Abstract 21651: X-Linked Inhibitor of Apoptosis Protein (XIAP)-Mediated Attenuation of Apoptosis Using a Novel Cardiac Enhanced Adeno-Associated Viral Vector
Background: Measurements of anti-apoptotic proteins, such as XIAP, in non-failing (NF) & failing (F) left ventricular (LV) samples have been inconsistent & complicate the development of anti-apoptotic gene therapies. The success of gene therapy for apoptosis requires both a transgene effective at attenuating injury & a highly efficient, safe vector. The goal of this study is to: 1) develop a highly efficient adeno-associated viral (AAV) vector for cardiac transduction & 2) compare levels of XIAP in high quality, well-characterized human NF & F LV samples.
Methods: Amino acid substitutions in the AAV2 & AAV3b capsid were assessed for improved cardiac transduction in vitro & in vivo. Caspase-3 (Cas-3) activity & XIAP protein abundance were quantified in LV tissue from unused human donor hearts (NF, n=7) & cardiac transplantation explants (F, n=8). A modified AAV3b (AAV-SASTG) was most efficient & used to generate a SASTG-XIAP viral vector. Following transduction with SASTG-XIAP or SASTG-GFP, rat neonatal cardiac myocytes (rNCM) underwent apoptosis induction (AI) by: a) PKCγ inhibition (chelerythrine (CHEL)), b) β-adrenergic agonism (isoproterenol (ISO)), or c) hypoxia/ischemia (H/I). Cas-3 activity was then measured.
Results: SASTG demonstrated 26-fold & 61-fold greater transduction than AAV1 or AAV6 in rNCM & 21-fold & 8.8-fold greater transduction than AAV1 or AAV9 in mouse hearts in vivo. Cas-3 activity was 267% higher (p<0.05) & XIAP abundance was 38% lower (p<0.05) in F vs. NF samples. SASTG-XIAP-treated rNCM exhibited lower Cas-3 activity vs. SASTG-GFP-treated rNCM following AI. (See figure.)
Conclusion: AAV-SASTG exhibits improved transduction efficiency in cardiac myocytes compared to other AAV serotypes. Our findings of increased Cas-3 activity with less XIAP in F humans & the anti-Cas-3 activity of SASTG-XIAP support the potential benefit of SASTG-XIAP gene therapy & warrant further study in isolated human myocytes & animal models of cardiac disease.
- © 2010 by American Heart Association, Inc.