Efficient Gene Delivery to Pig Airway Epithelia and Submucosal Glands Using Helper-Dependent Adenoviral Vectors

Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR). Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy. Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR). Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy. IntroductionGene therapy has been shown to offer clinical benefits to patients with diseases caused by genetic mutations such as Lebers congenital amaurosis, human X-linked retinitis pigmentosa, and adenosine deaminase-deficient severe combined immunodeficiency.1Cideciyan AV Hauswirth WW Aleman TS Kaushal S Schwartz SB Boye SL et al.Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year.Hum Gene Ther. 2009; 20: 999-1004Crossref PubMed Scopus (289) Google Scholar,2Bainbridge JW Smith AJ Barker SS Robbie S Henderson R Balaggan K et al.Effect of gene therapy on visual function in Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2231-2239Crossref PubMed Scopus (1594) Google Scholar,3Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (802) Google Scholar,4Cideciyan AV Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy.Prog Retin Eye Res. 2010; 29: 398-427Crossref PubMed Scopus (181) Google Scholar,5Beltran WA Cideciyan AV Lewin AS Iwabe S Khanna H Sumaroka A et al.Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.Proc Natl Acad Sci USA. 2012; 109: 2132-2137Crossref PubMed Scopus (189) Google Scholar However, efficient gene transfer to airway epithelial cells of cystic fibrosis (CF) patients has not been achieved. CF is the most common life-threatening autosomal recessive disease, caused by mutations in an epithelial chloride channel encoded by the CF transmembrane conductance regulator (CFTR) gene.6Riordan JR Rommens JM Kerem B Alon N Rozmahel R Grzelczak Z et al.Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5858) Google Scholar,7Rommens JM Iannuzzi MC Kerem B Drumm ML Melmer G Dean M et al.Identification of the cystic fibrosis gene: chromosome walking and jumping.Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2494) Google Scholar,8Davis PB Drumm M Konstan MW Cystic fibrosis.Am J Respir Crit Care Med. 1996; 154: 1229-1256Crossref PubMed Scopus (826) Google Scholar CFTR is a membrane protein expressed on the surface of multiple epithelial and submucosal of the and it as a chloride The of CF patients the genetic that a at the of the and the of and CFTR to the apical membrane to of chloride of CFTR and function in the for gene therapy of cystic J PubMed Scopus Google N A JR et the of cystic fibrosis transmembrane conductance regulator in the of on PubMed Google Scholar CFTR multiple lung is the of and in CF gene therapy strategies for CF on the of CFTR to airway epithelial cells and submucosal animal such as CF been for gene transfer and studies with to the clinical of gene delivery in CF patients due to in lung and anatomic between and of large animal for gene therapy strategies in human clinical as large animal in been for lung of lung and of et of the CFTR gene a model of cystic fibrosis in 2008; PubMed Scopus Google AS the to the the pig as a model of cystic fibrosis lung J 2008; PubMed Scopus Google Z J M R K and between human and PubMed Scopus Google Scholar of lung with and the pig and human CFTR in both the and the A of the cystic fibrosis transmembrane conductance regulator Natl Acad Sci USA. PubMed Scopus Google Scholar the of pig CFTR to with CFTR in and submucosal PB et lung as a model for cystic fibrosis.Am J 2008; PubMed Scopus Google of the cystic fibrosis transmembrane conductance PubMed Scopus Google S L N and of similarities to and human 2010; PubMed Scopus Google L Z A CFTR in and by airway submucosal J Google Scholar The of CF in pigs also to in in the airway and of in the S et fibrosis pigs lung and at Med. 2010; PubMed Scopus Google Scholar important of the pig model is to the of lung diseases and also to CF gene adenoviral vectors been in animal and clinical the clinical for CF M PB in and for cystic fibrosis lung Ther. 2012; 20: PubMed Scopus Google U G L A et of in a Ther. PubMed Scopus Google Scholar with for airway cells can efficiently both and cells. Because vectors due to the of a of helper-dependent was with of the not a also the vectors in systemic and the A K L of genetic in with a of helper-dependent adenoviral Natl Acad Sci USA. 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The human CFTR protein was expressed in pig lung epithelial cells at apical and the surface of submucosal cells, to human CFTR protein important insights the of clinical strategies for CF lung gene therapy in patients and for studies with the CF pig and pigs were for this of pigs was with utilized and in with the of by the for and the for the Care of by the of The Care of the were with with and with and for the of the The airway was by with a vectors and delivery to pig and of the vectors LacZ or hCFTR in this were G B J and airway helper-dependent adenoviral vectors with a Ther. PubMed Scopus Google U B G et of lung by a helper-dependent adenoviral in airway Natl Acad Sci USA. PubMed Scopus Google Scholar of was a an H K et delivery of an helper-dependent to lung an Gene Med. 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PubMed Scopus Google Scholar The for hCFTR were as and the hCFTR that and The and were for pig CFTR was in pig lung and pig were in and were a of were and were in for at in in for with of in for and for with CFTR CFTR and as well as procedure was by or cells cells in the was into the or lung in were on cells were for were by in was pig lung and cells. gene products were by was to as an The for gene in was to were as was with of hCFTR protein with and in pig and in of in pig lung The of for IntroductionGene therapy has been shown to offer clinical benefits to patients with diseases caused by genetic mutations such as Lebers congenital amaurosis, human X-linked retinitis pigmentosa, and adenosine deaminase-deficient severe combined immunodeficiency.1Cideciyan AV Hauswirth WW Aleman TS Kaushal S Schwartz SB Boye SL et al.Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year.Hum Gene Ther. 2009; 20: 999-1004Crossref PubMed Scopus (289) Google Scholar,2Bainbridge JW Smith AJ Barker SS Robbie S Henderson R Balaggan K et al.Effect of gene therapy on visual function in Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2231-2239Crossref PubMed Scopus (1594) Google Scholar,3Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (802) Google Scholar,4Cideciyan AV Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy.Prog Retin Eye Res. 2010; 29: 398-427Crossref PubMed Scopus (181) Google Scholar,5Beltran WA Cideciyan AV Lewin AS Iwabe S Khanna H Sumaroka A et al.Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.Proc Natl Acad Sci USA. 2012; 109: 2132-2137Crossref PubMed Scopus (189) Google Scholar However, efficient gene transfer to airway epithelial cells of cystic fibrosis (CF) patients has not been achieved. CF is the most common life-threatening autosomal recessive disease, caused by mutations in an epithelial chloride channel encoded by the CF transmembrane conductance regulator (CFTR) gene.6Riordan JR Rommens JM Kerem B Alon N Rozmahel R Grzelczak Z et al.Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5858) Google Scholar,7Rommens JM Iannuzzi MC Kerem B Drumm ML Melmer G Dean M et al.Identification of the cystic fibrosis gene: chromosome walking and jumping.Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2494) Google Scholar,8Davis PB Drumm M Konstan MW Cystic fibrosis.Am J Respir Crit Care Med. 1996; 154: 1229-1256Crossref PubMed Scopus (826) Google Scholar CFTR is a membrane protein expressed on the surface of multiple epithelial and submucosal of the and it as a chloride The of CF patients the genetic that a at the of the and the of and CFTR to the apical membrane to of chloride of CFTR and function in the for gene therapy of cystic J PubMed Scopus Google N A JR et the of cystic fibrosis transmembrane conductance regulator in the of on PubMed Google Scholar CFTR multiple lung is the of and in CF gene therapy strategies for CF on the of CFTR to airway epithelial cells and submucosal animal such as CF been for gene transfer and studies with to the clinical of gene delivery in CF patients due to in lung and anatomic between and of large animal for gene therapy strategies in human clinical as large animal in been for lung of lung and of et of the CFTR gene a model of cystic fibrosis in 2008; PubMed Scopus Google AS the to the the pig as a model of cystic fibrosis lung J 2008; PubMed Scopus Google Z J M R K and between human and PubMed Scopus Google Scholar of lung with and the pig and human CFTR in both the and the A of the cystic fibrosis transmembrane conductance regulator Natl Acad Sci USA. PubMed Scopus Google Scholar the of pig CFTR to with CFTR in and submucosal PB et lung as a model for cystic fibrosis.Am J 2008; PubMed Scopus Google of the cystic fibrosis transmembrane conductance PubMed Scopus Google S L N and of similarities to and human 2010; PubMed Scopus Google L Z A CFTR in and by airway submucosal J Google Scholar The of CF in pigs also to in in the airway and of in the S et fibrosis pigs lung and at Med. 2010; PubMed Scopus Google Scholar important of the pig model is to the of lung diseases and also to CF gene adenoviral vectors been in animal and clinical the clinical for CF M PB in and for cystic fibrosis lung Ther. 2012; 20: PubMed Scopus Google U G L A et of in a Ther. PubMed Scopus Google Scholar with for airway cells can efficiently both and cells. Because vectors due to the of a of helper-dependent was with of the not a also the vectors in systemic and the A K L of genetic in with a of helper-dependent adenoviral Natl Acad Sci USA. PubMed Scopus Google H N et with of vectors not with a helper-dependent Med. Google G B J and airway helper-dependent adenoviral vectors with a Ther. PubMed Scopus Google Scholar Because of the large gene carrying vectors can be to multiple with an epithelial vectors been shown to both and airway epithelial cells of and J and strategies for cystic fibrosis lung gene Ther. PubMed Scopus Google R B M et transgene for cystic fibrosis gene Ther. PubMed Scopus Google H K et delivery of an helper-dependent to lung an Gene Med. PubMed Scopus Google U B G et of lung by a helper-dependent adenoviral in airway Natl Acad Sci USA. PubMed Scopus Google H J et of helper-dependent adenoviral vectors to airway Ther. PubMed Scopus Google N R J et delivery of helper-dependent adenoviral into results in efficiency with Ther. Google Scholar human CFTR gene was shown to be localized in the in CFTR and the of to lung U B G et of lung by a helper-dependent adenoviral in airway Natl Acad Sci USA. PubMed Scopus Google for this was to a to efficiently human CFTR gene to pig efficient gene is such as and can be The and of pig and human it to lung gene delivery by to the and safety of gene transfer this we pigs as a large animal model for aerosolized airway gene transfer to the of to the and of and to the and systemic toxicity to the the we that human CFTR protein was expressed at the apical of pig airway epithelial cells and submucosal without systemic be for developing a and preclinical model of CFTR gene transfer with vectors that for in clinical

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