Depletion of Barrett's and Esophageal Adenocarcinoma Cells with CRISPR/Cas13d

使用 CRISPR/Cas13d 消除 Barrett 细胞和食管腺癌细胞

• 批准号: 10831233
• 负责人: Jianwen Que
• 金额: $ 16.45万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10831233
• 关键词: Address; Adenocarcinoma; Adenocarcinoma Cell; Administrative Supplement; Barrett Esophagus; Basal Cell; CDX2 gene; CRISPR/Cas technology; Cancer Center; Carcinogens; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Survival; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Country; Cytomegalovirus; Data; Defect; Development; Dysplasia; Epithelium; Esophageal Adenocarcinoma; Esophagogastric Junction; Foundations; Genetically Engineered Mouse; Glioblastoma; Goals; Grant; Growth; Guide RNA; Human; Human Cell Line; In Vitro; Incidence; Malignant Neoplasms; Malignant neoplasm of esophagus; Measures; Mediating; Messenger RNA; Methylnitrosourea; Modality; Modeling; Organoids; RNA; RNA Degradation; RNA Interference; Reporter; Reporting; Role; Specificity; Stomach; System; Techniques; Testing; Transcript; Transgenic Mice; cancer cell; cancer type; cell growth; design; in vivo; insight; interest; knock-down; member; mouse genetics; mouse model; neoplastic cell; novel; overexpression; response; stem cells; tool; transcription factor; transcription factor CDX2; transcriptome; tumor

Project Summary This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. The incidence of esophageal adenocarcinoma (EAC) has increased dramatically in Western countries and become the dominant esophageal cancer type in the U.S. The rapid increase in EAC is associated with Barrett’s esophagus (BE) which characteristically express high levels of the transcription factor CDX2. Our parental MPI R01 (R01CA272901) aims to address how stem/progenitor cells are involved in the formation of BE and EAC at the gastroesophageal junction (GEJ). This Administrative Supplement application is built on the R01 project while incorporating a novel CRISPR/Cas13 technique to explore avenues for killing BE and EAC cells. The application will combine the expertise of Dr. Que (MPI of the R01 grant, mouse genetics) and Dr. Wu (Columbia Cancer Center member, CRISPR/Cas13). The Que lab has successfully established a BE model by conditionally overexpressing human CDX2 at the GEJ. Significantly, invasive adenocarcinoma developed in the GEJ when CDX2 overexpression is combined with the treatment of the carcinogen N-methyl-N-nitrosourea (MNU). Intriguingly, the Wu lab recently reported that target RNA knockdown using a CRISPR/Cas13d system caused collateral degradation of the entire transcriptome in human cells, resulting in severe decrease in cell viability and proliferation. The collateral activity of the CRISPR/Cas13 was then used for selective elimination of glioblastoma cells expressing a reporter RNA in an in vitro setting. In this proposal, we together aim to test the utility of RNA-guided cancer cell elimination in our mouse models that overexpress CDX2. We hypothesize that targeting CDX2 in BE and EAC cells will lead to collateral transcriptome destruction and killing of BE and EAC cells. This is a proof-of-principle test and we formulate two specific aims to address the hypothesis. In Aim 1, we will optimize CRISPR/Cas13d gRNAs targeting CDX2 mRNA in human cell line models of BE and EAC and systematically evaluate the efficiency and specificity of CDX2 targeting gRNAs. In Aim 2, we will deliver Cas13 and the gRNA optimized in Aim 1 to BE and tumors in the SCJ of the transgenic mice that overexpress human CDX2. We will use the system to test whether we can selectively deplete CDX2-driven BE and tumor cells by using the collateral activity of CRISPR/Cas13. These studies will provide important findings for using the collateral activity of CRISPR/Cas13 for treating BE and EAC.

项目摘要 本申请是为了回应被确认为非CA-CA的特殊利益通知(NOSI)而提交的 23-045.食管腺癌(EAC)的发病率在西方国家急剧上升 并成为美国主要的食道癌类型。EAC的快速增加与 巴雷特食道(BE)，其特征是高水平表达转录因子CDX2。我们的 父母MPI R01(R01CA272901)旨在解决干细胞/祖细胞如何参与 BE和EAC位于胃食道交界处(GEJ)。此管理补充应用程序构建于 R01项目，同时采用新的CRISPR/Cas13技术来探索杀死BE和EAC的途径 细胞。该应用程序将结合Que博士(R01基金的MPI，小鼠遗传学)和Wu博士的专业知识 (哥伦比亚癌症中心成员，CRISPR/Cas13)。QUE实验室通过以下方式成功地建立了BE模型 在GEJ有条件地过表达人CDX2。值得注意的是，侵袭性腺癌发生在 CDX2过度表达与致癌物N-甲基-N-亚硝脲联合治疗的疗效观察 (MNU)。有趣的是，吴的实验室最近报告说，使用CRISPR/Cas13d系统靶向RNA击倒 导致人类细胞中整个转录组的侧枝降解，导致细胞严重减少 生存和增殖能力。然后，CRISPR/CAS13的附属活动被用于选择性地消除 体外实验中表达报告RNA的胶质母细胞瘤细胞。在这项提案中，我们共同致力于测试 在过度表达CDX2的小鼠模型中，RNA引导的癌细胞消除的实用性。我们假设 在BE和EAC细胞中靶向CDX2将导致BE和EAC的侧枝转录组破坏和杀伤 细胞。这是一个原则证明测试，我们制定了两个具体的目标来解决这个假设。在目标1中，我们 将在人BE和EAC细胞模型中优化针对CDX2 mRNA的CRISPR/Cas13d gRNAs和 系统评价CDX2靶向gRNA的效率和特异性。在目标2中，我们将交付Cas13 和目标1中优化的grna是和过表达人的转基因小鼠SCJ中的肿瘤。 CDX2。我们将使用该系统来测试我们是否可以通过以下方式选择性地消耗CDX2驱动的BE和肿瘤细胞 利用CRISPR/Cas13的抵押品活动。这些研究将为使用 CRISPR/Cas13治疗BE和EAC的侧支活性。