Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA.
Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA.
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DOI：
10.1016/j.omtn.2017.06.012
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发表时间：
2017-09-15
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影响因子：
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通讯作者：
Watts JK
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作者：
Pendergraff HM;Krishnamurthy PM;Debacker AJ;Moazami MP;Sharma VK;Niitsoo L;Yu Y;Tan YN;Haitchi HM;Watts JK
两种机制主导了基于多核苷酸的基因沉默的临床管道，即，募集RNA酶H切割靶RNA的反义方法和募集RISC复合物切割靶RNA的RNAi方法。可以使用多种化学设计来引出每种途径。我们使用四种类型的基因沉默剂比较了MRC-5肺成纤维细胞中哮喘易感基因ADAM 33的沉默，每种机制各使用两种：传统的双链小干扰RNA（SiRNA）、单链小干扰RNA（ss-SiRNA）、锁核酸（LNA）间隙体反义寡核苷酸（ASO）和ASO的新型十六烷基氧丙基缀合物。在这些设计中，gapmer ASO作为沉默ADAM 33表达的先导化合物出现：当用阳离子脂质转染时，几种gapmer ASO显示亚纳摩尔效力，当裸递送时，显示低微摩尔效力，无毒性。ADAM 33 mRNA对RNA酶H沉默的优先易感性可能与观察到的这种mRNA的高度核保留有关。动态光散射数据表明，十六烷氧基丙基阿索缀合物自组装成簇。这些缀合物相对于未缀合的ASO显示出降低的效力，除非使用生物可切割的连接将亲脂性尾部缀合至阿索。最后，基于来自（人）MRC-5细胞的前导ASO，我们开发了一系列具有优异活性的针对小鼠Adam 33的同源ASO。我们的工作证实了基于ASO的ADAM 33基因沉默是哮喘研究和治疗的有用工具。
Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.