RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.
RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.
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RNA 反式剪接靶向人红细胞中的内源性β-珠蛋白前信使 RNA。
DOI：
10.1089/hgtb.2016.077
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发表时间：
2017
影响因子：
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通讯作者：
Tisdale,JohnF
中科院分区：
文献类型：
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作者：
Uchida,Naoya;Washington,KareemN;Mozer,Brian;Platner,Charlotte;Ballantine,Josiah;Skala,LukeP;Raines,Lydia;Shvygin,Anna;Hsieh,MatthewM;Mitchell,LloydG;Tisdale,JohnF
镰状细胞病是由β-珠蛋白基因外显子1（共3个外显子）的点突变引起的。通过反式剪接用正常序列替换镰状β-珠蛋白外显子1（和外显子2）是一种潜在的治疗策略。因此，本研究试图开发人红系细胞中靶向β-珠蛋白前信使RNA的反式剪接。从随机β-珠蛋白序列中全面筛选结合结构域。六个候选者具有最佳结合，并且全部靶向内含子2。接下来，构建了编码RNA反式剪接分子的慢病毒载体，其中包含来自这些候选物的独特结合结构域、人工5′剪接位点和γ-珠蛋白cDNA，并在来自健康个体的CD 34+细胞衍生的红系细胞中评价反式剪接。慢病毒转导是有效的，载体拷贝数为9.7至15.3。在分子水平上检测到预期的反式剪接RNA产物，包括内源性β-珠蛋白和γ-珠蛋白的外显子3。通过更长的结合结构域，包括内含子2的5′剪接位点，反式剪接效率提高到0.07-0.09%。总之，进行筛选以选择用于反式剪接的有效结合结构域。在人红系细胞中获得了内源性β-珠蛋白RNA的可检测水平的反式剪接。这些方法为将来的反式剪接定向基因治疗提供了基础。
Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5′ splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07–0.09% by longer binding domains, including the 5′ splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.