Abstract 15274: High-Throughput, Genome-Wide siRNA Screening Identifies Novel Genes Regulating AAV Transduction of Cardiomyocytes and Other Post-Mitotic Cells
Viral vectors based on AAV have gained popularity for cardiovascular gene transfer due to their safety profile, lack of inflammatory response, prolonged transgene expression, relative genetic simplicity and high efficiency of transduction of post-mitotic tissues, such as muscle, heart, brain and retina. Given the molecular simplicity of AAV particles, the determinants of permissivity to vector transduction appear to reside within the host cell. We assessed the hypothesis that the cellular proteins of the DNA Damage Response (DDR) are key players in controlling AAV permissivity and further extended our investigation by performing a high-throughput screening (HTS). We observed that cardiomyocyte and skeletal muscle permissivity to AAV transduction correlates with the loss of expression of Mre11, Rad50 and Nbs1 (the MRN complex), which are downregulated upon terminal cell differentiation. In particular, the levels of these proteins, which bind the incoming AAV genomes and block its processing, fades in cardiomyocytes at 2 weeks after birth, coinciding with increased AAV permissivity. Accordingly, in vivo treatment of relatively poorly permissive tissues with siRNAs against members of MRN markedly increases AAV permissivity. To systematically identify novel cell factors involved in AAV internalization, intracellular trafficking, genome processing and gene expression, we performed a HTS using a genome-wide siRNA library (18,175 human gene targets) using a recombinant ssAAV2 vector expressing the firefly luciferase reporter gene. We identified 1528 genes that affect transduction of AAV vectors more than 4-fold (184 genes by more than 8-fold). Of these genes, 993 are inhibitors of AAV transduction, whereas 535 are required for efficient transduction by AAV vectors. Analysis of the inhibitor genes revealed a clear overrepresentation of genes related to DNA recombination and repair and cell cycle control. Of note, the top 10 siRNAs also significantly increased AAV transduction by other serotypes (e.g. AAV1, AAV5, AAV6, AAV9) and in various cell types in cell culture and in vivo. In conclusion, these results indicate that the transient inhibition of cellular factors repressing AAV transduction in vivo can be exploited to extend vector efficacy.
- © 2012 by American Heart Association, Inc.