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WO2016116768A1 - Agents neurothérapeutiques - Google Patents

Agents neurothérapeutiques Download PDF

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Publication number
WO2016116768A1
WO2016116768A1 PCT/GB2016/050145 GB2016050145W WO2016116768A1 WO 2016116768 A1 WO2016116768 A1 WO 2016116768A1 GB 2016050145 W GB2016050145 W GB 2016050145W WO 2016116768 A1 WO2016116768 A1 WO 2016116768A1
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Prior art keywords
sesn3
genes
module
expression
inventors
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Michael Johnson
Enrico Giuseppe PETRETTO
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Ip2ipo Innovations Ltd
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Imperial Innovations Ltd
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    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • 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]
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to diseases, disorders and conditions of the brain and particularly, although not exclusively, to the treatment and prevention of
  • the invention extends to agents that inhibit a particular gene implicated in the promotion of behavioural seizures.
  • Epilepsy is a serious neurological disorder affecting about i% of the world's population. Recently, a growing body of experimental and clinical data has implicated Toll-like receptor (TLR) signaling 1 and release of proconvulsant inflammatory molecules (i.e., IL- ⁇ ) in both seizure generation and epileptogenesis 2 ' 3 .
  • TLR Toll-like receptor
  • IL- ⁇ proconvulsant inflammatory molecules
  • the pathogenetic mechanisms linking these inflammatory processes with the development (and recurrence) of epileptic seizures in humans are unclear.
  • GWAS genome-wide association studies
  • exome sequencing approaches have so far provided limited insights into the genetic regulatory
  • transcriptional networks and pathways within pathologically relevant cells and tissues 13 ' 1 ⁇ Integrated analysis of transcriptional networks with genetic susceptibility data and phenotypic information allows specific transcriptional programmes to be connected to disease states, and thereby can identify disease pathways and their genetic regulators as new targets for therapeutic intervention ⁇ .
  • epilepsy surgery offers opportunities for gene expression profiling in ante-mortem brain tissue from pathophysiologically relevant brain structures such as the hippocampus 16 . This allows direct investigation of transcriptional programmes in brain tissue from living epilepsy patients.
  • the inventors have now integrated unsupervised network analysis of global gene expression in the hippocampi of patients with temporal lobe epilepsy (TLE) with GWAS data in a systems-genetics approach 1 ?. They have uncovered pathways and
  • the inventors have carried out validation experiments in independent in vitro and in vivo systems, which have confirmed the genetic regulation of the proconvulsant transcriptional program in epilepsy by Sestrin 3, therefore providing a first evidence of a function for SESN3 gene in disorders of the human brain.
  • the invention described herein is based upon the inventors' surprising discovery, in surgically acquired hippocampi from TLE patients, of a specialised, highly expressed transcriptional module encoding proconvulsive cytokines and TLR-signaling genes.
  • RNA-Seq analysis in a mouse model of TLE using epileptic and control hippocampi showed that the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy.
  • the inventors have mapped the irans-acting genetic control of this proconvulsive module to SESN3, and have demonstrated that SESN3 positively regulates the module in macrophages, microglia and neurons.
  • Morpholino-mediated sesn ⁇ knockdown in zebrafish confirmed the regulation of the transcriptional module, and attenuated chemically-induced behavioural seizures in vivo.
  • SESN3 positively regulates a proconvulsive gene co- expression module (Module 1 as described herein) and so inhibiting, reducing, knocking down or knocking out SESN3 gene expression or protein activity can ameliorate seizures.
  • an inhibitor of SESN3 gene expression or SESN3 protein activity can hence be used in therapy to treat neuroinflammation, and diseases, disorders and conditions in which neuroinflammation plays a part, such as epilepsy.
  • SESN3 gene and its protein product are known in the art, and their nucleic acid and amino acid sequences are thus publically available. Accordingly, in a first aspect, the invention provides an inhibitor of SESN3 gene expression for use in therapy.
  • the inhibitor of SESN3 gene expression is for use in a method of treating a disease, disorder or condition of the brain.
  • the inhibitor is for use in a method of treating neuroinflammation, or a disease, disorder or condition involving neuroinflammation.
  • the inhibitor is for use in a method of treating epilepsy.
  • the invention provides an inhibitor of SESN3 protein activity for use in therapy.
  • the inhibitor of SESN3 protein activity is for use in a method of treating a disease, disorder or condition of the brain. In a preferred embodiment, the inhibitor of SESN3 protein activity is for use in a method of treating
  • the inhibitor of SESN3 protein activity is for use in a method of treating epilepsy.
  • the inhibitor may be any agent capable of inhibiting SESN3 gene expression or SESN3 protein activity.
  • the agent may be a competitive or non-competitive antagonist of
  • the agent may be a biological agent, such as a protein or a nucleic acid, such as siRNA, or it may be a pharmaceutical agent.
  • “Inhibiting” can mean reducing the normal level of SESN3 gene expression or SESN3 protein activity by any amount.
  • the inhibitor may be an agent capable of reducing the level of SESN3 gene expression or protein activity by up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%.
  • Treating includes both preventing and ameliorating a disease, disorder or condition. Methods of prophylaxis and any and all methods that treat, reduce or help alleviate
  • the inhibitor may be administered to the subject to be treated on its own. It may be administered in a pharmaceutically acceptable vehicle.
  • the inhibitor according to the invention maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier.
  • Medicaments comprising agents of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
  • Agents according to the invention may also be incorporated within a slow- or delayed- release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or
  • SUBSTITUTE SHEET RULE 26 mode of administration the physiochemical properties of the agent, vaccine and medicament, and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the agent within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease, disorder or condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • a daily dose of between o.ooi g/kg of body weight and 10 mg/kg of body weight of agent or medicament according to the invention may be used for treating a disease, disorder or condition of the brain, depending upon which agent or medicament is used. More preferably, the daily dose is between o.oi ⁇ g/kg of body weight and ⁇ mg/kg of body weight, more preferably between o.i ⁇ g/kg and 100 ⁇ g/kg body weight, and most preferably between approximately o.i ⁇ g/kg and 10 ⁇ g/kg body weight.
  • the agent or medicament may be administered before, during or after onset of the disease, disorder or condition of the brain.
  • Daily doses maybe given as a single administration (e.g. a single daily injection).
  • the agent or medicament may require administration twice or more times during a day.
  • agents and medicaments may be administered as two (or more depending upon the severity of the disease, disorder or condition being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of agents, vaccines and medicaments according to the invention to a patient without the need to administer repeated doses.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents and medicaments according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of
  • a "disease, disorder or condition of the brain” includes any and all diseases, disorders and conditions that affect the brain.
  • the disease, disorder or condition involves neuroinflammation.
  • the disease, disorder or condition is epilepsy.
  • a "subject” may be a vertebrate, mammal, or domestic animal.
  • medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
  • livestock e.g. a horse
  • pets e.g. a human
  • the subject is a human being.
  • a “therapeutically effective amount” of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the disease, disorder or condition, or produce the desired effect.
  • the therapeutically effective amount of agent used may be from about o.ooi ng to about ⁇ mg, and preferably from about o.oi ng to about 100 ng. It is preferred that the amount of agent is an amount from about o.i ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention may be dissolved or suspended in a
  • liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of
  • compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • solutes or suspending agents for example, enough saline or glucose to make the solution isotonic
  • bile salts for example, enough saline or glucose to make the solution isotonic
  • acacia gelatin
  • sorbitan monoleate sorbitan monoleate
  • polysorbate 80 oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide
  • the agents used according to the invention can also be administered orally either in liquid or solid composition form.
  • Compositions suitable for oral administration include solid forms,
  • SUBSTITUTE SHEET RULE 26 elixirs, and suspensions forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the invention provides a method of treating a disease, disorder or condition of the brain in a subject comprising administering an inhibitor of SESN3 gene expression to the subject.
  • the invention provides a method of treating a disease, disorder or condition of the brain in a subject comprising administering an inhibitor of SESN3 protein activity to the subject.
  • the disease, disorder or condition of the brain, the subject, and the inhibitor may be as defined for the first and second aspects of the invention.
  • Figure 1 shows an identification of the TLE-network and functionally specialised transcriptional modules in human epileptic hippocampus,
  • Nodes represent genes and edges represent partial correlations between their expression profiles (FDR ⁇ 5%).
  • Node colour indicates the best GWAS P-value of association with focal epilepsy for SNPs within lookb of each gene (Supplementary Table 2). Boxes
  • Module-i and Module-2 details. The size of each node is proportional to its degree of inter-connectivity within each module. Light blue indicates genes showing nominal association with susceptibility to focal epilepsy. Numbers in parenthesis indicate multiple microarray probes representing the same gene, (d) KEGG pathways significantly enriched in Module-i (top) and Module-2 (bottom) (FDR ⁇ 5%). (e) Module-i is significantly highly expressed in the hippocampus of TLE patients.
  • Figure 3 shows that SESN3 is a trans-acting genetic regulator of Module-i in epileptic hippocampus, (a) Genome-wide mapping of genetic regulation of Module-i. For each autosome (horizontal axis), the strength of evidence for each SNP (filled dot) being a regulatory locus for the first PC of Module-i expression is measured by the logi 0 (Bayes
  • SUBSTITUTE SHEET RULE 26 Factor (vertical axis).
  • the Bayes Factor quantifies evidence in favor of genetic regulation versus no genetic control of module expression, and is reported as a ratio between the strengths of these models.
  • FDR i.e., logi 0 (Bayes Factor)>6, dashed line
  • SNP rsi050i829 nq2i, highlighted in red
  • Figure 4 shows that SESN3 regulates expression of Module-i genes in macrophages, microglial cells and neurons. Effect of siRNA-mediated knockdown of Sesn.3 as compared to control siRNA (siControl), showing significant inhibition of Sesn3 mRNA expression and downregulation of Module-i genes in murine LPS-stimulated (lhr) BMDM (a) and BV2 microglial cells (b), as well as in unstimulated BV2 microglial cells (c). Five independent biological replicates were used for BMDM experiments and at least three replicates in the BV2 microglia cells experiments. Data normalised to ⁇ - actin levels are shown as means relative to control ⁇ s.e.m. (d) SESN3
  • SESN3 cell fluorescence was assessed as follows: integrated density - (area of selected cell x mean fluorescence of background readings). SESN3 total cell fluorescence in TLE patients is significantly increased as compared to the SESN3 total cell fluorescence in autopsy samples (two-tailed Mann-Whitney test P ⁇ o.ooi).
  • Figure 5 shows that Sesn.3 modulates PTZ-induced c-fos expression, locomotor convulsions and Module-i genes in zebrafish.
  • sesn3 promotes convulsive locomotor response of zebrafish larvae exposed to PTZ.
  • Three dpf zebrafish larvae were incubated with and without 20mM PTZ for l-hr, during which locomotor activity was monitored continuously.
  • Larvae microinjected with sesn.3 morpholinos exhibited a sustained reduction in locomotor activity throughout the period of incubation with PTZ, in comparison with control morphant larvae.
  • sesn.3 morphant and control morphant larvae exhibited similarly low levels of locomotor activity in the absence of PTZ.
  • sesn3 morpholinos reduced the cumulative locomotor activity of zebrafish exposed to 2omM PTZ (black columns) without appreciably affecting basal locomotor activity of larvae incubated in the absence of PTZ (white columns)
  • (b) Co- injecting sesn3 morpholinos with synthetic sesn3 mRNA showed that sesn3 mRNA rescued the locomotor activity phenotype (total distance swam, y-axis). For each group, 16-18 larvae were analysed. Black bars, l-hr PTZ treatment (2omM).
  • Sesn3 morpholinos attenuate seizure-induced expression of the synaptic activity-regulated
  • Microarray probes were annotated using either the Human HT-12 v3 annotation file or Ensembl (release 72). All patients gave informed consent for use of their tissue and all procedures were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Bonn Medical Center.
  • GGMs Gene co-expression networks were reconstructed using GGMs, which use partial correlations to infer co-expression relationships between any microarray probe pair in
  • SUBSTITUTE SHEET RULE 26 the dataset, removing the effect of other probes 2 3.
  • the inventors used the empirical Bayes local FDR statistic 5 ⁇ to extract significant partial correlations (Supplementary Fig. 13), and which identified a large set of 2,124 inter-connected nodes belonging to the same connected component (TLE-network, Supplementary Data 2). Network extraction and identification of transcriptional modules are described in the
  • the inventors used Bayesian variable selection models 18 ' 29 to identify the genetic control points (regulatory 'hotspots') of transcriptional modules in the TLE patient cohort.
  • the inventors combined PC analysis 55 with multivariate regression approaches to prioritise genome-wide genomic regions associated with the module expression.
  • the inventors then analysed all genes of the module with all SNPs in the regulatory region using the hierarchical evolutionary stochastic search (HESS) algorithm 18 , where the module genes' expression are jointly considered. Further details are reported in Supplementary Methods.
  • HESS hierarchical evolutionary stochastic search
  • RNA-Seq analysis in whole hippocampus from 100 epileptic (pilocarpine model) 27 and 100 control na ' ive mice (NMRI) is detailed in Supplementary Methods. Briefly, raw reads were mapped to the reference mouse genome (mm 10) using TopHat version 2.0.8 56 and read counts per gene were normalised across all samples using the
  • siRNA knockdown experiments were performed in murine BMDMs and BV2 microglia cell lines using a mouse Sesn3 ON-TARGETplus SMARTpool siRNA (100 nM, ThermoFisher Scientific) and Dharmafect 1 (ThermoFisher Scientific) as transfection reagent, according to the manufacturer's recommendations.
  • LPS stimulation experiments the transfected cells were washed twice in DMEM and stimulated with LPS (Sigma, 100 ng/ml) for an hour.
  • LPS Long-generation Lentiviral Vectors
  • Embryos that were to be analysed by whole-mount in situ hybridisation were first treated with 1- phenyl-2 thiourea (PTU) at 23 h post-fertilisation (h.p.f.) to inhibit melanogenesis. At 3 days post-fertilisation (d.p.f.), larvae were treated for 1 h with 20 mM PTZ or left untreated, and all larvae were then fixed with paraformaldehyde immediately after the treatment period.
  • PTU 1- phenyl-2 thiourea
  • RNA in situ hybridisation analysis was carried out using a c-fos digoxigenin-labelled probe, which was prepared as recommended by the manufacturer of the in situ hybridisation reagents (Roche). Whole-mount in situ hybridisation was performed using standard procedures 58 . Analysis of zebrafish locomotor activity was carried out using the Viewpoint Zebrabox system (Viewpoint) as previously reported 34 . Rescue experiments were performed by co-injection of synthetic sesn3 RNA into one- cell stage AB wild type zebrafish embryos alone (2 nl of 0.3 ng/nl sesn3 mRNA) or in combination with sesn3 morpholinos. Additional details, including the quantitative
  • the inventors first assessed the degree of variation in gene expression between hippocampal subfields in TLE patients with hippocampal sclerosis (HS), and compared this with the total variation in gene expression measured both across subjects and between subfields (Supplementary Fig. l). The inventors found higher variability in gene expression across TLE subjects than between the hippocampal subfields alone, suggesting that variation in whole hippocampus expression can be used to infer co- expression networks in the hippocampus of TLE patients (Supplementary Fig. l).
  • the inventors investigated whether the transcriptome in the hippocampus of these 129 TLE patients was organised into discrete gene co-expression networks, and if these had functional implications for susceptibility to epilepsy.
  • Gene co-expression networks were reconstructed using Graphical Gaussian Models (GGMs) 23 , which identified a large co-expression network comprising 442 annotated genes (false discovery rate (FDR) ⁇ 5 , Fig. la and Supplementary Data 1).
  • GGMs Graphical Gaussian Models
  • This method interrogated high-confidence protein-protein interactions (PPI) to assess the physical connections among proteins encoded by the genes in the network.
  • gene expression may vary both as a cause and a consequence of disease, the inventors investigated the causal relationship between the TLE-network and epilepsy
  • SUBSTITUTE SHEET RULE 26 by integration with genetic susceptibility data.
  • DNA variation was used to infer causal relationships between the network and epilepsy by assessing whether the network as a whole was genetically associated with epilepsy.
  • the inventors used focal epilepsy GWAS data 6 ' 25 from a separate cohort of 1,429 cases (consisting mainly of patients with TLE) and 7,358 healthy controls.
  • Module-i was specifically enriched for gene ontology categories related to inflammatory mechanisms
  • Module-2 was enriched for cell-to-extracellular matrix adhesion processes (Fig. id and Supplementary Table 3), indicating functional sub-specialisation within the larger TLE-network.
  • the inventors observed that Module-i genes were significantly upregulated as compared with genes in the larger TLE-network, Module-2, or with respect to all other genes profiled in the hippocampus of TLE patients (Fig. le). This increased hippocampal expression of Module-i genes in TLE patients was not observed in separate gene expression data sets from the hippocampus of healthy subjects (i.e., individuals clinically classified as neurologically normal) (Supplementary Fig. 4).
  • the inventors investigated whether the TLE-network and Module-i genes, in particular, were conserved across-species and to this aim they carried out high- throughput sequencing of mRNA (RNA-Seq) in whole hippocampus from 100 epileptic (pilocarpine model) 2 ? and 100 control na ' ive mice (full details of this model are reported in the Supplementary Methods).
  • RNA-Seq mRNA
  • the inventors employed GGMs to assess the co- expression relationships between the 371 mouse orthologues of the human TLE- network genes, and found that 312 genes (84%) had significant co-expression
  • Example 3 - SESN3 is a genetic regulator of the pro-inflammatory network
  • the inventors set out to identify genetic variants that regulate the gene co-expression modules (i.e., regulatory 'hotspots') by employing genome-wide Bayesian expression QTL mapping approaches 18 2 ?. To this aim, the inventors have developed a multi-step strategy to identify SNPs that regulate the expression of a transcriptional module (or network) as a whole. As first step, the inventors summarised the expression of the gene co-expression modules (i.e., regulatory 'hotspots') by employing genome-wide Bayesian expression QTL mapping approaches 18 2 ?. To this aim, the inventors have developed a multi-step strategy to identify SNPs that regulate the expression of a transcriptional module (or network) as a whole. As first step, the inventors summarised the expression of the
  • PC principal component
  • This analysis prioritised genomic regions associated with variation in mRNA expression of the genes in each module.
  • the inventors regressed jointly the mRNA levels of module genes to all SNPs within the regulatory locus identified in the first step 18 . Given the functional specialisation within the large TLE-network (Fig. id), the inventors investigated the genetic regulation of both
  • Module-i and Module-2 by analysing 527,684 genome-wide SNPs in the TLE patient cohort.
  • Module-2 showed no significant genome-wide associations (Supplementary Fig. 6).
  • This analysis identified three additional SNPs (rs530i90, rs7i0766i and ⁇ 6483435) in the Best Model Visited (i.e., the best combination of SNPs predicting mRNA level of module genes, see Supplementary Methods) that were associated with the majority of genes of Module-i (58-74% of Module-i genes are predicted by individual SNPs, Fig. 3b).
  • the set of SNPs regulating in trans the expression of Module-i genes defined the boundaries of a minimal regulatory region spanning ⁇ 383kb (Fig. 3b).
  • SESN3 Sestrin 3
  • SESN3 Sestrin 3
  • Module-i gene expression remained significant following genome-wide correlation analysis in human hippocampus (P ⁇ o.00001, Supplementary Fig. 7).
  • SESN3 is the only gene within the minimal regulatory region and, when compared with all genes within a l-Mbp window around SNP rsi050i829, showed the strongest correlation with Module-i gene expression.
  • SESN3 as a candidate gene for the irans-acting genetic regulation of Module-i.
  • the inventors first carried out gene knockdown experiments followed by transcriptional analysis of Module-i genes by means of RNA interference using short interfering RNA (siRNA). Initially, they used murine bone-marrow-derived macrophages (BMDMs) and BV2 microglia cell line as an in vitro system as Module-i recapitulates the ATF3/AP1 transcriptional complex and IL-i signaling (Supplementary Fig. 8), known to be highly expressed in
  • BMDMs murine bone-marrow-derived macrophages
  • BV2 microglia cell line as an in vitro system as Module-i recapitulates the ATF3/AP1 transcriptional complex and IL-i signaling (Supplementary Fig. 8), known to be highly expressed in
  • LPS lipopolysaccharide
  • Fig. 4a-b Similar results were found in unstimulated BV2 microglial cells, suggesting that SESN3 can modulate expression of pro-inflammatory genes (e.g., IL- ⁇ , IL-iRN, IL-ia, TNFa) even in the absence of a strong inflammatory stimulus (Fig. 4c).
  • pro-inflammatory genes e.g., IL- ⁇ , IL-iRN, IL-ia, TNFa
  • Fig. 4c Within the human brain the inventors localised SESN3 expression to neurons by immunohistochemistry (Fig. 4d), and found that it is highly expressed in the hippocampus of TLE patients as compared with hippocampus from control autopsy samples (Fig.
  • SUBSTITUTE SHEET RULE 26 data reporting the activity of several inflammatory molecules in neuronal cells under pathological conditions 31 , including IL- ⁇ and its receptors 2 . Furthermore, the upregulation of proinflammatory genes in neurons supports the 'neurogenic inflammation' hypothesis, wherein neurons are proposed as triggers of innate and adaptive immune-cell activation in the central nervous system (CNS) (reviewed in Xanthos and Sandkuhler 33 ).
  • CNS central nervous system
  • Example 4 SESN3 regulates chemically-induced behavioural seizures
  • SESN3 is a positive regulator of Module-i.
  • inhibiting SESN3 would reduce the activity of genes in functional pathways enriched in Module-i, including proconvulsant signaling molecules, and thus by extension could have seizure- suppressing effects.
  • the inventors investigated the role of SESN3 in a zebrafish model of convulsant-induced seizures 34 ' 35 .
  • Sesn3 showed widespread expression in the brain of 3 and 4 days post fertilisation (d.p.f.) zebrafish larvae (Supplementary Fig. 11) and, following PTZ- treatment, the inventors found that sesn3 morphant zebrafish larvae exhibited significantly reduced locomotor activity as compared with control morphant larvae
  • Fig. 5a To test the specificity of the morpholino effect, the inventors co-injected the sesn3 morpholinos (Supplementary Fig. 12) along with synthetic sesn3 mRNA, which cannot be targeted by either of the splice-blocking morpholinos (see Supplementary Methods), and assessed whether the sesn3 mRNA could rescue the morphant phenotype. The inventors observed an almost complete rescue of the locomotor activity phenotype (only 10% difference between uninjected larvae and larvae co-injected with
  • SUBSTITUTE SHEET RULE 26 the TLE-hippocampus network with susceptibility to focal epilepsy. Within the TLE- network the inventors identified a functionally coherent and coordinated
  • SESN3 as a irans-acting genetic regulator of a proinflammatory transcriptional programme in the epileptic human hippocampus.
  • the positive regulation of this network by SESN3 was confirmed in vitro across different cell types by gene silencing (resulting in -50% reduction of Module-i gene expression) and overexpression experiments (resulting in ⁇ 2-7 fold activation of Module-i genes, Fig. 4), and in vivo using a zebrafish model of chemically-induced seizures (Fig. 5e).
  • SESN3 is a member of the Sestrin family of proteins that have been shown to decrease intracellular reactive oxygen species and to confer resistance to oxidative stress 19 .
  • Intrinsic antioxidant defenses are important for neuronal longevity and the genes that regulate these processes might well influence pathological processes associated with oxidative damage in the brain, a common feature of many neurodegenerative diseases including epilepsy 47 ' 48 . Therefore, the inventors hypothesised that SESN3 might regulate neuro-inflammatory molecules, previously implicated in epilepsy 1 ' 33 - 43 - 49 , through modulation of oxidative stress in the brain.
  • SUBSTITUTE SHEET RULE 26 the inventors used an experimental model of acute epileptic seizures 3 ⁇ ? and found that knockdown of sesn.3 attenuated chemical convulsant-induced locomotor activity and c- os expression, as well as modulating Module-i gene expression (Fig. 5).
  • the inventors' in vitro data in macrophages, BV2 microglial cells and primary neurons showed that SESN3 is a positive regulator of proinflammatory molecules (Fig. 4), including IL- ⁇ and TNF-a, major mediators of inflammation, which are capable of inducing changes in neuronal excitability 50 .
  • the inventors' data provide the first evidence of a function for SESN3 in regulating proconvulsant agents (e.g., TNF-a, ILi and TLR-signaling genes) in human epileptic hippocampus, and support the use of SESN3 as a new target for modulating brain inflammation 3 ⁇ and CNS excitability 53 .
  • proconvulsant agents e.g., TNF-a, ILi and TLR-signaling genes
  • SESN3 as a new target for modulating brain inflammation 3 ⁇ and CNS excitability 53 .
  • the inventors' systems genetics approach builds on and extends previous methods correlating individual genetic variation with disease susceptibility by identifying disease-associated gene networks, pathophysiological pathways and their upstream genetic regulators in human brain. References
  • Chemokine CXCLi enhances inflammatory pain and increases NMDA receptor activity and COX-2 expression in spinal cord neurons via activation of

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L'invention concerne des maladies, des troubles et des affections du cerveau, et des agents pour les traiter.
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