555. Distribution of AAV8 Particles in Cell Lysates and Supernatants Changes with Time and Is Dependent on the Vector Genome

Abstract

Introduction: AAV is a powerful gene delivery vehicle capable of safely transducing a variety of tissues to provide long-term expression. As a result, one AAV-based gene therapy has received regulatory approval thus far, with a number of clinical trials ongoing. However, producing AAV at scale for clinical trials poses a number of challenges and process optimization is essential. Here, we show that AAV production increases with time through 6 days post-transfection and over time the distribution of viral particles shifts more from cell lysate towards the supernatant. This shift in distribution is dependent on the vector genome.Methods: Small-scale two plasmid-transfections of 293T/17 cells were performed in 15 cm plates (n=3/group) and cells were cultured in DMEM with 10% FBS. Three different AAV expression cassettes were assayed, including Factor VIII (FVIII), Factor IX (FIX), and Protective Protein/Cathepsin A (PPCA). FIX and PPCA cassettes included a mutated ITR for packaging of self-complementary genomes. AAV8 particle titers were determined in cell lysates and supernatants using the Progen PRAAV8 ELISA Kit which detects only assembled AAV8 particles. Distribution of AAV was compared at days 3 and 6 post-transfection for all three genomes, while FVIII production and distribution was assayed at days 3, 5, 6, and 7 post-transfection.Results: At day 3 post-transfection, the majority of AAV8 particles were located in the supernatant for all three vector genomes that were packaged; however, while 76% of the FVIII particles were in the supernatant, only 63% of the FIX and PPCA particles were in the supernatant. FVIII showed an increase in production when culture was extended to 5, 6, and 7 days post-transfection, with day 5 showing a 9% increase over day 3 (p=0.08) and day 6 showing a 24% increase over day 3 (p<0.01). Interestingly, viral titer decreased from day 6 to 7, but was still 14% higher than day 3 (p<0.05). While 76% of FVIII AAV particles were in the supernatant at day 3, this proportion increased to 87% by day 5, 91% by day 6, and 94% by day 7 (all p<0.05 versus day 3). FIX and PPCA showed similar increases in production from day 3 to day 6 and a more dramatic shift in particle distribution, with 90% of AAV8 particles in the supernatant by day 6 for both vector genomes.Conclusions: Increasing the duration of culture post-transfection can significantly improve production of AAV and shift the distribution of particles more greatly towards the supernatant. This has important implications for large-scale production, during which the supernatant may be harvested alone, eliminating the more time-intensive processing of cell lysate, which contains 10% or less of the total particle yield. Additionally, the differences in particle distribution of different expression cassettes at day 3 post-transfection implies a genome-dependent processing mechanism which has not, to our knowledge, been previously described. Whether this relates to genome sequence, self-complementarity, or some other mechanism remains to be determined.