Increasing demand for recombinant adeno-associated virus (rAAV)-based gene therapies necessitates increased manufacturing production. Transient transfection of mammalian cells remains the most commonly used method to produce clinical-grade rAAVs due to its ease of implementation. However, transient transfection processes are often characterized by sub-optimal yields and low fractions of full-to-total capsids, both of which contribute to the high cost of goods of many rAAV-based gene therapies. Our previously developed mechanistic model for rAAV2/5 production indicated that the inadequate capsid filling is due to a temporal misalignment between viral DNA replication and capsid synthesis within the cells and the repression of later phase capsid formation by Rep proteins. We experimentally validated this prediction and showed that performing multiple, time-separated doses of plasmid increases the production of rAAV. In this study, we use the insights generated by our mechanistic model to develop an intensified process for rAAV production that combines continuous perfusion with high cell density re-transfection. We demonstrate that performing multiple, time-separated doses at high cell density boosts both cell-specific and volumetric productivity and improves plasmid utilization when compared to a single bolus at standard operating conditions. Our results establish a new paradigm for continuously manufacturing rAAV via transient transfection that improves productivity and reduces manufacturing costs.

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its non-pathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and non-dividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of pre-formed empty capsids and viral DNA copies per cell are not limiting to the capsid filling reaction. However, optimal expression of Rep proteins (< 240 ± 13 ag per cell) enables enrichment of the filled capsid population (> 12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.