# Engineering of High‐Yield Recombinant Adeno‐Associated Virus Producer Plasmids

## TL;DR

This study improves the production of gene therapy vectors by optimizing the design of plasmids used to make adeno-associated viruses.

## Contribution

The study identifies optimal configurations for AAV2 Rep gene clusters and helper plasmids to enhance virus yield and quality.

## Key findings

- Wild-type AAV2 Rep gene configurations yield 116-fold higher titers and reduce mispackaging.
- Modifications to Rep post-translational sites decrease empty capsid burden.
- A 7 kbp helper plasmid works for AAV2 but not AAV6 or AAV9 when Rep expression is optimized.

## Abstract

Recombinant Aadeno‐associated virus (rAAV) production lags demand with respect to quality and quantity. We report insights from producer plasmid engineering aimed at increasing yield and homogeneity of rAAV vectors obtained by HEK‐293 triple transfection. Miniaturized production and same‐day quantification streamlined the investigation. We demonstrate that modifications of the AAV2 Rep gene cluster reduces titers of currently circulating packaging plasmids. Revertants to wild type yielded 116‐fold higher titers of about 106 particles per cell and reduced mispackaging. Modifications of predicted Rep post‐translational modification sites decreased the empty capsid titer burden. A 7 kbp minimal helper plasmid lacking L4 22k maintained production capability upon optimized Rep expression for AAV2 but not for AAV6 and AAV9. Knockout of the egress protein MAAP increased rAAV yield from the cell pellet for convenient lysate processing. Together, these findings highlight the importance and potential tradeoffs of designing producer plasmids to obtain high titer systems.

Recombinant adeno‐associated viruses (AAV) are important in gene therapy but their production may be impaired by non‐optimized gene design. In this study, the authors demonstrate that production was best when the AAV replicase gene was in a wild‐type configuration while the complexity of other genetic elements could be reduced.