INVESTIGATING RECOMBINANT AND WILD-TYPE ADENO-ASSOCIATED VIRUS INTEGRATIONS IN THE MACAQUE AND HUMAN GENOMES FOLLOWING IN VIVO EXPOSURE
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Graduate group
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Genetics and Genomics
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Adeno-associated virus
Gene Therapy
Integration
rAAV
wtAAV
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Abstract
Adeno-associated virus (AAV) vectors are the predominant vehicles for in vivo gene therapies, utilized in over 200 clinical trials and three FDA-approved products. Integrations of both wild-type AAV (wtAAV) and recombinant AAV (rAAV) have been observed in host genomes for over two decades, raising concerns about genotoxicity and clonal expansion in rAAV-treated mice and dogs. Whether in vivo rAAV integration in primates mirrors non-pathogenic wtAAV integration or presents an elevated risk of genotoxicity and expansion remains uncertain. Despite rAAV-based therapies' extensive use and wtAAV's prevalence, the impact of integrations on short- and long-term safety and therapeutic efficacy remains uncertain, especially in primate models. In this study, we comprehensively characterize wtAAV and rAAV integrations following in vivo exposure in 253 macaques and 85 humans, using consistent methods of detection, analysis, and annotation. Both long-read and short-read approaches allowed for robust assessment of genomic locations, abundance, composition, expansion, and integrity of isolated chromosomal-AAV junctions. Our findings reveal that, while rAAV integrations are more frequent, no elevated risk of locational genotoxicity or expansion accompanies them compared to wtAAV integrations. Integration distributions for both types exhibit similar, broad distribution patterns enriched in DNA damage-prone regions. rAAV integration occurs early and maintains consistent distribution patterns. Although highly rearranged and truncated concatemers predominate at late timepoints, functional integrations can persist up to two years post-injection and may contribute to long-term transgene expression, particularly with non-immunogenic transgenes. The substantial similarity between our NHP and human cohorts underscores the relevance of primate-based AAV integration studies, endorsing their use in risk assessment over other biologically divergent preclinical models. This comprehensive AAV integration analysis bridges critical gaps in our understanding of AAV integration biology and holds important translational implications for the safety of rAAV as a gene therapy vector and highlights the clinical translatability of NHP AAV integration data.