Precision targeting of bladder cancer using CRISPR technology

摘要

ABSTRACT Chromosome rearrangements are common in cancers and typically occur early in carcinogenesis. They drive tumor development by altering the expression or function of oncogenes and tumor suppressor genes by copy number alterations, formation of oncogenic fusions or by alterations of DNA elements responsible for regulating cancer genes. Despite being a well-known feature of cancer, discovered over hundred years ago, this common cancer hallmark has not yet been therapeutically exploited. Now, for the first time, technological advancements in whole genome sequencing and the development of CRISPR technologies for precision targeting makes it possible to identify and target chromosome rearrangement junctions (CRJs) in tumor cells. In addition, recent breakthroughs in in vivo delivery of CRIPR reagents involving nanoparticles will open new avenues into clinical utilization. In this R21 application, we present a precision CRISPR approach that aims at targeting cancer-specific CRJs as an untapped Achilles heel common to all tumors. This CRISPR-based precision approach consists of pairs of CRJ-targeting guide RNAs (gRNAs) that bring together two parts of a dCas9-conjugated endonuclease, Fok1, leading to its activation and the induction of lethal DNA double strand breaks (DSBs) specifically in cancer cells. Regardless of the tumor-driving mechanism, the presence of CRJs will be exploited to selectively force these cancer cells to die. We have obtained proof-of-principle of the successful targeting of CRJs with this approach in both colon and bladder cancer cell lines. We have assembled a multi- disciplinary team with expertise in CRISPR technology and design, lipid nanoparticle delivery systems and bladder cancer mouse models. With this team in place and with strong evidence of efficient targeting of CRJs with Fok1-dCas9 in cancer cell lines, we are now primed to develop and test our approach in pre-clinical cancer models using recently developed breakthrough lipid nanoparticle delivery technologies. If successfully implemented, this paradigm-shifting precision- targeting cancer therapeutic platform could be transformative in designing uniform treatments for all cancer types irrespective of the biology of the tumor.