HEK293SF-3F6 cells were seeded at 1E+6 cells/mL the day prior to transfection to ensure that the cultures will attain at 2E+6 cells/mL when at transfection moment. The transfection mix consisted in cell culture media, DNA and transfection reagent, all adding up to 5% of the total working volume which was 20 mL and the transfection mix 1 mL. The cells were triple transfectected using FectoVIR®-AAV with the following plasmids: pXR2 (pRepCap), pXX6-80 (pHelper) and PF1451 (pGOI-GFP)/ PF1452_BDNF (pGOI-BDNF)/ PF1451_MSH2 (pGOI-MSH2).

Optimization of recombinant adeno-associated virus (rAAV) production is essential for effective gene therapy applications. However, multiple factors affect the rAAV productivity in mammalian cells, and often they interact with each other, making the optimization process highly challenging. In our previous work, we showed how coupling mixture design (MD) with face-centered central composite design (FCCD) was the most suitable design of experiments (DOE) approach for optimizing rAAV2 productivity and cell viability. In this study, we built on this method and demonstrate that combining MD with FCCD can be used to optimize the percentage of full capsids in rAAV2 upstream preparation. Additionally, we investigate the influence of the gene of interest (GOI) on the optimal conditions for viral particle production and packaging efficiency. By integrating MD and FCCD methodologies, we achieved an improvement of almost 100-fold in Log(Vp) in the case of egfp-expressing rAAV, and a 12-fold increase in bdnf-expressing full rAAV capsids, suggesting that this combined approach is a versatile and effective strategy for optimizing rAAV production processes. These findings emphasize the need for a comprehensive understanding of the factors influencing rAAV production to enhance the efficiency and efficacy of viral vector applications in gene therapy.