Data_Sheet_1_Cell-permeable peptide nucleic acid antisense oligonucleotide platform targeting human betacoronaviruses.pdf

IntroductionAntisense oligonucleotides (ASOs) with therapeutic potential have recently been reported to target the SARS-CoV-2 genome. Peptide nucleic acids (PNAs)-based ASOs have been regarded as promising drug candidates, but intracellular delivery has been a significant obstacle. Here, we present novel modified PNAs, termed OPNAs, with excellent cell permeability that disrupt the RNA genome of SARS-CoV-2 and HCoV-OC43 by introducing cationic lipid moiety onto the nucleobase of PNA oligomer backbone.
MethodsHCT-8 cells and Caco-2 cells were treated with 1 μM antisense OPNAs at the time of viral challenge and the Viral RNA levels were measured by RT-qPCR three days post infection.
ResultsNSP 14 targeting OPNA 5 and 11, reduced the viral titer to a half and OPNA 530, 531 and 533 lowered viral gene expression levels to less than 50% of control by targeting the 5’ UTR region. Several modifications (oligo size and position, etc.) were introduced to enhance the efficacy of selected OPNAs. Improved OPNAs exhibited a dose-dependent reduction in viral replication and nucleoprotein (NP) protein. When a mixture of oligomers was applied to infected cells, viral titer and NP levels decreased by more than eightfold.
DiscussionIn this study, we have developed a modified PNA ASO platform with exceptional chemical stability, high binding affinity, and cellular permeability. These findings indicate that OPNAs are a promising platform for the development of antivirals to combat future pandemic viral infections that do not require a carrier.

引言
近期有研究报道，具备治疗潜力的反义寡核苷酸（antisense oligonucleotides, ASOs）可靶向严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的基因组。基于肽核酸（peptide nucleic acids, PNAs）的ASOs已被视为极具前景的候选药物，但细胞内递送始终是制约其临床转化的关键障碍。本研究报道了一种新型修饰肽核酸，命名为OPNAs，其通过在PNA寡聚体骨架的核碱基上引入阳离子脂质基团，具备优异的细胞穿透能力，可破坏SARS-CoV-2与人类冠状病毒OC43（HCoV-OC43）的RNA基因组。

方法
将HCT-8细胞与Caco-2细胞在病毒攻毒时以1 μM浓度的反义OPNAs进行处理，于感染后3天通过逆转录实时荧光定量聚合酶链反应（RT-qPCR）检测病毒RNA水平。

结果
靶向非结构蛋白14（non-structural protein 14, NSP14）的OPNA 5与OPNA 11可将病毒滴度降至原有水平的一半；而靶向5’非翻译区（5’ untranslated region, 5’ UTR）的OPNA 530、OPNA 531与OPNA 533可使病毒基因表达水平降至对照组的50%以下。研究人员通过调整寡聚体长度、修饰位点等多种策略优化了筛选出的OPNAs的药效。优化后的OPNAs可呈剂量依赖性地抑制病毒复制与核蛋白（nucleoprotein, NP）表达。当将多种寡聚体混合物用于感染细胞时，病毒滴度与NP蛋白水平降幅均超过8倍。

讨论
本研究开发了一种修饰型PNA ASO平台，该平台具备优异的化学稳定性、高结合亲和力与细胞穿透能力。上述研究结果表明，OPNAs是一款极具前景的抗病毒药物开发平台，可用于研发无需递送载体、可对抗未来大流行性病毒感染的抗病毒疗法。