Thesis

Improved Plasmid-Based Muscle Gene Therapy

Muscular dystrophies are a group of inherited disorders characterized by progressive muscle weakness, and degeneration. One approach to treatment would be the replacement of the deficient protein via gene therapy. For effective gene therapy, both efficiency of gene delivery and stable expression of the transferred gene are important factors. Our goal was to determine which of the following mammalian expression vectors would be more useful (more stable) for muscle gene therapy; pAcGFP1-C1 and pEPito. We were also interested in switching/substituting both vectors' CMV promoter/enhancer region with the muscle specific promoter, Desmin (DES), to increase their stability for muscle gene therapy. This was accomplished by transfecting C2C12 myotubes with the aforementioned vectors. Both vectors showed relatively continuous GFP expression. Myotubes transfected with pEPito continued to express GFP till day 8. Cells transfected with pACGFP1-C1 also showed continuous GFP expression till day 6. Our results show that both vectors are promising candidates for gene therapy in muscle cells as they maintained stable gene expression of the GFP reporter gene for at least 6 days. Further studies should be done in order to determine the maximum duration that the myotubes would be able to maintain the plasmids and show continuous expression. Future studies could be done to assess stability of these vectors in mice which may lead to future gene therapy trials for muscle disorders and improving gene therapy strategies for other disorders.

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