An Engineered CRISPR System for Boosting Tumor Immunogenicity.

项目摘要 癌症免疫监视的发现和癌症免疫治疗的后续发展 是临床肿瘤学的重大突破免疫检查点抑制剂可以阻断 T细胞和肿瘤细胞之间的免疫抑制相互作用，从而激活免疫系统， 消除癌症。目前，全世界正在进行3,000多项临床试验，以评估T细胞 调节剂，约占所有肿瘤学试验的2/3。尽管它们具有显著的临床疗效， 免疫检查点抑制剂不能在大多数癌症患者中引起强烈的免疫应答。各种 已经提出了一些机制来解释对免疫检查点抑制剂的抗性，其中最广泛的是 公认的假设围绕免疫抑制肿瘤微环境，导致 肿瘤特异性T细胞的生成不足和功能不足。 先天性免疫反应的激活，尤其是2 '3'-环GMP-AMP（cGAMP）酶刺激剂 干扰素基因（cGAS-STING）途径的研究，提供了一个独特的途径来操纵肿瘤微环境。 cGAS-STING在非免疫细胞和免疫细胞中广泛表达，作为免疫细胞和非免疫细胞之间的直接介导物。 炎症和病原体感染。已证明cGAS-STING的小分子激动剂 在体外和小鼠模型中刺激肿瘤免疫原性。然而，小分子不能 编程与细胞特异性，这可能会导致长期的炎症和自身免疫性疾病， cGAS-STING信号传导的过度和持续激活。 在此，我们的目标是通过选择性地激活肿瘤细胞中的先天免疫应答来增强肿瘤免疫原性。 肿瘤微环境更具体地说，我们将设计一种新的RNA靶向CRISPR系统，CRISPR- RT，以选择性地和连续地在癌细胞中合成RNA：DNA异源双链体，这将通过 cGAS并促进cGAMP的合成。通过癌细胞的内在信号和外在串扰 与附近的免疫细胞一起，I型干扰素和其他促炎细胞因子将迅速产生， 从而根除肿瘤细胞。在这项提案中，我们将设计CRISPR-RT， 在活细胞中逆转录（Aim 1），将CRISPR-RT靶向癌症特异性转录物， 在癌细胞的细胞质中合成RNA：DNA异源双链体（Aim 2），并评估cGAS-STING 通过CRISPR-RT体外活化和使用小鼠异种移植模型（Aim 2和Aim 3）。我们设想选择性的 先天免疫应答的刺激将通过促进成熟改变肿瘤微环境， 各种肿瘤应答性免疫细胞如CD 8 + T细胞、自然杀伤细胞和树突细胞的浸润。 CRISPR-RT可单独或与免疫检查点抑制剂联合作用， 通过系统地重新连接肿瘤细胞之间的串扰的下一代癌症免疫疗法 和免疫系统。

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.