Puttaraju M, Jamison S F, Mansfield S G, Garcia-Blanco M A, Mitchell L G
Intronn LLC, Durham, NC 27701, USA.
Nat Biotechnol. 1999 Mar;17(3):246-52. doi: 10.1038/6986.
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 beta-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 trans-splicing. 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.
我们已经开发出了能够与靶信使RNA前体（前体mRNA）进行剪接体介导的RNA反式剪接反应的RNA分子。在HeLa细胞核提取物、培养的人类细胞以及无胸腺小鼠体内的H1299人肺癌肿瘤中均证实了靶向反式剪接。编码人绒毛膜促性腺激素基因6β亚基的癌症相关前体mRNA与前体反式剪接分子（PTM）RNA之间的反式剪接在体外和体内都是准确的。靶向反式剪接与非靶向反式剪接的比较显示出一定程度的特异性，通过添加包含PTM剪接位点的内部反向重复序列可提高该特异性。顺式剪接与反式剪接之间的竞争表明，反式剪接可以抑制顺式剪接。在细胞中，PTM可在无功能的lacZ转录本的剪接模型中实现RNA修复，从而恢复显著的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重新编程靶转录本序列的通用方法，为基因治疗提供了一种新途径。
