Using human tissue to translate microRNA-based therapies for epilepsy

Project Information

EpimiRTherapy

Grant agreement ID: 840262

DOI

10.3030/840262

Project closed

EC signature date 10 April 2019

Start date 1 December 2019

End date 30 November 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 184 590,72

EU contribution

€ 184 590,72

Coordinated by

ROYAL COLLEGE OF SURGEONS IN IRELAND
Ireland

Periodic Reporting for period 1 - EpimiRTherapy (Using human tissue to translate microRNA-based therapies for epilepsy)

Reporting period: 2019-12-01 to 2021-11-30

Summary of the context and overall objectives of the project

Problem to be addressed: The overall research aim of EpimiRTherapy is to elucidate the mechanism of a novel disease-modifying therapy for epilepsy in the human brain. MicroRNAs are short non-coding RNAs which regulate protein levels in the brain. Certain microRNAs are strongly associated with epilepsy and their knockdown, using antisense molecules called ‘antagomirs’, has anti-seizure effects in rodents. However, antagomirs to treat neurological disease have never been tested in humans. EpimiRTherapy, for the first time, fills this gap: I will use state-of-the-art techniques to produce human brain slices from tissue surgically resected during temporal lobectomies for epilepsy.

Why is it important for society: Epilepsy is a common neurological disorder which affects around 70 million people worldwide and manifests clinically as the susceptibility to recurrent spontaneous seizures. Roughly 25% of patients do not experience seizure freedom with available anti-epileptic drugs (AEDs). Additionally, adverse effects of AEDs are common due to their non-specific mechanisms of action, and no disease-modifying treatments are clinically available. Consequently, there is an urgent and unmet requirement for disease-modifying therapies in pharmaco-refractory epilepsy, to begin to alleviate the immense socio-economic burden caused by this disease. One of the most promising solutions is antisense knockdown of microRNAs (miRs). MiR levels are dysregulated in brain tissue from humans with epilepsy and in experimental rodent models, and correction of these alterations using antisense oligonucleotides (antagomirs) has disease-modifying anti-seizure effects in in vivo and in vitro (brain slice) models of the disease in rodents. The therapeutic effect of antagomirs has not been verified in human tissue and there remain several barriers to the translation of antagomir therapies for epilepsy to the clinic. Despite its immense promise, ant-134 is not currently being exploited or developed clinically, due to limited evidence of efficacy in humans.

What are the objectives?: 1: Are antimirs taken up in human tissue? (Establish sufficient transfection efficiency). 2: Which human genes and pathways are targeted by miR-134? 3: What are the biophysical effects of miR-134 in human tissue?

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

miR-134 in human tissue (Nov 2019 - March 2019; August 2021-Nov 2021): I established from scratch a new laboratory facility at Beaumont Hospital Dublin, for human brain tissue work. I created a collaborative network between neurosurgeons, neurologists, pathologists and scientists and wrote SOPs to facilitate this work. I then performed a series of trial experiments, in collaboration with J Cryan in neuropathology, to develop a method to transport live tissue to the research laboratory, without impacting neuropathological readouts. Using this technique, I began to create acute slice preparations of human neocortical tissues, incubating them for 24 hrs in either ant-134 or scr control. I used this tissue to test the molecular effects of ant-134 in human neocortex, and to begin to explore it's biophysical impacts on the tissue. Main results achieved: 1) Set-up of new lab facility and training of other users (A Lacey, A Sanfeliu). 2) Established method to transport viable human tissues in artificial CSF at Beaumont Hospital. 3) Bioanalyser analysis showed that samples processed in this way have far greater RNA quality than those transported with no solutions (as previously). 4) Incubation of slices for 24 hrs in 1 uM or 3 uM ant-134 inhibits functional miR-134 expression in human neocortex in a dose-dependent manner., as measured using qPCR 5) Despite the reduction in miR-134 expression, I saw no evidence for de-repression of key miR-134 targets, including LIMK1, DCX and CREB. 6) I performed initial work to explore the network biophysical impact of ant-134 in human neocortex. I established a method to induce seizure-like activity in human brain tissue treated with antimiRs. Dissemination: A manuscript is being prepared for dissemination of this work in an open access peer-reviewed journal. I authored review articles detailing the use of human tissues in brain research (https://doi.org/10.14573/altex.2007082) and prospects for using antimirs in neurological diseases (https://doi.org/10.1016/j.tips.2021.04.007).

miR-134 in rodent epilepsy models: I also performed pre-clinical experiments to interrogate the therapeutic potential of ant-134 in a number of rodent epilepsy models. Work in a P21 mouse model of epilepsy showed for the first time that ant-134 is a viable treatment for epilepsies in juveniles (https://doi.org/10.1038/s41598-020-79350-7). Building on this, work in mouse models of Dravet syndrome and Angelman syndrome, both genetic epilepsy syndromes) generated promising data showing a novel use for ant-134 to treat genetic epilepsies (both publications under review).

Other microRNA targets in epilepsy: As the molecular mechanisms of miR-134 were not conserved in human tissues, I also explored other potential therapeutic microRNA dysregulations in epilepsy models. Notably, my work showed that rational correction of upregulated microRNAs in TLE is therapeutic (http://dx.doi.org/10.1073/pnas.1919313117). More recent work identified miR-335 as a novel microRNA dysregulation in epilepsy. Unlike other upregulated miRs, miR-335 inhibition actually exacerbates seizure phenotypes. Through combined biophysical and molecular analysis, we showed that miR-335 likely acts via VGSC transcripts, and that miR-335 overexpression may be therapeutic.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

-First ever use of antimiRs in human brain tissue, and method to interrogate molecular and biophysical effects in human brain. Impact: Provides a key new resource to bridge the gap between preclinical and clinical antimiR research. Supports the development of a new class of molecules to the epilepsy clinic and a new treatment strategy for ~20 million people with drug-resistant epilepsy.

-Novel insights into the molecular mechanisms of miR-134 in human brain. Impact: Scientists better understand the functions of miR-134 in humans and the implications of this for clinical translation.

-Preclinical evidence that ant-134 is therapeutic in early-onset epilepsies and Angelman syndrome. Impact: new and more precise treatment options for children with seizure disorders and intractable genetic epilepsies.

-Identification of miRs 10, 21, 142 and 335 as therapeutic targets in epilepsy. Impact: In parallel with my work to bridge miR therapies to the epilepsy clinic, I also identified multiple new miR targets which can be progressed in parallel down the same pipeline.

-A novel machine learning approach to seizure detection. Impact: Researchers can quickly and accurately identify and quantify seizures in experimental models. This overcomes limitations of subjective bias and is a tool for higher throughput and more reliable epilepsy research.

-Demonstration of efficacy of BICS01, a new anti-seizure compound. Impact: BICS01 performed better than a frontline AED in seizure models, and therefore may be a highly promising new approach to treating seizures.

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