In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency.

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.

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.