Engineering and optimisation of mini-dystrophin constructs for Duchenne muscular dystrophy gene therapy

Abstract

Muscular dystrophies (MDs) are inherited disorders characterised by muscle weakness and atrophy. One of the most severe forms is Duchenne muscular dystrophy (DMD) which together with the milder allelic form Becker muscular dystrophy (BMD) are known as the dystrophinopathies and result from defects in the X-linked gene encoding dystrophin. Dystrophin is a structural protein of the muscle that connects the internal cytoskeleton of muscle fibres to the extracellular matrix. DMD is also amongst the most common forms of muscular dystrophy, affecting ~1 in 4000 live male birth and manifests as rapidly progressive muscle degeneration leading to loss of ambulation and death in the second or third decade from respiratory or cardiac failure. Currently, there is no cure for this devastating disease. Clinical management of symptoms and complications is limited to stabilising the condition, slowing deterioration over time and palliative care. Since discovery of the DMD gene in 1986, researchers have dedicated substantial effort into vector technologies, facilitating the use of gene therapy to reintroduce a functional copy of the dystrophin gene into muscle fibres, a potential approach to treat DMD patients. However, this approach poses additional challenges relative to many gene therapy approaches since the full-length dystrophin cDNA is ~14 kb, exceeding the packaging capacity of most viral vectors. A number of large internal in-frame dystrophin deletions have been identified in patients that produce a relatively mild phenotype with later age of onset and a slower rate of disease progression than classical DMD. This observation has inspired the construction of internally truncated, but largely functional versions of dystrophin suitable for gene transfer using viral vectors. So far the most widely used miniaturised dystrophin transgenes have been tested in AAV-mediated gene delivery which has identified several limitations indicating the use of more favourable transgenes that have smaller deletions, yet carrying more functional parts of dystrophin. In this study human mini-dystrophin constructs of 4.3-7.7 kb in size were designed that retain key functional elements of dystrophin molecule and their relative functionality investigated in mdx mice. The ultimate aim of this study is the characterisation and optimisation of these mini-dystrophin constructs for gene delivery studies via viral vectors as a therapeutic tool for treatment of Duchenne muscular dystrophy.