Engineered RNase P Ribozymes Effectively Inhibit the Infection of Murine Cytomegalovirus in Animals
Engineered RNase P Ribozymes Effectively Inhibit the Infection of Murine Cytomegalovirus in Animals
复制标题
工程化 RNase P 核酶可有效抑制动物体内鼠巨细胞病毒的感染。
DOI：
10.7150/thno.27776
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发表时间：
2018-01-01
期刊：
影响因子：
12.4
通讯作者：
Liu, Fenyong
中科院分区：
文献类型：
--
作者：
Li, Wei;Liu, Yujun;Liu, Fenyong
原理：基因靶向核酶是治疗应用中很有前途的基于核酸的基因干扰剂。我们之前使用体外选择程序设计了具有增强靶向活性的基于RNaseP的新型核酶变异体。方法与结果：本研究设计了一种新的工程核酶变异体R388-AS，用于靶向小鼠巨细胞病毒(MCMV)装配蛋白(AS)的mRNA，而装配蛋白(AS)是病毒后代生产所必需的。变异体R338-AS在体外对mRNA序列的切割效率至少是核酶M1-AS的200倍，核酶M1-AS来自野生型RNaseP催化RNA序列。在MCMV感染的培养细胞中，R338-AS显示出比M1-AS更好的抗病毒活性，并且病毒AS的表达减少了98-99%，病毒产量减少了15,000倍。在MCMV感染的小鼠中，R388-AS比M1-AS在抑制AS表达、阻断病毒复制和提高动物存活率方面更有活性。结论：我们的结果首次提供了直接证据，表明体外催化活性更高的新型RNase P核酶变异体在抑制动物体内病毒基因表达方面也更有效。此外，我们的研究表明，设计具有独特突变的新型RNase P核酶变体来提高核酶活性用于治疗应用的可能性。
Rationales: Gene-targeting ribozymes represent promising nucleic acid-based gene interference agents for therapeutic application. We previously used an in vitro selection procedure to engineer novel RNase P-based ribozyme variants with enhanced targeting activity. However, it has not been reported whether these ribozyme variants also exhibit improved activity in blocking gene expression in animals.Methods and Results: In this report, R388-AS, a new engineered ribozyme variant, was designed to target the mRNA of assemblin (AS) of murine cytomegalovirus (MCMV), which is essential for viral progeny production. Variant R338-AS cleaved AS mRNA sequence in vitro at least 200 times more efficiently than ribozyme M1-AS, which originated from the wild type RNase P catalytic RNA sequence. In cultured MCMV-infected cells, R338-AS exhibited better antiviral activity than M1-AS and decreased viral AS expression by 98-99% and virus production by 15,000 fold. In MCMV-infected mice, R388-AS was more active in inhibiting AS expression, blocking viral replication, and improving animal survival than M1-AS.Conclusions: Our results provide the first direct evidence that novel engineered RNase P ribozyme variants with more active catalytic activity in vitro are also more effective in inhibiting viral gene expression in animals. Moreover, our studies imply the potential of engineering novel RNase P ribozyme variants with unique mutations to improve ribozyme activity for therapeutic application.