Novel macrophage-tropic transmitted/founder SHIV model of CNS persistence to evaluate CRISPR/Cas9 gene editing

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Project Summary/Abstract. An eradicative HIV cure requires safe and effective clearance of replication competent virus from all reservoirs, including the brain. Characterization of the CNS reservoir and empiric testing of novel cure strategies require physiologically relevant animal models of HIV persistence in the brain. Here, we propose to integrate major advances from our groups in SHIV NHP models and AAV-delivered CRISPR/Cas9 editing to delineate key features of the CNS reservoir and determine the efficacy of CRISPR-based eradication in the brain. Transmitted/founder (TF) SHIVs, which encode minimally adapted TF HIV-1 Envs, represent a major advance in biologically relevant NHP models. TF SHIVs have demonstrated robust replication in rhesus macaques, with viral kinetics, cell tropism, and pathogenesis that mirror HIV-1 infection of humans. Further, they recently been shown to faithfully recapitulate virus – host interactions, persist through suppressive ART, and rebound with similar kinetics and clonality as HIV-1. Here, we will employ a novel, genetically barcoded TF SHIV model of CNS pathogenesis and persistence, based on TF SHIV.D.191859 (SHIV.D), which encodes a clade D TF HIV- 1 Env that is CCR5-tropic, efficiently replicates in CD4 T cells and monocyte-derived macrophages, and demonstrates consistent CNS replication, pathogenesis and persistence. Using this barcoded TF SHIV.D model CNS persistence, we will test a novel all-in-one AAV9-mediated CRISPR/Cas9 gene editing system. A recent first-in nonhuman-primate study of SIV-infected rhesus macaques demonstrated the tolerability and efficacy of this approach. The AAV9-CRISPR-Cas9 was broadly distributed across tissues, leading to cleavage and excision of the SIV genome and substantial reductions in the size of the proviral reservoir across tissues. Notably, the AAV was well distributed within CNS resulting in excision of provirus across brain regions. In this application, we will leverage advances in the macrophage-tropic barcoded SHIV.D model and AAV-delivered CRISPR/Cas9 editing to gain insight on CNS neuropathogenesis and persistence. Our hypothesis is that by (i) characterizing SHIV.D persistence in key CNS cells and tissues, (ii) optimizing AAV-delivered CRISPR-Cas9 approaches for SHIV.D persistence in the brain, and (iii) testing the effects of global and myeloid-targeting CRISPR approaches on CNS reservoir reduction in vivo, we will advance prospects for eradicating HIV from the brain. If this hypothesis is affirmed, the significance to HIV cure field would be substantial, since it would improve our understanding of the CNS reservoir, develop a robust model for HIV pathogenesis and persistence in the brain, and provide key pre-clinical data on the safety and efficacy of a promising CRISPR-based cure strategy.