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Cathy Cupples

Fellow – Cathy Cupples

Mentor – Charles Long

Project – Anti-myostatin gene therapy for muscle repair

Objective – To develop a muscle specific promoter capable of expressing green fluorescent protein (GFP) and a short hairpin RNA (shRNA) targeting myostatin in vitro.

Procedure – Recombinant lentiviral vectors (rLV) were designed to contain promoters for either mouse myogenin (mMp), bovine myogenin (bMp), or bovine muscle creatine kinase (bMCKp) and either a control shRNA or an shRNA targeting myostatin. The produced rLV were used to transduce bovine fetal muscle (BFM) cells, C2C12 mouse cells, and bovine fetal fibroblast (BFF) cells. Comparisons were made between promoters based on the amount of fluorescence using a fluorometer and fluorescence microscopy of GFP expression during muscle cell differentiation.

Results – bMp and bMCKp effectively expressed GFP in muscle cells, whereas mMp was ineffective in all cell types tested. bMp expressed GFP mainly in differentiated myotubes in BFM cells, whereas, bMCKp was less specific. The bMp and bMCKp both displayed significantly higher mean GFP intensity in BFM cells when compared to a non-transduced control on day nine of differentiation. bMCKp and bMp were less specific to differentiated myotubes in C2C12 cells and differences in GFP intensity for both promoters when compared to a non-transduced control was less evident on day nine post-differentiation. Both constructs expressed GFP in BFF cells.

Conclusions and Clinical Relevance – The bMp is more effective and specific in myotubes formed from BFM cells than bMCKp and mMp. The lack of specificity of the bMCKp may reflect the length of the promoter sequence isolated and used in this study. The pattern of GFP expression in C2C12 cells from bMp and bMCKp may reflect species differences in promoter usage.

Impact for Human Medicine – Once the most effective muscle specific promoter is determined, rLV can be designed that contain transgenes that target human and animal muscle cells in order to combat muscle damage and enhance muscle repair.