Dr. Mojgan Reza, Dr. Steven Hector Laval, Miss Stephanie Jan Carr, and Prof. Hanns Lochmüller
Human Gene Therapy Methods,
Duchenne muscular dystrophy is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered as a potential strategy for treatment of DMD, aiming to restore the missing protein. One of the major challenges of this method is the large size of the dystrophin cDNA of ~14 kb, exceeding the packaging capacity of conventional viral vectors. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterised in relation to their structure and function in vivo. In order to optimise a dystrophin cDNA construct for therapeutic application we designed and produced four human mini-dystrophins within the packaging capacity of lentiviral vectors. Two novel mini-dystrophins retained the centrally-located nNOS-anchoring domain in order to achieve sarcolemmal nNOS restoration which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of different constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.