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A - Viral Vector Development -> AAV Vectors – Clinical/Non-Human Primate Studies

78: AAV Serotype Tropism and Editing in Young Rhesus Monkeys

Type: Oral Abstract Session

Presentation Details
Session Title: AAV Vectors: Clinical and Non-Human Primate Studies
Location: Room 411
Start Time: 5/17/2023 16:45
End Time: 5/17/2023 17:00

Recombinant AAV vectors are at the forefront of current gene therapy clinical trials. For example, AAV5, AAV6, AAV8, and AAVrh10 have shown promise for liver-directed gene transfer in Hemophilia A and B clinical trials. In musculoskeletal/cardiovascular-directed gene therapies, AAV8, AAV9, and AAVrh74 have attained efficient gene transfer, and AAV9 has proven useful for expression in brain. Despite the abundant and occasionally discordant data describing AAV serotype tropism in animal models and humans, significant uncertainties remain with regard to the selection of an optimal serotype for targeting specific human tissues; thus, there is a need for a side-by-side comparative analysis of multiple AAV serotypes at a comparable dose. A multiplexed (barcode-based) approach was used that enabled comparative and comprehensive evaluation of AAV serotypes following intravenous (IV) administration in rhesus monkeys. The editing cassette included a ubiquitously expressed, mini-chicken beta actin (CBA) promoter driving SaCas9 with a unique barcode and an adjacent U6 promoter driving a guide RNA (gRNA) This study compared seven AAV serotypes using systemic (IV) administration and young rhesus monkeys (N=40). The AAV serotypes tested (3B, 5, 6, 8, 9, rh10, rh74) delivered S. aureus (Sa)Cas9 with gene editing measured by assay of indels or integration of the barcoded AAV at the PCSK9 (proprotein convertase subtilisin/kexin type 9) target site. The barcoding strategy provided a means to administer AAV vectors either individually or in single combined administrations of multiple serotypes (e.g., AAV9+AAVrh74; range 1x10e13-8x10e13 genome copies [gc]/kg). Infants were screened to confirm they were seronegative prior to vector administration, then monitored for 4 weeks post-administration (e.g., hematology, clinical chemistries, immune responses). Initial studies (N=6) tested vector safety and dose using a luciferase reporter (no editing). Overall, findings indicated: (i) all serotypes provided good levels of transduction for a wide range of tissues but limited selectivity was shown for target tissues based on vector tropism; (ii) RNA expression was noted in most tissues but transduction did not correlate with expression; (iii) systemic (IV) delivery resulted in low levels of editing (≤5%); (iv) adaptive immune responses to all AAV serotypes and/or SaCas9 were observed in all groups; and (v) all animals remained healthy and robust during the study period although transient liver toxicity was observed particularly with the highest dose studied (combined serotypes; total 8x10e13 gc/kg). These studies confirmed that editing was driven by the level of RNA expression, and that transduction is an imperfect predictor of editing. Studies also showed that AAV9 was preferential for many tissues when administered IV (e.g., AAV9>AAVrh10>AAVrh74 ≥AAV8>AAV6>AAV5~AAV3B). As a result of these and related studies where higher levels of editing was observed, we are addressing comparative tests of Cas9 expression cassettes for optimal editing.

Alice F. Tarantal1, Michele Martinez2, Lionel Sanz2, Charles Lee3, Henriette O'Geen4, Aravind Asokan5, Charles A. Gersbach6, David J. Segal7, Dennis J. Hartigan-O'Connor8

1University of California, Davis, Davis, CA,2Pediatrics, Cell Biology and Human Anatomy, and California National Primate Research Center, University of California, Davis, Davis, CA,3Cell Biology and Human Anatomy, and California National Primate Research Center, University of California, Davis, Davis, CA,4Genome Center, University of California, Davis, Davis, CA,5Surgery, Duke University, Durham, NC,6Biomedical Engineering, Duke University, Durham, NC,7Biochemistry and Molecular Medicine, and Genome Center, University of California, Davis, Davis, CA,8Medical Microbiology and Immunology, and California National Primate Research Center, University of California, Davis, Davis, CA
 A.F. Tarantal: None.

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