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bioRxiv · 10.1101/2022.04.05.487136

MicroRNA inhibition using antimiRs in acute human brain tissue sections

Morris, G.·Langa, E.·Fearon, C.·Conboy, K.·E-How, K. L.·Sanz-Rodriguez, A.·O'Brien, D. F.·Sweeney, K.·Lacey, A.·Delanty, N.·Beausang, A.·Brett, F. M.·Cryan, J.·Cunningham, M. O.·Henshall, D. C.
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Abstract

IntroductionAn emerging pre-clinical approach for the treatment of pharmacoresistant epilepsy is targeting the microRNA (miRNA) system. MiRNAs are short noncoding RNAs that suppress gene expression at the post-transcriptional level. Targeting miRNAs, which is possible using antisense oligonucleotide antimiRs can produce broad effects on gene expression suited to the complex pathophysiology in temporal lobe epilepsy. Potent anti-seizure and disease- modifying effects have been reported for antimiRs targeting microRNA-134 (antimiR-134). To date, however, pre-clinical testing has been performed using in vitro cell cultures and rodent models. It is uncertain how well this approach will translate to the clinic. Here, we develop an antimiR testing platform in human brain tissue sections. MethodologyHuman brain specimens were obtained with consent from patients undergoing resective surgery to treat focal drug-resistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF), dissected for clinical neuropathological examination, and unused material transferred for sectioning. Individual tissue sections were incubated in oxygenated ACSF, containing either antimiR-134 or a non-targeting control antimiR, for 24 hours at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels measured using RT-qPCR. ResultsACSF transport had no obvious impact on any clinical neurosurgical or neuropathological procedure and specimens were confirmed to be viable following this process. RNA was well- preserved by transportation of specimens in ACSF, with RNA integrity scores significantly higher than tissue transported without ACSF. AntimiR-134 mediated a specific and dose- dependent knockdown of miR-134 in human neocortical sections, with approximately 75% reduction of miR-134 at 1 {micro}M and 90% reduction at 3 {micro}M. These doses did not have off- target effects on expression of a selection of three other miRNAs (miR-10, miR-129 or miR- 132). SignificanceThis is the first demonstration of antimiR-134 effects in live human brain tissues. The findings lend further support to the preclinical development of miR-134 and offer a flexible platform for the pre-clinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain. Key pointsO_LIASO antimiRs are promising treatments for pharmacoresistant epilepsy C_LIO_LIWe developed a pipeline to preserve live human neocortical brain specimens from people undergoing resective surgery C_LIO_LIRNA integrity was sufficient to measure miRNA levels in human brain tissues transported in modified ACSF C_LIO_LIIncubation of acute human neocortical specimens in antimiR-134 resulted in potent and specific reduction in miR-134 levels C_LIO_LIAcute human brain slices are a promising model for testing ASOs C_LI

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Morris, G., Langa, E., Fearon, C., Conboy, K., E-How, K. L., Sanz-Rodriguez, A., O'Brien, D. F., Sweeney, K., Lacey, A., Delanty, N., Beausang, A., Brett, F. M., Cryan, J., Cunningham, M. O., Henshall, D. C.. 2022-04-07. MicroRNA inhibition using antimiRs in acute human brain tissue sections. https://doi.org/10.1101/2022.04.05.487136

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