Abstract

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by a variety of mutations in the dystrophin-encoding DMD gene that, spanning more than 2.4 Mb, is one of the largest human genes known. The length of the DMD coding sequence (11 kb) combined with the DNA packaging limitations of most commonly used viral vector systems has restricted viral vector-assisted targeted chromosomal insertion of transgenes encoding full-length dystrophin. We aim at developing an all-inclusive ex vivo DMD genetic therapy involving knocking-in a “healthy” copy of a full-length dystrophin-encoding transgene into the AAVS1 safe-harbour locus in human myogenic cells. To overcome size constrains, we investigated the feasibility of using tropism-modified high-capacity adenoviral vectors (HC-AdVs) as they can efficiently transduced human myogenic cells and host up to 36 kb of foreign DNA. HC-AdVs encoding full-length dystrophin or Cas9:gRNAAAVS1 complexes reached high titers and were shown to package intact recombinant vector DNA. Importantly, transduction efficiencies above 90% could readily be obtained in patient-derived myoblasts and HeLa cells as assessed through flow cytometry and immunofluorescence microscopy. Albeit varying in a cell type-dependent manner, HC-AdV delivery of donor templates consisting of the transgene flanked by large stretches of AAVS1 homologous sequences led to up to 60% of stable chromosomal insertion and expression of full-length dystrophin in the targeted cells. These results bode well for converting this HC-AdV platform into an ex-vivo genetic therapy for DMD aiming at complementing defective DMD alleles regardless of their mutation(s).