An Engineered CRISPR System for Boosting Tumor Immunogenicity.

用于增强肿瘤免疫原性的工程 CRISPR 系统。

• 批准号: 10354968
• 负责人: Jueqi Chen
• 金额: $ 24.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354968
• 关键词: Affect; Agonist; Animal Model; Architecture; Autoimmune Diseases; Biological Assay; Bone Marrow; CD8-Positive T-Lymphocytes; Cancer Model; Cancer Patient; Cell model; Cells; Chimeric Proteins; Clinical; Clinical Oncology; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Custom; Cyclic GMP; Cytoplasm; Cytosol; DNA; Dendritic Cells; Development; Embryo; Engineering; Foundations; Gap Junctions; Gene Activation; Generations; Growth; Guide RNA; Heteroduplex DNA; Human; Immune; Immune checkpoint inhibitor; Immune response; Immune signaling; Immune system; Immunologic Surveillance; Immunomodulators; Immunooncology; In Vitro; Infection; Infiltration; Inflammation; Innate Immune Response; Interferon Type I; Kidney; Lead; Malignant Neoplasms; Mediating; Mediator of activation protein; Melanoma Cell; Messenger RNA; Moloney Leukemia Virus; Mus; Names; Natural Killer Cells; Pathway interactions; Production; Proteins; RNA; RNA chemical synthesis; RNA-Directed DNA Polymerase; Resistance; Reverse Transcription; Route; Series; Signal Transduction; Specificity; Stimulator of Interferon Genes; System; T-Lymphocyte; Testing; Therapeutic; Tissues; Transcript; Tumor Suppression; Tumor-infiltrating immune cells; United States Food and Drug Administration; Xenograft Model; anti-cancer; base; cancer cell; cancer immunotherapy; cancer therapy; cell type; clinical efficacy; cytokine; design; immune checkpoint blockade; immunogenicity; in vivo; individual patient; innovative technologies; interest; mouse model; neoplastic cell; next generation; novel; nuclease; oncology trial; pathogen; personalized cancer therapy; recruit; small molecule; success; therapeutic development; tumor; tumor growth; tumor microenvironment; tumor xenograft; tumor-immune system interactions

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.

项目摘要 发现癌症免疫监测和随后的癌症免疫疗法的发展 代表临床肿瘤学的重大突破。免疫检查点抑制剂可以阻止 T细胞与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，全球正在进行3,000多次临床试验以评估T细胞 调节器，大约占所有肿瘤学试验的2/3。尽管它们具有显着的临床功效，但 免疫检查点抑制剂无法引起大多数癌症患者的强烈免疫反应。各种各样 已经提出了解释对免疫检查点抑制剂的耐药性的机制，最广泛 接受的假设以免疫抑制性肿瘤微环境为中心，这导致 肿瘤特异性T细胞的产生不足和功能不足。 先天免疫反应的激活，尤其是2'3'-循环GMP-AMP（CGAMP）合成酶刺激剂 干扰素基因（CGAS-STING）途径提供了一个独特的操纵肿瘤微环境的途径。 CGAS刺在非免疫和免疫细胞中广泛表达，作为直接介体 炎症和病原体感染。已经证明了CGAS丁的小分子激动剂 在体外和小鼠模型中刺激肿瘤免疫原性。但是，小分子不能 通过细胞特异性编程，这可能会导致长时间的炎症和自身免疫性疾病 CGAS插曲信号的过度和持续激活。 在此，我们旨在通过选择性地激活先天免疫反应来提高肿瘤免疫原性 肿瘤微环境。更具体地说，我们将设计一种新颖的RNA靶向CRISPR系统，CRISPR- RT，有选择地，连续合成RNA：癌细胞中的DNA异封纸，将通过 CGA并促进CGAMP的合成。通过癌细胞和外部串扰的固有信号传导 随着附近的免疫细胞，I型干扰素和其他促炎细胞因子将迅速产生， 从而消除肿瘤细胞。在此建议中，我们将设计CRISPR-RT以启用模板触发 活细胞中的逆转录（AIM 1），将CRISPR-RT靶向癌症特异性转录本 综合RNA：癌细胞细胞质中的DNA异源动链（AIM 2），并评估CGAS刺 CRISPR-RT在体外和使用小鼠异种移植模型（AIM 2和AIM 3）激活。我们设想选择性 刺激先天免疫反应将通过促进成熟和 各种肿瘤反应性免疫细胞（例如CD8+ T细胞，天然杀伤细胞和树突状细胞）浸润。 CRISPR-RT独立行动或与免疫检查点抑制剂结合起来，可以作为 下一代癌症免疫疗法通过系统地重新布线肿瘤细胞之间的串扰 和免疫系统。