Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.
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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DOI：
10.15252/emmm.201607284
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发表时间：
2018-03
影响因子：
11.1
通讯作者：
Tedesco FS
中科院分区：
文献类型：
--
作者：
Benedetti S;Uno N;Hoshiya H;Ragazzi M;Ferrari G;Kazuki Y;Moyle LA;Tonlorenzi R;Lombardo A;Chaouch S;Mouly V;Moore M;Popplewell L;Kazuki K;Katoh M;Naldini L;Dickson G;Messina G;Oshimura M;Cossu G;Tedesco FS
将大的或多个基因转移到原代人类干/祖细胞中受到载体容量限制的挑战，并且该障碍限制了基因治疗的成功。一个范例是杜氏肌营养不良症（DMD），这是一种由人类最大基因肌营养不良蛋白突变引起的无法治愈的疾病。大容量载体（如人类人工染色体（HAC））与干/祖细胞的组合可以克服这一限制。我们先前报道了移植了含有具有整个肌营养不良蛋白基因座的HAC（DYS‐HAC）的鼠肌肉祖细胞的小鼠中营养不良表型的改善。然而，将这种策略翻译成人肌肉祖细胞需要延长其增殖潜力以承受HAC转移后的克隆细胞扩增。在这里，我们表明，由慢病毒递送的可切除的hTERT和Bmi 1转基因介导的可逆细胞永生化延长了细胞增殖，使新型DYS-HAC能够转移到DMD卫星细胞衍生的成肌细胞和血管周围细胞衍生的中血管细胞中。遗传校正的细胞保持稳定的核型，不发生致瘤性转化，并保留其迁移能力。细胞在体外保持肌原性（自发或在MyoD诱导后），并在移植后植入鼠骨骼肌。最后，我们将上述功能结合到下一代HAC中，能够提供可逆的永生化，完全的遗传校正，额外的肌营养不良蛋白表达，诱导分化和可控的细胞死亡。这项工作建立了一个新的平台，复杂的基因转移到临床相关的人肌肉祖细胞的DMD基因治疗。
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.