Merkel Cell Carcinoma Therapeutic and Diagnostic Strategies Using CRISPR

Abstract

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen gene expression, post integration (~80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-based gene-editing to develop therapeutic and diagnostic tools for MCV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCV T antigens, we designed 13 gRNAs targeting the T antigen genomic region. We validated the MCV TAg targeting efficiency of our gRNAs by in vitro cleavage assays. To translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines as well. Our proof-of-concept study shows that 2 MCV targeting CRISPR/Cas gRNAs in combination can knock out MCV T antigen, thus, being of therapeutic importance. This CRISPR system can be potentially delivered in vivo using Adenovirus associated vectors (AAV) for advancing MCV positive MCC treatment in the future. To a pathologist both MCV positive and negative tumors are identical. However, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this knowledge of important diagnostic and prognostic value. Hence, we used the DETECTR method, pioneered by Chen et al., 2018, to create an in vitro Cas12a based molecular diagnostic test for MCV. Briefly, when RNA-guided Cas12a binds target MCV dsDNA it also leads to indiscriminate single-stranded DNA reporter cleavage. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. We show that DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and efficiently. This rapid MCV DNA detecting system is promising and can be coupled with histopathological and immunohistochemical studies to diagnose the viral status in MCC