<p>In recent years, recombinant adeno-associated virus (rAAV) vectors are the promising viral transfer tools for gene therapy and clinical trials, thanks to their favorable safety profile and long-term transgene expression. The increasing demand for rAAVs for gene therapy led to a rise in the amount of these vectors required for pre-clinical trials, clinical trials, and approved therapeutic applications. A majority of suspension HEK293 cell-based rAAV production protocols reported rely on a triple transfection at cell density below 2 × 10⁶ cells/mL. However, the low yield of such biomanufacturing challenges bioprocess engineers to develop more efficient strategies capable of increasing volumetric productivity. In this study, we developed a perfusion bioprocess to enable rAAV production efficiently at high cell density. We first optimized three key process parameters (the total DNA amount, ratio of polyethyleneimine (PEI) to DNA, and proportion of the three plasmids) of rAAV production at cell density of 2 × 10⁶ cells/mL by the design of experiment method, from which the robust setpoint (total DNA amount of 1.37 μg/mL, ratio of PEI to DNA of 1.52 μL/μg, the proportion of plasmids pHelper 24%, pRC 46%, pGOI 30%) was explored. We then developed a rAAV production process at a cell density of ~ 8 × 10⁶ cells/mL, with increasing DNA amount on a cell basis and optimizing transfection complex preparation. This approach was confirmed in a 5 L benchtop bioreactor connected with a perfusion system, resulting in a viral genomic titer of 7.28 × 10¹¹ vg/mL and a cell-specific viral genomic titer of 4.97 × 10⁴ vg/cell. This study demonstrates that the perfusion process coupled with optimized transfection complex preparation has the potential to improve manufacturing productivity.</p>