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. 2020 Aug 6;5(15):e135951.
doi: 10.1172/jci.insight.135951.

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

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

Meredith L Sosulski et al. JCI Insight. .

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.

Keywords: Gene therapy; Genetic diseases; Pulmonology; Therapeutics.

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Conflict of interest statement

Conflict of interest: RGC has equity in LEXEO, SMK is a consultant to LEXEO, and MLS, KMS, BPD, SMK, and RGC are participants in a patent disclosure (US20200102575A1) regarding oxidant-resistant gene therapy for alpha 1-antitrypsin deficiency.

Figures

Figure 1
Figure 1. Design of a second-generation gene therapy for AAT deficiency.
(A) Phylogenetic tree of AAT evolution (101, 102). M1(A213), M1(V213), M2, M3, and M4 are all normal variants. The common Z variant is derived from M1(A213). The less common S deficiency allele is derived from M1(V213). Since more than 95% of all AAT-deficient individuals are ZZ homozygotes, the M1(A213) normal variant was used as the base for the second-generation AAT gene therapy constructs to minimize immune responses to the therapy. (B) Three-dimensional schematic of human AAT with the A213 normal variant and active site methionine residues (M351, M358) indicated (red). (C) AAT variants tested.
Figure 2
Figure 2. In vitro comparison of the ability of modified variants of human AAT to inhibit NE and cathepsin G under normal and oxidizing conditions.
All data are presented as the percentage of WT inhibition in the absence of oxidizer. (A) NE inhibition by AAT variants under normal conditions (n = 6). (B) NE inhibition after exposure of AAT variants to 1 mM NCS for 20 minutes (n = 4). (C) NE inhibition after exposure of AAT variants to 250 mM H2O2 for 40 minutes (n = 4). (D) Cathepsin G inhibition by AAT variants under normal conditions (n = 2). (E) Cathepsin G inhibition after exposure of AAT variants to 1 mM NCS for 20 minutes (n = 2). (F) Cathepsin G inhibition after exposure of AAT variants to 250 mM H2O2 for 40 minutes (n = 2). Comparison for P value is between inhibition under normal conditions and inhibition with either NCS or H2O2 for each AAT variant. Each assay was performed in triplicate and averaged for each independent experiment. Statistical analysis was performed by ANOVA.
Figure 3
Figure 3. Comparison of in vivo–produced human AAT-modified variants’ ability to inhibit NE and cathepsin G.
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.
Figure 4
Figure 4. Association rates between human AAT and NE measured by surface plasmon resonance under normal and oxidizing conditions.
NE was coupled to the sensor chip, and serial 2-fold dilutions of AAT were flowed across and allowed to bind (AAT: 1000 nM shown in blue, 500 nM shown in green, 250 nM shown in red, 125 nM shown in yellow, 62.5 nM shown in purple). Curve fit to the Langmuir binding model (black line) (98). Binding curves are shown from 1 representative experiment of 3 independent experiments. (A) AAT-AMM binding to NE under normal conditions. (B) AAT-AVL binding to NE under normal conditions. (C) AAT-AMM binding to NE after exposure to 1 mM NCS for 20 minutes. (D) AAT-AVL binding to NE after exposure to 1 mM NCS for 20 minutes. See Table 1 for association rates and statistical comparisons.
Figure 5
Figure 5. In vivo levels of human AAT variants in serum over time following IV or IPL administration in male and female mice (n = 4–5/group).
C57BL/6 mice were administered 8/AMM, 8/AVL, or 8/Null (4 × 1011 gc) by IV and IPL routes. Serum was collected at 0, 4, 8, 12, 20, and 24 weeks, and human AAT was quantified by ELISA. (A) Male mice, IV. (B) Male mice, IPL. (C) Female mice, IV. (D) Female mice, IPL. At 24 weeks, there was no significant (ns) difference between 8/AMM and 8/AVL for either sex or administration route using Student’s t test.
Figure 6
Figure 6. Anti-NE activity of human AAT in serum from male mice administered AAV8 vectors under normal and oxidizing conditions.
C57BL/6 male mice were administered 8/AMM, 8/AVL, or 8/Null (4 × 1011 gc) by IV and IPL routes. Serum was collected at 4, 12, and 24 weeks, and human AAT was quantified by ELISA. Equal amounts (50 nM) of AAT were used in the NE inhibition assay. Data are presented as the percentage of WT (8/AMM) NE inhibition in the absence of oxidizer. (A) IV, 4 weeks. (B) IPL, 4 weeks. (C) IV, 12 weeks. (D) IPL, 12 weeks. (E) IV, 24 weeks. (F) IPL, 24 weeks. Assays were run in triplicate and averaged for samples from n = 4–5 mice/group, and statistical analysis was performed by ANOVA.
Figure 7
Figure 7. In vivo levels of human AAT in ELF over time following administration of the AAV8 vectors.
C57BL/6 mice were administered 8/AMM, 8/AVL, or 8/Null (4 × 1011 gc) by the IV and IPL routes (n = 4–5/group). Lung ELF was collected after sacrifice at 0, 4, 12, or 24 weeks, and human AAT was quantified by ELISA. (A) Male mice, IV. (B) Male mice, IPL. (C) Female mice, IV. (D) Female mice, IPL. At 24 weeks, there was no significant (ns) difference between 8/AMM and 8/AVL for either sex or administration route using Student’s t test. (E and F) Ratio of ELF to serum AAT. (E) Male mice. (F) Female mice. For E and F, data from all doses and time points were combined, and AAT levels (per mg protein) were collectively compared in ELF and serum for mice administered 8/AVL. Statistical analysis was performed using Student’s t test.
Figure 8
Figure 8. Anti-NE activity of human AAT in ELF from male mice administered AAV8 vectors under normal and oxidizing conditions.
C57BL/6 male mice were administered 8/AMM, 8/AVL, or 8/Null (4 × 1011 gc) by IV and IPL routes. ELF was collected at 24 weeks and human AAT quantified by ELISA. Equal amounts (50 nM) of AAT were used in the NE inhibition assay. Data are presented as the percentage of WT (8/AMM) NE inhibition in the absence of oxidizer. Assays were run in triplicate and averaged for samples from n = 4–5 mice/group. Statistical analysis was performed by ANOVA. (A) IV, 24 weeks. (B) IPL, 24 weeks.
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