par Loperfido, Mariana;Jarmin, Susan;Dastidar, Sumitava;Di Matteo, Mario;Perini, Ilaria;Moore, Marc;Nair, Nisha;Samara-Kuko, Ermira;Athanasopoulos, Takis;Tedesco, Francesco Saverio;Dickson, George;Sampaolesi, Maurilio;VandenDriessche, Thierry;Chuah Lay Khim, Marinee
Référence Nucleic acids research, 44, 2, page (744-760)
Publication Publié, 2016-01
Référence Nucleic acids research, 44, 2, page (744-760)
Publication Publié, 2016-01
Article révisé par les pairs
Résumé : | 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. |