Spliceosome-mediated RNA trans-splicing as a tool for gene therapy
Spliceosome-mediated RNA trans-splicing as a tool for gene therapy
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DOI：
10.1038/6986
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发表时间：
1999-03-01
影响因子：
46.9
通讯作者：
Mitchell, LG
中科院分区：
文献类型：
--
作者：
Puttaraju, M;Jamison, SF;Mitchell, LG
我们已经开发出能够影响剪接体介导的RNA反式剪接反应的RNA分子与靶信使RNA前体（前mRNA）。在HeLa细胞核提取物、培养的人细胞和无胸腺小鼠的H1299人肺癌肿瘤中证实了靶向反式剪接。编码人绒毛膜促性腺激素基因6的p-亚基的癌症相关前mRNA与前反式剪接分子（PTM）RNA之间的反式剪接在体外和体内都是准确的。靶向与非靶向反式剪接的比较显示了中等水平的特异性，这是通过添加包含PTM剪接位点的内部反向重复序列来改善的。顺式剪接和反式剪接之间的竞争表明，顺式剪接可以被反式剪接抑制。在非功能性lacZ转录物的剪接模型中，RNA修复在细胞中受到PTM的影响，PTM恢复了显着的β-半乳糖苷酶活性。这些观察结果表明，剪接体介导的RNA反式剪接可能代表了一种通用的方法重新编程的目标转录本的序列，提供了一种新的方法，基因治疗。
We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the p-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by transsplicing. RNA repair In a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.