Opportunities and challenges for microRNA-targeting therapeutics for epilepsy
Authors: Gareth Morris, Denis O’Brien, David C. Henshall
Journal: Trends in Pharmacological Sciences (2021)

MicroRNAs are small noncoding RNAs that suppress the translation of mRNAs and have emerged as therapeutic targets in epilepsy and other diseases.Inhibition of certain miRNAs with antisense oligonucleotides (ASOs) is therapeutic in preclinical temporal lobe epilepsy (TLE) models.Chemical modifications can be made to improve the therapeutic properties of ASOs.The blood–brain barrier is an obstacle in delivering ASOs as therapies in neurological disease, and ASOs currently have to be administered by direct central injection.miRNA-targeting ASOs are in clinical trials for other diseases, raising the prospect that they could also be safely applied in epilepsy. Epilepsy is a common and serious neurological disorder characterised by recurrent spontaneous seizures. Frontline pharmacotherapy includes small-molecule antiseizure drugs that typically target ion channels and neurotransmitter systems, but these fail in 30% of patients and do not prevent either the development or progression of epilepsy. An emerging therapeutic target is microRNA (miRNA), small noncoding RNAs that negatively regulate sets of proteins. Their multitargeting action offers unique advantages for certain forms of epilepsy with complex underlying pathophysiology, such as temporal lobe epilepsy (TLE). miRNA can be inhibited by designed antisense oligonucleotides (ASOs; e.g., antimiRs). Here, we outline the prospects for miRNA-based therapies. We review design considerations for nucleic acid-based approaches and the challenges and next steps in developing therapeutic miRNA-targeting molecules for epilepsy. Epilepsy is a common and serious neurological disorder characterised by recurrent spontaneous seizures. Frontline pharmacotherapy includes small-molecule antiseizure drugs that typically target ion channels and neurotransmitter systems, but these fail in 30% of patients and do not prevent either the development or progression of epilepsy. An emerging therapeutic target is microRNA (miRNA), small noncoding RNAs that negatively regulate sets of proteins. Their multitargeting action offers unique advantages for certain forms of epilepsy with complex underlying pathophysiology, such as temporal lobe epilepsy (TLE). miRNA can be inhibited by designed antisense oligonucleotides (ASOs; e.g., antimiRs). Here, we outline the prospects for miRNA-based therapies. We review design considerations for nucleic acid-based approaches and the challenges and next steps in developing therapeutic miRNA-targeting molecules for epilepsy. Epilepsy (see Glossary) is a common, often life-long brain disease characterised by recurrent, spontaneous seizures that are the result of hypersynchronous discharges of neurons [1.Thijs R.D. et al.Epilepsy in adults.Lancet (London, England). 2019; 393: 689-701Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. There are more than 20 different small-molecule drugs in clinical use that reduce or prevent seizures in people with epilepsy. This is a remarkable achievement, certainly relative to the lack of treatments for many neurological disorders, and is owed to a solid mechanistic understanding of how seizures arise through imbalances between excitation and inhibition and the availability of good disease models that identify molecules with antiexcitability properties [2.Bialer M. White H.S. Key factors in the discovery and development of new antiepileptic drugs.Nat. Rev. Drug Discov. 2010; 9: 68-82Crossref PubMed Scopus (356) Google Scholar]. 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Acquired forms of epilepsy, such as TLE, have a limited genetic basis and probably arise from an earlier brain insult that leads to select cell loss, gliosis, neuroinflammation, and vascular and microscopic as well as macroscopic reorganisation of brain networks [4.Klein P. et al.Commonalities in epileptogenic processes from different acute brain insults: do they translate?.Epilepsia. 2018; 59: 37-66Crossref PubMed Scopus (86) Google Scholar,5.Blümcke I. et al.International consensus classification of hippocampal sclerosis in temporal lobe epilepsy: a Task Force report from the ILAE Commission on Diagnostic Methods.Epilepsia. 2013; 54: 1315-1329Crossref PubMed Scopus (456) Google Scholar]. Because of this, single targets, such as ion channels, may be unsuitable or insufficient to overcome drug resistance, achieve disease modification, or prevent epileptogenesis [6.Löscher W. The holy grail of epilepsy prevention: preclinical approaches to antiepileptogenic treatments.Neuropharmacology. 2020; 167: 107605Crossref PubMed Scopus (26) Google Scholar] (Box 1). Novel approaches that can modify multiple targets may be necessary. Additionally, suitable target(s) may not reside on the outside of neurons but instead be intracellular. Both challenges could be solved by using RNA-based medicines such as ASOs, which offer virtually unlimited potential to target any gene, and small noncoding RNAs called microRNAs (miRNAs), which are 'multi-pathway' regulatory molecules (for a recent review on the general topic of RNA medicines, the reader is referred elsewhere [7.Dammes N. Peer D. Paving the road for RNA therapeutics.Trends Pharmacol. 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Current antiseizure drugs are symptomatic agents and do not modify the underlying pathophysiology. Disease-modifying drugs are ideally sought, which change the disease. Disease modification is defined as curing epilepsy, or at least modifying its severity or responsiveness to antiseizure drugs, by addressing the underlying disease pathophysiology. Prevention of epileptogenesis or antiepileptogenic drugs refers to agents that can prevent the development of epilepsy following a precipitating insult. Such drugs could be given to individuals at risk of epilepsy, for example, after traumatic brain injury, stroke, or infection. However, since only a percentage of such patients would normally develop epilepsy, biomarkers of epileptogenesis are needed so that only the at-risk patients are treated. The first miRNA medicine reached human trials in 2013 [8.Janssen H.L.A. et al.Treatment of HCV infection by targeting microRNA.N. Engl. J. 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Ambitious programmes, such as LifeTime to map the molecular basis of various common diseases at the single-cell level [11.Rajewsky N. et al.LifeTime and improving European healthcare through cell-based interceptive medicine.Nature. 2020; 587: 377-386Crossref PubMed Scopus (19) Google Scholar], will undoubtedly generate additional RNA targets for medicine that may synergise with advances in chemical modifications and delivery carriers to increase the scope for deployment in the future. In this review, we highlight the key advances, and also outstanding challenges, in the use of miRNA-targeting therapies in brain diseases with a specific focus on epilepsy. Mature miRNAs are generated via a multistep process. miRNAs are initially transcribed as relatively large (can be more than 1 kb) hairpin structures known as pri-miRNA. 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Because of this short seed requirement, individual miRNAs have dozens and potentially hundreds of potential targets. An individual miRNA can act upon several different pathways or a single mRNA target may contain sites for multiple miRNAs, which can increase the strength of negative regulation. Various mechanisms restrict the potential target pool, including cell-type-specific expression of miRNAs and mRNA and RNA compartmentalisation. Notably, several neuron-enriched miRNAs are concentrated at synapses, which brings them into close contact with mRNAs involved in shaping dendritic spines and synaptic plasticity [26.Brennan G.P. Henshall D.C. MicroRNAs as regulators of brain function and targets for treatment of epilepsy.Nat. Rev. Neurol. 2020; 16: 506-519Crossref PubMed Google Scholar,27.Schratt G.M. et al.A brain-specific microRNA regulates dendritic spine development.Nature. 2006; 439: 283-289Crossref PubMed Scopus (1337) Google Scholar]. 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However, these small molecules can saturate the miRNA pathway [49.Van Gestel M.A. et al.ShRNA-induced saturation of the microRNA pathway in the rat brain.Gene Ther. 2014; 21: 205-211Crossref PubMed Scopus (21) Google Scholar], can cause aberrant expression of the miRNA in cells where it is not usually present [50.Brennan G.P. et al.Dual and opposing roles of microRNA-124 in epilepsy are mediated through inflammatory and NRSF-dependent gene networks.Cell Rep. 2016; 14: 2402-2412Abstract Full Text Full Text PDF PubMed Google Scholar], and are associated with toxicity and mortality when delivered to the brain in vivo. As such, this approach has received less attention in the development of miRNA-based therapeutics. A potential solution may be the use of gene therapy vectors to deliver miRNAs in a more controlled and spatially restricted manner to specifically targeted cell types in the brain (as in [51.Christensen M. et al.Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo.Front. Neural Circuits. 2010; 3: 16PubMed Google Scholar] and see Delivery of miRNA-targeting therapeutics). The leading approach to reduce miRNA expression is using ASO antimiRs (see Table 1 for a summary of preclinical antimiR therapies in epilepsy to date). Oligonucleotide therapies offer lasting, but not permanent, gene targeting, require infrequent dosing due to their target binding and slow elimination, and are increasingly reaching the clinic [7.Dammes N. Peer D. Paving the road for RNA therapeutics.Trends Pharmacol. Sci. 2020; 41: 755-775Abstract Full Text Full Text PDF PubMed Google Scholar,52.Levin A.A. Treating disease at the RNA level with oligonucleotides.N. Engl. J. Med. 2019; 380: 57-70Crossref PubMed Scopus (106) Google Scholar]. In its most basic form, an antimiR is simply a synthetic ASO with perfect complementarity to a region (8-mer to 23-mer) of its miRNA target [53.Rooij E. Kauppinen S. Development of microRNA therapeutics is coming of age.EMBO Mol. Med. 2014; 6: 851-864Crossref PubMed Scopus (0) Google Scholar]. This basic antimiR unit can be stabilised by the addition of a terminal hairpin at either side of the targeting sequence [54.Vermeulen A. et al.Double-stranded regions are essential design components of potent inhibitors of RISC function.RNA. 2007; 13: 723-730Crossref PubMed Scopus (88) Google Scholar]. RNA sponges are larger constructs containing multiple miRNA binding sites [55.Ebert M.S. et al.MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.Nat. Methods. 2007; 4: 721-726Crossref PubMed Scopus (1494) Google Scholar]. 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Studies where antimiRs were given during or immediately after status epilepticus are considered pretreatment., bAbbreviations: chol, cholesterol; High K+, 9 mM potassium (slice model); IAKA, intra-amygdala kainic acid; icv, intracerebroventricular; ICVKA, intracerebroventricular kainic acid; LNA, locked nucleic acids; MOE, O-methoxyethyl; OMe, O-methyl; PILO, pilocarpine; PO, phosphodiester; PPS, perforant pathway stimulation; PS, phosphorothioate; PTZ, pentylenetetrazole.MicroRNA targetedSpeciesLinkage structureRibose modificationOther specificationsDoseDelivery methodTiming of treatment and seizure modelPrimary therapeutic outcomeRefs10a, 21a, 142aMousePO/PS mixLNA0.5 nmol in 2 μLicvPretreatment: IAKA, PILO, PPSPost-treatment: IAKA65% reduction in spontaneous recurrent seizure frequency[42.Venø M.T. et al.A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 15977-15988Crossref PubMed Google Scholar]134MousePSLNA3′-chol tag0.12 nmol in 1 μL (icv) / 0.24 nmol in 5 μL (intranasal)icv/intranasalPre- and post-treatment: IAKA87% reduction in spontaneous recurrent seizure frequency[88.Jimenez-Mateos E.M. et al.Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects.Nat. Med. 2012; 18: 1087-1094Crossref PubMed Scopus (306) Google Scholar]134RatPSLNA0.36 nmol in 6 μLicvPretreatment: PPS91% reduction in spontaneous recurrent seizure frequency[99.Reschke C.R. et al.Potent anti-seizure effects of locked nucleic acid antagomirs targeting miR-134 in multiple mouse and rat models of epilepsy.Mol. Ther. Nucleic Acids. 2017; 6: 45-56Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar]134MousePSLNA0.12 nmol in 2 μLicvPretreatment: PTZ69% in acute seizure power[99.Reschke C.R. et al.Potent anti-seizure effects of locked nucleic acid antagomirs targeting miR-134 in multiple mouse and rat models of epilepsy.Mol. Ther. Nucleic Acids. 2017; 6: 45-56Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar]134Rat (ex vivo slice model)PSLNA0.12 nmol in 1 μLicvPretreatment: High K+57% increase in time to acute seizure onset[99.Reschke C.R. et al.Potent anti-seizure effects of locked nucleic acid antagomirs targeting miR-134 in multiple mouse and rat models of epilepsy.Mol. Ther. Nucleic Acids. 2017; 6: 45-56Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar,100.Morris G. et al.Spared CA1 pyramidal neuron function and hippocampal performance following antisense knockdown of microRNA-134.Epilepsia. 2018; 59: 1518-1526Crossref PubMed Scopus (8) Google Scholar]134MousePSLNA3′-chol tag0.12 nmol in 2 μLicvPretreatment: PILO58% reduction in acute seizure power[98.Jimenez-Mateos E.M. et al.Antagomirs targeting microRNA-134 increase hippocampal pyramidal neuron spine volume in vivo and protect against pilocarpine-induced status epilepticus.Brain Struct. Funct. 2015; 220: 2387-2399Crossref PubMed Scopus (68) Google Scholar]134RatPO/PS mix2′-MOE0.12 nmol in 2 μLicvPretreatment: ICVKA44% reduction in spontaneous recurrent seizure frequency[103.Gao X. et al.Silencing microRNA-134 alleviates hippocampal damage and occurrence of spontaneous seizures after intraventricular kainic acid-induced status epilepticus in rats.Front. Cell. Neurosci. 2019; 13: 145Crossref PubMed Scopus (14) Google Scholar]135MousePSLNA3′-chol tag1 nmol in 2 μLicvPost-treatment: IAKA~60% reduction in spontaneous recurrent seizure frequency[101.Vangoor V.R. et al.Antagonizing increased miR-135a levels at the chronic stage of experimental TLE reduces spontaneous recurrent seizures.J. Neurosci. 2019; 39: 5064-5079Crossref PubMed Google Scholar]199aRatPSNot specified3′-chol tag1 nmol in 5 μLicvPretreatment: PILO~50% reduction in acute seizure amplitude[109.Wang D. et al.Targeting of microRNA-199a-5p protects against pilocarpine-induced status epilepticus and seizure damage via SIRT1-p53 cascade.Epilepsia. 2016; 57: 706-716Crossref PubMed Google Scholar]203MouseNot specified2′-OMe5 nmol in 24 μLintranasalPost-treatment: PILO72% reduction in spontaneous recurrent seizure frequency[87.Lee S.-T. et al.Inhibition of miR-203 reduces spontaneous recurrent seizures in mice.Mol. Neurobiol. 2017; 54: 3300-3308Crossref PubMed Google Scholar]324MousePO/PS mixLNA0.5 nmol in 2 μLicvPost-treatment: PILOAverage 50% reduction in spontaneous recurrent seizure frequency[34.Tiwari D. et al.MicroRNA inhibition upregulates hippocampal A-type potassium current and reduces seizure frequency in a mouse model of epilepsy.Neurobiol. Dis. 2019; 130: 104508Crossref PubMed Scopus (12) Google Scholar]a Studies that showed a therapeutic effect but did not specify antimiR details were not included. Studies where antimiRs were given during or immediately after status epilepticus are considered pretreatment.b Abbreviations: chol, cholesterol; High K+, 9 mM potassium (slice model); IAKA, intra-amygdala kainic acid; icv, intracerebroventricular; ICVKA, intracerebroventricular kainic acid; LNA, locked nucleic acids; MOE, O-methoxyethyl; OMe, O-methyl; PILO, pilocarpine; PO, phosphodiester; PPS, perforant pathway stimulation; PS, phosphorothioate; PTZ, pentylenetetrazole. Open table in a new tab ASOs have unconventional pharmacokinetic (PK) and pharmacodynamic (PD) properties that have important implications in relation to their efficacy and dosing as drugs [61.Khorkova O. Wahlestedt C. Oligonucleotide therapies for disorders of the nervous system.Nat. Biotechnol. 2017; 35: 249-263Crossref PubMed Scopus (70) Google Scholar]. These properties, and therefore the therapeutic potential of antimiR molecules, can be greatly impacted by chemical modifications to their molecular structure. DNA and RNA nucleotides are naturally linked by phosphodiester (PO) bonds. However, ASOs composed of only PO bonds with unmodified sugar structures are easily degraded by endo- and exonucleases, severely limiting their efficacy. Indeed, exposed ASOs can be degraded by a 3′ to 5′ exonuclease in serum in less than 30 minutes, and degradation occurs even faster in the presence of intracellular exo- and endonucleases [62.Eder P.S. et al.Substrate specificity and kinetics of degradation of antisen