High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer -: An alternative strategy for ex vivo gene therapy of primary myopathies
High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer -: An alternative strategy for ex vivo gene therapy of primary myopathies
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DOI：
10.1172/jci1505
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发表时间：
1998-05-15
影响因子：
15.9
通讯作者：
Cossu, G
中科院分区：
文献类型：
--
作者：
Lattanzi, L;Salvatori, G;Cossu, G
原发性肌病的离体基因治疗。基于遗传修饰的肌源性细胞的自体移植，受到可以分离、扩增、转导和重新植入患者肌肉中的原代肌源性细胞数量的严重限制。我们探讨了使用MyoD基因诱导非肌肉原代细胞以定量相关方式发生肌源性转化的可能性。用携带逆转录病毒长末端重复序列启动的MyoD cDNA的E1缺失腺病毒载体感染来自皮肤、肌肉或骨髓的原代人和小鼠成纤维细胞，MyoD的表达引起大多数细胞周期的不可逆退出和肌源性分化。（60 - 90%）的培养的成纤维细胞，如通过肌肉特异性基因的激活，融合成收缩性肌管，以及培养物中超微结构正常的肉瘤发生。在腺病毒暴露后24小时，将MyoD转化的培养物注射到免疫缺陷（严重联合免疫缺陷/米色）小鼠的再生肌肉中，在那里它们产生表达人肌球蛋白重链的P-半乳糖苷酶阳性的中央有核纤维。来源于转化成纤维细胞的纤维与通过注射lacZ转导的卫星细胞的对照培养物获得的纤维是不可区分的。MyoD转化的鼠成纤维细胞也参与了免疫活性同基因小鼠的肌肉再生。尽管这些小鼠的抗体在体外与腺病毒感染的细胞结合，但在整个3周的研究期间，移植部位不存在炎性浸润。这些数据支持了原发性肌病基因治疗的替代方法的可行性，该方法基于植入大量经遗传修饰的原代成纤维细胞，所述成纤维细胞通过离体MyoD的腺病毒递送而大规模转化为肌发生。
Ex vivo gene therapy of primary myopathies. based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA, Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to P-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.