title: piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.
abstract: Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium Center for Molecular & Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven 3000, Belgium.
authors.forename: Mariana
authors.initials: M
authors.lastname: Loperfido
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK.
authors.forename: Susan
authors.initials: S
authors.lastname: Jarmin
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium.
authors.forename: Sumitava
authors.initials: S
authors.lastname: Dastidar
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium Center for Molecular & Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven 3000, Belgium.
authors.forename: Mario
authors.initials: M
authors.lastname: Di Matteo
authors.affiliation: Translational Cardiomyology Laboratory, Embryo and Stem Cell Biology Unit, Department of Development and Regeneration, University of Leuven, Leuven 3000, Belgium.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Perini
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK.
authors.forename: Marc
authors.initials: M
authors.lastname: Moore
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium.
authors.forename: Nisha
authors.initials: N
authors.lastname: Nair
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium.
authors.forename: Ermira
authors.initials: E
authors.lastname: Samara-Kuko
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK Faculty of Science & Engineering, University of Wolverhampton, Wolverhampton, WV1 1LY, UK.
authors.forename: Takis
authors.initials: T
authors.lastname: Athanasopoulos
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, WC1E 6DE, UK.
authors.forename: Francesco Saverio
authors.initials: FS
authors.lastname: Tedesco
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK.
authors.forename: George
authors.initials: G
authors.lastname: Dickson
authors.affiliation: Translational Cardiomyology Laboratory, Embryo and Stem Cell Biology Unit, Department of Development and Regeneration, University of Leuven, Leuven 3000, Belgium.
authors.forename: Maurilio
authors.initials: M
authors.lastname: Sampaolesi
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium Center for Molecular & Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven 3000, Belgium.
authors.forename: Thierry
authors.initials: T
authors.lastname: VandenDriessche
authors.affiliation: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels, Brussels 1090, Belgium Center for Molecular & Vascular Biology, Department of Cardiovascular Sciences, University of Leuven, Leuven 3000, Belgium marinee.chuah@vub.ac.be.
authors.forename: Marinee K
authors.initials: MK
authors.lastname: Chuah
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D004285:Dogs; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D014162:Transfection
chemical_list: C484258:DMD protein, human; D004251:DNA Transposable Elements; D016189:Dystrophin; D049452:Green Fluorescent Proteins