Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy. Issue 2 (14th December 2017)

• Record Type: Journal Article
• Authors: Benedetti, Sara; Uno, Narumi; Hoshiya, Hidetoshi; Ragazzi, Martina; Ferrari, Giulia; Kazuki, Yasuhiro; Moyle, Louise Anne; Tonlorenzi, Rossana; Lombardo, Angelo; Chaouch, Soraya; Mouly, Vincent; Moore, Marc; Popplewell, Linda; Kazuki, Kanako; Katoh, Motonobu; Naldini, Luigi; Dickson, George; Messina, Graziella; Oshimura, Mitsuo; Cossu, Giulio; Tedesco, Francesco Saverio
• Is Part Of: EMBO molecular medicine. Volume 10:Issue 2(2018)
• Page Start: 254
• Page End: 275
• Publication Date: 2017-12-14
• Subjects: DMD -- gene therapy -- human artificial chromosomes -- human muscle stem/progenitor cells -- immortalisation; Molecular biology -- Periodicals; Medical genetics -- Periodicals; Pathology, Molecular -- Periodicals; 616.04205
• DOI: 10.15252/emmm.201607284
• Languages: English
• ISSNs: 1757-4676

Abstract

Abstract: 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. Synopsis: Reversible immortalisation of human dystrophic muscle progenitors with a novel human artificial chromosome (HAC) containing the entire dystrophin locus (DYS‐HAC2) enables genetic correction & provide evidence of translation of HAC technology into DMD muscle progenitors for ex vivo gene therapy. Lentivirally delivered hTERT and Bmi1 cDNAs extend proliferation of human muscle progenitors (both myoblasts and pericyte‐derived mesoangioblasts) and can be reverted by administering Cre recombinase and ganciclovir. Extension of the proliferative ability of muscle progenitors derived from Duchenne muscular dystrophy patients enables their genetic correction with a novel DYS‐HAC. A next‐generation DYS‐HAC containing multiple gene functions was engineered to enable simultaneous delivery of: (i) genomic integration‐free reversible immortalisation, (ii) genetic correction, (iii) inducible differentiation, (iv) controllable cell death. Abstract : Reversible immortalisation of human dystrophic muscle progenitors with a novel human artificial chromosome (HAC) containing the entire dystrophin locus (DYS‐HAC2) enables genetic correction & provide evidence of translation of HAC technology into DMD muscle progenitors for ex vivo gene therapy.