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

ABSTRACT: 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.

ORGANISM(S): Mus musculus synthetic construct Mus

PROVIDER: GSE269516 | GEO | 2024/12/05

REPOSITORIES: GEO

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Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

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

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