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

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.

Grant Number: 5R01MH128155-05
NIH Institute/Center: NIH
Principal Investigator: Katharine Bar