Transcriptional inactivation of the CNS HIV reservoir with Tat-targeting lipid nanoparticles.

Project Summary: Despite the success of antiretroviral therapy (ART), HIV remains an incurable infection, due to the presence of a persistent viral reservoir that is resistant to ART. This reservoir is widely disseminated throughout the body, including in solid tissues such as the central nervous system (CNS) where microglial cells constitute the primary reservoir. New cure approaches are needed that promote permanent silencing or inactivation of viral gene expression. In particular, the viral protein Tat plays a central role in driving viral gene expression, and secretion of Tat from infected microglia likely contributes to CNS toxicity and HIV-associated neurocognitive disorder (HAND). Thus, irreversibly inhibiting or inactivating Tat in brain resident microglia could represent an effective way to block viral reactivation and to limit virus-induced HAND. However, no antiviral drugs that target Tat or viral transcription are currently available for clinical use. In this proposal we aim to develop microglia-targeting lipid nanoparticles (LNPs) containing Cas9/sgRNA complexes that inactivate the viral Tat gene, leading to loss of Tat expression and permanent silencing of HIV proviruses. In a key breakthrough, we have recently identified a novel LNP formulation (MG-LNP) that is optimized for biomolecule delivery to microglia and we have demonstrated successful use of this formulation for CNS gene targeting in mice. MG- LNPs containing Cas9/sgRNAs against Tat will first be optimized in microglial models of HIV infection (Aim 1), followed by testing in ex vivo brain samples from SIV infected macaques (Aim 2). Finally, we will use a novel humanized mouse model of HIV CNS/microglia infection to test this approach in vivo, in the presence or absence of cannabinoid exposure (Aim 3). If successful, these tools will prevent the microglial reservoir from reactivating after therapy interruption and will also mitigate residual CNS pathogenesis from Tat expression during therapy.