title: Novel mini-dystrophin gene dual adeno-associated virus vectors restore neuronal nitric oxide synthase expression at the sarcolemma.
abstract: Six- to 8-kb mini-dystrophin genes are promising candidates for Duchenne muscular dystrophy (DMD) gene therapy. Several dual adeno-associated virus (AAV) mini-dystrophin vectors have been tested in dystrophin-deficient mice. Despite the encouraging preclinical results, none of the existing dual AAV vectors can restore sarcolemmal neuronal nitric oxide synthase (nNOS) expression. Localization of nNOS to the sarcolemma may greatly improve the therapeutic outcome in DMD (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635). In this study, we developed a series of dual AAV expression vectors to express a synthetic minigene that carries the nNOS localization domain. To help validate dual vector reconstitution, we also included a FLAG tag and a GFP reporter at different ends of the minigene. These dual AAV vectors were packaged in Y445F tyrosine mutant AAV-6 and tested in dystrophin-null mdx4cv mice by direct muscle injection. All dual vectors expressed GFP/FLAG-tagged mini-dystrophin and restored sarcolemmal nNOS. However, the reconstitution efficiency was significantly different among different sets. The dual vector set YZ27/YZ22 yielded the highest transduction efficiency (∼90%). Further development of this set dual vector may lead to more effective DMD gene therapy.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212, USA.
authors.forename: Yadong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Duan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007273:Injections, Intramuscular; D008297:Male; D051379:Mice; D018101:Mice, Inbred mdx; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology; D009137:Muscular Dystrophy, Animal / Q000201:enzymology / Q000473:pathology / Q000628:therapy; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012508:Sarcolemma / Q000201:enzymology* / Q000473:pathology
chemical_list: D016189:Dystrophin; D049452:Green Fluorescent Proteins; D052248:Nitric Oxide Synthase Type I