Progress Update: DUX4 mRNA silencing using CRISPR-Cas13

Lay progress update submitted by Afrooz Rashnonejad
See original grant DUX4 mRNA silencing using CRISPR-Cas13

In this report, we continued to study the ability of our CRISPR-Cas13b system to reduce DUX4 mRNA expression in both cells and animal models. Cas13b and gRNAs strongly reduced DUX4 protein levels in vitro. When tested in FSHD patient derived myoblasts, this system increased the number of viable cells. To increase the efficiency of this treatment we designed a new construct containing multiple gRNAs expressed under different promoters. We also demonstrated that gRNA expression alone is not toxic and will not induce cytokine expression, the molecules that regulate inflammation and immune responses, in human cells. This is an important safety aspect toward translating this therapy to the clinic. Finally, we tested the AAV.CRISPR-Cas13b system in an adult FSHD mouse model via intramuscular injections. Similar to what we detected in vitro, CRISPR-Cas13b successfully targeted DUX4 mRNA, reduced apoptotic nuclei, and protected the muscles from death. To investigate long-term expression of Cas13b and gRNA, another clinically relevant outcome measure, we injected AAV.Cas13b and scAAV.gRNA into mouse muscles and extracted the muscles in various intervals. Interestingly, we detected a continual increase in expression of both genes up to two months. Considering the shorter lifespan of approximately 1.5-2 years and a higher metabolism in mice, 2 months of a mouse life is roughly equivalent to 7-10 years of human life. For the next step, we intend to investigate gene expression at longer time points and perform more preclinical studies to successfully accomplish the main goal of this project, providing more pre-clinical data for faster clinical translation of this therapy.