Rational design of immune gene therapy combinations via in vivo CRISPR activation screening of tumor microenvironment modulators [APCM_Screen_CLCG4I]

The hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targeted in vivo CRISPR activation (CRISPRa) screens to uncover factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Through adeno-associated virus (AAV) delivery, we find that multiplexed activation of pooled immunoregulatory genes encoding antigen presentation, cytokine, and co-stimulation molecules (APCM) leads to enhanced anti-tumor immunity. A CRISPRa screen targeting APCM genes in metastatic tumors identifies CD80, TNFSF14, CXCL10, TNFSF18, TNFSF9, and IFNG as the top immunostimulatory candidates. Further optimization pinpoints 4-1BBL(TNFSF9) + IFNG + IL12B (4II) as a potent streamlined therapeutic combination. AAV delivered 4II has potent in vivo anti-tumor efficacy in multiple tumor models, by enhancing tumor antigen presentation while simultaneously promoting the infiltration, activation, and expansion of anti-tumor T cells. We further demonstrate that APCM therapy synergizes with CAR-T therapy against human solid tumors in vivo. CRISPRa screens and gene activation systems targeting APCM factors thus represent a powerful approach for rapid development of off-the-shelf immune gene therapies against solid tumors. Overall design: To generate metastatic lung tumors and perform in vivo screening under immune pressure, E0771-CLCG4I library cells were injected into 8-12 weeks old female C57BL/6J mice intravenously. E0771-1 and E0771-2 are cells before selection, E0771-Lung1-5 are lung tumors after selection; 6 sgRNA to profile