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

项目摘要 本申请是为了响应被标识为NOT-CA的特别利益通知（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 Barrett食管（BE），其特征性地表达高水平的转录因子CDX 2。我们 亲本MPI R 01（R 01 CA 272901）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。本行政补充申请是建立在 R 01项目，同时结合新的CRISPR/Cas 13技术，探索杀死BE和EAC的途径 细胞该应用程序将结合联合收割机博士Que（MPI的R 01赠款，小鼠遗传学）和博士吴 （哥伦比亚癌症中心成员，CRISPR/Cas 13）。Que实验室成功建立了BE模型， 在GEJ处条件性过表达人CDX 2。值得注意的是，浸润性腺癌发生在 当CDX 2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实验室最近报告说，使用CRISPR/Cas 13 d系统的靶RNA敲减 导致人类细胞中整个转录组的附带降解，导致细胞凋亡严重减少。 生存能力和增殖能力。然后，CRISPR/Cas 13的附带活性用于选择性消除 本发明涉及在体外环境中表达报告RNA的胶质母细胞瘤细胞。在本提案中，我们共同旨在测试 RNA引导的癌细胞消除在我们的过表达CDX 2的小鼠模型中的效用。我们假设 靶向BE和EAC细胞中的CDX 2将导致BE和EAC的侧支转录组破坏和杀伤 细胞这是一个原理验证测试，我们制定了两个具体的目标来解决这个假设。目标1： 将在BE和EAC的人类细胞系模型中优化靶向CDX 2 mRNA的CRISPR/Cas 13 d gRNA， 系统地评估靶向gRNA的CDX 2的效率和特异性。在目标2中，我们将提供Cas 13 以及在Aim 1中优化的gRNA对BE和过表达人源化的转基因小鼠的SCJ中的肿瘤的影响。 CDX2。我们将使用该系统来测试我们是否可以选择性地耗尽CDX 2驱动的BE和肿瘤细胞， 使用CRISPR/Cas 13的附带活性。这些研究将为使用 CRISPR/Cas 13治疗BE和EAC的副作用。

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.