Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene

Abstract

Specifically targeting genomic rearrangements and mutations in tumor cells has remained an elusive goal in cancer therapy. Here, we use Cas9-based genome editing to introduce the gene for the pro-drug converting enzyme ‘herpes simplex virus type 1 thymidine kinase’ (HSV1-tk) into the genome of cancer cells that carry unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135-CCDC67 and MAN2A1-FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and gRNAs targeting the breakpoint sequences and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed reduction of tumor burden with no mortality over the observation period. Our results suggest that Cas9-mediated suicide gene insertion might be a viable genotype-specific therapy for cancer.

Gene Therapy for Liver Cancers: Current Status from Basic to Clinics

“…The combinations of oncolytic virotherapy, other chemo-agents, and immune modifiers change the sensitivity of the tumor to the therapeutic options including immune checkpoint inhibition [77]. Thus, the suicide gene must be elicited in a tumor-specific manner using transcriptionally targeted retroviral replicating vectors [78], targeting genomic rearrangement in the tumor by genome-editing approach to insert the suicide gene [79]. One of the promising future targets includes diphtheria toxin A, an immunotoxin, which has been widely used in gene therapy for its roles in protein synthesis inhibition [80].…”

Clipping cancer with CRISPR

“…A separate study in Nature Biotechnology suggests that the CRISPR genomeediting technique can target gene fusions that initiate or accelerate tumor growth and invasiveness . To date, researchers have tallied thousands of cancer‐associated gene fusions.…”

“…A separate study in Nature Biotechnology suggests that the CRISPR genomeediting technique can target gene fusions that initiate or accelerate tumor growth and invasiveness. 3 To date, researchers have tallied thousands of cancer-associated gene fusions. "That certainly is providing us a thesaurus of targets to treat cancer patients," says study coauthor Jianhua Luo, MD, PhD, professor of pathology at the University of Pittsburgh School of Medicine and director of its High Throughput Genome Center.…”

The fourth annual BRDS on genome editing and silencing for precision medicines

“…This system has revolutionized targeted genome editing and hold potential to give rise to an entirely new class of therapeutics. Due to its precise specificity, the system can be applied to target genomic-breakpoints at any desired location [7], However, the off-target issues related to CRISPR-Cas system has been a concern, at least for using it as a therapeutic tool. Therefore, introducing a method to study the genome-wide off-target effects of CRISPR-Cas system is very promising.…”