Abstract 19195: New Insights into CRISPR/Cas9-Mediated Gene Therapy for Dystrophin-Deficient Cardiomyopathy
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophies caused by defective DMD gene, which encodes dystrophin. Dystrophin is a critical muscle protein that connects the cytoskeleton to the basal lamina through its association with the dystrophin-glycoprotein complex (DGC). The absence of dystrophin or other components of the DGC in the heart renders cardiomyocytes more sensitive to mechanical stress, Ca2+ overload and oxidative stress, resulting in progressive myocyte necrosis, cardiac fibrosis, and enlargement of the ventricle and ultimately heart failure. Although significant progress has been made in the last three decades to understand the pathogenesis of these devastating diseases, no effective treatment is currently available for them and there is no therapy to halt their progression into heart failure. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated gene editing technologies offer a promising opportunity to bypass the disadvantages associated with simple gene-replacement strategies. We and other groups recently showed that (CRISPR)-mediated deletion of mutant exon could restore dystrophin expression in the skeletal muscles of a mouse model of DMD and ameliorate the disease, thus highlighting the potential of CRISPR as a promising therapy for DMD-associated cardiomyopathy. However, this gene editing-based therapeutic approach faces some challenges and needs optimization. Therefore, we compared different approaches of delivering the CRISPR-Cas9/gRNA (two gRNA target sites were chosen from intron 20 and 23, respectively) packaged into a recombinant adeno-associated virus (rAAV) to the heart of mdx:utrophin+/– mice. Our immunofluorescence data showed that systemic administration of the CRISPR-Cas9 with gRNA via a systemic retro-orbital approach showed wide-spread restoration of dystrophin expression throughout the heart of mdx:utrophin+/– mice. Furthermore, PCR and western blotting analyses demonstrated that in-frame deletion of the genomic DNA covering exon 23 showed a truncated dystrophin expression in the heart of mdx:utrophin+/– mice. These data provide evidence of restoration of dystrophin expression in the hearts of DMD mice.
Author Disclosures: M.M. El Refaey: None. L. Xu: None. J. Xu: None. Y. Gao: None. R. Han: None.
- © 2016 by American Heart Association, Inc.