Gene therapy for alpha 1-antitrypsin deficiency with an oxidant-resistant human alpha 1-antitrypsin
Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal
Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal
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Research Article Pulmonology Therapeutics

Gene therapy for alpha 1-antitrypsin deficiency with an oxidant-resistant human alpha 1-antitrypsin

Abstract

Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell–mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease–inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.

Authors

Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal

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Figure 3

Comparison of in vivo–produced human AAT-modified variants’ ability to inhibit NE and cathepsin G.

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Comparison of in vivo–produced human AAT-modified variants’ ability to i...
AAV8 vectors expressing modified AAT variants were administered to C57BL/6 male mice (IV, 1011 gc), and serum was collected after 4 weeks. Human AAT levels were quantified by ELISA. An equal amount of each AAT variant was used in the protease inhibition assays. (A) Anti-NE activity. AAT variants were exposed to 1 mM NCS for 20 minutes before addition of NE and substrate. NE inhibition is presented as the percentage of WT (8/AMM) inhibition in the absence of oxidizer. (B) Anti–cathepsin G activity. AAT variants were exposed to 1 mM NCS for 20 minutes before addition of cathepsin G and substrate. Cathepsin G inhibition is presented as the percentage of WT (8/AMM) inhibition in the absence of oxidizer. Experiments were performed in triplicate and averaged for n = 3–5 mice per group, and statistical analysis was performed by ANOVA.