Multiplexed inhibition of immunosuppressive genes with Cas13d for on-demand combinatorial cancer immunotherapy

We devise Multiplex Universal Combinatorial Immunotherapy via Gene-silencing (MUCIG), as a versatile approach for combinatorial cancer immunotherapy. We harness CRISPR-Cas13d to efficiently target multiple endogenous immunosuppressive genes on demand, allowing us to silence various combinations of multiple immunosuppressive factors in the TME. Intra-tumoral AAV-mediated administration of MUCIG elicits significant anti-tumor activity with several Cas13d gRNA compositions (AAV-Cas13d-MUCIG). Optimization of TME targets by expression analysis led to a simplified off-the-shelf MUCIG targeting a four gene combination (PGGC: Pdl1/Cd274, Galectin9/Lgals9, Galectin3/Lgals3 and Cd47). AAV-Cas13d-PGGC showed significant in vivo anti-tumor efficacy in syngeneic tumor models. Single cell and flow profiling revealed that AAV-Cas13d-PGGC remodeled the TME by increasing CD8+ T cell infiltration and reducing neutrophils. MUCIG thus serves as a universal method to silence multiple immune genes in vivo, and can be delivered via AAV as a therapeutic approach. We devised an approach MUCIG (Multiplex Universal Combinatorial Immunotherapy via Gene-silencing) and designed MUCIG for targeting immunosuppressive genes at different scales of library pools. The first MUCIG pool included 313 genes selected based on these criteria. Subsequently, with a tiered approach, we designed three additional MUCIG pools (pool2: 152 genes; pool3: 55 genes; pool4: 19 genes). The distribution and coverage of gRNAs across all four pools were verified, confirming the robustness and specificity of this approach in the context of cancer immunosuppressive gene targeting therapy.