WO2024182357A1 - Compositions and methods for the chronic treatment of epilepsy and epileptogenesis - Google Patents

Abstract

A method of treating a patient in need of chronic treatment for epilepsy or epileptogenesis includes chronically administering to the patient an extracellular Sema4D peptide which has at least 80%, 85%, 90%, 95%, 98% or 99% identity to amino acid residues 22 to 734 of SEQ ID NO: 1, amino acid residues 22-690- of SEQ ID NO: 3, or amino acid residues 22-733 of SEQ ID NO: 5, and wherein the chronic administration reduces the incidence of seizures in the patient, slows disease progression, and/or reduces the risk of death in the patient.

Description

0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 COMPOSITIONS AND METHODS FOR THE CHRONIC TREATMENT OF EPILEPSY AND EPILEPTOGENESIS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application 63/487,322 filed on February 28, 2023, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT [0001] This invention was made with government support under NS102937, NS118799 and NS065856 awarded by the national Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING [0002] The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 13, 2024, is named “SEQ_LIST--107648037.xml” and is 17,155 bytes in size. The Sequence Listing does not go beyond the disclosure in the application as filed. FIELD OF THE DISCLOSURE [0003] The present disclosure is related to methods of treating patients in need of chronic treatment for epilepsy or epileptogenesis. BACKGROUND [0004] Status epilepticus (SE) is a life-threatening neurological emergency characterized by continuous seizure activity lasting greater than 5 minutes which can have serious long-term consequences including neuronal injury and death. First line treatment for SE is intravenous or intramuscular administration of benzodiazepines (BZD; e.g., diazepam). BZDs enhance the activity of GABAA receptor subunits, thereby affecting existing GABAergic synapses, and increasing inhibitory tone in the brain. Unfortunately, approximately 30% of SE patients do not respond to treatment with BZD plus at least one other anti-seizure medication, resulting in refractory SE (RSE) which has a mortality rate of 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 approximately 35%. Thus, the need for innovative anti-seizure medications to treat SE and in particular RSE is dire. [0005] U.S. Patent No.10,626,163 particularly focuses on the use of Sema4D, for example, for treatment for various neurological disorders, including seizures and certain forms of epilepsy. [0006] What is needed are additional methods of treatment, particularly for RSE. BRIEF SUMMARY [0007] In an aspect, a method of treating a patient in need of chronic treatment for epilepsy or epileptogenesis comprises chronically administering to the patient an extracellular Sema4D peptide which has at least 80%, 85%, 90%, 95%, 98% or 99% identity to amino acid residues 22 to 734 of SEQ ID NO: 1, amino acid residues 22-690 of SEQ ID NO: 3, or amino acid residues 22-733 of SEQ ID NO: 5, and wherein the chronic administration reduces the incidence of seizures in the patient, slows disease progression, and/or reduces the risk of death in the patient. BRIEF DESCRIPTION OF THE DRAWINGS [0008] Figures 1A-C show the extracellular domain of Sema4D protein decreases population spike amplitude in acute hippocampal slices. Sema4D-Fc treatment depressed population spike amplitude by approximately 50% while having no effect on fEPSP slope or paired-pulse facilitation (PPF) ratio. Acute hippocampal slices were isolated from rats of both sexes at 20-25 weeks old. Responses were recorded by low-frequency stimulation of the Schaffer-collateral commissural pathway by stimulating electrodes placed in the stratum radiatum and recording responses at the CA1 pyramidal/stratum oriens border. A stable baseline (1 hour) was obtained before drug application for 2hrs. Comparisons were made 3hrs post washout (i.e., at t=6hrs). 1A shows time plots representing the pooled effects (both n=8) of control Fc. (black) vs Sema4D-Fc (gray) application to evoked population spikes (Ai), fEPSP slope (Aii) and PPF (measured as the ratio of fEPSP slopes to 2 successive pulses delivered at a 50ms interval; Aiii). Shaded areas are the (bootstrapped) s.e.m. Traces above the time plots represent the raw data comprising averages of time points within each experiment taken at the time points indicated. Fc / Sema4D-Fc application are shown in light gray. For both conditions, vertical and horizontal scale lines indicate 1mV and 5ms, respectively. 1B shows raw values (points) and summary statistics (mean (bars) and SEM 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 (lines)) of population spike amplitude (Bi), fEPSP slope (Bii) and PPF ratio (Biii) shown at baseline and test time points as in A for control Fc (left, black) vs Sema4DFc (right, hatched). In the group receiving Sema4D-Fc treatment, the mean spike amplitude at test was significantly lower than its baseline (bootstrapped multiple comparisons test with Holm- Šidak correction); no other conditions differed significantly from one another; * indicates alpha < 0.05. 1C shows box plots to illustrate Cohen’s d (bootstrap) for population spike amplitude (Ci), fEPSP slope (Cii) and PPF ratio (Ciii). Mean and median effect sizes are represented by point and line, respectively. The interquartile range and 95% confidence interval are illustrated by box and whiskers, respectively. Significant effects from raw data (B) are also illustrated on this plot by asterisk for convenience. [0009] Figures 2A-I show Sema4D treatment suppresses epileptiform activity and restores diazepam sensitivity in vivo. 2A shows the experimental timeline. 2B shows the representative electrographic seizure activity from vehicle (top) and Sema4D-Fc-treated mice (bottom). 2C shows representative EEG power spectra from vehicle (top) and Sema4D-Fc treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. Sema4D-Fc was infused bilaterally into the CA1 region of the hippocampus 1hr prior to administration of kainic acid (20mg/kg, i.p.). Electrographic seizure activity was recorded for the entire 4hr period of the experiment. For the two hours following KA injection, the following characteristics were quantified 2D shows latency to onset of the first seizure. 2E shows cumulative epileptiform activity (% time). 2F shows the number of mice that died. 2G shows latency to SE. 2H shows latency to cessation of SE. 2I shows percent of diazepam insensitive mice which was quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 8 mice per experimental group; Student’s t-test was performed on data in panels D,E,G,H; * denotes p<0.05 using a Student’s t-test. N-1 corrected chi-squared test was performed on data in panels F,I; no significant difference was found. [0010] Figures 3A-D show validation of Sema4D-expressing adeno associated viruses in vitro. 3A is a schematic representing full-length or extracellular domain of Sema4D or CD4 protein encoded by the AAV viruses. 3B shows representative images of the CA1 principal cell layer in organotypic slices that were treated with Sema4D-Fc or Fc control proteins or infected with indicated AAV constructs; Sections are stained with an antibody that specifically recognizes GAD65 and DAPI. Scale bar represents 25μm. 3C shows the density of GAD65 puncta per CA1 pyramidal cell soma; error bars are SEM. n = 233-351 neurons per treatment condition (represented by individual points), 16- 40 neurons per slice, slices from 6-9 mice per treatment condition, 29 mice total. 3D shows box plot to illustrate 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 the effect size, Cohen’s d. Mean effect size is represented by single points, interquartile range (IQR) and 95% confidence interval (CI) are illustrated by box and whiskers, respectively for the following comparisons: Fc vs Sema4D-Fc; AAV-CD4-FL vs AAV- Sema4D-FL; AAV-CD4-ECD vs AAV-Sema4D-ECD. A mixed effect linear model was fitted to the data with treatment as the fixed effect and animal and slice as random effects in order to control for variability that arises within animals and problems associated with pseudoreplication within slices (modeled as random intercepts). Consequently, data is presented as the estimated marginal means obtained from the model. Effect sizes (Cohen’s d), estimates of s.e.m, IQR, CI and multiple comparisons tests are based on these estimates. * denotes p<0.05 by conducting an ANOVA on the linear mixed effects model with subsequent multiple comparisons tests using a Tukey correction. Significant effects from raw data in 3C are also indicated by an asterisk in 3D for convenience. [0011] Figures 4A-I show delivery of AAV-Sema4D-ECD to hippocampus reduces epileptiform activity and restores BZD sensitivity. 4A shows experimental timeline. 4B shows representative electrographic seizure activity from control AAV-CD4-ECD (top) and AAV-Sema4D-ECD treated mice (bottom). 4C shows representative EEG power spectra from control AAV-CD4-ECD (top) and AAV-Sema4D-ECD treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. AAV-Sema4D-ECD or control virus was injected into the DG region of the hippocampus 1wk prior to administration of KA (20mg/kg, i.p.). Electrographic activity was recorded for 1 hr prior to and 3hrs after KA injection which includes the 1 hour post diazepam administration (5mg/kg, i.p.). For the two hours following KA injection, the following characteristics were quantified. 4D shows latency to onset of the first seizure. 4E shows cumulative epileptiform activity (% time). 4F shows the number of mice that died (short hatched bar indicates 0 for AAV- Sema4D-ECD). 4G shows latency to SE. 4H shows latency to cessation of SE. 4I shows percent of diazepam insensitive mice quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 6-8 mice per experimental group; Student’s t-test was performed on data in panels D,E,G,H; N-1 corrected chi-squared test was performed on data in panels F,I. * denotes p<0.05 using a Student’s t-test; the chi squared test results were not significant [0012] The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. DETAILED DESCRIPTION 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 [0013] The number of patients suffering from pharmacoresistant epilepsy is expected to increase with the globally aging population. Alternative therapies to treat this disorder include resective surgery, deep-brain stimulation, and targeted laser ablation. All have disadvantages: surgeries/ablation are destructive and invasive; open-loop stimulations are only palliative seizure-reducing therapies. Chronically administered Sema4D could be introduced by microinjection of protein as a therapy for chronic intractable epilepsy and other chronic forms of epilepsy. The available evidence in rodent models of epilepsy suggests that, as a therapeutic, Sema4D has the potential to succeed where current AEDs fail. The ability of Sema4D to bypass mechanisms of pharmacoresistance combined with its potential to treat different seizure types, irrespective of etiology, in a minimally invasive fashion has the potential to be a disease modifying and life-altering therapy for the treatment of epileptic disorders. [0014] While the cellular pathophysiology underlying BZD resistance in RSE remains unclear, current models to explain this event include: aberrant removal of GABAARs from the neuronal cell surface, increased AMPA and NMDA receptor abundance at synapses, and dysfunction of the type-2 K+-Cl- cotransporter (KCC2) causing elevated intracellular Cl- concentrations, leading to depolarizing action of GABAARs. It has been demonstrated that the extracellular domain of Sema4D promotes the stabilization and development of GABAergic synapses on a rapid time scale (approximately 30 minutes); these synapses become functional within two hours. As described herein, treatment with the pro- synaptogenic molecule Sema4D could impart therapeutic effects in mouse models of this disease. [0015] In addition, previous studies using in vivo seizure models (intravenous pentylenetetrazol and electrical kindling) demonstrated that acute infusion of the purified Sema4D extracellular domain (Sema4D-Fc) into hippocampus increases GABAergic synapse density, increases time to seizure onset, and decreases seizure severity. Without being held to theory, a model is proposed whereby Sema4D treatment in hippocampus promotes GABAergic synapse formation, thus increasing inhibitory tone and suppressing seizures. [0016] First, described herein is the determination of the time course of Sema4D treatment on hippocampal circuit function. To accomplish this goal, extracellular recordings were used to assay population spike amplitude in the CA1 region of acute hippocampal slice before, during, and after 2 hours of Sema4D treatment. [0017] Second, described herein is the determination if a Sema4D protein infusion to the intact hippocampus could impede progression to SE and decrease the severity of seizures. 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 To address this question, KA-induced SE was employed which is an established rodent model of temporal lobe epilepsy that progresses to SE with development of diazepam insensitivity. Thus, purified Sema4D-Fc protein was delivered via cannula to the hippocampus of mice, and they were subjected to KA-induced SE and subsequent BZD treatment while monitoring seizure severity using EEG. [0018] Third, it was determined if chronic delivery of Sema4D protein via viral mediated gene transduction in the hippocampus could suppress seizure onset and severity similar to, or better than, the effect observed with acute infusion of purified Sema4D protein. This line of experimentation also allowed testing of the replacement of intrahippocampal infusion of Sema4D protein with virus-mediated gene transduction of Sema4D, a more generalizable and tractable delivery method for this potential therapeutic. To answer these questions, adeno associated virus expressing Sema4D-ECD was developed and validated. It was tested whether chronic Sema4D-ECD expression via viral mediated gene transduction could increase GABAergic synapse density. In addition, it was tested if viral delivery of Sema4D to intact hippocampus could suppress progression to and severity of SE using the KA model. [0019] In an aspect, a method of treating a patient in need of chronic treatment for epilepsy or epileptogenesis comprises chronically administering to the patient an extracellular Sema4D peptide which has at least 80%, 85%, 90%, 95%, 98% or 99% identity to amino acid residues 22 to 734 of SEQ ID NO: 1, amino acid residues 22-690 of SEQ ID NO: 3, or amino acid residues 22-733 of SEQ ID NO: 5, and wherein the chronic administration reduces the incidence of seizures in the patient, slows disease progression, and/or reduces the risk of death in the patient. [0020] U.S. Patent No.10,626,163 is incorporated herein by reference for its disclosure of Sema4D peptides, including extracellular Sema4D peptides. [0021] Human Sema4D isoform 1 is identified as SEQ ID NO: 1 (NP_006369; Uniprot Q928544). The coding sequence for human Sema4D isoform 1 is SEQ ID NO: 2 (NM_006378). Human Sema4D isoform 2 is identified as SEQ ID NO: 3 (NP_001135759). The coding sequence for human Sema4D isoform 1 is SEQ ID NO: 4 (NM_001142287). Mouse Sema4D is identified as SEQ ID NO: 5 (NP_038688.2). The coding sequence for mouse Sema4D is identified as SEQ ID NO: 6 (NM_013660). [0022] In an aspect, the human Sema4D extracellular domain is amino acid residues Met22-Arg734 of SEQ ID NO: 1. In an aspect, the human Sema4D extracellular domain is 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 amino acid residues of SEQ ID NO: 3 (Met22-Val690). In an aspect, the mouse Sema4D extracellular domain is amino acid residues 27 to 490 of SEQ ID NO: 5. [0023] As used herein, Sema4D polypeptides are highly conserved among species and the structures are known such that the skilled artisan would readily understand which regions of the Sema4D polypeptide can be altered without affecting a desired function mediated by the Sema4D. Percent sequence identity can be calculated using computer programs or direct sequence comparison. Exemplary computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, FASTA, BLASTP, and TBLASTN. The BLASTP and TBLASTN programs are publicly available from NCBI and other sources. The Smith Waterman algorithm may also be used to determine identity. [0024] As used herein, acute administration of a treatment for epilepsy is administration at the time of a seizure to manage the seizure. Acute administration is distinct from chronic administration. As used herein, chronic administration of a treatment for epilepsy is administration regardless of whether the subject is currently experiencing a seizure and is over a period of time such as greater than one month, greater than six months, greater than one year, to several years. Chronic administration may be daily administration or less frequent administration so long as the administration route releases the active agent over a period of time such that chronic administration is sustained. [0025] In an aspect, the patient is a human patient. [0026] In an aspect, the patient has suffered a brain damaging insult associated with epileptogenesis. As used herein epileptogenesis is a chronic process in which a previously normal brain network is altered to enhance the probability of spontaneous recurrent seizures (SRSs). Typically, epileptogenesis is associated with acquired epilepsy and refers to a latent period between a brain damaging insult such as traumatic brain injury, prolonged febrile seizure, stroke, intracerebral hemorrhage, infection or status epilepticus to the occurrence of spontaneous seizures and an epilepsy diagnosis. The brain damaging insult causes changes in the brain leading to chronic, spontaneous seizures. [0027] In an aspect, the patient is diagnosed with Dravet Syndrome. Dravet Syndrome is a genetic epilepsy syndrome that begins in infancy or early childhood and gives rise to seizures that are typically unresponsive to standard seizure medications. Anti-epileptic drugs (AEDs) such as oxcarbazepine, carbamazepine, phenytoin, and lamotrigine can make symptoms worse in Dravet’s patients. 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 [0028] In an aspect, the patient has chronic drug refractory epilepsy (CDRE) CDRE, also referred to as chronic intractable epilepsy, is a form of epilepsy that does not respond to available AEDs. CDRE has a mortality rate of about 35%. [0029] In another aspect, the patient has more than three generalized tonic-clonic seizures per year and is at risk for sudden unexpected death in epilepsy (SUDEP). Sudden unexpected death in epilepsy (SUDEP) occurs when a seemingly healthy person with epilepsy dies for no obvious reason. The greatest common risk factor for SUDEP is having more than three generalized tonic-clonic seizures per year. Other risk factors include medication non-compliance, having CDRE, being diagnosed with a genetic epilepsy including Dravet Syndrome, early age of epilepsy onset and uncontrolled or frequent seizures. [0030] In an aspect, the Sema4D peptide is delivered via viral mediated gene transduction, such as from a lentivirus an AAV such as an AAV9.hsyn. In an AAV virus for gene delivery, at least a portion of the viral DNA is replaced with DNA encoding the Sema4D peptide such that the AAV vector can infect cells and express the Sema4D polypeptide. [0031] Nucleic acids in vectors can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence. 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 [0032] An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a nucleic acid sequence that controls and regulates the transcription and/or translation of another nucleic acid sequence. [0033] In another aspect, the Sema4D peptide can be delivered in the form of an mRNA suitable for the expression of Sema4D. mRNAs can be delivered by means known in the art such as lipid nanoparticles, cationic nanoemulsions, cationic peptides and cationic polymers. The lipid-based nanoparticles used in the clinically successful COVID-19 vaccines may be employed. [0034] In other aspects, the Sema4D peptide is delivered in the form of a fusion polypeptide with a brain-targeting peptide, a nanoparticle delivery system such as lipid nanoparticles, liposomes, microemulsions, hydrogels, injectable polymers and the like. [0035] In an aspect, the Sema4D peptide or polynucleotide expressing the Sema4D peptide is delivered to the hippocampus, via intrathecal delivery or via intracerebroventricular delivery. [0036] In an aspect, the Sema4D peptide or polynucleotide expressing the Sema4D peptide is delivered in the form of an injectable or implantable pharmaceutical composition. [0037] The compositions described herein may further comprise a pharmaceutical excipient, carrier, buffer, or diluent, and may be formulated for administration to an animal, and particularly a human being. Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. [0038] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intra-articular, intramuscular administration and formulation. [0039] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active compound(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. The number of active compound(s) in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. [0040] In an aspect, the extracellular Sema4D peptide is co-administered, either simultaneously or sequentially, with an AED such as a benzodiazepine (diazepam, clorazepate, lorazepam, clonazepam, clobazam), sodium valproate, carmabazepine, lamotrigine, levitracetam, topiramate, gabapentin, primidone, rufinamide, stirpentol, vigabatrin, zonisamide, phenytoin, phenobarbital, oxcarbazepine, pregabalin, and lacosamide. [0041] The invention is further illustrated by the following non-limiting examples. EXAMPLES METHODS [0042] Animals: C57BL/6 male mice and adult Long-Evans male and female rats were purchased from the Charles River Laboratories and housed in the animal facility at Brandeis University or at Tufts University. Animals were maintained with a 12-hour light- dark cycle. Food and water were available ad libitum. Animal procedures were performed with approval from the Brandeis University Institutional Animal Care and Use Committee and the Tufts University School of Medicine Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (NRC). Animal studies were performed in compliance with ARRIVE guidelines as described in this methods section. [0043] Population spike recordings: Brains were isolated from Long-Evans rats of both sexes between 20-25 weeks old. The brain was rapidly dissected and placed in ice- chilled, oxygenated modified artificial cerebrospinal fluid (aCSF) comprising of (in mM): NaCl 124, KCl 3.7, NaHCO₃24.6, CaCl₂1, MgSO₄3, D-glucose 10, KH₂PO₄1.2 saturated with 95% O₂ / 5% CO₂. Parasagittal slices (400 μm thick) containing the hippocampal region were prepared in ice-chilled, oxygenated aCSF using a Leica VT1200 vibrating microtome (Leica Biosystems Inc.). [0044] Following equilibration for at least an hour, slices were transferred to a brain slice interface chamber (model BSC2, Scientific Systems Design, Inc.); slices rested on filter paper at the interface of the perfusing solution (0.4 ml min^-1) which comprised of standard aCSF saturated with 95% O₂ / 5% CO₂. Field recordings were obtained using a glass microelectrode (resistance approximately 2-4MΩ) containing 3M NaCl placed at the stratum 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 pyramidale/stratum oriens border. Responses were evoked by stimulating two electrodes placed within the Schaffer-collateral-commissural fibers. Baseline stimulation was every 150 seconds for each input with a 75 second interval between alternating inputs. Independence of inputs was assessed using a paired-pulse protocol. Stimulation intensity was adjusted such that the baseline population spike amplitude was approximately 40% of the maximum amplitude. Recordings were filtered at 3–10 kHz using an Axoclamp 2A Amplifier (Molecular Devices) and collected for online analysis at a sampling rate of 20kHz using WinLTP software. [0045] Virus production: AAV-Sema4D-ECD comprised an artificial N-terminal signal sequence and myc tag upstream of Sema4D NP_001268809.1 aa51 – aa733. These ORFs were cloned in place of GFP in the AAV9.hsyn1.GFP plasmid (Addgene #105539). Following cloning, the plasmid was packaged into AAV9 capsid and active virus was harvested and purified for use in vivo. Cloning and viral packaging and purification were performed by Vector Biolabs (Malvern, PA). [0046] Animal surgeries for EEG headmount attachment, cannula placement and AAV-Sema4D-ECD virus injection: Male mice (10-12 weeks of age) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine and a prefabricated headmount (part # 8201; Pinnacle Technology, Inc) was affixed to the skull with four screws and dental cement. Two of the screws serve as differential EEG leads, which were placed two bilaterally anterior and two posterior to bregma, and the other screws serve as the reference ground and the animal ground. For the Sema4D infusion experiments, mice were implanted with a guide cannula into the dorsal hippocampus (A/P: -2.0mm; M/L: ±1.5mm; D/V: -2mm) during EEG headmount attachment. The animals were allowed to recover for a minimum of 5 days before experimentation. For virally delivered Sema4D, 500nl of AAV-Sema4D (2.55 Gc/mL) or control virus was stereotaxically injected into the dorsal hippocampus during EEG headmount attachment 1 week prior to KA administration (20mg/kg, i.p.). [0047] KA seizure paradigm: Sema4D (100nM, 500nl) or vehicle (0.9% sterile injection saline, 500nl) was infused into the hippocampus 1 hr prior to KA administration (20mg/kg, i.p.). For both Sema4D infused animals and virus injected animals, 5mg/kg diazepam was administered 2hrs after KA administration as previously described in the art. [0048] Electroencephalogram: EEG recordings from male C57BL/6 mice were carried out as previously described in the art; recording and analyses were performed by two different experimenters with blinding to condition. Recordings were collected in awake, 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 behaving animals using a 100x gain preamplifier high pass filtered at 1.0 Hz (Pinnacle Technology, part #8202-SE) and tethered turnkey system (Pinnacle Technology, part #8200). [0049] The KA model of SE was employed for these studies because it is an acute, well-established model of TLE in which animals enter SE within an hour post-injection and develop seizures that do not respond to treatment with diazepam. Electrographic activity was recorded for 1hr before and 2hrs following KA administration. Diazepam was administered 2hr following KA administration and electrographic activity was recorded for an additional hour. In brief, epileptiform activity was considered to be paroxysmal activity having a sudden onset and an amplitude at least 2.5x the standard deviation of the baseline and a consistent change in the Power of the fast Fourier transform of the EEG. In the KA model, the animals typically enter SE by 1 hour post-KA administration. SE was defined as persistent, unremitting epileptiform activity lasting longer than 5 consecutive minutes. The definition of “epileptiform activity” includes both discrete ictal events and periods of SE. These criteria have been used previously in the art. Seizure latency was defined as the time elapsed from the KA injection to the start of the first electrographic seizure. The % time epileptiform activity was calculated as the cumulative time of all epileptiform activity during a 120-min recording period divided by 120 min. The latency to SE was calculated as the time elapsed from the KA injection to the start of the SE (first 5 mins of unremitting epileptiform activity) while SE cessation was identified as elimination of epileptiform activity lasting for at least 5 minutes. [0050] The diazepam sensitivity (as measured by a reduction in the power of the electrographic signal and cessation of epileptiform activity) was determined as previously described in the art. Mice were considered to be diazepam-insensitive if diazepam treatment failed to suppress epileptiform activity within 10 mins of administration. [0051] Immunohistochemistry and analysis for virus-injected animals: Male C57BL/6 mice were injected with AAV-Sema4D-ECD or AAV-CD4-ECD virus as above and sacrificed 2-3 weeks postinjection. The brains were removed and fixed in 4% paraformaldehyde for 24 hours, transferred to 10% sucrose for 24 hours, and then transferred to 30% sucrose for 24 hours. They were then embedded in Optimal Cutting Temperature (OCT) compound and frozen down for storage at - 80°C. The brains were sectioned at 40 μm using a cryostat (Leica) and slices were suspended in PBS at 4°C until being processed for immunohistochemistry. [0052] Representative slices from the hippocampus were chosen for staining. The sections were blocked in PBS with 10% normal goat serum (NGS) and 0.3% Triton^TM for one 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 hour at room temperature prior to incubation with the primary antibody mouse anti-GAD65 (EMD Millipore, MAB3551) at a concentration of 1:100 overnight at 4°C. The sections were then incubated in the secondary antibody (anti-Ms Alexa Fluor® 488, Invitrogen, A32766) at 1:200 for two hours at room temperature and mounted with Vectashield® Hardset^TM antifade mounting medium with DAPI (Vector Laboratories, H-1500). Imaging was performed on a Leica SP8 Confocal microscope using a 40x oil objective.2-3 images per hemisphere per section were taken of the hippocampus near A/P - 2.0 (the virus injection site). Microscope and laser settings were kept constant across all images. Quantification of GAD65 immunofluorescence was performed using ImageJ. An outline was traced around the CA1 region of the hippocampus and the fluorescence intensity in the region of interest was quantified. For each image, the CA1 mean intensity was normalized to the mean intensity of background. [0053] Organotypic slice culture: C57BL/6 mouse brains were dissected from P6-P8 animals of both sexes into cutting solution (126mM NaCl, 25mM NaHCO₃, 3mM KCl, 1mM NaH₂PO₄, 20mM dextrose, 2mM CaCl₂, 2mM MgCl₂ in deionized water at 315-319 mOsm). Coronal slices were taken at a thickness of 300 μm using a tissue chopper (Compresstome® VF-200, Precisionary Instruments Inc.). Individual slices were placed on cell culture inserts (0.4 um pore size, Millipore). [0054] Organotypic culture media (2mM Glutamax^TM, 1mM CaCl₂, 2mM MgSO₄, 12.9mM d-glucose, 0.08% ascorbic acid, 18 mM NaHCO₃, 35mM HEPES, 20% horse serum, 1 mg/mL insulin in minimum essential media) at pH 7.45 and 305 mOsm was added outside of the inserts. Slices were maintained for 6 days in vitro at 35°C and 5% CO₂ with media replacements every other day. [0055] One day after harvesting slices, a solution containing AAV9.hsyn viruses encoding either full length or the extracellular domain of either Sema4D or CD4 (designed and purified by VectorBiolabs) in combination with the same amount of AAV9.hsyn.GFP virus (Addgene, #105539-AAV9) was pipetted onto the hippocampus within the slice (1 μL on each hemisphere, each virus in the solution at 2.55 x 1012 Gc/mL). A subset of slices were infected with only AAV9.hsyn.GFP and at DIV6 were treated with human Sema4D-Fc ectodomain/human Fc fusion protein (Sema4D-Fc; R&D Systems, #7470-S4) or Fc control protein (R&D Systems, #110-HG) at a final concentration of 2 nM/well for 2 hours before being fixed immediately following. [0056] Immunofluorescence performed in organotypic slice cultures: To quantify synapse density at DIV6, organotypic slices were fixed in 4% paraformaldehyde/4% sucrose 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 for 20 minutes at 4°C. After 3 x 10-minute washes in PBS, slices were incubated overnight in permeabilization solution (0.1% Triton^TM-X in PBS) followed by an overnight incubation in blocking solution (20% bovine serum albumin with 0.1% Triton^TM-X in PBS) at 4°C. Next, slices were incubated in primary antibody anti-GAD65 (EMD Millipore, #MAB351) at 1:150 in blocking solution and incubated overnight at 4°C followed by 3 x 10-minute PBS washes and incubation with secondary antibody anti-mouse-Cy3 (Jackson Laboratories, #115-165-003) at 1:500 into secondary solution (blocking solution diluted 1:1000 in PBS) for 2 hours at room temperature. Following 3 x 10-minute PBS washes, slices were mounted (insert side down) on slides in Vectashield® + DAPI mounting media (Vector Laboratories). [0057] Imaging and analysis of organotypic slice cultures: 16-bit images of neurons were acquired on a Zeiss LSM880 Confocal microscope using a Plan- Apochromat 63x/1.40 Oil DIC M27 objective. Within each experiment (slices collected on the same day), images were acquired with identical settings for laser power, detector gain, and amplifier offset. Settings were initially optimized across multiple control slices to avoid oversaturation. Images were acquired as z-stacks (5-15 optical sections, 0.5 μm step size) for each of 4-5 fields of view per hemisphere (134.95 μm x 134.95 μm) containing the pyramidal cell layer in CA1 from each slice. Within each z-stack, the image with the highest fluorescent intensity for GAD65 signal was chosen for analysis and neurons were selected at random for analysis using only the GFP signal, thus blinding the experimenter to the GAD65 signal in the chosen neurons. [0058] Using the DAPI signal as a guide, the nuclei of these cells were traced and then a band expanding a uniform distance of 2 μm around each nucleus was drawn using the “draw band” function in ImageJ. Empirically, this band provides a close approximation of the cell soma boundary. Using ImageJ (NIH), the background signal was measured in three distinct places determined to lack true signal in each image, and the average mean background intensity was subtracted from the entire image. Finally, signal in the GAD65 channel was binarized, the adjustable watershed algorithm was applied, and GAD65 puncta within each band surrounding selected nuclei were quantified using the “analyze particles” function (size=0.1-10 and circularity=0.01-1.00). The number of puncta per band was divided by the area of each band to give a per soma density of GAD65 puncta. Between 16- 40 cells were analyzed per slice. A subset of neurons in the CA1 region exhibit very high GAD65 immunofluorescence which fills the cell soma. Such cells were excluded from analysis. 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 [0059] Statistical analysis: Statistical analyses were performed using the R programming language or SPSS. Linear mixed effects modeling was conducted in R utilized packages lmerTest and emmeans. Bootstrapped multiple comparisons tests were conducted using custom written code in R. Specific statistical tests are described in the text and figure legends. EXAMPLE 1: APPLICATION OF THE EXTRACELLULAR DOMAIN OF SEMA4D TO ACUTE HIPPOCAMPAL SLICES DECREASES EXCITABILITY [0060] Previously it was observed that the ECD of Sema4D fused to the Fc region of human IgG (Sema4DFc) mediates an increase in GABAergic synapse density as evidenced by immunostaining and seizure suppression in rodent hippocampus. In addition, using whole cell voltage-clamp, that Sema4D-Fc application caused an increase in mini Inhibitory Postsynaptic Currents (mIPSC) in dissociated hippocampal neuronal cultures and acute slice, consistent with an increase in the density of functional GABAergic synapses. Herein, the effect and time-course of Sema4D treatment on the hippocampal circuit were determined. A recording configuration and set-up were used that allow the recording of stimulation-evoked field hippocampal activity for long periods of time (> 12 hours). Recordings were performed in the CA1 region of acute hippocampal slices obtained from adult rats (aged postnatal day (P) 140-180). Evoked field responses comprise a positive-going field EPSP on which a negative-going population spike is superimposed. Following a stable 1-hour baseline, Sema4D-Fc or Fc control protein were bath-applied for 2 hours. A decrease in population spike amplitude was observed starting at approximately 1 hour after Sema4D-Fc perfusion began and lasted throughout the recording period, including for 3 hours post-washout of Sema4D-Fc (Figure 1A). This effect was quantified at 3 hours post-washout and a significant Sema4D-Fc-mediated decrease in population spike amplitude was observed (Figure 1Bi & Ci). [0061] In addition, no change in initial slope of the fEPSP was observed (Fig.1Aii- Cii), a measure of L-glutamate mediated excitatory synaptic input or paired-pulse facilitation (Fig.1Aiii-Ciii), a measure of presynaptic release dynamics, in the Sema4D-Fc treatment condition. These data suggest that the depression of evoked firing observed upon Sema4D- Fc treatment is not due to a presynaptic effect which would dampen down excitatory synaptic transmission. An effect of Sema4D on neuronal intrinsic excitability cannot be ruled out. Nonetheless, given previous studies demonstrating a rapid increase in GABAergic synapse formation in response to Sema4D application. Without being held to theory, it is believed 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 that the observed decrease in population spike amplitude is a Sema4D-dependent increase in evoked feedforward inhibitory transmission in the hippocampal slice. EXAMPLE 2: INTRAHIPPOCAMPAL INFUSION OF THE EXTRACELLULAR DOMAIN OF SEMA4D SUPPRESSES EPILEPTIFORM ACTIVITY AND PROGRESSION TO SE IN VIVO [0062] It was predicted that if a Sema4D-mediated decrease in evoked spiking activity in the hippocampus is the result of the formation/stabilization of new GABAergic synapses, then infusion of Sema4D-Fc into the hippocampus should suppress seizure activity and restore BZD sensitivity in pharmacoresistant epilepsy. The KA model of SE was selected to test this hypothesis because it is an acute, well-established model of TLE in which animals enter SE within an hour post-injection and develop seizures that do not respond to treatment with diazapam. Furthermore, diazepam-insensitivity increases with prolonged seizure activity, which mimics the manifestation of clinical pharmacoresistance. [0063] A cannula was inserted unilaterally into the CA1 region of hippocampus in 10- 12 week-old C57BL/6 male mice with hippocampal depth electrodes for EEG recordings affixed to their skulls. Two weeks later, 500nl of 100nM Sema4D-Fc or vehicle (Fc) was infused via cannula over five minutes, and after a one hour wait, induced seizures were induced by a 20mg/kg intraperitoneal (i.p.) injection of KA (Figure 2A). Seizure activity was monitored throughout the experiment using EEG. Epileptiform activity was considered to be paroxysmal activity having a sudden onset and an amplitude at least 2.5x the standard deviation of the baseline and a consistent change in the Power of the fast Fourier transform of the EEG (Fig.2B-C). In addition, SE was defined as persistent, unremitting epileptiform activity lasting longer than 5 consecutive minutes. All animals achieved SE by 1 hour post KA injection. [0064] Analysis of the 2-hour recording period before diazepam treatment indicated that Sema4D-Fc infusion increased the latency to onset of the first ictal event (Figure 2D). In addition, Sema4D infusion decreased the percentage of time the animals spent exhibiting epileptiform activity compared to control animals (Figure 2E), quantified as the cumulative time of all epileptiform activity during a 120-min recording period divided by 120 min. As is typical in SE experiments, a number of animals did not survive SE (Figure 2F); a N-1 corrected chi-squared test failed to reveal a difference in mortality between conditions (p=.54). Nonetheless an increased latency to SE was observed in the Sema4D-Fc treated group compared to control (Figure 2G) but no difference in latency to SE cessation (Figure 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 2H). Overall, this finding demonstrates that the duration of SE is decreased in Sema4D treated animals. [0065] Two hours after KA injection, diazepam (5mg/kg) was delivered via i.p. injection the mice were monitored for one hour. Diazepam sensitivity was defined as a reduction in the power of the electrographic signal and cessation of epileptiform activity as previously described in the art. Mice were considered to be pharmacoresistant if diazepam treatment did not suppress SE within 10 mins of administration. Thus, by analyzing the EEG recordings obtained for one hour post-diazepam injection it was observed that 2/6 animals in the Sema4D treatment group were insensitive to diazepam treatment compared to 6/7 animals in the control group (N-1 corrected chi-squared test p=.13) (Figure 2I). Taken together, these data indicate that hippocampal infusion of soluble Sema4D-Fc suppresses KA-induced seizure activity and impedes progression to SE. EXAMPLE 3: ADENO ASSOCIATED VIRUS EXPRESSING SEMA4D EXTRACELLULAR DOMAIN DRIVES FORMATION OF INHIBITORY BOUTONS IN VITRO [0066] The ability of Sema4D treatment to restore BZD sensitivity has important translational potential for the acute management of intractable seizures such as during SE. However, delivery of Sema4D-Fc by intrahippocampal infusion via a surgically implanted cannula is cumbersome and only allows for acute delivery of Sema4D. To circumvent these issues, virus-mediated delivery of the Sema4D ECD that would allow for both chronic and efficient delivery of the Sema4D protein was developed. [0067] Adeno-Associated Viruses (AAV) serotype 9 expressing either the full-length Sema4D protein (AAV-Sema4D-FL) or its ECD (AAV-Sema4D-ECD) and a control virus encoding full-length CD4 (AAV-CD4-FL), a transmembrane protein that has no effect on GABAergic synapse development or its ECD (AAV-CD4-ECD) (Figure 3A) was created. To selectively drive expression in neurons, all constructs are expressed under control of the human Synapsin I promoter. [0068] As an initial test of efficacy of these viruses, the ability of the viruses to increase GABAergic synapse density in coronal, organotypic brain slices containing anterior hippocampus obtained from C57BL/6 mouse pups ages P6-P8 was determined. This time point was chosen for initial verification because it is consistent with studies showing a pro- synaptogenic effect of purified Sema4D protein in ex vivo preparations. After 1 day in vitro (DIV1), slices were infected with a combination of AAV9.hsyn.GFP virus (Addgene) to 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 express GFP and one of the Sema4D- or CD4-expressing viruses. At DIV6, slices were fixed and immunostained with an antibody that detects GAD65, an enzyme localized to the GABAergic presynaptic bouton. (Figure 3B). As a positive control for the function of Sema4D, a subset of slices were infected with only AAV9.hsyn.GFP at DIV1 and treated with either Sema4D-Fc or Fc recombinant protein for two hours at DIV6 before processing the slices for immunostaining (Figure 3B). [0069] GABAergic synapse density was determined by imaging the pyramidal layer in the CA1 region of hippocampus and quantifying the density of GAD65-positive boutons formed onto the cell soma of GFP+ neurons similar to previous studies in the art. Neurons infected with AAV-Sema4D-ECD exhibited a significantly higher density of GAD65 puncta per soma as compared to the AAV-CD4-ECD control (Figure 3C, D). Notably, infection with AAVSema4D-FL did not increase the density of GAD65 puncta, perhaps indicating that soluble Sema4D-ECD is a more efficacious signaling moiety than membrane bound Sema4D. Importantly, the magnitude of change in GABAergic puncta density of approximately 20- 25% observed in this experiment is comparable to those observed using purified Sema4D-Fc protein in both this experiment (Figure 3C, D, Fc vs. Sema4D-Fc) and previous experiments in the art (see Figs.1A-C, 3A-D). Based on these results, AAV-Sema4D-ECD was tested in the KA model of SE, to determine whether chronic viral overexpression of Sema4D-ECD could reduce seizure symptoms and restore sensitivity to diazepam, similarly to infusion of purified Sema4D-Fc protein (Figure 2A-I). EXAMPLE 4: DELIVERY OF AAV-SEMA4D-ECD REDUCES EPILEPTIFORM ACTIVITY, DELAYS PROGRESSION TO SE, AND INCREASES SURVIVAL IN A KA MODEL OF SE [0070] AAV-Sema4D-ECD was tested in the KA model of SE to determine whether chronic viral overexpression of Sema4D-ECD could reduce seizure symptoms and impede progression to SE, similarly to infusion of purified Sema4D-Fc protein (Figure 2A-I). Bilateral injections of either AAV-Sema4D-ECD or control AAV-CD4-ECD virus into the CA1 region of hippocampus into 10-12 week-old C57BL/6 male mice were performed. Headmounts were affixed for EEG recording. Following a 1-week recovery period, a 20mg/kg i.p. injection of KA was performed to induce seizures. Seizure activity was monitored using EEG recording as described above (Fig.4A-C). Analysis of the two-hour recording period after KA administration and before diazepam treatment indicated no difference between AAV-CD4-ECD and AAVSema4D-ECD virus in latency to onset of the 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 first seizure (Figure 4D), which differs from results with infusion of purified Sema4D-Fc protein (Fig.2D above), and perhaps reflects differences in the levels of Sema4D-ECD at the time of seizure induction due to different delivery methods. However, it was observed that administration of AAV-Sema4D-ECD significantly reduced the amount of time spent in epileptiform activity as compared to mice treated with the AAV-CD4-ECD virus (Figure 4E), similar to the effect observed with Sema4D-Fc infusion (Fig.2E), and consistent with an increase in inhibition in the hippocampi of these animals. [0071] Interestingly, in this experiment only 5/8 animals in the AAV-Sema4D-ECD group achieved SE by 2 hours post KA (all 8 animals were subsequently subjected to diazepam administration; see below), while 8/8 animals in the AAV-CD4-ECD cohort achieved SE by 1 hour post KA injection. Further, 2/8 control animals died during SE compared to 0/8 for the AAV-Sema4D-ECD group, no significant difference in mortality was observed between conditions (N-1 corrected chi-squared test p=.14) (Figure 4F). In addition, progression to SE was delayed in the group treated with AAV-Sema4D-ECD (Figure 4G) while there was no difference in latency to seizure cessation compared to the control group (Figure 4H), demonstrating a decrease in the duration of SE with Sema4D treatment. Taken together, these data suggest a meaningful ability of chronic Sema4D expression to reduce progression to, and severity of, SE. Without being held to theory, it is speculated that the differences in severity of seizures post KA injection may be due to chronic expression of Sema4D in these animals, as opposed to the acute Sema4D-Fc treatment described above. [0072] Two hours after KA injection, diazepam (5mg/kg) was delivered via i.p. and the mice were monitored by EEG for an additional hour. Analysis of these recordings showed that 1/8 animals in the AAV-Sema4D-ECD treatment group were insensitive to diazepam treatment compared to 3/6 animals in the control group (N-1 corrected chi-squared test p=.14) (Figure 4I). To validate the targeting of the viral injections to hippocampus, post hoc immunostaining for GAD65 was performed on animals that had been injected with AAV-Sema4D-ECD or AAV-CD4-ECD (data not shown). AN~20% increase in GAD65 immunostaining fluorescence intensity was observed in the hippocampus of animals injected with AAV-Sema4D-ECD, consistent with correct targeting of the virus. Taken together, these results demonstrate that virally-mediated overexpression of Sema4D-ECD suppresses seizure activity, consistent with increased inhibition in the hippocampus. DISCUSSION 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 [0073] Described herein is the efficacy of Sema4D treatment using two different methods: 1) an acute, intrahippocampal infusion of purified Sema4D protein via cannula and 2) a chronic, virus mediated delivery of Sema4D ECD. One week of chronic Sema4D overexpression ameliorated epileptic activity and increased BZD efficacy. The ECD of Sema4D is approximately 700 amino acids and it is not expected to cross the Blood- Brain Barrier (BBB). Adeno Associated Virus (AAV) can be used to deliver therapeutics to the CNS. The results presented herein support the translational potential of virally delivered Sema4D as an antiseizure therapeutic for the treatment of both acute and chronic pharmacoresistant epilepsy. The present study advances the case for Sema4D-dependent GABAergic synapse formation as a new avenue for developing anti-seizure therapeutics based on the following observations. First, it was demonstrated that evoked CA1 excitability is suppressed by application of Sema4D protein to acute hippocampal slices and that this effect endures for at least 3 hrs, suggesting that Sema4D-mediated increased inhibition underlies the seizure suppression observed with in vivo application of Sema4D. Second, chronic delivery of Sema4D via viral transduction promotes increased GABAergic synapse density in ex vivo hippocampal organotypic slice cultures similar to purified Sema4D-Fc protein. Third, a chronic, less invasive method was developed to deliver Sema4D ECD using viruses. Chronic delivery of Sema4D via viral transduction promoted increased GABAergic synapse density in ex vivo hippocampal organotypic slice cultures similar to purified Sema4D-Fc protein (Figure 3A-D). Further, using the same KA model of SE, chronic expression of the Sema4D-ECD in hippocampus in vivo mitigated the progression to and severity of SE (Figure 4A-I). The current study builds upon the following previous findings: 1) Sema4D-Fc protein treatment increases the density of functional, GABAergic synapses containing the BZD-sensitive gamma subunit of the GABAA receptor in dissociated hippocampal neurons, 2) Sema4D-Fc protein treatment increases the density of functional, GABAergic synapses in acute slice, 3) Sema4D-Fc protein treatment stabilizes nascent, presynaptic GABAergic boutons in organotypic slice, 4) Sema4D-Fc infusion into the hippocampus of 6-8 week old mice both increases the density GAD65+ presynaptic termini in the hippocampus and suppresses seizures. All of these effects occur within a few hours of Sema4D treatment. It has now been shown that chronic administration of Sema4D is also effective to suppress seizures and relieve BZD resistance. [0074] The amount of functional inhibition in a neuronal network is determined by every aspect of synapse biology: e.g., the number of receptors available in the membrane at the postsynaptic specialization, the number of active zones in the presynapse, and the number 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 of synaptic contacts made between neurons. Described herein is the time course of the effect of Sema4D on population spike amplitude in CA1 in tissue taken from animals of an advanced age (Figure 1A-C). The observation that Sema4D treatment begins to suppress evoked population spike amplitude within one hour of treatment, taken together with immunohistochemistry data (Figure 3A-D), suggest that Sema4D promotes the formation of functional GABAergic synapses resulting in decreased hippocampal excitability. These data also support previous observations that the cellular machinery which responds to Sema4D signaling to promote GABAergic synapse formation remains accessible in adult animals. [0075] Studies in multiple model systems have demonstrated that Semaphorins and their receptors are critical mediators of synaptogenesis. Sema4D regulates GABAergic synapse formation via signaling through its high affinity receptor Plexin-B1. It is widely accepted that Sema4D binding to Plexin-B1 causes dimerization and activation of the PlexinB1 intracellular GTPase activating protein (GAP) domain, triggering downstream signal transduction events that regulate the actin cytoskeleton and cell morphology. [0076] The current model of Sema4D signaling posits that Sema4D/Plexin-B1 signaling modulates the scaffolding machinery present at the synapse. Specifically, live- imaging of fluorescently-labeled synaptic proteins revealed that Sema4D signaling changes the subcellular localization of the GABAergic scaffolding protein gephyrin at the postsynapse. Rapid “splitting” of gephyrin-GFP puncta within ten minutes of Sema4D application resulted in additional gephyrin puncta which may nucleate new sites for GABAergic synapses to form. Furthermore, the c-Met receptor tyrosine kinase, which regulates actin depolymerization downstream of Sema4D/Plexin-B1 signaling, has been implicated in Sema4D-mediated stabilization of presynaptic GABAergic boutons and is specifically required in the presynaptic neuron for this effect. However, the precise signal transduction mechanisms underlying Sema4D/Plexin-B1 mediated GABAergic synaptogenesis are yet to be determined. [0077] To design a novel route of Sema4D administration to the brain, the ability of AAV9 virus expressing different portions of the Sema4D protein to promote GABAergic synapse formation in ex vivo organotypic slice cultures was teste in the experiments described herein (Figure 3A-D). Interestingly, while overexpression of Sema4D-ECD promoted GABAergic synapse development similarly to application of the purified Sema4D-Fc protein, overexpression of Sema4D-FL did not. Although the extracellular domain of Sema4D is cleaved from the neuronal cell surface, these results were unexpected as previous studies demonstrate that Sema4D can signal as a transmembrane bound or soluble extracellular 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 domain. It is plausible that differences in protein stability, expression, localization, or membrane insertion between Sema4D-ECD and Sema4D-FL may account for this disparity in synapse-promoting activity. [0078] Both Sema4D and Plexin-B1 are expressed in inhibitory and excitatory neurons and glia in developing hippocampus. Functional studies show that Sema4D signals through Plexin-B1 expressed pre- and postsynaptically, suggesting that a bidirectional trans- synaptic signaling complex may regulate Sema4D-dependent GABAergic synapse formation. Since Sema4D-ECD expression was driven by a neuron-specific promoter in the AAV vector, these results demonstrate that overexpression of Sema4D-ECD in neurons is sufficient to increase the density of functional inhibitory synapses in hippocampus (Figure 4A-I). Without being held to theory, these data suggest a model whereby virally-delivered Sema4D- ECD, secreted from neurons, acts in both an autocrine and paracrine manner to regulate this process. [0079] Taken together the data presented herein suggest that Sema4D is uniquely suited to address problems of pharmacoresistance in SE, as Sema4D treatment in the hippocampus mitigates the BZD resistance observed in RSE, possibly by promoting GABAergic synapse development and/or stabilization. Based on the data presented herein, it is believed that chronic Sema4D treatment will be effective for seizure suppression. [0080] BZDs combat seizures and enhance inhibition by direct binding to GABAAR. However multiple groups showed that prolonged seizures are correlated with internalization or altered trafficking of GABAARs, with some studies citing decreased surface expression of the γ2 and β2/3 subunits of the GABAAR. Thus, therapeutics targeting SE that focus on methods to increase the density of GABAARs at the synapse in order to provide new sites for BZD binding could be effective as an add-on medication to BZD in RSE. [0081] The effect of ion flow through GABAARs can depolarize or hyperpolarize the neuron; this role changes throughout development and is mediated in large part by the constitutively active KCC2 and Cl- homeostasis. Thus, an alternative hypothesis regarding BZD resistance asserts that changes in KCC2 function interfere with BZD efficacy by altering the effect of net Cl- flow across the cellular membrane, reversing the electrochemical driving force and the direction of GABAA-mediated current. Interestingly, a recent study reported a proteomics approach which identified KCC2- interacting proteins. This study showed that gephyrin interacts with KCC2 and regulates its function by promoting KCC2 clustering at the membrane. It is possible that another mechanism by which Sema4D restores 0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 BZD sensitivity is via gephyrin splitting, which could cause a redistribution of gephyrin and therefore KCC2 at the membrane. [0082] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. [0083] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 CLAIMS 1. A method of treating a patient in need of chronic treatment for epilepsy or epileptogenesis, comprising chronically administering to the patient an extracellular Sema4D peptide which has at least 80%, 85%, 90%, 95%, 98% or 99% identity to amino acid residues 22 to 734 of SEQ ID NO: 1, amino acid residues 22-690 of SEQ ID NO: 3, or amino acid residues 22-733 of SEQ ID NO: 5, and wherein the chronic administration reduces the incidence of seizures in the patient, slows disease progression, and/or reduces the risk of death in the patient. 2. The method of claim 1, wherein the patient has suffered a brain damaging insult associated with epileptogenesis. 3. The method of claim 2, wherein the brain damaging insult is traumatic brain injury, prolonged febrile seizure, stroke, intracerebral hemorrhage, infection or status epilepticus. 4. The method of claim 1, wherein the patient is diagnosed with Dravet Syndrome. 5. The method of claim 1, wherein the patient has chronic drug refractory epilepsy (CDRE). 6. The method of claim 1, wherein the patient has more than three generalized tonic-clonic seizures per year and is at risk for sudden unexpected death in epilepsy (SUDEP). 7. The method of claim 6, wherein the subject has a SUDEP risk factor selected from medication non-compliance, having CDRE, being diagnosed with a genetic epilepsy, having early age of epilepsy onset, having uncontrolled or frequent seizures, or a combination thereof. 8. The method of any of the foregoing claims, wherein the Sema4D peptide is delivered via viral mediated gene transduction.

0107648.0037 BRANDEIS REF 2023-007, Tufts Ref. T002704 9. The method of claim 8, wherein the Sema4D peptide is expressed from an AAV virus. 10. The method of any of claims 1-7, wherein the Sema4D peptide is delivered in the form of a fusion polypeptide with a brain-targeting peptide, a nanoparticle delivery system, a liposome, a microemulsion, a hydrogel or an injectable polymer. 11. The method of any of claims 1-7, wherein the Sema4D peptide is delivered in the form of an mRNA. 12. The method of any of claims 1-7, wherein the Sema4D peptide or a polynucleotide expressing the Sema4 polypeptide is delivered to the hippocampus, via intrathecal delivery, via or via intracerebroventricular delivery. 13. The method of any of the foregoing claims, wherein the patient is a human patient.