Rational design of immune gene therapy combinations viain vivoCRISPR activation screen of tumor microenvironment modulators

Abstract

AbstractThe hostile tumor microenvironment (TME) is major challenge for cancer immunotherapies. Here, we design and perform TME-targetedin vivoCRISPR 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, multiplexed activation of pooled immunoregulatory genes encodingantigenpresentation,cytokine, and co-stimulationmolecules (APCM) leads to enhanced anti-tumor immunity. APCM screen in metastatic tumors identifiesCd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9, andIfngas the top immunostimulatory candidates. AAV-mediated delivery of these factors individually or in combination shows anti-tumor efficacy across different cancer models. Further optimization pinpointsIfng+Tnfsf9+Il12b(Il12/Il23)as a potent therapeutic combination, leading to increased IFN-γ+CD8+and tissue-resident memory T cells. APCM therapy synergizes with CAR-T cell therapy against human solid tumorsin vivo. APCM-based CRISPRa screen and gene activation systems can thus be leveraged for the rapid generation of off-the-shelf immune gene therapies against solid tumors.