In Vivo Gene Editing and Immunotherapy

T cells became the central focus of new cancer therapeutics. Immune checkpoint inhibitors targeting T cell signaling pathways and cellular therapeutics utilizing chimeric antigen receptors (CARs) have shown success in the clinic. Discovery of previously unknown genes that modulate T cell function is of urgent need to open different avenues for immunotherapies, as a large fraction of patients still do not respond to, or have undesired side effects to currently approved ones. Systematic approaches to identify new regulators of T cell functions in vivo can provide potentially orthogonal or complementary opportunities. We recently performed in vivo CRISPR screens in CD8 cytotoxic T cells, both in a genome-scale and in a focused manner, in tumor models of immunotherapy (Dong et al. 2019 Cell). Furthermore, we developed a novel AAV-SleepingBeauty system that enhanced the power of genetic screening in primary T cells (Ye et al. 2019 Nature Biotechnology). Our screen re-discovered prime immunotherapy targets such as PD-1, TIM-3 and LAG3, as well as previously undocumented targets. We characterized novel targets such as DHX37, ODC1, MGAT5 and PDIA3 for their activity in mouse and human CD8 T cells including CAR-Ts.

Current major types of immunotherapy include checkpoint blockade, adoptive cell transfer, human recombinant cytokines, and cancer vaccines. However, immunotherapy has met challenges in immunologically cold tumors. These challenges urge for new types of immunotherapies that are more potent and potentially less toxic. Recently, we have developed CRISPRa-mediated Multiplexed Activation of Endogenous Genes as an Immunotherapy (MAEGI) (Wang et al. 2019 Nature Immunology, in press). The CRISPR activation (CRISPRa) system uses a catalytically inactive Cas9 (dCas9), enabling simple and flexible gene expression regulation through dCas9-transcriptional activators paired with single guide RNAs (sgRNAs). This enables precise targeting of large gene pools of endogenous genes in a flexible manner. We demonstrate that MAEGI has therapeutic efficacy across three tumor types. Mechanistically, our preliminary work showed that MAEGI treatment elicits anti-tumor immune responses by recruiting effector T cells and remodeling the tumor microenvironment. We will perform advanced development, characterization and optimization of MAEGI, as a novel immune-gene therapy approach to elicit a potent and specific immune response to tumors based on their unique genetic composition.