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: 10674490
• 负责人: Jueqi Chen
• 金额: $ 16.4万
• 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/10674490
• 关键词: Affect; Agonist; Animal Model; Architecture; Autoimmune Diseases; Biological Assay; Bone Marrow; CD8-Positive T-Lymphocytes; CRISPR-mediated transcriptional activation; Cancer Model; Cancer Patient; Cell model; Cells; Chimeric Proteins; Clinical; Clinical Oncology; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; 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; Immunooncology; In Vitro; Infection; Infiltration; Inflammation; Inflammatory; Innate Immune Response; Interferon Type I; Kidney; Malignant Neoplasms; Mediating; Mediator; Melanoma Cell; Messenger RNA; Moloney Leukemia Virus; Mus; Names; Natural Killer Cells; Pathway interactions; Production; Proteins; RNA; RNA chemical synthesis; RNA-Directed DNA Polymerase; Repression; 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; cancer cell; cancer immunotherapy; cancer therapy; clinical efficacy; cytokine; design; immune cell infiltrate; immune checkpoint blockade; immune modulating agents; immunogenicity; immunoregulation; 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 eradication; 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.

项目概要 癌症免疫监视的发现以及癌症免疫治疗的后续发展 代表了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。目前全球正在进行超过 3,000 项临床试验来评估 T 细胞 调节剂，约占所有肿瘤学试验的 2/3。尽管其临床疗效显着， 免疫检查点抑制剂无法在大多数癌症患者中引起强烈的免疫反应。各种 已经提出了一些机制来解释对免疫检查点抑制剂的耐药性，其中最广泛的 公认的假设围绕免疫抑制肿瘤微环境，导致 肿瘤特异性 T 细胞生成不足且功能不足。 激活先天免疫反应，尤其是 2'3'-环 GMP-AMP (cGAMP) 合酶刺激剂 干扰素基因（cGAS-STING）途径，提供了操纵肿瘤微环境的独特途径。 cGAS-STING 广泛表达于非免疫细胞和免疫细胞中，作为细胞之间的直接介体 炎症和病原体感染。 cGAS-STING 的小分子激动剂已被证明可以 在体外和小鼠模型中刺激肿瘤免疫原性。但小分子不能 具有细胞特异性，这可能会导致长期炎症和自身免疫性疾病 cGAS-STING 信号传导过度且持续激活。 在这里，我们的目标是通过选择性地激活先天免疫反应来增强肿瘤的免疫原性。 肿瘤微环境。更具体地说，我们将设计一种新型 RNA 靶向 CRISPR 系统，CRISPR- RT，在癌细胞中选择性、连续地合成 RNA:DNA 异源双链体，并通过以下方法检测： cGAS并促进cGAMP的合成。通过癌细胞的内在信号传导和外在串扰 与附近的免疫细胞结合，会迅速产生I型干扰素和其他促炎细胞因子， 从而消灭肿瘤细胞。在本提案中，我们将设计 CRISPR-RT 以实现模板触发 活细胞中的逆转录（目标 1），将 CRISPR-RT 靶向癌症特异性转录物，选择性地 在癌细胞的细胞质中合成 RNA:DNA 异源双链体（目标 2），并评估 cGAS-STING 通过 CRISPR-RT 体外激活并使用小鼠异种移植模型（目标 2 和目标 3）。我们设想选择性 刺激先天免疫反应将通过促进成熟和改变肿瘤微环境 各种肿瘤反应性免疫细胞如CD8+T细胞、自然杀伤细胞和树突状细胞的浸润。 CRISPR-RT 可以单独发挥作用，也可以与免疫检查点抑制剂联合发挥作用。 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疗法 和免疫系统。