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Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated development of an improved model for metastatic renal cell carcinoma


Clear cell renal cell carcinoma (ccRCC) is responsible for tens of thousands of deaths annually. Immune-based therapies have yielded the only durable responses and some encouraging recent results. There are few immunocompetent murine models for this disease. Attempts to generate a ccRCC model through von Hippel Lindau (VHL) inactivation have produced modest results. None of these studies developed metastatic carcinoma. The RENCA cell line is the major murine immunocompetent model used, but it has wild type VHL. In order to create an improved immunocompetent murine model for ccRCC, we used the clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out VHL in the RENCA cell line. VHL knockout led to morphological and molecular changes indicative of an epithelial-mesenchymal transition (EMT). We went on to show that this leads to enhanced migratory and invasive potential in vitro as well as increased metastasis to the lungs in vivo. Double knockout studies using CRISPR revealed that these changes were hypoxia-inducible factor-1α (HIF-1α)-dependent. In performing our animal studies, we discovered that stable integration of our Cas9-containing expression cassette produced an immune rejection. Use of integrase deficient lentivirus followed by clonal selection allowed us to produce a VHL knockout cell line that was not expressing Cas9. We submitted our cell lines for RNA sequencing (RNA-Seq) and evaluated the genes and pathways that were upregulated upon VHL knockout. We found that the ten most upregulated genes in our cell lines corresponded to poor survival in The Cancer Genome Atlas (TCGA) data set. Further analysis reveals potential genes and pathways worthy of further study. Taken together, the studies presented herein demonstrate CRISPR-mediated disruption of VHL and the generation of a more aggressive, clinically relevant ccRCC model based on the RENCA cell line.

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