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

Abstract 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, multiplexed activation of pooled immunoregulatory genes encoding a ntigen p resentation, c ytokine, and co-stimulation m olecules (APCM) leads to enhanced anti-tumor immunity. APCM screen in metastatic tumors identifies Cd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9 , and Ifng as the top immunostimulatory candidates. AAV-mediated delivery of these factors individually or in combination shows anti-tumor efficacy across different cancer models. Further optimization pinpoints Ifng + 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 tumors in 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.