Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts
Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts
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DOI：
10.1371/journal.pone.0003468
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发表时间：
2008-10-22
期刊：
影响因子：
3.7
通讯作者：
Lorson, Christian L.
中科院分区：
文献类型：
--
作者：
Coady, Tristan H.;Baughan, Travis D.;Lorson, Christian L.
RNA模式正在发展成为重定向致病性前mRNA剪接事件的有力手段。提高这些分子在体内的效率是至关重要的，因为它们走向临床应用。脊髓性肌萎缩症（SMA）是由SMN 1缺失引起的。一个几乎相同的拷贝基因SMN 2由于选择性剪接而产生低水平的功能蛋白。我们以前报道了一种反式剪接RNA（tsRNA），重新定向SMN 2剪接。现在，我们表明，减少内源性剪接位点之间的竞争增强了反式剪接的效率。开发了表达SMN tsRNA和剪接位点阻断反义（ASO-tsRNA）的单一载体系统。ASO-tsRNA载体显著提高了SMA小鼠中枢神经系统内原代SMA患者成纤维细胞中的SMN水平，并增加了SMN依赖性的体外snRNP组装。这些结果表明，ASO-tsRNA策略提供了深入了解反式剪接机制和显着增强体内反式剪接活性的手段。
RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.