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Methods Matter -- Standard Production Platforms For Recombinant AAV Produce Chemically And Functionally Distinct Vectors

By Neil G. Rumachik, Stacy A Malaker, Nicole Poweleit, Lucy H. Maynard, Christopher M. Adams, Ryan D. Leib, Giana Cirolia, Dennis Thomas, Susan Stamnes, Kathleen Holt, Patrick Sinn, Andrew P May, Nicole K. Paulk

Posted 17 May 2019
bioRxiv DOI: 10.1101/640169 (published DOI: 10.1016/j.omtm.2020.05.018)

Different approaches are used in the production of recombinant AAV. The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches we have made two major discoveries: 1) rAAV capsids have post-translational modifications including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms. 2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data find that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (P < 0.05-0.0001), in various mouse tissues in vivo (P < 0.03-0.0001), and in human liver in vivo (P < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations. ### Competing Interest Statement The authors have declared no competing interest.

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