Rational design of immune gene therapy combinations via in vivo CRISPR activation screening of tumor microenvironment modulators

The hostile tumor microenvironment (TME) remains a major challenge for cancer immunotherapy. Here, we performed TME-targeted in vivo CRISPR activation (CRISPRa) screen to identify factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Multiplexed activation of genes encoding antigen presentation, T cell proliferation, co-stimulation, and migration (APCM) leads to enhanced anti-tumor responses. An APCM-focused CRISPRa screen in metastatic tumors identified CD80, TNFSF14, CXCL10, TNFSF18, TNFSF9, and IFNG as top immunostimulatory candidates. Further optimization pinpointed TNFSF9 (4-1BBL) + IFNG + IL12B (4II) as a potent therapeutic combination. AAV-4II enhanced antigen presentation, T cell activation, proliferation, cytotoxicity, and tumor infiltration. Preconditioning the TME with AAV-4II synergized with CAR-T and TCR-T cell therapies to suppress primary and metastatic solid tumors in vivo. These findings establish TME-targeted CRISPRa screening as a rapid route to develop immune gene therapy combinations against solid tumors.