A Mechanistic Model of rAAV Production in Synthetic Cell Lines

The recombinant adeno-associated virus (rAAV) is a widely used vector for gene therapy. Its manufacturing faces significant challenges in producing the large quantities of vectors needed for clinical applications and reducing empty particles. We have previously constructed synthetic cell lines that harbor all genes to enable scalable rAAV production. Through a series of design-construct-test cycles, productivity has increased to a level comparable to the traditional manufacturing method of triple plasmid transfection. In this work, we construct a mechanistic model of rAAV production in these synthetic cell lines to facilitate this design-construct-test cycle. The model was fit to the experimentally measured time profile of viral genome, transcripts, and proteins of viral components, capsid data, and packaged rAAV. The model recapitulates the trends in viral component dynamics with different inducer concentration time profiles, provides mechanistic insights into the effect of inducer profile on rAAV production, and predicts the effect of host cell genetic modifications. Finally, the model was used to optimize the inducer profile to increase vector genome production and full particle content in two separate cases. In both cases, the model-prescribed inducer concentration profile was experimentally performed and had the same trend of shift in VG and full particle content.