1Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
2Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.
Hum Gene Ther. 2019 Jul;30(7):841-854. doi: 10.1089/hum.2018.157. Epub 2019 May 28.
Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by an abnormal repetition of a CAG codon in the gene. This expansion translates into a long polyglutamine tract, leading to the misfolding of the mutant protein ataxin-3, which abnormally accumulates in the nucleus, thus leading to neurodegeneration in specific brain regions. No treatment able to modify the progression of the disease is available. However, it has previously been shown that specific silencing of mutant ataxin-3 by RNA interference with viral vectors is a promising therapeutic strategy for MJD. Nevertheless, reports of cytotoxic effects of this technology led to the safety profile of the previously tested lentiviral vectors encoding short hairpin (sh)RNAs (LV-shmutatx3) targeting mutant ataxin-3 upon brain injection being investigated. For this purpose, the vectors were injected in the mouse striata, and neuronal dysfunction, degeneration, gliosis, off-target effects, and saturation of the RNA interference machinery were evaluated. It was found that: (1) LV-shmutatx3 mediated stable and long-term expression of the shRNA in neurons of the mouse striatum; (2) neuronal dysfunction evaluated by darpp-32, NeuN, and cresyl violet staining, initially more pronounced, became indistinguishable from the phosphate-buffered saline group at 8 weeks and resolved within 20 weeks; (3) astrocytic activation was present, which resolved within 8 weeks; (4) microglial activity and proinflammatory cytokines release were present, which resolved and normalized within 20 weeks; and (5) there were no off-target effects or saturation of the endogenous RNA interference processing machinery in the mouse striatum. The data show that injection of lentiviral vectors encoding a shRNA targeting mutant ataxin-3 in the mouse brain induce transient dysfunctions, which resolve within 20 weeks. Importantly, long-term expression (up to 20 weeks post injection) of this shRNA (driven by H1 promoter) led to no toxic effect . This study thus constitutes an additional step in a future translation of gene silencing as a therapy for MJD.
脊髓小脑共济失调 3 型（Machado-Joseph disease，MJD）或 spinocerebellar ataxia type 3 是一种神经退行性疾病，由基因中 CAG 密码子的异常重复引起。这种扩展导致了突变蛋白 ataxin-3 的长聚谷氨酰胺链的错误折叠，导致突变蛋白在特定脑区的异常积累，从而导致神经退行性变。目前尚无能够改变疾病进展的治疗方法。然而，先前已经表明，通过病毒载体的 RNA 干扰特异性沉默突变型 ataxin-3 是治疗 MJD 的一种很有前途的治疗策略。然而，由于这项技术的细胞毒性作用的报道，以前测试的编码靶向突变型 ataxin-3 的短发夹 (sh)RNA 的慢病毒载体（LV-shmutatx3）的安全性，已经在脑内注射后进行了研究。为此，将载体注入小鼠纹状体，并评估神经元功能障碍、退化、神经胶质增生、脱靶效应和 RNA 干扰机制的饱和。结果发现：（1）LV-shmutatx3 介导的 shRNA 在小鼠纹状体神经元中稳定且长期表达；（2）通过 darpp-32、NeuN 和 Cresyl Violet 染色评估神经元功能障碍，最初更为明显，在 8 周时与磷酸盐缓冲盐水组无差异，并在 20 周时恢复；（3）星形胶质细胞激活存在，在 8 周内恢复；（4）小胶质细胞活性和促炎细胞因子释放存在，在 20 周内恢复并正常化；（5）在小鼠纹状体中没有脱靶效应或内源性 RNA 干扰处理机制的饱和。数据表明，在小鼠大脑中注射靶向突变型 ataxin-3 的慢病毒载体会导致短暂的功能障碍，这些障碍会在 20 周内恢复。重要的是，这种 shRNA（由 H1 启动子驱动）的长期表达（注射后长达 20 周）不会产生毒性作用。因此，这项研究是将基因沉默作为 MJD 治疗方法进行未来转化的又一步。
