Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa
Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa
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DOI：
10.1089/hum.1998.9.9-1359
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发表时间：
1998-06-10
影响因子：
4.2
通讯作者：
De Luca, M
中科院分区：
文献类型：
--
作者：
Dellambra, E;Vailly, J;De Luca, M
Laminin-5由三种不同的多肽组成，分别由LAMA3、lam3和LAMC2三个不同的基因编码。我们从一个表现为致命形式的大疱性结缔性表皮松解症的患者身上分离出表皮角质形成细胞，其特征是LAMB3基因的纯合突变，导致β 3多肽的完全缺失。在体外，β 3-空的角质形成细胞不能合成层粘连蛋白-5和组装半脂粒，其粘附性能受损，集落形成能力下降。用表达人β 3 cDNA的逆转录病毒构建物转导原代β 3-空角化细胞，克隆性β 3-空角化细胞转导效率为100%。β 3转导的角化细胞能够合成和分泌成熟的异三聚体层粘连蛋白-5，基因校正完全恢复了角化细胞粘附机制，包括适当半染色体组装的能力，并防止了集落形成能力的丧失，这表明对层粘连蛋白-5的粘附与角化细胞增殖能力之间存在直接联系。克隆分析表明，全息克隆永久表达转基因，表明表皮干细胞的稳定校正。由于培养的角质形成细胞通常用于为患有大面积皮肤或粘膜缺陷的患者制作自体移植物，因此，结构蛋白缺陷的原代人表皮干细胞的完全表型逆转为遗传性皮肤病的长期治疗开辟了新的前景。
Laminin-5 is composed of three distinct polypeptides, alpha 3, beta 3, and gamma 2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta 3 polypeptide. In vitro, beta 3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta 3 cDNA was used to transduce primary beta 3-null keratinocytes, Clonogenic beta 3-null keratinocytes were transduced with an efficiency of 100%. beta 3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5, Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.