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 5

AAV2H22-CFTRΔR transduces pig airway epithelia in vivo.

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AAV2H22-CFTRΔR transduces pig airway epithelia in vivo. (A–G) Gut-correc...
(A–G) Gut-corrected CFTR-null pigs (1 week old) were transduced with 1.96 × 1012 VG of AAV2H22-CFTRΔR. Two weeks later, pigs were studied and compared with age-matched WT and nontreated CFTR-null pigs. (A–D) Confocal images of CFTR expression. (A) WT. (B) CFTR-null control. (C) CFTR-null transduced with AAV2H22-CFTRΔR. (D) Confocal image from C superimposed on differential interference contrast (DIC) image to show cilia. Red, CFTR; green, ZO-1 in A and cadherin in B–D; blue, nuclei (DAPI). (E) Correction of cAMP-stimulated Isc in the trachea of CFTR-null pigs transduced with AAV2H22-CFTRΔR relative to untreated controls. (F) Short-circuit currents of nasal epithelia in a CFTR-null pig transduced with AAV2H22-CFTRΔR in the right nostril. Left nostril was used as control. Inhibition of Na+ current with amiloride (Amil.) (1 × 10−4 M) hyperpolarized apical membrane voltage and increased the driving force for Cl− secretion. Inhibition of non-CFTR Cl− channels with 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS) (1 × 10−4 M). CFTR activity stimulation (ΔIsccAMP) by cAMP levels elevated via forskolin (1 × 10−5 M) and IBMX (1 × 10−4 M). (G) Airway surface liquid (ASL) pH was measured in vivo using pH-sensitive planar optical probe placed on tracheal surface. (H) S. aureus–coated grids were placed on tracheal surface of WT and CFTR-null pigs transduced with AAV2H22-CFTRΔR or control. Data are percentage dead bacteria. For each pig, 2–3 grids were used for 1 minute, 5–16 fields were counted per grid, each field contained ~100–1,000 bacteria, and data from each field were averaged. For A–H, n = 3 CFTR-null pigs transduced with AAV2H22; n = 6 untreated CFTR-null pigs; n = 6 non-CF pigs. *P < 0.05 vs. untreated CFTR-null *P < 0.01, Mann-Whitney nonparametric t test. For F, n = 1 nostril with contralateral nostril as a control. The non-CF control pigs (E, G, and H) were shared with a companion manuscript by Cooney et al. (58).