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Corrected iPSC-Mediated DMD Therapy

“An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells” 

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are considered a possible treatment for Duchenne muscular dystrophy (DMD), a progressive and incurable neuromuscular disease. This would involve the ex vivo correction of the mutation in the Dystrophin gene which causes DMD, a strategy shown to be successful in mouse models of sickle cell anaemia (Hanna et al) and β-thalassaemia (Wang et al). In a recent report in Nature Communications, the laboratory of Rita C. R. Perlingeiro at the Lillehei Heart Institute, University of Minnesota, USA have  combined an ex vivo genetic correction strategy and their efficient protocol to generate skeletal muscle stem/progenitor cells with significant regeneration potential (Darabi et al) to produce cells which have the capacity to promote substantial muscle regeneration in vivo accompanied by functional improvement (Filareto et al).

iPSCs were generated from tail tip fibroblasts from the commonly used dystrophin/utrophin double knock-out mouse, which present a severe phenotype similar to human DMD patients (Deconinck et aland Grady et al). Retroviral transduction using Oct4, Klf-4 and Sox2 (Wernig et al) generated iPSCs which were subsequently corrected using a micro-utrophin (μUTRN) transgene using the non-viral Sleeping Beauty Transposon system. The μUTRN transgene resembles the dystrophin gene and has been shown to ameliorate the dystrophic phenotype (Tinsley et al 1996 and Tinsley et al 1998) and complements both the loss of dystrophin and utrophin (Sonnemann et al). Additionally, these cells were modified to allow for doxycycline-mediated Pax3 expression to enable differentiation of iPSCs to myogenic progenitors (Tinsley et al 1998) and doxycycline induced cells were subsequently isolated on expression of PDGFαR and lack of Flk-1. Cultivation of these cells in the presence of bFGF and doxycycline enabled the growth of proliferating myogenic precursors (immunopositive for Pax3, Myf5, M-cadherin, CD56, Vcam1, Synd-4 and Cxcr4), which could then be in vitro induced into final maturation to multinucleated myotubes with a high fusion index which expressed μUTRN as well as MyHC and MyoD.

Transplantation of μUTRN-corrected iPSC-derived myogenic precursors into the left tibialis anterior muscles of 3-week-old dystrophin/utrophin double knock-out mice led to substantial engraftment, as measured by utrophin expression with cells expressing β-dystroglycan, α1-syntrophin and neuronal nitric oxide synthase, components of the dystrophin-glycoprotein complex which are usually absent in the absence of dystrophin (Brenman et al, Deconinck et al, Grady et al and Ohlendieck and Campbell). Comparing the transplanted left leg with the untransplanted right leg demonstrated that engrafted muscles had markedly superior isometric tetanic force and increased absolute and specific force, although fatigue tests found no significant differences. Additionally, location of transplanted Pax7+ cells beneath the basal lamina suggested that these donor cells had become satellite cells, a muscle cell type which can respond to injury. This finding was confirmed by the presence of newly formed μUTRN+/embryonic MHC+ myofibers after injury, which also were positive for nicotinic acetylcholine receptors at the neuromuscular junctions, suggesting that these iPSC-derived cells were able to integrate within the neuromuscular system.

Overall, this study suggests that iPSC generation combined with ex vivo gene therapy synergise to provide a possible clinical approach to ameliorating dystrophic diseases by promoting muscle regeneration. The potential for long term success of the engrafting was demonstrated by the seeding of iPSC-derived cells into the satellite stem cell pool as well as promoting immediate muscle regeneration.   Moving this study forward into human studies, using patient-specific stem cells and covering a range of dystrophic diseases is a promising future avenue for this research.



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Study originally appeared in Nature Communications.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.