An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells

Abstract

The current therapies for treating tumors are lacking in efficacy and specificity. Synthetic biology principles may bring some new possible methods for curing cancer. Here we present a synthetic logic circuit based on the CRISPR/Cas9 system. The CRISPR/Cas9 technology has been applied in many biological fields, including cancer research. In this study, the expression of Cas9 nuclease was controlled indirectly by an enhanced hTERT promoter using the GAL4/upstream activating sequence (UAS) binding system. Cas9 was driven by 5XUAS, single guide RNA (sgRNA) was used to target mutant or wild-type HRAS, and the fusion gene GAL4-P65 was driven by the enhanced hTERT promoter. The system was tested in bladder cancer cells (T24 and 5637) and the results showed that the enhanced hTERT promoter could drive the expression of GAL4-P65 in these bladder cancer cell lines. Then all these devices were packed into lentivirus and the results of quantitative real-time PCR showed that the mRNA expression level of HRAS was selectively inhibited in the T24 and 5637 cells. The results of functional experiments suggested that the proliferation, cell migration and invasion were selectively suppressed, and that the apoptosis rate was increased in bladder cancer cells but not in human foreskin fibroblasts (HFF). In conclusion, we successfully constructed an enhanced hTERT promoter-driven CRISPR/Cas9 system and data showed that it could selectively suppress the progression of bladder cancer cells.

Crossroads of telomere biology and anticancer drug discovery

“…TERT promoter-driven activation of the CRISPR/Cas9 system is used for targeting the HRAS gene in bladder cancer cells. 47…”

Telomere Gene Therapy: Polarizing Therapeutic Goals for Treatment of Various Diseases

“…For example, human alpha fetoprotein, carcinoembryonic antigen, and MUC1 promoter can only target subsets of hepatocellular carcinoma, colorectal and lung cancers, or breast cancer, respectively [56]. The most common adaptation of TERT promoter-driven gene therapy simply expresses various therapeutic moieties (anticancer transgenes, miRNA, or the CRISPR/Cas9 system) in a cancer-selective manner [57,58,59,60,61,62].…”

“…Thus, the CRISPR/Cas9 system has emerged as a revolutionary tool for biomedical research, facilitating new possibilities for treating various diseases [69]. In 2017, the first report of a human TERT promoter-driven GAL4/upstream activating sequence (UAS) binding system for selective activation of CRISPR/Cas9 targeting the HRAS oncogene in bladder cancer cells was published [62]. Specifically, human TERT promoter drives GAL4 expression in a cancer-specific manner, and GAL4 subsequently binds to UAS to activate a promoter driving Cas9 nuclease expression, ultimately promoting preferential Cas9 expression in cancer cells through a genetic cascade.…”

Oncolytic Virus-Mediated RAS Targeting in Rhabdomyosarcoma

“…Although a growing number of studies have provided initial evidence that gene-editing technology could be used to target essential cancer genes, 4 , 5 , 6 , 7 several major obstacles remain before a cancer gene-editing therapy could be used in the clinic. The first is the development of high-efficiency gene-editing approaches that can target essential cancer genes with therapeutic efficiency and specificity.…”