Cystic Fibrosis Gene Therapy Trials and Tribulations
Authors: Pamela L. Zeitlin
Journal: Molecular Therapy (2000)

Gene therapy for cystic fibrosis has not progressed as rapidly or as successfully as initially projected, and the paper by Noone et al. in this issue of Molecular Therapy is the most recent example of the complexity of the problem (1Noone P.G. Hohneker K.W. Zhou Z. Johnson L.G. Foy C. Gipson C. Jones K. Noah T.L. Leigh M.W. Schwartzbach C. Efthimiou J. Pearlman R. Boucher R.C. Knowles M.R. Safety and biological efficacy of a lipid–CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis.Mol. Ther. 2000; 1: 105-114Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Noone and colleagues outline the progress of a carefully constructed nasal epithelial gene transfer study utilizing a cationic lipid delivery vehicle and high doses of plasmid CFTR cDNA in adult CF patients. Despite the promise of preclinical studies that established the optimal lipid/plasmid formulation in cell culture, confirmed the function of recombinant human CFTR protein after transfection, and assessed the safety and efficacy of single dosing to lungs of rhesus monkeys, the investigators were unable to detect recombinant CFTR mRNA in nasal epithelial biopsies or to detect CFTR-mediated chloride transport by nasal potential difference measurements. Previous studies of different cationic lipid formulations by other investigators (2Caplen N.J. Alton E.W. Middleton P.G. Dorin J.R. Stevenson B.J. Gao X. Durham S.R. Jeffery P.K. Hodson M.E. Coutelle C. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis [see comments].Nat. Med. 1995; 1: 39-46Crossref PubMed Scopus (709) Google Scholar, 3Porteous D.J. Dorin J.R. McLachlan G. Davidson-Smith H. Davidson H. Stevenson B.J. Carothers A.D. Wallace W.A. Moralee S. Hoenes C. Kallmeyer G. Michaelis U. Naujoks K. Ho L.P. Samways J.M. Imrie M. Greening A.P. Innes J.A. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.Gene Ther. 1997; 4: 210-218Crossref PubMed Scopus (289) Google Scholar, 4Zabner J. Cheng S.H. Meeker D. Launspach J. Balfour R. Perricone M.A. Morris J.E. Marshall J. Fasbender A. Smith A.E. Welsh M.J. Comparison of DNA–lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.J. Clin. Invest. 1997; 100: 1529-1537Crossref PubMed Scopus (150) Google Scholar, 5Alton E.W. Stern M. Farley R. Jaffe A. Chadwick S.L. Phillips J. Davies J. Smith S.N. Browning J. Davies M.G. Hodson M.E. Durham S.R. Li D. Jeffery P.K. Scallan M. Balfour R. Eastman S.J. Cheng S.H. Smith A.E. Meeker D. Geddes D.M. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: A double-blind placebo-controlled trial.Lancet. 1999; 353: 947-954Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar) have reported successful gene delivery by DNA-PCR and gene expression by RT-PCR, but only small changes in CFTR-mediated chloride conductance of 2–4 mV. Although all these studies were conducted with different formulations, study designs, and nasal potential difference protocols, the outcomes remain similar—transient and barely detectable changes in CFTR-mediated chloride transport and no clear evidence of a dose-dependent effect. Why is it so difficult to achieve CFTR gene transfer and CFTR-mediated ion transport? The nasal epithelium appears to be an ideal surrogate site for testing novel pulmonary gene transfer vectors or pharmaceuticals designed to restore airway epithelial ion transport balance. The inferior turbinate is a relatively safe site for testing agents that might trigger an unwanted inflammatory response. Regulated ion transport can be measured along the inferior turbinate relatively easily compared to the proximal airways by a technique called transepithelial potential difference. The nasal technique or nasal potential difference is a sensitive measure of sodium and chloride transport that can be reasonably specific for CFTR when the cAMP-dependent response is included in the protocol. It is not, however, completely specific, as alternative chloride conductances that respond to changes in intracellular calcium, cell volume, or extracellular pH, to name a few, could play a role whenever the chemical gradient for chloride exit is favored by a low chloride superfusate. The authors constructed their study so that one nares served as the placebo control for the treated nares in a double-blind, randomized design. The unexpected finding was a small increase in CFTR-mediated chloride conductance on the third baseline visit (day –1) suggesting that repeated daily measurements of nasal epithelial chloride transport induced changes of 2–3 mV. While the three predose measurements remained firmly within the CF range, the investigators point out that this magnitude of change is what has been reported as evidence of recombinant CFTR gene expression in other gene therapy trials. Are there comparable changes in CFTR function from pharmacological trials that might guide the interpretation of these results? What should we expect from sufficient CFTR gene expression in the nasal potential difference measurement? The magnitude of the change or repolarization of the potential difference after nasal superfusion of non-CF subjects with a chloride-free, amiloride-containing solution followed by the addition of a β-adrenergic agonist such as isoproterenol has been reported to be at least –1 mV with a mean response of –30 mV (6Middleton P.G. Geddes D.M. Alton E.W. Protocols for in vivo measurement of the ion transport defects in cystic fibrosis nasal epithelium [see comments].Eur. Respir. J. 1994; 7: 2050-2056Crossref PubMed Scopus (67) Google Scholar, 7Knowles M.R. Paradiso A.M. Boucher R.C. In vivo nasal potential difference: Techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis.Hum. Gene Ther. 1995; 6: 445-455Crossref PubMed Scopus (284) Google Scholar). CF patient volunteers ranged from –1 or –2 mV to about +10 mV with a mean of around +5 mV. This low chloride/isoproterenol response in CF subjects can be partially restored in specific situations. Pharmacologic correction of premature-translation termination stop codon mutants in CFTR (e.g., W1282X) by topical treatment with aminoglycosides to promote readthrough to a full-length CFTR has been reported to accomplish a change of 0 to –10 mV (8Wilschanski M. Famini C. Blau H. Rivlin J. Augarten A. Avital A. Kerem B. Kerem E. A pilot trial of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations.Am. J. Respir. Crit. Care Med. 1999; Google Scholar). Daily oral 4-phenylbutyrate therapy for 1 week to homozygous ΔF508 CFTR patients induced a –2- to –7-mV difference between pre- and postdrug values (9Rubenstein R.C. Zeitlin P.L. A pilot clinical trial of sodium 4-phenylbutyrate (Buphenyl) in delta F508-homozygous cystic fibrosis patients: Evidence of restoration of nasal epithelial CFTR function.Am. J. Respir. Crit. Care Med. 1998; 157: 484-490Crossref PubMed Scopus (317) Google Scholar). In the absence of any pharmacologic intervention, partially functional CFTR mutants such as those from Class IV (partial conduction) or V (reduced levels of CFTR) may be capable of generating small changes in potential difference. Several of the subjects in the paper by Noone et al. were compound heterozygotes with a potentially functional allele that might contribute variability to this measurement due to daily changes in nasal epithelial function or integrity. There are additional variables that might impact on detection of recombinant CFTR function by nasal potential difference measurement. Targeting gene transduction to as large a percentage of cells as possible, rather than a few highly expressing cells, may be critically important to the measurement of a change in transepithelial ion transport. There is a widely quoted study that at least 10% of epithelial cells in a monolayer cell culture need to be transduced with CFTR to favorably increase CFTR-mediated chloride conductance overall (10Johnson L.G. Olsen J.C. Sarkadi B. Moore K.L. Swanstrom R. Boucher R.C. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat. Genet. 1992; 2: 21-25Crossref PubMed Scopus (380) Google Scholar). Therefore, it may be necessary to distribute the vector evenly across the region to be tested. Gene delivery to the nasal epithelium should be relatively simple given the direct visual and anatomic access to the inferior turbinate. In this paper, the authors meticulously sprayed aliquots at intervals over several hours and had subjects avoid any positional changes that might disrupt prolonged contact of the vector with the epithelial surface. The DNA-PCR results confirm delivery of the vector to cells, but retention for only a short period of time. Growing attention by CF gene therapy investigators is being given to the issue of gene vector access to the pulmonary epithelium. For example, cell differentiation state and surface charge can affect cationic liposome gene delivery (11Matsui H. Johnson L.G. Randell S.H. Boucher R.C. Loss of binding and entry of liposome–DNA complexes decreases transfection efficiency in differentiated airway epithelial cells.J. Biol. Chem. 1997; 272: 1117-1126Crossref PubMed Scopus (178) Google Scholar). It has been demonstrated that viral receptors (utilized by adenoviral vectors) are found on the basolateral surface of epithelial cells beyond the tight junctions (12Parsons D.W. Grubb B.R. Johnson L.G. Boucher R.C. Enhanced in vivo airway gene transfer via transient modification of host barrier properties with a surface-active agent.Hum. Gene Ther. 1998; 9: 2661-2672Crossref PubMed Scopus (77) Google Scholar) and that CF sputum contains proteases and other factors that may interfere with viral vector entry (13Kitson C. Angel B. Judd D. Rothery S. Severs N.J. Dewar A. Huang L. Wadsworth S.C. Cheng S.H. Geddes D.M. Alton E.W. The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium.Gene Ther. 1999; 6: 534-546Crossref PubMed Scopus (124) Google Scholar). It is not known whether nasal secretions are a significant barrier for cationic liposome gene delivery. The low level of gene transfer efficiency and mRNA expression for the liposomal vector in this study was not unexpected (14Knoell D.L. Yiu I.M. Human gene therapy for hereditary diseases: A review of trials.Am. J. Health Syst. Pharm. 1998; 55: 899-904PubMed Google Scholar) and the investigators escalated to a relatively high dose (4 mg) in order to increase the likelihood of detecting a functional correction. No adverse local or systemic effects were seen. Had clear-cut evidence of functional epithelial CFTR been detected, it is likely to have been transient in nature, because the plasmid DNA vector would remain episomal. Similarly, the half-life of cell surface CFTR generated during adenoviral vector administration is transient and limited by the immune response. Although the half-life of nasal epithelial cells (15Ayers M.M. Jeffery P.K. Proliferation and differentiation in mammalian airway epithelium.Eur. Respir. J. 1988; 1: 58-80PubMed Google Scholar) is likely to be shortened in the event of chronic injury or inflammation, it is not known whether this would impact on a short-term gene therapy study. CF airway epithelial gene therapy still faces a number of obstacles—the epithelial barrier to gene delivery, chronic inflammation, and uncertainty over the surrogate end points. Nevertheless, the fundamental concepts remain valid and the solutions within reach. Nontraditional approaches may ultimately be required, and the safety of the patient volunteers must always be paramount.