ABSTRACT Recombinant A adeno‐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 10 6 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.
Radukic et al. (Thu,) studied this question.
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