Author:

Abstract:

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by mutations in the dystrophin gene that cause the absence of the dystrophin protein at the muscle fiber membrane of the affected patients. This leads to myofiber degeneration and progressive muscle wasting, ultimately resulting in significant morbidity and mortality. Currently, there is no treatment that prevents or reverses the disease progression. Genetically corrected stem/progenitor cells could potentially provide an effective treatment. However, due to its large size, commonly used viral vector technologies preclude efficient gene transfer of the full-length dystrophin coding DNA sequence (CDS; size: 11.1 Kb). In this study we validated a novel stem cell-based non-viral gene therapy approach for DMD with the use of piggyBac (PB) transposons. These plasmidbased non-viral vectors are able to stably integrate the gene of interest into the genome of the target cells leading to its sustained expression. Moreover, the large cargo capacity of these vectors could overcome one of the main bottlenecks in the field enabling gene therapy with full-length instead of truncated dystrophin. We have therefore generated PB transposons coding for either fulllength or truncated versions of the human dystrophin CDS. We demonstrated that this system enables stable non-viral gene delivery, with sustained expression of both full-length and truncated versions of dystrophin into murine myoblasts. We subsequently transferred PB transposons containing the fulllength human dystrophin CDS into dystrophic mesoangioblasts (MABs). These myogenic vessel associated stem/progenitor cells are capable of crossing the vessels and contribute to the regeneration of the dystrophic muscles upon intraarterial transplantation. The use of MABs has resulted to be relatively safe in a recently completed phase I/II clinical trial based on intra-arterial infusions at escalating doses of HLA-matched donor-derived MABs in DMD patients under immunosuppressive regimen (EudraCTno. 2011-000176-33). In our study, MABs were isolated from the muscles of a large animal model for DMD, the Golden Retriever muscular dystrophy (GRMD) dog. The genetically corrected GRMD MABs showed stable transposition and expression of the full-length human 12 dystrophin. Since MABs have a limited proliferative capacity, we have also investigated the possible use of MABs generated from induced pluripotent stem cells (iPSCs) of patients suffering from DMD, as an alternative (designated as human iPSC-derived mesoangioblast-like cells or HIDEMs). These cells can be expanded in culture to obtain a potentially unlimited supply of myogenic progenitors. HIDEMs derived from patients affected by DMD were then genetically corrected with PB transposons resulting in stable expression of the full-length human dystrophin CDS. These cells successfully engrafted into the muscles of immunodeficient/dystrophic mice (scid/mdx) leading to the in vivo expression of the PB-mediated full-length human dystrophin in the myofiber membrane. Taken together, these results showed for the first time the validity of a non-viral gene transfer approach based on PB transposons that allows for the sustained expression of the full-length human dystrophin in dystrophic MABs and DMD patient-specific iPSC-derived MABs. This study paves the way towards a novel stem/progenitor cell-based non-viral gene therapy for the treatment of DMD exploiting the potential of PB transposons to deliver large therapeutic genes.