Abstract：
Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5\'-cytosine-phosphate-guanine-3\' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.
摘要：
治疗性反义寡核苷酸(ASO)的免疫刺激潜力的早期表征是至关重要的。目前,关于第三代锁核酸（LNA）修饰的ASO的Toll样受体9（TLR9）介导的免疫刺激潜力知之甚少。在这项研究中,我们已经使用不同的小鼠和人类细胞培养系统系统地研究了LNA修饰的寡核苷酸的TLR9激活潜力。尽管有报道LNA修饰以及5'-胞嘧啶-磷酸-鸟嘌呤-3'（CpG）基序的胞嘧啶甲基化可以减少硫代磷酸酯（PTO）修饰的寡核苷酸对TLR9的刺激，我们鉴定了具有大量TLR9刺激活性的含CpG的LNA缺口体。我们进一步鉴定了没有CpG基序的免疫刺激LNA缺口体。出乎意料的是,仅CpG基序内的胞嘧啶甲基化不一定减少，甚至可以增加TLR9激活。相比之下,在大多数情况下，所有胞嘧啶的系统甲基化降低甚至消除了TLR9的激活。上下文相关，将LNA修饰引入侧翼可以增加或减少TLR9刺激。总的来说,我们的结果表明,TLR9依赖性免疫刺激潜能是寡核苷酸的个体特征,需要逐例进行研究.
