title: Optimization of Internally Deleted Dystrophin Constructs.
abstract: Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. 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 characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (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 various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.
authors.affiliation: 1 John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, University of Newcastle , Newcastle upon Tyne, United Kingdom .
authors.forename: Mojgan
authors.initials: M
authors.lastname: Reza
authors.affiliation: 1 John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, University of Newcastle , Newcastle upon Tyne, United Kingdom .
authors.forename: Steve H
authors.initials: SH
authors.lastname: Laval
authors.affiliation: 1 John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, University of Newcastle , Newcastle upon Tyne, United Kingdom .|2 Leibniz-Institut für Analytische Wissenschaften (ISAS) , Dortmund, Germany .
authors.forename: Andreas
authors.initials: A
authors.lastname: Roos
authors.affiliation: 1 John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, University of Newcastle , Newcastle upon Tyne, United Kingdom .
authors.forename: Stephanie
authors.initials: S
authors.lastname: Carr
authors.affiliation: 1 John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine, University of Newcastle , Newcastle upon Tyne, United Kingdom .
authors.forename: Hanns
authors.initials: H
authors.lastname: Lochmüller
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics / Q000627:therapeutic use; D016189:Dystrophin / Q000235:genetics* / Q000627:therapeutic use; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy*; D052248:Nitric Oxide Synthase Type I / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D016189:Dystrophin; D052248:Nitric Oxide Synthase Type I
keywords: assay development; disease models; muscle; vector production; vectors