Multiplexing AAV Serotype-Specific Neutralizing Antibodies in Preclinical Animal Models and Humans

The accurate assessment of AAV-specific pre-existing humoral immunity due to natural viral infection is critical for the efficient use of clinical gene therapy. The method described in the present study applies equivalent infection conditions to each AAV serotype (AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVAnc80L65). In the current study, we validated the assay by assessing AAV-neutralizing antibody titers in a limited cohort of random human donors and well-established preclinical large animal models, including dogs and non-human primates (NHPs). We achieved a rapid and accurate evaluation of neutralizing titers for each individual subject that can be used for clinical enrollment based on specific AAV serotypes and individualized selection of the most suitable AAV serotype for each specific patient

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca2+-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5–15 mg/dl) for 24 h did not affect l-[3H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC

SVIP overexpression induces vacuolization of cells.

<p><b>(A) AT01 cells were treated with</b> NT siRNA or 2 μM SVIP siRNA for 48 hours. siSVIP diminishes the SVIP-induced vacuoles in AT01 cell line. (B) vacuolization in the Hu339 cell line transfected with 2 μg SVIP plasmid.</p

SVIP is overexpressed in cells under prolonged ER stress.

<p>(A) Western blot of intracellular and extracellular (EC) levels of AAT and albumin expression level in cell lines. AT01 and Hu339 cells were lysed in RIPA buffer, and protein concentration was measured using Pierce BCA protein assay kit; 10 μg total lysate was resolved on 10% TGX electrophoresis gel. The volume of the media to detect EC AAT was correlated to total protein in corresponding cell lysate. (B) Western blot of SVIP and VCP protein expression levels and relative densities in the AT01 cells under prolonged ER stress and in Hu339 cells transfected with ZAAT plasmid. Cells were transfected with WT gp78 or pTR2-CB-ZAAT, as indicated. At 48 hours post transfection, total lysates were subjected to Western blot analysis. β-Actin levels were measured to demonstrate equal sample loading. (C) SVIP protein expression level and relative units in deficient and normal liver tissue samples. (D) SVIP mRNA expression as analyzed by quantitative PCR in cell lines AT01 and Hu339, with same treatments as panel B, and (E) in the liver tissue samples, respectively.</p

SVIP inhibits ERAD.

<p>(A) SVIP caused dose-dependent accumulation of ZAAT. AT01 cells were transfected with ZAAT-RFP and increasing amounts of PCMV6-entry-SVIP (0, 1, 2, 4 μg). The levels of AAT were determined by Western blotting with anti-AAT antibody. Actin was blotted as a loading control. (B) SVIP overexpression inhibited ZAAT degradation. AT01 and ZAAT-expressing stable Huh cells were co-transfected with Z-RFP and either with empty plasmid or PCMV6-entry-SVIP (2 μg). At 48 hours after transfection, the cells were radiolabeled with 500 μCi ³⁵S and chased for the indicated times. The levels of intracellular (IC) AAT were determined by SDS-PAGE autoradiography. (C) SVIP negatively regulates gp78-p97 mediated degradation of AAT. ZAAT-expressing stable Huh cells were transfected with NTsiRNA or siVCP/p97. 24h post silencing they were co transfected with gp78, gp78 and WT SVIP or SVIP mutant. The IC ZAAT, VCP, gp78 and SVIP were detected by western blott. Actin was used as loading control. (D) SVIP inhibited gp78-p97/VCP interaction. AT01 cells were transfected as indicated before and were processed for IP with anti-gp78 antibody for endogenous gp78. p97/VCP bound to gp78 was detected by Western blotting with anti-VCP antibody.</p

Functional interactions between gp78 and p97/VCP in AATD.

<p>(A) gp78 overexpression increased the level of ZAAT degradation in Huh 7.5 knockout cells stably expressing ZRFP. Cells were transfected with empty plasmid or WT gp78 as indicated. At 48 hours post transfection, cells were radiolabeled with 500 μCi ³⁵S and chased for the indicated times. The levels of intracellular (IC) and extracellular (EC) AAT were determined by SDS-PAGE autoradiography. (B) gp78 enhanced ZAAT degradation through interaction with p97/VCP. Cells were transfected with empty plasmid or gp78Δ135 as indicated. Experiemtns were performed as in A. (C) Expression level of AAT in liver tissue samples. (D) ZAAT accumulation enhanced the gp78-VCP interaction. Co-IP was performed on Hu339 and AT01 cells and liver tissue samples of three healthy controls and three AATD individuals; 100 μg of the total lysate was processed for IP with anti-gp78, followed by WB with anti-gp78 and anti-p97/VCP to detect the level of gp78 and associated p97/VCP.</p