Introduction
Prolonged persistence of HIV latently infected cells on antiretroviral therapy (ART) in people with HIV (PWH) serves as a major roadblock towards a cure for HIV infection. CRISPR-Cas13 can effectively degrade viral transcripts and could be exploited to eliminate viral reactivation after cessation of ART. We hypothesised that the programmable, RNA-targeting CRISPR-Cas13 system could knock down the essential HIV Tat protein, thus locking HIV into deep latent state.
Methods
We used a novel lipid nanoparticle (LNP) formulation to successfully deliver a nucleoside-modified Cas13 mRNA and crRNA in HIV latently infected cell lines, JLat A2 and ACH2 and measured Tat mRNA expression by RT-qPCR, and viral reactivation via flow cytometry-based assessment of GFP reporter and viral p24 expression. Additionally, we assessed the ability of Tat-targeting Cas13 mRNA to ablate viral infection in human CD4+ T cells infected with an HIV reporter virus via flow cytometry-based assays.
Results
Potent Tat-targeting crRNA resulted in over 75% silencing of Tat mRNA expression and abrogation of protein expression (n=3) in a transient transfection model. In JLat A2 latency cell model, LNP-delivered Tat-specific Cas13b mRNA led to 80%±5% reduction in Tat mRNA expression and 70%±5% reduction in viral reactivation (n=3). In the ACH2 cell model, Tat-targeting Cas13b mRNA resulted in a modest reduction of 40%±5% in viral p24 expression (n=2). Furthermore, in human CD4+ T cells infected with an HIV reporter virus, Tat-specific Cas13b mRNA treatment, resulted in a 40% reduction in productive infection and increase of 35% in latent population, consistent with enhancement of latent infection.
Conclusions
LNP-delivered Tat-targeting Cas13 mRNA effectively suppresses Tat expression in HIV latency cell models and HIV infected CD4+ T cells, locking HIV into deep latency. Future work will determine the effect of this approach in cells from PWH on ART and whether the suppression persists with cell division.