CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes
Benjamin Steines, … , David V. Schaffer, Joseph Zabner
Benjamin Steines, … , David V. Schaffer, Joseph Zabner
Published September 8, 2016
Citation Information: JCI Insight. 2016;1(14):e88728. https://doi.org/10.1172/jci.insight.88728.
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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 4

Surface-exposed mutations affect pig airway epithelial transduction.

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Surface-exposed mutations affect pig airway epithelial transduction. (A)...
(A) Diagram highlighting capsid mutations found in AAV2 or AAV2H22. Site-directed mutagenesis was used to introduce mutations to the AAV2 capsid (top) or to revert AAV2H22 to WT (AAV2) capsid sequence (bottom). (B and C) Effect of surface-exposed mutations on GFP transduction efficiency. Residues L598 and I708 were sufficient to augment AAV2 transduction efficiency (B) but were not required for AAV2H22 transduction (C). (D and E) Effect of non–surface-exposed mutations on GFP transduction efficiency. Residues A67 and V648, but not G207, in AAV2 were sufficient to augment GFP transduction (D). However, none were required for AAV2H22 transduction (E). n = 12; *P < 0.01 vs. AAV2 (B and D) or AAV2H22 (C and E). *P < 0.01, Mann-Whitney nonparametric t test.