(310b) RNA Interference High Throughput Screen for Improvement in Transduction Efficiency of Adeno-Associated Virus

Wallen, A. J., University of Pennsylvania
Barker, G. A., University of Pennsylvania
Jing, H., University of Pennsylvania
Fein, D. E., University of Pennsylvania
Diamond, S. L., University of Pennsylvania

Although viral-mediated gene therapy shows promise, there remains room for improvement in transduction efficiency. To investigate this issue, we have used a small-interfering RNA (siRNA) high-throughput screen in vitro approach to search for genes which may serve as barriers to adeno-associated viral gene delivery. The general protocol used was to reverse-transfect cells with siRNA for the target gene on Day 1, transduce the cells with adeno-associated virus type 2 (AAV2) containing a reporter gene on Day 2, and assay for the reporter protein on Day 3. In a primary screen, a druggable genome library consisting of 5,520 targets was tested with pools of three unique siRNA sequences per gene. Fifty genes which resulted in greater than 2.5-fold improvement in transduction were identified and each of the three siRNAs corresponding to those genes was further investigated individually. Overall, at least one siRNA sequence from each of the top ten genes provided approximately two-fold or greater improvement in transduction efficiency in larger scale experiments. The top three siRNAs, each from a unique gene, demonstrated improvements in the range of three- to seven-fold. The most promising results came from the solute carrier (SLC) group, the chloride intracellular channel (CLIC) group, and the G-protein coupled receptor (GPCR) group. The use of siRNA to explore improvements in viral transduction provides insight into the biology of AAV2, and suggests the possibility of enhanced adeno-associated viral gene therapy in vivo.