AAVHSCs, a Nuclease-Independent Approach for Transduction in Non-human Primate Brain and Retina and Editing of Retinal Cells in Human Organotypic Explants

Abstract

Purpose : We have previously shown that a subset of AAVHSCs (AAVHSC7, 15 and 17) cross the non-human primate blood-retinal barrier (BRB) and blood-brain barrier (BBB) following a single intravenous dose. We have now extended this analysis to a panel of 11 AAVHSC capsids. AAVHSCs can be used to transduce various cell types in the central nervous system and edit the genome of dividing cells in a nuclease-free manner. Here we investigate the tropism of AAVHSCs, specifically to the visual pathways, and investigate whether AAVHSCs can induce genome editing in human disease relevant retinal cells using ex vivo (human retinal explant cultures) models.

Methods : We delivered AAVHSCs intravenously (IV) to juvenile Cynomolgus macaques (non-human primates; NHPs), negative for neutralizing Clade F antibodies, with either formulation buffer (FB) or one of a total of 11 variant AAVHSC (4-7E13 vgs/kg, n = 25 macaques) vectors that contain a gene transfer construct expressing CBA.eGFP. We harvested brains and eyes at 2 weeks post-dose and monitored vector genomes (vgs) by ddPCR and eGFP expression by immunohistochemistry. In addition, two NHPs were dosed subretinally (SR @ 1E12 vgs/eye) with AAVHSC15-CBA.eGFP or FB. Human retinal explants were incubated with FB, or with (2.5E11vgs/explant) AAVHSC15-CMV.eGFP (n = 6), or AAVHSC15.B2M (n=12), a construct that contains homologous arms to edit the human beta-2-microglobulin (B2M) genomic locus.

Results : All AAVHSCs cross the BRB and BBB following a single-IV delivery in NHPs. The majority of AAVHSC capsids transduce retinal cells and retinofugal pathways including the retinogeniculate and retinotectal nuclei. Following a single-SR dose, AAVHSC15 transduces photoreceptors (PRs) and retinal pigment epithelial cells (RPE) in NHPs. Similarly, PRs are transduced in human retinal explants. Using next-generation sequencing, we confirm AAVHSC15.B2M-mediated integration into the human B2M locus.

Conclusions : In NHPs, AAVHSCs exhibit capsid selectivity along key relay points of the visual pathways. AAVHSCs are capable of transduction and genome editing in therapeutically relevant retinal cells based on results obtained in vivo (NHPs) and ex vivo (human). In human organotypic retinal explants, we demonstrate transduction and nuclease-free genome editing in disease relevant cell types.

This is a 2021 ARVO Annual Meeting abstract.