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135千碱基对人类FRDA基因组DNA位点的感染性递送和表达可补充人类细胞中的弗里德赖希共济失调缺陷。

Infectious delivery and expression of a 135 kb human FRDA genomic DNA locus complements Friedreich's ataxia deficiency in human cells.

作者信息

Gomez-Sebastian Silvia, Gimenez-Cassina Alfredo, Diaz-Nido Javier, Lim Filip, Wade-Martins Richard

机构信息

The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

出版信息

Mol Ther. 2007 Feb;15(2):248-54. doi: 10.1038/sj.mt.6300021.

Abstract

Friedreich's ataxia (FA) is the most common recessive ataxia, affecting 1-2 in 50,000 Caucasians, and there is currently no effective cure or treatment. FA results from a deficiency of the mitochondrial protein frataxin brought about by a repeat expansion in intron 1 of the FRDA gene. The main areas affected are the central nervous system (particularly the spinocerebellar system) and cardiac tissue. Therapies aimed at alleviating the neurological degeneration have proved unsuccessful to date. Here, we describe the construction and delivery of high capacity herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the entire 80 kb FRDA genomic locus, driven by the endogenous FRDA promoter and including all introns and flanking regulatory sequences within a 135 kb genomic DNA insert. FA patient primary fibroblasts deficient in frataxin protein and exhibiting sensitivity to oxidative stress were transduced at high efficiency by FRDA genomic locus vectors. Following vector transduction, expression of FRDA protein by immunofluorescence was shown. Finally, functional complementation studies demonstrated restoration of the wild-type cellular phenotype in response to oxidative stress in transduced FA patient cells. These results suggest the potential of the infectious bacterial artificial chromosome-FRDA vectors for gene therapy of FA.

摘要

弗里德赖希共济失调(FA)是最常见的隐性共济失调,在每50000名白种人中就有1至2人患病,目前尚无有效的治愈方法或治疗手段。FA是由FRDA基因内含子1中的重复序列扩增导致线粒体蛋白frataxin缺乏引起的。主要受影响的部位是中枢神经系统(特别是脊髓小脑系统)和心脏组织。迄今为止,旨在减轻神经退行性变的治疗方法均未取得成功。在此,我们描述了高容量1型单纯疱疹病毒(HSV-1)扩增载体的构建和递送,该载体由内源性FRDA启动子驱动,表达整个80 kb的FRDA基因组位点,包括135 kb基因组DNA插入片段内的所有内含子和侧翼调控序列。缺乏frataxin蛋白且对氧化应激敏感的FA患者原代成纤维细胞被FRDA基因组位点载体高效转导。载体转导后,通过免疫荧光显示了FRDA蛋白的表达。最后,功能互补研究证明,转导的FA患者细胞在氧化应激下恢复了野生型细胞表型。这些结果表明,感染性细菌人工染色体-FRDA载体在FA基因治疗中具有潜力。

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