Combinatorial gene therapy as an intervention for Duchenne muscular dystrophy

© 2015 Dr. Timothy Douglas ColganDuchenne muscular dystrophy (DMD) is a disease that deprives young children of the ability to walk and considerably reduces their quality of life. DMD is a progressive disorder with affected toddlers having difficulty in standing, children in early primary school struggling to keep up with their peers and teenage patients being confined to a wheelchair. Current medical practices using drug-based interventions, nutritional supplementation and exercise have helped reduce some of the symptoms of DMD but not address the underlying problem. Given the need for effective interventions, molecular therapies offer promise. Gene-based interventions can be developed to directly address the underlying pathology of the disease or treat the secondary pathologies. Therapeutics for DMD have been studied intensively, largely focusing on efforts to replace dystrophin, the gene mutated in this debilitating disease, and on building muscle strength to improve quality of life. The research described in this thesis unites one of the most efficient gene delivery methods, recombinant adeno-associated viruses (rAAVs), with genes that facilitate increases in muscle strength and lifespan, in a mouse model predicted to reflect the clinical severity of DMD, the severely affected dystrophin-/-:utrophin-/- double knockout (or dko) mouse. This approach uniquely combines two genes, microdystrophin and follistatin, to provide independent benefits to dystrophic muscles. Evaluating the therapeutic advantages of this approach will help underpin future clinical trials. The working hypothesis was that combinatorial treatment of severely dystrophic mice with both microdystrophin and follistatin would improve muscle morphology and function greater than the improvements conferred through microdystrophin alone. Since the effects of follistatin treatment alone in dystrophic muscles are unknown, this was first investigated at the intramuscular level in three strains: healthy, mildly dystrophic (mdx) and severely dystrophic (dko) mice, by administering an AAV carrying follistatin. While healthy and mildly dystrophic mice responded with increased myofibre diameter (168% and 114%, respectively), severely dystrophic dko mice exhibited an increase in myofibre number (33%), an unexpected result that accounted for their increase in muscle mass despite an unchanged mean myofibre diameter. Initial investigation into whole body physiological effects by systemically administrating follistatin resulted in an increased proportion of mice unable to reach experimental endpoint. We hypothesised that stabilising the myofibres with microdystrophin first, prior to inducing follistatin expression, would make the mouse healthier and circumvent this obstacle. Follistatin expression was delayed using an inducible promoter. This regulatory element controlling follistatin expression was manipulated by doxycycline, with the presence of doxycycline enabling gene transcription. The combined delivery of inducible follistatin and microdystrophin increased myofibre diameter in severely dystrophic dko mice, paralleling the response of mildly dystrophic mdx mice to an intramuscular delivery of follistatin. Additionally, peak force of tibialis anterior muscles when assessed in situ was increased to a level greater than that of dystrophic muscles treated solely with microdystrophin. While increasing muscle strength is an important aspect in potential therapies for DMD, there are currently few reliable methods to evaluate whether these treatments have any meaningful outcomes in terms of quality of life and survival. Here, we initiated a program of non-invasive assessments of body composition, gait and speed disturbances, and heart arrhythmia of dystrophic mice between 4-16 weeks of age, which revealed that the ability of a mouse to maintain locomotor speed can be a predictor of early death. This parameter can be used alongside other tests for future modelling of DMD therapeutics. In conclusion, follistatin has therapeutic potential in severely dystrophic mice, a finding that has significant translatable potential to ameliorate the DMD pathology and improve patient quality of life. Furthermore, a dual therapy of microdystrophin and delayed follistatin expression, which has advantages over delivering either gene alone, could prolong mobility and independence of DMD patients