548. Self-Complementary Adeno-Associated Virus 9 Encoding S100A1 Decreases Serum Levels of Proinflammatory Cytokines Compared To Single-Stranded AAV9 after Myocardial Delivery Post MI
Authors: Andrew P. Kendle, Michael G. Katz, Anthony S. Fargnoli, Nury M. Steuerwald, Charles R. Bridges
Journal: Molecular Therapy (2015)

Despite the cardiospecificity of recombinant vectors based on adeno-associated virus (AAV) serotype 9, transgene expression is limited. One important factor is AAV's single-stranded (ss) DNA genome that is transcriptionally inactive until uncoated, converted into double-stranded DNA, and transported to the nucleus. Self-complementary (sc) AAV enters the cardiomyocyte as double stranded DNA, eliminating the efficiency-limiting step of second-strand synthesis. Previous studies have demonstrated that scAAV9 vector has superior transduction in vivo and in vitro. The aim of this study is to assess the changes in pro- and anti-inflammatory cytokines after intramyocardial (IM) injection of gene in rats with ischemic heart failure. Heart failure was induced through myocardial infarction (MI) by ligation of the left anterior descending coronary artery in rats. Groups included sham thoracotomy (Sham, n=6), Saline IM injection MI (Saline, n=10), ssAAV.S100A1 IM injection MI (SS, n=10), and scAAV.S100A1 IM injection MI (SC, n=10). Serum inflammatory markers were analyzed 10 weeks post MI, well after any acute inflammatory response to surgery, injection or MI. Concentration of pro- and anti-inflammatory cytokines in sera were determined with Bio-Plex assay. Following washes, samples were incubated with biotinylated detection antibody. Captured analytes were detected by addition of streptavidin-phycoerythrin and quantified using a BioPlex array reader. Analyte concentrations were calculated (in pg/mL) with Bio-Plex software. Other data include electron microscopy, histology, gene expression assays and hemodynamic measurements by echocardiography. In addition to improved gene expression and hemodynamics in SC relative to SS groups, analysis of inflammatory markers generally showed significant inflammation relative to the Saline group in the SS group with only a trend in the SC group. Of highest importance, interferon gamma showed a 2.5-fold increase in expression in SS animals as compared to both SC and Saline groups (SS 263 ± 85, SC 108 ± 35, Saline 96 ± 25; p < 0.05) and tumor necrosis factor α showed a significant increase in only the SS group relative to Saline (SS 256 ± 50, SC 204 ± 79, Saline 122 ± 36; p < 0.05). In addition, monocyte chemoattractant protein 1 was significantly increased in both SS and SC (SS 848 ± 90, SC 819 ± 56, Saline 678 ± 42; p < 0.05). Anti-inflammatory cytokines (interleukins 4, 10 and 13) showed no significant differences between groups (p > 0.05). Self-Complimentary AAV, while limited in cloning capacity (2.4 kB for SC vs. 4.7 kB for SS), offers significant advantages in transduction efficiency and avoidance of an inflammatory response. Transfection with the SS vector resulted in higher levels of proinflammatory cytokines, which may result in an exacerbation of inflammatory-mediated cardiovascular pathology and a further reduction in transgene expression.