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 1

Selection of AAV2H22 with tropism for pig airway epithelia.

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Selection of AAV2H22 with tropism for pig airway epithelia. (A) After 3 ...
(A) After 3 rounds of in vitro selection to enrich for adeno-associated virus (AAV) with tropism for pig airway, an in vivo selection strategy was taken. Plasmids encoding an AAV viral capsid library were transfected into 293 cells, along with adenovirus helper plasmids. The viral library was aerosolized into the pig trachea, and 3 days later, the lungs were harvested and airway cells were isolated and infected with WT adenovirus (WT Ad). The capsid sequences for the successful AAVs were subcloned, and a new enriched viral library was made for each round. (B) Enrichment was monitored for in vivo rounds 4, 5, and 6 by assessing the number of viral genomes per cell after WT Ad amplification. (C) Pig airway epithelia were transduced with AAV2H22 that expressed GFP (blue line). Expression was monitored over time as GFP+ cells per high-power field (HPF, 20×). n = 6; *P < 0.01 vs. AAV2 (green line), Mann-Whitney nonparametric t test. (D) Comparison of transduction efficiency on large and small pig airway epithelia. AAV2H22 (blue) outperformed AAV1 (orange) and AAV6 (green). GFP+ cells per low power field (LPF, 10×). n = 3 epithelia from 3 different pigs, *P < 0.01 vs. AAV1 and AAV6, Mann-Whitney nonparametric t test. (E) AAV2H22-CFTRΔR–mediated correction of CF pig epithelia. CFTR-null epithelia lack CFTR activity stimulation (ΔIsccAMP) by cAMP levels elevated via forskolin (1 × 10−5 M) and IBMX (1 × 10−4 M). AAV2H22-CFTRΔR–corrected cAMP-regulated chloride current as compared with AAV2H22-GFP. n = 6; *P < 0.01, Mann-Whitney nonparametric t test.