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Development of stem cell-based gene therapy for Duchenne muscular dystrophy.

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
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
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.

Date:27 Sep 2010 →  16 Jun 2016
Keywords:Gene therapy, Dystrophin gene, Duchenne muscular dystrophy (DMD), Non-viral vector, Muscle progenitor cells, Stem cells, Cell therapy, iPS
Project type:PhD project