Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors
Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors
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DOI：
10.1093/hmg/ddg200
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发表时间：
2003-08-01
影响因子：
3.5
通讯作者：
Meneguzzi, G
中科院分区：
文献类型：
--
作者：
Baldeschi, C;Gache, Y;Meneguzzi, G
我们已经评估了逆转录病毒载体基因治疗隐性营养不良性大疱性表皮细胞瘤（RDEB）在表达突变的VII型胶原蛋白的狗的适用性。分离和分析的9 kb的狗VII型胶原蛋白的cDNA确定了致病的基因突变G1906 S，并公开了种间的VII型胶原蛋白的保护。使用来自LZRS-Ires-zeo和MSCV逆转录病毒的重组载体将野生型VII型胶原蛋白cDNA高效转移至犬RDEB和人原代RDEB VII型胶原蛋白缺失角质形成细胞，实现了转基因产物的持续和永久表达。重组VII型胶原蛋白的表达和翻译后修饰谱与野生型对应物的表达和翻译后修饰谱相当。人RDEB角质形成细胞和犬细胞中的重组犬VII型胶原蛋白纠正了细胞培养物和体外重建皮肤中由RDEB角质形成细胞引起的可观察到的缺陷。高运动性在人RDEB角质形成细胞中完全恢复，在狗RDEB细胞中强烈降低。这一观察结果表明，在突变基因产物存在下，不仅需要感染效率，而且需要高表达水平以确保治疗功效。我们的研究结果为遗传性皮肤病的第一个免疫活性大型动物模型的临床前基因治疗试验奠定了基础，并拓宽了逆转录病毒载体在上皮细胞中转移大型重组基因的临床前和临床应用范围。
We have assessed the suitability of retroviral vectors for gene therapy of recessive dystrophic epidermolysis bullosa (RDEB) in dogs expressing a mutated collagen type VII. Isolation and analysis of the 9 kb dog collagen type VII cDNA identified the causative genetic mutation G1906S and disclosed the interspecies conservation of collagen type VII. Highly efficient transfer of the wild-type collagen type VII cDNA to both dog RDEB and human primary RDEB collagen type VII-null keratinocytes using recombinant vectors derived from LZRS-Ires-zeo and MSCV retroviruses achieved sustained and permanent expression of the transgene product. The expression and post-translational modification profile of the recombinant collagen type VII was comparable to that of the wild-type counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Hypermotility was fully reverted in human RDEB keratinocytes, and strongly reduced in the dog RDEB cells. This observation suggests that not only infection efficiency but also high expression levels are required to ensure therapeutic efficacy in the presence of mutated gene products. Our results set the basis for preclinical gene therapy assays in the first immune-competent large animal model for an inherited skin disease and broaden the spectrum of preclinical and clinical applications of retroviral vectors in the transfer of large recombinant genes in epithelial cells.