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

Area of Science:

• Biotechnology
• Molecular Biology
• Virology

Background:

• High-throughput screening (HTS) methods for recombinant adeno-associated virus (rAAV) production are scarce, limiting research and bioproduction efficiency.
• RNA interference (siRNA) knockdown is a common strategy for pathway engineering in bioproduction, but its impact on rAAV production requires evaluation.

Purpose of the Study:

• To develop and validate a generalizable high-throughput relative rAAV titration method.
• To assess the method's utility within the context of an siRNA screening campaign.
• To evaluate the impact of siRNA transfection on rAAV production titers.

Main Methods:

• A 384-well scale method was established using quantitative PCR (qPCR) for genomic rAAV titer and transduction of COS7 cells for infectious rAAV titer.
• Crude samples from transfected HEK293T/17 cultures, including supernatant and lysed fractions, were analyzed.
• The method was tested on cultures undergoing siRNA reverse transfection followed by rAAV forward transfection to mimic screening conditions.

Main Results:

• The high-throughput titration method successfully assessed relative genomic and infectious rAAV titers.
• siRNA transfection measurably impacted rAAV titer, whereas delayed rAAV transfection did not affect siRNA activity in controls.
• Accurate differentiation of infectious titer levels depended on appropriate sample dilution, with consistent trends observed between qPCR and infectious assays.

Conclusions:

• A scalable and generalizable high-throughput method for rAAV titration has been demonstrated.
• The findings highlight the significant influence of siRNA transfection on rAAV production, a critical consideration for pathway engineering in bioproduction.
• This method provides a valuable tool for optimizing rAAV production through high-throughput screening.