Inducible rAAV producer cell lines yield vectors equivalent to transient transfection: a physicochemical and biological comparison.

Recombinant adeno-associated virus (rAAV) is one of the most widely used viral vectors in gene therapy. Currently, rAAV is commonly produced by plasmid transient transfection (TT) into host cells; however, the requirement for transfection limits scalability in manufacturing. To overcome this limitation, transfection-free production methods using inducible producer cell lines (PCLs)-in which essential genes for rAAV production are integrated into the host genome-have been actively developed. Despite the promise of PCL technology, comprehensive evidence showing that PCL-derived rAAV particles are equivalent to those produced by the conventional TT method has been lacking. In this study, we established an inducible rAAV PCL using a newly designed single plasmid harboring all genes necessary for rAAV production. Through a single selection step, we successfully isolated a high-yield single-cell clone. Physicochemical analyses of the produced rAAV, including capsid protein composition, genome packaging length, and sedimentation coefficient, confirmed equivalence between PCL-derived rAAV and that produced by conventional transient transfection. Furthermore, biological evaluation demonstrated that PCL-derived and transfection-derived rAAV exhibited comparable infectivity and transduction profiles both in HeLa cells in vitro and in the mouse brain in vivo. These results demonstrate that PCL-derived rAAV particles are equivalent to those from the TT method, highlighting that our inducible PCL approach is a promising platform for scalable rAAV production.