Altering VP1 and VP2 expression in trans affects the transduction efficiency of AAV9

Currently, adeno-associated virus (AAV) is one of the most reliable carrier for gene delivery in both proliferating and non-proliferating cells. Stable and long-lasting transgene expression has made this viral vector a key platform for the development of advanced therapy. Nevertheless, the widespread clinical use of AAV-based drugs remains limited due to their immunogenicity, low capsid capacity, and restricted tissue tropism. Tissue tropism depends largely on the the transduction efficiency of AAV capsids. In this study, we modified the standard three-plasmid transfection protocol to provide independent expression of VP1 or VP2 proteins from separate plasmids. Adjusting the ratio of these plasmids in the transfection mixture enabled alteration of the stoichiometric composition of the capsids, as SDS-PAGE and mass spectrometry confirmed. Increasing the amount of VP1 or VP2 in the capsid composition enhanced transduction efficiency, as demonstrated <i>in vitro</i> experiments on HEK293 cells. Obtained results contribute to a more comprehensive understanding of the AAV biology and have perspective of application in gene therapy.