An Engineered CRISPR System for Boosting Tumor Immunogenicity.

项目总结 癌症免疫监测的发现和癌症免疫治疗的后续发展 代表着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，从而激活免疫系统以 消灭癌症。目前，世界各地正在进行3000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发强烈的免疫反应。各种各样的 已经提出了解释免疫检查点抑制剂耐药性的机制，其中最广泛的 公认的假说以免疫抑制肿瘤微环境为中心，导致 肿瘤特异性T细胞生成不足，功能不全。 激活先天免疫反应，特别是2‘，3’-环GMP-AMP(CGAMP)合成酶刺激物 干扰素基因(cGAS-STING)途径为调控肿瘤微环境提供了一条独特的途径。 CGAS-STING在非免疫细胞和免疫细胞中广泛表达，在非免疫细胞和免疫细胞之间起直接中介作用。 炎症和病原体感染。CGAS-STING的小分子激动剂已被证明 在体外和小鼠模型中刺激肿瘤的免疫原性。然而，小分子不能 以细胞特异性编程，这可能通过以下途径导致长期炎症和自身免疫性疾病 CGAS-STING信号的过度和持续激活。 在这里，我们的目标是通过选择性地激活先天免疫反应来提高肿瘤的免疫原性。 肿瘤微环境。更具体地说，我们将设计一个新型的RNA靶向CRISPR系统，CRISPR- RT，在癌细胞中选择性地和连续地合成RNA：DNA异源双链，这将通过 CGAS，促进cGAMP的合成。通过癌细胞中的内在信号和外在串扰 有了附近的免疫细胞，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。在本提案中，我们将设计CRISPR-RT以启用模板触发 在活细胞中逆转录(AIM 1)，靶向CRISPR-RT以肿瘤特异性转录物为选择性 癌细胞胞浆中RNA：DNA异源双链的合成(AIM 2)及cGAS-STING CRISPR-RT体外激活和小鼠异种移植模型(AIM 2和AIM 3)。我们设想有选择性地 刺激先天免疫反应将通过促进肿瘤的成熟和成熟而改变肿瘤的微环境 CD8+T细胞、自然杀伤细胞、树突状细胞等多种肿瘤反应免疫细胞的浸润。 CRISPR-RT单独作用或与免疫检查点抑制剂联合作用可作为 通过系统地重新连接肿瘤细胞之间的串扰来进行下一代癌症免疫治疗 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.