Efficient and specific repair of sickle β-globin RNA by trans-splicing ribozymes
Efficient and specific repair of sickle β-globin RNA by trans-splicing ribozymes
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DOI：
10.1261/rna.5450203
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发表时间：
2003-10-01
期刊：
影响因子：
4.5
通讯作者：
Sullenger, BA
中科院分区：
文献类型：
--
作者：
Byun, J;Lan, N;Sullenger, BA
先前，我们证明了I组核酶可以在体外转录的核酶转染到哺乳动物细胞后进行反式剪接来修复镰状β -珠蛋白转录物。在这里，我们寻求开发在基因转移后能产生高水平活性核酶的表达盒。我们最初的表达结构被设计为产生与我们之前的RNA转染研究中使用的核酶相同的反式切片核酶，核酶含有6个核苷酸长的内部引导序列。然而，从这些磁带中表达的核酶被发现不能修复镰状β -珠蛋白rna。进一步的实验表明，两个额外的结构元件对核酶介导的RNA修复很重要：在核酶的5‘端和3’外显子开始之间形成的P10相互作用，以及在延伸的引导序列和底物RNA之间形成的额外的碱基配对相互作用。这些优化的表达盒产生的核酶能够在转染的哺乳动物细胞中修饰10%-50%的镰状β -珠蛋白rna。最后，与野生型β -珠蛋白rna相比，具有5bp延伸引导序列的核酶优先与镰状β -珠蛋白rna发生反应，尽管野生型β -珠蛋白转录物仅与核酶形成单一错配。这些结果表明，反式剪接核糖酶表达盒可以产生核糖酶，这些核糖酶可以修复哺乳动物细胞中镰状β -珠蛋白rna的临床相关部分，并且特异性大大提高。
Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.