Silencing of human α-synuclein in vitro and in rat brain using lentiviral-mediated RNAi
Silencing of human α-synuclein in vitro and in rat brain using lentiviral-mediated RNAi
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DOI：
10.1016/j.expneurol.2005.12.024
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发表时间：
2006-04-01
影响因子：
5.3
通讯作者：
Bohn, MC
中科院分区：
文献类型：
--
作者：
Sapru, MK;Yates, JW;Bohn, MC
已知人α-突触核蛋白过表达及其在神经元或神经胶质中的毒性积累在帕金森病和其他相关神经退行性突触核蛋白病的发病机制中起关键作用。α-突触核蛋白基因中的几个单点突变，以及基因重复和三倍，与家族性帕金森病有关。此外，α-突触核蛋白基因启动子的遗传变异性与特发性帕金森病相关。通过基于载体的RNA干扰（RNAi）沉默人α-突触核蛋白基因是用于突触核蛋白病的有希望的治疗方法。在这里，我们报告的人α-突触核蛋白的编码区，构成了一个有效的目标，通过RNAi强大的沉默和证明等位基因特异性沉默的A53 T突变体的21个核苷酸序列的鉴定。此外，我们已经开发了一种基于质粒载体的RNAi，用于在体外沉默人α-突触核蛋白。最后，使用共表达α-突触核蛋白靶向小发夹RNA（shRNA）和增强型绿色荧光蛋白（EGFP）作为标记基因的双盒慢病毒，我们证明了在体外人多巴胺能细胞系SH-SY 5 Y中内源性人α-突触核蛋白的有效沉默以及在大鼠脑中实验表达的人α-突触核蛋白的有效沉默。我们的研究结果表明，有效的沉默人α-突触核蛋白的表达在体外和体内的病毒载体为基础的RNAi和开发有效的基因沉默治疗突触核蛋白病，包括帕金森氏病的工具。(C)2006年爱思唯尔公司All rights reserved.
Human alpha-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the alpha-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the alpha-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human alpha-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human alpha-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human alpha-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human alpha-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an alpha-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human a-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human alpha-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human alpha-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease. (C) 2006 Elsevier Inc. All rights reserved.