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

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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.