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EMBO Mol Med. 2018 Feb;10(2):254-275. doi: 10.15252/emmm.201607284.

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

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Department of Cell and Developmental Biology, University College London, London, UK.
Great Ormond Street Institute of Child Health, University College London, London, UK.
Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Tottori, Japan.
Chromosome Engineering Research Center (CERC), Tottori University, Yonago, Tottori, Japan.
Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy.
San Raffaele Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute and Vita Salute San Raffaele University, Milan, Italy.
AIM/AFM Center for Research in Myology, Sorbonne Universités, UPMC Univ. Paris 06, INSERM UMRS974, CNRS FRE3617, Paris, France.
School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey, UK.
Department of Biosciences, University of Milan, Milan, Italy.
Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester, Manchester, UK
Department of Cell and Developmental Biology, University College London, London, UK


Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.


DMD ; gene therapy; human artificial chromosomes; human muscle stem/progenitor cells; immortalisation

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