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WO2014096418A2 - Micrornas as therapeutics and biomarkers for epilepsy - Google Patents

Micrornas as therapeutics and biomarkers for epilepsy Download PDF

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Publication number
WO2014096418A2
WO2014096418A2 PCT/EP2013/077836 EP2013077836W WO2014096418A2 WO 2014096418 A2 WO2014096418 A2 WO 2014096418A2 EP 2013077836 W EP2013077836 W EP 2013077836W WO 2014096418 A2 WO2014096418 A2 WO 2014096418A2
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mir
mmu
hsa
group
epilepsy
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PCT/EP2013/077836
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French (fr)
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WO2014096418A3 (en
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Bénédicte DANIS
Patrik FOERCH
Rafal M. KAMINSKI
Anita KRETSCHMANN
Franziska SIEGEL
Alexander Pfeifer
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Ucb Pharma Gbmh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of a miRNA molecule.
  • the invention further relates to a vaccine for use in the treatment or prevention of epilepsy, to a biomarker or group of biomarkers associated with epilepsy, a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, a method or assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
  • epilepsy The World Health Organization (WHO) and the International League against Epilepsy (ILAE) define epilepsy as a chronic, recurrent, repetitive neurological disorder consisting of paroxysmal phenomena caused by excessive and chaotic discharges in neuronal brain cells. Its incidence has two peaks: one in childhood and adolescence and a second more marked one over the age of 60. According to the International Bureau for Epilepsy (IBE), some 50 million people suffer from epilepsy worldwide, with 20-30% of them suffering more than one seizure per month (Forsgren et al., Eur J Neurol 2005;12:245-53).
  • IBE International Bureau for Epilepsy
  • Epilepsy is considered to comprise a diverse set of chronic neurological disorders characterized by seizures. These seizures may be recurrent and unprovoked, or may constitute single seizures combined with brain alterations increasing the chance of future seizures. Epileptic seizures typically result from abnormal, excessive or hypersynchronous neuronal activity in the brain.
  • the etiology of epilepsy can in certain cases be linked to mutations in several genes that code for protein sub units of voltage-gated and ligand-gated ion channels.
  • a further speculated mechanism for some forms of inherited predisposition for epilepsy are mutations of genes coding for sodium channel proteins. Accordingly modified or defective sodium channels may be opened for a prolonged period of time, leading to a hyper-excitability of neurons.
  • larger than normal amounts of the excitatory neurotransmitter glutamate can be released from the affected neurons and may trigger excessive calcium release in post-synaptic cells. The calcium release may, in turn, be neurotoxic to affected cell.
  • the hippocampus which comprises a large number of glutamatergic neurons is highly vulnerable to epileptic seizure, a subsequent spread of excitation, and possible neuronal death.
  • Other possible mechanisms involve mutations leading to a dysfunction of neurotransmitter GABA, as well as mutations in non-ion channel genes.
  • a consequence of the occurrence of epileptic seizures is an interference of physical, emotional, and social functioning. Further consequences of repeated seizures include neuronal loss, gliosis, parenchymal microhemorrhages, excess of starch bodies, leptomeningeal thickening, and periavascular atrophy.
  • Diagnosis of epilepsy is mainly based on detection of symptoms
  • MicroRNAs are a recently discovered class of endogenous, small non-coding RNAs. The first described microRNA, lin-4, was identified in C. elegans in 1993 (Lee et al., 1993, Cell 75(5): 843-85). Currently more than 2000 human mature microRNAs are annotated in databases (e.g. in www.mirBase.org). MicroRNAs regulate gene expression on the post-transcriptional level. They predominantly function as translational repressors by binding to the 3 ' UTR of their target gene mRNAs (He and Hannon, 2004, Nat Rev Genet 5(7): 522-531 ; Filipowicz et al. 2008 Nat Rev Genet 9(2): 102-114).
  • MicroRNAs are encoded in gene introns or in non-coding genomic regions. They are initially transcribed as long primary microRNA strands (pri- microRNA) that are processed to single hairpin precursors (pre-microRNA) by nuclear Drosha. Pre-microRNAs are typically transported to the cytoplasm by Exportin 5. Subsequently, the protein Dicer has been shown to cut the hairpin into the mature microRNA duplex (see Fig. 1 ). Both microRNA single strands may be incorporated into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC subsequently mediates binding of mature microRNAs to their respective recognition site, which is typically in the 3 ' UTR of a target gene, leading to translational repression (He and Hannon, 2004, Nat Rev Genet 5(7): 522-531 ; Filipowicz et al. 2008 Nat Rev Genet 9(2): 102-114; Huntzinger and Izaurralde 2011 , Nat Rev Genet 12(2): 99-1 10).
  • MicroRNAs mainly function as translation repressors, but can also mediate mRNA degradation by direct binding to the open reading frame (ORF) in mRNAs (see Fig. 1 ). Based on this mechanism, every single microRNA may not only inhibit one, but multiple target genes. In addition, most mRNAs contain binding sites for several microRNAs in their 3 ' UTRs, thus further increasing the complexity of the system. It is estimated that about 30% of the human proteome are regulated by microRNAs (Krek et al., 2005, Nat Genet 37(5): 495-500), thus underlining the importance of microRNAs for coordinated protein expression (Selbach et al., 2008, Nature 455(7209): 58-63).
  • microRNAs are abundant in the CNS, apparently coordinating neurogenesis, dendrite formation, synaptic plasticity and neuroinflammation (Christensen and Schratt, 2009, Neurosci Lett 466(2): 55-62; Schratt, 2009, Nat Rev Neurosci 10(12): 842-849). Although there is increasing evidence that microRNAs are implicated in neurodegenerative disorders such as Alzheimer ' s disease, Huntington's disease, Parkinson ' s disease or Prion disease (Saugstad, Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1564-1576), there is still relatively little known about the relevance of microRNAs in the etiology and development of epilepsy. McKiernan et al.
  • the present invention addresses this need and provides a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of Table 1 or 1a.
  • the present inventors could, in particular, identify significantly dysregulated microRNAs in preclinical animal models as well as in the biopsies of TLE patients, which were validated and analysed in a bioinformatic approach with regard to their target genes.
  • the microRNAs of the invention were further analyzed for their role in CNS and epileptogenesis in different in vitro assays as well as in in vivo models. In addition, expression of the respective target genes was determined.
  • hippocampal microRNA expression is significantly changed in epilepsy models and patients.
  • the detected changes appear to be modulated by factors like severity of disease (e.g. seizure frequency) as well as disease duration (e.g. acute vs. chronic epilepsy).
  • the miRNAs of the invention thus play a decisive regulatory role in the molecular mechanisms underlying epileptogenesis.
  • the miRNAS themselves or derivatives thereof, or antagonists thereof thus constitute a highly relevant therapeutic principle in the treatment or prevention of epilepsy. They are particularly considered as extremely valuable means for alleviating and/or reversing any effect of miRNA dysregulation observable in epilepsy.
  • said means as mentioned above comprises a nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 or in section A or H of Table 1 a, or a derivative, fragment or variant thereof, or an antagonist thereof, wherein said nucleic acid molecule or antagonist has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section B of Table 1 or in section B or H of Table 1a or to a complementary sequence thereof.
  • said nucleic acid molecule or antagonist comprises between at least 18 and 24 nucleotides, preferably 20 nucleotides, 21 nucleotides or 22 nucleotides.
  • the nucleic acid or antagonist is or comprises a DNA molecule or an RNA molecule, or a derivative thereof.
  • said RNA molecule as mentioned above is or comprises a pre-miRNA selected from section E of Table 1 or from section E or H of Table 1a, or a derivative, fragment or variant thereof, wherein said pre-miRNA molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section F of Table 1 or in section F or H of Table 1a or to a complementary sequence thereof.
  • said DNA molecule as mentioned above is or comprises a DNA molecule coding for a pre- miRNA, a miRNA or for an antagonist.
  • said DNA molecule is or comprises a double stranded DNA coding for at least one pre-miRNA selected from section E of Table 1 or from section E or H of Table 1a.
  • nucleic acid molecule as mentioned above comprised in a vector replicable in a subject.
  • nucleic acid molecule or antagonist is modified by a conjugate, preferably a linked conjugate.
  • nucleic acid molecule or antagonist comprises a chemically modified base.
  • said antagonist as mentioned above is an antagomir.
  • the nucleic acid molecule or antagonist as mentioned herein above may be modified by at least one modification selected from the group consisting of a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose and a 2'-methoxyethoxy ribose.
  • said nucleic acid molecule as mentioned herein above may comprise a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu- miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR- 193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-mi ' R-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR- 48a-3p, or miR-125a-5p molecule as defined in section A of Table 1 or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a
  • said nucleic acid molecule as mentioned herein above may comprise a hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa- miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p, mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR- 130a-3p or mmu-miR-191-5p molecule as defined in sections A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%,
  • said antagonist as mentioned herein above is an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR- 223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu- miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR- 138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129
  • said antagonist as mentioned herein above is an antagonist of a hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3, mmu-miR-129- 5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu- miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p or mmu-miR-222-3p molecule as defined in section A or H of Table 1 a, or of a derivative, fragment or variant thereof.
  • the present invention relates to a vaccine for use in the treatment or prevention of epilepsy, comprising a nucleic acid molecule or antagonist as mentioned herein above.
  • the present invention relates to an isolated biomarker or group of isolated biomarkers associated with epilepsy, wherein said isolated biomarker or group of isolated biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 or in section A or H of Table 1a, or a derivative, fragment or variant thereof.
  • the present invention relates to an isolated biomarker or group of isolated biomarkers, wherein the increase of expression (up-regulation) of at least one biomarker selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1- 3p, mmu-miR-2 9-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR- 132-3p, mmu-miR-222-3p, mmu-miR
  • the present invention relates to a biomarker or group of biomarkers, wherein the increase of expression (up- regulation) of at least one biomarker selected from the group of hsa-miR-129-5p, hsa- miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, or hsa-miR-494-3p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR- 29-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, or mmu- miR-494-3p as defined in section A of Table 1a, , when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
  • the present invention relates to a biomarker or group of biomarkers, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of mmu-miR- 24-3p, mmu-miR-124-5p, mmu- miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa- miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa- miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p, and miR-125a- 5p, when comparing the expression with the expression of a biomark
  • the present invention relates to a biomarker or group of biomarkers, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR- 34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR- 24-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR- 0-3p, mmu-miR- 30a-3p or mmu-miR
  • the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, and/or a probe specific for the biomarker or group of biomarkers as defined herein above.
  • the present invention relates to a method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined herein above.
  • the present invention relates to a method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample before and during an epilepsy treatment, optionally also after an epilepsy treatment.
  • said treatment is a treatment with a pharmaceutical composition as defined herein above, and/or with at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, zonisamide, and combinations thereof.
  • at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamaz
  • the determining step is accomplished by the measurement of the miRNA level(s) or by the determination of the biological effect of said biomarker or group of biomarkers.
  • the present invention relates to an assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
  • step (d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c) ,
  • testing steps are based on the use of a nucleic acid affinity ligand and/or a peptide affinity ligand for a biomarker or a group of biomarkers as EP2013/077836
  • the present invention relates to a method of identifying an individual eligible for an epilepsy therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to the levels of step (b);
  • the present invention relates to a diagnostic kit for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined herein above, wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, or a probe specific for the biomarker or group of biomarkers as defined herein above.
  • a detecting agent for a biomarker or group of biomarkers as defined herein above wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for
  • the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
  • the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
  • Fig. 1 provides an overview of micro NA biogenesis, showing that microRNAs hairpin precursors are transcribed and processed in the nucleus, that pre- microRNA (70-80nt hairpins) are exported to the cytoplasm and are finally incorporated in the RISC complex, which mediates microRNA function.
  • Fig. 2 shows an overview over the analytical steps described in the
  • Fig. 3 depicts the results of a bioinformatics analysis for the identification of some epilepsy-relevant microRNA target genes according to the invention.
  • Fig. 4 gives a schematic overview on functional in vitro assays and their relevance for the analysis of molecular mechanisms underlying epileptogenesis.
  • Fig. 5 provides a schematic presentation of lentiviral miRNA overexpression system used in vitro.
  • Fig. 6 shows fluorescence microscope photographs of mouse hippocampal neurons in culture at DIV13 and lentiviral constructs expressing several CMV-GFP-miRNAs 5 days upon transduction.
  • Fig. 7 shows the overexpression of several miRNAs in transduced hippocampal neurons. Values are calculated to scrambled miRNA sequence (control, miR-con) using a AA Ct method and the SN068 house keeping gene.
  • Fig. 8 depicts a graphical scheme of in vitro proliferation assay.
  • Fig. 9 shows fluorescence microscopy photographs of SH- SY5Y transduced with CMV-GFP-miRNA lentiviral constructs, showing GFP reporter expression upon 72 hours.
  • Fig. 10 shows a growth assay measured by MTT.
  • Fig. 11 depicts a graphical scheme of in vitro excitotoxicity induced by glutamate assay.
  • Fig. 12 shows effects of miRNA in excitotoxicity pathway induced by glutamate exposure in hippocampal neurons. Hippocampal neurons were transduced at DIV7 treated with 10uM glutamate for 24 hours at DIV13, measured by MTT assay
  • Fig. 13 shows a graphical scheme of in vitro activation of microglia assay.
  • Fig. 14 shows photographs of mouse primary microglia isolated from P3 brains: (A) non induced and (B) induced.
  • Fig. 15 shows secretion of IL-6.
  • Fig. 14 (A) provides absolute values of secreted IL-6.
  • levels of IL-6 were measured by ELISA assay in wt microglia in cells transduced with miRNA of interest.
  • Fig. 16 provides a graphical scheme of in vitro astrocytes growth assay.
  • Fig. 17 shows results of experiments with secondary mouse astrocytes.
  • Fig. 17 (A) provides fluorescence microscopy photographs of secondary mouse astroytes 3 to 5 days upon lentiviral transduction. Astrocytes expressing CMV-GFP- mi ' R constructs at MOM 00 differ in viability and size depending of expressed miRNA.
  • Fig. 17 (B) shows survival assay of secondary mouse astroytes 5 days upon LV transduction measured by MTT.
  • Fig. 18 shows the overlap of miRNA expression between the two chronic epilepsy mouse models (pilocarpine and SSSE) and the acute seizure mouse model (6 Hz) using Venn diagrams.
  • Fig. 19 shows fluorescence microscopy images of SH-SY5Y cells transduced with CMV-GFP-miRNA lentiviral constructs showing GFP reporter expression upon 72 hours.
  • Scale bar 20 left column; 10 m right column.
  • Fig. 20 shows growth assay measured by MTT.
  • SH-SY5Y cells expressing CMV-GFP-miR-124-3p and CMV-GFP-miR142-3p construct showed significantly decreased growth rate compared to wild-type and miR-control overexpressing SY5Y cells at the end of measurement.
  • Fig. 21 shows a graphical scheme of in vitro activation of primary microglial cells.
  • Fig. 22 depicts the results of a FACS analysis of the CD1 1 b + fraction. After isolation and immunomagnetic separation of the CD1 1b + cell population, cells were stained with anti-CD45 antibodies and analyzed by flow cytometry. CD11b and CD45-coexpressing populations were identified with red colour and the proportion of surviving cells was determined by propidium iodide (PI).
  • PI propidium iodide
  • A Total cell population isolated from brain tissues before immunomagnetic separation using CD11 b + beads.
  • B Negative fraction, a very low number of CD11 b + /CD45 + cells was detected in this fraction, whereas a high proportion of CD1 1b7CD45 + cells was sorted in the positive fraction (C).
  • D A tabel comparing the differences in CD1 b + /CD45 + cells between three fractions, suggesting high efficiency and specificity of the CD11b + cell separation from postnatal mouse brain tissues.
  • Fig. 23 shows the morphological characteristics of primary cultured mice microglial cells (P0-P3) 72 hours after transfection of miRNA-mimics.
  • Scale bar 20 ⁇ left column; 10 ⁇ right column.
  • Fig. 25 shows a graphical scheme of in vitro astrocytes viability assay.
  • Fig. 26 shows fluorescence microscopy images of secondary mouse astroytes 72 hours upon lentiviral transduction.
  • Astrocytes expressing CMV-GFP-miR constructs at MOI30 differ in viability and size depending of expressed miRNA.
  • Scale bar 20 pm left- ; 10 ⁇ right- column.
  • Fig. 27 shows representative images of cultured mice astrocytes (POPS) after 72 hours lentviral transduction. Double immunofluorescence staining for GFAP (red) and GFP (green).
  • A-C Wild-type astrocytes show absence of GFP expression, but GFAP immunoreactivity.
  • D-l Astrocytes transduced with CMV-GFP- miR constructs showing abundant GFP expression colocalized with the astrocytic marker GFAP. Scale bar: 10 pm.
  • the present invention relates to a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of miRNAs and corresponding biomarkers and methods for using them, as well as suitable kits and compositions.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of ⁇ 20 %, preferably ⁇ 15 %, more preferably ⁇ 10 %, and even more preferably ⁇ 5 %.
  • the present invention concerns in one aspect a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of the following Table 1 :
  • the present invention concerns human orthologues of mouse miRNA molecules as defined hin Table 1 , e.g. for all miRNA molecules starting with mmu. Such human orthologues can be obtained by database comparisons, or by homology searches.
  • the present invention relates to human orthologue miRNA molecules of murine miRNAs which can be obtained by performing amplification reactions on human RNA or DNA or human samples obtained from several different stages or sources, e.g. epilectic patiens, tissue samples etc. as define herein with primers which have been derived from the mmu miRNA sequences.
  • these primer sequences may further be adapted to known functional or structural differences between the hsa and mmu miRNA molecules.
  • the present invention accordingly envisages the use of human miRNA molecules which are structurally and/oir functionally homologous to murine miRNA molecules as describe in Table 1 , or derivable form Table 32, or from any other Table disclosed herein.
  • human orthologue refers to a molecule which is structurally and/or functionally equivalent to another miRNA molcule, in particular to a murine miRNA molecule. Particularly preferred are human orthologues of mmu-miR- 129-5p, mmu-miR-142-3p, mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, mmu-miR- 494-3p mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, or mmu-miR-2137.
  • the present invention concerns a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A or section H of the following Table 1a:
  • microRNA as used in section A of Table 1 or in section A or H of Table 1a relates to a mature microRNA molecule as identified by the indicated designation. The designation is based on the nomenclature provided in relevant databases such as mirbase.org as of 10 December 2012 or later version thereof.
  • the mature microRNA of section A of Table 1 or 1 a or of section H of Table 1a is further defined by a corresponding sequence as identified in section B of Table 1 or 1a or in section H of Table 1a.
  • the effect of dysregulation of a miRNA may, in further embodiments, also be an effect detectable on the level of pre-miRNA. Such an effect may be a dysregulation of pre-miRNA molecules as identified in section E of Table 1 or 1a.
  • pre-miRNAs are based on the nomenclature provided in relevant databases such as mirbase.org as of 10 December 2012 or later versions thereof.
  • the pre-microRNA of section E of Table 1 or 1a or of section H of Table 1a is further defined by a corresponding sequence as identified in section F of Table 1 or 1a of in section H of Table 1a.
  • the effect of dysregulation of a miRNA may, in specific embodiments, be correlated to the regulatory behavior of mature miRNAs as observed in analyses described in the Examples of the invention.
  • the effect of dysregulation may be a up-regulation of a mature miRNA as indicated by a "+” in section C of Table 1 or 1a, or a down-regulation as indicated by a "+” in section D of Table 1 or 1a.
  • the micro-RNAs mmu-miR-34b-5p and mmu-miR-191-5p as mentioned in Table 1 a show an up-regulation in specific models, while they show a down- regulation in other specific models. This behavior is apparently dependent on the model and in particular on the phase analysed (see also the examples portion).
  • the level of dysregulation may further be derived from indications in Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43, e.g.
  • FC fold change
  • the fold change may vary according to the model.
  • FC values may in specific embodiments at least be comparable within the model, e.g. within one of Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43. It is preferred using miRNAs with a high FC value as indicated in Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43 e.g. the first 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of Tables 1 to 25 or 27 to 31 , 33 to 39 and 42 to 43. It is particularly preferred using miRNAs as shown in Table 1 a.
  • the present invention also relates to corresponding DNA sequences of any of SEQ ID NO: 1 to 445 as defined in Table 1 , 1 a and/or Table 32, or of the miRNAs as described in any other part of the description.
  • the present invention relates to a composition or compositions which is/are capable of inhibiting or antagonizing an upregulated miRNA as indicated by a "+” in section C of Table 1 or 1a, or by a "+” in section “upreguiation” of Table 32, or which is able to reduce the amount or activity of an upregulated miRNA as indicated by a "+” in section C of Table 1 or 1 a, or in section "upreguiation” of Table 32.
  • Such an increase may, for example, be provided by the presence of antagonist of upregulated miRNAs in the composition, as described herein.
  • the present invention relates to a composition or compositions which is/are capable of increasing the amount of a downregulated miRNA as indicated by a "+" in section D of Table 1 or 1 a, or by a "+” in section “downregulation” of Table 32.
  • Such an increase may, for example, be provided by the presence of the downregulated miRNAs in the composition itself, as described herein.
  • the present invention relates to at least one miRNA selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-711 , mmu-miR-7 0, mmu-miR-882, mmu-miR-20a-5p, mmu-miR-335-3p, mmu-miR-219-5p, mmu-miR-140-5p, mmu-miR-294-5p, mmu-miR-690, mmu-miR-207, mmu-miR-338-3p, mmu-miR-34b-3p, mmu-miR-465b-5p, mmu-let-7f-5p, mmu-miR-1983, mmu-miR- 302a-3p, mmu-let-7b-5p, mmu-miR-290-5p, mmu-miR-3
  • the present invention relates to at least one miRNA selected from the group of mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR- 191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-18 b-5p, hsa-mi ' R-34b-5p, hsa-miR- 676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p, mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-494-3p, mmu-mRNA
  • the present invention relates to the group of miRNAs as presented in Table 3, or at least one of the miRNAs presented in Table 3. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (24 h, 2 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 4, or at least one of the miRNAs presented in Table 4. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (24h, 2 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 5, or at least one of the miRNAs presented in Table 5. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (28 d, 1 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 6, or at least one of the miRNAs presented in Table 6. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (28 d, 1 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 7, or at least one of the miRNAs presented in Table 7. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (28 d, 2 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 8, or at least one of the miRNAs presented in Table 8. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (28 d, 2 h Diazepam) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 9, or at least one of the miRNAs presented in Table 9. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the SSSE study (24 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 10, or at least one of the miRNAs presented in Table 10. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the SSSE study (24 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 11 , or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being up regulated in the SSSE study (28 d) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 12, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being down regulated in the SSSE study (28 d) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 13, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (3 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 14, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (3 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 15, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (6 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 16, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (6 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 17, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (24 h) or at least one of the miRIMAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 18, or at least one of the miRNAs presented in Table 18. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (24 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 19, or at least one of the miRNAs presented in Table 19. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (72 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 20, or at least one of the miRNAs presented in Table 20. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (72 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 21 , or at least one of the miRNAs presented in Table 21. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (3 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 22, or at least one of the miRNAs presented in Table 22. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Audiogenic study (3 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 23, or at least one of the miRNAs presented in Table 23. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (6 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 24, or at least one of the miRNAs presented in Table 24. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (72 h) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 25, or at least one of the miRNAs presented in Table 25. In a further embodiment, the present invention relates to the group of miRNAs being qRT-PCR validated in animal models or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 27, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being up regulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 28, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being down regulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 29, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being qRT-PCR validated in hippocampi of human TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 30, or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling (acute phase) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 31 , or at least one of the miRNAs presented in Table
  • the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling (chronic phase) or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 32, or at least one of the miRNAs presented in Table 32.
  • the present invention in particular also refers to miRNAs and pre-miRNAs as defined in SEQ ID NO: 322 to 375 or 383, 384, 385, 386, 387, 389, 390, 392, 393, 395, or 401 to 435 as indicated in said Table 32.
  • the present invention relates to the group of miRNAs as presented in Table 33, or at least one of the miRNAs presented in Table 33. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the pilocarpine model at the timepoints 24h and 28d or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 34, or at least one of the miRNAs presented in Table 34. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the SSSE model at the timepoint 24h or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 35, or at least one of the miRNAs presented in Table 35. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the SSSE model at the timepoint 28d or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 36, or at least one of the miRNAs presented in Table 36. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 3h following seizure or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 37, or at least one of the miRNAs presented in Table 37. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 6h following seizure or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 38, or at least one of the miRNAs presented in Table 38. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 24h following seizure or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 39, or at least one of the miRNAs presented in Table 39. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and down regulated miRNAs in the 6 Hz model at the timepoint 72h following seizure or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 40, or at least one of the miRNAs presented in Table 40. In a further embodiment, the present invention relates to the group of miRNAs being commonly deregulated miRNAs in the pilocarpine and SSSE model at 24 h and 28d following SE or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 42, or at least one of the miRNAs presented in Table 42. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 43, or at least one of the miRNAs presented in Table 43. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 43, or at least one of the miRNAs presented in Table 43. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
  • the present invention relates to the group of miRNAs as presented in Table 44, or at least one of the miRNAs presented in Table 44. In a further embodiment, the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling or at least one of the miRNAs of this group.
  • the miRNAs may be one, two, three, 4 or all or the mature miRNA molecules miR-2137, miR-124-3p, miR-142-3p, miR-184, miR-132-3p, or of corresponding pre-miRNAs as defined in Table 1.
  • miR-2137 is a mouse specific miRNA identified by tissue sequencing. Target genes and physiological relevance of this miRNA are unknown. It is situated in the first intron of Zfp185 protein located on the X chromosome. As can be derived from the Examples, miR-2137 is highly up-regulated in acute phase of mouse epilepsy models only. Currently, no ortholog of this miRNA is familiar in human genome. Nevertheless, its specific seed sequence might be of importance in biochemical pathways conserved between mouse and human.
  • miR-124 is deeply conserved and shows CNS-enriched expression in all animals examined from nematode to human (see Stark et al., 2005; Cao et al., 2007, Genes Dev.; 21 (5):531-6; Kapsimali et al., 2007, Genome Biol. 2007;8(8): R173; Makeyev et al., 2007, Mol Cell. 2007, 27(3):435-48; Visvanathan et al., 2007, Genes Dev; 21 (7):744-9; Rajasethupathy et al., 2009; Neuron; 63(6):803-17; and Shkumatava et al., 2009, Genes Dev; 23(4):466-81 ).
  • pre-miR-124a loci there are three pre-miR-124a loci (pre-miR- 124a-1 , pre-miR-124a-2 and pre-miR-124a-3) in the mouse and human genomes.
  • pre-miR-124a-1 is located on chromosome 8p23.1.
  • chromosomal duplication, deletion or mutation of the 8p23.1 region have been reported to be involved in cerebral development and neuropsychiatric disorders, including autism, bipolar disorder, schizophrenia, learning difficulties, epilepsy and microcephaly (see Tabares-Seisdedos et al., 2009, Mol. Psychiatry 14, 563-589; Baulac et al., 2008, Arch. Neurol.
  • miR-124* and miR-124 originating from the same precursor transcripts are acutely down-regulated in mouse model of epilepsia (pilocarine model) with the rate of 12% and 10%, respectively.
  • miR- 124 shows same tendency and is down-regulated in patients with AHS compared to patients suffering from nonAHS form of epilepsy.
  • These two groups of patients show different clinical picture, of which the most striking feature is death of neurons in severe neuronal cell loss and gliosis in the hippocampus, specifically in the CA-1 (Cornu Ammonis area 1 ) and subiculum of the hippocampus in AHS patients.
  • the most striking feature is death of neurons in severe neuronal cell loss and gliosis in the hippocampus, specifically in the CA-1 (Cornu Ammonis area 1 ) and subiculum of the hippocampus in AHS patients.
  • CA-1 Cornu Ammonis area 1
  • AHS patients group is more severe/pharmacoresistant.
  • miR-142 is associated with a variety diseases especially with cancer. It is also expressed in the CNS. miR-142 has further been shown to target the transcripts of key neuronal genes encoding the D1 dopamine receptor (DRD1 ) which plays an important role in neurons. As can be derived from the Examples, miR-142 is up regulated in the mouse and human miRNA profiling.
  • D1 dopamine receptor D1 dopamine receptor
  • miR-184 is a single copy gene and evolutionarily conserved at the nucleotide level from flies to humans. In humans, miR-184 is located within region 25.1 on the q-arm of chromosome 15, and its corresponding transcript is comparatively small (84 bp) which is not encoded near other clustered miRNAs. Mature miR-184 is particularly enriched in the brain and testis. miR-184 is identified as a novel contributor to neuronal survival following both mild and severe seizures. As can also be derived from the Examples, miR-184 is down-regulated to 13% comparing AHS and nonAHS human samples and its deregulation is detected only in human form of disease, pointing to this miRNA as potentially highly specific biomarker for human epileptic brain.
  • miR-132 is enriched in neuronal cells and several targets for miR-132 have been described, including mediators of neurological development, synaptic transmission, inflammation and angiogenesis. Furthermore, for miR-132 neuroprotective functions have been shown after Status Epilepticus. miR-132 arises from the miR-212/132 cluster located in the intron of a non-coding gene on mouse chromosome 11. The transcription of this cluster was found to be enhanced by the transcription factor CREB (cAMP-response element binding protein) (see Wanet et al., 2012, Nucleic Acids Res.,40(1 1 ): 4742-53). The data provided in the Examples show that miR-132 is upregulated in mouse models of epilepsy. Taking into consideration role of miR- 32 in dendritic sprouting, this miRNA may play a significant role in brain during disease.
  • CREB cAMP-response element binding protein
  • miR-135b has been described as being upregulated after the induction of oxidative stress in mouse hippocampal neurons. It was further described that human miR-135b regulates the expression of DISC-1 (Disrupted-in-schizophrenia-1 ), a gene which is known to play an important role in different neuropsychiatric phenotypes (shizophrenia, depression). The data provided in the Examples show miR-135b-5p is significantly up regulated in the chronic phase of both mouse chronic epilepsy models (SSSE and Pilocarpine). miR -222 has been found to be expressed in the brain and was shown to to regulate endogenous ICAM-1 expression.
  • SSSE mouse chronic epilepsy models
  • Pilocarpine Pilocarpine
  • miR-222 targets PTEN, a phosphatase and negative regulator of the PI3K signalling pathway, which is important for central axon growth.
  • the data provided in the Examples show that the expression of miR-222-3p was upregulated at 28 days in both chronic mouse epilepsy models.
  • miR-129 has two loci in humans and in mice. In humans the gene encoding for mir-129-1 is located on Chromosome 7 and the gene for mir-129-2 is located on chromosome 11. For mice it is chromosome 6 and chromosome 2, respectively. Both stem loop sequences express the same mature sequence of miR- 129-5p. It was demonstrated that F Rl is a target of miR-129-5p. While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. The data provided in the Examples show that mi ' R-129-5p was significantly upregulated in both mouse models and in human patient samples in the comparison AHS versus nonAHS.
  • FXS Fragile X syndrome
  • miR-1298 has not been described so far in the literature.
  • the data provided in the Examples show that miR-1298 is a highly relevant biomarker.
  • miR-181 b two loci in humans and in mice could be identified.
  • the gene encoding for mir-181 b-1 is located on Chromosome 1 and the gene for mir- 81 b-2 is located on chromosome 9.
  • the gene encoding for mir-181 b-1 is located on chromosome 1 and and the gene for mir-181 b-2 is located on chromosome 2, respectively. It could be shown that a downregulation of miR-181 leads to a pro-inflammatory response in astrocytes.
  • the data provided in the Examples show that miR-181 b-5p was significantly downregulated in human patient samples in the comparison AHS plus nonAHS versus post mortem. In addition, it was downregulated in both chronic epilepsy mouse models at 24 hours.
  • miR-34b was shown to be downregulated in patients with Parkinson ' s disease.
  • the data provided in the Examples show that miR-34b-5p was significantly downregulated in human patient samples in the comparison AHS plus nonAHS versus post mortem.
  • miR-676 was identified in an expression profiling approach of a murine TH-MYCN neuroblastoma model, which revealed similarities with human tumors.
  • Mmu-miR-140-3p was identified in a approach to elucidtate early mechanisms of pathobiology by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice.
  • miR-130a was shown to be expressed in gfiobSastoma.
  • miR-191 was described that as being differentially expressed following induction of neuronal activity, indicating its possible role in neuronal development and neuronal activity.
  • miR-494-3p was described as being involved in Parkinson ' s disease.
  • Epilepsy refers to a disease comprising a diverse set of chronic neurological disorders characterized by seizures. Such seizures may be recurrent and unprovoked. They may alternatively constitute single seizures combined with brain alterations and thus increase the chance of future seizures. Epileptic seizures may, for example, result from abnormal, excessive or hypersynchronous neuronal activity in the brain. Epilepsy may further be classified according to seizure types or forms. Seizure types are typically organized according to whether the source of the seizure within the brain is localized ⁇ partial or focal onset seizures) or distributed (generalized seizures). Epilepsy comprising partial seizures may further be divided on the extent to which awareness is affected. If awareness is unaffected, then it is an epilepsy comprising simple partial seizure.
  • a partial seizure may typically spread within the brain, i.e. lead to secondary generalization.
  • Generalized seizures typically involve loss of consciousness and may further be divided according to the effect on the body. Examples include absence (petit mal), myoclonic, clonic, tonic, tonic-clonic (grand mal), and atonic seizures.
  • the term "means for alleviating or reversing the effect of a dysregulation of at least one miRNA” as used herein refers to a compound, which is capable of overcoming or influencing an effect caused by an aberrant expression of a miRNA, preferably a mature miRNA as defined in Table 1 or 1 a, or a pre-miRNA as defined in Table 1 or 1 a.
  • the aberrant expression may be a decreased or increased expression of the miRNA, preferably a mature miRNA as defined in Table 1 or 1a, or a pre-miRNA as defined in Table 1 or 1a in comparison to an expression situation in a healthy or normal subject.
  • the effect may additionally or alternatively also be a secondary effect, e.g.
  • a dysregulation of one or more target genes of a miRNA may be an increase or decrease of expression of the target gene, or the presence or absence of the proteineous gene product of the target gene, a mislocalization of the protein, a modification of interaction with further proteins etc.
  • the pharmaceutical compositions of the present invention are capable of reducing or reversing the effect of a dysregulation by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95%, 96%, 97%, 98, 99% or 100% when compared to an untreated control.
  • the use of a pharmaceutical composition as defined herein leads to a reconstitution of the miRNA expression state of a healthy or normal subject.
  • the means for alleviating or reversing the effect of a dysregulation of at least one miRNA may comprise, in one embodiment, a nucleic acid molecule comprising a mature miRNA molecule as defined in section A of Table 1 or 1a or section H of Table 1 a.
  • a "nucleic acid molecule" as used herein is a polynucleotide or oligonucleotide having, comprising or consisting of a nucleotide sequence. It may comprise at least two deoxy ribonucleotides or ribonucleotides in either single- or double-stranded form.
  • the nucleic acid may be DNA, RNA, PNA, CNA, HNA, LNA or ANA molecule, preferably a DNA or RNA molecule.
  • the DNA may be in the form of, e.g. A-DNA, B-DNA or Z-DNA.
  • the RNA may be in the form of, e.g. p-RNA, i.e. pyranosysl-RNA or structurally modified forms like hairpin RNA or a stem-loop RNA.
  • PNA peptide nucleic acid
  • the PNA backbone is typically composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • the various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds.
  • PNAs are generally depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
  • CAN relates to an aminocyclohexylethane acid nucleic acid. Furthermore, the term relates to a cyclopentane nucleic acid, i.e. a nucleic acid molecule comprising for example 2'-deoxycarbaguanosine.
  • HNA hexitol nucleic acids
  • DNA analogues which are built up from standard nucleobases and a phosphorylated 1 ,5-anhydrohexitol backbone.
  • LNA locked nucleic acids
  • a locked nucleic acid is a modified and thus inaccessible RNA nucleotide.
  • the ribose moiety of an LNA nucleotide may be modified with an extra bridge connecting the 2' and 4' carbons. Such a bridge locks the ribose in a 3'-endo structural conformation.
  • the locked ribose conformation enhances base stacking and backbone pre-organization. This may significant!y increase the thermal stability, i.e. melting temperature of the oligonucleotide.
  • ANA relates to arabinoic nucleic acids or derivatives thereof.
  • a preferred ANA derivative in the context of the present invention is a 2'-deoxy-2 - fluoro-beta-D-arabinonucleoside (2'F-ANA).
  • nucleic acid molecules may comprise a combination of any one of DNA, RNA, PNA, CNA, HNA, LNA and ANA. Particularly preferred are mixtures of LNA nucleotides with DNA or RNA bases.
  • nucleic acid molecules as defined herein above may be in the form of short oligonucleotides, long oligonucleotides or polynucleotides.
  • nucleic acid molecules as defined herein above may be single- stranded or double-stranded.
  • single-stranded nucleic acid relates to nucleic acid molecules which comprise a single sugar-phosphate backbone and/or are not organized in a helical form. Preferably these nucleic acid molecules exhibit no secondary structures or intermolecular associations.
  • double stranded nucleic acid relates to nucleic acid molecules which comprise two sugar-phosphate backbones. In a preferred embodiment the double-stranded nucleic acids are organized in a double helical form.
  • double-stranded nucleic acids according to the present invention may be composed of different types of nucleic acid molecules, e.g.
  • They may alternatively also be composed of combinations of stretches of any of the above mentioned nucleotide variants.
  • nucleotide refers to a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases may include purines and pyrimidines, which may further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, or synthetic derivatives of purines and pyrimidines, which may further include modifications which place new reactive groups, e.g. amines, alcohols, thiols, carboxylates, and alkylhalides.
  • the DNA may further be in the form of plasmid DNA, pre-condensed DNA, a PGR product, a vector (e.g. PI, PAC, BAC, YAC, artificial chromosomes), an expression cassettes, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups.
  • a vector e.g. PI, PAC, BAC, YAC, artificial chromosomes
  • an expression cassettes e.g. PI, PAC, BAC, YAC, artificial chromosomes
  • Nucleic acid molecules may, in specific embodiments, include or comprise known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring, and which may have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, or 2'-0-methyl ribonucleotides.
  • a nucleic acid molecule according to the invention may further encompass nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • a particular nucleic acid sequence also implicitly encompass conservatively modified variants thereof (e.g., codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • a nucleic acid molecule according to the invention may comprise, essentially consist of or consist of a miRNA molecule as defined herein, e.g. a mature miRNA molecule as defined in sections A or B of Table 1 or 1a, or in section H of Table 1 a, and/or may comprise, essentially consist of or consist of a portion a mature miRNA molecule as defined in sections A and B of Table 1 or 1 a, or in section H of Table 1a.
  • a nucleic acid molecule may comprise, essentially consist of or consist of a pre-miRNA molecule as defined herein, e.g.
  • a per-miRNA molecule as defined in sections E or F of Table 1 or 1a, or in section H of Table 1 a and/or may comprise, essentially consist of or consist of a portion a pre-miRNA molecule as defined in sections E or F of Table 1 or 1 a or in section H of Table 1a.
  • a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule, e.g. genomic DNA molecule, providing the sequence of, or encoding a miRNA as defined herein, e.g. a mature miRNA as defined in sections A or B or Table 1 or 1a, or in section H of Table 1 a.
  • a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule, e.g. genomic DNA molecule, providing the sequence of or encoding a pre-miRNA molecule as defined in section E, or comprising, essentially consisting or consisting of the sequence of any of the SEQ ID NOs. indicated in section F of Table 1 or 1a, or in section H of Table 1a.
  • a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule providing the sequence of or encoding an antagonist according to the invention, e.g. an antisense molecule.
  • the DNA molecule may accordingly be equipped with promoter or transcription elements allowing for the provision of an antisense nucleic acid molecule as defined herein.
  • the DNA molecule may be single-stranded, or preferably double-stranded.
  • the nucleic acid molecule may further comprise, essentially consist of or consist of a portion or fragment of such a DNA molecule, e.g. genomic DNA.
  • the nucleic acid molecule may further comprise, essentially consist of, or consist of an RNA molecule derived or transcribed from said DNA molecule, e.g. genomic DNA molecule, in an early, medium or late stage of processing into a miRNA molecule.
  • nucleic acid molecules constituting DNA sequences complementary to such RNA molecules, or antisense molecules thereof.
  • the nucleic acid molecule may comprise or be a stem-loop forming nucleic acid molecule.
  • Such stem-loop forming nucleic acid molecule may serve as Dicer-substrates, leading to the provision of pre- miRNAs and finally to mature miRNA molecules.
  • Stem-loop forming nucleic acids may be identified according to the information provided in sections A, B, E and F of Table 1 or 1 a, or in section H of Table 1 a.
  • a stem-loop forming nucleic acid may have a length of between 35 to 3500 nucleotides, preferably of between 35 to 2000 nucleotides, more preferably of between 35 to 120 nucleotides.
  • a stem-loop forming nucleic acid molecule may, in specific embodiments, include pre-miRNAs, or may have be larger or less processed than a pre-miRNA.
  • synthetic or artificial stem-loop forming nucleic acid molecules may be designed which are also suitable for producing or obtaining a miRNAs of the invention as defined herein above.
  • the nucleic acid molecule may, in further specific embodiments, also comprise homologous or heterologous flanking regions, e.g. derived from other genetic contexts or other organisms, plasmids, genes, open reading frames etc. at the 5' or 3' terminus of the molecule as defined herein above.
  • the nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 or 1 a or in section H of Table 1 a may have a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1a or in section H of Table 1a, e.g.
  • nucleic acid molecule comprising a miRNA molecule, e.g.
  • a pre-miRNA molecule as defined in section E of Table 1 or 1a or in section H of Table 1a may have a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section F of Table 1 or 1a or in section H of Table 1a, e.g.
  • nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • the query sequence may be an entire sequence or any fragment as described herein.
  • nucleic acid molecule is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., 1990, Comp. App. Biosci. 6: 237-245.
  • RNA sequence In a nucleotide sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5'and 3'of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage may then be subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at the 5' end.
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5'and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5' or 3' end of the subject sequence, which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3'of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • a derivative of the nucleic acid molecule comprising a miRNA molecule as defined herein above.
  • the term "derivative" as used herein refers to a nucleic acid molecule that may differ from the nucleic acid molecule it refers to in that one or more nucleotides of the original molecule are substituted by other nucleotides and/or modified, e.g. chemically modified, by methods known to the skilled person.
  • the derivative may, in particularly preferred embodiments, be a derivative of a DNA or RNA molecule as defined herein. Preferably, the derivative may still be capable of fulfilling its respective function.
  • a “derivative” may comprise, for example, nucleotide substitutions, deletions or insertions, e.g.
  • the derivate may comprise or contain modified bases or base moieties.
  • it may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methyla
  • the molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the derivative molecule may comprise alternatively or additionally at least one modified phosphate backbone, e.g. a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an a Iky I phosphotriester, and a formacetal or analog thereof.
  • the nucleic acid molecule may comprise a chemically modified base.
  • chemically modified base means that an oligonucleotide and/or nucleic acid molecule comprises a 2'-modified nucleotide containing ol igodeoxy n ucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance.
  • the presence of methylphosphonate modifications may increase the affinity of the oligonucleotide for its target molecule and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide.
  • nucleic acid molecules may further include nucleobase oligomers containing modified backbones or non-natural internucleoside linkages.
  • the nucleic acid molecule or antagonist may be modified by the introduction or conversion of a nucleotide or bond in said nucleic acid molecule or antagonist molecule into a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose or a 2'-methoxyethoxy ribose.
  • modifications comprising more than one of the mentioned introductions or conversion, or more than one different of the mentioned introductions or conversion.
  • the modification may be provided by carrying out chemical reactions with a molecule of the invention, or by employing one of the modified entities during the synthesis or production of the nucleic acid molecule or antagonist.
  • fragment refers to a short polynucleotide having, comprising or consisting of a nucleic acid sequence, which is a portion of the sequence contained in the nucleotide sequence of any one of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440 as defined in section B of Table 1 or 1a or any one of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116 in section H of Table 1a, or any one of SEQ ID NOs: 150 to 321 or 384, 387, 390, 393, 438 or 441 of section F of Table 1 or 1a or any one of SEQ ID NO.
  • a nucleotide fragments according to the invention may be at least about 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt of the nucleotide sequence of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or it may be about at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16
  • a fragment of "at least 15 nt in length” may, for example, include 15 or more contiguous bases from the sequence contained in the nucleotide sequence of SEQ ID NOs: 1 to 375 or 383 to 435 or the complementary strand thereto.
  • representative examples of polynucleotide fragments of the invention include, for example, fragments comprising, or alternatively consisting of a sequence from about nucleotide number 1-10, 5-15, 11-20 of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or from about nucleotide number 1-10, 5-15, 1 1 -20, 16 to 25, 21-30, 26-35, 31-40, 36-45, 41 to 50, 46 to 55, 51 to 60, 56 to 65 etc.
  • a particularly preferred fragment of a miRNA molcule is a seed sequence of the miRNA.
  • seed sequence refers to a sequence of about 6-8 nucleotides in length at the 5' end of an animal miRNA which is an important determinant of target specificity.
  • the seed sequence is typically situated from nucleotide position 2 to nucleotide position 7-9 in a miRNA molecule.
  • 6 nt, 7 nt and 8 nt molecules comprising the seed sequence which are preferred by the present invention, may have an influence on the binding of the miRNA to its target, e.g. mRNA.
  • molecules comprising seed sequences, or sub-fragments thereof e.g.
  • the seed sequence is perfectly complementary to the target sequences, e.g. a target mRNA.
  • the sequence may, for example, be derived from the SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440 as mentioned in Table 1 or 1a, or from the SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16 as mentioned in Table 1a. Further details may be derived from suitable literature sources such as Lewis BP et al, 2005. Cell 120(1 ): 15-20. Additional information on seed regions may further be derived from suitable databases such as the Targetscan database (http://www.targetscan.org/ faqs.htm).
  • a nucleic acid molecule e.g. a miRNA molecule, an RNA molecule, a DNA molecule, or an antagonist, e.g. antisense molecule
  • a nucleic acid molecule may have a length of at least about 10 to 50 nt. It is particularly preferred that the length is at least 18 to 24 nt. For example, the length may be 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt or 24 nt.
  • variants refers to a polynucleotide which differs from the polynucleotide of the present invention, e.g. the polynucleotide of SEQ ID NOs: 1 to 375, or 383 to 435, but retains essential properties thereof.
  • variants are overall closely similar, and, in many regions, identical to the polynucleotide of the present invention.
  • Variants according to the present invention may contain alterations in the stem region of a pre-miRNA molecule, in the loop region of a pre-miRNA, in the 3' terminus of a mature miRNA, in the 5' region of a mature miRNA, or any combination thereof. Further variants may be combination of two or more, e.g. 3, 4, 5, 6, 7, 8, 9, 10 or miRNA molecules on a nucleic acid molecule. Such combinations may, for example, be partially overlapping and accordingly comprise a common overlapping region and specific termini. In specific embodiment, a variant may comprise one or more seed sequences as defined herein above.
  • an antagonist of a nucleic acid molecule comprising a miRNA molecule as defined herein above.
  • the term "antagonist of a nucleic acid molecule comprising a miRNA molecule” as used herein refers to any molecule or compound which is capable of reducing the amount or stability of nucleic acid molecule comprising a miRNA molecule of the present invention, or of reducing or abolishing the binding capability of a miRNA molecule or a nucleic acid molecule comprising a miRNA molecule.
  • the term particularly refers to a molecule or compound which is capable of reducing the amount or stability or binding capability of a mature miRNA molecule as defined in section A of Table 1 or 1a or in section H of Table 1a, or the amount or stability or binding capability of a pre-miRNA molecule as defined in section E of Table 1 or 1a or in section H of Table 1a.
  • reducing the amount of a nucleic acid molecule comprising a miRNA molecule means a diminishment of the amount of the nucleic acid molecule comprising a miRNA moleculet by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in comparison to a control situation in which no antagonist is used.
  • reducing the stability of a nucleic acid molecule comprising a miRNA molecule means that the half-life of an nucleic acid molecule comprising a miRNA molecule is diminished, preferably by factor 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000 or 0000 in comparison to a control situation in which no antagonist is used.
  • reducing the binding capability of a nucleic acid molecule comprising a miRNA molecule means a diminishment of the binding capability of the nucleic acid molecule comprising a miRNA molecule to a target by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in comparison to a control situation in which no antagonist is used.
  • said antagonist of a nucleic acid molecule comprising a miRNA molecule according to the present invention is an antisense molecule against the nucleic acid molecule comprising comprising a miRNA molecule, more preferably against a nucleic acid comprising the sequence of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390,
  • antisense molecule refers to the complementary strand of a nucleic acid corresponding to the sequences comprised in SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326,
  • the antisense molecule may also be a double-stranded molecule able to form a triple-helix structure.
  • the antisense molecule of the invention comprises a sequence complementary to at least a portion of a nucleic acid molecule comprising a miRNA molecule according to the present invention.
  • an antisense molecule may also be based on untranscribed or regulatory sequences surrounding the genomic locus of expression of a miRNA molecule as defined in sections A or E of Table 1 or 1a.
  • antisense technology may be used to control, i.e. reduce or terminate miRNA gene expression through antisense DNA or RNA, or through triple-helix formation.
  • an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence.
  • a vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention.
  • Such a vector would contain a sequence encoding the antisense nucleic acid of the invention.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule.
  • Corresponding vectors can be constructed by recombinant DNA technology methods known to the person skilled in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.
  • the antisense molecule may be separately administered.
  • the 5' coding portion of a nucleic acid according to the present invention e.g. of the sequence of SEQ ID NO: 1 to 149, or SEQ ID NO; 150 to 321 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or any one of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327,
  • 398, 400, 266, 443, 445 or 284 may be used to design an antisense RNA or DNA oligonucleotide, e.g. of a length of 5 to 25, 5 to 50, or 5 to 80 nt.
  • the antisense oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.
  • the antisense nucleic acids of the invention may comprise a sequence complementary to at least a portion the nucleic acid comprising a miRNA molecule according to the present invention.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion the nucleic acid comprising a miRNA molecule" as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the molecule, e.g. forming a stable duplex triplex formation in the case of double stranded antisense nucleic acids. The ability to hybridize may depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • the larger the hybridizing nucleic acid the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex or triplex.
  • a person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the antagonist e.g. antisense molecule
  • the antagonist may be a nucleic acid molecule comprising, essentially consisting of or consisting of a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1 a, e.g.
  • the antagonist e.g. antisense molecule
  • the antagonist may be a nucleic acid molecule comprising, essentially consisting of or consisting of a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section F of Table 1 or 1a, e.g.
  • the antagonist or antisense molecule may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (ca rboxy hyd roxy I methyl ) uracil, 5-carboxymethyl- aminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N6-adenine, 7-methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouraci
  • the antisense molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose.
  • the antisense molecule may comprise alternatively or additionally at least one modified phosphate backbone, e.g. a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an a Iky I phosphotriester, and a formacetal or analog thereof for example, to improve stability of the molecule, hybridization, etc.
  • the antagonist or antisense molecule may be chemically modified as defined herein above, e.g. comprising 2'- modifiednucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance.
  • An antisense molecule according to the present invention may preferably be a DNA molecule, or an RNA molecule, or a chimeric molecule, or a mixtures of DNA and RNA, or a mixture of DNA, RNA and/or chimeric molecules, or of derivatives or modified versions thereof.
  • Further envisaged antagonists include an aptamer specific for a miRNA molecule according to the present invention.
  • An "aptamer specific for specific for a miRNA molecule" as used herein refers to a short nucleic acid molecule, e.g.
  • An antagonists according to the invention may further be a catalytic RNA molecule or a riboyzme.
  • catalytic RNA or “ribozyme” refers to a non-coding RNA molecule, which is capable of specifically binding to a target RNA and of cutting or degrading said target RNA, e.g.
  • RNA molecules comprising the nucleotide sequence of any one of SEQ ID NO: 1 to 149, or SEQ ID NO: 150 to 321 , or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16, or SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284.
  • ribozymes cleave RNA molecules at site specific recognition sequences and may be used to destroy RNAs.
  • a preferred example of ribozymes are hammerhead ribozymes.
  • Hammerhead ribozymes cleave RNAs at locations dictated by flanking regions that form complementary base pairs with the target RNA.
  • the construction and production of hammerhead ribozymes is known in the art and is described in Haseloff and Gerlach, 1988, Nature, 334: 585-591.
  • the ribozyme may be engineered so that the cleavage recognition site is located near the 5' end of the RNA corresponding to the miRNAs of the invention.
  • Ribozymes or catalytic RNAs of the invention can be composed of modified oligonucleotides and may be delivered to cells, which express the polynucleotides of the invention in vivo.
  • DNA constructs encoding a ribozyme or catalytic RNA according to the present invention may be introduced into the cell according to suitable methods known to the person skilled in the art.
  • a preferred method of delivery involves using a DNA construct encoding the ribozyme under the control of a strong constitutive promoter so that transfected cells will produce sufficient quantities of the ribozyme to destroy targeted messages and inhibit translation.
  • An antagonists according to the invention may, in a particularly preferred embodiment, be an antagomir of a miRNA molecule as defined in sections A or E of Table 1 or 1a, or of section H of Table 1a.
  • the term "antagomir” as used herein relates to a chemically engineered oligonucleotide, which is specifically used to silence a microRNA.
  • An antagomir typically is a small synthetic RNA that is highly, e.g. 95%, 96%, 97%, 98%, 99% complementary, or preferably perfectly complementary to a specific miRNA target, e.g.
  • nucleic acid molecule comprising the sequence of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a.
  • antagomir typically involves a mispairing at the cleavage site of the Argonaut-2 activity (Ago-2) or a base modification to inhibit Argonaut-2 cleavage. It is preferred that an antagomir according to the invention has a modification which renders it more resistant to degradation. Particularly preferred are the presence of 2' methoxi groups and phosphothioates.
  • Antagomirs may preferably be used to constitutively inhibit the activity of a miRNA according to the invention, e.g. a miRNA as defined in section A or C or Table 1 or 1a, or in section H of Table 1a.
  • the nucleic acid molecule of the invention as defined herein above, or an antangonistic sequence as defined herein above may be comprised in a vector.
  • vector refers to a nucleic acid molecule, for example, a plasmid, cosmid, or bacteriophage, that is capable of replication in a host cell.
  • a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell.
  • expression may be under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
  • Transducing viral (e.g., retroviral, adenoviral, lentiviral and adeno- associated viral) vectors may, for example, be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression. It is particularly preferred that the vector allows for the production of RNA molecules, e.g. single stranded RNA molecules or double stranded RNA molecules, e.g. stem-loop structures of miRNA molecules.
  • the vector may, for example, be a eukaryotic expression vector such as e.g.
  • Vectors may preferably be used for replication and/or expression in vertebrate cells.Further details on vectors and suitable methods for the provision and manipulation of vectors whould be known to the skilled person, or can be derived from suitable literature sources such as (Sambrook and Russell 2001 , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press 3rd ed; Pfeifer and Hofmann, 2009 , Methods Mol Biol 530: 391-405).
  • a vector or a portion thereof may be transcribed, producing a nucleic acid comprising a miRNA molecule, a miRNA molecule, a pre-miRNA molecule, or an antisense nucleic acid of the invention.
  • the vector may accordingly comprise a sequence as defined in any one of SEQ ID NO: 1 to 149, or SEQ ID NO: 150 to 321 , or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a.
  • the vector may remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired nucleic acid.
  • Polynucleotides according to the present invention may be joined, for example, to a vector, which contains a selectable marker for propagation in a host.
  • the polynucleotide insert may be operably linked to an appropriate promoter for the host cell in which the vector is to be used, e.g. phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs.
  • an appropriate promoter for the host cell in which the vector is to be used e.g. phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs.
  • Particularly preferred is the use of the CMV, CamKII, U6 and/or H1 promoter, or of derivatives or variants thereof.
  • Other suitable promoters are known
  • the expression constructs may further contain sites for transcription initiation and termination.
  • the efficiency of expression may further be enhanced by the inclusion of appropriate transcription enhancer elements etc.
  • Selectable marker may be, for instance, dihydrofolate reductase, G418, hygromycin or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for cu!turing in E. coli and other bacteria.
  • Further selection markers include the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase.
  • Suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia.
  • An example of a preferred expression vector is pl_enti6/V5-DEST (available e.g. from Invitrogen).
  • a further preferred example of a suitable expression vector which allows for the production of dsRNA directly in the target cell is vector pSUPER (available e.g. from OligoEngine). Further information on the vector and the mechanism how the dsRNA can be produced by using said vector may be derived from Brummelkamp et al., 2002, Science, Vol. 296, 550-553.
  • vector pSilencer available, e.g. from Ambion. Further details on the vector may be derived from Sui et al., 2002, Proc. Natl. Acad. Sci. Vol. 99, 5515-5520.
  • the vector is a vector for transducing a mammalian cell, e.g. a human cell, which provides the nucleotide sequence of a miRNA molecule as defined in section A of Table 1 or 1 a, or in section H of Table 1 a.
  • the vector preferably further comprises one or more regulatory sequences operably linked thereto to allow transcription of said sequence in said cell.
  • a termination sequence may be present.
  • the vector may comprise a promoter being functional in a mammalian cell, e.g. in a human cell, operably linked thereto a first nucleic acid molecule which comprises a sequence as defined in SEQ ID NO: 1 to 149, or in SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16 of section H of Table 1a.
  • the vector may comprise a second nucleic acid, which comprises a sequence, which is reverse complementary to the first sequence, i.e.
  • a termination sequence may be present.
  • nucleic acid molecule or the antagonist as defined herein may include other appended groups such as peptides, or agents facilitating transport across the cell membrane or the blood-brain barrier, hybridization triggered cleavage agents or intercalating agents. Also envisaged is a conjugation of the nucleic acid molecule or the antagonist to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting molecule.
  • a preferred moiety is a cholesterol moiety or a lipid moiety or a bile acid.
  • a further moiety for conjugation may be a carbohydrate, a phospholipid, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or a dye.
  • a conjugate group may be attached directly to a nucleic acid molecule, antagonist or derivative thereof as defined herein.
  • a conjugate group may be attached to a nucleic acid molecule antagonist or derivative thereof as defined herein via a suitable linker moiety.
  • Envisaged linker moieties may, for example, be selected from amino hydroxy I, carboxylic acid, thiol, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 -carboxylate (SMCC), 6-aminohexanoic acid (ALEX or AHA), substituted C1 -C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl.
  • ADO 8-amino-3,6-dioxaoctanoic acid
  • SMCC succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 -carboxylate
  • ALEX or AHA 6-aminohexanoic acid
  • substituted C1 -C10 alkyl substituted or unsubstitute
  • a linker may be selected from a hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl or alkynyl group. Also envisaged is a conjugation to a polymer, an antibody or an RNA molecule.
  • a nucleic acid molecule, antagonist or derivative thereof as defined herein may be conjugated to one or more stabilizing groups that are attached to one or both termini of the molecule to enhance properties such as, for example, nuclease stability.
  • Preferred examples or such stabilizing groups are cap structures which typically protect a modified nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • the cap may be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3 -cap) of the molecule, or can be present on both termini.
  • Cap structures may include, for example, inverted deoxy abasic caps.
  • Suitable, envisaged cap structures may be selected from a 4',5'- methylene nucleotide, a 1 -(beta-D-erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1 ,5-anhydrohexitol nucleotide, an L-nucleotide, an alpha- nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo- pentofuranosyl nucleotide, an acyclic 3' ,4'-seco nucleotide, an acyclic 3,4- dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety,
  • a pharmaceutical composition of the invention comprises a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR- 298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b- 3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa- miR-221-3p, miR-767-5p, hsa-miR-148a-3p or miR-125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof
  • a pharmaceutical composition of the invention comprises a nucleic acid molecule which comprises one or more mature miRNAs selected from mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR- 140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa- mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191-5p molecule as defined in sections A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has
  • mmu-miR- 24-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p, and miR- 125a-5p could be identified to be down-regulated in epilepsy models as described in the Examples, a nucleic acid molecule comprising such a miRNA is assumed to counter-act the down-regulation, thus reconstituting
  • mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR- 130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181b-5p, hsa- miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191- 5p could be shown to be down-regulated in epilepsy models as described in the Examples, or are assumed to be down regulated, a nucleic acid molecule comprising such a miRNA is assumed to counter-act the down-regulation, thus reconstituting a normal or healthy miRNA
  • a down- regulation of mmu-miR-34b-5p was shown for specific phases, e.g. based on a mouse acute model as described in the Examples.
  • a down-regulation of mmu-miR- 191-5p was shown only for specific phases, e.g. based on a Pilocarpine/SSEE mouse model as described in the Examples.
  • a nucleic acid molecule comprising mmu-miR-34b-5p, hsa-miR-34b-5p, mmu-miR-191-5p or hsa- miR-191 -5p may be assumed to counter-act the down-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation in accordance with these specific phases or similar phases. This may advantageously be reflected by certain time windows for administration or a phase dependent administration or activation scheme.
  • the pharmaceutical composition may further comprise any derivative, variant, fragment, conjugate, linked conjugate, vector, vector portion, pre-miRNA, genomic encoding DNA molecules or sequences, stem-loop forming molecules or sequences etc.
  • mmu- miR-124-5p as defined herein above of one or more of mmu- miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140- 3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir- 181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p, or optionally of mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR- 130a-3p, mmu-m
  • the pharmaceutical composition comprises a vector or entity which allows the expression or provision of a mmu-miR-124-5p, mmu-miR-181 b- 5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu- miR-191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa- miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191 -5p; or optionally of a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p,
  • a pharmaceutical composition of the invention comprises an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219- 5p, mmu-miR-135b-5p, mmu-miR-129-1 -3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-2 a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR- 222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR
  • a pharmaceutical composition of the invention comprises an antagonist of a mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR- 184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa- miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 molecule as defined in section A or H of Table 1a, or of a derivative, fragment or variant thereof.
  • mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR-494-3p, hsa-miR-129-5p, hsa-miR- 142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, and hsa-miR-494-3p could be identified to be up-regulated in epilepsy models as described in the Examples, or are assumed to be up-regulated, an antagonist against such a miRNA is assumed to counter-act the up- regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation.
  • an antagonist against mmu-miR-34b-5p, hsa- miR-34b-5p, mmu-miR-191 -5p or hsa-miR-191 -5p may be assumed to counter-act the up-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation in accordance with these specific phases or similar phases. This may advantageously be reflected by certain time windows for administration or a phase dependent administration or activation scheme.
  • the pharmaceutical composition may, for example, comprise an antisense nucleic acid molecule of, or an aptamer, an antagomir, or a ribozyme against any one of mature mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR- 494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3; or optionally of any
  • a pharmaceutical composition may also, e.g. optionally, comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • a carrier is pharmaceutically acceptable, i.e. is generally non-toxic to a recipient at the dosage and concentration employed. It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as provided by a sucrose solution.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.
  • Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of, e.g., solutions, suspensions, emulsion, powders, sustained- release formulations and the like.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Some other examples of substances which can serve as pharmaceutical carriers are sugars, such as glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; cranberry extracts and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations
  • wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers and anti-oxidants can also be present. It is also envisaged by the present invention to administer the active ingredients of the pharmaceutical composition in encapsulated form, e.g. as cellulose encapsulation, in gelatine, with polyamides, niosomes, wax matrices, with cyclodextrins or liposomally encapsulated.
  • active ingredients of the pharmaceutical composition in encapsulated form, e.g. as cellulose encapsulation, in gelatine, with polyamides, niosomes, wax matrices, with cyclodextrins or liposomally encapsulated.
  • the ingredients may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical composition may be formulated in accordance with routine procedures adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Preferred is a direct injection into the brain.
  • the composition may accordingly be formulated for a brain injection, e.g. comprising suitable buffers, diluents, stabilizers etc.; further details would be known to the skilled person.
  • an administration via the nose e.g. intranasal.
  • the composition may accordingly be formulated as inhalation composition e.g. based on an aerosolizing agent and/or provided via an inhaler, nebulizer, or dispenser; further details would be known to the skilled person.
  • Another envisaged administration is administration via the eye.
  • the composition may accordingly be formulated as eye drops or droplet formulation; further details would be known to the skilled person.
  • the formulation of a composition in the form of nanoparticles are also envisaged by the present invention.
  • a nanoparticle may be lipid- based and comprise or be bound with a miRNA molecule or an antagonist thereof.
  • the nanoparticle may subsequently be administered to the brain, e.g. via direct injection or on any suitable alternative route.
  • peptides which may be bound to a miRNA molecule or an antagonist and which may be provided to the brain, e.g. directly, or indirectly via any suitable administration route.
  • a short peptide derived from rabies virus glycoprotein (RVG), preferably RVG-9R may be used, e.g. as described in Kumar et al., 2007, Nature, 448(7149):39-43.
  • a miRNA molecule or antagonist as defined herein may be provided on or linked to a carrier.
  • Suitable carriers are, for example, cationic cell penetrating peptides (CPP), cation ic polymers, dentrimers, cationic lipids.
  • CPP cationic cell penetrating peptides
  • cation ic polymers dentrimers
  • cationic lipids e.g., a combination with an aptameric structure.
  • Lipid or nanoparticle structures as mentioned herein may comprise more than one carrier element as define herein. Further details may be derived from suitable literature sources, e.g. from Wang et al., 2010, The AAPS Journal, 12(4), 492- 503.
  • SNALPs stable nucleic acid lipid particles
  • LDL particles LDL particles
  • suitable literature sources e.g. from Davidson and McCray, 2011 , Nature Reviews Genetics, 12, 329-340.
  • the use of 'brain homing' peptides or antibodies linked to liposomes or nanoparticles comprising miRNAs or antagonists is envisaged. Further details may be derived from suitable literature sources, e.g. from Boudreau et al., Human Molecular Genetics, 201 1 , 20, (1 ): R21-R27.
  • the present invention further comprises and may use any new development in this area, in particular, new stabilizing components which may be linked or combined with miRNAs or antagonists as defined herein, or new carriers or carrier-like elements, in particular carriers allowing to cross the blood-brain barrier and/or to enter the brain.
  • the pharmaceutical composition may also be formulated as neutral or salt form.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the pharmaceutical composition of the present invention can also comprise a preservative.
  • Preservatives according to certain compositions of the invention include buty!paraben, ethylparaben, imidazolidinyl urea, methylparaben, O- phenylphenol, propylparaben, quaternium-14, quaternium-15, sodium dehydroacetate, zinc pyrithione, thimerosal and the like.
  • the preservatives may be used, for example, in amounts effective to prevent or retard microbial growth. Generally, the preservatives are used in amounts of about 0.1 % to about 1 % by weight of the total composition with about 0.1 % to about 0.8% being preferred and about 0.1 % to about 0.5% being most preferred. It is particularly preferred including a protective agent for RNA molecules, e.g. an RNAse inhibitor.
  • the pharmaceutical composition may comprise or be mixed with at least one suitable adjuvant.
  • Adjuvants may be used to enhance the effectiveness of the pharmaceutical composition.
  • adjuvants include, for example, chloroquine, protic polar compounds, such as propylene glycol, polyethylene glycol, glycerol, EtOH, 1 -methyl L-2-pyrrolidone or their derivatives, or aprotic polar compounds such as dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane, dimethylformamide, dimethylacetamide, tetra-methylurea, acetonitrile or their derivatives.
  • DMSO dimethylsulfoxide
  • DMSO dimethylsulfoxide
  • di-n-propylsulfoxide dimethylsulfone
  • sulfolane dimethylformamide
  • dimethylacetamide tetra-methylurea
  • a pharmaceutical composition according to the present invention may be administered to a patient, subject or individual with the help of any suitable delivery system known to the person skilled in the art, e.g., via encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction may be any suitable method known, including topical, enteral or parenteral introduction.
  • the methods of introduction may also include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalational, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents.
  • infusion or bolus injection by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents.
  • Administration can be systemic or local. It is particularly preferred to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may, for example, be facilitated by an intraventricular catheter, preferably attached to a reservoir, such as an Ommaya reservoir. Also envisaged are further suitable forms of direct brain injection. Pulmonary or inhalational administration can be employed, e.g., via the use of an inhaler or nebulizer, and a concomitant formulation with an aerosolizing agent.
  • a preferred method of local administration is by direct injection.
  • any ingredient of the pharmaceutical composition of the present invention as defined herein above may be complexed with a delivery vehicle to be administered by direct injection into or locally within the area of arteries.
  • Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
  • Another method of local administration is to contact a pharmaceutical composition of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the pharmaceutical composition can be coated on the surface of tissue inside the wound or the pharmaceutical composition can be injected into areas of tissue inside the wound.
  • ingredients of the pharmaceutical composition of the present invention as defined herein above can be complexed to a targeted delivery vehicle.
  • Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
  • Envisaged methods of systemic administration include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using standard methods in the art (see, for example, Stribling et al., 1992, PNAS, 189: 1 1277-11281 ).
  • Oral delivery can be performed by complexing ingredients of the pharmaceutical composition of the present invention as defined herein above to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.
  • a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.
  • Such carriers include plastic capsules or tablets, such as those known in the art.
  • Topical delivery can be performed, for instance, by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
  • a lipophilic reagent e.g., DMSO
  • the pharmaceutical composition may be delivered directly to internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the site of interest.
  • the pharmaceutical composition may also be administered to disease sites at the time of surgical intervention.
  • the pharmaceutical composition may be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249, 1527- 1533; Treat et al., 1989 in Liposomes in the Therapy of Infectious Disease and Cancer,
  • the composition can be delivered in a controlled release system.
  • a pump may be used.
  • polymeric materials can be use.
  • a controlled release system can be placed in proximity of a therapeutic target, e.g. the brain, lymphatic organs etc. thus requiring only a fraction of the systemic dose.
  • composition of the present invention can be administered to a mammal.
  • mammal as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, the term “mammal” encompasses human beings. Also encompassed are mammals such as dogs, cats, mice, rats, monkeys, rabbits, goats, sheep, pigs, guinea pigs, cattle, horses etc.
  • administered means administration of a therapeutically effective dose of the pharmaceutical composition.
  • therapeutically effective amount is meant a dose that produces the effects for which it is administered, preferably this effect is a reduction or prevention of epileptic seizures.
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • the pharmaceutical composition may be used in both human therapy and veterinary therapy, preferably in human therapy.
  • the compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein.
  • the concentration of the therapeutically active compound in the formulation may vary from about 0.00001-100 wt %.
  • concentration of the active ingredients or compounds of a pharmaceutical composition according to the present invention may be further adjusted to the intended dosage regimen, the intended usage duration, the exact amount and ratio of all ingredients of the composition and further factors and parameter known to the person skilled in the art.
  • Assays may optionally be employed to help identify optimal ratios and/or dosage ranges for ingredients of pharmaceutical compositions of the present invention.
  • the precise dose and the ratio between the ingredients of the pharmaceutical composition as defined herein above to be employed in the formulation will also depend on the route of administration, and the exact type of disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses or ingredient ratios may be extrapolated from dose-response curves derived from in vitro or (animal) model test systems.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • a pharmaceutical composition may preferably be given once a week, more preferably 2 times, 3 times, 4 times, 5 times or 6 times a week and most preferably daily and or 2 times a day or more often, unless otherwise indicated.
  • the dosages may be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., several times a day.
  • the present invention relates to a vaccine for use in the treatment of epilepsy, comprising a nucleic acid molecule as defined herein above, or an antagonist as defined herein above.
  • the vaccine may comprise a nucleic acid molecule comprising or encoding a miRNA molecule as identified in section A of Table 1 or 1a, or in section H of Table 1 a, or may comprise a nucleic acid molecule comprising or encoding a pre-miRNA molecule as identified in section E of Table 1 or 1 a, or in section H of Table 1 a.
  • the vaccine may comprise a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR- 298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b- 3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa- miR-221-3p, miR-767-5p, hsa-miR-148a-3p, or miR-125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, wherein
  • the vaccine may comprise a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-5p, mmu-miR-181 b-5p, mmu- miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191- 5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676- 3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191 -5p molecule as defined in section A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has
  • the vaccine may comprise a nucleic acid molecule giving yield to an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-22
  • the vaccine may comprise a nucleic acid molecule giving yield to an antagonist of a mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191- 5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu- miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR
  • a vaccine according to the present invention may, for example, comprise expression vectors capable of providing miRNA molecules, or antisense molecule, e.g. DNA plasmid vectors, viral vectors etc. as described herein above. Also envisaged is the presence of host cells expressing such miRNAs or antisense molecules.
  • the components of the vaccine may be delivered using one or more vehicles. Additionally, the components may be organized in the form of a kit, e.g. comprising additional kit ingredients, e.g. a leaflet etc.
  • Nucleic acid molecules in a vaccine as defined herein above may be provided in the vaccine in the form of vectors, e.g. DNA plasmids or viral vectors.
  • Vectors in particular viral vectors may be capable of replication or replication-impaired or non-replicating.
  • non-replicating or “replication-impaired” as used herein means that the vector is not capable of replication to any significant extent in the majority of normal mammalian cells or normal human cells.
  • Viruses which are non- replicating or replication-impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication.
  • Suitable viral vectors for use in a vaccine according to the present invention include non- replicating adenoviruses such as E1 deletion mutants, vectors based on herpes virus and Venezuelan equine encephalitis virus (VEE).
  • Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA (see Darji et a!., 1997, Cell 91 : 765-775).
  • Alternative suitable non-viral vectors include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage.
  • a vaccinia virus vector such as MVA or NYVAC may be used.
  • MVA vaccinia strain modified virus ankara
  • MVA is a replication impaired vaccinia strain with a good safety record. In most cell types and normal human tissues, MVA does not replicate.
  • Alternatives to vaccinia vectors include pox virus vectors, e.g. avipox vectors such as fowl pox or canarypox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC, and strains derived therefrom.
  • the vaccine as described herein above, or a nucleic acid molecule allowiong the expression of a miRNA molecule or of an antagonist as described herein above may be provided in the form of a genetherapy vehicle. It is accordingly envisaged introducing the genetherapy vehicle permanently or transiently into a cell of the human body, allowing it to express and generate miRNA molecules or miRNA antagonists as defined herein.
  • biomarker or group of biomarkers associated with epilepsy wherein said biomarker or group of biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 or 1a, or in section H of Table 1a or a derivative, fragment or variant thereof.
  • biomarker associated with epilepsy means that the expression level of the biomarker or group of biomarkers is modified in a subject afflicted by epilepsy, e.g. increased or decreased, when comparing to the expression of said biomarker or group of biomarkers in a control level, e.g. a healthy subject, in particular in a subject which has been diagnosed or confirmed not to be affected by epilepsy.
  • control level relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, i.e. epilepsy is/are known.
  • the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the miRNAs of the biomarker or group of biomarkers according to Table 1 or 1a, or in section H of Table 1a in samples from subjects whose disease state, i.e. epilepsy, is known.
  • the control level can be derived from a database of expression levels or patterns from previously tested subjects or cells.
  • control level may be multiple control levels determined from multiple reference samples.
  • the control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art.
  • a different type of control level can be determined from a reference sample derived from a healthy subject, e.g. a subject who has been diagnosed or confirmed not to suffer from epilepsy.
  • sample refers as used herein relates to any biological material obtained via suitable methods known to the person skilled in the art from an individual.
  • the sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.
  • the sample material is a bodily fluid.
  • bodily fluid refers to whole blood, serum, plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, liquid stool, fluid harvested from a site of an immune response, fluid harvested from a pooled collection site, bronchial lavage, and urine.
  • material such as biopsy material, e.g. from all suitable organs, e.g. the lung, the muscle, brain, liver, skin, pancreas, stomach, etc., a nucleated cell sample, a fluid associated with a mucosal surface, hair, or skin may be used.
  • biopsy material e.g. from all suitable organs, e.g. the lung, the muscle, brain, liver, skin, pancreas, stomach, etc.
  • a nucleated cell sample e.g. the material that samples, a fluid associated with a mucosal surface, hair, or skin
  • the material is typically homogenized and/or resuspended in a suitable buffer solution.
  • a sample may contain a cell extract derived from or a cell population including a brain cell. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. Samples, in particular after initial processing, may be pooled. However, also non-pooled samples may be used.
  • bodily fluid or sample material as mentioned herein above may be processed by adding chemical or biological reactants. This may be performed in order to stabilize the sample material, to remove sample components, or to avoid interaction in samples.
  • chemical or biological reactants for example, EDTA or heparin may be used to stabilize blood samples.
  • the content of a sample may be submitted to an enrichment step.
  • a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types functionalized for example with magnetic particles.
  • the material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.
  • blood i.e. whole blood, serum or cerebrospinal liquid samples.
  • control level determined from a biological sample that is known not to associated with epilepsy is called "normal control level".
  • the control level can be from biological sample associated with epilepsy, e.g. a sample from a subject for which epilepsy was diagnosed independently, it may be designated as “epilepsy control level”.
  • reference samples may comprise material derived from epilepsy models, e.g. as described in the Examples of the present invention.
  • Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art.
  • comparing a control level to a measured expression level a modification of the expression may be registered. For such comparison processes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed.
  • isolated biomarker or “biomarker”, as used herein, particularly relates to an isolated nucleic acid molecule comprising a miRNA as defined in section A of Table 1 or 1 a, or in section H of Table 1a, a precursor version of said miRNA, e.g. a pre-miRNA as defined in section E of Table 1 or 1 a, or in section H of Table 1 a, an encoding genetic unit, or a genomic sequence associated with said miRNA.
  • nucleic acid molecules comprising a nucleotide sequence at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1 a or in section H of Table 1 a, e.g.
  • isolated refers to a molecule which has been removed and/or prepared from a sample or tissue as defined herein. Methods for isolation are known to the skilled personl. These methods may include steps of amplification, hybridization, or cloning of the nucleic acids.
  • the biomarker may further comprise a derivative, fragment or variant of a miRNA molecule as defined above.
  • Such derivatives, fragments or variants correspond to the derivatives, fragments or variants as defined in the context of the pharmaceutical compositions as provided herein above.
  • expression level refers to the amount of any miRNA molecule, or derivative or precursor etc. thereof derivable from a defined number of cells or a defined tissue portion, preferably to the amount of miRNA molecule, or derivative or precursor etc. obtainable in a standard nucleic acid (in particular RNA) extraction procedure. Suitable extraction methods are known to the person skilled in the art. The amount may also be determined indirectly via suitable binding assays etc.
  • modified or “modified expression level” in the context of the present invention thus denotes a change in the expression level.
  • Expression levels are deemed to be “changed” when the miRNA expression the biomarker according to Table 1 or 1a, e.g. in a sample to be analyzed, differs by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, e.g. a sample derived from a healthy or normal subject, or when it differs at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level, e.g. a sample derived from a healthy or normal subject.
  • modified as used throughout the specification relates preferably to a decrease or down-regulation or an increase or up-regulation of the expression level of the biomarker or group of biomarkers according to Table 1 or 1a, or a complete inhibition of the expression of the biomarker or group of biomarkers according to Table 1 or 1a if a test sample is compared to a control level.
  • the term “increased” or “increased expression level” or “up-regulated expression level” or “increase of expression level” in the context of the present invention thus denotes a raise in the expression level of the biomarker or group of biomarkers according to Table 1 or 1a between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from a healthy state. This may further be compared to the expression in any suitable epilepsy state known to the person skilled in the art. Expression levels are deemed to be “increased” when the expression of the biomarker or group of biomarkers according to Table 1 or 1a, e.g.
  • a control level differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level.
  • the term “reduced” or “reduced expression level” or “down-regulated expression level” or “decrease of expression level” thus denotes a reduction of the expression level of the biomarker or group of biomarkers according to Table 1 or 1 a between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could, for example be a control level, e.g. a control level derivable from a healthy state. This may further be compared to the expression in any suitable epilepsy state known to the person skilled in the art.
  • Expression levels are deemed to be “reduced” or “down-regulated” when the expression of the biomarker or group of biomarkers according to Table 1 or 1a decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level.
  • the biomarker or group of biomarkers comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more or all biomarkers indicated with a "+” in section C of Table 1 or 1 a.
  • the biomarker or group of biomarkers comprises the first 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers indicated with a "+” in section C of Table 1 or 1a.
  • the biomarker or group of biomarkers comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more or all biomarkers indicated with a "+” in section D of Table 1 or 1 a.
  • the biomarkers or group of biomarkers comprises the first 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers indicated with a "+” in section D of Table 1 or 1 a.
  • the group of biomarkers may comprise markers which are up-regulated or markers which are down-regulated, or markers which are up-regulated and which are down-regulated, preferably at least one marker with an increased expression level and one marker with a decreased expression level.
  • the group of biomarker may comprise at least one biomarker indicated with a + in section C of Table 1 or 1a and at least one biomarker indicated with a + in section D of Table 1 or 1a.
  • the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-2137, mmu- miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR- 129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-mi ' R-92b-3p
  • the bio marker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-129-5p, mmu-miR-142-3p, mmu- miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184- 3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR- 34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 as defined in section A or H of Table 1a.
  • the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-124-3p, mmu- miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu- miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa- miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a- 3p, and miR-125a-5p, as defined in section A of Table 1.
  • the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p as defined in section A or H of Table 1 a.
  • the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1 -3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3
  • the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR- 30a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR- 25a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR- 148a-3p, and miR-125a-5p.
  • the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-129-5p, mmu-miR- 142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR-494-3p, hsa-miR-129-5p, hsa- miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, and hsa-miR-494-3p.
  • the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p.
  • the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy, or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or group of biomarkers as defined as defined in Table 1 or 1a, a peptide affinity ligand for a biomarker or group of biomarkers as defined in Table 1 or 1a, an oligonucleotide specific for the biomarker or group of biomarkers as defined in Table 1 or 1a and/or a probe specific for the biomarker or group of biomarkers as defined in Table 1 or 1a.
  • the composition may further comprise probes or oligonucleotides for the detection of a reference analyte, e.g. a gene and/or miRNA known to be associated with epilepsy.
  • nucleic acid affinity ligand for the expression product of a biomarker or group of biomarkers refers to a nucleic acid molecule being able to specifically bind to a miRNA molecule, or derivative, fragment or variant thereof of said biomarker or a group of biomarkers as defined above, preferably to the a miRNA molecule depicted in section A of Table 1 or 1a, or in section H of Table 1a or to DNA copy thereof.
  • the nucleic acid affinity ligand may also be able to specifically bind to a miRNA molecule being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B of Table 1 or 1a, or in section H of Table 1a or a pre-miRNA molecule being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section F of Table 1 or 1a, or in section H of Table 1 a or to any fragments of said sequences.
  • peptide affinity ligand for the biomarker or group of biomarkers refers to a peptide molecule being able to specifically bind to the miRNA biomarker or group of biomarkers according to section A of Table 1 or 1a, or in section H of Table 1a.
  • the peptide molecule may preferably be able to specifically bind to miRNA molecule comprising a sequence as set forth in section B of Table 1 or 1a, or in section H of Table 1a, or to a DNA copy thereof.
  • the peptide affinity ligand may also be able to specifically bind to a miRNA molecule being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B of Table 1 or 1a, or in section H of Table 1a, or pre-miRNA molecule being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section F of Table 1 or 1a, or in section H of Table 1a, or to any fragments of said sequences.
  • peptide in the context of the affinity ligand of the present invention refers to any type of amino acid sequence comprising more than 4 amino acids, e.g. polypeptide structures, protein structures or functional derivatives thereof. Furthermore, the peptide may be combined with further chemical moieties or functionalities.
  • oligonucleotide specific for the biomarker or group of biomarkers refers to a nucleotide sequence which is complementary to the miRNA biomarker or group of biomarkers according to Table 1 or 1a, or to its antisense nucleic acid.
  • the oligonucleotide is complementary to the sequence shown in section B or D of Table 1 or 1 a, or in section H of Table 1 a, or to the complementary sequence of the sequence shown in section B or D of Table 1 or 1a, or in section H of Table 1 a.
  • the oligonucleotide sequence may also be complementary to a sequence being at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B or D of Table 1 or 1 a, or in section H of Table 1a.
  • the oligonucleotide may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section B or D of Table 1 or 1 a, or in section H of Table 1 a or its complement.
  • the oligonucleotide may have a length of between 8 and 60 nucleotides, preferably of between 10 and 35 nucleotides, more preferably a length of 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33 nucleotides.
  • Oligonucleotide sequences specific for the miRNA biomarker or group of biomarkers according to Table 1 or 1a may be defined with the help of software tools known to the person skilled in the art.
  • probe specific for the biomarker or group of biomarkers means a nucleotide sequence which is complementary to the miRNA biomarker or group of biomarkers according to Table 1 or 1a.
  • the probe is complementary to the sequence(s) depicted in section B or D of Table 1 or 1 a, or in section H of Table 1 a, or to the complementary sequence of the sequence(s) shown in section B or D of Table 1 or 1a, or in section H of Table 1a.
  • the probe sequence may also be complementary to a sequence being at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in the sequences indicated in section B or D of Table 1 or 1a, or in section H of Table 1 a.
  • the probe may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section B or D or its/their complement.
  • the probe may have a length of between 30 and 300 nucleotides, preferably of between 30 and 150 nucleotides, more preferably a length of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 70, 90, 100, 110, 120, 130, 140 or 150 nucleotides.
  • Probe sequences specific for the biomarker or group of biomarkers according to Table 1 or 1a may be defined with the help of software tools known to the person skilled in the art.
  • the probe may preferably be labeled with a suitable label, e.g. radioactive, fluorescent or dye label.
  • the present invention relates to a method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample.
  • diagnosis epilepsy means that a subject or individual may be considered to be suffering epilepsy, when the expression level of a biomarker or the group of biomarkers of the present invention is modified, e.g. increased/up-regulated or reduced/down-regulated, compared to the expression level of a control level as defined herein above.
  • diagnostic also refers to the conclusion reached through that comparison process.
  • An expression level may be deemed to be modified, when the expression level of a biomarker or group of biomarkers as defined herein above differs by, for example, between about 1 % and 50%, e.g.
  • control level as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to such a control level.
  • the modification may be an increase or a reduction of said expression level.
  • an additional similarity in the overall gene expression pattern of a group of biomarkers according to the present invention between a sample obtained from a subject and a control sample as described herein above, may indicate that the subject is suffering epilepsy.
  • the diagnosis may be combined with the elucidation of additional epilepsy markers, e.g. clinical markers, EEG results, brain imaging results or any combination thereof.
  • detecting epilepsy means that epilepsy may be identified in an organism.
  • the identification of epilepsy may be accomplished by a comparison of the expression level of the biomarker or group of biomarkers of the present invention in a sample from a patient or individual to be analyzed and the expression level of a control level, wherein said control level corresponds to the expression level of said biomarker or group of biomarkers in a healthy subject.
  • epilepsy may be detected if the expression level the biomarker or group of biomarkers of a sample or subject to be analyzed is similar to an expression level of a control sample derived from a subject known to be afflicted by epilepsy.
  • the expression level of the epilepsy sample may also independently be established, e.g. from sample depositories, value databases or the like.
  • graduating epilepsy means that the clinical stage, phase, grade or any other suitable sub-step of epilepsy disease related features, e.g. suitable parameters of epilepsy in an organism may be determined in an organism.
  • the graduating of epilepsy may be accomplished by a comparison of the expression level of the biomarker or group of biomarkers according to Table 1 or 1 a of the present invention in a sample from a patient or individual to be analyzed and a control level as defined herein above.
  • monitoring epilepsy as used herein relates to the accompaniment of a diagnosed or detected epilepsy during a certain period of time, typically during 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time.
  • accommodation means that states of disease as defined herein and, in particular, changes of these states of disease may be detected by comparing the expression level of the biomarker or group of biomarkers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established epitleptical sample, or to corresponding database values in any type of a periodical time segment, e.g.
  • the term relates to the accompaniment of a diagnosed epilepsy.
  • prognosticating epilepsy refers to the prediction of the course or outcome of a diagnosed or detected epilepsy, e.g. during a certain period of time, during a treatment or after a treatment. The term also refers to a determination of chance of survival, deterioration or recovery from the disease, as well as to a prediction of the expected survival time of a subject.
  • a prognosis may, specifically, involve establishing the likelihood for survival or deterioration of a subject during a period of time into the future, such as 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time.
  • predisposition for developing epilepsy is a state of risk of developing epilepsy.
  • a predisposition for developing epilepsy may be present in cases in which the expression level of the biomarker or group of biomarker of the present invention as defined herein above is above or below a normal control level as defined herein above, i.e. a reference expression level derived from tissues or samples of a subject which are evidently healthy.
  • the term “above” in this context relates to an expression level of the biomarker of group of biomarkers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.
  • the term “below” in this context relates to an expression level of the biomarker or group of biomarkers which is decreased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.
  • a predisposition for developing epilepsy in the context of the present invention may be given in situations in which the expression level of the biomarker or group of biomarkers as defined herein above is above a normal control level and in which further, alternative epilepsy markers show no modification or alteration.
  • Suitable further epilepsy markers e.g. clinical marker, EEG or imaging marker etc. would be known to the person skilled in the art.
  • determining the level of a biomarker or group of biomarkers refers to the determination of the presence or amount of expression product(s) of the biomarker or biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a, e.g. miRNA molecule.
  • level of the biomarker or group of biomarkers thus means the presence or amount of (an) expression product(s) of the biomarker or group of biomarkers according to Table 1 or 1a, e.g. miRNA molecule according to Table 1 or 1a.
  • the determination of the presence or amount of (an) expression product(s) of the biomarker or group of biomarkers according to Table 1 or 1a may be accomplished by any means known in the art.
  • the determination of the presence or amount of the expression products of the biomarker or group of biomarkers as mentioned herein is accomplished by the measurement of miRNA levels or by the determination of the biological effect of said biomarker or group of biomarkers.
  • the measurement of the miRNA level of the expression of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1a may be assessed by separation of nucleic acid molecules (e.g. RNA or cDNA) obtained from the sample in agarose or polyacrylamide gels, followed by hybridization with biomarker specific oligonucleotide probes as defined herein above, e.g.
  • oligonucleotide probes comprising fragments of the sequences indicated in section B or D of Table 1 or 1a, or in section H of Table 1 a, or complementary sequences thereof.
  • the expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples may be placed on the gel such that patient and control or standard nucleic acid are in adjacent lanes. Comparison of expression levels may be accomplished visually or by means of a densitometer. Further methods for the detection of RNA products are known to the person skilled in the art.
  • the miRNA level of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a may be detected in a DNA array or microarray approach.
  • sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label.
  • such nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a of the present invention or known biomarker genes.
  • Suitable means for carrying out microarray analyses are known to the person skilled in the art.
  • the present invention also envisages a corresponding microarray.
  • a microarray comprises immobilized high-density probes to detect a number of miRNAs.
  • the probes on the array are complementary to one or more parts of the sequence of the miRNA, or to a pre-miRNA.
  • any type of biomarker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the biomarker expression and the expression of other genes.
  • cDNAs, PGR products, and oligonucleotides may be used as probes.
  • a fragment comprising 5'- or 3'-portions of the biomarkers or group of biomarkers as mentioned herein above or according to Table 1 or 1a e.g. of the sequences indicated in section B or D of Table 1 or 1 a, or in section H of Table 1a, may be used as a probe.
  • the microarray may comprise probes of one or more of the biomarker of Table 1 or 1a, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 etc. or all of the biomarkers or any combination of said markers.
  • any type of fragment or sub-portion of any of the markers sequences may be combined with any further fragment or sub-portion of any of the markers sequences of Table 1 or 1 a.
  • biomarkers In addition to the determination of the expression of biomarkers according to Table 1 or 1a also the determination of the expression of other markers, e.g. additional biomarker is envisaged by the present invention.
  • a marker can be represented by two or more probes, the probes hybridizing to different parts of a gene. Probes are designed for each selected marker gene.
  • Such a probe is typically an oligonucleotide comprising 5-50 nucleotide residues. Longer DNAs can be synthesized by PCR or chemically. Methods for synthesizing such oligonucleotides and applying them on a substrate are well known in the field of micro-arrays.
  • the nucleic acid level of expression of the biomarker or group of biomarkers according to Table 1 or 1 a may be detected in a quantitative RT- PCR approach, preferably in a real-time PCR approach following the reverse transcription of the RNA of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a.
  • a miRNA is reverse transcribed into a cDNA molecule according to any suitable method known to the person skilled in the art.
  • a quantitative or real-time PCR approach may subsequently be carried out based on a first DNA strand obtained as described above.
  • Taqman or Molecular Beacon probes as principal FRET-based probes of this type may be used for quantitative PCR detection.
  • the determination of the biological effect of said biomarker or group of biomarkers may be performed on the basis of a biological function of one or more target genes of a miRNA as identified in Table 1 or 1a.
  • Target genes or their expression products may accordingly be analysed by RNA expression analysis, protein expression analysis, cellular localization or other suitable methods known to the skilled person.
  • the present invention relates to a method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample before and during an epilepsy treatment.
  • monitoring epilepsy therapy as used herein relates to the determination of the level of biomarkers before and during a treatment procedure.
  • the period of time may depend on the treatment period or treatment cycle.
  • the monitoring may be carried out for 2 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time.
  • epilepsy states as defined herein and, in particular, changes of these states of epilepsy may be detected by comparing the expression level of the biomarker or group of biomarkers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established epileptical control sample, or to corresponding database values, or most preferably to one or more initially taken samples or earlier samples of the same subject, e.g. a sample take at the onset of therapy, taken a week, a month, a year etc. before the actual sample taking.
  • the comparison may be carried out in any type of a periodical time segment, e.g.
  • the monitoring may be continued after the therapy has been terminated, e.g. 2 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other time value in between these values after the termination of a therapy.
  • the determination may be carried out as described herein above.
  • the treatment may be a treatment with a suitable epileptical therapeutic or anticonvulsant.
  • suitable epileptical therapeutic or anticonvulsant examples or such medicaments include lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, and zonisamide.
  • the suitable anticonvulsant may be a pharmaceutical composition as defined herein above. Further envisaged is a combination of any of these medicaments with additional, non-epileptic medicaments.
  • the present invention relates to an assay for detecting, diagnosing, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
  • step (d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c).
  • the assay is preferably performed with a nucleic acid affinity ligand for a biomarker or group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above or a probe specific for the biomarker or group of biomarkers as defined herein above.
  • the testing for expression of the biomarker or group of biomarkers according to Table 1 or 1a may be carried out according to steps as defined herein above.
  • the testing may be carried out as measurement of miRNA levels of the biomarker or group of biomarkers according to Table 1 or 1a, more preferably according to the herein above described options for such measurements.
  • controls or control samples controls as defined herein above may be used.
  • the testing steps may be based on the use of nucleic acid affinity ligand, a peptide affinity ligand, a probe or an oligonucleotide specific for a the biomarker or group of biomarkers of Table 1 or 1a as laid out above.
  • Epilepsy may be diagnosed or prognosticated in said assay according to the corresponding definitions provided herein above in the context of the biomarker or group of biomarkers according to Table 1 or 1a.
  • the present invention relates to a method of identifying an individual eligible for an epilepsy therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to the levels of step (b);
  • the level of a biomarker or group of biomarkers may be determined on the miRNA expression level as described herein above.
  • concentration of primers and/or probe oligonucleotides may be modified.
  • concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
  • a control sample as used in the context of the method of identifying an individual eligible for an epilepsy therapy may be a control sample as defined herein above, e.g. from the same individual as the test sample, or a control sample derived from a different source or individual.
  • the control sample may further be either a sample derived from the same tissue, preferably brain tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are muscle tissue, or blood, urine etc.
  • the testing of a reference gene or miRNA is envisaged, e.g. a miRNA with known expression pattern and/or function.
  • the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a biomarker or group of biomarkers may be combined.
  • the term further means that the expression in a test sample for a biomarker and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
  • an individual may be considered to be eligible for an epilepsy therapy when the expression level of an up-regulated biomarker as defined herein above, or as indicated in section C of Table 1 or 1 a, or the expression level of a group of biomarkers as defined herein above is increased.
  • the expression level is deemed to be "increased" when the biomarker expression, or the expression of the group of biomarkers the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding biomarker expression, or to the expression of the corresponding group of biomarkers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the biomarker expression, or the expression of the group of biomarkers in a control sample.
  • an individual may also be considered to be eligible for an epilepsy therapy when the expression level of a down-regulated biomarker as defined herein above, or as indicated in section D of Table 1 or 1a, or the expression level of a group of biomarkers as defined herein above is decreased.
  • the expression level is deemed to be "decreased" when the biomarker expression, or the expression of the group of biomarkers in the test sample is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding biomarker gene expression, or to the expression of the corresponding group of biomarkers in a control sample, or lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the biomarker expression, or to the expression of the group of biomarkers in a control sample
  • the expression of a reference gene may be used to normalize or adjust the expression of a biomarker or group of biomarkers as defined herein.
  • the therapy to be carried out on a subject who is identified eligible for an epilepsy therapy may be any suitable epilepsy therapy.
  • Preferred is the administration of a pharmaceutical composition as defined herein above, or of an anticonvulsant selected from lacosamide, leveti race tarn, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, and zonisamide, or any combination of the mentioned anticonvulsants.
  • the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined herein above.
  • the diagnostic kit of the present invention may accordingly comprise any suitable agent allowing the specific detection of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a.
  • the kit may comprise at least one component selected from a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, or a probe specific for the biomarker or group of biomarkers as defined herein above. It is also envisaged that more than one component may be present in the kit.
  • oligonucleotides specific for the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a together with suitable enzymatic activities and other ingredients necessary for the detection. Further enivsaged is the presence of labeled probes as defined herein, e.g. fluorescently labeled probes.
  • a diagnostic kit of the present invention contains detection reagents for expression product(s) of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a.
  • detection reagents comprise, for example, buffer solutions, labels or washing liquids etc.
  • the kit may comprise an amount of a known nucleic acid molecule, which can be used for a calibration of the kit or as an internal control.
  • a diagnostic kit for the detection of expression product(s) of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc.
  • a diagnostic kit may, in specific embodiments, also comprise accessory ingredients like secondary affinity ligands, detection dyes and any other suitable compound or liquid necessary for the performance of nucleic acid detection or amplification.
  • accessory ingredients like secondary affinity ligands, detection dyes and any other suitable compound or liquid necessary for the performance of nucleic acid detection or amplification.
  • Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.
  • kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
  • the kit may further comprise probes or oligonucleotides for the detection of a reference analyte, e.g. a gene and/or miRNA known to be associated with epilepsy.
  • a reference analyte e.g. a gene and/or miRNA known to be associated with epilepsy.
  • the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
  • the marker may accordingly be used for a diagnosis activity as described above, for a detection activity as described above, for graduating a stage or form of epilepsy as described above, or for monitoring epilepsy, or an epilepsy treatment as described above.
  • a biomarker or a group of biomarkers as defined herein above for the prognosis of epilepsy development, or the prognosis of epilepsy development during epilepsy treatments.
  • a biomarker or a group of biomarkers as defined herein above for detecting a predisposition for epilepsy as described above.
  • the present invention relates to the use of a biomarker or group of biomarkers as defined herein above for identifying a pharmaceutically active agent useful in the treatment or prevention of epilepsy.
  • identifying a pharmaceutically active agent refers to a screening for potentially agonistic or antagonistic molecules which either increase a reduced miRNA expression level, or reduce an increased miRNA expression level according to the indications of Table 1 or 1a, in particular sections C and D. I.e.
  • the screening is for potentially agonistic or antagonistic molecules leading to an increase of miRNA levels in case of down-regulated miRNAs indicated with a "+" in section D of Table 1 or 1a, or leading to a decrease of miRNA levels in case or up-regulated miRNAs indicated with a "+” in section C of Table 1 or 1 a.
  • the present invention also relates to a corresponding method, as well as agonistic and antagonistic molecules obtained by said method.
  • the screening for such molecules involves producing appropriate cells or organism models which express the miRNAs.
  • Cells expressing the miRNAs may subsequently be contacted with a potential antagonist or agonist to observe binding, stimulation, and/or inhibition of miRNA expression and/or of subsequent cell regulatory activity.
  • step a) one may use a cell obtained from a healthy human being of from a patient suffering from epilepsy. Also envisaged is the use of cultured cell lines. In a preferred embodiment, these cells and cell lines are outside the human body.
  • step c) one may subject such cells and cell lines to libraries of potentially pharmaceutically active agents. Such libraries may be small molecule libraries, aptamer libraries, antibody libraries, siRNA libraries, peptide libraries etc. Determining expression levels in steps b) and d) may be undertaken as is common in the art. Correspondingly obtained agents may be used directly to treat or prevent epilepsy, or they may be used as hits or lead compounds for further drug development.
  • bodily fluids preferably of blood, serum or cerebrospinal fluid for screening approaches as defined herein.
  • Such bodily fluids may, for example, for used in order to detect interacting partners or elements having an influence on miRNA expression and/or regulation.
  • Item 1 A pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of Table 1.
  • Item 2 The pharmaceutical composition of item 1 , wherein said means comprises a nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, or an antagonist thereof, wherein said nucleic acid molecule or antagonist has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section B of Table 1 or to a complementary sequence thereof.
  • Item 3 The pharmaceutical composition of item 2, wherein said nucleic acid molecule or antagonist comprises between at least 18 and 24 nucleotides, preferably 20 nucleotides, 21 nucleotides or 22 nucleotides.
  • Item 4 The pharmaceutical composition of item 2 or 3, wherein said nucleic acid or antagonist is or comprises a DNA molecule or an RNA molecule, or a derivative thereof.
  • Item 5 The pharmaceutical composition of item 4, wherein said RNA molecule is or comprises a pre-miRNA selected from section E of Table 1 , or a derivative, fragment or variant thereof, wherein said pre-miRNA molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section F of Table 1 or to a complementary sequence thereof.
  • Item 6 The pharmaceutical composition of item 3, wherein said DNA molecule is or comprises a DNA molecule coding for a pre-miRNA, a miRNA or for an antagonist.
  • Item 7 The pharmaceutical composition of item 6, wherein said DNA molecule is or comprises a double stranded DNA coding for at least one pre-miRNA selected from section E of Table 1.
  • Item 8 The pharmaceutical composition of any one of items 2 to 4, 6 or 7, wherein said nucleic acid molecule is comprised in a vector replicable in a subject.
  • Item 9 The pharmaceutical composition of any one of items 2 to 8, wherein said nucleic acid molecule or antagonist is modified by a conjugate, preferably a linked conjugate.
  • Item 10 The pharmaceutical composition of any one of items 2 to 9, wherein said nucleic acid molecule or antagonist comprises a chemically modified base.
  • Item 11 The pharmaceutical composition of any one of items 2, 3, 4, 9 or 10, wherein said antagonist is an antagomir.
  • Item 12 The pharmaceutical composition of item 10 or 11 , wherein said nucleic acid molecule or antagonist is modified by at least one modification selected from the group consisting of a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose and a 2'- methoxyethoxy ribose.
  • Item 13 The pharmaceutical composition of any one of items 2 to 12, wherein said nucleic acid molecule comprises a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p or miR- 125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, wherein said nucle
  • Item 14 The pharmaceutical composition of any one of items 2 to 12, wherein said antagonist is an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-
  • Item 15 A vaccine for use in the treatment or prevention of epilepsy, comprising a nucleic acid molecule or antagonist as defined in any one of items 2 to 14.
  • Item 16 A biomarker or group of biomarkers associated with epilepsy, wherein said biomarker or group of biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 , or a derivative, fragment or variant thereof.
  • Item 17 The biomarker or group of biomarkers of item 16, wherein the increase of expression (up-regulation) of at least one biomarker selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b- 5p, mmu-miR- 29-1 -3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR- 221 -3p
  • Item 18 The biomarker or group of biomarkers of item 16, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa- miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221- 3p, miR-767-5p, hsa-miR-148a-3p, and miR-125a-5p as defined in section A of Table 1 , when comparing
  • Item 19 A composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18.
  • Item 20 A method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 in a sample.
  • Item 21 A method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 in a sample before and during an epilepsy treatment, optionally also after an epilepsy treatment.
  • Item 22 The method of item 21 , wherein said treatment is a treatment with a pharmaceutical composition as defined in any one of items 1 to 14, and/or with at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, zonisamide, and combinations thereof.
  • at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam,
  • Item 23 The method of any one of items 20 to 22, wherein the determining step is accomplished by the measurement of the miRNA level(s) or by the determination of the biological effect of said biomarker or group of biomarkers.
  • Item 24 An assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
  • step (d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c) ,
  • testing steps are based on the use of a nucleic acid affinity ligand and/or a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18.
  • Item 25 A method of identifying an individual eligible for an epilepsy therapy comprising: (a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined in item 17 or 18;
  • step (c) classifying the levels of expression of step (a) relative to the levels of step (b);
  • identifying the individual as eligible to receive an epilepsy therapy where the individual's sample is classified as having an increased level of expression of a biomarker or a group of biomarkers as defined in item 17, or where the individual's sample is classified as having a decreased level of expression of a biomarker or a group of biomarkers as defined in item 18.
  • Item 26 A diagnostic kit for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined in any one of items 16 to 18, wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18.
  • Item 27 Use of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
  • Item 28 Use of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
  • microRNA profiling in epilepsy animal models was performed.
  • microRNA screening was performed in 4 animal models of epilepsy (see Example 2, infra).
  • Hippocampal microRNAs were analyzed at an acute timepoint (24h after induction of seizure) as well as in the chronic phase (28d) to monitor changes in expression levels during disease progression.
  • Profiling was performed using the miRCURYTM LNA Array microRNA Profiling Service by Exiqon A S according to Exiqon
  • RNA quality was analyzed using the Agilent Bioanalyzer Lab-on-a-Chip System.
  • SE Status Epilepticus
  • Table 2 Animal models of epilepsy used for profiling of hippocampal mlcroRNAs.
  • the Table shows control- and treatment groups as well as animal numbers per group and the timepoints of analysis for the (A) Pilocarpine, (B) SSSE, (C) 6 Hertz and (D) Audiogenic animal model.
  • MicroRNA profiling data obtained from the miRCURYTM LNA Array microRNA Screen are summarized in the following Tables. The microarrays provided a complete coverage of miRBase v14. Data were regarded as valid, when the microRNA gave a stable signal across all arrays per group.
  • the Profiling data obtained from the animal studies were normalized using LOWESS normalization. For each model differences in microRNA expression were assessed based on the calculation of fold changes (FC) between the control groups and the treatment groups (see criteria below). In addition, special attention was paid to differences in hippocampal microRNA profiles during the acute phase (24 h post seizure) and the chronic phase (28d post seizure) to identify miRNAs dysregulated during Epilepsy progression.
  • Table 5 Up regulated miRNAs in the Pilocarpine study (28 d, 1 h Diazepam).
  • Table 7 Up regulated miRNAs In the Pilocarpine study (28 d, 2 h Diazepam).
  • Table 9 Up regulated miRNAs in the SSSE study (24 h). miRBase v14 annotation.
  • Table 10 Down regulated miRNAs in the SSSE study (24 h). miRBase v14 annotation.
  • Table 11 Up regulated miRNAs in the SSSE study (28 d). miRBase v14 annotation.
  • Table 12 Down regulated miRNAs in the SSSE study (28 d). miRBase v14 annotation.
  • Table 13 Up regulated mIRNAs in the 6 Hertz study (3 h). mi'RBase v14 annotation.
  • Table 14 Down regulated miRNAs in the 6 Hertz study (3 h). miRBase vi4 annotation.
  • mmu-miR-706 1.20 mmu-miR-30a 1.20 mmu-miR-106a 1.20 mmu-miR-325 1.20 mmu-miR-127* 1.19 mmu-miR-337-5p 1.19 mmu-let-7d 1.19 mmu-miR-433 1.19 mmu-miR-667 1.19 mmu-miR-434-5p 1.19 mmu-miR-181 c 1.19 mmu-miR-487b 1.19 mmu-miR-495 1.19 mmu-miR-455 1.18 mmu-mlR-1949 1.18 mmu-miR-129-3p 1.18 mmu-miR-30b 1.18 mmu-miR-410 1.18 mmu-miR-380-3p 1.18 mmu-miR-218 1.18 mmu-miR-543 1.18 mmu-miR-668 1.18
  • mmu-miR-741 1.15 mmu-miR-137 1.15 mmu-miR-382 1.15 mmu-miR-351 1.15 mmu-miR-345-5p 1.15 mmu-miR-2 37 1.15 mmu-miR-24-1 * 1.15 mmu-miR-124* 1.15 mmu-miR-341 1.15 mmu-miR-500 1.15 mmu-miR-300* 1.15 mmu-miR-328 1.15 mmu-miR-29a 1.15 mmu-miR-26a 1.15 mmu-miR-29b 1.15 mmu-mi ' R-384-5p 1.15 mmu-miR-129-5p 1.15 mmu-miR-100 1.15
  • Table 15 Up regulated miRNAs in the 6 Hertz study (6 h). miRBase v14 annotation.
  • Table 16 Down regulated miRNAs in the 6 Hertz study (6 h). miRBase v14 annotation.
  • Table 17 Op regulated miRNAs in the 6 Hertz study (24 h). miRBase v14 annotation.
  • Table 18 Down regulated miRNAs in the 6 Hertz study (24 h). miRBase v14 annotation.
  • Table 19 Up regulated miRNAs in the 6 Hertz study (72 h). miRBase v14 annotation.
  • Table 20 Down regulated miRNAs in the 6 Hertz study (72 h). miRBase v14 annotation.
  • Table 21 Up regulated miRNAs In the Audiogenic study (3 h). miRBase v14 annotation.
  • Table 22 Down regulated miRNAs in the Audiogenic study (3 h). miRBase v14 annotation.
  • mmu-miR-21 1.14
  • Table 23 Up regulated miRNAs to the Audiogenic study (6 h). miRBase v14 annotation.
  • Table 24 Up regulated miRNAs in the Audiogenic study (72 h). miRBase v14 annotation. No significantly down regulated miRNAs were identified at the 72 h timepoint.
  • microRNA candidates were validated in the mouse samples by single-assay qRT-PCR.
  • miRNA candidates were selected predominantly based on the models of chronic Epilepsy (pilocarpine, SSSE) as the more relevant mouse models to compare with human Epilepsy.
  • MicroRNA expression analyses were performed on the treatment and naive groups of the Pilocarpine and SSSE model samples using the Universal cDNA Synthesis Kit, SYBR Green master mix, and pre-designed LNATM-enhanced microRNA qPCR primer sets (all by Exiqon) according to manufacturers ' instructions. MicroRNA candidates were regarded as validated when showing a FC>1.20fold/ ⁇ 0.80fold and consistent with the profiling data.
  • Table 25 summarizes most relevant microRNA candidates and qRT- PCR validation results.
  • Table 25 qRT-PCR validation of microRNAs from Epilepsy animal models.
  • the table includes the fold-change as detected in the microRNA profiling, the timepoint of detection (either in the acute (24h) or chronic (28d) phase, or generally dys regulated), and the qRT-PCR validation status. (V confirmed).
  • RNA profiling in human samples The study included patients diagnosed with TLE and either AHS positive or negative. Hippocampal post mortem samples served as control. RNA quality was analyzed using the Agilent Bioanalyzer Lab-on-a-
  • the small RNA fraction (19-29 bases) from all human RNA samples was isolated.
  • the small RNAs were poly(A) tailed and ligated to RNA adapter.
  • RNA adapter After cDNA synthesis ail cDNA were pooled and PCR amplified.
  • the cDNA pools were sequenced on a lllumina HiSeq 2000 system using a read length of 36 bp (single reads) and approximately 5 mio reads per cDNA. All reads were aligned to miRBase v18 to identify expressed miRNAs. This alignment was done by InteRNA Genomics. The aim was to get a more detailed insight into expressional changes in the human samples.
  • MicroRNA candidate was significantly (p ⁇ 0.05, ttest) dysregulated in human samples.
  • MicroRNA candidate shows a fold change FC>1.14 in case of up regulation and FC ⁇ 0.88 in case of down regulation.
  • Table 27 MicroRNAs up regulated in hippocampal biopsies of TLE patients. The microRNAs were identified In a Deep Sequencing approach. The Tables indicate FC for 2 different comparisons: AHS versus nonAHS as well as AHS and nonAHS versus post mortem.
  • Table 28 microRNAs down regulated in hippocampal biopsies of TLE patients.
  • microRNAs were identified in a Deep Sequencing approach.
  • the Tables indicate FC for 2 different comparisons: AHS versus nonAHS as well as AHS and nonAHS versus post mortem.
  • the table displays the fold-changes in microR A levels as detected in the Deep Sequencing profiling in the indicated comparisons (AHS versus nonAHS).
  • qRT-PCR validation status is shown in the last column. (V confirmed; ⁇ upregulated in qRT-PCR; ⁇ downregulated in qRT-PCR; (-) not changed in qRT-PCR, (ns) not significant in qRT-PCR).
  • miRNAs were identified as being expressed overlapping manner in the mouse and human approaches.
  • Table 30 MicroRNA candidates overlapping in mouse models and human profiling (acute phase).
  • the bioinformatical analysis included target gene prediction followed by GO enrichment analysis.
  • the predicted target genes for the microRNA candidates were compiled from the databases (mouse samples: Target Scan and Microcosm database; human study: ExprTarget DB).
  • mouse miRNA candidates predicted target genes identified in both databases (Targe Scan and Microcosm) were used for further analysis. Afterwards.for each miRNA the 100 top hits of the target gene prediction were used fo a GO enrichment analysis done with the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/ home.jsp). Predicted pathways were selected for the target genes of each miRNA based on the prediction score.
  • FIGURE 3 shows a number of enriched target genes for a selected number of microRNAs. All these target genes are putatively relevant for different cellular processes during epileptogenesis.
  • Example 10
  • epileptogenesis brain is affected by severe changes characterized as sudden and uncontrolled firing of neurons, onset of astrogliosis and activation of microglia. All these events have unknown or partially known molecular mechanisms in neurons, astrocytes, microglia and other brain cells, that underlie pathophysiology of disease.
  • Pathophyisological changes of brain from initial insult to emergence of spontaneous seizures can be divided into three major phases: 1 ) activation of immediate early genes and activation of ion channels; 2) transcriptional activation, neuronal cell death, set of inflammation; 3) neurogenesis, sprouting of neurons, gliosis and neuronal network reorganization.
  • Each of these assays addresses changes present in epileptic brain. In this way, it was achieved to gain insight into roles of the identified miRNAs in cell growth, neuroprotection, activation of microglia and calcium release (see FIGURE 4).
  • lentiviral microRNA expression system and transduction of different cell types
  • LV lentiviral
  • Precursor miRNA was cloned using Xhol and BamHI restriction sites at the 3' end of eGFP (enhanced Green Fluorescent Protein) under CMV promoter (human cytomegalovirus immediate early promoter).
  • lentiviral system of gene delivery to cells is the controlled and precise amount of delivered particles, expressed as multiplicity of infection, MOI (number of viral particles infecting a single cell). See FIGURE 5 for lentiviral vectors used.
  • lentivirus virus began with cloning the gene of interest (goi), in this case precursor microRNA into an entry plasmid pLENTI6.4- promoter-transgene (pl_ENTI6.4-CMV-premiRNA).
  • the expression plasmid was co- transfected into HEK293T producer cells with three supercoiled packaging plasmids (pLP1 , pLP2 and pLPA/SV-G) which supply helper functions and viral proteins in trans.
  • the supernatant containing the viral particles
  • lentiviral vectors can be used to transduce the mammalian cell line of choice.
  • High-titer lentivector preparations are essential to achieve high transgenesis rates (Pfeifer A, Hofmann A, 2009).
  • lentiviral vectors were diluted in cell medium. The rate of dilution highly depends on the desired transgenesis rates (MOI, number of viral integrants per cell) and in this case was MO1100. After overnight incubation cell medium was changed and expression was monitored upon 48-96 hrs. First, expression of eGFP in cells was confirmed using a fluorescence microscope (see FIGURE 6). Then, miRNA expression was confirmed using q-RT-PCR method (see FIGURE 7) and miRNA specific primers purchased from Exiqon (http://www.exiqon.com/mirna-pcr- primer).
  • Generated LV system enabled easy and precise delivery of gene of interest at MOI100 rate, in our case pre-miRNAs, therefore enabling their overexpression and processing in mammalian cells, as shown for primary mouse hippocampal neurons (see FIGURE 7).
  • all miRNA could be very well expressed in primary mouse hippocampal neurons, but the level of their expression detected by q-RT-PCR differed. This effect is due to already present native miRNA level recognized as well by specific miRNA primers used. Native miRNAs level are cell and tissue specific and differ among SH-SY5Y cell line, mouse hippocampal neurons, cortical neurons, astrocytes and microglia.
  • Transduced miRNA showed either no significant effect on growth of SH- SY5Y cells, showed higher growth rates than control or showed slower growth rate than control. These effects fit to present knowledge of miRNAs as molecular brakes in certain cancer cells (hepatocitoma). Here we show, that miRNA-124 drastically inhibits cell growth in neuroblastoma! cells (see FIGURE 10).
  • SH-SY5Y was transduced with a lentiviral system, expressing precursor miRNAs miR-124-3p, miR- 142-3p miR-184 and miR-con linked to a GFP as reporter protein at OI100 72 hours upon transduction, GFP positive cells were selected and sorted into 96 well plates using FACS. After this growth rates of transgenic SH-SY5Y cells expressing the miR- 124-3p, miR-142-3p and miR-184 were measured by applying 12 m MTT for 2 hours at 37 °C and detection of absorbance level at OD 540/600 nm. The growth rates of transgenic cell lines were measured over a time period of 9 days.
  • the expression of the GFP reporter protein of different precursor miRNAs was detectable in SH-SY5Y cells after transduction with the lentiviral vectors (see FIGURE 19).
  • Excitotoxicity assay induced by glutamate Epilepsy is characterized by sudden and uncontrolled firing of neuronal circuits that leads to extensive neuronal death due to uncontrolled synaptic glutamate release.
  • miRNA miRNA during glutamate excitotoxicity release
  • Primary hippocampal neurons were prepared from postnatal day zero or postnatal day one mouse brains (P0/P1), strain C57BL/6J. First, hippocampus was dissected out, gathered and washed in IxHBSS solution. Single cell suspension was achieved using trypsin based neural tissue dissociation kit (T) (Miltenyibiotec, Cat.#130-093-231) and cells were placed on PLL coated plates (0,33 mg/mL Poly-L- Lysine, Sigma) in plating medium. Upon 3 hrs medium was changed to growth medium. Cultures were obtained for period of 2 at 37C 5% C0 2 by changing half of growth medium two times per week.
  • T trypsin based neural tissue dissociation kit
  • hippocampal neurons were placed on PLL coated plates (Poly- L-Lysine, Sigma) and transduced with lentiviral constructs carrying miRNAs of interests at MOM 00 at DIV7 (day in vitro). At DIV13 hippocampal neurons were subjected to 10 ⁇ and 100 ⁇ final glutamate concentration for 24 hours, upon which survival rates were measured using MTT test.
  • microglia play an important role in neuroinflammation during and after SE.
  • Microglia can be activated using INF-g and LPS. Following activation they secrete IL-6. This approach was used to analyze the influence of miRNA overexpression on IL-6 secretion in primary microglia.
  • Obtained cells were plated into 96 well plates and transduced using lentiviral constructs carrying miRNA of interest at DIV1.
  • Medium used was DMEM/F12 (Gibco, Cat. #31765) supplemented with 10% FBS (Gibco, Cat. # 10082139) and 1 % Penicillin/Streptomicin and changed every two days.
  • DIV4 cell were induced using LPS and IFN-g at concentrations 4 ug/mL and 10 ng/mL (LPS Enzo Life Science, Cat. # 581-007-LOOZ, lot L28106; IFN-g Miltenyi Biotec, MACS Cytokines, Cat. # 130-096- 872) as activating stimulus for 24 hours. After that supernatant was collected and levels of secreted cytokines IL-6 were measured by ELISA sandwich kit (BD, Cat. # 550950) (see FIGURE 13 and FIGURE 14).
  • miRNAs in primary microglia cells could be shown to lead to a change of pattern in secreted cytokines.
  • miR-124 leads to decreased secretion of IL-6 and silencing of immune response also shown by Ponomarev et al. 2011 whereas miR-132, miR-124 and miR-2137 lead to high level of IL-6 expression in non-induced conditions (see FIGURE 15).
  • miRNA-mimic mediated by lipid concoctions as reagents (Invitrogen, Cat. # 13778-150). Obtained cells were plated into 96 well plates and transfected with miRNA-mimic of interest at DIV7. MiRNA-mimic is artificially synthesized, double- stranded RNA which mimic mature endogenous miRNA after transfection into cells. In contrast to lentiviral approach to delivering miRNA to the cells, miRNA-mimic transfection is simple to use, high efficient, relatively low or non-cytotoxicity and inflammatory responses to the primary microglia.
  • the growth medium used was DMEM/F12 (Gibco, Cat. #11320-074) supplemented with 10% FBS (Gibco, Cat. # 10082139), 0,1 % nonessential amino acids (Gibco, Cat. # 1 1 40-050), 0, 1 % GlutaMAX (Gibco, Cat. # 35050-038) and 1 % Penidllin/Streptomicin and changed every two or three days (see FIGURE 21 ).
  • the cells were induced using LPS and IFN- ⁇ at concentrations of 4 ug/mL and 10 ng/mL, respectively (LPS Enzo Life Science, Cat. # 581-007-LOOZ, lot L28106; IFN- ⁇ Miltenyi Biotec, MACS Cytokines, Cat. # 30-096-872) as activating stimulus for 24 hours. Afterwards the supernatant was collected and the level of the secreted cytokine IL-6 was measured by ELISA sandwich kit (BD, Cat. # 550950).
  • miRNAs mediated by the miRNA-mimic transfection in primary microglial cells induced a change of pattern in secreted cytokines.
  • the IL-6 level was significantly reduced in miR- 124-3p overexpressing cells alone in comparison to the wild-type after LPS and INF- ⁇ stimulation. Therefore, it seems likely that miR-124-3p might have an anti-inflammatory effect as suggested in the previous paper by Ponomarev et al.; 2011.
  • Astrocytes viability During progression of epilepsy and as response to dying neurons astrocytes increase in their number. This process is known as astrogliosis. To analyse this in vitro miRNAs of interest were overexpressed in primary astrocytes and the proliferation of these cells was measured using MTT.
  • astrocytes were prepared from postnatal day zero or postnatal day one mouse brains (P0/P1 ), strain C57BL/6J using mechanical dissociation method (BD Nylon-strainer, Cat.# 352340) and grown in ten centimeter dishes.
  • Medium used was DMEM/F12 (Gibco, Cat.# 31765) supplemented with 10% FBS (Gibco, Cat.# 10082139) and 1 % Penicillin/ Streptomicin, changed one day upon plating and then every two days.
  • primary astrocytes were prepared from postnatal mouse brains (P0-P3), strain C57BL/6J using mechanical dissociation method (BD Nylon-strainer, Cat. # 352340) and grown in T25 flask pro brain.
  • the medium used was DMEM GlutaMax (Gibco, Cat. # 31966-021 ) supplemented with 10%FBS FBS (Gibco, Cat.# 10082139) and 1 % Penicillin/Streptomicin, changed the medium every three days upon plating. A confluent layer (70-80%) of cells was generated after 3-4 days.
  • Primary astrocytes were trypsinized and raised in T75 flask for the next 5-7 days.
  • FIGURE 26 Fluorescence microscopy images of secondary mouse astroytes 72 hours upon lentiviral transduction is shown in FIGURE 26. Representative images of cultured mice astrocytes (P0-P3) after 72 hours lentviral transduction are depicted in FIGURE 27. The MTT assay was used to evaluate viability of astrocytes in vitro after 72 hours of lentiviral transduction. The overexpression of miR-124-3p in primary astrocytes leads to a significant decrease in astrocyte proliferation in comparison to wild-type and miR-control transduced cells (see FIGURE 28).
  • miRNAs dysregulated during acute and chronic phase of epilepsy play an important role in above described events.
  • miR-124-3p and miR-142-3p decreased SH-SY5Y cell proliferation, respectively.
  • the presented excitotoxicity assay results suggest that overexpression of miR-124-3p has a neuroprotective effect in primary hippocampal neurons.
  • miR-124-3p mimic overexpressing microglia showed significantly reduced secretion of IL-6 in comparison to wild-type cells. This result indicates that miR- 24-3p might have an antiinflammatory effect on primary microglia.
  • lentivirus mediated miR-124-3p overexpression in astrocytes significantly decrease the proliferation compared to wild- type and miR-control overexpressing cells

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Abstract

The present invention relates to a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of a miRNA molecule. The invention further relates to a vaccine for use in the treatment or prevention of epilepsy, to a biomarker or group of biomarkers associated with epilepsy, a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, a method or assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy. Also envisaged is a method for monitoring epilepsy therapy, a method of identifying an individual eligible for an epilepsy therapy, a corresponding diagnostic kit and the use of a biomarker or a group of biomarkers for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.

Description

MICRORNAS AS THERAPEUTICS AND BIOMARKERS FOR EPILEPSY
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of a miRNA molecule. The invention further relates to a vaccine for use in the treatment or prevention of epilepsy, to a biomarker or group of biomarkers associated with epilepsy, a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, a method or assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy. Also envisaged is a method for monitoring epilepsy therapy, a method of identifying an individual eligible for an epilepsy therapy, a corresponding diagnostic kit and the use of a biomarker or a group of biomarkers for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
BACKGROUND OF THE INVENTION
The World Health Organisation (WHO) and the International League Against Epilepsy (ILAE) define epilepsy as a chronic, recurrent, repetitive neurological disorder consisting of paroxysmal phenomena caused by excessive and chaotic discharges in neuronal brain cells. Its incidence has two peaks: one in childhood and adolescence and a second more marked one over the age of 60. According to the International Bureau for Epilepsy (IBE), some 50 million people suffer from epilepsy worldwide, with 20-30% of them suffering more than one seizure per month (Forsgren et al., Eur J Neurol 2005;12:245-53).
The number of new epilepsy cases per year worldwide ranges between 24 and 53 cases per 100,000 inhabitants. In Europe, from the prevalence studies carried out in different countries and years, it was calculated that 0.9 million children and adolescents, 1.9 million adults of an age between 20 and 64 years, and 0.6 million older person of 65+ years are afflicted by epilepsy (Forsgren et al., Eur J Neurol 2005;12:245-53).
Epilepsy is considered to comprise a diverse set of chronic neurological disorders characterized by seizures. These seizures may be recurrent and unprovoked, or may constitute single seizures combined with brain alterations increasing the chance of future seizures. Epileptic seizures typically result from abnormal, excessive or hypersynchronous neuronal activity in the brain.
The etiology of epilepsy can in certain cases be linked to mutations in several genes that code for protein sub units of voltage-gated and ligand-gated ion channels. A further speculated mechanism for some forms of inherited predisposition for epilepsy are mutations of genes coding for sodium channel proteins. Accordingly modified or defective sodium channels may be opened for a prolonged period of time, leading to a hyper-excitability of neurons. In consequence, larger than normal amounts of the excitatory neurotransmitter glutamate can be released from the affected neurons and may trigger excessive calcium release in post-synaptic cells. The calcium release may, in turn, be neurotoxic to affected cell. Typically, the hippocampus, which comprises a large number of glutamatergic neurons is highly vulnerable to epileptic seizure, a subsequent spread of excitation, and possible neuronal death. Other possible mechanisms involve mutations leading to a dysfunction of neurotransmitter GABA, as well as mutations in non-ion channel genes.
A consequence of the occurrence of epileptic seizures is an interference of physical, emotional, and social functioning. Further consequences of repeated seizures include neuronal loss, gliosis, parenchymal microhemorrhages, excess of starch bodies, leptomeningeal thickening, and periavascular atrophy.
Diagnosis of epilepsy is mainly based on detection of symptoms
(seizures), electrophysiological measurements (e.g. EEG) and imaging studies of the brain (e.g. MRI, MRS, SPECT, PET). Molecular marker for epilepsy, as well as corresponding pharmaceutical compositions are largely lacking.
One class of molecular or biomarkers, which is considered helpful in several desease classes are microRNAs. MicroRNAs are a recently discovered class of endogenous, small non-coding RNAs. The first described microRNA, lin-4, was identified in C. elegans in 1993 (Lee et al., 1993, Cell 75(5): 843-85). Currently more than 2000 human mature microRNAs are annotated in databases (e.g. in www.mirBase.org). MicroRNAs regulate gene expression on the post-transcriptional level. They predominantly function as translational repressors by binding to the 3'UTR of their target gene mRNAs (He and Hannon, 2004, Nat Rev Genet 5(7): 522-531 ; Filipowicz et al. 2008 Nat Rev Genet 9(2): 102-114).
MicroRNAs are encoded in gene introns or in non-coding genomic regions. They are initially transcribed as long primary microRNA strands (pri- microRNA) that are processed to single hairpin precursors (pre-microRNA) by nuclear Drosha. Pre-microRNAs are typically transported to the cytoplasm by Exportin 5. Subsequently, the protein Dicer has been shown to cut the hairpin into the mature microRNA duplex (see Fig. 1 ). Both microRNA single strands may be incorporated into the RNA-induced silencing complex (RISC). The RISC subsequently mediates binding of mature microRNAs to their respective recognition site, which is typically in the 3'UTR of a target gene, leading to translational repression (He and Hannon, 2004, Nat Rev Genet 5(7): 522-531 ; Filipowicz et al. 2008 Nat Rev Genet 9(2): 102-114; Huntzinger and Izaurralde 2011 , Nat Rev Genet 12(2): 99-1 10).
MicroRNAs mainly function as translation repressors, but can also mediate mRNA degradation by direct binding to the open reading frame (ORF) in mRNAs (see Fig. 1 ). Based on this mechanism, every single microRNA may not only inhibit one, but multiple target genes. In addition, most mRNAs contain binding sites for several microRNAs in their 3'UTRs, thus further increasing the complexity of the system. It is estimated that about 30% of the human proteome are regulated by microRNAs (Krek et al., 2005, Nat Genet 37(5): 495-500), thus underlining the importance of microRNAs for coordinated protein expression (Selbach et al., 2008, Nature 455(7209): 58-63).
It was shown that several microRNAs are abundant in the CNS, apparently coordinating neurogenesis, dendrite formation, synaptic plasticity and neuroinflammation (Christensen and Schratt, 2009, Neurosci Lett 466(2): 55-62; Schratt, 2009, Nat Rev Neurosci 10(12): 842-849). Although there is increasing evidence that microRNAs are implicated in neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, Parkinson's disease or Prion disease (Saugstad, Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1564-1576), there is still relatively little known about the relevance of microRNAs in the etiology and development of epilepsy. McKiernan et al. (PLoS One, 2012; 7(5): e35921 ), for example, disclose that a loss of Dicer activity and failure of mature miRNA expression may be a feature of the pathophysiology of Hippocampal sclerosis in patients with temporal lobe epilepsy (TLE). There is thus a need for effective biomarkers and corresponding pharmaceutical compositions, which are able to image and reflect the complex regulations of epileptic diseases. OBJECTS AND SUMMARY OF THE INVENTION
The present invention addresses this need and provides a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of Table 1 or 1a. The present inventors could, in particular, identify significantly dysregulated microRNAs in preclinical animal models as well as in the biopsies of TLE patients, which were validated and analysed in a bioinformatic approach with regard to their target genes. The microRNAs of the invention were further analyzed for their role in CNS and epileptogenesis in different in vitro assays as well as in in vivo models. In addition, expression of the respective target genes was determined. It could, in particular be revealed that hippocampal microRNA expression is significantly changed in epilepsy models and patients. The detected changes appear to be modulated by factors like severity of disease (e.g. seizure frequency) as well as disease duration (e.g. acute vs. chronic epilepsy). The miRNAs of the invention thus play a decisive regulatory role in the molecular mechanisms underlying epileptogenesis. The miRNAS themselves or derivatives thereof, or antagonists thereof thus constitute a highly relevant therapeutic principle in the treatment or prevention of epilepsy. They are particularly considered as extremely valuable means for alleviating and/or reversing any effect of miRNA dysregulation observable in epilepsy.
In a preferred embodiment of the present invention said means as mentioned above comprises a nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 or in section A or H of Table 1 a, or a derivative, fragment or variant thereof, or an antagonist thereof, wherein said nucleic acid molecule or antagonist has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section B of Table 1 or in section B or H of Table 1a or to a complementary sequence thereof.
In a further preferred embodiment, said nucleic acid molecule or antagonist comprises between at least 18 and 24 nucleotides, preferably 20 nucleotides, 21 nucleotides or 22 nucleotides. In yet another preferred embodiment the nucleic acid or antagonist is or comprises a DNA molecule or an RNA molecule, or a derivative thereof.
It is further preferred that said RNA molecule as mentioned above is or comprises a pre-miRNA selected from section E of Table 1 or from section E or H of Table 1a, or a derivative, fragment or variant thereof, wherein said pre-miRNA molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section F of Table 1 or in section F or H of Table 1a or to a complementary sequence thereof.
In another preferred embodiment of the present invention, said DNA molecule as mentioned above is or comprises a DNA molecule coding for a pre- miRNA, a miRNA or for an antagonist.
In yet another preferred embodiment of the present invention, said DNA molecule is or comprises a double stranded DNA coding for at least one pre-miRNA selected from section E of Table 1 or from section E or H of Table 1a.
It is also preferred that said nucleic acid molecule as mentioned above comprised in a vector replicable in a subject.
In a further preferred embodiment of the present invention said nucleic acid molecule or antagonist is modified by a conjugate, preferably a linked conjugate.
Also preferred is that the nucleic acid molecule or antagonist comprises a chemically modified base.
In a particularly preferred embodiment of the present invention said antagonist as mentioned above is an antagomir.
In yet another preferred embodiment, the nucleic acid molecule or antagonist as mentioned herein above may be modified by at least one modification selected from the group consisting of a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose and a 2'-methoxyethoxy ribose.
In an embodiment, said nucleic acid molecule as mentioned herein above may comprise a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu- miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR- 193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-mi'R-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR- 48a-3p, or miR-125a-5p molecule as defined in section A of Table 1 or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in section B of Table 1. In a particularly preferred embodiment, said nucleic acid molecule as mentioned herein above may comprise a hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa- miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p, mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR- 130a-3p or mmu-miR-191-5p molecule as defined in sections A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in sections B or H of Table 1a.
In a further embodiment, said antagonist as mentioned herein above is an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR- 223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu- miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR- 138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR-142-3p, or hsa-miR- 24-3p molecule as defined in section A of Table 1 , or of a derivative, fragment or variant thereof. In a particularly preferred embodiment, said antagonist as mentioned herein above is an antagonist of a hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3, mmu-miR-129- 5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu- miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p or mmu-miR-222-3p molecule as defined in section A or H of Table 1 a, or of a derivative, fragment or variant thereof.
In a further aspect, the present invention relates to a vaccine for use in the treatment or prevention of epilepsy, comprising a nucleic acid molecule or antagonist as mentioned herein above.
In another aspect, the present invention relates to an isolated biomarker or group of isolated biomarkers associated with epilepsy, wherein said isolated biomarker or group of isolated biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 or in section A or H of Table 1a, or a derivative, fragment or variant thereof.
In an embodiment the present invention relates to an isolated biomarker or group of isolated biomarkers, wherein the increase of expression (up-regulation) of at least one biomarker selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1- 3p, mmu-miR-2 9-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR- 132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa- miR-142-3p, and hsa-miR-124-3p as defined in section A of Tab!e 1 , when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy. In a particularly preferred embodiment the present invention relates to a biomarker or group of biomarkers, wherein the increase of expression (up- regulation) of at least one biomarker selected from the group of hsa-miR-129-5p, hsa- miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, or hsa-miR-494-3p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR- 29-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, or mmu- miR-494-3p as defined in section A of Table 1a, , when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
In another embodiment the present invention relates to a biomarker or group of biomarkers, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of mmu-miR- 24-3p, mmu-miR-124-5p, mmu- miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa- miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa- miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p, and miR-125a- 5p, when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy. In a particularly preferred embodiment the present invention relates to a biomarker or group of biomarkers, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR- 34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR- 24-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR- 0-3p, mmu-miR- 30a-3p or mmu-miR-191 -5p as defined in section A of Table 1 a,when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
In a further aspect the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, and/or a probe specific for the biomarker or group of biomarkers as defined herein above.
In another aspect the present invention relates to a method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined herein above.
In further aspect the present invention relates to a method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample before and during an epilepsy treatment, optionally also after an epilepsy treatment.
In a preferred embodiment of the method for monitoring epilepsy therapy, said treatment is a treatment with a pharmaceutical composition as defined herein above, and/or with at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, zonisamide, and combinations thereof.
In yet another preferred embodiment of the method for diagnosing or for monitoring epilepsy therapy, the determining step is accomplished by the measurement of the miRNA level(s) or by the determination of the biological effect of said biomarker or group of biomarkers.
In a further aspect, the present invention relates to an assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
(a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined herein above;
(b) testing in a control sample for the expression of the same biomarker or group of biomarkers as in (a);
(c) determining the difference in expression of the biomarker or group of biomarkers of steps (a) and (b); and
(d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c) ,
wherein said testing steps are based on the use of a nucleic acid affinity ligand and/or a peptide affinity ligand for a biomarker or a group of biomarkers as EP2013/077836
defined herein above.
In a further aspect the present invention relates to a method of identifying an individual eligible for an epilepsy therapy comprising:
(a) testing in a sample obtained from an individual for the expression an up- or down-regulated biomarker or a group of such biomarkers as defined herein above;
(b) testing in a control sample for the expression of an up- or down- regulated biomarker or a group of such biomarkers as mentioned above;
(c) classifying the levels of expression of step (a) relative to the levels of step (b); and
(d) identifying the individual as eligible to receive an epilepsy therapy where the individual's sample is classified as having an increased level of expression of an up-regulated biomarker or a group of biomarkers as herein above, or where the individual's sample is classified as having a decreased level of expression of a down- regulated biomarker or a group of biomarkers as defined herein above.
In a further aspect the present invention relates to a diagnostic kit for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined herein above, wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, or a probe specific for the biomarker or group of biomarkers as defined herein above.
In yet another aspect the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
In yet another aspect the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 provides an overview of micro NA biogenesis, showing that microRNAs hairpin precursors are transcribed and processed in the nucleus, that pre- microRNA (70-80nt hairpins) are exported to the cytoplasm and are finally incorporated in the RISC complex, which mediates microRNA function.
Fig. 2 shows an overview over the analytical steps described in the
Examples.
Fig. 3 depicts the results of a bioinformatics analysis for the identification of some epilepsy-relevant microRNA target genes according to the invention.
Fig. 4 gives a schematic overview on functional in vitro assays and their relevance for the analysis of molecular mechanisms underlying epileptogenesis.
Fig. 5 provides a schematic presentation of lentiviral miRNA overexpression system used in vitro.
Fig. 6 (A to E) shows fluorescence microscope photographs of mouse hippocampal neurons in culture at DIV13 and lentiviral constructs expressing several CMV-GFP-miRNAs 5 days upon transduction.
Fig. 7 (A to E) shows the overexpression of several miRNAs in transduced hippocampal neurons. Values are calculated to scrambled miRNA sequence (control, miR-con) using a AACt method and the SN068 house keeping gene.
Fig. 8 depicts a graphical scheme of in vitro proliferation assay.
Fig. 9 (A to F) shows fluorescence microscopy photographs of SH- SY5Y transduced with CMV-GFP-miRNA lentiviral constructs, showing GFP reporter expression upon 72 hours.
Fig. 10 shows a growth assay measured by MTT. SH-SY5Y cells expressing CMV-GFP-miR-124 construct demonstrate significantly slower growth rate compared to control (experiments done in N=6 replicates, measurement n=5 per condition).
Fig. 11 depicts a graphical scheme of in vitro excitotoxicity induced by glutamate assay.
Fig. 12 shows effects of miRNA in excitotoxicity pathway induced by glutamate exposure in hippocampal neurons. Hippocampal neurons were transduced at DIV7 treated with 10uM glutamate for 24 hours at DIV13, measured by MTT assay
Fig. 13 shows a graphical scheme of in vitro activation of microglia assay.
Fig. 14 (A and B) shows photographs of mouse primary microglia isolated from P3 brains: (A) non induced and (B) induced.
Fig. 15 shows secretion of IL-6. Fig. 14 (A) provides absolute values of secreted IL-6. Fig. 14 (B) provides relative values of secreted IL-6 (experiments performed N=3). Upon LPS/SFN-g induction, levels of IL-6 were measured by ELISA assay in wt microglia in cells transduced with miRNA of interest.
Fig. 16 provides a graphical scheme of in vitro astrocytes growth assay.
Fig. 17 shows results of experiments with secondary mouse astrocytes. Fig. 17 (A) provides fluorescence microscopy photographs of secondary mouse astroytes 3 to 5 days upon lentiviral transduction. Astrocytes expressing CMV-GFP- mi'R constructs at MOM 00 differ in viability and size depending of expressed miRNA. Fig. 17 (B) shows survival assay of secondary mouse astroytes 5 days upon LV transduction measured by MTT.
Fig. 18 shows the overlap of miRNA expression between the two chronic epilepsy mouse models (pilocarpine and SSSE) and the acute seizure mouse model (6 Hz) using Venn diagrams.
Fig. 19 shows fluorescence microscopy images of SH-SY5Y cells transduced with CMV-GFP-miRNA lentiviral constructs showing GFP reporter expression upon 72 hours. Scale bar: 20 left column; 10 m right column.
Fig. 20 shows growth assay measured by MTT. SH-SY5Y cells expressing CMV-GFP-miR-124-3p and CMV-GFP-miR142-3p construct showed significantly decreased growth rate compared to wild-type and miR-control overexpressing SY5Y cells at the end of measurement. Experiments done in N=3 replicates, measurement n=4 per condition (* < 0,05).
Fig. 21 shows a graphical scheme of in vitro activation of primary microglial cells.
Fig. 22 depicts the results of a FACS analysis of the CD1 1 b+ fraction. After isolation and immunomagnetic separation of the CD1 1b+ cell population, cells were stained with anti-CD45 antibodies and analyzed by flow cytometry. CD11b and CD45-coexpressing populations were identified with red colour and the proportion of surviving cells was determined by propidium iodide (PI). (A) Total cell population isolated from brain tissues before immunomagnetic separation using CD11 b+ beads. (B) Negative fraction, a very low number of CD11 b+/CD45+ cells was detected in this fraction, whereas a high proportion of CD1 1b7CD45+ cells was sorted in the positive fraction (C). (D) A tabel comparing the differences in CD1 b+/CD45+ cells between three fractions, suggesting high efficiency and specificity of the CD11b+ cell separation from postnatal mouse brain tissues.
Fig. 23 (A and B) shows the morphological characteristics of primary cultured mice microglial cells (P0-P3) 72 hours after transfection of miRNA-mimics. Non-transfected wild-tpye ramified primary microglial cell with round to oval small somata and a long primary process, whereas miRNA overexpressing primary microglia undergo several dramatic morphological changes, inculding large variably shaped cell body often loaded with debris, ruffling and relative short, thick extensions of processes. Scale bar: 20 μηι left column; 10 μιη right column.
Fig. 24 illustrates the release of IL-6 in primary cultured microglia. Histogramm showing the absolute values of released IL-6 before and after induction with LPS/IFN-γ. The levels of IL-6 were measured by ELISA assay in wild-type microglia and cells transfected with miRNA-mimics of interest. Experiments done in N=4 replicates, measurement n=2 per condition (* < 0,05, ** < 0,01 ; error bar mean + s.e).
Fig. 25 shows a graphical scheme of in vitro astrocytes viability assay.
Fig. 26 shows fluorescence microscopy images of secondary mouse astroytes 72 hours upon lentiviral transduction. Astrocytes expressing CMV-GFP-miR constructs at MOI30 differ in viability and size depending of expressed miRNA. Scale bar: 20 pm left- ; 10 μπη right- column.
Fig. 27 shows representative images of cultured mice astrocytes (POPS) after 72 hours lentviral transduction. Double immunofluorescence staining for GFAP (red) and GFP (green). (A-C) Wild-type astrocytes show absence of GFP expression, but GFAP immunoreactivity. (D-l) Astrocytes transduced with CMV-GFP- miR constructs showing abundant GFP expression colocalized with the astrocytic marker GFAP. Scale bar: 10 pm.
Fig. 28 illustrates the effect of miRNA overexpression on the viability of cultured mice astrocytes. Viability of astrocytes was measured by using MTT assay after viral transduction for 72 hours. Astrocytes overexpressing miRNA-124-3p showed significantly reduced growth rate compared to miR-control transduced cells. Experiments done in N=5 replicates, measurement n=4 per condition with the exception of miR-135b-5p and miR-222-3p (* < 0,05; error bar mean + s.e). DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of miRNAs and corresponding biomarkers and methods for using them, as well as suitable kits and compositions.
Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.
Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.
It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the", include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a polypeptide" or "a ribosome" includes one or more of such polypeptides or ribosomes, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.
It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group, which preferably consists of these embodiments only.
Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ii)", "(iii)", "(iv)", "(v)", "(vi)", "(vii)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ii)", "(iii)", "(iv)", "(v)", "(vi)", "(vii)" etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application or claims as set forth herein above or below.
It is to be understood that this invention is not limited to the particular methodology, protocols, proteins, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
As has been set out above, the present invention concerns in one aspect a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of the following Table 1 :
Table 1
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
In further embodiments, the present invention concerns human orthologues of mouse miRNA molecules as defined hin Table 1 , e.g. for all miRNA molecules starting with mmu. Such human orthologues can be obtained by database comparisons, or by homology searches. In specific embodiments the present invention relates to human orthologue miRNA molecules of murine miRNAs which can be obtained by performing amplification reactions on human RNA or DNA or human samples obtained from several different stages or sources, e.g. epilectic patiens, tissue samples etc. as define herein with primers which have been derived from the mmu miRNA sequences. Optionally these primer sequences may further be adapted to known functional or structural differences between the hsa and mmu miRNA molecules. The present invention accordingly envisages the use of human miRNA molecules which are structurally and/oir functionally homologous to murine miRNA molecules as describe in Table 1 , or derivable form Table 32, or from any other Table disclosed herein.
The term "human orthologue" as used herein refers to a molecule which is structurally and/or functionally equivalent to another miRNA molcule, in particular to a murine miRNA molecule. Particularly preferred are human orthologues of mmu-miR- 129-5p, mmu-miR-142-3p, mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, mmu-miR- 494-3p mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, or mmu-miR-2137.
In a preferred embodiment, the present invention concerns a pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A or section H of the following Table 1a:
Table 1a
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
The term "miRNA" as used in section A of Table 1 or in section A or H of Table 1a relates to a mature microRNA molecule as identified by the indicated designation. The designation is based on the nomenclature provided in relevant databases such as mirbase.org as of 10 December 2012 or later version thereof. The mature microRNA of section A of Table 1 or 1 a or of section H of Table 1a is further defined by a corresponding sequence as identified in section B of Table 1 or 1a or in section H of Table 1a. The effect of dysregulation of a miRNA may, in further embodiments, also be an effect detectable on the level of pre-miRNA. Such an effect may be a dysregulation of pre-miRNA molecules as identified in section E of Table 1 or 1a. The designation of pre-miRNAs is based on the nomenclature provided in relevant databases such as mirbase.org as of 10 December 2012 or later versions thereof. The pre-microRNA of section E of Table 1 or 1a or of section H of Table 1a is further defined by a corresponding sequence as identified in section F of Table 1 or 1a of in section H of Table 1a. The effect of dysregulation of a miRNA may, in specific embodiments, be correlated to the regulatory behavior of mature miRNAs as observed in analyses described in the Examples of the invention. Preferably, the effect of dysregulation may be a up-regulation of a mature miRNA as indicated by a "+" in section C of Table 1 or 1a, or a down-regulation as indicated by a "+" in section D of Table 1 or 1a. The micro-RNAs mmu-miR-34b-5p and mmu-miR-191-5p as mentioned in Table 1 a show an up-regulation in specific models, while they show a down- regulation in other specific models. This behavior is apparently dependent on the model and in particular on the phase analysed (see also the examples portion). The level of dysregulation may further be derived from indications in Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43, e.g. via the value of fold change (FC) in the measured expression. The fold change may vary according to the model. Thus, FC values may in specific embodiments at least be comparable within the model, e.g. within one of Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43. It is preferred using miRNAs with a high FC value as indicated in Tables 1 to 25 and 27 to 31 , 33 to 39 and 42 to 43 e.g. the first 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of Tables 1 to 25 or 27 to 31 , 33 to 39 and 42 to 43. It is particularly preferred using miRNAs as shown in Table 1 a. The present invention also relates to corresponding DNA sequences of any of SEQ ID NO: 1 to 445 as defined in Table 1 , 1 a and/or Table 32, or of the miRNAs as described in any other part of the description. In further embodiments, the present invention relates to a composition or compositions which is/are capable of inhibiting or antagonizing an upregulated miRNA as indicated by a "+" in section C of Table 1 or 1a, or by a "+" in section "upreguiation" of Table 32, or which is able to reduce the amount or activity of an upregulated miRNA as indicated by a "+" in section C of Table 1 or 1 a, or in section "upreguiation" of Table 32. Such an increase may, for example, be provided by the presence of antagonist of upregulated miRNAs in the composition, as described herein. In further embodiments, the present invention relates to a composition or compositions which is/are capable of increasing the amount of a downregulated miRNA as indicated by a "+" in section D of Table 1 or 1 a, or by a "+" in section "downregulation" of Table 32. Such an increase may, for example, be provided by the presence of the downregulated miRNAs in the composition itself, as described herein.
In a specific embodiment the present invention relates to at least one miRNA selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-711 , mmu-miR-7 0, mmu-miR-882, mmu-miR-20a-5p, mmu-miR-335-3p, mmu-miR-219-5p, mmu-miR-140-5p, mmu-miR-294-5p, mmu-miR-690, mmu-miR-207, mmu-miR-338-3p, mmu-miR-34b-3p, mmu-miR-465b-5p, mmu-let-7f-5p, mmu-miR-1983, mmu-miR- 302a-3p, mmu-let-7b-5p, mmu-miR-290-5p, mmu-miR-369-5p, mmu-miR-374b-5p, mmu-miR-23b-3p, mmu-miR-709, mmu-let-7a-5p, mmu-miR-330-3p, mmu-miR-148a- 3p, mmu-miR-582-5p, mmu-miR-491 -5p, mmu-miR-1961 , mmu-miR-125b-5p, mmu- miR-1 a-3p, mmu-miR-409-5p, mmu-miR-582-3p, mmu-miR-1 192, mmu-miR-337-3p, mmu-miR-127-3p, mmu-miR-496a-3p, mmu-miR-378a-5p, mmu-let-7c-5p, mmu-miR- 200b-3p, mmu-mi"R-24-3p, mmu-miR-767, mmu-miR-337-3p, mmu-miR-127-3p, mmu- miR-496a-3p, mmu-miR-378a-5p, mmu-let-7c-5p, mmu-let-7c-5p, mmu-miR-377-3p, mmu-miR-431-3p, mmu-miR-541-5p, mmu-miR-98-5p, mmu-miR-194-5p, mmu-miR- 467b-5p, mmu-miR-30c-5p, mmu-miR-30c-5p, mmu-miR-1839-3p, mmu-miR-30a-5p, mmu-miR-106a-5p, mmu-miR-325-3p, mmu-miR-338-5p, mmu-miR-144-3p, mmu-miR- 135a-5p, mmu-miR-669c-5p, mmu-miR-467e-3p, mmu-miR-455-5p, mmu-miR-24-2- 5p, mmu-miR-1971 , mmu-miR-466i-3p, mmu-miR-1929-5p, mmu-miR-467a-3p, mmu- miR-34c-5p, mmu-miR-34b-5p, mmu-miR-691 , mmu-miR-1952, mmu-miR- 93a-3p, mmu-miR-467b-3p, mmu-miR-297a-3p, mmu-miR-219-5p, mmu-miR-124-3p, mmu- miR-124-5p, hsa-miR-1298, hsa-m'iR-191 -5p, hsa-miR-211-5p, hsa-miR-548a-3p, hsa-miR-1323, hsa-miR-34c-5p, hsa-miR-193b-3p, hsa-miR-1258, hsa-miR-21 16-3p, hsa-miR-34b-5p, hsa-miR-4780, hsa-miR-204-5p, hsa-miR-4749-5p, hsa-miR-375, hsa-miR-1254, hsa-miR-1292-5p, hsa-miR-577, hsa-miR-4775, hsa-miR-548s, hsa- miR-3928, hsa-mi'R-449a, hsa-miR-192-5p, hsa-miR-3 73-5p, mi'R-219-5p, hsa-mi'R- 190b, hsa-miR-514a-3p, hsa-miR- 47b, hsa-miR-329, hsa miR-320d, hsa-miR-1976, hsa-miR-655, hsa-miR-584-5p, hsa-miR-654-3p, hsa-miR-5581-3p, hsa-miR-495-3p, hsa-miR-346, miR-767-5p, hsa-miR-582-5p, hsa-miR-380-5p, hsa-miR-656, hsa-miR- 320b, hsa-miR-4640-3p, hsa-miR-556-5p, hsa-miR-505-3p, hsa-miR-4763-5p, hsa- miR-1299, hsa-miR-1910, hsa-miR-3653, hsa-miR-429, hsa-miR-410, hsa-miR-539-3p, hsa-miR-885-5p, hsa-miR-3615, hsa-miR-223-3p, miR-125a-5p, hsa-miR-27b-3p, hsa- miR-379-5p, hsa-miR-36 3-5p, hsa-miR-124-3p, hsa-miR-29c-3p, hsa-miR-1468, hsa- miR-148a-3p, mmu-miR-130a-3p, mmu-miR-551 b-3p, mmu-miR-187-3p, mmu-miR- 92b-3p, miR-767,and mmu-miR-125a-5p, as defined in Table 1 or Table 32.
In a specific, preferred embodiment the present invention relates to at least one miRNA selected from the group of mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR- 191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-18 b-5p, hsa-mi'R-34b-5p, hsa-miR- 676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR-191-5p, mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR- 135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-494-3p, hsa- miR-142-5p, hsa-miR-184-3 as defined in Table 1 a.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 3, or at least one of the miRNAs presented in Table 3. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (24 h, 2 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 4, or at least one of the miRNAs presented in Table 4. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (24h, 2 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 5, or at least one of the miRNAs presented in Table 5. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (28 d, 1 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 6, or at least one of the miRNAs presented in Table 6. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (28 d, 1 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 7, or at least one of the miRNAs presented in Table 7. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Pilocarpine study (28 d, 2 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 8, or at least one of the miRNAs presented in Table 8. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Pilocarpine study (28 d, 2 h Diazepam) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 9, or at least one of the miRNAs presented in Table 9. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the SSSE study (24 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 10, or at least one of the miRNAs presented in Table 10. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the SSSE study (24 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 11 , or at least one of the miRNAs presented in Table
11. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the SSSE study (28 d) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 12, or at least one of the miRNAs presented in Table
12. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the SSSE study (28 d) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 13, or at least one of the miRNAs presented in Table
13. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (3 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 14, or at least one of the miRNAs presented in Table
14. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (3 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 15, or at least one of the miRNAs presented in Table
15. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (6 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 16, or at least one of the miRNAs presented in Table
16. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (6 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 17, or at least one of the miRNAs presented in Table
17. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (24 h) or at least one of the miRIMAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 18, or at least one of the miRNAs presented in Table 18. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (24 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 19, or at least one of the miRNAs presented in Table 19. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the 6 Hertz study (72 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 20, or at least one of the miRNAs presented in Table 20. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the 6 Hertz study (72 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 21 , or at least one of the miRNAs presented in Table 21. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (3 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 22, or at least one of the miRNAs presented in Table 22. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in the Audiogenic study (3 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 23, or at least one of the miRNAs presented in Table 23. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (6 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 24, or at least one of the miRNAs presented in Table 24. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in the Audiogenic study (72 h) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 25, or at least one of the miRNAs presented in Table 25. In a further embodiment, the present invention relates to the group of miRNAs being qRT-PCR validated in animal models or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 27, or at least one of the miRNAs presented in Table
27. In a further embodiment, the present invention relates to the group of miRNAs being up regulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 28, or at least one of the miRNAs presented in Table
28. In a further embodiment, the present invention relates to the group of miRNAs being down regulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 29, or at least one of the miRNAs presented in Table
29. In a further embodiment, the present invention relates to the group of miRNAs being qRT-PCR validated in hippocampi of human TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 30, or at least one of the miRNAs presented in Table
30. In a further embodiment, the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling (acute phase) or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 31 , or at least one of the miRNAs presented in Table
31. In a further embodiment, the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling (chronic phase) or at least one of the miRNAs of this group.
In a further specific embodiment, the present invention relates to the group of miRNAs as presented in Table 32, or at least one of the miRNAs presented in Table 32. The present invention in particular also refers to miRNAs and pre-miRNAs as defined in SEQ ID NO: 322 to 375 or 383, 384, 385, 386, 387, 389, 390, 392, 393, 395, or 401 to 435 as indicated in said Table 32.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 33, or at least one of the miRNAs presented in Table 33. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the pilocarpine model at the timepoints 24h and 28d or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 34, or at least one of the miRNAs presented in Table 34. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the SSSE model at the timepoint 24h or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 35, or at least one of the miRNAs presented in Table 35. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the SSSE model at the timepoint 28d or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 36, or at least one of the miRNAs presented in Table 36. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 3h following seizure or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 37, or at least one of the miRNAs presented in Table 37. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 6h following seizure or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 38, or at least one of the miRNAs presented in Table 38. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 24h following seizure or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 39, or at least one of the miRNAs presented in Table 39. In a further embodiment, the present invention relates to the group of miRNAs being significantly up- and down regulated miRNAs in the 6 Hz model at the timepoint 72h following seizure or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 40, or at least one of the miRNAs presented in Table 40. In a further embodiment, the present invention relates to the group of miRNAs being commonly deregulated miRNAs in the pilocarpine and SSSE model at 24 h and 28d following SE or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 42, or at least one of the miRNAs presented in Table 42. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 43, or at least one of the miRNAs presented in Table 43. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 43, or at least one of the miRNAs presented in Table 43. In a further embodiment, the present invention relates to the group of miRNAs being deregulated in hippocampal biopsies of TLE patients or at least one of the miRNAs of this group.
In a further embodiment, the present invention relates to the group of miRNAs as presented in Table 44, or at least one of the miRNAs presented in Table 44. In a further embodiment, the present invention relates to the group of miRNAs being overlappingly expressed in mouse models and human profiling or at least one of the miRNAs of this group.
In one embodiment, the miRNAs may be one, two, three, 4 or all or the mature miRNA molecules miR-2137, miR-124-3p, miR-142-3p, miR-184, miR-132-3p, or of corresponding pre-miRNAs as defined in Table 1.
Particularly preferred are one, two, three, 4 or all or the mature miRNA molecules mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR- 191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR- 676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191-5p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b- 5p, hsa- miR-191-5p, or hsa-mi'R-494-3p or of corresponding pre-miRNAs as defined in Table 1a.
miR-2137 is a mouse specific miRNA identified by tissue sequencing. Target genes and physiological relevance of this miRNA are unknown. It is situated in the first intron of Zfp185 protein located on the X chromosome. As can be derived from the Examples, miR-2137 is highly up-regulated in acute phase of mouse epilepsy models only. Currently, no ortholog of this miRNA is familiar in human genome. Nevertheless, its specific seed sequence might be of importance in biochemical pathways conserved between mouse and human.
miR-124 is deeply conserved and shows CNS-enriched expression in all animals examined from nematode to human (see Stark et al., 2005; Cao et al., 2007, Genes Dev.; 21 (5):531-6; Kapsimali et al., 2007, Genome Biol. 2007;8(8): R173; Makeyev et al., 2007, Mol Cell. 2007, 27(3):435-48; Visvanathan et al., 2007, Genes Dev; 21 (7):744-9; Rajasethupathy et al., 2009; Neuron; 63(6):803-17; and Shkumatava et al., 2009, Genes Dev; 23(4):466-81 ). There are three pre-miR-124a loci (pre-miR- 124a-1 , pre-miR-124a-2 and pre-miR-124a-3) in the mouse and human genomes. In humans, pre-miR-124a-1 is located on chromosome 8p23.1. Notably, chromosomal duplication, deletion or mutation of the 8p23.1 region have been reported to be involved in cerebral development and neuropsychiatric disorders, including autism, bipolar disorder, schizophrenia, learning difficulties, epilepsy and microcephaly (see Tabares-Seisdedos et al., 2009, Mol. Psychiatry 14, 563-589; Baulac et al., 2008, Arch. Neurol. 65, 943-951 ; and Glancy et al., 2009, Eur. J. Hum. Genet. 17, 37-43). In individuals with temporal lobe epilepsy and model animals, aberrantly sprouting mossy fibers are often observed (see Koyama et al., 2004, Curr. Neurovasc. Res. 1 , 3-10). However, the molecular mechanisms remain unknown. Recently, miR-124 has been shown to promote microglia quiescence (see Ponomarev et al., 2011 , Nat Med 17(1 ): 64-70). As can be derived from the Examples, miR-124* and miR-124 originating from the same precursor transcripts are acutely down-regulated in mouse model of epilepsia (pilocarine model) with the rate of 12% and 10%, respectively. In human samples, miR- 124 shows same tendency and is down-regulated in patients with AHS compared to patients suffering from nonAHS form of epilepsy. These two groups of patients show different clinical picture, of which the most striking feature is death of neurons in severe neuronal cell loss and gliosis in the hippocampus, specifically in the CA-1 (Cornu Ammonis area 1 ) and subiculum of the hippocampus in AHS patients. In addition, the
AHS patients group is more severe/pharmacoresistant.
miR-142 is associated with a variety diseases especially with cancer. It is also expressed in the CNS. miR-142 has further been shown to target the transcripts of key neuronal genes encoding the D1 dopamine receptor (DRD1 ) which plays an important role in neurons. As can be derived from the Examples, miR-142 is up regulated in the mouse and human miRNA profiling.
miR-184 is a single copy gene and evolutionarily conserved at the nucleotide level from flies to humans. In humans, miR-184 is located within region 25.1 on the q-arm of chromosome 15, and its corresponding transcript is comparatively small (84 bp) which is not encoded near other clustered miRNAs. Mature miR-184 is particularly enriched in the brain and testis. miR-184 is identified as a novel contributor to neuronal survival following both mild and severe seizures. As can also be derived from the Examples, miR-184 is down-regulated to 13% comparing AHS and nonAHS human samples and its deregulation is detected only in human form of disease, pointing to this miRNA as potentially highly specific biomarker for human epileptic brain.
miR-132 is enriched in neuronal cells and several targets for miR-132 have been described, including mediators of neurological development, synaptic transmission, inflammation and angiogenesis. Furthermore, for miR-132 neuroprotective functions have been shown after Status Epilepticus. miR-132 arises from the miR-212/132 cluster located in the intron of a non-coding gene on mouse chromosome 11. The transcription of this cluster was found to be enhanced by the transcription factor CREB (cAMP-response element binding protein) (see Wanet et al., 2012, Nucleic Acids Res.,40(1 1 ): 4742-53). The data provided in the Examples show that miR-132 is upregulated in mouse models of epilepsy. Taking into consideration role of miR- 32 in dendritic sprouting, this miRNA may play a significant role in brain during disease.
miR-135b has been described as being upregulated after the induction of oxidative stress in mouse hippocampal neurons. It was further described that human miR-135b regulates the expression of DISC-1 (Disrupted-in-schizophrenia-1 ), a gene which is known to play an important role in different neuropsychiatric phenotypes (shizophrenia, depression). The data provided in the Examples show miR-135b-5p is significantly up regulated in the chronic phase of both mouse chronic epilepsy models (SSSE and Pilocarpine). miR -222 has been found to be expressed in the brain and was shown to to regulate endogenous ICAM-1 expression. In addition, it was shown that miR-222 targets PTEN, a phosphatase and negative regulator of the PI3K signalling pathway, which is important for central axon growth. The data provided in the Examples show that the expression of miR-222-3p was upregulated at 28 days in both chronic mouse epilepsy models.
miR-129 has two loci in humans and in mice. In humans the gene encoding for mir-129-1 is located on Chromosome 7 and the gene for mir-129-2 is located on chromosome 11. For mice it is chromosome 6 and chromosome 2, respectively. Both stem loop sequences express the same mature sequence of miR- 129-5p. It was demonstrated that F Rl is a target of miR-129-5p. While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. The data provided in the Examples show that mi'R-129-5p was significantly upregulated in both mouse models and in human patient samples in the comparison AHS versus nonAHS.
miR-1298 has not been described so far in the literature. The data provided in the Examples show that miR-1298 is a highly relevant biomarker.
For miR-181 b two loci in humans and in mice could be identified. In humans the gene encoding for mir-181 b-1 is located on Chromosome 1 and the gene for mir- 81 b-2 is located on chromosome 9. In mice the gene encoding for mir-181 b-1 is located on chromosome 1 and and the gene for mir-181 b-2 is located on chromosome 2, respectively. It could be shown that a downregulation of miR-181 leads to a pro-inflammatory response in astrocytes. The data provided in the Examples show that miR-181 b-5p was significantly downregulated in human patient samples in the comparison AHS plus nonAHS versus post mortem. In addition, it was downregulated in both chronic epilepsy mouse models at 24 hours.
miR-34b was shown to be downregulated in patients with Parkinson's disease. The data provided in the Examples show that miR-34b-5p was significantly downregulated in human patient samples in the comparison AHS plus nonAHS versus post mortem.
miR-676 was identified in an expression profiling approach of a murine TH-MYCN neuroblastoma model, which revealed similarities with human tumors.
Mmu-miR-140-3p was identified in a approach to elucidtate early mechanisms of pathobiology by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice. miR-130a was shown to be expressed in gfiobSastoma.
miR-191 was described that as being differentially expressed following induction of neuronal activity, indicating its possible role in neuronal development and neuronal activity.
miR-494-3p was described as being involved in Parkinson's disease.
"Epilepsy" as used herein refers to a disease comprising a diverse set of chronic neurological disorders characterized by seizures. Such seizures may be recurrent and unprovoked. They may alternatively constitute single seizures combined with brain alterations and thus increase the chance of future seizures. Epileptic seizures may, for example, result from abnormal, excessive or hypersynchronous neuronal activity in the brain. Epilepsy may further be classified according to seizure types or forms. Seizure types are typically organized according to whether the source of the seizure within the brain is localized {partial or focal onset seizures) or distributed (generalized seizures). Epilepsy comprising partial seizures may further be divided on the extent to which awareness is affected. If awareness is unaffected, then it is an epilepsy comprising simple partial seizure. If awareness is affected, it is an epilepsy comrpising complex partial or psychomotor seizure. A partial seizure may typically spread within the brain, i.e. lead to secondary generalization. Generalized seizures typically involve loss of consciousness and may further be divided according to the effect on the body. Examples include absence (petit mal), myoclonic, clonic, tonic, tonic-clonic (grand mal), and atonic seizures.
The term "means for alleviating or reversing the effect of a dysregulation of at least one miRNA" as used herein refers to a compound, which is capable of overcoming or influencing an effect caused by an aberrant expression of a miRNA, preferably a mature miRNA as defined in Table 1 or 1 a, or a pre-miRNA as defined in Table 1 or 1 a. The aberrant expression may be a decreased or increased expression of the miRNA, preferably a mature miRNA as defined in Table 1 or 1a, or a pre-miRNA as defined in Table 1 or 1a in comparison to an expression situation in a healthy or normal subject. The effect may additionally or alternatively also be a secondary effect, e.g. a dysregulation of one or more target genes of a miRNA according to the invention. Such a dysregulation of a target gene may be an increase or decrease of expression of the target gene, or the presence or absence of the proteineous gene product of the target gene, a mislocalization of the protein, a modification of interaction with further proteins etc. In a specific embodiment of the present invention the pharmaceutical compositions of the present invention are capable of reducing or reversing the effect of a dysregulation by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95%, 96%, 97%, 98, 99% or 100% when compared to an untreated control. In a preferred embodiment, the use of a pharmaceutical composition as defined herein leads to a reconstitution of the miRNA expression state of a healthy or normal subject.
The means for alleviating or reversing the effect of a dysregulation of at least one miRNA may comprise, in one embodiment, a nucleic acid molecule comprising a mature miRNA molecule as defined in section A of Table 1 or 1a or section H of Table 1 a. A "nucleic acid molecule" as used herein is a polynucleotide or oligonucleotide having, comprising or consisting of a nucleotide sequence. It may comprise at least two deoxy ribonucleotides or ribonucleotides in either single- or double-stranded form. The nucleic acid may be DNA, RNA, PNA, CNA, HNA, LNA or ANA molecule, preferably a DNA or RNA molecule. The DNA may be in the form of, e.g. A-DNA, B-DNA or Z-DNA. The RNA may be in the form of, e.g. p-RNA, i.e. pyranosysl-RNA or structurally modified forms like hairpin RNA or a stem-loop RNA.
The term "PNA" relates to a peptide nucleic acid, i.e. an artificially synthesized polymer similar to DNA or RNA which is used in biological research and medical treatments, but which is not known to occur naturally. The PNA backbone is typically composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are generally depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
The term "CAN" relates to an aminocyclohexylethane acid nucleic acid. Furthermore, the term relates to a cyclopentane nucleic acid, i.e. a nucleic acid molecule comprising for example 2'-deoxycarbaguanosine.
The term "HNA" relates to hexitol nucleic acids, i.e. DNA analogues which are built up from standard nucleobases and a phosphorylated 1 ,5-anhydrohexitol backbone.
The term "LNA" relates to locked nucleic acids. Typically, a locked nucleic acid is a modified and thus inaccessible RNA nucleotide. The ribose moiety of an LNA nucleotide may be modified with an extra bridge connecting the 2' and 4' carbons. Such a bridge locks the ribose in a 3'-endo structural conformation. The locked ribose conformation enhances base stacking and backbone pre-organization. This may significant!y increase the thermal stability, i.e. melting temperature of the oligonucleotide.
The term "ANA" relates to arabinoic nucleic acids or derivatives thereof. A preferred ANA derivative in the context of the present invention is a 2'-deoxy-2 - fluoro-beta-D-arabinonucleoside (2'F-ANA).
In certain embodiments nucleic acid molecules may comprise a combination of any one of DNA, RNA, PNA, CNA, HNA, LNA and ANA. Particularly preferred are mixtures of LNA nucleotides with DNA or RNA bases. In a further preferred embodiment the nucleic acid molecules as defined herein above may be in the form of short oligonucleotides, long oligonucleotides or polynucleotides.
The nucleic acid molecules as defined herein above may be single- stranded or double-stranded. The term "single-stranded nucleic acid" relates to nucleic acid molecules which comprise a single sugar-phosphate backbone and/or are not organized in a helical form. Preferably these nucleic acid molecules exhibit no secondary structures or intermolecular associations. The term "double stranded nucleic acid" relates to nucleic acid molecules which comprise two sugar-phosphate backbones. In a preferred embodiment the double-stranded nucleic acids are organized in a double helical form. In a specific embodiment double-stranded nucleic acids according to the present invention may be composed of different types of nucleic acid molecules, e.g. of DNA and RNA, DNA and PNA, DNA and CNA, DNA and HNA, DNA and LNA, DNA and ANA, or RNA and CNA, RNA and PNA, RNA and CNA, RNA and HNA, RNA and LNA, RNA and ANA, or PNA and CNA, PNA and HNA, PNA and LNA, PNA and ANA or CNA and HNA, CNA and LNA, CNA and ANA, or HNA and LNA, HNA and ANA, or LNA and ANA. They may alternatively also be composed of combinations of stretches of any of the above mentioned nucleotide variants. The term "nucleotide" refers to a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" may include purines and pyrimidines, which may further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, or synthetic derivatives of purines and pyrimidines, which may further include modifications which place new reactive groups, e.g. amines, alcohols, thiols, carboxylates, and alkylhalides.
The DNA may further be in the form of plasmid DNA, pre-condensed DNA, a PGR product, a vector (e.g. PI, PAC, BAC, YAC, artificial chromosomes), an expression cassettes, a chimeric sequence, a chromosomal DNA, or derivatives and combinations of these groups.
Nucleic acid molecules may, in specific embodiments, include or comprise known nucleotide analogs or modified backbone residues or linkages, which may be synthetic, naturally occurring, or non-naturally occurring, and which may have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, or 2'-0-methyl ribonucleotides. A nucleic acid molecule according to the invention may further encompass nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. A particular nucleic acid sequence also implicitly encompass conservatively modified variants thereof (e.g., codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
In specific embodiments, a nucleic acid molecule according to the invention may comprise, essentially consist of or consist of a miRNA molecule as defined herein, e.g. a mature miRNA molecule as defined in sections A or B of Table 1 or 1a, or in section H of Table 1 a, and/or may comprise, essentially consist of or consist of a portion a mature miRNA molecule as defined in sections A and B of Table 1 or 1 a, or in section H of Table 1a. In further embodiments, a nucleic acid molecule may comprise, essentially consist of or consist of a pre-miRNA molecule as defined herein, e.g. a per-miRNA molecule as defined in sections E or F of Table 1 or 1a, or in section H of Table 1 a, and/or may comprise, essentially consist of or consist of a portion a pre-miRNA molecule as defined in sections E or F of Table 1 or 1 a or in section H of Table 1a. In further specific embodiments, a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule, e.g. genomic DNA molecule, providing the sequence of, or encoding a miRNA as defined herein, e.g. a mature miRNA as defined in sections A or B or Table 1 or 1a, or in section H of Table 1 a. In a further preferred embodiment a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule, e.g. genomic DNA molecule, providing the sequence of or encoding a pre-miRNA molecule as defined in section E, or comprising, essentially consisting or consisting of the sequence of any of the SEQ ID NOs. indicated in section F of Table 1 or 1a, or in section H of Table 1a. In a further preferred embodiment a nucleic acid molecule may comprise, essentially consist of or consist of a DNA molecule providing the sequence of or encoding an antagonist according to the invention, e.g. an antisense molecule. The DNA molecule may accordingly be equipped with promoter or transcription elements allowing for the provision of an antisense nucleic acid molecule as defined herein. The DNA molecule may be single-stranded, or preferably double-stranded.
The nucleic acid molecule may further comprise, essentially consist of or consist of a portion or fragment of such a DNA molecule, e.g. genomic DNA. The nucleic acid molecule may further comprise, essentially consist of, or consist of an RNA molecule derived or transcribed from said DNA molecule, e.g. genomic DNA molecule, in an early, medium or late stage of processing into a miRNA molecule. Also envisaged are nucleic acid molecules constituting DNA sequences complementary to such RNA molecules, or antisense molecules thereof.
In a further specific embodiment, the nucleic acid molecule may comprise or be a stem-loop forming nucleic acid molecule. Such stem-loop forming nucleic acid molecule may serve as Dicer-substrates, leading to the provision of pre- miRNAs and finally to mature miRNA molecules. Stem-loop forming nucleic acids may be identified according to the information provided in sections A, B, E and F of Table 1 or 1 a, or in section H of Table 1 a. A stem-loop forming nucleic acid may have a length of between 35 to 3500 nucleotides, preferably of between 35 to 2000 nucleotides, more preferably of between 35 to 120 nucleotides. A stem-loop forming nucleic acid molecule may, in specific embodiments, include pre-miRNAs, or may have be larger or less processed than a pre-miRNA. In an alternative embodiment, synthetic or artificial stem-loop forming nucleic acid molecules may be designed which are also suitable for producing or obtaining a miRNAs of the invention as defined herein above.
The nucleic acid molecule may, in further specific embodiments, also comprise homologous or heterologous flanking regions, e.g. derived from other genetic contexts or other organisms, plasmids, genes, open reading frames etc. at the 5' or 3' terminus of the molecule as defined herein above.
The nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 or 1 a or in section H of Table 1 a may have a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1a or in section H of Table 1a, e.g. any one of SEQ ID NO: 1 to 149, or 383, 386, 389, 392 or 440, or any one of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116. Alternatively, a nucleic acid molecule comprising a miRNA molecule, e.g. a pre-miRNA molecule as defined in section E of Table 1 or 1a or in section H of Table 1a, may have a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section F of Table 1 or 1a or in section H of Table 1a, e.g. any one of SEQ ID NO: 150 to 321 , or 384, 387, 390, 393, 438 or 441 , or any one of SEQ ID NO. 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284.
By a nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence or any fragment as described herein.
Whether any particular nucleic acid molecule is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., 1990, Comp. App. Biosci. 6: 237-245.
In a nucleotide sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix-Unitary, k-tuple=4, Mismatch PenaSty-1 , Joining PenaSty-30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5'and 3'of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage may then be subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5'and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' end of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. However, only bases 5' and 3'of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
Further envisaged is the use of a derivative of the nucleic acid molecule comprising a miRNA molecule as defined herein above. The term "derivative" as used herein refers to a nucleic acid molecule that may differ from the nucleic acid molecule it refers to in that one or more nucleotides of the original molecule are substituted by other nucleotides and/or modified, e.g. chemically modified, by methods known to the skilled person. The derivative may, in particularly preferred embodiments, be a derivative of a DNA or RNA molecule as defined herein. Preferably, the derivative may still be capable of fulfilling its respective function. A "derivative" may comprise, for example, nucleotide substitutions, deletions or insertions, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitution, deletions or insertions. Furthermore, the derivate may comprise or contain modified bases or base moieties. For example, it may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-th iou racil , 3-(3-amino-3-N-2- carboxypropyl) uracil, and 2,6-diaminopurine. The molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. In another embodiment, the derivative molecule may comprise alternatively or additionally at least one modified phosphate backbone, e.g. a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an a Iky I phosphotriester, and a formacetal or analog thereof.
In a preferred embodiment, the nucleic acid molecule may comprise a chemically modified base. The term "chemically modified base" as used herein means that an oligonucleotide and/or nucleic acid molecule comprises a 2'-modified nucleotide containing ol igodeoxy n ucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications may increase the affinity of the oligonucleotide for its target molecule and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed to enhance the stability or efficacy of a nucleic acid molecule. Such nucleic acid molecules may further include nucleobase oligomers containing modified backbones or non-natural internucleoside linkages.
In further particularly preferred embodiment the nucleic acid molecule or antagonist may be modified by the introduction or conversion of a nucleotide or bond in said nucleic acid molecule or antagonist molecule into a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose or a 2'-methoxyethoxy ribose. Also envisaged are modifications comprising more than one of the mentioned introductions or conversion, or more than one different of the mentioned introductions or conversion. The modification may be provided by carrying out chemical reactions with a molecule of the invention, or by employing one of the modified entities during the synthesis or production of the nucleic acid molecule or antagonist.
Further envisaged is the use of a fragment of a nucleic acid molecule comprising a miRNA molecule as defined herein above. The term "fragment" as used in this context refers to a short polynucleotide having, comprising or consisting of a nucleic acid sequence, which is a portion of the sequence contained in the nucleotide sequence of any one of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440 as defined in section B of Table 1 or 1a or any one of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116 in section H of Table 1a, or any one of SEQ ID NOs: 150 to 321 or 384, 387, 390, 393, 438 or 441 of section F of Table 1 or 1a or any one of SEQ ID NO. 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a or the complementary strand thereto. A nucleotide fragments according to the invention may be at least about 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt of the nucleotide sequence of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or it may be about at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt or 75 nt of the nucleotide sequence of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or it may be about at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt or 75 nt of the nucleotide sequence of SEQ ID NOs: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or it may be about at least 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 ηί, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt or 75 nt of the nucleotide sequence of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284.
A fragment of "at least 15 nt in length" may, for example, include 15 or more contiguous bases from the sequence contained in the nucleotide sequence of SEQ ID NOs: 1 to 375 or 383 to 435 or the complementary strand thereto. Moreover, representative examples of polynucleotide fragments of the invention include, for example, fragments comprising, or alternatively consisting of a sequence from about nucleotide number 1-10, 5-15, 11-20 of SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or from about nucleotide number 1-10, 5-15, 1 1 -20, 16 to 25, 21-30, 26-35, 31-40, 36-45, 41 to 50, 46 to 55, 51 to 60, 56 to 65 etc. of SEQ ID NOs: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or from about nucleotide number 1-10, 5-15, 1 1 -20, 16 to 25, 21-30, 26-35, 31-40, 36-45, 41 to 50, 46 to 55, 51 to 60, 56 to 65 etc. of SEQ ID NOs: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 or SEQ ID NOs: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or the complementary strand thereto.
A particularly preferred fragment of a miRNA molcule is a seed sequence of the miRNA. The term "seed sequence" refers to a sequence of about 6-8 nucleotides in length at the 5' end of an animal miRNA which is an important determinant of target specificity. The seed sequence is typically situated from nucleotide position 2 to nucleotide position 7-9 in a miRNA molecule. 6 nt, 7 nt and 8 nt molecules comprising the seed sequence, which are preferred by the present invention, may have an influence on the binding of the miRNA to its target, e.g. mRNA. Further preferred are molecules comprising seed sequences, or sub-fragments thereof, e.g. fragments of 3, 4, or 5 nt lengths being located within the seed sequence. It is preferred that the seed sequence is perfectly complementary to the target sequences, e.g. a target mRNA. The sequence may, for example, be derived from the SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440 as mentioned in Table 1 or 1a, or from the SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16 as mentioned in Table 1a. Further details may be derived from suitable literature sources such as Lewis BP et al, 2005. Cell 120(1 ): 15-20. Additional information on seed regions may further be derived from suitable databases such as the Targetscan database (http://www.targetscan.org/ faqs.htm).
In a preferred embodiment, a nucleic acid molecule, e.g. a miRNA molecule, an RNA molecule, a DNA molecule, or an antagonist, e.g. antisense molecule, may have a length of at least about 10 to 50 nt. It is particularly preferred that the length is at least 18 to 24 nt. For example, the length may be 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt or 24 nt.
Further envisaged is the use of a variant of a nucleic acid molecule comprising a miRNA molecule as defined herein above. The term "variant" as used in the context of the present invention refers to a polynucleotide which differs from the polynucleotide of the present invention, e.g. the polynucleotide of SEQ ID NOs: 1 to 375, or 383 to 435, but retains essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide of the present invention. Variants according to the present invention may contain alterations in the stem region of a pre-miRNA molecule, in the loop region of a pre-miRNA, in the 3' terminus of a mature miRNA, in the 5' region of a mature miRNA, or any combination thereof. Further variants may be combination of two or more, e.g. 3, 4, 5, 6, 7, 8, 9, 10 or miRNA molecules on a nucleic acid molecule. Such combinations may, for example, be partially overlapping and accordingly comprise a common overlapping region and specific termini. In specific embodiment, a variant may comprise one or more seed sequences as defined herein above.
Further envisaged is the use of an antagonist of a nucleic acid molecule comprising a miRNA molecule as defined herein above. The term "antagonist of a nucleic acid molecule comprising a miRNA molecule" as used herein refers to any molecule or compound which is capable of reducing the amount or stability of nucleic acid molecule comprising a miRNA molecule of the present invention, or of reducing or abolishing the binding capability of a miRNA molecule or a nucleic acid molecule comprising a miRNA molecule. The term particularly refers to a molecule or compound which is capable of reducing the amount or stability or binding capability of a mature miRNA molecule as defined in section A of Table 1 or 1a or in section H of Table 1a, or the amount or stability or binding capability of a pre-miRNA molecule as defined in section E of Table 1 or 1a or in section H of Table 1a. The term "reducing the amount of a nucleic acid molecule comprising a miRNA molecule" means a diminishment of the amount of the nucleic acid molecule comprising a miRNA moleculet by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in comparison to a control situation in which no antagonist is used. The term "reducing the stability of a nucleic acid molecule comprising a miRNA molecule" means that the half-life of an nucleic acid molecule comprising a miRNA molecule is diminished, preferably by factor 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000 or 0000 in comparison to a control situation in which no antagonist is used. The term "reducing the binding capability of a nucleic acid molecule comprising a miRNA molecule" means a diminishment of the binding capability of the nucleic acid molecule comprising a miRNA molecule to a target by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in comparison to a control situation in which no antagonist is used.
In one embodiment of the invention said antagonist of a nucleic acid molecule comprising a miRNA molecule according to the present invention is an antisense molecule against the nucleic acid molecule comprising comprising a miRNA molecule, more preferably against a nucleic acid comprising the sequence of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390,
393, 438 or 441 of sections B or F of Table 1 or 1 a, respectively, or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or any one of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a. The term "antisense molecule" refers to the complementary strand of a nucleic acid corresponding to the sequences comprised in SEQ ID NOs: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326,
394, 399, 439, 442, 444 or 1 16, or any one of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1 a. In specific embodiments, the antisense molecule may also be a double-stranded molecule able to form a triple-helix structure. Preferably, the antisense molecule of the invention comprises a sequence complementary to at least a portion of a nucleic acid molecule comprising a miRNA molecule according to the present invention. In further embodiments, an antisense molecule may also be based on untranscribed or regulatory sequences surrounding the genomic locus of expression of a miRNA molecule as defined in sections A or E of Table 1 or 1a.
It is generally envisaged that antisense technology may be used to control, i.e. reduce or terminate miRNA gene expression through antisense DNA or RNA, or through triple-helix formation. In one embodiment, an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence. A vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule. Corresponding vectors can be constructed by recombinant DNA technology methods known to the person skilled in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.
In another embodiment, the antisense molecule may be separately administered. As an example, the 5' coding portion of a nucleic acid according to the present invention, e.g. of the sequence of SEQ ID NO: 1 to 149, or SEQ ID NO; 150 to 321 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or any one of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327,
398, 400, 266, 443, 445 or 284may be used to design an antisense RNA or DNA oligonucleotide, e.g. of a length of 5 to 25, 5 to 50, or 5 to 80 nt. Preferably, the antisense oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.
The antisense nucleic acids of the invention may comprise a sequence complementary to at least a portion the nucleic acid comprising a miRNA molecule according to the present invention. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion the nucleic acid comprising a miRNA molecule" as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the molecule, e.g. forming a stable duplex triplex formation in the case of double stranded antisense nucleic acids. The ability to hybridize may depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex or triplex. A person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
In a preferred embodiment, the antagonist, e.g. antisense molecule, may be a nucleic acid molecule comprising, essentially consisting of or consisting of a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1 a, e.g. SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or in section H of Table 1a, e.g.SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394,
399, 439, 442, 444 or 116, or with a complementary sequence thereof. Alternatively, the antagonist, e.g. antisense molecule, may be a nucleic acid molecule comprising, essentially consisting of or consisting of a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section F of Table 1 or 1a, e.g. SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or in section H of Table 1a, e.g. SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 or with a complementary sequence thereof.
In a further embodiment of the invention, the antagonist or antisense molecule may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (ca rboxy hyd roxy I methyl ) uracil, 5-carboxymethyl- aminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N6-adenine, 7-methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6- diaminopurine. The antisense molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In another embodiment, the antisense molecule may comprise alternatively or additionally at least one modified phosphate backbone, e.g. a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an a Iky I phosphotriester, and a formacetal or analog thereof for example, to improve stability of the molecule, hybridization, etc.
In a particularly preferred embodiment the antagonist or antisense molecule may be chemically modified as defined herein above, e.g. comprising 2'- modifiednucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance.
An antisense molecule according to the present invention may preferably be a DNA molecule, or an RNA molecule, or a chimeric molecule, or a mixtures of DNA and RNA, or a mixture of DNA, RNA and/or chimeric molecules, or of derivatives or modified versions thereof. Further envisaged antagonists include an aptamer specific for a miRNA molecule according to the present invention. An "aptamer specific for specific for a miRNA molecule" as used herein refers to a short nucleic acid molecule, e.g. an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule or any other suitable nucleic acid format known to the person skilled in the art, being capable of very specifically binding to the miRNA molecule as defined in section A of Table 1 or 1a, or in section H of Table 1a preferably to a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a.
An antagonists according to the invention may further be a catalytic RNA molecule or a riboyzme. The term "catalytic RNA" or "ribozyme" refers to a non-coding RNA molecule, which is capable of specifically binding to a target RNA and of cutting or degrading said target RNA, e.g. a molecule comprising the nucleotide sequence of any one of SEQ ID NO: 1 to 149, or SEQ ID NO: 150 to 321 , or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16, or SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284. Typically, ribozymes cleave RNA molecules at site specific recognition sequences and may be used to destroy RNAs. A preferred example of ribozymes are hammerhead ribozymes. Hammerhead ribozymes cleave RNAs at locations dictated by flanking regions that form complementary base pairs with the target RNA. The construction and production of hammerhead ribozymes is known in the art and is described in Haseloff and Gerlach, 1988, Nature, 334: 585-591. There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme may be engineered so that the cleavage recognition site is located near the 5' end of the RNA corresponding to the miRNAs of the invention.
Ribozymes or catalytic RNAs of the invention can be composed of modified oligonucleotides and may be delivered to cells, which express the polynucleotides of the invention in vivo. DNA constructs encoding a ribozyme or catalytic RNA according to the present invention may be introduced into the cell according to suitable methods known to the person skilled in the art. A preferred method of delivery involves using a DNA construct encoding the ribozyme under the control of a strong constitutive promoter so that transfected cells will produce sufficient quantities of the ribozyme to destroy targeted messages and inhibit translation.
An antagonists according to the invention may, in a particularly preferred embodiment, be an antagomir of a miRNA molecule as defined in sections A or E of Table 1 or 1a, or of section H of Table 1a. The term "antagomir" as used herein relates to a chemically engineered oligonucleotide, which is specifically used to silence a microRNA. An antagomir typically is a small synthetic RNA that is highly, e.g. 95%, 96%, 97%, 98%, 99% complementary, or preferably perfectly complementary to a specific miRNA target, e.g. to a nucleic acid molecule comprising the sequence of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440, or SEQ ID NO: 150 to 321 or 384, 387, 390, 393, 438 or 441 , or of SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a. The function of an antagomir typically involves a mispairing at the cleavage site of the Argonaut-2 activity (Ago-2) or a base modification to inhibit Argonaut-2 cleavage. It is preferred that an antagomir according to the invention has a modification which renders it more resistant to degradation. Particularly preferred are the presence of 2' methoxi groups and phosphothioates. Antagomirs may preferably be used to constitutively inhibit the activity of a miRNA according to the invention, e.g. a miRNA as defined in section A or C or Table 1 or 1a, or in section H of Table 1a.
In a further embodiment, the nucleic acid molecule of the invention as defined herein above, or an antangonistic sequence as defined herein above, may be comprised in a vector. The term "vector" as used herein refers to a nucleic acid molecule, for example, a plasmid, cosmid, or bacteriophage, that is capable of replication in a host cell. Preferably, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression may be under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers. Transducing viral (e.g., retroviral, adenoviral, lentiviral and adeno- associated viral) vectors may, for example, be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression. It is particularly preferred that the vector allows for the production of RNA molecules, e.g. single stranded RNA molecules or double stranded RNA molecules, e.g. stem-loop structures of miRNA molecules. The vector may, for example, be a eukaryotic expression vector such as e.g. a plasmid, a minichromosome, a cosmid, a bacterial phage, a viral, a retroviral vector or any other vector known to the skilled person, or a prokaryotic expression vector, or a shuttle vector allowing pro- and eukaryotic expression. Vectors may preferably be used for replication and/or expression in vertebrate cells.Further details on vectors and suitable methods for the provision and manipulation of vectors whould be known to the skilled person, or can be derived from suitable literature sources such as (Sambrook and Russell 2001 , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press 3rd ed; Pfeifer and Hofmann, 2009 , Methods Mol Biol 530: 391-405).
In preferred embodiments a vector or a portion thereof may be transcribed, producing a nucleic acid comprising a miRNA molecule, a miRNA molecule, a pre-miRNA molecule, or an antisense nucleic acid of the invention. The vector may accordingly comprise a sequence as defined in any one of SEQ ID NO: 1 to 149, or SEQ ID NO: 150 to 321 , or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116, or SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284 of section H of Table 1a. The vector may remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired nucleic acid.
Polynucleotides according to the present invention may be joined, for example, to a vector, which contains a selectable marker for propagation in a host. Furthermore, the polynucleotide insert may be operably linked to an appropriate promoter for the host cell in which the vector is to be used, e.g. phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs. Particularly preferred is the use of the CMV, CamKII, U6 and/or H1 promoter, or of derivatives or variants thereof. Other suitable promoters are known to the person skilled in the art. The expression constructs may further contain sites for transcription initiation and termination. The efficiency of expression may further be enhanced by the inclusion of appropriate transcription enhancer elements etc. Selectable marker may be, for instance, dihydrofolate reductase, G418, hygromycin or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for cu!turing in E. coli and other bacteria. Further selection markers include the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds, 2007, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York. Suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. An example of a preferred expression vector is pl_enti6/V5-DEST (available e.g. from Invitrogen). A further preferred example of a suitable expression vector which allows for the production of dsRNA directly in the target cell is vector pSUPER (available e.g. from OligoEngine). Further information on the vector and the mechanism how the dsRNA can be produced by using said vector may be derived from Brummelkamp et al., 2002, Science, Vol. 296, 550-553. Another preferred vector which may be used in the context of the preset invention is vector pSilencer (available, e.g. from Ambion). Further details on the vector may be derived from Sui et al., 2002, Proc. Natl. Acad. Sci. Vol. 99, 5515-5520.
In a specific embodiment, the vector is a vector for transducing a mammalian cell, e.g. a human cell, which provides the nucleotide sequence of a miRNA molecule as defined in section A of Table 1 or 1 a, or in section H of Table 1 a. The vector preferably further comprises one or more regulatory sequences operably linked thereto to allow transcription of said sequence in said cell. Optionally, a termination sequence may be present.
In a further preferred embodiment, the vector may comprise a promoter being functional in a mammalian cell, e.g. in a human cell, operably linked thereto a first nucleic acid molecule which comprises a sequence as defined in SEQ ID NO: 1 to 149, or in SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 1 16 of section H of Table 1a. Additionally, the vector may comprise a second nucleic acid, which comprises a sequence, which is reverse complementary to the first sequence, i.e. to a sequence defined in SEQ ID NO: 1 to 149 or in SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116 of section H of Table 1a. Optionally, a termination sequence may be present.
In a further preferred embodiment of the present invention the nucleic acid molecule or the antagonist as defined herein may include other appended groups such as peptides, or agents facilitating transport across the cell membrane or the blood-brain barrier, hybridization triggered cleavage agents or intercalating agents. Also envisaged is a conjugation of the nucleic acid molecule or the antagonist to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting molecule. A preferred moiety is a cholesterol moiety or a lipid moiety or a bile acid. A further moiety for conjugation may be a carbohydrate, a phospholipid, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or a dye. In certain embodiments, a conjugate group may be attached directly to a nucleic acid molecule, antagonist or derivative thereof as defined herein. In preferred embodiments, a conjugate group may be attached to a nucleic acid molecule antagonist or derivative thereof as defined herein via a suitable linker moiety. Envisaged linker moieties may, for example, be selected from amino hydroxy I, carboxylic acid, thiol, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 -carboxylate (SMCC), 6-aminohexanoic acid (ALEX or AHA), substituted C1 -C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In further embodiments, a linker may be selected from a hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl or alkynyl group. Also envisaged is a conjugation to a polymer, an antibody or an RNA molecule.
In further embodiments, a nucleic acid molecule, antagonist or derivative thereof as defined herein may be conjugated to one or more stabilizing groups that are attached to one or both termini of the molecule to enhance properties such as, for example, nuclease stability. Preferred examples or such stabilizing groups are cap structures which typically protect a modified nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3 -cap) of the molecule, or can be present on both termini. Cap structures may include, for example, inverted deoxy abasic caps. Suitable, envisaged cap structures may be selected from a 4',5'- methylene nucleotide, a 1 -(beta-D-erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1 ,5-anhydrohexitol nucleotide, an L-nucleotide, an alpha- nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo- pentofuranosyl nucleotide, an acyclic 3' ,4'-seco nucleotide, an acyclic 3,4- dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety, a 3'-2'-in verted nucleotide moiety, a 3'-2'inverted abasic moiety, a 1 ,4-butanediol phosphate, a 3 -phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3'-phosphate, a 3 -phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, a non-bridging methylphosphonate moiety 5'-amino-alkyl phosphate, a 1 ,3-diamino-2-propyl phosphate, a 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1 ,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a S'-phosphoramidate, a 5'-phosphorothioate, and a 5 -mercapto moiety.
In one embodiment, a pharmaceutical composition of the invention comprises a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR- 298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b- 3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa- miR-221-3p, miR-767-5p, hsa-miR-148a-3p or miR-125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in section B of Table 1. In a particularly preferred embodiment, a pharmaceutical composition of the invention comprises a nucleic acid molecule which comprises one or more mature miRNAs selected from mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR- 140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa- mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191-5p molecule as defined in sections A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in sections B or H of Table 1a. Since mmu-miR- 24-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p, and miR- 125a-5p could be identified to be down-regulated in epilepsy models as described in the Examples, a nucleic acid molecule comprising such a miRNA is assumed to counter-act the down-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation. Further, since mmu-miR-124-5p, mmu-miR-181b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR- 130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181b-5p, hsa- miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191- 5p could be shown to be down-regulated in epilepsy models as described in the Examples, or are assumed to be down regulated, a nucleic acid molecule comprising such a miRNA is assumed to counter-act the down-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation. A down- regulation of mmu-miR-34b-5p was shown for specific phases, e.g. based on a mouse acute model as described in the Examples. Similarly, a down-regulation of mmu-miR- 191-5p was shown only for specific phases, e.g. based on a Pilocarpine/SSEE mouse model as described in the Examples. In very specific embodiments a nucleic acid molecule comprising mmu-miR-34b-5p, hsa-miR-34b-5p, mmu-miR-191-5p or hsa- miR-191 -5p may be assumed to counter-act the down-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation in accordance with these specific phases or similar phases. This may advantageously be reflected by certain time windows for administration or a phase dependent administration or activation scheme. The pharmaceutical composition may further comprise any derivative, variant, fragment, conjugate, linked conjugate, vector, vector portion, pre-miRNA, genomic encoding DNA molecules or sequences, stem-loop forming molecules or sequences etc. as defined herein above of one or more of mmu- miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140- 3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir- 181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p, or optionally of mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR- 130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR- 184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR- 655, miR-219-5p, hsa-miR-221 -3p, miR-767-5p, hsa-miR-148a-3p, or miR-125a-5p. It is particularly preferred that the pharmaceutical composition comprises a vector or entity which allows the expression or provision of a mmu-miR-124-5p, mmu-miR-181 b- 5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu- miR-191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa- miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191 -5p; or optionally of a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR- 92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a- 3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR- 767-5p, hsa-miR-148a-3p, or miR-125a-5p mature miRNA molecule in situ, e.g. in a cell.
In a further embodiment a pharmaceutical composition of the invention comprises an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219- 5p, mmu-miR-135b-5p, mmu-miR-129-1 -3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-2 a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR- 222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR-142-3p, or hsa- miR-124-3p molecule as defined in section A of Table 1 , or of a derivative, fragment or variant thereof. In a particularly preferred embodiment, a pharmaceutical composition of the invention comprises an antagonist of a mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR- 184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa- miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 molecule as defined in section A or H of Table 1a, or of a derivative, fragment or variant thereof. Since mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR- 223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu- miR-135b-5p, mmu-miR-129-1 -3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR- 138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-mi'R-92b-3p, hsa-miR-129-5p, hsa-miR-142-3p, and hsa-miR- 124-3p could be identified to be up-regulated in epilepsy models as described in the Examples, an antagonist against such a miRNA is assumed to counter-act the up- regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation. Further, since mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR-494-3p, hsa-miR-129-5p, hsa-miR- 142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, and hsa-miR-494-3p could be identified to be up-regulated in epilepsy models as described in the Examples, or are assumed to be up-regulated, an antagonist against such a miRNA is assumed to counter-act the up- regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation. An up-regulation of mmu-miR-34b-5p was shown for specific phases, e.g. based on a 6 Hz seizure model (at the 3 hours timepoint) as described in the Examples. Similarly, an up-regulation of mmu-miR-191 -5p was shown only for specific phases, e.g. based on a 6 Hz mouse model as described in the Examples. In very specific embodiments an antagonist against mmu-miR-34b-5p, hsa- miR-34b-5p, mmu-miR-191 -5p or hsa-miR-191 -5p may be assumed to counter-act the up-regulation, thus reconstituting a normal or healthy miRNA expression pattern or underlying molecular situation in accordance with these specific phases or similar phases. This may advantageously be reflected by certain time windows for administration or a phase dependent administration or activation scheme. The pharmaceutical composition may, for example, comprise an antisense nucleic acid molecule of, or an aptamer, an antagomir, or a ribozyme against any one of mature mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR- 494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3; or optionally of any of mature mmu-miR-2137, mmu- miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR- 129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR- 129-5p, hsa-miR-142-3p, or hsa-miR-124-3p. The pharmaceutical composition may further comprise any derivative, variant, fragment, conjugate, linked conjugate, vector, vector portion, encoding DNA molecule or sequence etc. of one or more of said miRNAs as defined herein above.
In addition to the ingredients mentioned above, a pharmaceutical composition may also, e.g. optionally, comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such a carrier is pharmaceutically acceptable, i.e. is generally non-toxic to a recipient at the dosage and concentration employed. It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as provided by a sucrose solution. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Suitable pharmaceutical excipients include starch, glucose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ion, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of, e.g., solutions, suspensions, emulsion, powders, sustained- release formulations and the like.
Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Some other examples of substances which can serve as pharmaceutical carriers are sugars, such as glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; cranberry extracts and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations such as Vitamin C, estrogen and echinacea, for example. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers and anti-oxidants can also be present. It is also envisaged by the present invention to administer the active ingredients of the pharmaceutical composition in encapsulated form, e.g. as cellulose encapsulation, in gelatine, with polyamides, niosomes, wax matrices, with cyclodextrins or liposomally encapsulated.
Generally, the ingredients may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
In a specific embodiment, the pharmaceutical composition may be formulated in accordance with routine procedures adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Preferred is a direct injection into the brain. The composition may accordingly be formulated for a brain injection, e.g. comprising suitable buffers, diluents, stabilizers etc.; further details would be known to the skilled person. Also envisaged is an administration via the nose, e.g. intranasal. The composition may accordingly be formulated as inhalation composition e.g. based on an aerosolizing agent and/or provided via an inhaler, nebulizer, or dispenser; further details would be known to the skilled person. Another envisaged administration is administration via the eye. The composition may accordingly be formulated as eye drops or droplet formulation; further details would be known to the skilled person. Also envisaged by the present invention is the formulation of a composition in the form of nanoparticles. For example, a nanoparticle may be lipid- based and comprise or be bound with a miRNA molecule or an antagonist thereof. The nanoparticle may subsequently be administered to the brain, e.g. via direct injection or on any suitable alternative route. Further envisaged is the use of peptides which may be bound to a miRNA molecule or an antagonist and which may be provided to the brain, e.g. directly, or indirectly via any suitable administration route. For example, a short peptide derived from rabies virus glycoprotein (RVG), preferably RVG-9R, may be used, e.g. as described in Kumar et al., 2007, Nature, 448(7149):39-43.
In further specific embodiments of the present invention, a miRNA molecule or antagonist as defined herein may be provided on or linked to a carrier. Suitable carriers are, for example, cationic cell penetrating peptides (CPP), cation ic polymers, dentrimers, cationic lipids. Also envisaged is a combination with an aptameric structure. Lipid or nanoparticle structures as mentioned herein may comprise more than one carrier element as define herein. Further details may be derived from suitable literature sources, e.g. from Wang et al., 2010, The AAPS Journal, 12(4), 492- 503.
Also preferably envisaged is the employment of stable nucleic acid lipid particles (SNALPs), LDL particles, and HDL particles linked to miRNAs or antagonists as defined herein. Further details may be derived from suitable literature sources, e.g. from Davidson and McCray, 2011 , Nature Reviews Genetics, 12, 329-340.
In further specific embodiments, the use of 'brain homing' peptides or antibodies linked to liposomes or nanoparticles comprising miRNAs or antagonists is envisaged. Further details may be derived from suitable literature sources, e.g. from Boudreau et al., Human Molecular Genetics, 201 1 , 20, (1 ): R21-R27. The present invention further comprises and may use any new development in this area, in particular, new stabilizing components which may be linked or combined with miRNAs or antagonists as defined herein, or new carriers or carrier-like elements, in particular carriers allowing to cross the blood-brain barrier and/or to enter the brain.
The pharmaceutical composition may also be formulated as neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. The pharmaceutical composition of the present invention can also comprise a preservative. Preservatives according to certain compositions of the invention include buty!paraben, ethylparaben, imidazolidinyl urea, methylparaben, O- phenylphenol, propylparaben, quaternium-14, quaternium-15, sodium dehydroacetate, zinc pyrithione, thimerosal and the like. The preservatives may be used, for example, in amounts effective to prevent or retard microbial growth. Generally, the preservatives are used in amounts of about 0.1 % to about 1 % by weight of the total composition with about 0.1 % to about 0.8% being preferred and about 0.1 % to about 0.5% being most preferred. It is particularly preferred including a protective agent for RNA molecules, e.g. an RNAse inhibitor.
In a further embodiment of the present invention the pharmaceutical composition may comprise or be mixed with at least one suitable adjuvant. Adjuvants may be used to enhance the effectiveness of the pharmaceutical composition. Such adjuvants include, for example, chloroquine, protic polar compounds, such as propylene glycol, polyethylene glycol, glycerol, EtOH, 1 -methyl L-2-pyrrolidone or their derivatives, or aprotic polar compounds such as dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane, dimethylformamide, dimethylacetamide, tetra-methylurea, acetonitrile or their derivatives.
A pharmaceutical composition according to the present invention may be administered to a patient, subject or individual with the help of any suitable delivery system known to the person skilled in the art, e.g., via encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction may be any suitable method known, including topical, enteral or parenteral introduction. The methods of introduction may also include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalational, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents.
Administration can be systemic or local. It is particularly preferred to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may, for example, be facilitated by an intraventricular catheter, preferably attached to a reservoir, such as an Ommaya reservoir. Also envisaged are further suitable forms of direct brain injection. Pulmonary or inhalational administration can be employed, e.g., via the use of an inhaler or nebulizer, and a concomitant formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, local infusion, e.g. during surgery, topical application, e. g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
A preferred method of local administration is by direct injection. Preferably, any ingredient of the pharmaceutical composition of the present invention as defined herein above may be complexed with a delivery vehicle to be administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries. Another method of local administration is to contact a pharmaceutical composition of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the pharmaceutical composition can be coated on the surface of tissue inside the wound or the pharmaceutical composition can be injected into areas of tissue inside the wound.
For systemic administration, ingredients of the pharmaceutical composition of the present invention as defined herein above can be complexed to a targeted delivery vehicle. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. Envisaged methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using standard methods in the art (see, for example, Stribling et al., 1992, PNAS, 189: 1 1277-11281 ).
Oral delivery can be performed by complexing ingredients of the pharmaceutical composition of the present invention as defined herein above to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed, for instance, by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
In another embodiment of the present invention the pharmaceutical composition may be delivered directly to internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the site of interest.
The pharmaceutical composition may also be administered to disease sites at the time of surgical intervention.
In another embodiment, the pharmaceutical composition may be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249, 1527- 1533; Treat et al., 1989 in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327). In yet another embodiment, the composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be use. In yet another embodiment, a controlled release system can be placed in proximity of a therapeutic target, e.g. the brain, lymphatic organs etc. thus requiring only a fraction of the systemic dose.
The composition of the present invention can be administered to a mammal. The term "mammal" as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, the term "mammal" encompasses human beings. Also encompassed are mammals such as dogs, cats, mice, rats, monkeys, rabbits, goats, sheep, pigs, guinea pigs, cattle, horses etc.
The term "administered" as used herein means administration of a therapeutically effective dose of the pharmaceutical composition. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered, preferably this effect is a reduction or prevention of epileptic seizures. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
The pharmaceutical composition may be used in both human therapy and veterinary therapy, preferably in human therapy. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. The concentration of the therapeutically active compound in the formulation may vary from about 0.00001-100 wt %. The concentration of the active ingredients or compounds of a pharmaceutical composition according to the present invention may be further adjusted to the intended dosage regimen, the intended usage duration, the exact amount and ratio of all ingredients of the composition and further factors and parameter known to the person skilled in the art.
Assays, e.g. those based on experimental tests described herein or derivable from known and qualified textbooks of the prior art, may optionally be employed to help identify optimal ratios and/or dosage ranges for ingredients of pharmaceutical compositions of the present invention. The precise dose and the ratio between the ingredients of the pharmaceutical composition as defined herein above to be employed in the formulation will also depend on the route of administration, and the exact type of disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses or ingredient ratios may be extrapolated from dose-response curves derived from in vitro or (animal) model test systems.
Typically, the attending physician and clinical factors may determine the dosage regimen. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. For example, a pharmaceutical composition may preferably be given once a week, more preferably 2 times, 3 times, 4 times, 5 times or 6 times a week and most preferably daily and or 2 times a day or more often, unless otherwise indicated. During progression of the treatment the dosages may be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., several times a day.
In another aspect the present invention relates to a vaccine for use in the treatment of epilepsy, comprising a nucleic acid molecule as defined herein above, or an antagonist as defined herein above. In a particular embodiment, the vaccine may comprise a nucleic acid molecule comprising or encoding a miRNA molecule as identified in section A of Table 1 or 1a, or in section H of Table 1 a, or may comprise a nucleic acid molecule comprising or encoding a pre-miRNA molecule as identified in section E of Table 1 or 1 a, or in section H of Table 1 a. In one embodiment, the vaccine may comprise a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR- 298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b- 3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa- miR-221-3p, miR-767-5p, hsa-miR-148a-3p, or miR-125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in section B of Table 1. In a preferred embodiment, the vaccine may comprise a nucleic acid molecule which comprises one or more mature miRNAs selected from a mmu-miR-124-5p, mmu-miR-181 b-5p, mmu- miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191- 5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676- 3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa- miR-191 -5p molecule as defined in section A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in section B or H of Table 1 a. Further preferred is the use of a vector or DNA construct encoding one or more of said miRNAs, or leading to the expression of one or more of said miRNAs.
Ina further embodiment, the vaccine may comprise a nucleic acid molecule giving yield to an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR- 142-3p, or hsa-miR-124-3p molecule as defined in section A of Table 1 , or of a derivative, fragment or variant thereof. Further preferred is the use of a vector or DNA construct encoding one or more of antisense molecules of said miRNAs, or leading to the expression of one or more of said antisense molecules. In a particularly preferred embodiment, the vaccine may comprise a nucleic acid molecule giving yield to an antagonist of a mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191- 5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu- miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 molecule as defined in section A or H of Table 1a, or of a derivative, fragment or variant thereof. Further preferred is the use of a vector or DNA construct encoding one or more of antisense molecules of said miRNAs, or leading to the expression of one or more of said antisense molecules. Thus, a vaccine according to the present invention may, for example, comprise expression vectors capable of providing miRNA molecules, or antisense molecule, e.g. DNA plasmid vectors, viral vectors etc. as described herein above. Also envisaged is the presence of host cells expressing such miRNAs or antisense molecules. The components of the vaccine may be delivered using one or more vehicles. Additionally, the components may be organized in the form of a kit, e.g. comprising additional kit ingredients, e.g. a leaflet etc.
Nucleic acid molecules in a vaccine as defined herein above may be provided in the vaccine in the form of vectors, e.g. DNA plasmids or viral vectors. Vectors, in particular viral vectors may be capable of replication or replication-impaired or non-replicating. The term "non-replicating" or "replication-impaired" as used herein means that the vector is not capable of replication to any significant extent in the majority of normal mammalian cells or normal human cells. Viruses which are non- replicating or replication-impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. Suitable viral vectors for use in a vaccine according to the present invention include non- replicating adenoviruses such as E1 deletion mutants, vectors based on herpes virus and Venezuelan equine encephalitis virus (VEE). Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA (see Darji et a!., 1997, Cell 91 : 765-775). Alternative suitable non-viral vectors include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage. In further embodiments, a vaccinia virus vector such as MVA or NYVAC may be used. Most preferred is the vaccinia strain modified virus ankara (MVA) or a strain derived therefrom. MVA is a replication impaired vaccinia strain with a good safety record. In most cell types and normal human tissues, MVA does not replicate. Alternatives to vaccinia vectors include pox virus vectors, e.g. avipox vectors such as fowl pox or canarypox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC, and strains derived therefrom.
The vaccine as described herein above, or a nucleic acid molecule allowiong the expression of a miRNA molecule or of an antagonist as described herein above may be provided in the form of a genetherapy vehicle. It is accordingly envisaged introducing the genetherapy vehicle permanently or transiently into a cell of the human body, allowing it to express and generate miRNA molecules or miRNA antagonists as defined herein.
In a further aspect the present invention relates to a biomarker or group of biomarkers associated with epilepsy, wherein said biomarker or group of biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 or 1a, or in section H of Table 1a or a derivative, fragment or variant thereof. The term "biomarker associated with epilepsy" as used herein means that the expression level of the biomarker or group of biomarkers is modified in a subject afflicted by epilepsy, e.g. increased or decreased, when comparing to the expression of said biomarker or group of biomarkers in a control level, e.g. a healthy subject, in particular in a subject which has been diagnosed or confirmed not to be affected by epilepsy.
The term "control level" as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, i.e. epilepsy is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the miRNAs of the biomarker or group of biomarkers according to Table 1 or 1a, or in section H of Table 1a in samples from subjects whose disease state, i.e. epilepsy, is known. Furthermore, the control level can be derived from a database of expression levels or patterns from previously tested subjects or cells. Moreover, the control level may be multiple control levels determined from multiple reference samples. The control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art. In a specific embodiment of the present invention, a different type of control level can be determined from a reference sample derived from a healthy subject, e.g. a subject who has been diagnosed or confirmed not to suffer from epilepsy.
The term "sample" as used herein refers as used herein relates to any biological material obtained via suitable methods known to the person skilled in the art from an individual. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.
In a specific embodiment the sample material is a bodily fluid. The term "bodily fluid" as used herein refers to whole blood, serum, plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, liquid stool, fluid harvested from a site of an immune response, fluid harvested from a pooled collection site, bronchial lavage, and urine.
In further embodiments also material such as biopsy material, e.g. from all suitable organs, e.g. the lung, the muscle, brain, liver, skin, pancreas, stomach, etc., a nucleated cell sample, a fluid associated with a mucosal surface, hair, or skin may be used. For such a testing, the material is typically homogenized and/or resuspended in a suitable buffer solution.
Furthermore, a sample may contain a cell extract derived from or a cell population including a brain cell. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. Samples, in particular after initial processing, may be pooled. However, also non-pooled samples may be used.
In further embodiments bodily fluid or sample material as mentioned herein above may be processed by adding chemical or biological reactants. This may be performed in order to stabilize the sample material, to remove sample components, or to avoid interaction in samples. For example, EDTA or heparin may be used to stabilize blood samples.
In a specific embodiment of the present invention the content of a sample may be submitted to an enrichment step. For instance, a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types functionalized for example with magnetic particles. The material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.
It is particularly preferred using blood, i.e. whole blood, serum or cerebrospinal liquid samples.
In the context of the present invention, a control level determined from a biological sample that is known not to associated with epilepsy is called "normal control level". In another embodiment of the present invention, the control level can be from biological sample associated with epilepsy, e.g. a sample from a subject for which epilepsy was diagnosed independently, it may be designated as "epilepsy control level". Alternatively, reference samples may comprise material derived from epilepsy models, e.g. as described in the Examples of the present invention. Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art. By comparing a control level to a measured expression level a modification of the expression may be registered. For such comparison processes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed.
The term "isolated biomarker" or "biomarker", as used herein, particularly relates to an isolated nucleic acid molecule comprising a miRNA as defined in section A of Table 1 or 1 a, or in section H of Table 1a, a precursor version of said miRNA, e.g. a pre-miRNA as defined in section E of Table 1 or 1 a, or in section H of Table 1 a, an encoding genetic unit, or a genomic sequence associated with said miRNA. The term further refers to nucleic acid molecules comprising a nucleotide sequence at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section B of Table 1 or 1 a or in section H of Table 1 a, e.g. any one of SEQ ID NO: 1 to 149 or 383, 386, 389, 392 or 440 or SEQ ID NO: 104, 105, 436, 385, 135, 388, 391 , 326, 394, 399, 439, 442, 444 or 116; or a nucleic acid molecule comprising a miRNA molecule, e.g. a pre- miRNA molecule as defined in section E of Table 1 or 1a, or in section H of Table 1a, comprising a nucleotide sequence which is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical with a reference sequence as indicated in section F of Table 1 or 1 a, or in section H of Table 1a, e.g. any one of SEQ ID NO: 150 to 321 , or 384, 387, 390, 393, 438 or 441 or any one of SEQ ID NO: 264, 265, 266, 437, 395, 435, 304, 396, 397, 327, 398, 400, 266, 443, 445 or 284. The term "isolated" refers to a molecule which has been removed and/or prepared from a sample or tissue as defined herein. Methods for isolation are known to the skilled personl. These methods may include steps of amplification, hybridization, or cloning of the nucleic acids.
The biomarker may further comprise a derivative, fragment or variant of a miRNA molecule as defined above. Such derivatives, fragments or variants correspond to the derivatives, fragments or variants as defined in the context of the pharmaceutical compositions as provided herein above.
The term "expression level" as used herein refers to the amount of any miRNA molecule, or derivative or precursor etc. thereof derivable from a defined number of cells or a defined tissue portion, preferably to the amount of miRNA molecule, or derivative or precursor etc. obtainable in a standard nucleic acid (in particular RNA) extraction procedure. Suitable extraction methods are known to the person skilled in the art. The amount may also be determined indirectly via suitable binding assays etc.
The term "modified" or "modified expression level" in the context of the present invention thus denotes a change in the expression level. Expression levels are deemed to be "changed" when the miRNA expression the biomarker according to Table 1 or 1a, e.g. in a sample to be analyzed, differs by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, e.g. a sample derived from a healthy or normal subject, or when it differs at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level, e.g. a sample derived from a healthy or normal subject.
The term "modified" as used throughout the specification relates preferably to a decrease or down-regulation or an increase or up-regulation of the expression level of the biomarker or group of biomarkers according to Table 1 or 1a, or a complete inhibition of the expression of the biomarker or group of biomarkers according to Table 1 or 1a if a test sample is compared to a control level.
The term "increased" or "increased expression level" or "up-regulated expression level" or "increase of expression level" (which may be used synonymously) in the context of the present invention thus denotes a raise in the expression level of the biomarker or group of biomarkers according to Table 1 or 1a between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from a healthy state. This may further be compared to the expression in any suitable epilepsy state known to the person skilled in the art. Expression levels are deemed to be "increased" when the expression of the biomarker or group of biomarkers according to Table 1 or 1a, e.g. in a sample to be analyzed, differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level.
The term "reduced" or "reduced expression level" or "down-regulated expression level" or "decrease of expression level" (which may be used synonymously) in the context of the present invention thus denotes a reduction of the expression level of the biomarker or group of biomarkers according to Table 1 or 1 a between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could, for example be a control level, e.g. a control level derivable from a healthy state. This may further be compared to the expression in any suitable epilepsy state known to the person skilled in the art. Expression levels are deemed to be "reduced" or "down-regulated" when the expression of the biomarker or group of biomarkers according to Table 1 or 1a decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level.
In a specific embodiment of the present invention the biomarker or group of biomarkers comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more or all biomarkers indicated with a "+" in section C of Table 1 or 1 a. In a further specific embodiment of the present invention the biomarker or group of biomarkers comprises the first 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers indicated with a "+" in section C of Table 1 or 1a.
In a further specific embodiment of the present invention the biomarker or group of biomarkers comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more or all biomarkers indicated with a "+" in section D of Table 1 or 1 a. In a further specific embodiment of the present invention the biomarkers or group of biomarkers comprises the first 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers indicated with a "+" in section D of Table 1 or 1 a.
In a further embodiment of the present invention the group of biomarkers may comprise markers which are up-regulated or markers which are down-regulated, or markers which are up-regulated and which are down-regulated, preferably at least one marker with an increased expression level and one marker with a decreased expression level. For example, the group of biomarker may comprise at least one biomarker indicated with a + in section C of Table 1 or 1a and at least one biomarker indicated with a + in section D of Table 1 or 1a.
In one embodiment, the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-2137, mmu- miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR- 129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-mi'R-92b-3p, hsa-miR- 129-5p, hsa-miR-142-3p, and hsa-miR-124-3p as defined in section A of Table 1. In a preferred embodiment, the bio marker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-129-5p, mmu-miR-142-3p, mmu- miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184- 3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR- 34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 as defined in section A or H of Table 1a. In a further embodiment the increase of expression (up- regulation) of one biomarker or a group of biomarkers selected from mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR- 148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR- 142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa- miR-129-5p, hsa-miR-142-3p, hsa-miR-124-3p, when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy. In a particularly preferred embodiment the increase of expression (up- regulation) of one biomarker or a group of biomarkers selected from the group of mmu- miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494- 3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p, mmu-miR-222-3p, hsa- miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3 as defined in section A or H of Table 1 a when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy
In a further embodiment, the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-124-3p, mmu- miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu- miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa- miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a- 3p, and miR-125a-5p, as defined in section A of Table 1. In a preferred embodiment, the biomarker or group of biomarkers is or comprises at least one biomarker selected from the group of mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191-5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p as defined in section A or H of Table 1 a. In a further embodiment the decrease of expression (down-regulation) of at least one biomarker or a group of biomarkers selected from the group of mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR- 148a-3p, and miR-125a-5p, when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy. In a particularly preferred embodiment the decrease of expression (down-regulation) of at least one biomarker or a group of biomarkers selected from the group of mmu-miR- 124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b- 5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
In a further specific embodiment, the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1 -3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR- 142-3p, and hsa-miR-124-3p. In yet another specific embodiment, the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR- 30a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR- 25a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR- 148a-3p, and miR-125a-5p. In a preferred embodiment, the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-129-5p, mmu-miR- 142-3p, mmu-miR-34b-5p, mmu-miR-191 -5p, mmu-miR-494-3p, hsa-miR-129-5p, hsa- miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, and hsa-miR-494-3p. In another preferred embodiment, the group of biomarkers comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 biomarkers from mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR- 676-3p, mmu-miR-140-3p, mmu-miR-130a-3p, mmu-miR-191 -5p, hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181 b-5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, and hsa- miR-191-5p.
In another aspect the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy, or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or group of biomarkers as defined as defined in Table 1 or 1a, a peptide affinity ligand for a biomarker or group of biomarkers as defined in Table 1 or 1a, an oligonucleotide specific for the biomarker or group of biomarkers as defined in Table 1 or 1a and/or a probe specific for the biomarker or group of biomarkers as defined in Table 1 or 1a. In a specific embodiment, the composition may further comprise probes or oligonucleotides for the detection of a reference analyte, e.g. a gene and/or miRNA known to be associated with epilepsy.
The term "nucleic acid affinity ligand for the expression product of a biomarker or group of biomarkers" as used herein refers to a nucleic acid molecule being able to specifically bind to a miRNA molecule, or derivative, fragment or variant thereof of said biomarker or a group of biomarkers as defined above, preferably to the a miRNA molecule depicted in section A of Table 1 or 1a, or in section H of Table 1a or to DNA copy thereof. The nucleic acid affinity ligand may also be able to specifically bind to a miRNA molecule being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B of Table 1 or 1a, or in section H of Table 1a or a pre-miRNA molecule being at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section F of Table 1 or 1a, or in section H of Table 1 a or to any fragments of said sequences.
The term "peptide affinity ligand for the biomarker or group of biomarkers" as used herein refers to a peptide molecule being able to specifically bind to the miRNA biomarker or group of biomarkers according to section A of Table 1 or 1a, or in section H of Table 1a. The peptide molecule may preferably be able to specifically bind to miRNA molecule comprising a sequence as set forth in section B of Table 1 or 1a, or in section H of Table 1a, or to a DNA copy thereof. The peptide affinity ligand may also be able to specifically bind to a miRNA molecule being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B of Table 1 or 1a, or in section H of Table 1a, or pre-miRNA molecule being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section F of Table 1 or 1a, or in section H of Table 1a, or to any fragments of said sequences. The term "peptide" in the context of the affinity ligand of the present invention refers to any type of amino acid sequence comprising more than 4 amino acids, e.g. polypeptide structures, protein structures or functional derivatives thereof. Furthermore, the peptide may be combined with further chemical moieties or functionalities. The term "oligonucleotide specific for the biomarker or group of biomarkers" as used herein refers to a nucleotide sequence which is complementary to the miRNA biomarker or group of biomarkers according to Table 1 or 1a, or to its antisense nucleic acid. Preferably, the oligonucleotide is complementary to the sequence shown in section B or D of Table 1 or 1 a, or in section H of Table 1 a, or to the complementary sequence of the sequence shown in section B or D of Table 1 or 1a, or in section H of Table 1 a. The oligonucleotide sequence may also be complementary to a sequence being at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section B or D of Table 1 or 1 a, or in section H of Table 1a. The oligonucleotide may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section B or D of Table 1 or 1 a, or in section H of Table 1 a or its complement. Typically, the oligonucleotide may have a length of between 8 and 60 nucleotides, preferably of between 10 and 35 nucleotides, more preferably a length of 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33 nucleotides. Oligonucleotide sequences specific for the miRNA biomarker or group of biomarkers according to Table 1 or 1a may be defined with the help of software tools known to the person skilled in the art.
The term "probe specific for the biomarker or group of biomarkers" as used herein means a nucleotide sequence which is complementary to the miRNA biomarker or group of biomarkers according to Table 1 or 1a. Preferably, the probe is complementary to the sequence(s) depicted in section B or D of Table 1 or 1 a, or in section H of Table 1 a, or to the complementary sequence of the sequence(s) shown in section B or D of Table 1 or 1a, or in section H of Table 1a. The probe sequence may also be complementary to a sequence being at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in the sequences indicated in section B or D of Table 1 or 1a, or in section H of Table 1 a. The probe may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section B or D or its/their complement. Typically, the probe may have a length of between 30 and 300 nucleotides, preferably of between 30 and 150 nucleotides, more preferably a length of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 70, 90, 100, 110, 120, 130, 140 or 150 nucleotides. Probe sequences specific for the biomarker or group of biomarkers according to Table 1 or 1a may be defined with the help of software tools known to the person skilled in the art. The probe may preferably be labeled with a suitable label, e.g. radioactive, fluorescent or dye label.
In a further aspect the present invention relates to a method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample.
The term "diagnosing epilepsy" as used herein means that a subject or individual may be considered to be suffering epilepsy, when the expression level of a biomarker or the group of biomarkers of the present invention is modified, e.g. increased/up-regulated or reduced/down-regulated, compared to the expression level of a control level as defined herein above. The term "diagnosing" also refers to the conclusion reached through that comparison process. An expression level may be deemed to be modified, when the expression level of a biomarker or group of biomarkers as defined herein above differs by, for example, between about 1 % and 50%, e.g. 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 40% from a control level as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to such a control level. The modification may be an increase or a reduction of said expression level.
In a further embodiment, an additional similarity in the overall gene expression pattern of a group of biomarkers according to the present invention between a sample obtained from a subject and a control sample as described herein above, may indicate that the subject is suffering epilepsy. In another embodiment of the present invention, the diagnosis may be combined with the elucidation of additional epilepsy markers, e.g. clinical markers, EEG results, brain imaging results or any combination thereof.
The term "detecting epilepsy" as used herein means that epilepsy may be identified in an organism. The identification of epilepsy may be accomplished by a comparison of the expression level of the biomarker or group of biomarkers of the present invention in a sample from a patient or individual to be analyzed and the expression level of a control level, wherein said control level corresponds to the expression level of said biomarker or group of biomarkers in a healthy subject. In a further embodiment of the present invention epilepsy may be detected if the expression level the biomarker or group of biomarkers of a sample or subject to be analyzed is similar to an expression level of a control sample derived from a subject known to be afflicted by epilepsy. The expression level of the epilepsy sample may also independently be established, e.g. from sample depositories, value databases or the like.
The term "graduating epilepsy" as used herein means that the clinical stage, phase, grade or any other suitable sub-step of epilepsy disease related features, e.g. suitable parameters of epilepsy in an organism may be determined in an organism. In a preferred embodiment the graduating of epilepsy may be accomplished by a comparison of the expression level of the biomarker or group of biomarkers according to Table 1 or 1 a of the present invention in a sample from a patient or individual to be analyzed and a control level as defined herein above.
The term "monitoring epilepsy" as used herein relates to the accompaniment of a diagnosed or detected epilepsy during a certain period of time, typically during 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time. The term "accompaniment" means that states of disease as defined herein and, in particular, changes of these states of disease may be detected by comparing the expression level of the biomarker or group of biomarkers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established epitleptical sample, or to corresponding database values in any type of a periodical time segment, e.g. every week, every 2 weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month, every 1.5 year, every 2, 3, 4, 5, 6, 7, 8,9 or 10 years, during any period of time, e.g. during 2 weeks, 3 weeks, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, respectively. In a preferred embodiment of the present invention the term relates to the accompaniment of a diagnosed epilepsy.
The term "prognosticating epilepsy" as used herein refers to the prediction of the course or outcome of a diagnosed or detected epilepsy, e.g. during a certain period of time, during a treatment or after a treatment. The term also refers to a determination of chance of survival, deterioration or recovery from the disease, as well as to a prediction of the expected survival time of a subject. A prognosis may, specifically, involve establishing the likelihood for survival or deterioration of a subject during a period of time into the future, such as 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time.
The term "predisposition for developing epilepsy" as used in the context of the present invention is a state of risk of developing epilepsy. Preferably a predisposition for developing epilepsy may be present in cases in which the expression level of the biomarker or group of biomarker of the present invention as defined herein above is above or below a normal control level as defined herein above, i.e. a reference expression level derived from tissues or samples of a subject which are evidently healthy. The term "above" in this context relates to an expression level of the biomarker of group of biomarkers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%. The term "below" in this context relates to an expression level of the biomarker or group of biomarkers which is decreased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.
Alternatively, a predisposition for developing epilepsy in the context of the present invention may be given in situations in which the expression level of the biomarker or group of biomarkers as defined herein above is above a normal control level and in which further, alternative epilepsy markers show no modification or alteration. Suitable further epilepsy markers, e.g. clinical marker, EEG or imaging marker etc. would be known to the person skilled in the art.
The term "determining the level of a biomarker or group of biomarkers" refers to the determination of the presence or amount of expression product(s) of the biomarker or biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a, e.g. miRNA molecule. The term "level of the biomarker or group of biomarkers" thus means the presence or amount of (an) expression product(s) of the biomarker or group of biomarkers according to Table 1 or 1a, e.g. miRNA molecule according to Table 1 or 1a. The determination of the presence or amount of (an) expression product(s) of the biomarker or group of biomarkers according to Table 1 or 1a may be accomplished by any means known in the art. In a preferred embodiment of the present invention the determination of the presence or amount of the expression products of the biomarker or group of biomarkers as mentioned herein is accomplished by the measurement of miRNA levels or by the determination of the biological effect of said biomarker or group of biomarkers. For example, the measurement of the miRNA level of the expression of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1a may be assessed by separation of nucleic acid molecules (e.g. RNA or cDNA) obtained from the sample in agarose or polyacrylamide gels, followed by hybridization with biomarker specific oligonucleotide probes as defined herein above, e.g. oligonucleotide probes comprising fragments of the sequences indicated in section B or D of Table 1 or 1a, or in section H of Table 1 a, or complementary sequences thereof. Alternatively, the expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples may be placed on the gel such that patient and control or standard nucleic acid are in adjacent lanes. Comparison of expression levels may be accomplished visually or by means of a densitometer. Further methods for the detection of RNA products are known to the person skilled in the art.
Alternatively, the miRNA level of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a may be detected in a DNA array or microarray approach. Typically, sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label. Subsequently, such nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a of the present invention or known biomarker genes. Suitable means for carrying out microarray analyses are known to the person skilled in the art.
In a specific embodiment the present invention also envisages a corresponding microarray. In a standard setup such a microarray comprises immobilized high-density probes to detect a number of miRNAs. The probes on the array are complementary to one or more parts of the sequence of the miRNA, or to a pre-miRNA. In the present invention, any type of biomarker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the biomarker expression and the expression of other genes. Typically, cDNAs, PGR products, and oligonucleotides may be used as probes. For example, a fragment comprising 5'- or 3'-portions of the biomarkers or group of biomarkers as mentioned herein above or according to Table 1 or 1a e.g. of the sequences indicated in section B or D of Table 1 or 1 a, or in section H of Table 1a, may be used as a probe. The microarray may comprise probes of one or more of the biomarker of Table 1 or 1a, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 etc. or all of the biomarkers or any combination of said markers. Furthermore, any type of fragment or sub-portion of any of the markers sequences may be combined with any further fragment or sub-portion of any of the markers sequences of Table 1 or 1 a. In addition to the determination of the expression of biomarkers according to Table 1 or 1a also the determination of the expression of other markers, e.g. additional biomarker is envisaged by the present invention. There is no limitation on the number of probes corresponding to the marker genes used, which are spotted on a DNA array. Also, a marker can be represented by two or more probes, the probes hybridizing to different parts of a gene. Probes are designed for each selected marker gene. Such a probe is typically an oligonucleotide comprising 5-50 nucleotide residues. Longer DNAs can be synthesized by PCR or chemically. Methods for synthesizing such oligonucleotides and applying them on a substrate are well known in the field of micro-arrays.
Alternatively, the nucleic acid level of expression of the biomarker or group of biomarkers according to Table 1 or 1 a may be detected in a quantitative RT- PCR approach, preferably in a real-time PCR approach following the reverse transcription of the RNA of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a. Typically, as first step, a miRNA is reverse transcribed into a cDNA molecule according to any suitable method known to the person skilled in the art. A quantitative or real-time PCR approach may subsequently be carried out based on a first DNA strand obtained as described above. Preferably, Taqman or Molecular Beacon probes as principal FRET-based probes of this type may be used for quantitative PCR detection.
The determination of the biological effect of said biomarker or group of biomarkers may be performed on the basis of a biological function of one or more target genes of a miRNA as identified in Table 1 or 1a. Target genes or their expression products may accordingly be analysed by RNA expression analysis, protein expression analysis, cellular localization or other suitable methods known to the skilled person.
In a further aspect the present invention relates to a method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined herein above in a sample before and during an epilepsy treatment. The term "monitoring epilepsy therapy" as used herein relates to the determination of the level of biomarkers before and during a treatment procedure. The period of time may depend on the treatment period or treatment cycle. For example, the monitoring may be carried out for 2 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time. Accordingly, epilepsy states as defined herein and, in particular, changes of these states of epilepsy may be detected by comparing the expression level of the biomarker or group of biomarkers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established epileptical control sample, or to corresponding database values, or most preferably to one or more initially taken samples or earlier samples of the same subject, e.g. a sample take at the onset of therapy, taken a week, a month, a year etc. before the actual sample taking. The comparison may be carried out in any type of a periodical time segment, e.g. every week, every 2 weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 month, every 1.5 year, every 2, 3, 4, 5, 6, 7, 8,9 or 10 years, during any period of time, e.g. during 2 weeks, 3 weeks, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years etc. In a specific embodiment, the monitoring may be continued after the therapy has been terminated, e.g. 2 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other time value in between these values after the termination of a therapy. The determination may be carried out as described herein above.
In a preferred embodiment, the treatment may be a treatment with a suitable epileptical therapeutic or anticonvulsant. Examples or such medicaments include lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, and zonisamide. Also envisaged is the use of any combinations of the mentioned anticonvulsants. In a specific embodiment, the suitable anticonvulsant may be a pharmaceutical composition as defined herein above. Further envisaged is a combination of any of these medicaments with additional, non-epileptic medicaments.
In another aspect the present invention relates to an assay for detecting, diagnosing, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
(a) testing in a sample obtained from an individual for the expression of a biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1a;
(b) testing in a control sample for the expression of the same biomarker or group of biomarkers as in (a);
(c) determining the difference in expression of the biomarker or group of biomarkers of steps (a) and (b); and
(d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c). The assay is preferably performed with a nucleic acid affinity ligand for a biomarker or group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above or a probe specific for the biomarker or group of biomarkers as defined herein above.
The testing for expression of the biomarker or group of biomarkers according to Table 1 or 1a may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of miRNA levels of the biomarker or group of biomarkers according to Table 1 or 1a, more preferably according to the herein above described options for such measurements. As controls or control samples controls as defined herein above may be used.
In a particularly preferred embodiment the testing steps may be based on the use of nucleic acid affinity ligand, a peptide affinity ligand, a probe or an oligonucleotide specific for a the biomarker or group of biomarkers of Table 1 or 1a as laid out above. Epilepsy may be diagnosed or prognosticated in said assay according to the corresponding definitions provided herein above in the context of the biomarker or group of biomarkers according to Table 1 or 1a.
In a further aspect the present invention relates to a method of identifying an individual eligible for an epilepsy therapy comprising:
(a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined herein above;
(b) testing in a control sample for the expression of a biomarker or a group of biomarkers as defined herein above;
(c) classifying the levels of expression of step (a) relative to the levels of step (b); and
(d) identifying the individual as eligible to receive an epilepsy therapy where the individual's sample is classified as having an increased level of expression of a biomarker or a group of biomarkers as defined herein above, or where the individual's sample is classified as having a decreased level of expression of a biomarker or a group of biomarkers as defined herein above.
The level of a biomarker or group of biomarkers may be determined on the miRNA expression level as described herein above. For the performance of the multiplex detection the concentration of primers and/or probe oligonucleotides may be modified. Furthermore, the concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
A control sample as used in the context of the method of identifying an individual eligible for an epilepsy therapy may be a control sample as defined herein above, e.g. from the same individual as the test sample, or a control sample derived from a different source or individual. The control sample may further be either a sample derived from the same tissue, preferably brain tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are muscle tissue, or blood, urine etc. In addition, in specific embodiment, the testing of a reference gene or miRNA is envisaged, e.g. a miRNA with known expression pattern and/or function. Furthermore, the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a biomarker or group of biomarkers may be combined.
The term "classifying the levels of expression of step (a) relative to levels of step (b)" as used herein means that the expression in a test sample for a biomarker or group of biomarkers according to the invention and the expression in a control sample for a biomarker or group of biomarkers as defined herein are compared, e.g. after normalization against a suitable normalization references. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample. The term further means that the expression in a test sample for a biomarker and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
According to the classification of the expression results an individual may be considered to be eligible for an epilepsy therapy when the expression level of an up-regulated biomarker as defined herein above, or as indicated in section C of Table 1 or 1 a, or the expression level of a group of biomarkers as defined herein above is increased. The expression level is deemed to be "increased" when the biomarker expression, or the expression of the group of biomarkers the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding biomarker expression, or to the expression of the corresponding group of biomarkers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the biomarker expression, or the expression of the group of biomarkers in a control sample. According to the classification of the expression results an individual may also be considered to be eligible for an epilepsy therapy when the expression level of a down-regulated biomarker as defined herein above, or as indicated in section D of Table 1 or 1a, or the expression level of a group of biomarkers as defined herein above is decreased. The expression level is deemed to be "decreased" when the biomarker expression, or the expression of the group of biomarkers in the test sample is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding biomarker gene expression, or to the expression of the corresponding group of biomarkers in a control sample, or lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the biomarker expression, or to the expression of the group of biomarkers in a control sample
In a specific embodiment, the expression of a reference gene may be used to normalize or adjust the expression of a biomarker or group of biomarkers as defined herein.
The therapy to be carried out on a subject who is identified eligible for an epilepsy therapy may be any suitable epilepsy therapy. Preferred is the administration of a pharmaceutical composition as defined herein above, or of an anticonvulsant selected from lacosamide, leveti race tarn, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, and zonisamide, or any combination of the mentioned anticonvulsants.
In another aspect the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined herein above. The diagnostic kit of the present invention may accordingly comprise any suitable agent allowing the specific detection of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a. In preferred embodiments, the kit may comprise at least one component selected from a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined herein above, a peptide affinity ligand for a biomarker or a group of biomarkers as defined herein above, an oligonucleotide specific for the biomarker or group of biomarkers as defined herein above, or a probe specific for the biomarker or group of biomarkers as defined herein above. It is also envisaged that more than one component may be present in the kit. Specifically envisaged is the presence of a set of oligonucleotides specific for the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a together with suitable enzymatic activities and other ingredients necessary for the detection. Further enivsaged is the presence of labeled probes as defined herein, e.g. fluorescently labeled probes.
Preferably, a diagnostic kit of the present invention contains detection reagents for expression product(s) of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a. Such detection reagents comprise, for example, buffer solutions, labels or washing liquids etc. Furthermore, the kit may comprise an amount of a known nucleic acid molecule, which can be used for a calibration of the kit or as an internal control. Typically, a diagnostic kit for the detection of expression product(s) of the biomarker or group of biomarkers as mentioned herein above or according to Table 1 or 1 a may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc. A diagnostic kit may, in specific embodiments, also comprise accessory ingredients like secondary affinity ligands, detection dyes and any other suitable compound or liquid necessary for the performance of nucleic acid detection or amplification. Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.
Additionally, the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
In a specific embodiment, the kit may further comprise probes or oligonucleotides for the detection of a reference analyte, e.g. a gene and/or miRNA known to be associated with epilepsy.
In another aspect the present invention relates to the use of a biomarker or a group of biomarkers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy. The marker may accordingly be used for a diagnosis activity as described above, for a detection activity as described above, for graduating a stage or form of epilepsy as described above, or for monitoring epilepsy, or an epilepsy treatment as described above. Also envisaged is the use of a biomarker or a group of biomarkers as defined herein above for the prognosis of epilepsy development, or the prognosis of epilepsy development during epilepsy treatments. Also envisaged is the use of a biomarker or a group of biomarkers as defined herein above for detecting a predisposition for epilepsy as described above.
In another aspect the present invention relates to the use of a biomarker or group of biomarkers as defined herein above for identifying a pharmaceutically active agent useful in the treatment or prevention of epilepsy. The term "identifying a pharmaceutically active agent" as used herein refers to a screening for potentially agonistic or antagonistic molecules which either increase a reduced miRNA expression level, or reduce an increased miRNA expression level according to the indications of Table 1 or 1a, in particular sections C and D. I.e. the screening is for potentially agonistic or antagonistic molecules leading to an increase of miRNA levels in case of down-regulated miRNAs indicated with a "+" in section D of Table 1 or 1a, or leading to a decrease of miRNA levels in case or up-regulated miRNAs indicated with a "+" in section C of Table 1 or 1 a. The present invention also relates to a corresponding method, as well as agonistic and antagonistic molecules obtained by said method.
Preferably, the screening for such molecules involves producing appropriate cells or organism models which express the miRNAs. Cells expressing the miRNAs may subsequently be contacted with a potential antagonist or agonist to observe binding, stimulation, and/or inhibition of miRNA expression and/or of subsequent cell regulatory activity.
In a specific embodiment the present invention relates to a method for identifying pharmaceutically active agents which can be used to treat or prevent epilepsy comprising at least the steps of:
a) providing a cell;
b) determining expression of a biomarker or a group of biomarkers as described herein in said cell;
c) subjecting said cell to at least one potentially pharmaceutically active agent, e.g. agonist or antagonist;
d) determining expression of a biomarker or a group of biomarkers as described herein in said cell which has been subjected to at least one potentially pharmaceutically active agent, e.g. agonist or antagonist;
e) comparing the expression level of step b) and d); f) classifying the at least one potentially pharmaceutically active agent as an agent that either increases or decreases expression of a biomarker or a group of biomarkers as described herein. In step a) one may use a cell obtained from a healthy human being of from a patient suffering from epilepsy. Also envisaged is the use of cultured cell lines. In a preferred embodiment, these cells and cell lines are outside the human body. In step c) one may subject such cells and cell lines to libraries of potentially pharmaceutically active agents. Such libraries may be small molecule libraries, aptamer libraries, antibody libraries, siRNA libraries, peptide libraries etc. Determining expression levels in steps b) and d) may be undertaken as is common in the art. Correspondingly obtained agents may be used directly to treat or prevent epilepsy, or they may be used as hits or lead compounds for further drug development.
Also envisaged is the use of bodily fluids, preferably of blood, serum or cerebrospinal fluid for screening approaches as defined herein. Such bodily fluids may, for example, for used in order to detect interacting partners or elements having an influence on miRNA expression and/or regulation.
In a further aspect the present invention relates to the following embodiments:
Item 1 : A pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A of Table 1.
Item 2: The pharmaceutical composition of item 1 , wherein said means comprises a nucleic acid molecule comprising a miRNA molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, or an antagonist thereof, wherein said nucleic acid molecule or antagonist has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section B of Table 1 or to a complementary sequence thereof.
Item 3: The pharmaceutical composition of item 2, wherein said nucleic acid molecule or antagonist comprises between at least 18 and 24 nucleotides, preferably 20 nucleotides, 21 nucleotides or 22 nucleotides.
Item 4: The pharmaceutical composition of item 2 or 3, wherein said nucleic acid or antagonist is or comprises a DNA molecule or an RNA molecule, or a derivative thereof.
Item 5: The pharmaceutical composition of item 4, wherein said RNA molecule is or comprises a pre-miRNA selected from section E of Table 1 , or a derivative, fragment or variant thereof, wherein said pre-miRNA molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section F of Table 1 or to a complementary sequence thereof.
Item 6: The pharmaceutical composition of item 3, wherein said DNA molecule is or comprises a DNA molecule coding for a pre-miRNA, a miRNA or for an antagonist.
Item 7: The pharmaceutical composition of item 6, wherein said DNA molecule is or comprises a double stranded DNA coding for at least one pre-miRNA selected from section E of Table 1.
Item 8: The pharmaceutical composition of any one of items 2 to 4, 6 or 7, wherein said nucleic acid molecule is comprised in a vector replicable in a subject.
Item 9: The pharmaceutical composition of any one of items 2 to 8, wherein said nucleic acid molecule or antagonist is modified by a conjugate, preferably a linked conjugate.
Item 10: The pharmaceutical composition of any one of items 2 to 9, wherein said nucleic acid molecule or antagonist comprises a chemically modified base.
Item 11 : The pharmaceutical composition of any one of items 2, 3, 4, 9 or 10, wherein said antagonist is an antagomir.
Item 12: The pharmaceutical composition of item 10 or 11 , wherein said nucleic acid molecule or antagonist is modified by at least one modification selected from the group consisting of a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose and a 2'- methoxyethoxy ribose.
Item 13: The pharmaceutical composition of any one of items 2 to 12, wherein said nucleic acid molecule comprises a mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa-miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221-3p, miR-767-5p, hsa-miR-148a-3p or miR- 125a-5p molecule as defined in section A of Table 1 , or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in section B of Table 1.
Item 14: The pharmaceutical composition of any one of items 2 to 12, wherein said antagonist is an antagonist of a mmu-miR-2137, mmu-miR-212-3p, mmu- miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b-5p, mmu-miR-129-1-3p, mmu-miR-129-5p, mmu- miR-135a-5p, mmu-miR-138-5p, mmu-miR-21 a-5p, mmu-miR-142-3p, mmu-miR-132- 3p, mmu-miR-222-3p, mmu-miR-221-3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR- 142-3p or hsa-miR-124-3p molecule as defined in section A of Table 1 , or of a derivative, fragment or variant thereof.
Item 15: A vaccine for use in the treatment or prevention of epilepsy, comprising a nucleic acid molecule or antagonist as defined in any one of items 2 to 14.
Item 16: A biomarker or group of biomarkers associated with epilepsy, wherein said biomarker or group of biomarkers comprises at least one biomarker selected from the miRNAs identified in section A of Table 1 , or a derivative, fragment or variant thereof.
Item 17: The biomarker or group of biomarkers of item 16, wherein the increase of expression (up-regulation) of at least one biomarker selected from the group of mmu-miR-2137, mmu-miR-212-3p, mmu-miR-142-5p, mmu-miR-223-3p, mmu-miR-767, mmu-miR-148a-3p, miR-138-1-3p, mmu-miR-219-5p, mmu-miR-135b- 5p, mmu-miR- 29-1 -3p, mmu-miR-129-5p, mmu-miR-135a-5p, mmu-miR-138-5p, mmu-miR-21a-5p, mmu-miR-142-3p, mmu-miR-132-3p, mmu-miR-222-3p, mmu-miR- 221 -3p, hsa-miR-92b-3p, hsa-miR-129-5p, hsa-miR-142-3p, and hsa-miR-124-3p as defined in section A of Table 1 , when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
Item 18: The biomarker or group of biomarkers of item 16, wherein the decrease of expression (down-regulation) of at least one biomarker selected from the group of mmu-miR-124-3p, mmu-miR-124-5p, mmu-miR-130a-3p, mmu-miR-298-5p, mmu-miR-92b-3p, miR-767, mmu-miR-125a-5p, hsa-miR-184, hsa-miR-193b-3p, hsa- miR-514a-3p, hsa-miR-329, hsa-miR-222-3p, hsa-miR-655, miR-219-5p, hsa-miR-221- 3p, miR-767-5p, hsa-miR-148a-3p, and miR-125a-5p as defined in section A of Table 1 , when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
Item 19: A composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18.
Item 20: A method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 in a sample.
Item 21 : A method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 in a sample before and during an epilepsy treatment, optionally also after an epilepsy treatment.
Item 22: The method of item 21 , wherein said treatment is a treatment with a pharmaceutical composition as defined in any one of items 1 to 14, and/or with at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, zonisamide, and combinations thereof.
Item 23: The method of any one of items 20 to 22, wherein the determining step is accomplished by the measurement of the miRNA level(s) or by the determination of the biological effect of said biomarker or group of biomarkers.
Item 24: An assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps
(a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined in any one of items 16 to 18;
(b) testing in a control sample for the expression of the same biomarker or group of biomarkers as in (a);
(c) determining the difference in expression of the biomarker or group of biomarkers of steps (a) and (b); and
(d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c) ,
wherein said testing steps are based on the use of a nucleic acid affinity ligand and/or a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18.
Item 25: A method of identifying an individual eligible for an epilepsy therapy comprising: (a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined in item 17 or 18;
(b) testing in a control sample for the expression of a biomarker or a group of biomarkers as defined in item 17 or 18;
(c) classifying the levels of expression of step (a) relative to the levels of step (b); and
identifying the individual as eligible to receive an epilepsy therapy where the individual's sample is classified as having an increased level of expression of a biomarker or a group of biomarkers as defined in item 17, or where the individual's sample is classified as having a decreased level of expression of a biomarker or a group of biomarkers as defined in item 18.
Item 26: A diagnostic kit for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined in any one of items 16 to 18, wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of items 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of items 16 to 18.
Item 27: Use of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
Item 28: Use of a biomarker or a group of biomarkers as defined in any one of items 16 to 18 for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
The following examples and figures are provided for illustrative purposes. It is thus understood that the example and figures are not to be construed as limiting. The skilled person in the art will clearly be able to envisage further modifications of the principles laid out herein. EXAMPLES
Example 1 Outline of the experiments carried out
To identify microRNAs that are dysregulated in Epilepsy steps as outlined in FIGURE 2 were carried out. In particular a microRNA profiling in epilepsy animal models was performed.
In order to identify microRNAs with a function in epileptogenesis as putative novel drug targets a genome-wide microRNA screening was performed in 4 animal models of epilepsy (see Example 2, infra).
Hippocampal microRNAs were analyzed at an acute timepoint (24h after induction of seizure) as well as in the chronic phase (28d) to monitor changes in expression levels during disease progression. Profiling was performed using the miRCURY™ LNA Array microRNA Profiling Service by Exiqon A S according to Exiqon
SOPs.RNA quality was analyzed using the Agilent Bioanalyzer Lab-on-a-Chip System.
All samples analyzed had a RIN-number >7.
Example 2
Animal Study overview Changes in hippocampal microRNA expression patterns were analyzed in different animal models of epilepsy:
(A) the Pilocarpine model, (B) the SSSE model, (C) the 6Hertz model and (D) the Audiogenic model A) Pilocarpine model (chemical induction of generalized epileptic seizure by
Pilocarpine):
Animals were i.p. injected first with N-Methylscopolamine 1 mg/kg and after 30min with Pilocarpine (300mg/kg). After 10 to 45min the animals display generalized seizures that develop into Status Epilepticus (SE) (characterized by a long lasting generalized seizure that can continue for 5-6 h without interruption). In order to limit the extreme brain damage but to obtain mice chronically epileptic (that means mice that display spontaneous recurrent seizures (SRSs) after some days), SE is interrupted after 1-2h by i.p. administration of Diazepam (10mg/kg). Naive animals and animals injected with N-Methylscopolamine served as controls. Animals were sacrificed and hippocampi were dissected for analyses either 24h or 28d post SE.
B) SSSE (sustained status epilepticus) model (electrically stimulated model):
Animals were implanted with electrodes for electrical stimulation of seizures. Sham-implanted animals served as controls. MicroRNA profiles were assessed in the hippocampus at 24h and at 28d after electrical induction of seizures.
C) 6 Hertz model (electrically stimulated model):
Animals received a 3 sec corneal stimulation that induces partial seizures. Only limbic structures are affected by these seizures. Here, non-stimulated animals served as control. The following timepoints were used for microRNA profiling: 3 h, 6 h, 24 h, and 72 h after stimulation.
D) Audiogenic mode! (acoustically stimulated model)
In this model secondary generalized seizures are induced by exposure of the animals to a loud sound of 90 dB. Non-stimulated animals served as controls. The following timepoints were used for microRNA profiling: 3 h, 6 h, 24 h, and 72 h after stimulation.
6 Hz and Audiogenic models were used because in these models seizures are induced without chemical injections and electrode implantation. Therefore, the variability between the animals is lower in these models compared to the Pilocarpine and SSSE models. Using these models a more direct seizure-related analysis of the microRNA profiling pattern appeared possible.
Treatment conditions and n-numbers in are summarized in the following
Table 2:
Figure imgf000096_0001
Figure imgf000097_0001
(D) Audiogenic Naive
Table 2: Animal models of epilepsy used for profiling of hippocampal mlcroRNAs. The Table shows control- and treatment groups as well as animal numbers per group and the timepoints of analysis for the (A) Pilocarpine, (B) SSSE, (C) 6 Hertz and (D) Audiogenic animal model.
Example 3
Epilepsy animal models - microRNA Profiling Data MicroRNA profiling data obtained from the miRCURY™ LNA Array microRNA Screen are summarized in the following Tables. The microarrays provided a complete coverage of miRBase v14. Data were regarded as valid, when the microRNA gave a stable signal across all arrays per group.
The Profiling data obtained from the animal studies were normalized using LOWESS normalization. For each model differences in microRNA expression were assessed based on the calculation of fold changes (FC) between the control groups and the treatment groups (see criteria below). In addition, special attention was paid to differences in hippocampal microRNA profiles during the acute phase (24 h post seizure) and the chronic phase (28d post seizure) to identify miRNAs dysregulated during Epilepsy progression.
Criteria for dysregulated microRNAs were the following:
MicroRNA was significantly (p<0.05, ttest) dysregulated in one of the animal models (Pilocarpine, SSSE, 6Hz and Audiogenic) MicroRNA shows a fold change FC>1.14 in case of up regulation and FCO.88 in case of down regulation
Data are always represented as delta log median ratio (ALMR) and fold- change (FC), based on the median expression values of each animal group (n=8 or n=4). Tables are sorted for FCs.
Figure imgf000098_0001
mmu-miR-21 2.63
mmu-miR-212 1.92
mmu-miR-711 1 81
mmu-miR-882 1.71
mmu-miR-142-5p 1.46
mmu-miR-132 1.44
mmu-miR-142-3p 1.43
Figure imgf000099_0001
mmu-miR-223 1.39
mmu-miR-709 1.24
mmu-miR-706 1.21
mmu-miR-294* 1.20
mmu-miR-193 1.19
mmu-miR-22 1.18
mmu-miR-23a 1.17
mmu-miR-1897-5p 1.16
mmu-miR-29b* 1.16
mmu-miR-494 1.15
mmu-miR-126-3p 1.14
Up regulated miRNAs in the Pilocarpine study (24h, 2 h Diazepam). miRBase v14 annotation
Figure imgf000099_0002
mmu-miR-204 0.76
mmu-miR-1952 0.76
mmu-miR-181 b 0.82
mmu-miR-409-5p 0.86
mmu-miR-466c-5p 0.86
mmu-miR-219 0.87
mmu-miR-434-5p 0.87
mmu-miR-338-5p 0.87
mmu-miR-124 0.87
mmu-miR-669c 0.87
mmu-miR-323-3p 0.87
mmu-miR-365 0.87
mmu-miR-136* 0.87
mmu-m!R-409-3p 0.87
mmu-miR-669i 0.87
mmu-miR-136 0.88
mmu-miR-124* 0.88
mmu-miR-138 0.88
mmu-miR-384-5p 0.88
mmu-miR-301a 0.88
mmu-miR-30a 0.88
Figure imgf000100_0001
mmu-miR-30e
Table 4: Down regulated miRNAs in the Pilocarpine study (24h, 2 h
Diazepam). miRBase v14 annotation.
Figure imgf000100_0002
mmu-miR-135b 1.79
mmu-miR-14 1.44
mmu-miR-135a 1.39
mmu-miR-132 1.37
mmu-miR-23a 1.31
mmu-miR-129-3p 1.30
mmu-miR-467e* 1.25
mmu-miR-24-2* 1.23
mmu-miR-21 1.23
mmu-miR-455 1.22
mmu-miR-129-5p 1.21
mmu-miR-669c 1.21
mmu-miR-142-3p 1.19
mmu-miR-466d-5p 1.19
mmu-miR-708 1.18
mmu-miR-27a 1.18
mmu-miR-466a-5p 1.17
mmu-miR-1952 1.16
mmu-miR-598 1.16
mmu-miR-669l 1.16
mmu-miR-669o 1.15
mmu-miR-669f 1.15
mmu-miR-23b 1.15
mmu-miR-337-3p 1.15
mmu-miR-22* 1.14
mmu-miR-487b 1.14
mmu-miR-1971 1.14
Table 5: Up regulated miRNAs in the Pilocarpine study (28 d, 1 h Diazepam).
miRBase v14 annotation.
Figure imgf000101_0001
mmu-miR-130a 0.78
mmu-miR-551 b 0.80
mmu-miR-187 0.80
mmu-miR-298 0.80
mmu-miR-674 0.84
mmu-let-7d* 0.85
mmu-miR-330* 0.85
mmu-miR-676 0.85
mmu-miR-191 0.86
mmu-miR-875-3p 0.87
mmu-miR-709 0.87
mmu-miR-181 a 0.87
mmu-miR-125a-5p 0.88
mmu-miR-431* 0.88
mmu-miR-767 0.88
Table 6: Down regulated miRNAs in the Pilocarpine study (28 d, 1 h
Diazepam). miRBase v14 annotation.
Figure imgf000101_0002
mmu-miR-132 1.29
mmu-miR-221 1.23
mmu-miR-222 1.19
mmu-miR-467e* 1.18
mmu-miR-129-5p 1.17
mmu-miR-669o 1.17
mmu-miR-720 1.15
mmu-miR-494 1.15
Table 7: Up regulated miRNAs In the Pilocarpine study (28 d, 2 h Diazepam).
miRBase v14 annotation.
Figure imgf000101_0003
mmu-miR-551 b
mmu-miR-338-3p
Figure imgf000102_0001
mmu-miR-376b* 0.86
mmu-miR-194 0.87
mmu-miR-92b 0.87
mmu-miR-382 0.88
Table 8: Down regulated miRNAs in the Pilocarpine study (28 d, 2 h
Diazepam). miRBase v14 annotation.
Figure imgf000102_0002
mmu-miR-2137 6.41
mmu-miR-21 2.78
mmu-miR-212 1.76
mmu-miR-710 1.69
mmu-miR-132 1.58
mmu-miR-294* 1.50
mmu-miR-882 1.49
mmu-miR-20a 1.44
mmu-miR-142-3p 1.33
mmu-miR-706 1.28
mmu-miR-675-5p 1.27
mmu-miR-17 1.19
mmu-miR-146a 1.17
mmu-miR-1900 1.17
mmu-miR-7a 1.14
mmu-miR-146b 1.14
mmu-miR-351 1.14
mmu-miR-298 1.14
mmu-miR-29a* 1.14
Table 9: Up regulated miRNAs in the SSSE study (24 h). miRBase v14 annotation.
Figure imgf000102_0003
Figure imgf000103_0001
mmu-miR-
297a7mmu-miR-
297b-3p/mmu-miR-
297c*
mmu-miR-467a- 17mmu-miR-467d*
mmu-miR-669i
mmu-miR-669h-3p
mmu-miR-669m
mmu-miR-466i
mmu-miR-466a- 3p/mmu-miR-466b- 3p/mmu-miR-466c- 3p/mmu-miR-466e- mmu-miR-669f 0.84
mmu-miR-1839-3p 0.85
mmu-miR-30c 0.85
mmu-miR-467e 0.85
mmu-miR-29b 0.85
mmu-miR-466b-5p 0.85
mmu-miR-467g 0.85
mmu-miR-150 0.85
mmu-miR-181 0.86
mmu-miR-466f-5p 0.87
mmu-miR-466d-3p 0.87
mmu-miR-466a-5p 0.87
mmu-miR-1903 0.87
mmu-miR-466f-3p 0.88
Table 10: Down regulated miRNAs in the SSSE study (24 h). miRBase v14 annotation.
Figure imgf000103_0002
mmu-miR-221 1.52
mmu-miR-669c 1.40
mmu-miR-222 1.38
mmu-miR-21 1.35
mmu-miR-669! 1.31
mmu-miR-455 1.30
mmu-miR-467e* 1.29 6
Figure imgf000104_0001
mmu-miR-2137 1.26
mmu-miR-494 1.25
mmu-miR-669f 1.23
mmu-miR-1971 1.23
mmu-miR-466i 1.22
mmu-miR-1929 1.21
mmu-miR-138 1.21
mmu-miR-467g 1.20
mmu-miR-467a- 1.20
1*/mmu-miR-467d*
mmu-miR-466f-3p 1.18
mmu-mi -574-5p 1.14
Table 11 : Up regulated miRNAs in the SSSE study (28 d). miRBase v14 annotation.
Figure imgf000104_0002
mmu-miR-378 0.85
mmu-miR-7b 0.85
mmu-miR-497 0.86
mmu-miR-30a 0.87
mmu-miR-2183 0.88
mmu-miR-150 0.88
mmu-miR-218-2* 0.88
mmu-miR-543 0.88
mmu-miR-345-5p 0.88
Table 12: Down regulated miRNAs in the SSSE study (28 d). miRBase v14 annotation.
Figure imgf000104_0003
mmu-miR-335-3p 1.46
mmu-miR-690 1.31
mmu-miR-207 1.30
mmu-miR-34b-3p 1.29
mmu-miR-720 1.27
mmu-miR-1983 1.27
mmu-miR-302a 1.25
mmu-miR-582-3p 1.23 T EP2013/077836
Figure imgf000105_0001
mmu-miR-212 1.21
mmu-miR-24 1.21
mmu-miR-767 1.21
mmu-miR-34a 1.21
mmu-miR-210 1.21
mmu-let-7b 1.20
mmu-miR-124 1.20
mmu-miR-125a-5p 1.20
mmu-miR-132 1.19
mmu-miR-503* 1.19
mmu-miR-126-3p 1.19
mmu-miR-125b-5p 1.18
mmu-miR-181 d 1.18
mmu-miR-290-5p 1.18
mmu-miR-23b 1.18
mmu-miR-138* 1.18
mmu-miR-1961 1.17
mmu-miR-431* 1.17
mmu-miR-455 1.16
mmu-miR-138 1.16
mmu-miR-181 a 1.16
mmu-miR-185 1.16
mmu-miR-1949 1.16
mmu-miR-1929 1.16
mmu-miR-872* 1.16
mmu-miR-222 1.15
mmu-miR-49 1.15
mmu-miR-181c 1.15
mmu-miR-330* 1.15
mmu-miR-2137 1.14
mmu-miR-674 1.14
mmu-miR-675-5p 1.14
mmu-miR-325 1.14
Table 13: Up regulated mIRNAs in the 6 Hertz study (3 h). mi'RBase v14 annotation.
Figure imgf000105_0002
mmu-miR-1895 0.86
mmu-miR-7b 0.88
mmu-miR-598 0.88
Table 14: Down regulated miRNAs in the 6 Hertz study (3 h). miRBase vi4 annotation.
Figure imgf000106_0001
mmu-miR-335-3p 1.42
mmu-miR-140 1.36
mmu-miR-212 1.33
mmu-miR-302a 1.30
mmu-miR-299* 1.29
mmu-let-7f 1.28
mmu-let-7b 1.27
mmu-miR-207 1.26
mmu-miR-290-5p 1.26
mmu-miR-720 1.26
mmu-miR-369-5p 1.25
mmu-miR-374 1.25
mmu-miR-767 1.25
mmu-miR-23b 1.25
mmu-miR-582-3p 1.25
mmu-miR-222 1.24
mmu-let-7a 1.24
mmu-miR-330* 1.24
mmu-miR-24 1.23
mmu-miR-148a 1.23
mmu-miR-582-5p 1.23
mmu-miR-491 1.23
mmu-miR-1961 1.23
mmu-miR-125b-
5p 1.23
mmu-miR-1 1.23
mmu-miR-409-5p 1.23
mmu-miR-1983 1.22
mmu-miR-337-3p 1.22
mmu-miR-127 1.22
mmu-miR-690 1.22
mmu-miR-132 1.22
mmu-miR-496 1.22
mmu-miR-378 1.22
Figure imgf000107_0001
mmu-miR-125a-
5p 1.22 mmu-!et-7c 1.22 mmu-miR-34fa-3p 1.21 mmu-miR-377 1.21 mmu-miR-431 * 1.21 mmu-mlR-541 1.21 mmu-m!R-98 1.21 mmu-miR-138* 1.20 mmu-miR-194 1.20 mmu-miR-467b 1.20 mmu-miR-30c 1.20 mmu-miR-1839-
1.20
3p
mmu-miR-706 1.20 mmu-miR-30a 1.20 mmu-miR-106a 1.20 mmu-miR-325 1.20 mmu-miR-127* 1.19 mmu-miR-337-5p 1.19 mmu-let-7d 1.19 mmu-miR-433 1.19 mmu-miR-667 1.19 mmu-miR-434-5p 1.19 mmu-miR-181 c 1.19 mmu-miR-487b 1.19 mmu-miR-495 1.19 mmu-miR-455 1.18 mmu-mlR-1949 1.18 mmu-miR-129-3p 1.18 mmu-miR-30b 1.18 mmu-miR-410 1.18 mmu-miR-380-3p 1.18 mmu-miR-218 1.18 mmu-miR-543 1.18 mmu-miR-668 1.18 mmu-miR-494 1.17 mmu-miR-329 1.17 mmu-miR-27b 1.17 mmu-miR-379 1.17 mmu-let-7e 1.17 mmu-miR-191 1.17
Figure imgf000108_0001
mmu-miR-361 1.17 mmu-miR-331-3p 1.17 mmu-miR-9* 1.17 mmu-miR-185 1.17 mmu-miR-301 a 1.17 mmu-miR-24-2* 1.17 mmu-miR-652 1.17 mmu-miR-376b 1.17 mmu-miR-92b 1.16 mmu-miR-30d 1.16 mmu-miR-421 1.16 mmu-miR-770-3p 1.16 mmu-miR-744 1.16 mmu-miR-124 1.16 mmu-miR-503* 1.16 mmu-miR-674* 1.16 mmu-miR-41 1* 1.16 mmu-miR-872* 1.16 mmu-miR-339-5p 1.16 mmu-miR-466a-
3p/mmu-miR-
466b-3p/mmu-
1 16 miR-466c-
3p/mmu-miR-
466e-3p
mmu-miR-741 1.15 mmu-miR-137 1.15 mmu-miR-382 1.15 mmu-miR-351 1.15 mmu-miR-345-5p 1.15 mmu-miR-2 37 1.15 mmu-miR-24-1 * 1.15 mmu-miR-124* 1.15 mmu-miR-341 1.15 mmu-miR-500 1.15 mmu-miR-300* 1.15 mmu-miR-328 1.15 mmu-miR-29a 1.15 mmu-miR-26a 1.15 mmu-miR-29b 1.15 mmu-mi'R-384-5p 1.15 mmu-miR-129-5p 1.15 mmu-miR-100 1.15
Figure imgf000109_0001
mmu-miR-150 1.15
mmu-miR-674 1.15
mmu-miR-433* 1.15
mmu-miR-99b 1.14
mmu-miR-350 1.14
mmu-miR-30e 1.14
mmu-miR-151-5p 1.14
mmu-miR-376b* 1.14
mmu-miR-148b 1.14
mmu-let-7g 1.14
mmu-miR-149 1.14
mmu-miR-103 1.14
mmu-miR-128 1.14
mmu-miR-497 1.14
Table 15: Up regulated miRNAs in the 6 Hertz study (6 h). miRBase v14 annotation.
Figure imgf000109_0002
mmu-miR-709 0.78
mmu-miR-193 0.79
mmu-miR-1895 0.85
mmu-miR-669a 0.85
mmu-miR-669e 0.88
Table 16: Down regulated miRNAs in the 6 Hertz study (6 h). miRBase v14 annotation.
Figure imgf000109_0003
mmu-miR-335-3p 1.23
mmu-miR-218 1.19
mmu-miR-302a 1.15
mmu-miR-374 1.15
mmu-miR-431* 1.15
mmu-miR-137 1.14 mmu-let-7b 1.14
mmu-miR-138 1.14
mmu-miR-582-3p 1.14
mmu-miR-767 1.14
Table 17: Op regulated miRNAs in the 6 Hertz study (24 h). miRBase v14 annotation.
Figure imgf000110_0001
mmu-miR-466f-3p
mmu-m>R-466i
Table 18: Down regulated miRNAs in the 6 Hertz study (24 h). miRBase v14 annotation.
Figure imgf000110_0002
mmu-miR-335-3p 1.38
mmu-miR-465b-5p 1.29
mmu-miR-212 1.16
mmu-miR-222 1.15
mmu-miR-33 1.14
Table 19: Up regulated miRNAs in the 6 Hertz study (72 h). miRBase v14 annotation.
Figure imgf000110_0003
mmu-miR-34c 0.65
mmu-miR-34b-5p 0.67
mmu-miR-193 0.78
mmu-miR-338-5p 0.86
mmu-miR-365 0.88
Table 20: Down regulated miRNAs in the 6 Hertz study (72 h). miRBase v14 annotation.
Figure imgf000110_0004
Figure imgf000111_0001
mmu-miR-338-3p 1.30
mmu-miR-135a 1.20
mmu-miR-338-5p 1.20
mmu-miR-34a 1.20
mmu-miR-677 1.16
mmu-miR-365 1.16
Table 21 : Up regulated miRNAs In the Audiogenic study (3 h). miRBase v14 annotation.
Figure imgf000111_0002
mmu-miR-143 0.81
mmu-miR-7a 0.85
mmu-miR-341 0.86
mmu-miR-222 0.86
mmu-miR-706 0.87
Table 22: Down regulated miRNAs in the Audiogenic study (3 h). miRBase v14 annotation.
Figure imgf000111_0003
mmu-miR-219 1.32
mmu-miR-338-3p 1.28
mmu-miR-135a 1.23
mmu-miR-200b 1.22
mmu-miR-34a 1.22
mmu-miR-15a 1.16
mmu-miR-338-5p 1.16
mmu-miR-677 1.15
mmu-miR-20a 1.15
mmu-miR-181 a 1.14
mmu-miR-16 1.14
mmu-miR-17 1.14
mmu-miR-21 1.14 Table 23: Up regulated miRNAs to the Audiogenic study (6 h). miRBase v14 annotation.
At the 6 h timepoint no significantly down regulated miRNAs were identified in the Audiogenic mouse model. In addition, no significantly dysreguiated miRNAs were identified at the 24 h timepoint.
Figure imgf000112_0001
mmu-miR-200b 1.19
Table 24: Up regulated miRNAs in the Audiogenic study (72 h). miRBase v14 annotation. No significantly down regulated miRNAs were identified at the 72 h timepoint.
Example 4
Validation of identified microRNA candidates
A selected number of the microRNA candidates were validated in the mouse samples by single-assay qRT-PCR. Here, miRNA candidates were selected predominantly based on the models of chronic Epilepsy (pilocarpine, SSSE) as the more relevant mouse models to compare with human Epilepsy.
Criteria for qPCR selection were the following:
1. Selection based on the FC shown in the miRNA profiling (most up and most downregulated miRNAs).
2. Up or downregulation of a certain miRNA candidate specific to acute or chronic time point in the Pilocarpine and SSSE mouse model.
3. Known expression and/or function of miRNAs in the CNS and Epilepsy based predominantly on Pubmed database search.
4. Dysregulated miRNAs identified in the mouse and human profiling.
MicroRNA expression analyses were performed on the treatment and naive groups of the Pilocarpine and SSSE model samples using the Universal cDNA Synthesis Kit, SYBR Green master mix, and pre-designed LNA™-enhanced microRNA qPCR primer sets (all by Exiqon) according to manufacturers' instructions. MicroRNA candidates were regarded as validated when showing a FC>1.20fold/ <0.80fold and consistent with the profiling data.
Table 25 summarizes most relevant microRNA candidates and qRT- PCR validation results.
Figure imgf000114_0001
Table 25: qRT-PCR validation of microRNAs from Epilepsy animal models.
The table includes the fold-change as detected in the microRNA profiling, the timepoint of detection (either in the acute (24h) or chronic (28d) phase, or generally dys regulated), and the qRT-PCR validation status. (V confirmed).
Example 5
MicroRNA profiling in hippocampal biopsies of epilepsy patients In a parallel approach a genome-wide microRNA profiling in hippocampal biopsies from patients that underwent curative hippocampectomy was performed at the university hospital Bonn (Clinic of Neurosurgery, Dr. M. von Lehe). All patients were diagnosed with temporal lobe epilepsy (TLE) and suffered from severe recurrent generalized epileptic seizures. One patient subgroup showed the pathophysiological hallmarks of Ammon's horn sclerosis (AHS), indicating loss of pyramidal neurons and gliosis. The other patient group was AHS negative (nonAHS). Hippocampal specimens from post mortem samples served as controls. Table 26, infra, gives an overview on human patients' samples included in this project:
Figure imgf000115_0001
MicroRNA profiling in human samples. The study included patients diagnosed with TLE and either AHS positive or negative. Hippocampal post mortem samples served as control. RNA quality was analyzed using the Agilent Bioanalyzer Lab-on-a-
Chip System. All samples analyzed had a RIN-number >7. Furthermore, all human samples were subjected to a deep sequencing analysis using the Sequencing Service of Vertis GmbH.
In detail, the small RNA fraction (19-29 bases) from all human RNA samples was isolated. Next, the small RNAs were poly(A) tailed and ligated to RNA adapter. After cDNA synthesis ail cDNA were pooled and PCR amplified. The cDNA pools were sequenced on a lllumina HiSeq 2000 system using a read length of 36 bp (single reads) and approximately 5 mio reads per cDNA. All reads were aligned to miRBase v18 to identify expressed miRNAs. This alignment was done by InteRNA Genomics. The aim was to get a more detailed insight into expressional changes in the human samples.
Example 6
MicroRNAs dysregulated in human samples
For data analysis we did an initial comparison of the profiling data obtained with the microRNA arrays vs. the Deep Sequencing approach. Since Deep Sequencing revealed a much higher sensitivity with respect to detection levels as well as to detection of expression changes, we focused on these data for further analysis. For each patients group the in microRNA expression was assessed based on the calculation of fold changes (FC) between the control groups (nonAHS and post mortem, respectively) and the treatment group (AHS). We calculated FC and p-value for 2 different comparisons: AHS versus nonAHS and AHS and nonAHS versus post mortem. Criteria for microRNA candidate selection were the following:
1. MicroRNA candidate was significantly (p<0.05, ttest) dysregulated in human samples.
2. Sufficient high read-number (> 10) in the Deep Sequencing
3. MicroRNA candidate shows a fold change FC>1.14 in case of up regulation and FC<0.88 in case of down regulation.
Lists of identified microRNA candidates that are significantly up and down regulated in hippocampal biopsies of Epilepsy patients are found in Table 27 and Table 28, infra:
Figure imgf000117_0001
Table 27: MicroRNAs up regulated in hippocampal biopsies of TLE patients. The microRNAs were identified In a Deep Sequencing approach. The Tables indicate FC for 2 different comparisons: AHS versus nonAHS as well as AHS and nonAHS versus post mortem.
Figure imgf000118_0001
Figure imgf000119_0001
Table 28: microRNAs down regulated in hippocampal biopsies of TLE patients.
The microRNAs were identified in a Deep Sequencing approach. The Tables indicate FC for 2 different comparisons: AHS versus nonAHS as well as AHS and nonAHS versus post mortem.
In the human profiling the majority of dysregulated miRNAs was down regulated.
Example 7
Validation of identified microRNA candidates For qRT-PCR validation of human miRNA candidates only the AHS vs. nonAHS comparison was considered. Dysregulated miRNAs in the AHS and nonAHS versus post mortem were not used for validation because of the low post mortem sample number. Due to the higher variability in the human sample groups which is caused e.g. by differences in disease progression or disease duration also non- significantly (p>0.05) dysregulated miRNAs were added to the candidate list.
Criteria for microRNA candidate selection were the following:
1. Selection based on the FC shown in the miRNA profiling (most up and most down regulated miRNAs.
2. Known expression and/or function of miRNAs in the CNS and Epilepsy based predominantly on Pubmed database search.
3. Dysregulated miRNAs identified in the mouse and human profiling.
Validation of identified human microRNA candidates was carried out by single-assay qRT-PCR on the human samples. MicroRNA expression analyses were performed on the AHS, nonAHS and post mortem groups using the Universal cDNA Synthesis Kit, SYBR Green master mix, and pre-designed LNA™-enhanced microRNA qPCR primer sets (all by Exiqon) according to manufacturers" instructions.
The following Table 29 summarizes most relevant microRNA candidates and qRT-PCR validation results.
Figure imgf000120_0001
Figure imgf000121_0001
qRT-PCR validation of microRNAs identified as dys regulated in hippocampi of human TLE patients.
The table displays the fold-changes in microR A levels as detected in the Deep Sequencing profiling in the indicated comparisons (AHS versus nonAHS). qRT-PCR validation status is shown in the last column. (V confirmed; † upregulated in qRT-PCR; \ downregulated in qRT-PCR; (-) not changed in qRT-PCR, (ns) not significant in qRT-PCR). Example 8
Identification of overlapping microRNA candidates (mouse/human)
The following miRNAs were identified as being expressed overlapping manner in the mouse and human approaches.
Figure imgf000122_0001
Table 30: MicroRNA candidates overlapping in mouse models and human profiling (acute phase).
Figure imgf000122_0002
Table 31 : MicroRNA candidates overlapping in mouse models and human profiling (chronic phase). Example 9
Bioinformatic evaluation of microRNA candidates
(Target Gene Identification / Pathway analyses)
In order to identify target genes and pathways regulated by the candidate miRNAs a bioinformatical analysis was performed. The aim of this analysis was the prediction of epilepsy relevant target genes and pathways.
Based on the miRNA profilings 11 mouse and 11 human miRNA candidates were selected for this analysis. The criteria for the miRNA candidate selection were the following.
Criteria of miRNA candidate selection for bioinformatical analysis:
1. FC of miRNA expression in mouse and human profiling. The most up and down regulated miRNA candidates were selected.
2. Known expression/functions in the CNS, reported expression/functions in Epilepsy.
3. Known and experimentally validated target genes of a miRNA candidate.
The bioinformatical analysis included target gene prediction followed by GO enrichment analysis.
The predicted target genes for the microRNA candidates were compiled from the databases (mouse samples: Target Scan and Microcosm database; human study: ExprTarget DB). For the mouse miRNA candidates predicted target genes identified in both databases (Targe Scan and Microcosm) were used for further analysis. Afterwards.for each miRNA the 100 top hits of the target gene prediction were used fo a GO enrichment analysis done with the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/ home.jsp). Predicted pathways were selected for the target genes of each miRNA based on the prediction score.
FIGURE 3 shows a number of enriched target genes for a selected number of microRNAs. All these target genes are putatively relevant for different cellular processes during epileptogenesis. Example 10
Overview in vitro validation of microRNAs
During epileptogenesis brain is affected by severe changes characterized as sudden and uncontrolled firing of neurons, onset of astrogliosis and activation of microglia. All these events have unknown or partially known molecular mechanisms in neurons, astrocytes, microglia and other brain cells, that underlie pathophysiology of disease.
Pathophyisological changes of brain from initial insult to emergence of spontaneous seizures can be divided into three major phases: 1 ) activation of immediate early genes and activation of ion channels; 2) transcriptional activation, neuronal cell death, set of inflammation; 3) neurogenesis, sprouting of neurons, gliosis and neuronal network reorganization.
The analysis was focused on studying effects of selected miRNAs dysregulated in Epilepsy, that are obtained from our miRNA sequencing screen described above. In order to do so, several in vitro assay systems were set up:
proliferation of neuroblastoma! cell line,
excitotoxicity induced by glutamate,
activation of microglia,
viability and growth of astrocytes, and
Each of these assays addresses changes present in epileptic brain. In this way, it was achieved to gain insight into roles of the identified miRNAs in cell growth, neuroprotection, activation of microglia and calcium release (see FIGURE 4).
Example 11
Lentiviral microRNA expression system and transduction of different cell types In order to investigate effects of miRNA in different mammalian systems, generated lentiviral (LV) expression constructs were generated for stable overexpression of micro RNAs in established cell lines as well as in primary mouse hippocampal neurons, astrocytes and microglia. Precursor miRNA (pre-miRNA) was cloned using Xhol and BamHI restriction sites at the 3' end of eGFP (enhanced Green Fluorescent Protein) under CMV promoter (human cytomegalovirus immediate early promoter).
In addition a linker sequence of approximately 200bp upstream and downstream of pre-miRNA sequence was cloned to enable good processing by miRNA protein machinery. The microRNA precursor was therefore co-expressed with an eGFP reporter gene, which served as control of infection, and further processed by Dicer/Ago machinery into 21-22 nts duplexes. Advantage of using lentiviral system of gene delivery to cells is the controlled and precise amount of delivered particles, expressed as multiplicity of infection, MOI (number of viral particles infecting a single cell). See FIGURE 5 for lentiviral vectors used.
The following sequences of precursor miRNA have been used:
Pre-miR-124: >hsa-mir-124-1 MI0000443
AGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCCA TACAATTAAGGCACGCGGTGAATGCCAAGAATGGGGCTG (SEQ ID NO: 376)
Pre-miR-130a:>hsa-mir-130a Ml 0000448
TGCTGCTGGCCAGAGCTCTTTTCACATTGTGCTACTGTCTGCACCT GTCACTAGCAGTGCAATGTTAAAAGGGCATTGGCCGTGTAGTG (SEQ ID NO: 377)
Pre-miR-132:>hsa-mir-132 MI0000449
CCGCCCCCGCGTCTCCAGGGCAACCGTGGCTTTCGATTGTTACTG TGGGAACTGGAGGTAACAGTCTACAGCCATGGTCGCCCCGCAGCACGCCCACGC GC (SEQ ID NO: 378) Pre-miR-142:>hsa-mir-142 MI0000458
GACAGTGCAGTCACCCATAAAGTAGAAAGCACTACTAACAGCACTGGAGGGTGTA GTGTTTCCTACTTTATGGATGAGTGTACTGTG (SEQ ID NO: 379) Pre-miR-184:>hsa-mir-184 MI0000481
CCAGTCACGTCCCCTTATCACTTTTCCAGCCCAGCTTTGTGACTGT AAGTGTTGGACGGAGAACTGATAAGGGTAGGTGATTGA (SEQ ID NO: 380)
Pre-miR-221 :>hsa-mir-221 MI0000298
TGAACATCCAGGTCTGGGGCATGAACCTGGCATACAATGTAGATTT CTGTGTTCGTTAGGCAACAGCTACATTGTCTGCTGGGTTTCAGGCTACCTGGAAA CATGTTCTC (SEQ ID NO: 381 )
Pre-miR-2137:>mmT-mir-2137 I0010750
GTAGTATACCTCCTTCCTGCTGCCTTGTTGGCTTGCCGGCGGGAG CCCCAGGGAGTAGAGCATTGC (SEQ ID NO: 382)
Preparation of infectious reagent, virus, was a multistep process. An optimized mixture containing all plasmids was prepared in a sterile reaction tube in order to facilitate viral packaging of our pLenti expression vector
followingcotransfection into HEK293T producer cells.
The following mixture was used:
270 mg lentivector (pLENTI6.4-promoter-transgene),
175 mg pLP1 encoding for Gag-Pol sequence,
68 mg pLP2 encoding for rev transcriptase gene,
95 mg pLP-VSVG encoding for VSV-G envelope.
All plasmids were purchased from Invitrogen and their vector maps are available under http://products.invitrogen.com.
The production of a lentivirus virus began with cloning the gene of interest (goi), in this case precursor microRNA into an entry plasmid pLENTI6.4- promoter-transgene (pl_ENTI6.4-CMV-premiRNA). The expression plasmid was co- transfected into HEK293T producer cells with three supercoiled packaging plasmids (pLP1 , pLP2 and pLPA/SV-G) which supply helper functions and viral proteins in trans. At 48-72 hours post-transfection, the supernatant (containing the viral particles) was harvested and clarified. At this point lentiviral vectors can be used to transduce the mammalian cell line of choice. High-titer lentivector preparations are essential to achieve high transgenesis rates (Pfeifer A, Hofmann A, 2009).
For transduction, lentiviral vectors were diluted in cell medium. The rate of dilution highly depends on the desired transgenesis rates (MOI, number of viral integrants per cell) and in this case was MO1100. After overnight incubation cell medium was changed and expression was monitored upon 48-96 hrs. First, expression of eGFP in cells was confirmed using a fluorescence microscope (see FIGURE 6). Then, miRNA expression was confirmed using q-RT-PCR method (see FIGURE 7) and miRNA specific primers purchased from Exiqon (http://www.exiqon.com/mirna-pcr- primer).
Generated LV system enabled easy and precise delivery of gene of interest at MOI100 rate, in our case pre-miRNAs, therefore enabling their overexpression and processing in mammalian cells, as shown for primary mouse hippocampal neurons (see FIGURE 7). As seen in FIGURE 6, all miRNA could be very well expressed in primary mouse hippocampal neurons, but the level of their expression detected by q-RT-PCR differed. This effect is due to already present native miRNA level recognized as well by specific miRNA primers used. Native miRNAs level are cell and tissue specific and differ among SH-SY5Y cell line, mouse hippocampal neurons, cortical neurons, astrocytes and microglia.
Example 12
Proliferation assay Human neuroblastoma Sine, SH-SY5Y was transducted with lentiviral system, expressing precursor mi'RNA of interested linked to GFP reporter at MOM 00. 72 hours upon transductions, GFP positive cells were selected and sorted into 96 well plates using FACS. After this growth rates of transgenic SH-SY5Y cells expressing miRNA of interest were measured by applying 12m MTT for 1 hour at 37 °C and measuring absorbance level at 560 nm. The growth rates of transgenic cell lines were measured over the time period of 10 days (see FIGURE 8 and FIGURE 9).
Transduced miRNA showed either no significant effect on growth of SH- SY5Y cells, showed higher growth rates than control or showed slower growth rate than control. These effects fit to present knowledge of miRNAs as molecular brakes in certain cancer cells (hepatocitoma). Here we show, that miRNA-124 drastically inhibits cell growth in neuroblastoma! cells (see FIGURE 10).
In a second approach human neuroblastoma cell line, SH-SY5Y was transduced with a lentiviral system, expressing precursor miRNAs miR-124-3p, miR- 142-3p miR-184 and miR-con linked to a GFP as reporter protein at OI100 72 hours upon transduction, GFP positive cells were selected and sorted into 96 well plates using FACS. After this growth rates of transgenic SH-SY5Y cells expressing the miR- 124-3p, miR-142-3p and miR-184 were measured by applying 12 m MTT for 2 hours at 37 °C and detection of absorbance level at OD 540/600 nm. The growth rates of transgenic cell lines were measured over a time period of 9 days.
The expression of the GFP reporter protein of different precursor miRNAs was detectable in SH-SY5Y cells after transduction with the lentiviral vectors (see FIGURE 19).
It could be shown that lentiviral overexpression of miR-124-3p and miR- 142-3p drastically inhibited cell growth in neuroblastoma cells, whereas lentiviral overexpression of miR-184 did not inhibit cell growth (see FIGURE 20). Example 13
Excitotoxicity assay induced by glutamate Epilepsy is characterized by sudden and uncontrolled firing of neuronal circuits that leads to extensive neuronal death due to uncontrolled synaptic glutamate release. In order to investigate effects of miRNA during glutamate excitotoxicity release, we have performed in vitro assays on transduced hippocampal neurons and measured their viability 24 upon exposure by MTT (see FIGURE 11).
Primary hippocampal neurons were prepared from postnatal day zero or postnatal day one mouse brains (P0/P1), strain C57BL/6J. First, hippocampus was dissected out, gathered and washed in IxHBSS solution. Single cell suspension was achieved using trypsin based neural tissue dissociation kit (T) (Miltenyibiotec, Cat.#130-093-231) and cells were placed on PLL coated plates (0,33 mg/mL Poly-L- Lysine, Sigma) in plating medium. Upon 3 hrs medium was changed to growth medium. Cultures were obtained for period of 2 at 37C 5% C02 by changing half of growth medium two times per week.
Primary hippocampal neurons were placed on PLL coated plates (Poly- L-Lysine, Sigma) and transduced with lentiviral constructs carrying miRNAs of interests at MOM 00 at DIV7 (day in vitro). At DIV13 hippocampal neurons were subjected to 10μΜ and 100μΜ final glutamate concentration for 24 hours, upon which survival rates were measured using MTT test.
There was no significant effect in neuronal viability during increased glutamate conditions for our candidate miRNAs, although miR-124 showed tendency toward protective effect. Since in vitro assay presents limited tool due to absence of other brain cells, protective effect of miR-124 during glutamate excitotoxicity might play significant role in animal models (see FIGURE 12).
Exam le 14
Microglia activation
Activated microglia play an important role in neuroinflammation during and after SE. Microglia can be activated using INF-g and LPS. Following activation they secrete IL-6. This approach was used to analyze the influence of miRNA overexpression on IL-6 secretion in primary microglia.
Primary microglia were isolated from postnatal day three mouse brains
(P3), strain C57BL/6J using CD11 b magnetic microbeads from Miltenyibiotec (Microglia MicroBeads, Cat.# 130-093-634). Brain tissue was first dissociated using papin based neural tissue dissociation kit (P) (Miltenyibiotec, Cat. # 130-092-628) from Miltenyibiotec and cells expressing CD1 1 b, microglia marker, were enriched on the magnetic column (Miltenyi, Cat. # 130-042-201 ) according to the manufacturer instructions.
Obtained cells were plated into 96 well plates and transduced using lentiviral constructs carrying miRNA of interest at DIV1. Medium used was DMEM/F12 (Gibco, Cat. #31765) supplemented with 10% FBS (Gibco, Cat. # 10082139) and 1 % Penicillin/Streptomicin and changed every two days. At DIV4 cell were induced using LPS and IFN-g at concentrations 4 ug/mL and 10 ng/mL (LPS Enzo Life Science, Cat. # 581-007-LOOZ, lot L28106; IFN-g Miltenyi Biotec, MACS Cytokines, Cat. # 130-096- 872) as activating stimulus for 24 hours. After that supernatant was collected and levels of secreted cytokines IL-6 were measured by ELISA sandwich kit (BD, Cat. # 550950) (see FIGURE 13 and FIGURE 14).
Overexpression of miRNAs in primary microglia cells could be shown to lead to a change of pattern in secreted cytokines. In activated microglia, miR-124 leads to decreased secretion of IL-6 and silencing of immune response also shown by Ponomarev et al. 2011 whereas miR-132, miR-124 and miR-2137 lead to high level of IL-6 expression in non-induced conditions (see FIGURE 15).
Another approach for the overexpression of miRNA into cells is transfection with miRNA-mimic mediated by lipid concoctions as reagents (Invitrogen, Cat. # 13778-150). Obtained cells were plated into 96 well plates and transfected with miRNA-mimic of interest at DIV7. MiRNA-mimic is artificially synthesized, double- stranded RNA which mimic mature endogenous miRNA after transfection into cells. In contrast to lentiviral approach to delivering miRNA to the cells, miRNA-mimic transfection is simple to use, high efficient, relatively low or non-cytotoxicity and inflammatory responses to the primary microglia. All the miRNA-mimic of interest were provided by Ambion and used at final concentration of 60 nM, respectively. The growth medium used was DMEM/F12 (Gibco, Cat. #11320-074) supplemented with 10% FBS (Gibco, Cat. # 10082139), 0,1 % nonessential amino acids (Gibco, Cat. # 1 1 40-050), 0, 1 % GlutaMAX (Gibco, Cat. # 35050-038) and 1 % Penidllin/Streptomicin and changed every two or three days (see FIGURE 21 ). At DIV9 the cells were induced using LPS and IFN-γ at concentrations of 4 ug/mL and 10 ng/mL, respectively (LPS Enzo Life Science, Cat. # 581-007-LOOZ, lot L28106; IFN-γ Miltenyi Biotec, MACS Cytokines, Cat. # 30-096-872) as activating stimulus for 24 hours. Afterwards the supernatant was collected and the level of the secreted cytokine IL-6 was measured by ELISA sandwich kit (BD, Cat. # 550950).
A FACS analysis was carried out with the CD1 1 b+ fraction (see FIGURE
22). Furthermore, the morphological characteristics of primary cultured mice microglial cells (P0-P3) 72 hours after transfection of miRNA-mimics were determined (see FIGURE 23).
The overexpression of miRNAs mediated by the miRNA-mimic transfection in primary microglial cells induced a change of pattern in secreted cytokines. As shown in FIGURE 24, the IL-6 level was significantly reduced in miR- 124-3p overexpressing cells alone in comparison to the wild-type after LPS and INF-γ stimulation. Therefore, it seems likely that miR-124-3p might have an anti-inflammatory effect as suggested in the previous paper by Ponomarev et al.; 2011.
Example 15
Astrocytes viability During progression of epilepsy and as response to dying neurons astrocytes increase in their number. This process is known as astrogliosis. To analyse this in vitro miRNAs of interest were overexpressed in primary astrocytes and the proliferation of these cells was measured using MTT.
Primary astrocytes were prepared from postnatal day zero or postnatal day one mouse brains (P0/P1 ), strain C57BL/6J using mechanical dissociation method (BD Nylon-strainer, Cat.# 352340) and grown in ten centimeter dishes. Medium used was DMEM/F12 (Gibco, Cat.# 31765) supplemented with 10% FBS (Gibco, Cat.# 10082139) and 1 % Penicillin/ Streptomicin, changed one day upon plating and then every two days.
Upon reaching 90% confluence, primary astrocytes were trypsin ized and plated in 96 well plates. 72 hours upon lentiviral transduction at MOI100 growth rates of secondary astrocytes were measured by MTT assay in period of 10 days (see FIGURE
16).
Overexpression of miR-2137 in primary astrocytes leads to a significant decrease in astrocyte proliferation (see FIGURE 17).
In a further approach primary astrocytes were prepared from postnatal mouse brains (P0-P3), strain C57BL/6J using mechanical dissociation method (BD Nylon-strainer, Cat. # 352340) and grown in T25 flask pro brain. The medium used was DMEM GlutaMax (Gibco, Cat. # 31966-021 ) supplemented with 10%FBS FBS (Gibco, Cat.# 10082139) and 1 % Penicillin/Streptomicin, changed the medium every three days upon plating. A confluent layer (70-80%) of cells was generated after 3-4 days. Primary astrocytes were trypsinized and raised in T75 flask for the next 5-7 days. Trypsinized astrocytes after about two weeks (70-80% confluence) and plated into 96 well plates. 72 hours upon lentiviral transduction at MOI30 growth rates of secondary astrocytes were measured by MTT assay over a period of 9 days (see FIGURE 25).
Fluorescence microscopy images of secondary mouse astroytes 72 hours upon lentiviral transduction is shown in FIGURE 26. Representative images of cultured mice astrocytes (P0-P3) after 72 hours lentviral transduction are depicted in FIGURE 27. The MTT assay was used to evaluate viability of astrocytes in vitro after 72 hours of lentiviral transduction. The overexpression of miR-124-3p in primary astrocytes leads to a significant decrease in astrocyte proliferation in comparison to wild-type and miR-control transduced cells (see FIGURE 28).
Conclusions
The in vitro findings of Examples 12 to 15 underline an important role for micro RNAs in primary neurons, astrocytes, microglia cells and human neuroblastoma! cells SH-SY5Y. Neuronal toxicity, astrogliosis, microglia activation and neuroblastoma proliferation are regulated by finely tuned transcriptional networks and biochemical pathways. It remains to be determined how exactly the onset of pathophysiological changes in epilepsy is regulated.
It could be shown that miRNAs dysregulated during acute and chronic phase of epilepsy play an important role in above described events. In detail, miR-124-3p and miR-142-3p decreased SH-SY5Y cell proliferation, respectively. The presented excitotoxicity assay results suggest that overexpression of miR-124-3p has a neuroprotective effect in primary hippocampal neurons. At the same time miR-124-3p mimic overexpressing microglia showed significantly reduced secretion of IL-6 in comparison to wild-type cells. This result indicates that miR- 24-3p might have an antiinflammatory effect on primary microglia. In addition, lentivirus mediated miR-124-3p overexpression in astrocytes significantly decrease the proliferation compared to wild- type and miR-control overexpressing cells
Exampte l.6
A summary of the experimental results of examples 1 to 15, as well as of examples 17 to 20 is provided in the following Table 32, providing information on the identified miRNAs and underlying molecule results:
Figure imgf000135_0001

Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
GO
Figure imgf000139_0001
Figure imgf000140_0001
o
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
2013/077836
Example 17
Refined analysis of the epilepsy mouse models A more precise and refined analysis was performed on the mouse epilepsy samples using a more stringent analysis to identify the miRNAs commonly deregulated between the models. The two chronic mouse models considered as the most relevant were compared to the 6Hz single acute seizure model. The data was processed as follows:
Normalization and Preprocessing
Data is preprocessed and analyzed using bioconductor® (Gentleman et al., 2004) and R® (R Development Core Team, 201 1 ) tools. Red and green intensities and their respective background values are extracted from two-channel arrays. In order to avoid negative corrected intensities and to reduce variability of low intensity log- ratios the norm exponential convulsion method is used for background correction (Ritchie et al., 2007; Silver, Ritchie, & Smyth, 2009; Smyth, 2005). Background- corrected values are then normalized and summarized to average log intensities and intensity log-ratios using loess normalization for within array normalization and quantile normalization for between array normalization, considering spot quality weights (Rao et al., 2008; Risso, Massa, Chiogna, & Romualdi, 2009; Smyth & Speed, 2003; Yang et al., 2002). A rigorous quality assessment confirmed the quality of the chips. The GAL file from Exiqon together with the 20th release of miRBase® (Griffiths-Jones, 2004; Kozomara & Griffiths-Jones, 20 1 ) were used for chip annotation.
Differential Expression Analysis
Differential expression analyses for miRNAs were performed using "limma" Linear models for Microarray Data (Smyth, 2005) utilizing the empirical Bayes method (Casella, 1985). Statistical dependencies of samples between different conditions and replicates were considered via a factorial design matrix in "limma" using a "condition-replicate" factor. Contrasts are considered for interaction effects. Correlations between the technical quadruplicates in the chips are taken into consideration and Spot quality weights are used (manually flagged spots, empty, poor and negative spots are down-weighted with 0.7, 0.4, 0.2 and 0.1 factors respectively). Corrections for multiple testing was done using Benjamini & Hochberg's method (Benjamini & Hochberg, 1995). Significant differentially expressed miRNAs are reported at FDRBH < 0.05. In the differential expression analyses, we compared pilocarpine-treated mice samples to their counterpart wild type samples at 24 hours and 28 days respectively. SSSE samples at 24 hours and 28 days are compared to a single SSSE control group. In 6 hertz data, samples at the subsequent time points (3, 6, 24 and 72 hours) are compared to the samples at the initial time point 0 hours.
Up- and down regulated miRNAs Comparing the individual time points within each model to their corresponding control groups we identified number of differentially expressed miRNAs. All significantly changed miRNAs (Benjamini & Hochberg corrected p value < 0.05) displaying a fold change (FC) of > 1 were defined as up-regulated miRNAs, whereas miRNAs with a FC of < 1 were defined as down-regulated. Tables 33 to 39, below, summarize the annotations of these differentially expressed miRNAs in all three models analyzed.
Pilocarpine 24 h
miRNA FC p-value FDR.BH Regulation mmu-miR-2137 11.66 1.20E-64 2.17E-60 up mmu-miR-21-5p 2.80 9.97E-59 9.00E-55 up
mmu-miR-711 1.94 7.79E-25 2.01 E-21 up
mmu-miR-212-3p 1.91 5.99E-52 3.60E-48 up mmu-miR-882 1.90 2.54E-24 5.73E-21 up mmu-miR-1947-5p 1.72 6.54E-04 3.91 E-02 up
mmu-miR-21-3p 1.65 1.04E-15 8.15E-13 up mmu-miR-142-5p 1.58 4.10E-10 1.35E-07 up mmu-miR-467d-5p 1.55 5.65E-04 3.46E-02 up
mmu-miR-132-3p 1.52 6.26E-23 1.03E-19 up rnmu-miR-710 1.50 2.09E-19 2.70E-16 UP
mmu-miR-712-5p 1.48 9.37E-18 9.40E-15 up mmu-miR-223-3p 1.47 1.78E-23 3.57E-20 up
mmu-miR-142-3p 1.41 3.09E- 5 2.32E-12 UP
mmu-miR-706 1.39 5.36E-12 2.76E-09 UP
mmu-miR-691 1.32 2.68E-10 9.66E-08 up mmu-miR-294-5p 1.31 5.41 E-15 3.91E-12 UP
mmu-miR-709 1.30 7.20E-08 1.30E-05 up mmu-miR-22-3p 1.29 3.00E-10 1.04E-07 up
mmu-miR-29a-3p 1.28 5.82E-05 5.17E-03 up mmu-miR-431-5p 1.27 6.06E-17 5.21 E-14 up
mmu-miR-126-3p 1.25 2.80E-08 5.69E-06 up mmu-miR-29b-1-5p 1.25 3.77E-12 2.00E-09 up
mmu-miR-483-3p 1.24 1.06E-05 1.18E-03 up mmu-mtR-29b-3p 1.24 3.04E-04 2.06E-02 up mmu-miR-1892 1.23 1.31E-04 1.03E-02 UP mmu-miR-1957 1.22 2.87E-04 1.95E-02 up mmu-let-7a-5p 1.22 1.95E-04 1.42E-02 up mmu-miR-23a-3p 1.21 1.10E-09 3.00E-07 up mmu-let-7e-5p 1.19 5.25E-07 7.58E-05 up mmu-miR-19a-3p 1.19 3.12E-05 3.06E-03 up mmu-miR-494-3p 1.19 1.74E-08 3.61 E-06 up mmu-miR-24-2-5p 1.17 1.37E-05 1.49E-03 up mmu-miR-335-3p 1.17 4.81 E-04 2.99E-02 up mmu-miR-146b-5p 1.16 3.98E-09 1.02E-06 up mmu-miR-203-3p 1.16 9.59E-06 1.09E-03 up mmu-miR-875-3p 1.16 1.87E-07 2.98E-05 up mmu-miR-1983 1.15 1.74E-04 1.30E-02 up mmu-miR-1897-5p 1.15 3.75E-04 2.42E-02 up mmu-miR-17-5p 1.12 3.88E-05 3.64E-03 up mmu-miR-146a-5p 1.12 1.27E-04 1.00E-02 up mmu-miR-1895 1.12 4.10E-05 3.77E-03 up mmu-miR-290-5p 1.11 1.85E-06 2.32E-04 UP mmu-miR-674-5p 1.11 4.88E-04 3.00E-02 up mmu-miR-1935 1.11 7.03E-05 6.05E-03 up mmu-mlR-331-3p 0.93 3.61E-04 2.37E-02 down mmu-let-7d-3p 0.92 3.35E-04 2.24E-02 down mmu-miR-181 c-5p 0.92 3.58E-04 2.36E-02 down mmu-miR-324-5p 0.92 7.04E-05 6.05E-03 down mmu-let-7b-3p 0.91 3.13E-04 2.11 E-02 down mmu-miR-194-5p 0.91 2.19E-04 1.57E-02 down mmu-miR-409-5p 0.91 1.31 E-04 1.03E-02 down mmu-miR-125b-2-3p 0.90 2.59E-04 1.81 E-02 down mmu-miR-1941 -3p 0.90 2.21 E-06 2.74E-04 down mmu-miR-467d-3p 0.90 2.86E-04 1.95E-02 down mmu-miR-433-3p 0.90 5.23E-05 4.67E-03 down mmu-miR-30a-5p 0.89 4.37E-04 2.78E-02 down mmu-miR-149-5p 0.89 3.68E-07 5.54E-05 down mmu-miR-668-3p 0.89 1.64E-05 1 69E-03 down mmu-miR-873-5p 0.89 2.78E-04 1.91 E-02 down mmu-miR-181d-5p 0.89 5.50E-07 7.87E-05 down mmu-miR-467e-5p 0.88 1.08E-04 8.82E-03 down mmu-miR-181 b-5p 0.88 2.67E-07 4.19E-05 down mmu-miR-466d-3p 0.88 1.64E-05 1.69E-03 down mmu-miR-1839-3p 0.87 1.22E-08 2.72E-06 down mmu-miR-124-5p 0.87 4.22E-08 8.11 E-06 down mmu-miR-186-5p 0.87 1.62E-08 3.44E-06 down mmu-miR-224-5p 0.87 4.82E-05 4.40E-03 down mmu-miR-218-2-3p 0.87 1.28E-05 1.41 E-03 down mmu-miR-881-3p 0.87 3.91 E-05 3.66E-03 down mmu-miR-330-5p 0.86 3.07E-08 6.15E-06 down
mmu-miR-491-5p 0.86 1.13E-06 1.52E-04 down mmu-miR-337-5p 0.86 5.86E-08 1.09E-05 down mmu-miR-380-3p 0.86 3.91 E-08 7.67E-06 down mmu-miR-542-3p 0.85 6.90E-04 4.08E-02 down mmu-miR-361-5p 0.85 2.75E-08 5.64E-06 down mmu-miR-181 a-1 -3p 0.84 1.86E-05 1.90E-03 down mmu-miR-669h-3p 0.84 1.55E-07 2.57E-05 down mmu-miR-1224-5p 0.84 3.32E-05 3.20E-03 down mmu-miR-374-5p/
mmu-miR-374c-5p 0.83 1.34E-05 1.47E-03 down mmu-miR-466c-5p 0.82 6.09E-08 1.12E-05 down mmu-miR-208a-3p 0.81 4.74E-04 2.96E-02 down
mmu-miR-425-3p 0.79 7.55E-05 6.34E-03 down
mmu-miR-466g 0.79 4.95E-05 4.49E-03 down mmu-miR-760-3p 0.78 1.11 E-06 1.52E-04 down mmu-miR-673-5p 0.78 5.85E-04 3.56E-02 down mmu-miR-301 b-3p 0.77 1.44E-05 1.55E-03 down mmu-miR-742-3p 0.77 6.99E-05 6.05E-03 down mmu-miR-488-5p 0.76 7.95E-04 4.55E-02 down mmu-miR-216b-5p 0.76 8.48E-04 4.78E-02 down mmu-miR-761 0.75 1.89E-04 1.39E-02 down mmu-miR-499-5p 0.74 2.58E-04 1.81 E-02 down mmu-miR-1 b-5p 0.73 1.49E-04 1.14E-02 down mmu-miR-449b 0.70 3.79E-05 3.58E-03 down mmu-miR-211-5p 0.68 1.63E-04 1.24E-02 down mmu-miR-703 0.66 7.51 E-04 4.36E-02 down mmu-miR-339-3p 0.66 6.31 E-04 3.78E-02 down mmu-miR-466l-3p 0.61 1.31 E-07 2.22E-05 down mmu-miR-302b-5p 0.59 1.12E-04 9.10E-03 down
Table 33: Significantly up- and downregulated miRNAs in the pilocarpine model at the timepoints 24h and 23d (p < 0.05 and FDR < 0.05). Bold = commonly deregulated in the SSSE and Pilocarpine model; bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz model. SSSE 24
mi NA FC p-value FDR.BH Regulation mmu-miR-2137 6.58 1.21E-47 7.27E-44 up mmu-miR-21-5p 2.68 4.35E-56 7.84E-52 up mmu-miR-711 1.86 2.95E-23 5.32E-20 up mmu-miR-212-3p 1.83 6.22E- 8 5.61 E-44 up mmu-miR-21-3p 1.74 7.74E-20 9.97E-17 UP mmu-miR-712-5p 1.66 3.79E-26 8.55E-23 UP mmu-miR-132-3p 1.58 1.14E-26 2.95E-23 up mmu-miR-882 1.55 1.52E-13 1.10E-10 up mmu-mlR-142-5p 1.44 6.B1E-07 1.49E-04 up mmu-miR-710 1.41 1.43E-15 1.36E-12 up mmy-miR-142-3p 1.39 1.24E-14 1.07E-11 UP mmu-miR-431 -5p 1.39 3.94E-27 1.19E-23 UP mmu-miR-762 1.36 4.10E-08 1.14E-05 up mmu-miR-1892 1.35 5.38E-08 1.43E-05 P mmu-miR-20a-5p 1.35 1.82E-06 3.81 E-04 up mmu-miR-291 -5p 1.34 2.51 E-05 4.20E-03 up mmu-miR-29b-1-5p 1.29 1.86E-15 1.68E-12 up mmu-fniR-294-5p 1.27 1.48E-12 9.90E-10 UP mmu-miR-1899 1.27 1.48E-05 2.67E-03 up mmu-miR-455-3p 1.25 1.47E-08 4.58E-06 up mmu-miR-706 1.25 2.01 E-06 4.12E-04 up mmu-miR-665-3p 1.24 6.90E-11 2.89E-08 up mmu-miR-291a-5p 1.23 1.17E-04 1.59E-02 up mmu-miR-675-5p 1.22 4.88E-08 1.33E-05 up mmu-miR-1934-5p 1.21 1.57E-04 1.98E-02 up mmu-miR-183-3p 1.21 1.15E-04 1.58E-02 up mmu-miR-18a-5p 1.20 9.04E-05 1.31 E-02 up mmu-miR-129-5p 1.20 1.17E-08 3.69E-06 up mmu-miR-203-3p 1.18 1.89E-06 3.92E-04 up mmu-miR-146a-5p 1.18 3.25E-08 9.30E-06 up mmu-miR-19a-3p 1.16 2.92E-04 3.31 E-02 up mmu-miR-187-3p 1.16 8.90E-05 1.29E-02 up mmu-miR-17-5p 1.15 3.46E-07 8.11E-05 up mmu-miR-298-5p 1.14 2.08E-04 2.47E-02 up mmu-miR-674-5p 1.13 3.40E-05 5.58E-03 up mmy-miR-146b-§p 1.13 2.68E-06 5.37E-04 up mmu-miR-494-3p 1.12 1.38E-04 1.81 E-02 up mmu-miR-770-3p 1.11 4.62E-04 4.96E-02 up mmu-let-7f-1-3p 1.09 2.44E-04 2.84E-02 up mmu-miR-290-5p 1.09 2.51 E-04 2.90E-02 up mmu-miR-324-5p 0.92 1.73E-04 2.11 E-02 down mmu-miR-328-3p 0.91 1.06E-04 1.48E-02 down mmu-miR-181b-5p 0.91 8.50E-05 1.25E-02 down mmu-mlR-331-3p 0.90 1.35E-06 2.89E-04 down mmu-mi'R-362-3p 0.90 3.31 E-04 3.73E-02 down mmu-miR-677-5p 0.90 9.24E-05 1.32E-02 down mmu-miR-124-5p 0.89 5.86E-06 1.12E-03 down mmu-miR-214-3p 0.89 1.63E-04 2.02E-02 down mmu-miR-485-3p 0.89 1.44E-06 3.06E-04 down mmu-miR-741-3p 0.89 4.10E-04 4.46E-02 down mmu-miR-151 -3p 0.89 3.80E-04 4.20E-02 down mmu-miR-297c-5p 0.89 1.35E-04 1.79E-02 down mmu-miR-881-3p 0.88 4.13E-04 4.46E-02 down mmu-miR-669a-5p/
mmu-miR-669p-5p 0.88 1 26E-04 1.70E-02 down mmu-miR-466b-3-3p 0.88 2.03E-07 4.88E-05 down mmu-miR-466f-3p 0.88 3.50E-04 3.92E-02 down mmu-miR-181 d-5p 0.88 9.75E-08 2.48E-05 down mmu-miR-378-3p/
mmu-miR-378b 0.88 7.18E-06 1.36E-03 down mmu-miR-30a-5p 0.88 5.32E-05 8.28E-03 down mmu-miR-150-5p 0.88 8.53E-08 2.20E-05 down mmu-miR-467f 0.88 1.52E-04 1.96E-02 down mmu-miR-99a-5p 0.87 2.08E-05 3.60E-03 down mmu-miR-30b-3p 0.87 1.19E-05 2.19E-03 down mmu-miR-669n 0 87 1.53E-04 1.96E-02 down mmu-miR-466b-5p 0.87 1.02E-04 1.43E-02 down mmu-miR-466f-5p 0.87 1.91 E-05 3.35E-03 down mmu-miR-1187 0.86 2.23E-05 3.80E-03 down mmu-miR-1839-3p 0.86 5.87E-10 2.30E-07 down mmu-miR-30c-5p 0.86 2.01 E-04 2.41 E-02 down mmu-miR- 903 0.86 4.70E-11 2.07E-08 down mmu-miR-468-3p 0.86 1.1 E-07 2.89E-05 down mmu-miR-467g 0.85 1.09E-07 2.74E-05 down mmu-miR-297a-5p 0.85 2.32E-08 7.09E-06 down mmu-miR-466a-5p/
mmu-miR-466p-5p 0.85 1.01 E-05 1 88E-03 down mmu-miR-466c-5p 0.83 4.30E-07 9.95E-05 down mmu-miR-466a-3p/
mmu-miR-466e-3p 0.83 1.07E-11 5.36E-09 down mmu-miR-669o-5p 0.83 2.13E-04 2.52E-02 down mmu-miR-669f-3p 0.83 5.02E-08 1.35E-05 down mmu-miR-669m-3p 0.82 3.27E-09 1.18E-06 down mmu-miR-466i-3p 0.81 8.19E-12 4.35E-09 down mmu-miR-297a-3p/
mmu-miR-297b-3p/
mmu-miR-297c-3p 0.81 8.52E-12 4.39E-09 down mmu-miR-467d-3p 0.81 5.26E-12 3.06E-09 down mmu-miR-669i 0.80 2.29E-11 1.06E-08 down mmu-miR-669h-3p 0.80 1.65E-11 7.83E-09 down mmu-mIR-467b-3p 0.79 3.14E-12 1.89E-09 down
mmu-miR- 66d-3p 0.78 3.10E-1 2.43E-11 down
mmu-miR-1192 0.78 1.51 E-13 1.10E-10 down
Table 34: Significantly up- and down regulated miRNAs in the SSSE model at the timepoint 24h (p < 0.05 and FDR < 0.05). Bold = commonly deregulated in the SSSE and Pilocarpine model; bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz model.
Figure imgf000156_0001
mlRNA FC p-value FDR.BH Regulation
mmu-miR-135b-5p 1.74 3.47E-20 6.26E-16 up
mmu-miR-21-5p 1.43 1.97E-13 8.87E-10 up
mmu-miR-1971 1.34 2.54E-07 1.97E-04 up
mmu-miR-221-3p 1.34 7.51 E-20 6.78E-16 up
mmu-miR-455-3p 1.32 7.63E-12 2.29E-08 up
mmu-miR-222-3p 1.27 1.33E-16 8.02E-13 UP
mmu-miR-669c-5p 1.25 2.06E-05 6.41 E-03 up
mmu-miR-466f-3p 1.25 2.21 E-09 4.44E-06 up
mmu-miR-466g 1.25 1.72E-04 3.26E-02 up
mmu-miR-669l-5p 1.22 6.04E-06 2.60E-03 up
mmu-miR-106b-3p 1.21 2.50E-04 4.47E-02 up
mmu-miR-1907 1.21 1.05E-05 4.01 E-03 up
mmu-miR-669m-3p 1.21 1.41 E-08 1.69E-05 up
mmu-miR-466d-5p/
mmu-miR-466n-5p 1.20 1.74E-07 1.65E-04 up
mmu-miR-494-3p 1.20 3.50E-09 6.32E-06 up
mmu-miR-669f-3p 1.20 2.31 E-07 1.89E-04 up
mmu-miR-881-5p 1.20 8.34E-07 5.37E-04 up
mmu-miR-467f 1.19 9.37E-07 5.83E-04 up
mmu-miR-574-5p 1.17 2.28E-07 1.89E-04 up
mmu-rniR-669h-3p 1.17 1.60E-06 8.48E-04 up
mmu-miR-1929-5p 1.17 1.00E-08 1.40E-05 up
mmu-miR-467g 1.17 2.97E-07 2.06E-04 up
mmu-miR-466i-3p 1.17 2 62E-07 1.97E-04 up
mmu-miR-669a-5p/
mmu-miR-669p-5p 1.16 5.85E-06 2.60E-03 up
mmu-miR- 67c-3p/
mmu-miR-467e-3p 1.16 1.24E-05 4.49E-03 up
mmu-miR-669n 1.16 9.98E-05 2.12E-02 up
mmu-miR-466c-5p 1.16 4.53E-05 1.22E-02 up
mmu-miR-883a-5p 1.15 5.48E-05 1.41E-02 up mmu-miR-669i 1.15 1.80E-05 6.02E-03 up
mmu-miR-881-3p 1.15 1.44E-04 2.93E-02 up
mmu-miR-467b-3p 1.15 2.91 E-05 8.68E-03 up
mmu-miR-467e-5p 1.14 3.17E-05 8.93E-03 up
mmu-miR-467d-3p 1.14 1.91 E-05 6.17E-03 up
mmu-miR-1192 1.14 7.12E-05 1.65E-02 up
mmu-miR-466d-3p 1.14 2.95E-05 8.68E-03 up
mmu-miR-146a-5p 1.13 2.24E-05 6.85E-03 up
mmu-miR-466a-3p/
mmu-miR-466e-3p 1.12 1.23E-05 4.49E-03 up
mmu-miR-297a-3p/
mmu-miR-297b-3p/
mmu-miR-297c-3p 1.12 1.56E-04 3.05E-02 up
mmu-miR-883b-5p 1.12 1.90E-04 3.54E-02 up
mmu-miR-466b-3-3p 1.12 6.00E-06 2.60E-03 up
mmu-miR-1935 1.11 8.73E-05 1.90E-02 up
mmu-miR-1839-3p 0.91 6.12E-05 1.47E-02 down
mmu-miR-191-5p 0.91 1.91 E-05 6.17E-03 down
mmu-miR-S76-3p 0.90 5.11 E-05 1.34E-02 down
mmu-miR-195-5p 0.90 2.54E-04 4.49E-02 down
mmu-miR-674-3p 0.90 4.58E-06 2.18E-03 down
mmu-miR-378-3p/
mmu-miR-378b 0.89 8.57E-05 1.88E-02 down
mmu-miR-346-5p 0.89 7.33E-05 1.65E-02 down
mmuHmiR-140-3p 0.88 9.28E-06 3.68E-03 down
mmu-miR-30b-5p 0.88 1.24E-05 4.49E-03 down
mmu-miR-130a-3p 0.88 1.63E-05 5.55E-03 down
mmu-miR-150-5p 0.87 6.03E-08 6.40E-05 down
mmu-miR-351-5p 0.87 2.82E-07 2.03E-04 down
mmu-miR-7b-5p 0.87 9.32E-06 3.66E-03 down
mmu-miR-30a-5p 0.87 7.68E-06 3.22E-03 down
mmu-miR-148a-3p 0.85 3.86E-05 1.05E-02 down
mmu-miR-99a-5p 0.85 3.88E-07 2.59E-04 down
Table 35: Significantly up- and downregulated miRNAs in the SSSE model at the timepoint 28d (p < 0.05 and FDR < 0.05). Bold = commonly deregulated in the SSSE and Pilocarpine model; bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz model. 6Hz 3h
miRNA FC p-value FDR.BH Regulation mmu-mi'R-448-3p 1.74 3.71 E-05 3.02E-03 up mmu-miR-1298-5p 1.72 2.95E-05 2.47E-03 up mmu-miR-34c-3p 1.71 7.95E-07 9.20E-05 up mmu-miR-34b-5p 1.60 1.12E-04 8.32E-03 up mmu-miR-34c-5p 1.56 3.95E-04 2.63E-02 up mmu-miR-335-3p 1.52 2.34E-10 5.56E-08 up mmu-miR-204-5p 1.50 1.98E-04 1.39E-02 up mmu-miR-142-5p 1.46 4.94E-07 5.93E-05 up mmu-miR-106b-3p 1.45 1.36E-04 9.84E-03 up mmu-miR-1957 1.43 6.62E-06 6.36E-04 up mmu-miR-184-3p 1.38 7.70E-05 5.97E-03 up mmu-miR-34b-3p 1.38 3.20E-10 7.40E-08 up mmu-miR-219-2-3p 1.28 1.37E-08 2.17E-06 up mmu-miR-207 1.28 4.04E-18 3.94E-14 up mmu-miR-133b-3p 1.28 6.17E-08 8.77E-06 up mmu-miR-1961 1.26 3.01 E-05 2.50E-03 up mmu-miR-1192 1.25 3.31 E-07 4.11 E-05 up mmu-miR-1983 1.24 1.10E-09 2.25E-07 up mmu-miR-133a-3p 1.24 3.06E-04 2.10E-02 up mmu-miR-302a-3p 1.24 6.65E-12 2.35E-09 up mmu-miR-5097 1.23 1.30E-12 5.34E-10 up mmu-miR-31-5p 1.23 2.80E-05 2.36E-03 up mmu-miR-212-3p 1.23 2.08E-11 6.24E-09 up mmu-miR-125a-5p 1.23 2.03E-06 2.20E-04 up mmu-miR-883b-5p 1.22 6.61 E-04 4.20E-02 up mmu-miR-690 1.22 4.99E-10 1.13E-07 up mmu-miR-491 -3p 1.21 2.03E-15 3.53E-12 up mmu-miR-132-3p 1.21 8.27E-13 3.55E-10 up mmu-miR-34a-5p 1.21 7.1 E-07 8.39E-05 up mmu-miR-532-5p 1.21 1.23E-04 9.00E-03 up mmu-miR-24-3p 1.21 2.89E-09 5.42E-07 up mmu-miR-135b-5p 1.21 2.52E-04 1.75E-02 up mmu-miR-1929-5p 1.21 5.42E-05 4.27E-03 up mmu-miR-126-3p 1.20 6.75E-09 1.17E-06 up mmu-miR-210-3p 1.20 6.91 E-05 5.37E-03 up mmu-miR-767 1.20 9.82E-08 1.35E-05 up mmu-miR-125b-5p 1.20 2.05E-06 2 20E-04 up mmu-miR-338-3p 1.20 1 93E-05 1.70E-03 up mmu-miR-431-3p 1.19 1.27E-04 9.22E-03 up mmu-miR-181d-5p 1.19 5.35E-11 1.46E-08 up mmu-miR-290-5p 1.19 2.25E-07 2.88E-05 up mmu-miR-124-3p 1.19 7.17E-07 8.40E-05 up mmu-let-7b-5p 1.19 1.22E-08 2.03E-06 up mmu-mi'R-1839-5p 1.19 4.53E-04 2.96E-02 up mmu-miR-494-3p 1.19 3.08E-04 2.1 1 E-02 up mmu-let-7a-5p 1.18 9.39E-06 8.77E-04 up mmu-miR-668-3p 1.18 3.74E-06 3.69E-04 up mmu-miR-491-3p 1.18 1.31 E-08 2.12E-06 up mmu-miR-872-3p 1.18 1 35E-08 2.15E-06 up mmu-miR-455-3p 1.17 1.38E-04 9.89E-03 up mmu-miR-181 a-5p 1.17 1.19E-08 2.01 E-06 up mmu-miR-222-3p 1.17 7.54E-07 8.77E-05 up mmu-let-7c-5p 1.17 2.17E-05 1.89E-03 up mmu-miR-185-5p 1.16 2.50E-08 3.77E-06 up mmu-miR-466d-3p 1.16 9.80E-05 7.43E-03 up mmu-miR-181 c-5p 1.16 3.23E-04 2.20E-02 up mmu-miR-138-1-3p 1.16 4.08E-07 5.01 E-05 up mmu-miR-330-3p 1.15 2.32E-06 2.43E-04 up mmu-miR-675-5p 1.15 2.60E-06 2.69E-04 up mmu-miR-503-3p 1.15 7.94E-08 1.11 E-05 up mmu-miR-129-2-3p 1.15 8.59E-06 8.07E-04 up mmu-miR-30a-5p 1.15 1.49E-06 1.66E-04 up mmu-miR-192-5p 1.15 4.32E-04 2.84E-02 up mmu-miR-409-5p 1.15 9.95E-05 7.51 E-03 up mmu-let-7i-5p 1.14 1.95E-06 2.12E-04 up mmu-miR-27b-3p 1.14 1.72E-05 1.54E-03 up mmu-miR-140-3p 1.14 5.49E-08 7.93E-06 up mmu-miR-331 -3p 1.14 9.53E-10 2.00E-07 up mmu-miR-103-3p 1.14 1.14E-05 1.04E-03 up mmu-miR-491-5p 1.14 3.71 E-06 3.67E-04 up mmu-miR-369-5p 1.14 5.67E-04 3.64E-02 up mmu-miR-300-5p 1.14 1.74E-05 1.54E-03 up mmu-miR-16-5p 1.14 3.34E-05 2.72E-03 up mmu-let-7f-5p 1.14 9.47E-05 7.24E-03 up mmu-let-7e-5p 1.14 4.63E-05 3.68E-03 up mmu-miR-667-3p 1.13 2.11 E-06 2.26E-04 up
mmu-miR-107-3p 1.13 1.28E-07 1.72E-05 up
mmu-miR-128-3p 1.13 1.69E-04 1.19E-02 up
mmu-miR-23b-3p 1.13 7.19E-06 6.83E-04 up
mmu-miR-129-1-3p 1.13 1.20E-Q4 8.79E-03 up
mmu-miR-100-5p 1.12 4.76E-05 3.77E-03 up
mmu-miR-361-5p 1.12 1.24E-04 9.02E-03 up
mmu-miR-106a-5p 1.12 2.66E-06 2.73E-04 up
mmu-miR-138-5p 1.12 2.01 E-05 1.75E-03 up
mmu-miR-652-3p 1.11 1.67E-04 1.19E-02 up
mmu-miR-376b-3p 1.11 7.69E-04 4.82E-02 up
mmu-miR-127-3p 1.11 8.48E-06 8.01 E-04 up
mmu-mi'R-744-5p 1.11 3.53E-06 3.52E-04 up
mmu-miR-106 -5p 1.10 4.47E-05 3.57E-03 up
mmu-miR-1900 1.10 1.69E-04 1.19E-02 up
mmu-miR-132-5p 1.10 1.56E-04 1.11 E-02 up
mmu-miR-434-5p 1.10 4.83E-04 3.14E-02 up
mmu-miR-328-3p 1.09 2.25E-05 1.94E-03 up
mmu-miR-26a-5p 1.09 6.99E-04 4.41 E-02 up
mmu-miR-30d-5p 1.09 2.58E-05 2.19E-03 up
mmu-miR-326-3p 1.08 3.01 E-04 2.07E-02 up
mmu-miR-139-3p 0.86 4.15E-04 2.74E-02 down
mmu-miR-488-3p 0.85 4.24E-04 2.79E-02 down
mmu-miR-1895 0.82 4.96E-07 5.93E-05 down
Table 36: Significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 3h following seizure (p < 0.05 and FDR.BH < 0.05).
Bold and italic - commonly deregulated in SSSE, Pilocarpine and 6Hz.
6Hz 6h
miRNA FC p-value FDR.BH Regulation mmu-miR-375-3p 2.55 6.19E-05 4.06E-03 up
mmu-miR-200c-3p 2.25 1.04E-04 6.48E-03 up
mmu-miR-760-5p 1.74 2.70E-05 1.93E-03 up
mmu-miR-140-5p 1.56 5.84E-05 3.85E-03 up
mmu-miR-184-3p 1.51 6.42E-07 6.19E-05 up
mmu-miR-142-5p 1.51 7.20E-08 8.28E-06 up
mmu-miR-106 b-3p 1.50 3.50E-05 2.43E-03 up
mmu-miR-1957 1.50 5.54E-07 5.44E-05 up
mmu-miR-7a-5p 1.47 3.79E-06 3.21 E-04 up
mmu-miR-335-3p 1.42 6.44E-08 7.45E-06 up
mmu-miR-299-3p 1.38 3 96E-04 2.17E-02 up
mmu-miR-1961 1.35 1.78E-07 1.90E-05 up mmu-miR-145-3p 1.33 9.66E-04 4.71 E-02 up mmu-miR-299-5p/mmu-miR-299b-5p 1.31 4.22E-05 2.85E-03 up mmu-miR-212-3p 1.31 2.20E-16 3.72E-13 up mmu-miR-323-3p 1.29 3.48E-05 2.43E-03 up mmu-miR-1839-5p 1.29 3.97E-07 4.02E-05 up mmu-miR-374-5p/mmu-miR-374c-5p 1.28 1.24E-07 1.34E-05 up mmu-miR-369-5p 1.28 9.26E-10 1.42E-07 up mmu-miR-496-3p 1.27 1.08E-06 9.91 E-05 up mmu-miR-148a-3p 1.27 7.58E-06 6.17E-04 up mmu-miR-5097 1.27 3.54E-15 2.56E-12 up mmu-miR-668-3p 1.26 2.60E-10 4.59E-08 up mmu-miR-133a-3p 1.26 1.03E-04 6.46E-03 up mmu-miR-290-5p 1.26 5.41E-11 1.13E-08 up mmu-miR-409-5p 1.26 6.83E-10 1.09E-07 up mmu-miR-302a-3p 1.25 5.96E-13 1.89E-10 up mmu-let-7f-5p 1.25 7.05E-11 1.35E-08 up mmu-miR-207 1.25 1.52E-15 1.34E-12 up mmu-miR-384-5p 1.24 4.30E-07 4.29E-05 up mmu-miR-34b-3p 1.24 1.54E-05 1.18E-03 up mmu-miR-376c-3p 1.24 1.03E-08 1.31 E-06 up mmu-miR-98-5p 1.24 4.03E-08 4.81 E-06 up mmu-miR-706 1.24 1.43E-04 8.67E-03 up mmu-let-7a-5p 1.23 4.27E-08 5.06E-06 up mmu-miR-431-3p 1.23 9.08E-06 7.25E-04 up mmu-miR-30a-5p 1.23 7.39E-12 1.85E-09 up mmu-miR-335-5p 1.23 2 26E-04 1.31 E-02 up mmu-let-7c-5p 1.22 5.84E-08 6.88E-06 up mmu-miR-27b-3p 1.22 4.59E-10 7.81 E-08 up mmu-miR-125b-5p 1.22 1.66E-07 1.78E-05 up mmu-miR-24-3p 1.22 5.21 E-10 8.55E-08 up mmu-miR-1 a-3p 1.22 1.24E-04 7.59E-03 up mmu-miR-431 -5p 1.22 4.82E-05 3.22E-03 up mmu-miR-222-3p 1.22 1.61 E-09 2.36E-07 up mmu-miR-125a-5p 1.22 5.52E-06 4.59E-04 up mmu-miR-1983 1.21 3.25E-08 3.96E-06 up mmu-miR-330-3p 1.21 4.84E-10 8.15E-08 up mmu-miR-23b-3p 1.21 3.39E-11 7.36E-09 up mmu-miR-133b-3p 1.21 2.48E-05 1.80E-03 up mmu-miR-543-3p 1.20 3.19E-06 2.74E-04 up mmu-miR-24-2-5p 1.20 3.42E-04 1.91 E-02 up mmu-miR-767 1.20 7.98E-08 9.06E-06 up mmu-miR-337-5p 1.20 2.47E-07 2.57E-05 up mmu-miR-132-3p 1.20 8.40E-12 2.05E-09 up mmu-miR-667-3p 1.20 5.78E-11 1.15E-08 up mmu-let-7b-5p 1.20 3.17E-09 4.46E-07 up mmu-miR-221-5p 1.20 5.19E-05 3.44E-03 up mmu-miR-218-2-3p 1.19 6.80Έ-05 4.44E-03 up mmu-miR-181 c-5p 1.19 2.45E-05 1.79E-03 up mmu-miR-137-3p 1.19 8.73E-06 7.03E-04 up mmu-miR-582-5p 1.19 6.49E-04 3.34E-02 up mmu-miR-491-5p 1.19 3.49E-09 4.88E-07 up mmu-miR-301a-3p 1.19 3.83E-08 4.61 E-06 up mmu-miR-425-5p 1.19 1.02E-05 8.05E-04 up mmu-let-7a-2-3p 1.19 1.88E-04 1.11 E-02 up mmu-miR-455-3p 1.19 5.54E-05 3.66E-03 up mmu-miR-31-5p 1.18 7.37E-04 3.73E-02 up mmu-miR-872-3p 1.18 4.66E-09 6.14E-07 up mmu-miR-30c-5p 1.18 7.00E-07 6.72E-05 up mmu-miR-532-5p 1.18 8.83E-04 4.36E-02 up mmu-miR-127-3p 1.18 6.44E-11 1.25E-08 up mmu-miR-361 -5p 1.18 9.28E-08 1.04E-05 up mmu-miR-185-5p 1.17 5.65E-09 7.33E-07 up mmu-miR-138-5p 1.17 2.16E-09 3.12E-07 up mmu-miR-487b-3p 1.17 1.18E-05 9.18E-04 up mmu-miR-376b-3p 1.17 1.21 E-06 1.11 E-04 up mmu-let-7e-5p 1.17 7.80E-07 7.37E-05 up mmu-miR-497-5p 1.17 3.28E-04 1.85E-02 up mmu-miR-30e-3p 1.17 7.02E-05 4.57E-03 up mmu-miR-467b-5p 1.17 1.11 E-04 6.87E-03 up mmu-miR-138-1 -3p 1.17 9.80E-08 1.09E-05 up mmu-miR-140-3p 1.17 4.18E-10 7.19E-08 up mmu-iet-7f-1-3p 1.16 4.51 E-04 2.43E-02 up mmu-miR-491 -3p 1.16 5.93E-10 9.56E-08 up mmu-miR-331 -3p 1.15 5.60Έ-1 1 1.14E-08 up mmu-miR-9-3p 1.15 1.91 E-04 1.13E-02 up mmu-miR-218-5p 1.15 7.18E-07 6.86E-05 up mmu-miR-192-5p 1.15 3.47E-04 1.93E-02 up mmu-miR-194-5p 1.15 2.94E-05 2.07E-03 up mmu-miR-690 1.15 6.98E-06 5.70E-04 up mmu-miR-23a-3p 1.15 3.72E-05 2.55E-03 up mmu-miR-103-3p 1.15 3.06E-06 2.66E-04 up mmu-miR-434-5p 1.15 5.84E-07 5.67E-05 up mmu-miR-337-3p 1.15 4.46E-07 4.42E-05 up mmu-miR-106a-5p 1.15 1.08E-08 1.35E-06 up mmu-let-7g-5p 1.15 1.97E-04 1.16E-02 up mmu-miR- 07-3p 1.15 1.72E-08 2.14E-06 up mmu-miR-154-5p 1.14 2.84E-05 2.02E-03 up mmu-miR-541-5p 1.14 6.75E-06 5.54E-04 up mmu-miR-1839-3p 1.14 8.68E-06 7.02E-04 up mmu-miR-377-3p 1.14 1.76E-04 1.05E-02 up mmu-let-7d-5p 1.14 7.18E-05 4.66E-03 up mmu-miR-127-5p 1.14 3.54E-06 3.02E-04 up mmu-miR-380-3p 1.14 4.51 E-06 3.76E-04 up mmu-miR-491-3p 1.13 6.23E-06 5.16E-04 up mmu-mi'R-30b-5p 1.13 2.51 E-05 1.81 E-03 up mmu-miR-29a-3p 1.13 5.69E-04 2.99E-02 up mmu-miR-382-5p 1.13 3.58E-04 1.98E-02 up mmu-miR-378-3p/mmu-miR-378b 1.13 3.39E-04 1.91 E-02 up mmu-miR-433-3p 1.13 2.95E-05 2.07E-03 up mmu-miR-130a-3p 1.13 3.88E-04 2.13E-02 up mmu-miR-181 d-5p 1.13 4.16E-06 3.51 E-04 up mmu-!et-7i-5p 1.13 2.48E-05 1.80E-03 up mmu-miR-124-3p 1.13 5.42E-04 2.88E-02 up mmu-miR-191 -5p 1.12 1.96E-06 1 75E-04 up mmu-miR-341-3p 1.12 1.49E-04 8.97E-03 up mmu-miR-129-2-3p 1.12 1.73E-04 1.03E-02 up mmu-miR-148b-3p 1.12 1.01 E-04 6.37E-03 up mmu-miR-381-3p 1.12 8.53E-04 4.24E-02 up mmu-miR-221-3p 1.12 3.08E-06 2.68E-04 up mmu-miR-99a-5p 1.12 1.17E-04 7.22E-03 up mmu-miR-26b-5p 1.12 1.36E-04 8.28E-03 up mmu-miR-300-5p 1.12 2.09E-04 1.22E-02 up mmu-miR-693-5p 1.12 3.86E-04 2.12E-02 up mmu-miR-129-1-3p 1.12 4.37E-04 2.36E-02 up mmu-miR-16-5p 1.11 5.89E-04 3.07E-02 up mmu-miR-744-5p 1.11 1.56E-06 1.41 E-04 up mmu-miR-328-3p 1.11 9.49E-07 8.78E-05 up mmu-miR-30d-5p 1.11 5.21 E-07 5.14E-05 up mmu-miR-652-3p 1.10 4.91 E-04 2.63E-02 up mmu-miR-150-5p 1.10 1.86E-04 1.10E-02 up mmu-miR-350-3p 1.10 7.01 E-04 3.56E-02 up mmu-miR-26a-5p 1.10 3.53E-04 1.96E-02 up mmu-miR-100-5p 1.10 9.78E-04 4.76E-02 up mmu-miR-503-3p 1.09 8.33E-04 4.16E-02 up mmu-miR-22-5p 1.08 1.27E-04 7.77E-03 up mmu-miR-149-5p 1.07 8.36E-04 4.17E-02 up mmu-miR-33-5p 0.87 1.22E-04 7 50E-03 down mmu-miR-466i-3p 0.84 7.57E-04 3.81 E-02 down mmu-miR-193-3p 0.81 7.62E-05 4.91 E-03 down mmu-miR-1895 0.80 3.73E-08 4.52E-06 down mmu-miR-466f-3p 0.79 1.11 E-05 8.67E-04 down mmu-miR-709 0.79 4.34E-06 3.64E-04 down mmu-miR-1249-3p 0.78 4.79E-04 2.57E-02 down mmu-miR-691 0.75 5.07E-09 6.63E-07 down Table 37: Significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 6h following seizure (p < 0.05 and FDR.BH < Θ.05). Bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz.
Figure imgf000164_0001
miRNA FC p-value FDR.BH Regulation
mmu-miR-760-5p 1.65 1.42E-04 4.76E-02 up
mmu-miR-142-5p 1.41 4.14E-06 7.47E-03 up
mmu-miR-184-3p 1.40 4.19E-05 2.37E-02 up
mmu-miR-335-3p 1.32 9.73E-06 1.12E-02 up
mmu-miR-218-2-3p 1.19 9.12E-05 3.50E-02 up
mmu-miR-137-3p 1.18 1.82E-05 1.49E-02 up
mmu-miR-27b-3p 1.17 2.94E-07 2.65E-03 up
mmu-miR-218-5p 1.17 2.29E-08 4.14E-04 up
mmu-miR-376c-3p 1.16 3.02E-05 1.88E-02 up
mmu-miR-138-5p 1.13 1.07E-06 3.22E-03 up
mmu-miR-30a-5p 1.13 1 95E-05 1.49E-02 up
mmu-miR-302a-3p 1.13 4 41 E-05 2.41 E-02 up
mmu-let-7b-5p 1.12 7.51 E-05 3.04E-02 up
mmu-miR-434-5p 1.11 6.58E-05 2.83E-02 up
mmu-miR-107-3p 1.10 3.39E-05 2.04E-02 up
mmu-miR-221 -3p 1.10 9.66E-05 3.56E-02 up
mmu-miR-331-3p 1.09 5.54E-05 2.63E-02 up
Table 38: Significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 24h following seizure (p < Θ.Θ5 and FDR.BH < 0.05). Bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz.
Figure imgf000164_0002
miRNA FC p-value FDR.BH Regulation
mmu-miR-335-3p 1.44 1.61 E-08 9.68E-05 up
mmu-miR-212-3p 1.15 4.60E-06 1.53E-02 up
mmu-miR-138-5p 1.12 1.19E-05 3.06E-02 up
mmu-miR-21 -3p 1.24 2.25E-05 4.51 E-02 up
Table 39: Significantly up- and downregulated miRNAs in the 6 Hz model at the timepoint 72h following seizure (p < 0.Θ5 and FDR.BH < 0.05). Bold and italic = commonly deregulated in SSSE, Pilocarpine and 6Hz. Example 18
Overlapping miRNA expression profiles in seizure models To identify miRNAs potentially associated with chronic form of epilepsy, we investigated the overlap of miRNA expression between the two chronic epilepsy models (pilocarpine and SSSE) and the acute seizure model (6 Hz) using Venn diagrams (FIGURE 18). Again, the analysis was based on all differentially expressed miRNAs (p value < 0.05) between epileptic and control animals within each model. First, the data for the 24 h time-point were compared between the three models. The Venn diagram showed overlap of three miRNAs between the acute and chronic models at the 24 h time point (Fig. 5A). Although the three miRNAs (miR-142-5p, miR-331-3p and miR-30a-5p) are differentially regulated, only miR-142-5p is consistently up- regulated in all three models. In contrast, miR-331-3p and miR-30a-5p are up-regulated in the chronic models and down-regulated in the 6Hz model at 24h. Two miRNAs are specifically overlapping between the pilocarpine and the 6Hz model. MiR-335-3p is consistently up-regulated while levels for miR-218-2-3p, show different regulation in both models (FIGURE 18A). Taken together, the number of overlapping miRNAs between the acute and chronic models is lower compared to the corresponding miRNAs between the two chronic models.
Furthermore, a detailed comparison of both chronic models is shown in FIGURE 18B. Among all the significant deregulated miRNAs of both chronic models only miR-494-3p is consistently up-regulated in all experimental conditions. Commonly deregulated miRNAs at the early time point (24 h) are higher (36 miRNAs) versus the late time point (28 d) with only 15 miRNAs. The annotations for these miRNA are shown in the table 1. The 36 miRNAs deregulated at the acute time point represent 36,4% and 41 ,4% of all the deregulated miRNAs of the pilocarpine and SSSE model respectively. Similarly the 15 miRNAs from the late time point represent 29,4% and 26,3% of all the deregulated miRNAs of the pilocarpine and SSSE model respectively. This indicates a larger overlap of the models at the early time-point relative to the more chronic stage of the model.
Analysis of the different time points in the acute 6 Hz seizure model showed that only early time points (3 h and 6 h) displayed substantial more miRNA deregulation, while at later time points less miRNAs are deregulated (24 h and 72 h) FIGURE 18C). The majority of the miRNAs (146) was significantly deregulated at the 6 h time-point. 73 miRNAs were identified to overlap at the earliest time points representing 73,7% and 50% of the miRNAs of the 3 h and 6h time-point. Of note, two miRNAs are commonly deregulated across all time points; miR-335-3p and miR-138- 5p. Taken together, the herein presented data show that the majority of changes in differential miRNA expression were detected before 24 h in the acute seizure model.
Common elements in Common elements in
"Ρ.2ΛΗ" and "S.24H": -P.28D" and "S.28D":
mmu-miR-2137 mmu-miR-135b-5p
mmu-miR-71 1 mmu-miR-676-3p
mmu-miR-882 mmu-miR-221-3p
mmu-miR-710 mmu-miR-140-3p
mmu-miR-712-5p mmu-miR-222-3p
mmu-miR-431 -5p mmu-miR-130a-3p
mmu-miR-21 -3p mmu-miR-467c-3p/
mmu-miR-142-3p mmu-miR-467e-3p
mmu-miR-294-5p mmu-miR-191-5p
mmu-miR-706 mmu-miR-669c-5p
mmu-miR-142-5p
mmu-miR-146b-5p
mmu-miR-124-5p
mmu-miR-181 b-5p
mmu-miR-181 d-5p
mmu-miR-290-5p
mmu-miR-19a-3p
mmu-miR-17-5p
mmu-miR-1892
Common elements in Common elements in
"P.24H", "S.24H" and "P.28D", "S.28D" and
P.28D: P.24H:
mmu-miR-212-3p mmu-miR-467e-5p
mmu-miR-132-3p
mmu-miR-29b-1 -5p
mmu-miR-203-3p
mmu-miR-324-5p
mmu-miR-331 -3p
mmu-miR-674-5p
Common elements in Common elements in
"P.24H", "S.24H" and "P.28D", "S.28D" and
S.28D: S.24H:
mmu-miR-21 -5p mmu-miR-455-3p
mmu-miR-1839-3p mmu-miR-669f-3p
mmu-miR-466c-5p mmu-miR-467g
mmu-miR-669h-3p mmu-miR-466f-3p mmu-miR-466d-3p
mmu-miR-881-3p
mmu-miR-146a-5p
mmu-miR-467d-3p
mmu-miR-30a-5p
Common elements in
"P.24H", "S.24H", "P.28D" and S.28D:
mmu-miR-494-3p
Table 40: Commonly deregulated miRNAs in the pilocarpine and SSSE model at 24h and 28d following SE
Example 19
Refined MicroRNA profiling analysis in hippocampal biopsies of Epilepsy patients A more precise and refined analysis was performed on a subset of the human patient samples using a more stringent analysis. The patients samples were described herein above. Table 41 , below gives an overview of the exact number of patients' samples included in this new analysis. As this analysis is considered as more stringent the identified deregulated miRNAs are believed to be more relevant.
Figure imgf000168_0001
Table 41 : MicroRNA profiling in human samples. The refined study included patients diagnosed with TLE and either AHS positive or negative. Hippocampal post mortem samples served as control
Description of the Deep Sequencing procedure was provided above. Using the Deep Sequencing data deregulated miRNAs were identified with the new analysis as follow: Analysis of the sequencing data was performed by miR-lntess small RNA analysis pipeline (InteRNA Technologies B.V., Netherlands). Reads were preprocessed to trim the adapter sequences and mapped against the human genome assembly GRCh37. Annotations of the mapped loci were retrieved from Ensemble database (v. 65) and from miRBase (v. 18) and aligned reads were classified according to these annotations. Prediction of novel miRNA candidates was performed by miR- Intess as described previously (Berezikov et al. 2010). Differentially expressed microRNA were identified using the Bioconductor edgeR package and applying an exact test for the negative binomial distribution (Robinson et al. 2010). Correction for multiple testing was done according to Benjamini and Hochberg (1995). Two different comparisons were performed: AHS versus nonAHS and AHS plus nonAHS versus post mortem. MiRNA candidates for which the false discovery rate (FDR) is lower than 0.05 were finally selected.
Lists of identified microRNA candidates that are significantly (FDR < 0.05) up and down regulated in hippocampal biopsies of Epilepsy patients are found in Table 42 and Table 43, below.
AHS versus non-AHS
miRNA FC FDR Regulation
hsa-mir-184-3p 0.10 0.0016 down
hsa-mir-129-5p 5.12 0.0127 up microRNA candidates deregulated in hippocampal biopsies of TLE patients. The microRNAs were identified in a Deep Sequencing approach. Comparison AHS versus nonAHS
AHS + non-AHS versus post-mortem
miRNA FC FDR Regulation
hsa-mir-1298-5p 0.03 0.0000 down
hsa-mir-1911-5p 0.02 0.0000 down
hsa-mir-181b-5p 0.36 0.0010 down
hsa-mir-34b-5p 0.08 0.0012 down microRNA candidates deregulated in hippocampal biopsies of TLE patients. The microRNAs were identified in a Deep Sequencing approach. Comparison AHS and non-AHS versus post-mortem
MiR As miR-184-3p and miR-129-5p were previously validated using qPCR (see also above). Example 20
Overlapping miRNAs based on the new analysis
The identified deregulated miRNA in the refined mouse and human analysis were compared to each other. MiRNAs deregulated in both species were identified regardless of their FC. Overall, only some miRNA candidates are similar regulated in the mouse and human profiling such as miR-129-5p and miR-181b-5p.
The following Table 44 summarizes the overlap between the 3 mouse models and human profiling.
Figure imgf000170_0001
Table 44: MicroRNA candidates overlapping in mouse models and human profiling

Claims

1. A pharmaceutical composition for use in the treatment or prevention of epilepsy, comprising a means for alleviating or reversing the effect of a dysregulation of at least one miRNA selected from section A or H of Table 1a.
2. The pharmaceutical composition of claim 1 , wherein said means comprises a nucleic acid molecule comprising a miRNA molecule as defined in section
A or H of Table 1a, or a derivative, fragment or variant thereof, or an antagonist thereof, wherein said nucleic acid molecule or antagonist has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section B or H of Table 1 a or to a complementary sequence thereof.
3. The pharmaceutical composition of claim 2, wherein said nucleic acid molecule or antagonist comprises between at least 18 and 24 nucleotides, preferably 20 nucleotides, 21 nucleotides or 22 nucleotides.
4. The pharmaceutical composition of claim 2 or 3, wherein said nucleic acid or antagonist is or comprises a DNA molecule or an RNA molecule, or a derivative thereof.
5. The pharmaceutical composition of claim 4, wherein said RNA molecule is or comprises a pre-miRNA selected from section E or H of Table 1a, or a derivative, fragment or variant thereof, wherein said pre-miRNA molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence identified in section F or H of Table 1 a or to a complementary sequence thereof.
6. The pharmaceutical composition of claim 3, wherein said DNA molecule is or comprises a DNA molecule coding for a pre-miRNA, a miRNA or for an antagonist.
7. The pharmaceutical composition of claim 6, wherein said DNA molecule is or comprises a double stranded DNA coding for at least one pre-miRNA selected from section E or H of Table 1 a.
8. The pharmaceutical composition of any one of claims 2 to 4, 6 or 7, wherein said nucleic acid molecule is comprised in a vector replicable in a subject.
9. The pharmaceutical composition of any one of claims 2 to 8, wherein said nucleic acid molecule or antagonist is modified by a conjugate, preferably a linked conjugate.
10. The pharmaceutical composition of any one of claims 2 to 9, wherein said nucleic acid molecule or antagonist comprises a chemically modified base.
11. The pharmaceutical composition of any one of claims 2, 3, 4, 9 or 10, wherein said antagonist is an antagomir.
12. The pharmaceutical composition of claim 10 or 11 , wherein said nucleic acid molecule or antagonist is modified by at least one modification selected from the group consisting of a 2'-0-methyl-ribonucleotide, a phosphorothioate bond, a N3'-P5' phosphoroamidate bond, a peptide-nucleic acid bond, a C-5 thiazole uracil, a C-5 propynyl-cytosine, a phenoxazine-modified cytosine, a 2'-0-propyl ribose and a 2'- methoxyethoxy ribose.
13. The pharmaceutical composition of any one of claims 2 to 12, wherein said nucleic acid molecule comprises a hsa-miR-124-5p, hsa-miR-1298, hsa-mir-181b- 5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, or hsa-miR- 191-5p, mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu-miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p or mmu-miR-191-5p molecule as defined in sections A or H of Table 1a, or a derivative, fragment or variant thereof, wherein said nucleic acid molecule has a nucleotide sequence at least 70%, 80%, 90% or 95% identical to the corresponding sequence as defined in sections B or H of Table 1a.
14. The pharmaceutical composition of any one of claims 2 to 12, wherein said antagonist is an antagonist of a hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b- 5p, hsa- miR-191-5p, hsa-miR-494-3p, hsa-miR-142-5p, hsa-miR-184-3, mmu-miR- 129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, mmu-miR-494-3p, mmu-miR-142-5p, mmu-miR-184-3p, mmu-miR-135b-5p or mmu-miR-222-3p molecule as defined in section A or H of Table 1a, or of a derivative, fragment or variant thereof.
15. A vaccine for use in the treatment or prevention of epilepsy, comprising a nucleic acid molecule or antagonist as defined in any one of claims 2 to 14.
16. An isolated biomarker or group of isolated biomarkers associated with epilepsy, wherein said isolated biomarker or group of isolated biomarkers comprises at least one isolated biomarker selected from the miRNAs identified in section A of Table 1a, or a derivative, fragment or variant thereof.
17. The isolated biomarker or group of isolated biomarkers of claim 16, wherein the increase of expression (up-regulation) of at least one isolated biomarker selected from the group of hsa-miR-129-5p, hsa-miR-142-3p, hsa-miR-34b-5p, hsa- miR-191-5p, or hsa-miR-494-3p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR-129-5p, mmu-miR-142-3p, mmu-miR-34b-5p, mmu-miR-191-5p, or mmu-miR-494-3p as defined in section A of Table 1a, when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
18. The isolated biomarker or group of isolated biomarkers of claim 16, wherein the decrease of expression (down-regulation) of at least one isolated biomarker selected from the group of hsa-miR-124-5p, hsa-miR- 298, hsa-mir- 81b- 5p, hsa-miR-34b-5p, hsa-miR-676-3p, hsa- miR-140-3p, hsa-miR-130a-3p, hsa- miR- 191-5p as defined in section H of Table 1a, or optionally selected from the group of human orthologues of mmu-miR-124-5p, mmu-miR-181 b-5p, mmu-miR-34b-5p, mmu- miR-676-3p, mmu-miR-140-3p, mmu-miR-130a-3p or mmu-miR-191-5pas defined in section A of Table 1a, when comparing the expression with the expression of a healthy subject, is indicative for epilepsy, or a predisposition for epilepsy.
19. A composition for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of claims 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of claims 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of claims 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of claims 16 to 18.
20. A method for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the step of determining the level of a biomarker or a group of biomarkers as defined in any one of claims 16 to 18 in a sample.
21. A method for monitoring epilepsy therapy comprising the step of determining the level of a biomarker or a group of biomarkers as defined in any one of claims 16 to 18 in a sample before and during an epilepsy treatment, optionally also after an epilepsy treatment.
22. The method of claim 21 , wherein said treatment is a treatment with a pharmaceutical composition as defined in any one of claims 1 to 14, and/or with at least one anticonvulsant selected from the group of lacosamide, levetiracetam, brivaracetam, phenobarbital, primidone, midazolam, clonazepam, topiramate, carbamazepine, oxcarbazepine, eslicarbazepine, mesuximide, ethosuximide, valproic acid and salts thereof, tiagabine, vigabatrine, gabapentin, pregabalin, phenytoin, lamotrigine, sultiam, felbamate, retigabine, zonisamide, and combinations thereof.
23. The method of any one of claims 20 to 22, wherein the determining step is accomplished by the measurement of the miRNA level(s) or by the determination of the biological effect of said biomarker or group of biomarkers.
24. An assay for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy comprising at least the steps (a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined in any one of claims 16 to 18;
(b) testing in a control sample for the expression of the same biomarker or group of biomarkers as in (a);
(c) determining the difference in expression of the biomarker or group of biomarkers of steps (a) and (b); and
(d) deciding on the presence of epilepsy or predisposition for epilepsy based on the results obtained in step (c) ,
wherein said testing steps are based on the use of a nucleic acid affinity ligand and/or a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of claims 16 to 18.
25. A method of identifying an individual eligible for an epilepsy therapy comprising:
(a) testing in a sample obtained from an individual for the expression of a biomarker or a group of biomarkers as defined in claim 17 or 18;
(b) testing in a control sample for the expression of a biomarker or a group of biomarkers as defined in claim 17 or 18;
(c) classifying the levels of expression of step (a) relative to the levels of step (b); and
(d) identifying the individual as eligible to receive an epilepsy therapy where the individual's sample is classified as having an increased level of expression of a biomarker or a group of biomarkers as defined in claim 17, or where the individual's sample is classified as having a decreased level of expression of a biomarker or a group of biomarkers as defined in claim 18.
26. A diagnostic kit for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy, comprising a detecting agent for a biomarker or group of biomarkers as defined in any one of claims 16 to 18, wherein said detecting agent is a nucleic acid affinity ligand for a biomarker or a group of biomarkers as defined in any one of claims 16 to 18, a peptide affinity ligand for a biomarker or a group of biomarkers as defined in any one of claims 16 to 18, an oligonucleotide specific for the biomarker or group of biomarkers as defined in any one of claims 16 to 18, or a probe specific for the biomarker or group of biomarkers as defined in any one of claims 16 to 18.
27. Use of a biomarker or a group of biomarkers as defined in any one of claims 16 to 18 as a marker for diagnosing, detecting, graduating, monitoring or prognosticating epilepsy or a predisposition for epilepsy.
28. Use of a biomarker or a group of biomarkers as defined in any one of claims 16 to 18 for identifying pharmaceutically active agents useful in the treatment or prevention of epilepsy.
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