A robust gene-editing technology for facioscapulohumeral muscular dystrophy therapy based on CRISPR-Cas13d

Topic:

Pre-Clinical Research

Poster Number: T349

Author(s):

Manal Ali, PhD, Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Kate Neal, Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital, Scott Harper, PhD, Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children’s Hospital

Background
Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating neuromuscular disease affecting roughly 1 in 8,000 individuals. Clinical weakness typically manifests in the second or third decade of life and ~20% of patients ultimately require a wheelchair. FSHD is caused by de-repression of the myotoxic DUX4 gene in muscle, leading to myofiber death. There are currently no cures or therapy for FSHD, so effective treatment is critically needed.
Objective
Given its central role in FSHD, we hypothesize that FSHD therapies should focus on DUX4 inhibition. We are developing CRISPR-based approaches to accomplish this goal and are focused on using Cas13 enzymes to knockdown DUX4 mRNA, instead of the more commonly used Cas9-based systems that cut DNA and pose safety risks for FSHD patients. In this study, we developed a CRISPR-Cas13d system to inhibit DUX4, due to its high activity and small size, which makes it a promising platform for AAV vector delivery systems.
Methods
We designed fifty guide RNAs (gRNAs) targeting DUX4 mRNA and developed a luciferase screening assay to test efficacy in vitro. Specifically, we used a dual luciferase assay in which full-length DUX4 was inserted as the 3’ UTR of Renilla Luciferase. We co-transfected the luciferase reporter plasmid along with Cas13d and gRNA expression cassettes to identify gRNAs with high DUX4 silencing based on potent reduction of Renilla luciferase activity in vitro. We then selected lead gRNAs and confirmed silencing using a DUX4-responsive GFP reporter followed by western blotting to assess reduction of DUX4 protein levels in vitro.
Results
All 50 gRNAs caused significant DUX4 knockdown compared to non-targeting controls in the luciferase assay. We then selected 11 leads, which individually reduced DUX4-activated GFP expression by 58-77%. Using western blot, we further narrowed our lead candidates to select 3 gRNAs, that significantly diminished DUX4 protein production by 80-90% for in vivo efficacy and safety testing in FSHD mouse models.
Conclusions
We conclude that Cas13d is a robust DUX4 RNA-targeting tool in vitro and is a promising strategy for DUX4 mRNA silencing. Animal studies are ongoing.