WO2016116768A1 - Neurotherapeutic agents - Google Patents

Abstract

The invention relates to diseases, disorders and conditions of the brain, and agents for treatment of the same.

Description

Neurotherapeutic Agents

Field of the Invention

The present invention relates to diseases, disorders and conditions of the brain and particularly, although not exclusively, to the treatment and prevention of

neuroinflammation, especially epilepsy. The invention extends to agents that inhibit a particular gene implicated in the promotion of behavioural seizures.

Background of the Invention

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) signaling¹ and release of proconvulsant inflammatory molecules (i.e., IL-ιβ) in both seizure generation and epileptogenesis²'³. However, the pathogenetic mechanisms linking these inflammatory processes with the development (and recurrence) of epileptic seizures in humans are unclear. Despite high heritability of epilepsy^⁶, both genome-wide association studies (GWAS) and exome sequencing approaches have so far provided limited insights into the genetic regulatory

mechanisms underlying inflammatory pathways in epilepsy etiology?-¹¹, and traditional single-variant association approaches are likely to be underpowered to detect complex gene network interactions that underlie disease susceptibility¹².

As the molecular processes driving complex disease usually affect sets of genes acting in concert, the inventors' have used systems-level approaches to investigate

transcriptional networks and pathways within pathologically relevant cells and tissues¹³'¹^ 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^. Almost uniquely among disorders of the human brain, epilepsy surgery offers opportunities for gene expression profiling in ante-mortem brain tissue from pathophysiologically relevant brain structures such as the hippocampus¹⁶. This allows direct investigation of transcriptional programmes in brain tissue from living epilepsy patients.

SUBSTITUTE SHEET RULE 26 Summary of the Invention

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 approach¹?. They have uncovered pathways and

transcriptional programmes associated with epilepsy that are conserved in mouse epileptic hippocampus, including a proconvulsant gene network encoding 1Τ-ιβ3 and TLR-signaling genes¹ previously implicated in epilepsy. Using genome-wide Bayesian expression QTL mapping¹⁸ the inventors have probed the genome for key genetic regulators of the network in human brain. They have pinpointed an unexpected gene, Sestrin 3 (SESN3) whose protein product controls the intracellular response to reactive oxygen species¹? ²², as a irans-acting genetic regulator of the proconvulsant gene network in human epileptic hippocampus. 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.

Detailed Description

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. In the TLE patients, 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.

As explained further herein, 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. This means that 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.

SUBSTITUTE SHEET RULE 26 Both the 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.

In an embodiment, the inhibitor of SESN3 gene expression is for use in a method of treating a disease, disorder or condition of the brain. In a preferred embodiment, the inhibitor is for use in a method of treating neuroinflammation, or a disease, disorder or condition involving neuroinflammation. In a very preferred embodiment, the inhibitor is for use in a method of treating epilepsy.

In a second aspect, the invention provides an inhibitor of SESN3 protein activity for use in therapy.

In an embodiment, 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

neuroinflammation, or a disease, disorder or condition involving neuroinflammation. In a very preferred embodiment, 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

SESN3. 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. For example, 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

SUBSTITUTE SHEET RULE 26 symptoms associated with said disease, disorder or condition are therefore expressly included.

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. Thus, for example, 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. It will be appreciated that 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). In a preferred embodiment, 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

subcutaneous (bolus or infusion), or intradermal (bolus or infusion). It will be appreciated that the amount of the agent or medicament that is required is determined by its biological activity and bioavailability, which in turn depends on the

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.

Generally, 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 μg/kg of body weight and ι mg/kg of body weight, more preferably between o.i μg/kg and 100 μg/kg body weight, and most preferably between approximately o.i μg/kg and 10 μg/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). Alternatively, the agent or medicament may require administration twice or more times during a day. As an example, 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 μg 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. Alternatively, 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

administration).

SUBSTITUTE SHEET RULE 26 A "disease, disorder or condition of the brain" includes any and all diseases, disorders and conditions that affect the brain. In a preferred embodiment, the disease, disorder or condition involves neuroinflammation. In a very preferred embodiment, the disease, disorder or condition is epilepsy.

A "subject" may be a vertebrate, mammal, or domestic animal. Hence, 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. Most preferably, 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. For example, the therapeutically effective amount of agent used may be from about o.ooi ng to about ι 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.

In one embodiment, 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. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, 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. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

SUBSTITUTE SHEET RULE 26 However, 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

pharmaceutically acceptable 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,

preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of 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.

glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, 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

administration using sterile water, saline, or other appropriate sterile injectable medium.

The agents and 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. 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, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups,

SUBSTITUTE SHEET RULE 26 elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

It will be appreciated that administration, into a subject to be treated, of an agent or medicament according to the invention will result in the inhibition of SESN3 gene expression or SESN3 protein activity, and that this inhibition will aid in treating a disease, disorder or condition of the brain, such as neuroinflammation, or a disease, disorder or condition involving neuroinflammation, and preferably epilepsy. In a third aspect, 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.

In a fourth aspect, 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.

In these aspects of the invention, 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.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:

Figure 1 shows an identification of the TLE-network and functionally specialised transcriptional modules in human epileptic hippocampus, (a) Gene co-expression network identified in the hippocampus of TLE patients (TLE-network). 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

SUBSTITUTE SHEET RULE 26 mark two transcriptional modules within the network, (b) KEGG pathways

significantly enriched in the TLE-network (FDR<5%). The fold enrichment for each KEGG pathway is reported on the side of each bar. (c) 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. mRNA expression of Module-i (n=8o probes, representing 69 unique annotated genes) as compared with Module-2 (n=6o probes, representing 54 unique annotated genes), other network genes (n=37i probes, representing 319 unique annotated genes) and all other probes represented on the microarray (n=48,256). **P=3.8xio^~4; ***P<io^~10, Mann-Whitney test, two-tailed. Figure 2 shows TLE-network conservation in mouse epileptic hippocampus, (a)

Human TLE-network genes that are conserved and co-expressed (84%) in the mouse hippocampus. Each node in the network represents a transcript that had partial correlation with at least another transcript in the network (5% FDR). Conserved Module-i and Module-2 genes are indicated in blue and green, respectively, (b) Distribution of partial correlations (FDR<5%) between pairs of transcripts from 10,000 bootstrap permutation samples in epileptic (top) and control (bottom) mouse hippocampus. In each case, the red line indicates the actual number of significant partial correlations (FDR<5%) between all genes in the network. The number of significant partial correlations observed in control hippocampus was no different from chance expectation (P=o.659). In contrast, the number of significant partial correlations detected in epileptic hippocampus was significantly higher than expected by chance (P=o.ooi). (c) Differential expression of Module-i genes between control and epileptic mouse hippocampus shows specific enrichment for TLR-signaling and cytokine genes amongst the upregulated genes. Stars denote significant fold changes between epileptic and control mouse hippocampus (FDR<5%); blue bars indicate TLR- signaling and cytokine genes.

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 logi₀(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. At 5% FDR (i.e., logi₀(Bayes Factor)>6, dashed line), SNP rsi050i829 (nq2i, highlighted in red) was significantly correlated with Module-i expression, (b) Joint mRNA levels-SNPs analysis within the iMbp region centered on SNP rsi050i829, comprising 178 SNPs genotyped in the TLE-patient cohort. The inventors carried out multivariate Bayesian regression modeling¹⁸ of all Module-i probes (n=89) and all SNPs (n=i78) to identify the most informative SNPs in the region predicting Module-i expression. For each SNP the inventors report the proportion of associated genes in Module-i (vertical axes): four SNPs (rsi050i829, rs530i90, rs7i0766i and ^6483435) that are individually associated with 58% to 74% of Module- 1 genes are highlighted. The grey box indicates the boundaries of the associated regulatory region (delimited by SNPs rs530i90 and ^6483435), spanning 383kb. (c) For each candidate gene at the iMbp regulatory locus the inventors report the average Pearson correlation (r; ± s.e.m.) with the expression of Module-i genes (y- axes) and its statistical significance for deviation from r=o (x-axes). Two-tailed P- values are reported on a negative log scale and were calculated using one sample Wilcoxon Signed Rank test. Two genes {ENDODi and MTMR2) were represented by two microarray probes and were analysed separately, (d) Association between increased Sesn.3 mRNA expression and upregulation of Module-i genes in epileptic mouse hippocampus. For each gene the inventors report its logi₀(fold change) in epilepsy vs control (y-axes) and its correlation with Sesn.3 mRNA expression (x-axes). The 95% confidence interval of the slope of the regression line is indicated. TLR- signaling and cytokines genes are highlighted in blue.

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 β- actin levels are shown as means relative to control ±s.e.m. (d) SESN3

immunofluorescence of human hippocampal slices from TLE patients: co- immunostainings with NeuN (green) antibody showed that SESN3 (red) is localised in neurons. Scale bar=ioc^m. (e) Quantification of SESN3 expression in human

SUBSTITUTE SHEET RULE 26 hippocampal tissue by immunofluorescence analysis. Maximum intensity projections of confocal z-stack images of immunohistochemical stainings with antibody against SESN3 were used. For determination of SESN3 cell fluorescence as a measure of SESN3 expression level, SESN3 expressing cells in the CA2 region of the hippocampus in both TLE patients samples (n=7) and autopsy samples (n=8) were measured using ImageJ software. 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).

Fluorescence intensity data are reported as means±s.e.m. (f) Effect of lentiviral- mediated Sesn.3 overexpression on Module-i genes in primary hippocampal neurons. Left, relative levels of Sesn.3 mRNA in transduced neurons (LV-CMV-Sesn3) compared with the levels in mock transduction (Mock). Right, relative mRNA levels of Module-i genes and a control gene not in the network (Hprt) in transduced neurons compared with levels in mock transduction. Data normalised to Gapdh levels are shown as means relative to control ±s.e.m. Four (Mock group) and twelve (LV-CMV-Sesn3 group) replicates were used in neuronal cell experiments. Statistical significance of the differences (P-value) between s SESN3 (or LV-CMV-Sesn3) and siControl (or Mock) was assessed by i-test (2-tailed) and adjusting for unequal variances across different groups. *P<0.05; **P<o.oi; ***P<o.ooi; ****p <o.oooi; ns, not significant (P>o.05).

Figure 5 shows that Sesn.3 modulates PTZ-induced c-fos expression, locomotor convulsions and Module-i genes in zebrafish. (a) Left, 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. Both sesn.3 morphant and control morphant larvae (n=i2) exhibited similarly low levels of locomotor activity in the absence of PTZ. Right, 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). (c) Sesn3 morpholinos attenuate seizure-induced expression of the synaptic activity-regulated

SUBSTITUTE SHEET RULE 26 gene c-fos. Left and central images, dorsal views of the brains of 3 dpf control morphant (top) and sesn.3 morphant larvae (bottom) maintained for l-hr in the absence and presence of 2omM PTZ, after which larvae were fixed and analysed for c-fos expression by whole mount in situ hybridisation. Red arrowheads, position of the transverse sections of the brains. Following PTZ treatment (20mM, l-hr), qPCR analysis revealed that sesn.3 morphant larvae exhibited significantly lower mRNA expression of c-fos in the brain than control morphant larvae (c, right panel), (d) PTZ- induced transcriptional response of Module-i genes was significantly lower in sesn3 morphants as compared with uninjected larvae; six samples were used in the qPCR experiments (one sample= 15-20 pooled larvae), (e) Up-regulation of Module-i genes upon injection of synthetic mRNA (ing) in zebrafish embryos (n=3o) as compared with uninjected control embryos (n=3o). Total RNA was extracted 28 hours post fertilisation and qPCR experiments were performed for the 2 pools of embryos using 6 technical replicates. Data reported as means ± s.e.m. were determined by the 2^_ΔΔα method and normalised to the housekeeping gene beta-actin. P-values calculated by t- test (2-tailed) adjusting for unequal variances across different groups, ns, not significant (P>0.05).

Examples

The materials and methods employed in the studies described in the Examples were as follows, unless where otherwise indicated:

Gene expression profiling in the human hippocampus

All 129 patients considered in this study had mesial TLE and all tissue samples were from indistinguishable hippocampal tissue portions. Sample preparation and microarray analysis of human hippocampi are detailed in Supplementary Methods.

Expression data were analysed using Illumina's GenomeStudio Gene Expression

Module and normalised by quantile normalisation with background subtraction.

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.

Gene co-expression network analysis

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 probes²3. The inventors used the empirical Bayes local FDR statistic⁵^ 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

Supplementary Methods.

Mapping the genetic control of networks

The inventors used Bayesian variable selection models¹⁸'²⁹ to identify the genetic control points (regulatory 'hotspots') of transcriptional modules in the TLE patient cohort. First, the inventors combined PC analysis⁵⁵ 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) algorithm¹⁸, where the module genes' expression are jointly considered. Further details are reported in Supplementary Methods.

Genetic association of the TLE-network with epilepsy

To test if TLE-network genes are likely to be causally involved in the disease process, the inventors assessed whether network genes are enriched for SNP variants associated to focal epilepsy by GWAS⁶<²⁵. Full details on the GWAS of focal epilepsy are reported in Supplementary Methods. Briefly, in the GWAS-enrichment analysis each gene of the TLE-network was assigned a GWAS significance value consisting of the smallest P- value of all SNPs mapped to it. The inventors used the hypergeometric distribution test to assess whether SNPs close to (< 100 kb from) any network gene were more likely to associate with epilepsy by GWAS than SNPs close to genes not in the TLE-network. Empirical GWAS-enrichment P-values were generated by 1,000,000 randomly selected gene-sets and are reported in Supplementary Table 2 (see Supplementary Methods for additional details).

RNA-Seq analysis in the mouse hippocampus

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 2.0.8⁵⁶ and read counts per gene were normalised across all samples using the

'trimmed mean of M-value' (TMM) approach⁵⁷. Differential expression analysis was

SUBSTITUTE SHEET RULE 26 performed using the edgeRs? and a threshold of 5% FDR was used to identify significant gene expression changes. Experimental animals were used only once for each study. All experimental procedures complied with the guidelines of the European Union Directive 2010/63/EU. A local ethical committee approved the experimental protocol.

In vitro studies

Details on all cell cultures used in these studies are reported in Supplementary

Methods. 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. For LPS stimulation experiments, the transfected cells were washed twice in DMEM and stimulated with LPS (Sigma, 100 ng/ml) for an hour. For overexpression experiments, a third- generation Lentiviral Vectors (LV) was used to transduce murine primary hippocampal neuronal culture. Additional information and details on Sesn.3 siRNA target sequences, the real-time quantitative PCR for Module-i genes and primer sequences are given in Supplementary Methods and Supplementary Table 4. The relative expression levels normalised to Beta-actin (or Gapdh as indicated) gene expression were then

determined by the 2^_ΔΔα method.

In vivo studies

To study the function of sestrin3 in response to PTZ-induced seizures, two different morpholinos were designed to block the normal splicing of the zebrafish sesn.3 primary transcript (see Supplementary Fig. 12 and Supplementary Methods). 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. 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 procedures⁵⁸. Analysis of zebrafish locomotor activity was carried out using the Viewpoint Zebrabox system (Viewpoint) as previously reported³⁴. 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

SUBSTITUTE SHEET RULE 26 PCR analyses of c-fos and Module-i genes, primer sequences are reported in

Supplementary Methods. All experimental procedures involving zebrafish were performed in compliance with the UK Animal (Scientific Procedures) Act and approved by the University of Sheffield Animal Welfare Ethical Review Board.

Example ι - Identification of a gene network associated with epilepsy

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). After excluding subjects with incomplete clinical data or non-HS pathology, whole-genome expression profiles in surgically resected hippocampi from 129 TLE patients (median age at surgery 35 years, range 1-64 years, male:female ratio of 1.2:1; Supplementary Table 1) were available for gene co-expression network analysis.

The inventors then 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). To investigate whether the protein products of the TLE-hippocampus derived transcriptional network (TLE-network) genes had a shared function at the protein level, the inventors used the DAPPLE algorithm²^. This method interrogated high-confidence protein-protein interactions (PPI) to assess the physical connections among proteins encoded by the genes in the network. Genes comprising the TLE-network were found to have increased protein- protein interconnectivity as compared with random PPI networks (P = 9.9 x io^~5, Supplementary Fig. 2). This provided evidence that proteins encoded by the co- expressed genes in the TLE-network interacted physically, supporting the validity of the gene network topography. As 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. Here, 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. To this aim, the inventors used focal epilepsy GWAS data⁶'²⁵ from a separate cohort of 1,429 cases (consisting mainly of patients with TLE) and 7,358 healthy controls. Although no Single

Nucleotide Polymorphism (SNP) achieved genome-wide significance in the epilepsy GWAS (Supplementary Fig. 3), the inventors found that the TLE-network as a whole was highly enriched for genetic associations to focal epilepsy compared with genes not in the network (P = 2 x ιο^~?; Fig. la and Supplementary Table 2). These integrated analyses of co-expression network and genetic susceptibility data from a focal epilepsy GWAS provided independent evidence to support the causal involvement of the TLE- network in epilepsy aetiology.

Example 2 - Conservation and functional specialisation of the network

The TLE-network was significantly enriched for genes belonging to several biological pathways involving cell-to-extracellular matrix adhesion including 'extracellular matrix-receptor interaction' (P = 3.9 x 10 ⁵), 'focal adhesion' (P = 1.4 x 10 ⁴), and inflammation, such as the 'cytokine-cytokine receptor interaction' (P = 2.4 x 10 ⁵) and 'TLR signaling' (P = 3.9 x 10 ⁵; Fig. lb). The observation that the TLE-network was enriched for multiple pathways led the inventors to investigate whether the network contained functionally homogenous transcriptional modules (i.e., sub-networks of highly correlated genes) with implications for epilepsy etiology. Using unsupervised agglomerate clustering approaches (see Supplementary Methods), the inventors identified two transcriptional modules comprising 69 (Module-i) and 54 (Module-2) unique genes, respectively (Fig. IC and Supplementary Data 1). Module-i was specifically enriched for gene ontology categories related to inflammatory mechanisms, whereas 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).

Notably, Module-i was enriched for highly expressed inflammatory cytokines (16-fold enrichment, P = 6.6 x lcr^) many of which belong to the IL-i signaling cascade (IL-ιβ,

SUBSTITUTE SHEET RULE 26 IL-iRN, IL-ia, TNFa), and the TLR-signaling pathway (11-fold enrichment, P = 1.4 x lo ⁶), previously implicated in epileptogenesis¹ and brain inflammation²^

(Supplementary Fig. 5). Taken together these data indicated the presence of a coordinated transcriptional program (Module-i), encompassing TLR activation and release of proinflammatory cytokines (including IL-ιβ), in chronic human epileptic hippocampus as previously hypothesised³'²⁶.

The inventors then 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). 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

(FDR<5%) with at least another network gene in mouse epileptic hippocampus (Fig. 2a). The conserved TLE-network genes formed 1,119 significant partial correlations in mouse epileptic hippocampus, which is significantly higher than expected by chance (P=o.ooi by 10,000 bootstrap permutations; Fig. 2b). In contrast, only 615 significant partial correlations between the same 312 genes were detected in healthy hippocampus (P=o.659 by 10,000 bootstrap permutations), suggesting that the TLE-network is specifically conserved in the epileptic mouse hippocampus (Fig. 2b). In keeping with the high expression of proinflammatory genes observed in the hippocampus of TLE patients (Supplementary Fig. 5), the mouse orthologues of Module-i genes that were significantly upregulated in epileptic hippocampus were enriched for TLR-signaling and cytokines (gene set enrichment analysis²⁸, P = 9.03 x io^~4, Fig. 2c). These comparative genomics analyses revealed that, to a large extent, the hippocampal TLE- network is conserved across-species, and confirm that genes for TLR-signaling and proinflammatory cytokines within the TLE-network are upregulated in chronic epileptic hippocampus.

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

SUBSTITUTE SHEET RULE 26 genes in each module using principal component (PC) analysis and detected regulatory 'hotspots' using a Bayesian regression model at the genome-wide level²?. This analysis prioritised genomic regions associated with variation in mRNA expression of the genes in each module. As a second step, to refine the genetic mapping results, the inventors regressed jointly the mRNA levels of module genes to all SNPs within the regulatory locus identified in the first step¹⁸. 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. In the first step, they identified a single locus on chromosome nq2i centered on SNP rsi050i829, which was significantly associated with the first PC of Module-i expression (FDR<5%; Fig. 3a). Module-2 showed no significant genome-wide associations (Supplementary Fig. 6). In the second step, the inventors investigated in detail the locus on chromosome nq2i regulating Module-i and carried out joint mRNA levels-SNPs analysis of all genes in Module-i and all SNPs genotyped within a l-Mbp region centered on SNP rsi050i829. 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).

The larger l-Mbp region centered on SNP rsi050i829 contained eight annotated protein-coding genes (Fig. 3b). To further prioritise candidate genes, the inventors carried out co-expression analysis between each of these genes and all genes in Module- 1, and found that Sestrin 3 (SESN3) was, on average, most strongly and positively correlated with Module-i gene expression (P = 1.7 x 10 ¹³, Fig. 3c). The positive association between SESN3 and Module-i gene expression remained significant following genome-wide correlation analysis in human hippocampus (P<o.00001, Supplementary Fig. 7). In summary, 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. Similarly, in the epileptic mouse hippocampus, the inventors found that increased Sesn3 mRNA expression was also significantly associated with up regulation of Module- 1 genes (P = 5.4 x io^~6, Fig. 3d), therefore providing independent, cross-species

SUBSTITUTE SHEET RULE 26 evidence supporting SESN3 as a positive regulator of Module-i genes in epileptic hippocampus.

Taken together, these data prioritise SESN3 as a candidate gene for the irans-acting genetic regulation of Module-i. To test this hypothesis, 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

lipopolysaccharide (LPS)-stimulated macrophages3°. Consistent with the positive correlation of Module-i genes with SESN3 mRNA expression (Fig. 3c-d and

Supplementary Fig. 7), the inventors observed decreased expression of Module-i genes after siRNA-mediated knockdown of SESN3 in both LPS-stimulated BMDMs and BV2 microglia cells (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-ιβ, IL-iRN, IL-ia, TNFa) even in the absence of a strong inflammatory stimulus (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. 4e and Supplementary Fig. 9). In keeping with this, the inventors found increased Sesn.3 mRNA expression in the mouse hippocampus after pilocarpine- induced status epilepticus (Supplementary Fig. 10), suggesting an association between SESN3 gene expression and epilepsy that is conserved across species. The inventors then tested whether Module-i genes are upregulated when SESN3 is overexpressed in neurons. To address this aim the inventors used an integrating lentiviral vector for gene overexpression in primary murine neurons (see Supplementary Methods) and quantitative PCR analysis showed that the relative levels of Sesn.3 mRNA were markedly increased in transduced neurons compared with the levels observed in mock transduction (Fig. 4 ). Consistent with the observed positive correlation between increased Sesn3 expression and Module-i genes in the hippocampus (Fig. 3c-d), lentiviral-mediated overexpression of Sesn3 resulted in significant upregulation of Module-i genes in hippocampal neurons (Fig. 4Ϊ). These in vitro experiments show that Sesn3 is capable of regulating Module-i gene expression in different cell types and in particular of inducing upregulation of proinflammatory genes in hippocampal neuronal cells. The inventors' findings in primary neurons are in keeping with previous

SUBSTITUTE SHEET RULE 26 data reporting the activity of several inflammatory molecules in neuronal cells under pathological conditions³¹, including IL-ιβ and its receptors². 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 Sandkuhler³³).

Example 4 - SESN3 regulates chemically-induced behavioural seizures

The in vitro data, combined with the positive association between SESN3 and Module-i gene expression in human and mouse epileptic hippocampus, indicated that SESN3 is a positive regulator of Module-i. The inventors hypothesised that 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. To test this hypothesis in vivo, the inventors investigated the role of SESN3 in a zebrafish model of convulsant-induced seizures³⁴'³⁵. In this model, exposure of 2- or 3-day-old zebrafish larvae to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic activity-regulated genes in the central nervous system and causes vigorous episodes of calcium flux in muscle cells as well as intense locomotor activity characteristic of epileptic seizures³⁴ _' ³⁵. This acute seizure model has been primarily used to investigate the anti-/proconvulsant activity of compounds³⁶ and for in vivo drug discovery³⁴. In particular, molecular and behavioral phenotypes in the zebrafish PTZ-induced seizure model have been employed to identify compounds that attenuate seizure activity³⁷. The inventors employed this model to correlate the locomotor responses with gene network dynamics, i.e., transcriptional activation of the neuronal activity-regulated gene 0/os³⁸ and an additional subset of

Module-i genes in response to PTZ treatment in sesn3 morphant and control morphant larvae. 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 sesn3 morpholinos and sesn.3 mRNA), with no significant differences in the locomotor activities between the uninjected larvae and the larvae injected with synthetic sesn.3 mRNA alone (Fig. 5b). To further confirm sesn^-dependent modification of neuronal response to PTZ, the inventors measured transcriptional activation of the neuronal activity-regulated gene c- fos, an important regulator for cellular mechanisms mediating neuronal excitability and survivals⁸. Consistent with the behavioral assay (Fig. 5a), there was decreased expression of c-fos in the brain (mostly in forebrain and midbrain) following PTZ exposure in sesn3 morphant larvae as compared with control morphant larvae (Fig. 5c and Supplementary Fig. 12). As silencing of Sesn3 resulted in downregulation of Module-i genes in vitro (Fig. 4a-c), the inventors tested whether inhibiting sesn3 could similarly reduce the activity of Module-i genes following PTZ exposure in vivo. The inventors observed significant reduction in the PTZ-induced mRNA expression of Module-i genes in the sesn3 morphant as compared with control morphant larvae (Fig. 5d), and they also found that transient overexpression of sesn3 in zebrafish larvae increased expression of Module-i genes independently of PTZ treatment (Fig. 5e). Taken together, these data show that sesn3 knockdown attenuates both PTZ-induced locomotor convulsive behaviour and the transcriptional responses of c-fos and Module- 1 genes to treatment with PTZ. These findings in the zebrafish model supported the evidence from the inventors' studies of human and mouse epileptic hippocampus and primary murine neurons that SESN3 positively regulates expression of preconvulsive molecules (Module-i genes). Discussion & Concluding Remarks

Deciphering the complex regulatory processes of pathophysiological pathways in human brain has previously remained a challenge due to the inaccessibility of ante- mortem tissue, but it could have important mechanistic and therapeutic implicationss^. In the study reported herein, the inventors used surgical hippocampal tissue samples and employed systems-genetics approaches¹⁷ to investigate transcriptional networks for epilepsy and their genetic regulation. The inventors identified a large gene co- expression network in the human epileptic hippocampus that was conserved in mouse epileptic hippocampus and was enriched for GWAS genetic signals of focal epilepsy. In keeping with similar network-based studies of complex disease such as Type-i diabetes*³ and autism spectrum disorder^^ the inventors' approach leverages the combined evidence from genetic susceptibility variants across multiple genes^ to link

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

transcriptional program (Module-i), which was over-expressed in the hippocampus of TLE patients, and which encoded epileptogenic IL1²'³'⁴³ and TLR-signaling pathways¹. The inventors confirmed the upregulation TLR-signaling genes and proinflammatory cytokines in chronic epilepsy by RNA-Seq analysis in 200 mouse hippocampi.

Preclinical studies in experimental models of epilepsy have consistently shown that individual pro-inflammatory cytokines such as IL-ιβ or TNF-a are over-expressed in brain areas of seizure generation and propagation⁴⁴. Therefore, targeting TLR and ILi signaling has been proposed as a possible avenue for therapeutic intervention in epilepsy and antiepileptogenesis¹ _' ³, including reduction of acute seizures^ and drug resistant chronic epileptic activity⁴⁶. The identification of genetic regulators of these pathways in the human epileptic brain might suggest opportunities for novel targets for disease modification. To investigate the genetic regulation of the TLE-network and the proinflammatory module therein, the inventors employed Bayesian expression QTL mapping approaches¹⁸, which identified 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 stress¹⁹.

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 epilepsy⁴⁷'⁴⁸. Therefore, the inventors hypothesised that SESN3 might regulate neuro-inflammatory molecules, previously implicated in epilepsy¹'³³-⁴³-⁴⁹, through modulation of oxidative stress in the brain.

The inventors' systems-genetics analysis in the human hippocampus, combined with in vitro and in vivo data, revealed SESN^-dependent regulation of epileptogenic IL-ιβ³ and TLR-signaling genes¹. The upstream genetic control of the proconvulsant transcriptional program by SESN3 in human TLE-hippocampus suggested a role for this gene in modulating seizures. To test the potential functional role of SESN3 in vivo

SUBSTITUTE SHEET RULE 26 the inventors used an experimental model of acute epileptic seizures³^? 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-ιβ and TNF-a, major mediators of inflammation, which are capable of inducing changes in neuronal excitability⁵⁰. The finding of reduced severity of PTZ-induced seizures upon knockdown of sesn.3 in the zebrafish model is consistent with previous studies in rodents describing the effects of pro-inflammatory cytokines on seizures. In the context of pre-existing brain inflammation, antibody-mediated antagonism of TNF-a function inhibited susceptibility to PTZ-induced seizures in rats⁵¹, whereas

administration of exogenous TNF-a increased susceptibility to PTZ-induced seizures⁵². The inventors' findings in zebrafish are therefore in keeping with a role for SESN3 in regulating pro-inflammatory cytokines and their downstream effect on CNS excitability and seizure susceptibility.

Taken together, 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 inflammation³^ and CNS excitability⁵³. 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

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SUBSTITUTE SHEET RULE 26

Claims

Claims

I. An inhibitor of SESN3 gene expression for use in therapy.

2. An inhibitor of SESN3 gene expression for use in a method of treating a disease, disorder or condition of the brain.

3. An inhibitor of SESN3 gene expression as claimed in claim 2, wherein the disease, disorder or condition of the brain involves neuroinflammation.

4. An inhibitor of SESN3 gene expression as claimed in claim 2 or claim 3, wherein the disease, disorder or condition of the brain is epilepsy.

5. An inhibitor of SESN3 protein activity for use in therapy.

6. An inhibitor of SESN3 protein activity for use in a method of treating a disease, disorder or condition of the brain.

7. An inhibitor of SESN3 protein activity as claimed in claim 6, wherein the disease, disorder or condition of the brain involves neuroinflammation.

8. An inhibitor of SESN3 protein activity as claimed in claim 6 or claim 7, wherein the disease, disorder or condition of the brain is epilepsy.

9. A method of treating a disease, disorder or condition of the brain in a subject comprising administering an inhibitor oiSESN3 gene expression to the subject.

10. 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.

II. An inhibitor as claimed in any one of claims 1-8, or a method as claimed in claim 9 or claim 10, wherein said inhibitor is a biological agent, a protein, a nucleic acid, an siRNA, or a pharmaceutical agent.