O.8DUX4 mRNA silencing with CRISPR-Cas13 gene therapy as a prospective treatment for Facioscapulohumeral muscular dystrophy

Abstract

DUX4 de-repression causes Facioscapulohumeral muscular dystrophy (FSHD), which is associated with progressive muscle wasting and weakness that may lead to wheelchair dependence. There are currently no treatments that alter the course of FSHD and therapy development is an unmet need in the field. DUX4 genome editing using the CRISPR-Cas9 approach is promising but also has challenges, partly because the DUX4 gene is present in potentially hundreds of copies, embedded within identical or nearly identical D4Z4 repeats. The CRISPR-Cas13 system does not cleave DNA, but can be specifically directed to a RNA transcript of interest using a sequence-specific guide RNA (gRNA), thereby reducing the risk of permanent DNA damage. Here, the goal is to develop a new Cas13/CRISPR-mediated DUX4 mRNA silencing method as an effective therapeutic strategy for FSHD, without altering host cell DNA. We designed several Cas13b-gRNAs targeting different parts of DUX4 mRNA and demonstrated their ability to suppress DUX4 and prevent cell death in vitro and in vivo. We detected >90% reduction of DUX4 protein in treated cells, and improved viability in these cells. DUX4 mRNA levels were markedly decreased in treated FSHD myotubes, as determined by RNAscope in situ hybridization. This reduction was accompanied by complete or near complete reduction in levels of DUX4-responsive biomarkers in treated patient myoblasts/myotubes. In our first in vivo studies in tibialis anterior muscles of TIC-DUX4 mouse model, our lead constructs caused nearly 100% reduction of DUX4 expression. As of this writing, additional in vivo studies are underway, including testing the capabilities of our AAV-delivered CRISPR/Cas13 system to suppress DUX4 long-term in our new TIC-DUX4 mouse model, and improve histopathological, functional, and molecular outcomes. This study provides proof of concept for silencing DUX4 expression using the RNA targeting CRISPR-Cas13b system and has implications for future FSHD therapy.