metabolism of the DNA-lipid complexes, especially at these high doses, are unknown. To further cloud the issues, in vitro models have shown that DNA alone seems to have the same transfection efficiency as DNAliposome complexes, raising the obvious question about the utility of the c o m p l e ~ e s . ~ ~ Other disadvantages to liposome delivery are endosomal degradation of the DNAliposome complexes and lack of nuclear targeting of DNA. The manufacturing of DNAlipid complexes is difficult, with little unanimity about the proper ratios and the solution chemistry of the assembled complex. FUTURE DIRECTIONS
The achievements in the past year have included improvements in vectoring “immune escape” for adenoviral vectors, completion of AAV trials, and extension of the DNA liposome trials. Perhaps the most pressing research question for the future will be measurement of ionic content of airway surface liquid (from glands and from small airways). Two competing models for handling of ions in the respiratory tract depend upon this mea-
IS DNA DESTINY?
surement, as does the development of the airway defensin story. Progress toward gene therapy for CF has been slow but steady, and important insights into pulmonary biology have been gained by the effort. References 1. Alton EW, Geddes D Gene therapy for cystic fibrosis: Steady progress, should do well. Eur Respir J 10257259, 1997 2. Alton EW, Middleton F, Caplen P, et al: Noninvasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nat Genet 5:135-142, 1993 3. Armentano D, Zabner J, Sacks C, et al: Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J Virol 71:2408-2416, 1997 4. Bellon G, Michel-Calemard L, Thouvenot D, et al: Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: A phase I clinical trial. Hum Gene Ther 8:15-25, 1997 5. Berkner K Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 6:61&629, 1988 6. Caplen N, Alton E, Middleton P, et al: Liposomemediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1:3946,1995 7. Crystal RG, Mastrangeli A, Sanders A, et al: Evaluation of repeat administration of a replication deficient, recombinant adenovirus containing the normal cystic fibrosis transmembrane conductance regulator cDNA to the airways of individuals with cystic fibrosis. Hum Gene Ther 6:667-703, 1995 8. Crystal RG, McElvaney NG, Rosenfeld MA, et al: Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42-71, 1994 9. Engelhardt J, Yankaskas J, Emst S, et al: Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 2240-248, 1992 10. FitzSimmons S: Cystic Fibrosis Foundation Patient Registry Annual Data Report 1996. Bethesda, MD, 1997 11. Flotte T, Afione S, Conrad C, et al: Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adenoassociated virus vector. Proc Natl Acad Sci 90:10613-10617, 1993 12. Goldman M, Wilson JM: Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J Virol 69:5951-5958, 1995 13. Goldman M, Anderson G, Stolzenberg E, et al: Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88:553560, 1997 14. Hay JG, McElvaney NG, Herena J, et al: Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther 6:1487-1496, 1995 15. Horwitz M Adenoviridae and their replication. In Fields B (ed): Virology. New York, Raven Press, 1990, pp 1679-1721 16. Howard M, DuVall MD, Devor DC, et al: Epitope
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tagging permits cell surface detection of functional CETR. Am J Physiol 269:C1565-1576, 1995 17. Hyde S, Gill D, Higgins C, et al: Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 362250-255, 1993 18. Johnson LG, Boyles SE, Wilson J, et al: Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. J Clin Invest 95:1377-1382, 1995 19. Knowles MR, Hohneker KW, Zhou Z, et al: A controlled study of adenoviral-vector-mediatedgene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333:823-831, 1995 20. Knowles MR, Paradiso AM, Boucher R In vivo nasal potential difference: Techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum Gene Ther 6:445-455, 1995 21. Leigh M, Kylander J, Yankaskas J, et al: Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients. Am J Respir Cell Mol Biol 12:605412, 1995 22. Mastrangeli A, Harvey B, Yao J, et al: "Sero-switch adenovirus-mediated in vivo gene transfer: Circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype. Hum Gene Ther 779-87, 1996 23. McElvaney N, Crystal R IL-6 release and airway administration of human CFTR cDNA adenovirus vector. Nat Med 1:182-184, 1995 24. Pilewski JM, Latoche JD, Arcasoy S, et al: Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo. Am J Physiol 273:L25&L263, 1997 25. Porteous DJ, Dorin JR, McLachlan G, et a1 Evidence for safety and efficacy of DOTAP cationic lipsome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 4210-218, 1997 26. Rich D, Couture L, Cardoza L, et a1 Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum Gene Ther 4461476, 1993 27. Riordan JR, Rommens J, Kerem B, et al: Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:106&1073, 1989 28. Rommens J, Iannuzzi M, Kerem 8, et al: Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059-1065, 1989 29. Rosenfeld M, Collins FS: Gene therapy for cystic fibrosis. Chest 109:241-252, 1996 30. Rubinstein R, McVeigh U, Flotte T, et al: CFTR gene transduction in neonatal rabbits using an adeno-associated virus vector. Gene Ther 43384-392, 1997 31. Samulski R Adeno-associated virus: Integration at a specific chromosomal locus. Curr Opin Genet Dev 3:74-80, 1993 32. Scaria A, St. George J, Gregory R Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway. Gene Ther 4:611417, 1997 33. Southern KW, Hyde SC, Fitzjohn EM, et al: Repeated nasal administration of liposome-mediated CFTR gene transfer reagents: The clinical and immunologi-
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34. 35.
36.
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cal consequences. Pediatr Pulmonol Suppl 14:A209, 1997 Smith J, Travis S, Greenberg E, et al: Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229-236, 1996 Stem M, Munkonge FM, Caplen NJ, et al: Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects. Gene Ther 2:766774, 1995 Teramoto S, Johnson L, Huang W, et al: Effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells. Hum Gene Ther 6:1045-1053, 1995 Wagner J, Moran M, Messner A, et al: Efficient and persistent gene transfer of AAV-CFTR in the maxillary sinus of CF patients with antrostomies [abstr]. Pediatr Pulmonol Suppl 14, A216, 1997
38. Wilson J M Gene therapy for cystic fibrosis: Challenges and future directions. J Clin Invest 96:25472554, 1995 39. Yang Y, Li Q, Ertl H, et al: Cellular and humoral immune response to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 69:2004-2015, 1995 40. Yang Y, Nunes F, Berencsi K, et al: Cellular immunity to viral antigens limits El-deleted adenovimses for gene therapy. Proc Natl Acad Sci 91:4407-4411, 1994 41. Zabner J, Cheng SH, Meeker D: Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J Clin Invest 100:1529-1537, 1997 42. Zabner J, Couture LA, Gregory RJ, et al: Adenovirusmediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 75:207-216, 1993
Address reprint requests to Cynthia B. Robinson, MD SmithKline-Beechman Pharmaceuticals Presbyterian Hospital University of Pennsylvania Health System 51 N 39th Street Philadelphia, PA 19104
CYSTIC FIBROSIS
+ .OO
IS DNA DESTINY? A Cure for Cystic Fibrosis Cynthia B. Robinson, MD
CYSTIC FIBROSIS GENE THERAPY UPDATE
Time, for cystic fibrosis (CF) patients, has been forever divided into the dark years "before" and the bright promise of "after." The singular event that cleaved time in two was the discovery of the gene responsible for CF 28 Before the discovery of the gene, in 1989.27, only the natural history and the genetic nature of the disease were well characterized. After the discovery of the gene, the basic defect became understood and curing the disease became a tangible possibility.', 29 The resultant effort in gene therapy for CF has produced important new insights into lung biology that benefit the pulmonary community at large. This article updates the status of gene therapy for CF, highlights the remaining hurdles, and provides insights into lung biol-
ogy. GENERAL CONSIDERATIONS
For many reasons, CF remains an attractive target for gene therapy. CF is an autosomal recessive single gene defect. This means that
to correct the defect, one does not need to "silence" the existing genes and that provision of a single copy of the normal gene is sufficient. Furthermore, there must be only one gene principally responsible for the disease. Supplying a single normal copy of the gene to all cells of the lung would be a daunting task, but in vitro work has shown that only 7% to 10% of cells need to be corrected to overcome some of the ion transport abnormalities.ls Although the ciliated epithelial cells express cystic fibrosis transmembrane conductance regulator (CFTR), abundant expression is found in submucosal glands of the large airway^.^ It is not known which cell type is most important to target for gene therapy, but aerosol delivery of drug to the epithelium is more feasible. In all phase I trials, the principal outcome measure is safety assessment. Incorporation of pharmacodynamic endpoints, however, is desirable, even in phase I trials. A variety of techniques have been employed to demonstrate gene expression. The most basic demonstration of gene expression is to show evidence of transgene DNA by DNA amplification via polymerase chain reaction (PCR)
From the Department of Clinical Pharmacology and Adult Cystic Fibrosis Program, University of Pennsylvania; and SmithKline Beecham Pharmaceuticals, Presbyterian Hospital, University of Pennsylvania Health System, Philadelphia, Pennsylvania
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in harvested cells. DNA PCR cannot distinguish between exogenously administered DNA (i.e., the dose of DNA) from DNA that has been successfully incorporated into cells, however. A higher level of proof of successful transfection is reverse transcription PCR (RTPCR) from CFTR messenger RNA. In this situation, the only source of CFTR RNA is from the successfully incorporated transgene. In situ hybridization using the radiolabeled DNA (or RNA) antisense strand also requires uptake by the cells of the exogenously provided transgene. Demonstration of protein expression by immunohistochemistry is probably the strongest level of proof of successful gene transfer. Immunohistochemistry, however, is far less sensitive than any of the other aforementioned molecular biology techniques. The second pharmacodynamic endpoint used in several phase I trials for gene therapy in CF has been demonstration of CFTR function. Two distinct parameters of CFTR function have been described in respiratory epithelium-a baseline hyperpolarization caused by excessive sodium absorption and a lack of depolarization (by chloride secretion) in response to CAMP agonists.19 Restoration of the electrical potentials toward the normal response is becoming accepted as proof of concept. It is not clear, however, which electrical potential is the most important to correct-the baseline sodium hyperabsorption or the chloride secretory response to cyclic adenosine monophosphate (CAMP).Another ex vivo method of demonstrating intact CFTR function is to load harvested cells with a fluorescent dye-SPQ (6-methoxy-N-[3sulfopropyl]quinolinium).16~ 35 SPQ's signal is quenched in the proximity of anions like chloride. When chloride secretion is stimulated by CAMP, the SPQ signal is dequenched and increasing fluorescence is measured. Extent of CFTR correction can be ascertained from the magnitude of the change in fluorescence and the number of cells exhibiting this change. The newest measure of protein function is whether defective CF airway defensin function has been restored as a consequence of gene therapy. Airway defensins are primitive antimicrobial peptides that work against a variety of organisms. In high salt environ-
ments, however, the defensins are inoperative, suggesting that the CF airway milieu is hypert~nic.'~, 34 Although numerous measurements have been attempted, it is not clear whether CF airway surface fluid is hypertonic. The ionic content of airway surface fluid is susceptible to changes during measurement. Furthermore, it is likely that the ionic content of glandular secretions is different from the pericellular water layer, confusing the analysis. The ability of CF cells to resist bacterial colonization may be another demonstration of effective CFTR protein function. The clinical trials to date for CF gene therapy have addressed some difficult problems in drug development. At a minimum, these trials have sought to profile the safety and the presence of gene expression. ADENOVIRAL VECTORS
Promises Most of the early experience with gene therapy for CF has been with adenovirus vectors. Adenovirus has a natural tropism for respiratory epithelium and can be grown to high titers in permissive cell lines5,15, 26 Use of adenoviral vectors has been made possible by the ability to modify specific portions of the viral genome that govern viral replication and immune escape while preserving the ability to make intact virions. In the place of the deleted viral genes, an expression cassette comprising the transgene (CFTR) and its promoter is inserted into the viral backbone by recombination. The replication permissive cell lines package the intact virion with the new transgene. Clinical Trials
The first trial (National Institutes of Health/Cornell University) was initiated in April of 1993 and a total of eight CF patients were treated with an adenovirus 5 El, E3deleted vector delivered bronchoscopically into their lungs.8 To date, approximately 33 patients have been treated with adenoviral
IS DNA DESTINY?
vectors.1°An aerosol trial has been completed, as has a repeat-dosing regimen.4 Both the nose and the lung have been dosed. Details of the completed trials are shown in Table 1. In general, adenoviral vectors can achieve scant, patchy gene expression, as shown by RT-PCR, immunohistochemistry, and in situ hybridization in the nose and the lung. A modest dose-proportionality response in the number of patients exhibiting expression is seen, but no dose-response relationship exists for the number of cells transduced. Duration of transgene expression has not been formally evaluated in patients because of the difficulty in obtaining samples and sampling error. Functional correction has been difficult to show. The results from the large, placebocontrolled, dose-rising study from the University of North Carolina showed a trend toward normalization of the baseline hyperpolarization when comparing the highest dose cohort with the lowest dose cohort.19 The Cornell trial also assessed the electrical potentials of nasal epithelium following a single administration of an adenoviral vector containing CFTR to one nares. In this unblinded study, baseline potential difference, amiloride response, and response to both low chloride and cAMP agonist were statistically different from the patients' untreated side ( P
529
= 0.01, P = 0.02, P = 0.05, P = 0.05, respectively).l4 Overall, the safety profile of adenoviral vectors is quite good. In the first trial, at a total dose of 2 X lo9 pfu (plaque forming unit), a marked inflammatory response occurred, with fever, hypotension, and pulmonary infiltrates.8 This syndrome was attributed to cytokine release.23 The cytokine release syndrome was attributed, in part, to the high volume of instillate (20 mL) used. High volumes of instillates promote alveolarization of airway materials, including bacteria. High fevers after bronchoalveolar lavage are quite common, even in non-CF individuals. Both the humoral and cytotoxic responses to adenoviral vectors are significant barriers to successful gene transfer. Elaboration of neutralizing antibodies to adenoviral coat proteins, particularly in the pulmonary parenchyma, would prevent adhesion and subsequent internalization of the vector. To date, modest increases in serum-neutralizing antibodies have been reported in some individuals after bronchoscopic instillation but not in patients who received aerosol delivery or nasal instillation (J Wilson, personal communication, 1997).4, Host defense against de novo viral infection involves antigen processing and presen-
Table 1. COMPLETED CLINICAL TRIALS OF ADENOVIRAL VECTORS Vector
Lung
2
x lo9 pfu'
Nose Lung
2 2
x x
Ad5 AEl, A E3
Nose
2
x 1Olopfu
Ad2 AEl, AE4, E3 Ad5 AEl, AE3
Nose
5
Nose Lung (aerosol)
4 x lo8 pfu 5.4 x 108 pfu
National Institutes of Health/Cornell
Ad5 AEl, AE3
University of Pennsylvania
Ad5 AEl, AE3
University of North Carolina University of Iowa
pfu 10' pfu
Expression
+ in situ in 1 of 3 + basal PD, amil, cAMP + in situ in 1 of 8 +
RT-PCR in 5 of 12 in situ basal mean PD + / - cyclic adenosine monophosphate
Reference Crystal RG7 Hay J14 Wilson JM personal communication, 1997 Knowles MR19
-
+
Lyon Transgene S.A.
Highest Dose
Site
x lo7 pfu
+
RT-PCR in 4 of 6
+ immunohisto in 6 of 6 - RT-PCR in 6 of 6 + immuno in 2 of 6
Zabner J42 Bellon G4
'pfu = Plaque-forming units; PD = Potential difference. For the RT-PCR and immunohistochemistry expression data, any positive data at any time point are included. For nasal potential data, only statistically significant results are presented.
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ROBINSON
tation via major histocompatibility (MHC) class I to T cells (CD8 ). Virally infected cells display parts of these foreign epitopes via MHC class I, a process that involves viral protein synthesis. Once these antigens are presented to stimulated T cells, infected cells are targeted for destruction by a cytotoxic T lymphocyte (CTL) response supported by yinterferon (IFN) elaboration. In animals, CTL response has been demonstrated against both the vector and the t r a n ~ g e n e .It~is ~ ,too ~ ~early to determine whether a CTL response has been generated against CFTR in the patients. Cytokine profiles have been highly variable and unpredictive of toxicity (J Wilson, personal communication, 1997). None of the studies has been powered sufficiently to examine effects on pulmonary function tests but no substantial declines in spirometry have been observed. At very high doses in the nose, unilateral nasal congestion, earache, and odynophagia were ob~erved.'~
+
Hurdles to Development
The principal hurdles for development of adenoviral vectors are immunologic responses to viral and transgene proteins and limitations on infectability of differentiated respiratory epithelium. The viral protein "coat" contains receptor ligands for specific respiratory cell surface markers. Integrins-specifically the aVP5 subtype-appear to mediate viral attachment to specific respiratory cell types.12 This integrin is displayed on the lower airway surface epithelium but not in the nasal epithelium, explaining the difficulty in transducing the nasal epithelium. In vitro studies and animal work have repeatedly shown that it is relatively difficult to transduce ciliated airway epithelial cells and quite easy to transduce the basal cells after Adenoviral receptor expression may be greater on basal cells than on ciliated epithelial cells. The viral protein coat presents receptor ligands for viral penetration into infectable cells and provides antigen to the host immune system. Indeed, approximately 97% of all humans over 3 years of age have antibod-
ies against these adenoviral coat proteins. If these antibodies are neutralizing, then reinfection of the host cells will be difficult, if not impossible, because attachment will be prevented. Readministration (i.e., repeat dosing) of the same adenoviral serotype therefore may not be possible. Because the immunodominant epitopes are the viral coat proteins (late gene products), readministration of transgene using different adenoviral serotypes has been in~estigated.~, 22 The host response to viral infections is mediated principally by CTL-mediated destruction of cells that exhibit processed viral antigen. CTL-mediated attack on virally infected, transgene-bearing cells limits the duration of expression of the t r a n ~ g e n e 40 . ~Initiation ~, of a CTL response requires participation of CD8 + lymphocytes, antigen-presenting cells, and costimulatory molecules, a process supported by y-IFN. By manipulating antigen presentation (or y-IFN elaboration), it should be possible to decrease CTL attack and thereby decrease inflammation and prolong transgene expression. One approach to manipulating antigen presentation is to modify the viral genes that promote viral "escape" from antigen presentation. Recent research has demonstrated the importance of adenoviral protein E4 in modulating antigen pre~entation.~, 38 Another approach to diminishing CTL attack on virally infected cells is to interfere with costimulatory molecule presentation (CTLa B7 for example). Recent animal work has validated this approach to reducing CTL response to both the transgene and the viral proteins by reducing costimulatory molecular i n t e r a ~ t i o nTransient .~~ immunosuppression either by vectoring or by medicating may be important to the success of using adenoviral vectors. When the virion infects cells, viral proteins help direct the synthesis of the new transgene and viral proteins. The viral DNA remains episomally, however, and is lost when the cell replicates. For rapidly dividing cells, rapid loss of transgene therefore would be expected, also limiting transgene expression. The ongoing airway inflammation may make CF airways easier to transduce but the rapid turnover may limit the duration of transgene expression (Table ,).,I, 36
IS DNA DESTINY?
531
Table 2. COMPARISON BETWEEN VECTORS CURRENTLY IN CLINICAL TRIALS FOR GENE THERAPY IN CYSTIC FIBROSIS ~
Vector
~~~
Advantages
Adenovirus
High titers, genome characterized, has internal markers
Adenoassociated virus
Host genome integration
Liposomes
No immunologic response
ADENOASSOCIATED VIRUS Promises
One approach to improving duration of transgene expression is to use adenoassociated virus (AAV) as a delivery vector. AAV is a much simpler and smaller virus that requires assistance from adenovirus to infect cells.31AAV has signal sequences that direct integration into a specific place in the host chromosomes. When the host cell replicates, the integrated transgene will also replicate and not be lost. Preclinical work on AAV (at doses up to 5 x 1O1O particles) in neonatal rabbits has shown RT-PCR expression of human CFTR at 3 weeks but not at 6 months after inoculation.ll,30
Clinical Trials
A human phase I trial was begun in 1996 and a total of 19 patients have been treated (T Flotte, personal communication, 1997). Both the nose and the lung were dosed. The contralateral nares served as a vehicle control. An interim analysis after 13 patients showed scant evidence of gene transfer by either potential difference or by DNA PCR and six additional patients were dosed at a higher level (up to lo6 infectious units or approximately lo8 to lo9 particles). No safety concerns arose during the trial. A complementary trial using AAV in the maxillary sinus in 12 patients was completed
Disadvantages
Clinical Implications
Poor transfection of differentiated cells, cytotoxic T lymphocyte and antibody response No markers, difficult purification to make sufficient quantity, low efficiency of transfection
May not be able to give repeat doses, may not be able to transfect correct cells No control over integration poses question of risk of oncogenesis, unable to compare results because of marker absence Impractical to make sufficient quantity
lneff icient, formulation inconsistencies
recently.37An unusual feature of this trial was performance of sinus potential difference, modeled after the technique of nasal potential difference. At doses over 5 X lo4 replication units, evidence of gene transfer was seen by DNA PCR. At lo5 replication units, a trend toward hyperpolarization at baseline was seen in the treated side. Sinus potential difference is a new technique that has not been validated in normal subjects or in disease controls, however, so it is difficult to interpret the physiologic data. Hurdles to Development
Because of its small size, insertion of very large therapeutic gene cassettes (such as CFTR) can decrease efficient packaging by permissive cell lines. To improve packaging of CFTR, the signal sequences that govern site-specific integration have been deleted. When those signal sequences are deleted, random insertion into host DNA can occur. Random integration may pose a concern about carcinogenicity of an AAV vector. Because the AAV backbone is so small, the incorporation of transgene-specific markers suitable for RTPCR amplification has been difficult. It therefore is impossible to compare the relative efficiencies of transduction of adenoviral vectors and AAV vectors using RT-PCR. Growth of AAV in permissive cell lines requires coinfection with adenovirus. Subsequent purification of large titers of AAV from adenovirus has been difficult. It is not clear how the
532
ROBINSON
dose of AAV should be expressed or how to compare the doses between AAV and adenovirus. For AAV vectors to succeed as delivery vehicles, control over integration sites and improvements in production need to be achieved.
CATIONIC LIPOSOMES Promises
More recently, cationic liposomes complexed with DNA have undergone testing for efficiency and safety of gene transfer. Cationic liposomes are highly positively charged lipophilic complexes that associate with the highly negatively charged DNA. By neutralizing the charge on DNA, more efficient penetration into cells would be predicted. The chief advantage to this nonviral vector system is the absence of an immunologic response to vector and transgene. Preclinical work in CF mutant and transgenic mice demonstrated that cationic liposomes could correct the cAMP defective chloride secretory defect in vivo without convincing evidence of histopathology2,l7 Clinical Trials
The first trial conducted in the United Kingdom evaluated the effects on nasal epithelium of a single administration of DCcholesterol/DOPE and CFTR cDNA (highest dose of DNA was 300 mg) in nine CF patients and six CF patients treated with DCcholesterol/DOPE alone.6* 25 No abnormal histopathology was observed. Nasal potential difference was measured serially and a 20% improvement in the low chloride response was observed at approximately the third day. No changes in basal potential difference or cAMP response were noted in this trial. The majority of patients had molecular evidence of gene transfer by either DNA PCR or RTPCR. Improvements in vectoring design have been incorporated into another trial, in which repeat nasal dosing was administered at monthly intervals for 3 months in 12 patients.
Multiple endpoints were sampled after each dose. DNA PCR was positive after the third dose in eight of the patients. In contrast, all samples were below sensitivity for in situ hybridization. By immunohistochemistry, 2% to 5% of ciliated cells were positive in six of nine analyzed patients. Interestingly, all of the positive immunohistochemistry samples were also positive for SPQ fluorescence. A positive SPQ fluorescence was scored as peak fluorescence of 33% of cells at lox magnification after examining between 50 and 200 ciliated cells. To treat a small area of the nose required 400 p,g of total DNA and 2.4 FM dose of cationic liposomes.”, 33 An aerosol trial using 42 mg of DNA and 229 mg of liposomes in 16 mL has been initiated.
Hurdles to Development
It is not clear how practical DNA-lipid complexes will be for commercial production to treat the lung. The pulmonary toxicity and metabolism of the DNA-lipid complexes, especially at these high doses, are unknown. To further cloud the issues, in vitro models have shown that DNA alone seems to have the same transfection efficiency as DNAliposome complexes, raising the obvious question about the utility of the c o m p l e ~ e s . ~ ~ Other disadvantages to liposome delivery are endosomal degradation of the DNAliposome complexes and lack of nuclear targeting of DNA. The manufacturing of DNAlipid complexes is difficult, with little unanimity about the proper ratios and the solution chemistry of the assembled complex. FUTURE DIRECTIONS
The achievements in the past year have included improvements in vectoring “immune escape” for adenoviral vectors, completion of AAV trials, and extension of the DNA liposome trials. Perhaps the most pressing research question for the future will be measurement of ionic content of airway surface liquid (from glands and from small airways). Two competing models for handling of ions in the respiratory tract depend upon this mea-
IS DNA DESTINY?
surement, as does the development of the airway defensin story. Progress toward gene therapy for CF has been slow but steady, and important insights into pulmonary biology have been gained by the effort. References 1. Alton EW, Geddes D Gene therapy for cystic fibrosis: Steady progress, should do well. Eur Respir J 10257259, 1997 2. Alton EW, Middleton F, Caplen P, et al: Noninvasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice. Nat Genet 5:135-142, 1993 3. Armentano D, Zabner J, Sacks C, et al: Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J Virol 71:2408-2416, 1997 4. Bellon G, Michel-Calemard L, Thouvenot D, et al: Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: A phase I clinical trial. Hum Gene Ther 8:15-25, 1997 5. Berkner K Development of adenovirus vectors for the expression of heterologous genes. Biotechniques 6:61&629, 1988 6. Caplen N, Alton E, Middleton P, et al: Liposomemediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Nat Med 1:3946,1995 7. Crystal RG, Mastrangeli A, Sanders A, et al: Evaluation of repeat administration of a replication deficient, recombinant adenovirus containing the normal cystic fibrosis transmembrane conductance regulator cDNA to the airways of individuals with cystic fibrosis. Hum Gene Ther 6:667-703, 1995 8. Crystal RG, McElvaney NG, Rosenfeld MA, et al: Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8:42-71, 1994 9. Engelhardt J, Yankaskas J, Emst S, et al: Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat Genet 2240-248, 1992 10. FitzSimmons S: Cystic Fibrosis Foundation Patient Registry Annual Data Report 1996. Bethesda, MD, 1997 11. Flotte T, Afione S, Conrad C, et al: Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adenoassociated virus vector. Proc Natl Acad Sci 90:10613-10617, 1993 12. Goldman M, Wilson JM: Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J Virol 69:5951-5958, 1995 13. Goldman M, Anderson G, Stolzenberg E, et al: Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88:553560, 1997 14. Hay JG, McElvaney NG, Herena J, et al: Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther 6:1487-1496, 1995 15. Horwitz M Adenoviridae and their replication. In Fields B (ed): Virology. New York, Raven Press, 1990, pp 1679-1721 16. Howard M, DuVall MD, Devor DC, et al: Epitope
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tagging permits cell surface detection of functional CETR. Am J Physiol 269:C1565-1576, 1995 17. Hyde S, Gill D, Higgins C, et al: Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 362250-255, 1993 18. Johnson LG, Boyles SE, Wilson J, et al: Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. J Clin Invest 95:1377-1382, 1995 19. Knowles MR, Hohneker KW, Zhou Z, et al: A controlled study of adenoviral-vector-mediatedgene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333:823-831, 1995 20. Knowles MR, Paradiso AM, Boucher R In vivo nasal potential difference: Techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum Gene Ther 6:445-455, 1995 21. Leigh M, Kylander J, Yankaskas J, et al: Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients. Am J Respir Cell Mol Biol 12:605412, 1995 22. Mastrangeli A, Harvey B, Yao J, et al: "Sero-switch adenovirus-mediated in vivo gene transfer: Circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype. Hum Gene Ther 779-87, 1996 23. McElvaney N, Crystal R IL-6 release and airway administration of human CFTR cDNA adenovirus vector. Nat Med 1:182-184, 1995 24. Pilewski JM, Latoche JD, Arcasoy S, et al: Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo. Am J Physiol 273:L25&L263, 1997 25. Porteous DJ, Dorin JR, McLachlan G, et a1 Evidence for safety and efficacy of DOTAP cationic lipsome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 4210-218, 1997 26. Rich D, Couture L, Cardoza L, et a1 Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum Gene Ther 4461476, 1993 27. Riordan JR, Rommens J, Kerem B, et al: Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:106&1073, 1989 28. Rommens J, Iannuzzi M, Kerem 8, et al: Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059-1065, 1989 29. Rosenfeld M, Collins FS: Gene therapy for cystic fibrosis. Chest 109:241-252, 1996 30. Rubinstein R, McVeigh U, Flotte T, et al: CFTR gene transduction in neonatal rabbits using an adeno-associated virus vector. Gene Ther 43384-392, 1997 31. Samulski R Adeno-associated virus: Integration at a specific chromosomal locus. Curr Opin Genet Dev 3:74-80, 1993 32. Scaria A, St. George J, Gregory R Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway. Gene Ther 4:611417, 1997 33. Southern KW, Hyde SC, Fitzjohn EM, et al: Repeated nasal administration of liposome-mediated CFTR gene transfer reagents: The clinical and immunologi-
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38. Wilson J M Gene therapy for cystic fibrosis: Challenges and future directions. J Clin Invest 96:25472554, 1995 39. Yang Y, Li Q, Ertl H, et al: Cellular and humoral immune response to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 69:2004-2015, 1995 40. Yang Y, Nunes F, Berencsi K, et al: Cellular immunity to viral antigens limits El-deleted adenovimses for gene therapy. Proc Natl Acad Sci 91:4407-4411, 1994 41. Zabner J, Cheng SH, Meeker D: Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo. J Clin Invest 100:1529-1537, 1997 42. Zabner J, Couture LA, Gregory RJ, et al: Adenovirusmediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell 75:207-216, 1993
Address reprint requests to Cynthia B. Robinson, MD SmithKline-Beechman Pharmaceuticals Presbyterian Hospital University of Pennsylvania Health System 51 N 39th Street Philadelphia, PA 19104