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P29

Lipid-nanoparticle co-delivery of rAAV genomes and mRNA for AAV integrase Rep78 replicates wildtype AAV genome integration as assessed by targeted sequencing

M T Radukic(1) T Krassuski(1) K M Müller(1)

1:Bielefeld University, Faculty of Technology, Cellular and Molecular Biotechnolgy, Bielefeld, Germany

Recombinant AAV vector genome integration, occurring randomly at low frequency after rAAV gene therapy, is currently seen as a double-edged sword: It may ensure long-term gene expression but raises concerns about potential genotoxicity. In contrast, wildtype AAV integrates its genome mediated by its integrases Rep68/78, and AAV genetic safety stands the test of time. We, therefore, propose to embrace wildtype-like AAV vector genome integration by co-delivery of Rep78 mRNA. For a proof of concept, we transfected a cell culture model using a lipid nanoparticle (LNP)-based system with synthetic rAAV genomes and Rep78 mRNA at different ratios. We found that addition of Rep78 mRNA significantly increased transgene expression already at 0.2% molar fraction of total nucleic acids delivered, and the increase was highest with more than 2-fold more expression at 1.6% mRNA. Transgene expression from LNP-delivered AAV genomes sustained for the tested time periods of up to 17 days potentially through the formation of AAV genome-like episomes and integration. We then investigated vector genome integration by a Cas9-guided direct sequencing approach. 99.7% of recombinant AAV genomes remained episomal nine days after co-transfection. Still, 27 distinct integration events were observed, none of which located in known cancer genes. The integration pattern closely resembled published wildtype AAV patterns with integration at the known site AAVS1 and at other sites distant from assumed Rep recognition motifs. These findings provide an intriguing first step to a fully synthetic option for AAV-like gene therapy with LNP-based delivery of AAV genomes and, optionally, Rep78 mRNA.

Sekretariat der DG-GT e.V.
Institut für Experimentelle Hämatologie
Hildegard Büning
Carl-Neuberg-Str. 1
30625 Hannover

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© 2021 Die Deutsche Gesellschaft für Gentherapie e.V.

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