Molecular Tools to Evaluate Pathogenesis and Treatment of Canine Muscular Dystrophy

Abstract

Duchenne (DMD) and golden retriever (GRMD) muscular dystrophy are genetically homologous conditions caused by mutations in the DMD gene, with loss of the protein dystrophin. There’s currently no cure for DMD, although some gene therapy approaches have been studied in the GRMD model. However, studies of these potential therapies would be greatly facilitated by a standardized cell line. Moreover, with any treatment, effects must be studied at the subcellular level to identify biomarkers that track with disease.

In this dissertation, two different cluster regularly interspaced short palindromic repeats (CRISPR) guides, A and B, and one set of transcription activator-like effector nucleases (TALEN) were tested with homologous directed repair (HDR) in vitro and in GRMD dogs. Separately, a normal immortalized myoblast cell line (MyoK9) was developed and characterized by studying its RNA and protein expression through differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) was performed on the cell line to characterize the muscle proteome in culture to better quantify dystrophin and other proteins in gene therapy treated GRMD dogs.

Dystrophin expression varied among the CRISPR treatments used, with guide sgRNA A being the most effective at the DNA and protein level in two dogs treated. There was no dystrophin expression in either of the two TALEN treated dogs. No adverse effects were detected in dogs with either CRISPR or TALEN treatments. The immortalized cell line displayed delayed differentiation. However, more than 95% of proteome at the single cell myoblast level was labeled and allowed for muscle specific proteins to be identified. SILAC labeling was performed to quantify proteome changes after gene therapy of GRMD dogs.

Based on these preliminary results, CRISPR is more effective than TALEN when using HDR to edit the DMD gene in GRMD dogs. Our creation and characterization of an immortalized primary canine myoblast cell line, together with SILAC labeling, provide additional tools to quantify dystrophin expression and whole proteome changes in future studies.