549. Fine Tuning of Transduction Efficiency of rAAV Vectors via Modulation of Capsid Composition

Recombinant Adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. Even though the industry is poised for the expansion into several application areas represented by orphan diseases, a simple and scalable rAAV production technology is still lacking. We have recently developed the OneBac system to allow scalable, high-titer production of the full range of rAAV serotypes by infection of stable insect Sf9 cell lines with a single baculovirus. Some of the serotypes produced in this system, however, were characterized by a low transduction efficiencies compared to HEK 293-derived vectors. Here we describe an approach for resolving this drawback by modulating the ratios of VP1:VP2:VP3 capsid composition to derive particles with a higher VP1 content. This is accomplished by modifying a canonical Kozak sequence preceding VP1 ATG start codon. A range of Kozak sequences has been tested and the most favorable have been identified for AAV5 and AAV9 serotypes. These newly designed capsid genes were incorporated into Sf9 stable cell lines mediating packaging of AAV5 and AAV9 vectors which exceeded transduction efficiencies of HEK 293-derived counterparts by 2-3 folds. Curiously, the optimal ratios of VP1:VP2:VP3 were serotype-specific requiring fine-tuning to achieve a compromise between higher VP1 content mediating higher transduction potencies vs. lower packaging efficiencies impeding production yield. In summary, we have developed a novel approach of significantly enhancing biological potencies of AAV vectors derived from baculovirus system thus facilitating translational applications.