Direct and Indirect Targeting of HIV Tat for Selective RNA Degradation and Protein Suppression - Project Summary/Abstract: The persistence of latent reservoirs of HIV-infected CD4+ T cells presents a critical barrier to achieving a cure. These reservoirs harbor replication-competent HIV proviruses that evade immune detection and viral cytopathic effects, enabling long-term survival despite antiretroviral therapy (ART). Innovative strategies are needed to disrupt latency, suppress viral protein expression, and selectively eliminate infected cells. We aim to develop and evaluate novel CRISPR-Cas13-based therapeutic approaches to target HIV-infected cells and sensitize them to cell death through direct viral RNA degradation and host pathway modulation. The overarching goal of this study is to evaluate and optimize a dual-pronged approach combining CRISPR-Cas13d-mediated degradation of HIV Tat mRNA and CRISPR-Cas13b-mediated targeting of host PI3K pathway components to sensitize infected cells to apoptosis. The specific aims are as follows: AIM1. Assess the efficiency and specificity of CRISPR-RfxCas13d in degrading Tat mRNA in CD4+ T cells from people living with HIV on suppressive ART. We will use a lipid nanoparticle (LNP)-based delivery platform capable of transfecting CD4+ T cells with high efficiency. Single-cell RNA-seq (HIV-seq), and digital PCR will measure changes in HIV RNA and protein expression. Additionally, novel Cas13 orthologs with enhanced collateral activity will be characterized and test their ability to selectively deplete HIV-infected cells. AIM2. Develop strategies to sensitize HIV-infected cells to apoptosis through PI3K pathway targeting. The PI3K pathway plays a key role in maintaining HIV latency and infected cell survival. Using CRISPR-Cas13b, this aim will target specific PI3K effectors, such as PI3K p110, p110 and TNFAIP8, to induce apoptosis in infected cells. Synergistic effects of PI3K inhibition and Cas13d-mediated Tat mRNA degradation will be evaluated to determine the optimal strategy to test in an animal model. AIM3. Validate the efficacy of the combined approach in HIV-infected humanized mice. The candidate therapeutic strategy will be tested in a humanized mouse model of HIV infection (HIV-HIS) allowing for preclinical evaluation of Tat RNA and protein degradation, reservoir reduction, and delay in viral rebound following ART cessation. LNP-delivered CRISPR constructs will be optimized for dose and targeting specificity and their effects on HIV RNA, protein expression, and reservoir size will be characterized. This study integrates cutting-edge RNA-editing technology with enhanced activity, leveraging a novel LNP platform, to disrupt HIV latency and promote infected cell elimination. The work will advance our understanding of mechanisms of Tat mRNA degradation and PI3K inhibition for synergistic viral suppression and reservoir reduction. This project has the potential to significantly advance curative strategies for HIV by developing a novel therapeutic approach that directly targets both viral and host factors critical for latency and persistence, offering a promising pathway toward durable HIV remission.
