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.

项目概要 本申请是为了响应被识别为 NOT-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增加。 并成为美国主要的食管癌类型。EAC 的快速增加与 巴雷特食管 (BE) 的特征是表达高水平的转录因子 CDX2。 亲本 MPI R01 (R01CA272901) 旨在解决干/祖细胞如何参与形成 胃食管交界处 (GEJ) 的 BE 和 EAC 本行政补充应用程序基于 R01项目同时结合了一种新颖的CRISPR/Cas13技术来探索杀死BE和EAC的途径 该应用程序将结合 Que 博士（R01 资助的 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 实验室已成功建立了 BE 模型。 显着地，在 GEJ 处条件性地过度表达人 CDX2。 当 CDX2 过表达与致癌物 N-甲基-N-亚硝基脲治疗相结合时 GEJ (MNU) 有趣的是，Wu 实验室最近报道了使用 CRISPR/Cas13d 系统进行靶向 RNA 敲除。 引起人类细胞中整个转录组的附带降解，导致细胞数量严重减少 然后利用 CRISPR/Cas13 的附带活性来选择性消除 在本提案中，我们共同致力于测试在体外表达报告基因 RNA 的胶质母细胞瘤细胞。 在我们过度表达 CDX2 的小鼠模型中，RNA 引导的癌细胞消除的效用。 在BE和EAC细胞中靶向CDX2将导致BE和EAC的附带转录组破坏和杀死 这是一个原理验证测试，我们在目标 1 中制定了两个具体目标。 将在 BE 和 EAC 人类细胞系模型中优化靶向 CDX2 mRNA 的 CRISPR/Cas13d gRNA， 明确评估 CDX2 靶向 g​​RNA 的效率和特异性 在目标 2 中，我们将提供 Cas13。 以及在 Aim 1 中优化为 BE 的 gRNA 以及过度表达人类的转基因小鼠 SCJ 中的肿瘤 我们将使用该系统来测试是否可以通过以下方式选择性地消除 CDX2 驱动的 BE 和肿瘤细胞。 利用 CRISPR/Cas13 的附带活性，这些研究将为使用 CRISPR/Cas13 提供重要的发现。 CRISPR/Cas13 治疗 BE 和 EAC 的附带活性。