Gene Therapy for Cystic Fibrosis: Lessons Learned and Paths Forward
Authors: Soon H. Choi, John F. Engelhardt
Journal: Molecular Therapy (2021)

Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in an anion channel known as the CF transmembrane conductance regulator (CFTR). Although CF is a multi-organ disease, the primary cause of patient mortality is chronic bacterial infections of the lung, and thus this organ has been the primary target of gene therapy. For 90% of patients, small-molecule drugs that correct the basic defects in the mutant CFTR channel (i.e., CFTR modulators) are an effective treatment option. However, CF patients harboring CFTR mutations that produce little or no protein will require gene therapy. The CF gene therapy effort began shortly after the CFTR gene was identified in 1989, with clinical trials by four groups targeting the nose and lung using first-generation recombinant adenovirus (Ad). Although gene replacement therapy appeared straightforward at the time, these attempts had to be terminated due to viral immune responses1Crystal R.G. McElvaney N.G. Rosenfeld M.A. Chu C.S. Mastrangeli A. Hay J.G. Brody S.L. Jaffe H.A. Eissa N.T. Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.Nat. Genet. 1994; 8: 42-51Crossref PubMed Scopus (779) Google Scholar and the discovery that the Ad receptor resided in the poorly accessible basolateral membrane of airway epithelia. The next major approach into the clinic was liposome-mediated plasmid delivery, and it was popular for the following decade. However, transfection from the apical membrane of the differentiated airway epithelium is inefficient. This approach was more recently revisited in a 2015 consortium clinical trial performed in the United Kingdom, but failure to achieve the primary clinical endpoint placed liposomes on the shelf once again. From 1998–2007, recombinant adeno-associated virus (rAAV) was evaluated as a CFTR delivery vector. At the time, the number of rAAV serotypes was limited and there were challenges to producing vector in quantities sufficient for treating the entire human lung. The Targeted Genetics Corporation led the effort testing a rAAV2-CFTR vector, performing several clinical studies in the maxillary sinus and lung.2Wagner J.A. Reynolds T. Moran M.L. Moss R.B. Wine J.J. Flotte T.R. Gardner P. Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus.Lancet. 1998; 351: 1702-1703Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,3Moss R.B. Milla C. Colombo J. Accurso F. Zeitlin P.L. Clancy J.P. Spencer L.T. Pilewski J. Waltz D.A. Dorkin H.L. et al.Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial.Hum. Gene Ther. 2007; 18: 726-732Crossref PubMed Scopus (203) Google Scholar Despite intensive efforts, the primary lung function endpoint in a lung repeat-dosing trial was not met and transgene-derived CFTR mRNA was absent in airway brushing.3Moss R.B. Milla C. Colombo J. Accurso F. Zeitlin P.L. Clancy J.P. Spencer L.T. Pilewski J. Waltz D.A. Dorkin H.L. et al.Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial.Hum. Gene Ther. 2007; 18: 726-732Crossref PubMed Scopus (203) Google Scholar Nevertheless, these studies were the first to demonstrate that delivery of aerosolized rAAV2 to the human CF lung is safe, and they set the stage for future studies by other companies. With CF gene therapies tarnished by 15 years of unsuccessful clinical studies, around the time of the 2008 stock market crash, corporations moved dollars into developing CFTR modulators—and clinical testing of CF gene therapies entered nearly a decade of silence. This hiatus was not necessarily a bad thing for the field, because CFTR modulators were a major breakthrough for many CF patients and there were critical tools and knowledge the CF gene therapy field needed to acquire to enable future success. Collectively, the early CF gene therapy attempts had taught the field several important lessons. First, development of an effective gene therapy would require a clear understanding of vector biology in the target cell type. In the context of the Targeted Genetics studies, subsequent research on the intracellular trafficking of rAAV2 in airway epithelial cells revealed that this virus (and most other serotypes) encounters a post-entry block in transport to the nucleus4Duan D. Yue Y. Yan Z. Yang J. Engelhardt J.F. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.J. Clin. Invest. 2000; 105: 1573-1587Crossref PubMed Scopus (298) Google Scholar and that this block could be overcome by enhancing capsid ubiquitination with proteasome inhibitors.5Yan Z. Zak R. Luxton G.W. Ritchie T.C. Bantel-Schaal U. Engelhardt J.F. Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors.J. Virol. 2002; 76: 2043-2053Crossref PubMed Scopus (172) Google Scholar Second, preclinical models used to evaluate the performance of viral vectors can be misleading. For example, preclinical studies in rhesus macaque had demonstrated that rAAV2 can efficiently transduce the airways in this species, but subsequent in vitro studies revealed that transduction of airway epithelium in this species is 100-fold more effective than in humans, presumably due to differences in the receptors/co-receptors on the apical surfaces.6Liu X. Luo M. Trygg C. Yan Z. Lei-Butters D.C. Smith C.I. Fischer A.C. Munson K. Guggino W.B. Bunnell B.A. Engelhardt J.F. Biological Differences in rAAV Transduction of Airway Epithelia in Humans and in Old World Non-human Primates.Mol. Ther. 2007; 15: 2114-2123Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Third, it became clear that an animal model recapitulating the human CF phenotype was needed both for testing gene therapies and identifying the pathophysiologically relevant target cell types. The past decade has seen tremendous advances in CF research and vector development, paving the way for gene therapy to reenter the clinic soon. The development of larger CF animal models, which are clarifying the mechanisms underlying lung disease pathophysiology and defining the cellular targets for gene therapy, has been critical to these advances. For two decades, CF mice had been the only animal model available in which to develop therapeutics, but they did not spontaneously develop lung disease because their lung cell biology differs from that of humans.7Keiser N.W. Engelhardt J.F. New animal models of cystic fibrosis: what are they teaching us?.Curr. Opin. Pulm. Med. 2011; 17: 478-483Crossref PubMed Scopus (84) Google Scholar More recently, a Noah's Ark of new CF animal models was developed, including pigs, ferrets, sheep, rabbits, and rats. Among these, the ferret and pig models have been studied extensively and reproduce the multiorgan CF disease phenotype seen in humans, including the lethal bacterial colonization of the lung.8Stoltz D.A. Meyerholz D.K. Pezzulo A.A. Ramachandran S. Rogan M.P. Davis G.J. Hanfland R.A. Wohlford-Lenane C. Dohrn C.L. Bartlett J.A. et al.Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth.Sci. Transl. Med. 2010; 2: 29ra31Crossref PubMed Scopus (357) Google Scholar,9Sun X. Olivier A.K. Liang B. Yi Y. Sui H. Evans T.I. Zhang Y. Zhou W. Tyler S.R. Fisher J.T. et al.Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets.Am. J. Respir. Cell Mol. Biol. 2014; 50: 502-512Crossref PubMed Scopus (72) Google Scholar These newer CF models have illuminated aspects of lung pathophysiology that are highly relevant to gene therapies for the CF lung.10Tang Y. Yan Z. Engelhardt J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.Hum. Gene Ther. 2020; 31: 524-537Crossref PubMed Scopus (6) Google Scholar For example, they revealed that impaired secretions from CFTR-rich airway submucosal glands (SMGs) contribute to CF airway defects in mucociliary clearance and bacterial killing. Studies in CF ferrets also demonstrated that defective innate immunity and a mucoinflammatory state are distinct pathophysiologic disease-causing events in the CF lung.11Rosen B.H. Evans T.I.A. Moll S.R. Gray J.S. Liang B. Sun X. Zhang Y. Jensen-Cody C.W. Swatek A.M. Zhou W. et al.Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.Am. J. Respir. Crit. Care Med. 2018; 197: 1308-1318Crossref PubMed Scopus (68) Google Scholar Studies in CF pigs revealed that CFTR delivery using two vector systems, rAAV and lentivirus, can restore key airway functions of CFTR, including surface liquid pH, chloride transport, and bacterial killing.12Yan Z. McCray Jr., P.B. Engelhardt J.F. Advances in gene therapy for cystic fibrosis lung disease.Hum. Mol. Genet. 2019; 28: R88-R94Crossref PubMed Scopus (28) Google Scholar A potentially important question that remains includes the identification of airway cell types capable of reversing disease progression once CFTR is expressed.10Tang Y. Yan Z. Engelhardt J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.Hum. Gene Ther. 2020; 31: 524-537Crossref PubMed Scopus (6) Google Scholar CFTR is expressed at widely divergent levels in a handful of cell types in the lung, and how each contributes to the coordination of mucociliary clearance and innate immunity remains to be determined. New single-cell transcriptomic technologies (single cell RNA sequencing [scRNA-seq]) have aided in defining the cell types that express CFTR as well as their frequencies in the lung.10Tang Y. Yan Z. Engelhardt J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.Hum. Gene Ther. 2020; 31: 524-537Crossref PubMed Scopus (6) Google Scholar For example, although ciliated cells were long considered a good target for CF gene therapy, scRNA-seq demonstrated that CFTR expression is generally undetectable in these cells, but abundant in a newly identified cell type called the pulmonary ionocyte, and moderate in secretory cells and a subset of basal cells. Thus, it remains unclear whether ectopic expression of CFTR in ciliated cells can prevent or reverse disease in the CF lung. It also remains unclear whether pulmonary ionocytes, which express ∼50% of the CFTR in the proximal airways but constitute only 1% of cells in the epithelium, are a required target for CF gene therapy.10Tang Y. Yan Z. Engelhardt J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.Hum. Gene Ther. 2020; 31: 524-537Crossref PubMed Scopus (6) Google Scholar In vitro models of polarized CF airway epithelium suggest that reconstituting the precise cellular expression patterns of CFTR in the airway may not be required for disease complementation. Furthermore, expression of CFTR in only 6%–10% of airway epithelial cells can give rise to normal chloride current. However, given that CFTR cooperates with other channels to move fluid and ions across an epithelium, some of its in vivo functions that have yet to be discovered might be cell-autonomous and require gene-editing approaches to reconstitute endogenous CFTR expression patterns.10Tang Y. Yan Z. Engelhardt J.F. Viral Vectors, Animal Models, and Cellular Targets for Gene Therapy of Cystic Fibrosis Lung Disease.Hum. Gene Ther. 2020; 31: 524-537Crossref PubMed Scopus (6) Google Scholar New methods of genetic engineering in ferrets13Yu M. Sun X. Tyler S.R. Liang B. Swatek A.M. Lynch T.J. He N. Yuan F. Feng Z. Rotti P.G. et al.Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus.Sci. Rep. 2019; 9: 1971Crossref PubMed Scopus (8) Google Scholar that enable conditional knockout and knockin of CFTR in specific cell types are expected to provide definitive answers regarding appropriate cellular targets for CF lung gene therapy. Fortunately for CF patients, and to the credit of the Cystic Fibrosis Foundation campaign to "leave no patient behind," corporate and academic interest in developing gene-replacement and gene-editing therapies for the CF lung is currently considerable. Vector systems being advanced for CF lung gene therapy include rAAV, lentivirus, helper-dependent Ad (HDAd), and nanoparticle mRNA. Translate Bio is the most recent company to enter the CF clinic and the first to test a CFTR mRNA gene delivery approach in the lung. Another vector with potential includes a chimeric vector composed of human bocavirus type-1 (HBoV1) capsid and AAV2 genomes. Each of these approaches has its advantages and disadvantages. rAAV has been extensively tested in clinical trials but lacks a payload sufficient for delivery of the intact CFTR cDNA with a strong promoter like that of HBoV1. However, the development of a CFTR minigene and a synthetic enhancer/promoter14Yan Z. Sun X. Feng Z. Li G. Fisher J.T. Stewart Z.A. Engelhardt J.F. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.Hum. Gene Ther. 2015; 26: 334-346Crossref PubMed Scopus (33) Google Scholar has positioned rAAV vector systems for clinical studies. Furthermore, the development of AAV evolution technologies has provided new paths for creating vectors with a high tropism for the airway cell types that must be targeted to complement CFTR function.15Excoffon K.J. Koerber J.T. Dickey D.D. Murtha M. Keshavjee S. Kaspar B.K. Zabner J. Schaffer D.V. Directed evolution of adeno-associated virus to an infectious respiratory virus.Proc. Natl. Acad. Sci. USA. 2009; 106: 3865-3870Crossref PubMed Scopus (112) Google Scholar In this case, selecting the most effective species for capsid evolution will be critical, and cross-species panning will likely be required to avoid the generation of species-specific vectors. Viral approaches must also tackle the immunologic issues that accompany repeated dosing, which will likely be required for non-integrated vectors like rAAV and HDAd. Whereas CFTR mRNA delivery might circumvent immunologic issues, the need for frequent dosing to achieve durable CFTR expression will come with other costs. CF gene-replacement strategies will likely be the first clinical successes and will enable the application of vector systems for gene editing with capabilities of correcting cell-autonomous functions of CFTR within a complex epithelium. CF is a complex disease and thus developing gene therapies for it will involve additional challenges and setbacks. However, many companies and academic laboratories have contributed significant resources and talent to tackling these challenges and ensuring success. Finally, as was the case for CFTR modulators, when genetic therapies for CF patients emerge, they will define a path for the treatment of other complex devastating diseases.