Scaling Down for Big Impact: Streamlined High-throughput Recombinant Adeno-associated Virus Production.

Abstract

In recent years, recombinant adeno-associated viral (rAAV) vectors have emerged as a leading platform for gene therapy applications. However, the cumbersome production and purification processes remain significant bottlenecks in drug development. High-throughput, small-scale rAAV production without labor-intensive and time-consuming purification procedures offers a valuable alternative strategy to accelerate preclinical research and early-stage therapeutic screening. This approach enables the rapid generation of diverse rAAV variants or therapeutic constructs with sufficient quality for initial in vitro and in vivo evaluation. This protocol introduces two methods distinguished by cell type and production scale: microscale and miniscale rAAV production. Microscale production is carried out in a 6-well format using adherent human embryonic kidney (HEK) 293T cells (micro-6-well), while miniscale production utilizes suspension cells in 24-well plates (mini-HT24). Vectors generated through these methods undergo downstream analyses to optimize cell culturing conditions and/or are used to assess the biological activity of transgenes. Key analytical readouts include vector genome titration (vg/mL), capsid titer determination (vp/mL), western blot analysis, and cell transduction assays. Additionally, vectors produced at the miniscale and semi-purified using affinity resins are compared with midiscale preparations purified via cesium chloride density gradients. The streamlined methods described here demonstrate significant potential for refining cell culture parameters, identifying lead candidates during screenings (i.e., optimized transgene cassettes or superior AAV capsids), and enhancing transgene expression. Collectively, the presented workflows support the establishment of a robust and efficient platform for iterative development in the gene therapy pipeline that can be readily implemented in any research lab.