MicroRNA inhibition using antimiRs in acute human brain tissue sections

Abstract

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 −1 and 90% reduction at 3 μmol L −1 . 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 effectsThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

MicroRNAs – small RNAs with a big influence on brain excitability

“…To overcome this, studies in the epilepsy field have used surgically resected specimens in order to characterise MTIs in the human seizure focus (Heiland et al., 2022). A recent method (Morris et al., 2022) allows for the application of antimiRs to non‐diseased acutely resected human temporal neocortex (note – this tissue was removed during resective epilepsy surgery to give access to the seizure focus within the temporal lobe; whilst the neocortical tissue is assumed healthy, it was taken from the brain of a person with epilepsy and should be interpreted in this context). Induced pluripotent stem cell‐based models can also be used to study MTIs in a human‐derived preparation (e.g.…”

Can in vitro studies aid in the development and use of antiseizure therapies? A report of the ILAE/AES Joint Translational Task Force

“…Human brain specimens can be sectioned for acute slice recordings and typically have longer viability than rodent slices. An adapted slice storage method can be used to extend their viability for up to 72 h, 107 permitting the screening of ASO‐based therapies in human brain 108 . For longer term use, including the application of viral vectors, human brain slices can be maintained in organotypic culture 109,110 .…”

“…An adapted slice storage method can be used to extend their viability for up to 72 h, 107 permitting the screening of ASO-based therapies in human brain. 108 For longer term use, including the application of viral vectors, human brain slices can be maintained in organotypic culture. 109,110 This approach has been extended recently to the study of the developing human brain.…”

“…This is a hugely costly strategy in terms of time, money, and also ethically, owing to the animals used. Recent advances in the use of human-derived tissues for translational epilepsy research are invaluable to bridge this gap 105,108,109,131 and to provide relatively cheap and fast human-based screens of new therapeutics, before they begin further pre-clinical validation. For small molecules, acutely resected human epileptic tissue can be used as an in vitro seizure screen.…”

Cross Talk proposal: Human‐derived brain tissue is a better epilepsy model than animal‐based approaches

“…miR-134), as antagomirs take a considerable period of time (c. 12 h) to alter mRNA levels. This would permit an electrophysiological assessment of this therapy, which to date has only been assessed in preclinical animal models, for epileptiform activity in human tissue in vitro (Morris et al, 2019(Morris et al, , 2022. Animal models (e.g.…”