Replacement of huntingtin exon 1 by trans-splicing.
Replacement of huntingtin exon 1 by trans-splicing.
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DOI：
10.1007/s00018-012-1083-5
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发表时间：
2012-12
影响因子：
8
通讯作者：
Lorson, Christian L.
中科院分区：
文献类型：
--
作者：
Rindt, Hansjoerg;Yen, Pei-Fen;Thebeau, Christina N.;Peterson, Troy S.;Weisman, Gary A.;Lorson, Christian L.
亨廷顿病（Huntington's disease，HD）是一种由亨廷顿蛋白（Huntingtin，HTT）氨基端多聚谷氨酰胺（polyglutamine）扩增引起的常染色体显性遗传性神经退行性疾病。HD为有希望的基于RNA的治疗方法提供了独特的机会，旨在减少突变HTT表达，因为HD突变被认为是一种“功能获得性”突变。保留野生型等位基因表达并降低突变蛋白水平的等位基因特异性策略将特别令人感兴趣。在这里，我们进行了概念验证研究，以证明剪接体介导的反式剪接是一种可行的分子策略，特异性修复HTT等位基因。我们采用了一个双质粒转染系统组成的前mRNA反式剪接模块（PTM）含有HTT外显子1和HTT minigene，以证明HTT外显子1可以被替换的反式。我们检测到存在的反式剪接的RNA，其中PTM外显子1正确地连接到minigene外显子2和3。此外，PTM的外显子1在培养细胞以及疾病相关模型中反式剪接至内源性HTT前mRNA，包括HD患者成纤维细胞和来自先前描述的HD小鼠模型的原代神经元。这些结果表明，HTT的重复扩增不仅可以在合成小基因的背景下成功修复，而且可以在HD神经元的背景下修复。因此，前体mRNA反式剪接可能是治疗HD和其他显性遗传性疾病的一种有前途的方法。
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a “gain-of-function” mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.
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