CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes
Benjamin Steines, David D. Dickey, Jamie Bergen, Katherine J.D.A. Excoffon, John R. Weinstein, Xiaopeng Li, Ziying Yan, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Linda S. Powers, Nicholas D. Gansemer, Lynda S. Ostedgaard, John F. Engelhardt, David A. Stoltz, Michael J. Welsh, Patrick L. Sinn, David V. Schaffer, Joseph Zabner
Benjamin Steines, David D. Dickey, Jamie Bergen, Katherine J.D.A. Excoffon, John R. Weinstein, Xiaopeng Li, Ziying Yan, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Linda S. Powers, Nicholas D. Gansemer, Lynda S. Ostedgaard, John F. Engelhardt, David A. Stoltz, Michael J. Welsh, Patrick L. Sinn, David V. Schaffer, Joseph Zabner
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Research Article Pulmonology

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

Abstract

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl– transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Authors

Benjamin Steines, David D. Dickey, Jamie Bergen, Katherine J.D.A. Excoffon, John R. Weinstein, Xiaopeng Li, Ziying Yan, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Linda S. Powers, Nicholas D. Gansemer, Lynda S. Ostedgaard, John F. Engelhardt, David A. Stoltz, Michael J. Welsh, Patrick L. Sinn, David V. Schaffer, Joseph Zabner

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Figure 3

AAV2H22 is less reliant than AAV2 on heparan sulfate proteoglycan (HSPG) as a receptor in CHO and HeLa cells.

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AAV2H22 is less reliant than AAV2 on heparan sulfate proteoglycan (HSPG)...
(A) The parental cell line CHO-K1, which expresses HSPG, and mutant cell line pgsD-677, which does not express HSPG, were transduced with AAV2-GFP, AAV5-GFP, or AAV2H22-GFP, and the number of GFP+ cells was determined. Data were quantified as a percentage of GFP+ CHO-K1 cells. n = 6; *P < 0.01 vs. AAV5 *P < 0.01, Mann-Whitney nonparametric t test. (B) The effect of heparinase was determined in HeLa cells treated with heparinase I or heparinase III and inoculated with AAV5, AAV2H22, or AAV2. n = 6; *P < 0.01 vs. untreated *P < 0.01, Mann-Whitney nonparametric t test. (C) AAV2, AAV5, or AAV2H22 were incubated with increasing concentrations of heparin for 30 minutes at 37°C, followed by transduction on HeLa cells. The number of GFP+ cells was determined and quantified as the % change relative to untreated. n = 6; *P < 0.01 vs. untreated *P < 0.01, Mann-Whitney nonparametric t test.