The objective of this project was to evaluate the alleviation of muscle wasting phenotypes by hepatic delivery and expression of a recombinant mutated myostatin propeptide mini gene in wild type mice. Initial work focused on using naked plasmid DNA as a vector for delivery of the therapeutic gene. Various β-galactosidase reporter plasmids were tested including pCMVβ, pCAGGSβ, pA2β and a liver specific pSV40 Albuminβ for systemic gene transfer to liver tissue. Following hydrodynamic intravenous administrations, pCAGGSβ proved to be the optimum plasmid and a cDNA encoding a mutated form of the myostatin propeptide (ProFcD/A) was subsequently cloned into a CAGG based plasmid (pAAVCAGGProFcD/A). The expression and bioactivity of the propeptide were assessed using in vitro cell proliferation experiments which showed the ProFcD/A was capable of increasing both proliferation and differentiation in a range of cell types. An initial in vivo study involved intravenous hydrodynamic injections of pAAVCAGGProFcD/A into MF-1 male mice and monitoring growth rates over time. A significant increase was observed in growth rate of the treated mice compared to controls with an accompanying increase in tibialis anterior (TA) muscle mass. Fibre analysis revealed pAAVCAGGProFcD/A treated mice to contain significantly larger fibres than controls owing to muscle hypertrophy. Transgene product levels were detected strongly 7 days post injection in the serum, which declined steadily over the following 21 days. By day 28, levels of transgene product were barely detectable. There was no indication response to either the vector or transgene product. The pAAVCAGGProFcD/A was then used to produce an AAV serotype 8 viral vector containing the ProFcD/A gene (AAV8ProFc) and assessed in initial intravenous tail vein injections of AAV8ProFc into MF-1 male mice. Growth rates were measured over four weeks with a significant increase in the AAV8ProFc treated mice compared to the pCAGG empty plasmid controls. In addition, significant weight increases in both TA and gastrocnemius muscles were seen in AAV8ProFc treated mice over the pCAGG control mice at week 4. Serum showed high levels of transgene expression after 7 days which was sustained up to 28 days post injection with no indication of immune response. Levels of the myogenic protein MyoD also remained higher in AAV8ProFc than pCAGG treated mice. Future studies may involve intravenous injection of both pAAVCAGGProFcD/A and AAV8ProFc into mdx mice, with subsequent monitoring of growth rates, creatine kinase levels, tissue necrosis and muscle strength over time.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:497826 |
| Date | January 2008 |
| Creators | Kawar, Susannah Louise |
| Publisher | Royal Holloway, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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