Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

Jinhong Meng,Steven H Laval,John R Counsell,Francesco Muntoni,Hanns Lochmüller,Olivier Danos,Mojgan Reza,Jennifer E Morgan,Adrian Thrasher

doi:10.1038/srep19750

Jinhong Meng, Steven H Laval + Show 7 more

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https://doi.org/10.1038/srep19750

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Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

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Lentiviruses are able to transduce non-dividing and dividing cells, leading to stable and long-term gene expression[9], but they only have a small cloning capacity[10] and cannot accommodate the cDNA of full length dystrophin (>11 Kb)
We show that cells derived from skeletal muscle of a Duchenne Muscular Dystrophy (DMD) patient[6] can be transduced with lentiviruses expressing dystrophin constructs
In our cell transplantation mouse model, we have restored functional dystrophin in regenerated muscle fibres derived from DMD patient-derived stem cells that had been lentivirally-transduced with two novel dystrophin constructs

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Lentiviruses are able to transduce non-dividing and dividing cells, leading to stable and long-term gene expression[9], but they only have a small cloning capacity[10] and cannot accommodate the cDNA of full length dystrophin (>11 Kb) To circumvent this limitation, truncated forms of dystrophin have been developed, retaining dystrophin domains thought to serve essential functions[11,12]. C1 contains the nNOS binding site-spectrin repeats 16 and 17 These constructs were lentivirally-transduced into human muscle stem cells and their efficacy compared. The large size of the hDesmin promoter (1.8 Kb) presented a technical drawback To circumvent this limitation and enable efficient packaging of larger dystrophin constructs, we produced lentiviral vectors expressing dystrophin under the control of the much smaller SFFV promoter (412 bp). In fibres expressing dystrophin C1, nNOS was present at the sarcolemma, suggesting that the C1 dystrophin construct is superior to C2 in that it can restore the nNOS signaling pathway in vivo

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