Abstract
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.
Highlights
Duchenne muscular dystrophy (DMD) is a muscle wasting disease affecting 1 out of ~5,000 males worldwide
We extended this work by injecting gene-editing plasmids intramuscularly in a total of six golden retriever muscular dystrophy dog (GRMD) dogs
Expanding upon prior work in the mdx mouse and DMD cultured cells [6,7,8, 10, 17, 33], homology-directed repair (HDR)-mediated gene editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9 or transcription activator-like effector nucleases (TALEN) to restore dystrophin expression was employed for the first time in the phenotypically relevant GRMD model of DMD
Summary
Duchenne muscular dystrophy (DMD) is a muscle wasting disease affecting 1 out of ~5,000 males worldwide. Mutations in the DMD gene eliminate expression of dystrophin protein [1]. SML’s salary was funded by SOLID Biosciences. SOLID Biosciences provided support in the form of salaries for SML, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors are articulated in the ‘author contributions’ section
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