Chronic exposure to HIV antigens in people living with HIV leads to exhaustion of HIV-specific CD8 T cells, primarily driven by upregulation of the immune checkpoint programmed death-1 (PD-1), resulting in reduced immunological function and compromised ability to achieve immune-mediated control of HIV. To reverse HIV-specific T-cell exhaustion, we aim to use the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas13b ortholog to transiently reduce expression of PD-1. Cas13b is a programmable RNA nuclease that complexes with guide RNAs to mediate specific mRNA knockdown inside cells.
We designed guide RNAs predicted to bind the PD-1 mRNA sequence, and co-delivered the CRISPR-Cas13b mRNA and PD-1-targeting guide RNA via a novel lipid nanoparticle (LNP) formulation that efficiently delivers mRNA to resting T cells, called LNP-X. To quantify the silencing potency of the PD-1 gRNA, we assessed the downregulation of endogenous PD-1 expression in cell lines via flow cytometry. To further increase PD-1 knockdown, we evaluated the effects of Cas13b timing and dosage.
In initial experiments in 293T cells transfected with a PD-1-expressing plasmid, we identified a guide RNA capable of degrading PD-1 mRNA. Subsequently, we found that Cas13b complexed with this guide RNA targeting PD-1 leads to ~50% reduction in PD-1 expression compared to a non-targeting control guide RNA in the Jurkat T-cell line. Knockdown peaked at 48-hours, and decreased to ~20% knockdown at 96-hours. This reduction was dose-dependent, increasing with larger RNA dose. No increased PD-1 knockdown was observed following multiple dosing of Cas13b LNPs.
Our findings confirm that CRISPR-Cas13b can degrade PD-1 mRNA and reduce PD-1 expression on T cells. The transient nature of this knockdown decreases the risk of immune-mediated adverse events, thereby demonstrating its potential as a novel HIV immunotherapeutic strategy. Ongoing experiments will focus on examining the impact of this knockdown in primary T-cells and on T-cell function.