Research Project
10354968
PROJECT SUMMARY The discovery of cancer immunosurveillance and the subsequent development of cancer immunotherapy represent a major breakthrough in clinical oncology. Immune checkpoint inhibitors can block the immunosuppressive interaction between T cells and tumor cells, thereby activating the immune system to eliminate cancer. Currently more than 3,000 clinical trials are undergoing around the world to evaluate T cell modulators, which accounts for approximately 2/3 of all oncology trials. Despite their remarkable clinical efficacy, immune checkpoint inhibitors fail to elicit strong immune response in the majority of cancer patients. A variety of mechanisms have been proposed to explain the resistance to immune checkpoint inhibitors, with the most widely accepted hypothesis centered around an immunosuppressive tumor microenvironment which results in insufficient generation and inadequate function of tumor-specific T cells. Activation of innate immune response, especially the 2'3'-cyclic GMP-AMP (cGAMP) synthase-stimulator of interferon genes (cGAS-STING) pathway, provides a distinct route to manipulate the tumor microenvironment. cGAS-STING is broadly expressed in non-immune and immune cells, serving as a direct mediator between inflammation and pathogen infection. Small-molecule agonists of cGAS-STING have been demonstrated to stimulate tumor immunogenicity both in vitro and in mouse models. However, small molecules cannot be programmed with cell specificity, which may lead to prolonged inflammation and autoimmune disorders through excessive and persistent activation of cGAS-STING signaling. Herein, we aim to boost tumor immunogenicity by activating innate immune response selectively in the tumor microenvironment. More specifically, we will engineer a novel RNA-targeting CRISPR system, CRISPR- RT, to selectively and continuously synthesize RNA:DNA heteroduplex in cancer cells, which will be detected by cGAS and promote synthesis of cGAMP. Through both intrinsic signaling in cancer cells and extrinsic crosstalk with nearby immune cells, type I interferons and other proinflammatory cytokines will be rapidly produced, thereby eradicating tumor cells. In this proposal, we will engineer CRISPR-RT to enable template-triggered reverse transcription in live cells (Aim 1), target CRISPR-RT to cancer-specific transcripts to selectively synthesize RNA:DNA heteroduplex in the cytoplasm of cancer cells (Aim 2), and evaluate cGAS-STING activation by CRISPR-RT in vitro and using mouse xenograft models (Aim 2 and Aim 3). We envision selective stimulation of innate immune response will alter the tumor microenvironment by promoting the maturation and infiltration of various tumor-responsive immune cells such as CD8+ T cells, natural killer cells, and dendritic cells. Acting independently or in combination with immune checkpoint inhibitors, CRISPR-RT may serve as next-generation cancer immunotherapy by systematically rewiring the crosstalk between tumor cells and the immune system.
