Amgen Research, Translational Safety and Bioanalytical Sciences, Amgen, Inc., Thousand Oaks, California, USA.
Metrion Biosciences Ltd, Granta Center, Cambridge, United Kingdom.
Nucleic Acid Ther. 2023 Apr;33(2):132-140. doi: 10.1089/nat.2022.0043. Epub 2022 Dec 26.
In accord with International Conference on Harmonization S7B guidelines, an human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.
根据国际协调会议 S7B 指南，人 ether-a-go-go 相关基因 (hERG) 测定是延迟心室复极综合风险评估的一个组成部分。hERG 的功能可能受到直接（急性）机制或间接（慢性）机制的影响。一些已批准的寡核苷酸疗法已向监管机构提交了 hERG 数据，这些数据均使用与小分子测试相同的方案（孵育时间 <20 分钟；急性）收集，但寡核苷酸具有独特的作用机制和作用时间（间接）。为了重新构建沉默 RNA (siRNA) 的 hERG 测试策略，进行了一项研究以评估 siRNA 介导的 hERG 功能和基因表达抑制的时间过程。通过自动化电生理学和聚合酶链反应，在稳定表达 hERG 的细胞系中，用全细胞电压钳评估了商业可得的 hERG siRNA。在急性 hERG 研究中，用 100 nM siRNA 处理 20 分钟后未观察到作用。在转染后 8-48 小时内评估和记录了 100 nM siRNA 对 hERG 功能的慢性影响。在 8 小时时没有显著影响，而在 48 小时时观察到 77%的减少。hERG mRNA 水平的测量表明，hERG mRNA 在 8 和 48 小时时分别降低了 79%和 93%，与 hERG 转录抑制一致。结果表明，抗 hERG siRNA 需要在 hERG 测定中长时间暴露（48 小时）才能使 hERG 电流最大减少；短暴露（20 分钟-8 小时）没有效果。这些发现表明，新型寡核苷酸的脱靶分析可能受益于使用长孵育时间的 hERG 方案来降低 hERG 通道的潜在脱靶（间接）影响。如果这些发现用于支持 QTc（心率校正后的 QT 间期持续时间）延长的综合非临床-临床风险评估，则这种 hERG 测定的修改可能很重要。
