Myogenic stem cells for the therapy of primary myopathies: wishful thinking or therapeutic perspective?
Myogenic stem cells for the therapy of primary myopathies: wishful thinking or therapeutic perspective?
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DOI：
10.1172/jci10376
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发表时间：
2000-06-01
影响因子：
15.9
通讯作者：
Mavilio, F
中科院分区：
文献类型：
--
作者：
Cossu, G;Mavilio, F
原发性肌病的体外基因治疗方法旨在克服至少一些与成肌细胞移植相关的免疫问题。在这种策略中，细胞从肌肉活检中分离出来，在体外扩增，用编码治疗基因的适当载体进行转导，最后重新注射到最初分离出细胞的患者的一块或多块肌肉中（12）。然而，这种方法面临两个额外的问题：首先，难以产生合适的整合载体来容纳非常大的肌营养不良蛋白或营养不良蛋白cdna；其次，从营养不良患者身上分离的肌源性细胞寿命有限。在出生后的早期生长过程中，人类肌源性前体的增殖潜力显著下降，与此同时，端粒长度在生命的前20年逐渐减少（13）。考虑到DMD患者的肌源性细胞在体内的各种纤维变性和再生周期中会经历额外的细胞分裂次数，它们的复制能力在儿童早期急剧下降，并在生命的第一个十年中继续下降。这些细胞从肌肉活检中恢复的数量很少，在体外生长不良，并且迅速衰老，尽管它们可以被逆转录病毒载体转导，其效率与正常细胞相当（14,15）。这使得在体外获得合理数量的转基因细胞变得非常困难，因此降低了这种策略可能成为现实的期望。解决有限寿命问题的尝试依赖于肌源性细胞的永生化或具有更高增殖潜力的非肌源性细胞的募集。几十年来，致癌基因对哺乳动物细胞的永活化已经取得了成功，在SV40大T抗原或多瘤病毒等分子的情况下，永活化仍然与向成熟肌肉纤维的一定程度分化相兼容（16,17）。然而，到目前为止，与永生化细胞基因组中活性癌基因持续存在相关的安全性问题阻碍了该策略的任何临床应用。为了克服这些问题，我们设计了一种表达野生型猿猴液泡形成病毒40 （SV40）大T抗原的逆转录病毒载体，并在长末端重复序列中包含两个lox位点。用该载体转导的人原代肌源性细胞寿命延长，并保持其分化能力。Cre重组酶的瞬时表达使得整个原病毒可以在bbb90 %的转导细胞中被切除，然后这些细胞在体内进行终末分化，其效率与未处理的原代肌生成细胞相当（18）。因此，可逆永生化允许原代成肌细胞在培养中扩增而不影响其在体内的分化能力，这可能是一种安全的方法，可以增加这些细胞在临床应用中的可用性。根据类似的思路，可以设想其他的策略，例如表达活性端粒酶或抑制抗癌基因的表达。为了增加原发性成肌细胞的可用性，我们和其他人也探索了使用MyoD基因诱导非肌肉细胞、自体细胞（如皮肤成纤维细胞）的成肌转化的可能性，这些细胞的生长潜力在所有原发性肌病中都不受影响。成纤维细胞可以从许多可获得的来源获得（如皮肤），易于扩增，并在培养中进行基因修饰。如果用MyoD高效转导，例如用瞬态…
The ex vivo approach to gene therapy of primary myopathies was designed to overcome at least some of the immunological problems linked to myoblast transplantation. In this strategy, cells are isolated from a muscle biopsy, expanded in vitro, transduced with an appropriate vector encoding the therapeutic gene, and finally reinjected into one or more muscles of the patient from which they had been initially isolated (12). This approach, however, faces two additional problems: first, the difficulty of producing an appropriate, integrating vector to accommodate the very large dystrophin or utrophin cDNAs; and second, the limited life-span of myogenic cells isolated from dystrophic patients. The proliferative potential of human myogenic precursors declines considerably during early postnatal growth, in parallel with the progressive reduction in telomere length which occurs in the first 2 decades of life (13). Given the extra number of cell divisions that myogenic cells from DMD patients undergo in vivo during the various cycles of fiber degeneration and regeneration, their replication capacity is dramatically decreased during early childhood and continues to drop during the first decade of life. These cells are recovered in low number from muscle biopsies, grow poorly in vitro, and rapidly undergo senescence, even though they can be transduced by retroviral vectors with an efficiency comparable to that of normal cells (14, 15). This makes it very difficult to obtain reasonable numbers of genetically modified cells ex vivo and consequently lowers the expectations that this type of strategy might become practical. Attempts to solve the problem of the limited life-span have relied on either immortalization of myogenic cells or recruitment of nonmyogenic cells with a higher proliferative potential. Immortalization of mammalian cells by oncogenes has been successful for decades, and in the case of molecules such as the large T antigen of SV40 or polyoma virus, immortalization is still compatible with a certain degree of differentiation into mature muscle fibers (16, 17). Nevertheless, safety concerns related to the persistence of an active oncogene in the genome of the immortalized cells have precluded, so far, any clinical application of this strategy. In order to overcome these concerns, we designed a retroviral vector expressing the wild-type simian vacuolating virus 40 (SV40) large T antigen and harboring two lox sites in the long terminal repeats. Human primary myogenic cells transduced with this vector showed extended life-spans and retained their differentiation capacity. Transient expression of Cre recombinase allowed the entire provirus to be excised in> 90% of transduced cells, which then underwent terminal differentiation in vivo with an efficiency comparable to that of untreated, primary myogenic cells (18). Reversible immortalization thus allows primary myoblasts to be expanded in culture without compromising their ability to differentiate in vivo and could represent a safe means to increase the availability of these cells for clinical application. Alternative strategies might be conceived along similar lines, such as expressing active telomerase or inhibiting the expression of anti-oncogenes.To increase the availability of primary myoblasts, we and others have also explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, autologous cells such as skin fibroblasts, the growth potential of which is uncompromised in all primary myopathies. Fibroblasts can be obtained from a number of accessible sources (eg, skin), easily expanded, and genetically modified in culture. If transduced at high efficiency with MyoD, for instance by transient …