Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.
Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.
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α-l-LNA修饰的磷酸硫酸磷酸盐反义寡核苷酸的结构活性关系。
DOI：
10.1038/mtna.2012.34
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发表时间：
2012-09-18
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--
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中科院分区：
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我们报道了α- l锁定核酸（LNA）修饰的短14聚硫代缺口反义寡核苷酸（ASOs）及其靶向磷酸酶和紧张素同源物（PTEN）信使RNA的相关修饰在小鼠中的结构活性关系。α-L-LNA代表对映体LNA的α-异位体，修饰后的寡核苷酸对互补RNA表现出与LNA类似的结合亲和力。与序列匹配LNA的gapmer ASOs显示血浆丙氨酸转氨酶（ALT）水平升高表明肝毒性相反，α-L-LNA及其相关类似物修饰的gapmer ASOs在肝组织中显示PTEN信使RNA的显著下调，而不引起血浆ALT水平升高。然而，α-L-LNA ASO表现出中等剂量依赖性的肝脏和脾脏重量增加，表明其具有较高的免疫刺激倾向。有趣的是，用R-5 ' - me -α-L-LNA取代3 ' -和5 ' -侧翼的α-L-LNA核苷酸，而不是R-6 ' - me -或3 ' - me -α-L-LNA核苷酸，逆转了药物引起的器官重量增加。对二核苷酸单元结构模型的检查表明，5 ' -Me基团增加了靠近硫代磷酸酯骨架的空间体积，或在骨架构象上产生细微的变化，从而干扰了假定的免疫受体对ASO的识别。我们的数据表明，在糖-磷酸主链的5 '位置引入立体体可能是减轻寡核苷酸药物免疫刺激谱的一般策略。在临床环境中，促炎作用表现为注射部位反应和流感样症状。因此，缓解这些影响可以增加患者使用aso治疗时的舒适度和依从性。
We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.
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