Neuroscience, Physiology, and Pharmacology, University College London, London, UK.
Department of Physiology and Medical Physics, RCSI University of Medicine & Health Sciences, Dublin, Ireland.
Epilepsia. 2022 Aug;63(8):e92-e99. doi: 10.1111/epi.17317. Epub 2022 Jun 20.
Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 μmol L and 90% reduction at 3 μmol L . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.
反义寡核苷酸抑制 microRNA 是一种新兴的耐药性癫痫的临床前治疗方法。一个主要的候选物是针对 microRNA-134（ant-134）的“antimiR”，但迄今为止的测试都使用了啮齿动物模型。在这里，我们在人脑组织切片中开发了一种 antimiR 测试平台。脑标本取自接受切除手术以治疗耐药性癫痫的患者。将新皮质标本浸入改良的人工脑脊液（ACSF）中，并进行临床神经病理学检查，然后将未使用的材料转移进行切片。将单个切片在含氧 ACSF 中孵育，在室温下分别孵育含有 ant-134 或非靶向对照 antimiR 的 ACSF24 小时。使用 BioAnalyzer 处理评估 RNA 完整性，并使用定量逆转录聚合酶链反应测量个别 miRNA 水平。在 ACSF 中运输的标本可用于神经病理学诊断，并且具有良好的 RNA 完整性。Ant-134 介导 miR-134 的剂量依赖性下调，在 1μmol/L 时 miR-134 减少约 75%，在 3μmol/L 时减少约 90%。这些剂量对另外三种 miRNA 的表达没有脱靶作用。这是首次在活体人脑组织中证明 ant-134 的作用。这些发现进一步支持了针对 miR-134 的治疗的临床前开发，并为在人脑组织中进行 antimiR 和其他反义寡核苷酸治疗剂的临床前测试提供了一个灵活的平台。
