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

David Nathan Quan1, Joseph Shiloach1

  • 1NIDDK Biotechnology Core, NIDDK, National Institutes of Health, Bethesda, Maryland, USA.

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|July 30, 2021
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Summary

This study presents a new high-throughput method for screening recombinant adeno-associated virus (rAAV) production. The developed assay effectively measures both genomic and infectious rAAV titers, crucial for bioproduction advancements.

Keywords:
AAVhigh-throughputproductionscale-downscreening

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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.