rAAV Production and Titration at the Microscale for High-Throughput Screening.

In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples. To evaluate relevant differences in titer for conditions that could be used in an actual screen, cultures subjected to an siRNA reverse transfection and subsequent rAAV forward transfection were also tested. The delayed forward rAAV triple-plasmid transfection was not seen to affect the siRNA activity of tested controls, while siRNA transfection was shown to measurably impact rAAV titer. Effective differentiation between infectious titer levels was dependent upon the choice of sample dilution, but trends between qPCR and infectious titer assays were consistent across sample sets.