Viral therapy for the treatment of MEF2c haploinsufficiency syndrome

Abstract

MEF2C haploinsufficiency syndrome (MCHS) is a neurodevelopmental disorder with profound impacts on the affected children and their families. Deletion or mutation of one copy of a MEF2C gene, which codes for an activity-regulated transcription factor, causes MEF2C Haploinsufficiency syndrome. Described herein are nucleic acid molecules comprising an expression cassette comprising a MEF2C isoform coding sequence and a neuronal specific promoter that is operably linked to the MEF2C isoform coding sequence. Recombinant adeno-associated virus vectors comprising the nucleic acid molecules, and self-complementary forms of adeno-associated virus vectors comprising the nucleic acid molecules. Also provided are pharmaceutical compositions for treating MEF2C haploinsufficiency syndrome, methods of treating MEF2C haploinsufficiency syndrome in a patient in need thereof, and methods of inducing transgenic expression of MEF2C in a subject.

Description

[0001] DESCRIPTION

[0002] VIRAL THERAPY FOR THE TREATMENT OF MEF2C HAPLOINSUFFICIENCY SYNDROME

[0003] PRIORITY CLAIM

[0004] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 682,122, filed August 12, 2024, the entire contents of which are hereby incorporated by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0006] This invention was made with government support under grant no. R01MH111464 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO A SEQUENCE LISTING

[0008] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on August 11, 2025, is named MESCP0144WO.xml and is 33,203 bytes in size.

[0009] BACKGROUND

[0010] 1. Field of the Disclosure

[0011] The present disclosure relates generally to the fields of medicine, genetics, and molecular biology. More particular, the disclosure relates to compositions and methods of treating MEF2C haploinsufficiency syndrome.

[0012] 2. Background

[0013] MEF2C haploinsufficiency syndrome (MCHS) is a neurodevelopmental disorder with profound impacts on the affected children and their families. Deletion or mutation of one copy of MEF2C gene, which codes for an activity-regulated transcription factor, causes MEF2C Haploinsufficiency syndrome (MCHS). MCHS is commonly associated with severe language

[0014] -1-

[0015] 4900-4314-0443, v. 1 deficits, social impairments, repetitive behaviors, reduced muscle tone, intellectual disability, sensory abnormalities, sleep problems, variable types of seizures, hyperactivity, immune system dysregulation, and motor coordination challenges. There are two copies of the MEF2C gene in our cells on chromosome 5ql4.3, and each gene copy contributes -50% of the total protein needed for MEF2C to carry out its normal function(s) in cells. As such, it appears that loss of one normally functioning copy of MEF2C in human development reduces overall MEF2C levels and causes a myriad symptoms associated with MCHS. In addition to the numerous gene deletions, the inventors found that MCHS patient missense mutations, where one or more amino acids are changed to a different residue, produced profound loss of DNA binding and / or reduced MEF2C levels, suggesting that these are predominantly loss-of- function (LOF) mutations. Even in the few cases of small base-pair duplications, they occurred in a highly conserved part of the MEF2C protein and appear to decrease protein stability and / or MEF2C function.

[0016] To date, there are more than 400 patients worldwide with MCHS, and with the increasing affordability and accessibility of genetic sequencing technology, the number of individuals with documented or suspected MCHS is growing almost daily. Some have speculated that the prevalence of MCHS might eventually emerge as >10 times the rate of well- known Rett syndrome. Indeed, one study of more than 300 individuals with idiopathic intellectual disability revealed that nearly 2% had genetic variation in the MEF2C gene, suggesting that MEF2C mutations underlie a staggering proportion of individuals with a neurodevelopmental disorder of unknown cause. As such, understanding and treating MEF2C haploinsufficiency could have a large impact on afflicted individuals.

[0017] -2-

[0018] 4900-4314-0443, v. 1 SUMMARY

[0019] Thus, in accordance with the present disclosure, there is provided a nucleic acid molecule comprising an expression cassette comprising a MEF2C isoform coding sequence and a neuronal-specific promoter that is operably linked to the MEF2C isoform coding sequence. In some embodiments, the MEF2C isoform coding sequence described above does not comprise a gamma-domain. In some embodiments, the MEF2C isoform coding sequence described above does comprise a gamma-domain. In some embodiments, the MEF2C isoform coding sequence described above includes a miniature intron to allow alternative splicing to both include and exclude the gamma-domain. In some embodiments, the neuronal specific promoter described above is a synapsin promoter (hSyn). In some embodiments, the promoter might include promoter or enhancer sequences from human MEF2C. In some embodiments, the promoter might include alternative promoter regulatory sequences that confer brain expression patterns that closely approximate expression of endogenous MEF2C mRNA or protein in human brain.

[0020] In some embodiments, MEF2C may comprise at least one of the following: the alphal domain, the beta domain, and the gamma domain sequence. In some embodiments, MEF2C may comprise the alpha 2 domain. In some embodiments, MEF2C may not comprise the alphal domain. In some embodiments, MEF2C may not comprise the alpha2 domain. In some embodiments, MEF2C may not comprise the beta domain. In some embodiments, MEF2C may not comprise the gamma domain.

[0021] In some embodiments, there is provided a recombinant adeno-associated virus (rAAV) vector comprising any of the nucleic acid molecules described above. In some embodiments, there is provided a self-complementary form adeno-associated virus (scAAV) vector comprising any of the nucleic acid molecules described above.

[0022] In some embodiments, there is provided a pharmaceutical composition for treating MEF2C haploinsufficiency syndrome in a patient in need thereof, the pharmaceutical composition comprising any one of the nucleic acid molecules described above. In some embodiments, there is provided a pharmaceutical composition for treating MEF2C haploinsufficiency syndrome in a patient in need thereof, the pharmaceutical composition comprising any of the recombinant adeno-associated virus (rAAV) vectors described above.

[0023] In some embodiments, there is provided a method of treating MEF2C haploinsufficiency syndrome in a patient in need thereof comprising administering an effective amount of any of the pharmaceutical compositions described above. In some embodiments,

[0024] -3-

[0025] 4900-4314-0443, v. 1 there is provided a method of inducing transgenic expression of MEF2C in a patient comprising administering an effective amount of any of the pharmaceutical compositions described above. In some embodiments, the methods of treating described above comprise improving one or more symptoms selected from the group consisting of autism spectrum disorder, intellectual disability, speech disability, hyperactivity, epilepsy, attention-deficit / hyperactivity disorder, and schizophrenia. In some embodiments, administering comprises injection of the pharmaceutical composition to bilateral transverse sinus regions. In some embodiments, administering comprises oral, intravenous, subcutaneous, intrathecal, or topical administration. In some embodiments, the patient is between 0 and 5, 0 and 10, 0 and 15, 0 and 20, or 0 and 25 years of age. In some embodiments, any of the pharmaceutical compositions described above are administered in combination with a second MEF2C haploinsufficiency syndrome treatment. In some embodiments, the rAAV or scAAV vectors are derived from serotype AAV9.

[0026] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0027] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0028] -4-

[0029] 4900-4314-0443, v. 1 BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIGS. 1A-C. Coronal brain slices of a P28 mouse displaying AAV9-hSyn- EGFP injected through neonatal transverse sinus injections at P0-1. This image was taken under a Leica THUNDER epifluorescence microscope at 20x. (FIG. 1A) A brain section displaying robust GFP expression in the prefrontal cortex and motor areas. (FIG. IB) GFP expression is present in the dentate gyrus of the hippocampus and the somatosensory cortex. (FIG. 1C) GFP is induced in caudal cortical regions.

[0032] FIGS. 2A-C. (FIG. 2A) (top) Prefrontal cortex area that was imaged, (bottom) Somatosensory cortex (SSC) area that was imaged. (FIG. 2B) Proportion of virally infected neurons in the superficial (n=2 animals, 8 images per animal) vs. deeper layers (n=2, 9 images for Animal 1, 8 images for Animal 2) of the SSC by immunohistochemistry for GFP and NeuN (neuronal nuclear protein) and Leica Stellaris 5 confocal imaging (63X oil). Deeper cortical layers were preferentially infected. (FIG. 2C) Based on two animals (17 images for Animal 1, 16 images for Animal 2), the average proportion of neurons infected in the SSC cortex was .45. The average proportion in the PFC was .50 (17 images for Animal 1, 13 images for Animal 2). Statistical significance was determined by unpaired t-test for B. **pc.0L

[0033] FIGS. 3A-B. (FIG. 3A) GFP and PV immunohistochemistry in the somatosensory cortex (SSC) and (FIG. 3B) prefrontal cortex (PFC) to assess PV cell targeting. AAV9-hSyn-EGFP targets PV cells, a subtype of inhibitory neuron. Images were acquired using a Leica Stellaris 5 Confocal microscope (40X oil).

[0034] FIG. 4. AAV9-hSyn-EGFP targets cerebellar Purkinje cells, a subtype of inhibitory neuron that expresses MEF2C, as evidenced by co-localization between GFP and GAD67, which is expressed in GABA containing neurons.

[0035] -5-

[0036] 4900-4314-0443, v. 1 Images were acquired using a Leica Stellaris 5 Confocal microscope (40X oil). GAD67= glutamate decarboxylase 67, SSC= somatosensory cortex.

[0037] FIGS. 5A-C. Plasmids of (FIG. 5A) control and the (FIGS. 5B-C) predominant MEF2C isoforms (with and without the gamma domain) generated. These plasmids were packaged by the University of South Carolina Viral Vector Core and validated in the immunohistochemistry images (right) using the Leica Stellaris 5 Confocal microscope (63X oil).

[0038] FIG. 6. Vector map of an exemplary pAAV vector with a human synapsin promoter plus mouse MEF2C open-reading-frame (alpha2 = beta domain + gamma domain) with a 3X Flag epitope tag + P2A cleavage site + mCherry ORF and hGH polyA sequence.

[0039] FIGS. 7A-D. (FIG. 7A) An embodiment of preclinical and clinical vector constructs and the constitutive elements (alpha 1, beta, and gamma domains) which comprise each; the unique mini-intron sequence permits alternative splicing events to occur for the production of MEF2C isoforms which may (i) or may not (ii) contain the gamma domain. Version 1 is utilized in preclinical studies to detect vector derived expression of MEF2C protein fused to a FLAG epitope tag with the ultimate goal of implementing version 2 in clinical treatment with administration via a human compatible capsid with no FLAG tag within the sequence. (FIG. 7B) The MEF2C isoforms produced from the alternative splicing of the preclinical vector construct may (i) or may not (ii) contain the gamma domain at a ratio of one to one. (FIG. 7C) Semi-quantitative RT-PCR analysis of Mef2c mRNA transcripts produced by mouse neuroblastoma cells (N2A) transfected with the preclinical plasmid construct indicates the expression of the ‘plus gamma’ (+y; 344 bp) and ‘minus gamma’ (-y; 248 bp) isoforms. (FIG. 7D) Western blots of protein from the brain tissue of untreated C57 BL / 6J (left panel) demonstrating MEF2C isoform expression in the wildtype and N2A cells (right panel) transfected with the preclinical plasmid construct producing vector-derived MEF2C protein fused to the FLAG tag.

[0040] -6-

[0041] 4900-4314-0443, v. 1 FIG. 8. Vector map of an exemplary pAAV vector with a human synapsin I promoter plus human MEF2C open reading frame (alpha2 = beta domain + gamma domain) with a 3X FLAG epitope tag and hGH polyA sequence. The location of the unique mini-intron sequence which allows inclusion or exclusion of the gamma domain is indicated by the broken line in the diagram.

[0042] FIGS. 9A-H. Coronal brain slices of a Pl 6 mouse demonstrating AAV.PhP.eB- hSyn-MEF2C-FLAG vector expression following neonatal intracerebroventricular injection at PO-1. The expression levels of MEF2C (FIG. 9A, FIG. 9C, FIG. 9E, and FIG. 9G) and FLAG (FIG. 9B, FIG. 9D, FIG. 9F, and FIG. 9H) are shown for the somatosensory cortex (dotted outline region in FIG. 9A and FIG. 9B) and the prefrontal cortex (dotted outline region in FIG. 9C and FIG. 9D). Exemplar instances of cell types that are expressing MEF2C alone (open arrowheads, Mef2c+FLAG-) or both MEF2C and FLAG (filled arrowheads, Mef2c+FLAG+) are indicated for each region. Images were acquired using a Leica Stellaris 5 confocal microscope at 20X (FIGS. 9A-D) and 40X (FIGS. 9E- H).

[0043] FIGS. 10A-H. Higher magnification images from the somatosensory cortex (SSC, FIG. 10A and FIG. 10B) and the prefrontal cortex (PFC, FIG. 10E and FIG. 10F) displaying expression for Mef2c protein (FIG. 10A and FIG. 10E) and FLAG epitope tag (FIG. 10B and FIG. 10F) showing cell types that are expressing MEF2C alone (open arrowheads, Mef2c+FLAG-) or both MEF2C and FLAG (filled arrowheads, Mef2c+FLAG+). Quantitative analysis of mean fluorescence intensity for MEF2C protein in cell types that are expressing MEF2C alone (FLAG-) or both MEF2C and FLAG (FLAG+) are shown as boxplots for the SSC (FIG. IOC) and PFC (FIG. 10G). The proportion of MEF2C expressing cells which also express the FLAG epitope tag (indicating vector derived expression) is also shown for the SSC (FIG. 10D) and the PFC (FIG. 10H). Images were acquired using a Leica Stellaris 5 confocal microscope at 40X (oil) with a zoom factor of ~2x. Number of animals = 3. Asterisks in FIG. IOC and FIG. 10G represent a p- value < 0.0001 (Student’s unpaired t-test).

[0044] -7-

[0045] 4900-4314-0443, v. 1 FIG. 11. Schematic of Mef2c RNA splicing. Mef2c transcripts undergo tissuespecific alternative mRNA splicing at different sites. All transcripts contain either the al or a2 (exon 3) domain, and -50% of the transcripts will also express the alternatively spliced y-domain (located in exon 9).

[0046] FIGS. 12A-D. Tissue-specific expression of different MEF2C isoforms. Quantitative RT-PCR using primers specific for Mef2c was used to examine expression of Mef2c isoforms in tissue from wild-type (C57BL6 / J) mice. (FIGS. 12A-B) qPCR analysis of Mef2c isoforms in adult (~p56) tissue. (FIG. 12A) Using a primer set common to all Mef2c isoforms, we show that Mef2c is highly expressed in cortical tissue, compared to other tissue examined. (FIG. 12B) Using primers specific to each splice domain of Mef2c, we show that neuronal Mef2c isoforms express al and 0 domains, and about half of the isoforms express the y-domain. Domain expression was normalized to total Mef2c expression from (FIG. 12A). (FIGS. 12C-D) qPCR analysis of Mef2c isoforms expressed in the cortex throughout development in C57BL6 / J mice. (FIG. 12C) Mef2c is highly expressed early in development (P0-P7) and is expressed in adult cortical tissue. (FIG. 12D) Relative expression of Mef2c domains remains consistent throughout development, showing that cortical Mef2c express al and 0 domains (-100%) and y-domain (-50%). N=3 for each experiment.

[0047] -8-

[0048] 4900-4314-0443, v. 1 DETAILED DESCRIPTION

[0049] The fundamental cause of MCHS is an inadequate amount of normally functioning MEF2C protein following mutation or deletion of one MEF2C gene copy. The inventors’ initial therapeutic approaches seek to increase MEF2C levels from the non-mutated gene copy, which holds promise to reduce, or even eliminate, symptoms. The human brain is extraordinarily adaptable and has cell machinery to change and adapt to our lifelong experiences. This brain plasticity provides tremendous hope that restoring sufficient MEF2C levels in individuals with MCHS will provide the needed ingredients for the brain and body to restore neurotypical functionality.

[0050] These and other aspects of the disclosure are described below.

[0051] I. MEF2C and MCHS

[0052] Myocyte-specific enhancer factor 2C also known as MADS box transcription enhancer factor 2, polypeptide C is a protein that in humans is encoded by the MEF2C gene. MEF2C is a transcription factor in the Mef2 family. The gene is located at 5ql4.3 on the minus (Crick) strand and is 200,723 bases in length. The encoded protein has 473 amino acids with a predicted molecular weight of 51.221 kD. Three isoforms have been identified. Several post translational modifications have been identified including phosphorylation on serine-59 and serine-396, sumoylation on lysine-391, acetylation on lysine-4 and proteolytic cleavage.

[0053] MEF2C has been shown to interact with EP300, HDAC4, HDAC7, HDAC9, MAPK7, SOX18, SP1, TEAD1, and SETD1A. This gene is involved in cardiac morphogenesis and myogenesis and vascular development. It may also be involved in neurogenesis and in the development of cortical architecture. Mice without a functional copy of the Mef2c gene die before birth and have abnormalities in the heart and vascular system. It is one of the targets of an oncomiR, MIRN21. The MEF2C -binding site is associated with minor allele of SNP rs630923, associated with the risk of multiple sclerosis, and responsible for reduced CXCR5 gene promoter activity in B -cells during activation, that could lead to decreased autoimmune response.

[0054] MEF2C exists as two main isoforms in the brain and at multiple developmental timepoints. Due to alternative splicing of Mef2c mRNA, a small polypeptide, called the gamma domain, is either included in or absent from MEF2C protein (FIG. 11). This gamma domain may influence transcriptional activation. These isoforms may exist in about equal parts within

[0055] -9-

[0056] 4900-4314-0443, v. 1 the brain (FIG. 12B and FIG. 12D). MEF2C variants including the gamma-domain may be particularly sensitive to activation by membrane depolarization.

[0057] Mef2A, C and D encode protein variants by virtue of alternative splicing of primary transcripts, and these genes have similar structures and alternative splicing patterns that are conserved across evolution. The alternative splicing involves mutually exclusive exons (alphal and alpha2), a cassette exon (beta), and alternative splice acceptors that flank a short region (gamma) (FIG. 11). The corresponding short polypeptide domains encoded by these alternative segments are nested within the Mef2 carboxy-termini and are structurally conserved across isotypes. These domains confer specific functions, including splicing variant-specific functional interactions with co-activators; potent transactivation by an "acid blob” (beta); and transrepression that is mediated by SUMOylation (gamma) and that is under control of various signaling events that modify Mef2 and act in cis to control steady-state MEF2 SUMOylation. As one aspect of an effort to elucidate the roles of Mef2 alternative splicing variants, the inventors have developed an RT-PCR long fragment assay in which all eight Mef2C mRNA isoforms can be simultaneously monitored in cell and tissue samples. This assay is used to confirm and extend prior observations of regulated Mef2 alternative splicing among tissues, during development and during muscle differentiation. The technique is well suited for rapid qualitative evaluation of splicing variant expression, and could be effectively used for candidates with established splicing variants or for the validation of findings observed with “next generation” sequencing. Importantly, this strategy uniquely allows for the evaluation of co-variations in multiple alternative splicing events for primary transcripts of a given gene. Sciabica et al (Sciabica, K., Ramachandran, B., Wu, Y., Yowanto, H., & Gulick, T. (n.d.). Evaluation of splicing event co-variation with a strategy for the simultaneous detection of alternatively spliced MEF2C transcripts) is hereby incorporated by reference in its entirety.

[0058] In humans, mutations of this gene result in autosomal dominant mental retardation 20 (MRD20). It is also called MEF2C haploinsufficiency syndrome (MCHS). Mutations in the MEF2C gene that cause this syndrome are rare. More than 400 individuals have been reported in the medical literature or have self-identified to the MEF2C family group (Facebook), but it is likely that there are many more undiagnosed individuals. The disease is characterized by severe psychomotor impairment, periodic tremor and an abnormal motor pattern with mirror movement of the upper limbs observed during infancy, hypotonia, abnormal EEG, epilepsy, absence of speech, autistic behavior, bruxism, and mild dysmorphic features, mild thinning of the corpus callosum and delay of white matter myelination in the occipital lobes.

[0059] -10-

[0060] 4900-4314-0443, v. 1 Much of what is known about MEF2C function comes from fundamental research in mice. The inventors and others have generated and characterized mice lacking one functional copy of MEF2C (Me / 2<?+ / _), and these mice displayed numerous MCHS-related behavioral phenotypes, including deficits in social behavior, sensory sensitivity abnormalities, motor hyperactivity, repetitive behavior, reduced sensitivity to pain and sound, altered sleep patterns. The brains of the MCHS mice show alterations in structure and profound changes in the way neurons transmit information in the brain. As a transcription factor, or protein that binds to the genomic DNA and regulates other genes, the inventors found that MCHS mouse brains have hundreds of dysregulated genes, including gene products with known links to risk for autism spectrum disorder (ASD) and with known functions in regulating the electrochemical nodes of communication in the brain, or synapses. The inventors’ recent research also reveals that MCHS in mice disrupts the normal functions of the brain’s major immune cell population (called microglia). These microglia are known to play critical roles in pruning away excess or inappropriate synapses during brain development. Moreover, the inventors find that MEF2C haploinsufficiency in both excitatory and inhibitory neurons are sufficient to reproduce many of the symptoms of MCHS, which is perhaps not surprising since MEF2C is found at high levels in these neurons during development and into adulthood, and most of the symptoms of MCHS are consistent with altered neurological function.

[0061] Taken together, these findings paint a clear picture that multiple brain cell populations are affected by reduced levels of MEF2C, and that successful therapeutic approaches might need to target multiple cell populations.

[0062] IL Neuronal Specific Promoters

[0063] In a particular embodiment, it may be desirable to use a neuronal- specific promoter to achieve neuron-specific expression of a desired polynucleotide sequence. Illustrative examples of neuronal specific promoters include, but are not limited to: a glial fibrillary acidic protein (GFAP) promoter (astrocyte expression), a synapsin promoter (neuron expression), and calcium / calmodulin-dependent protein kinase II (neuron expression) , tubulin alpha I (neuron expression) , neuron- specific enolase (neuron expression), platelet-derived growth factor beta chain (neuron expression), a TRPVl promoter (neuron expression), a Navl.7 promoter (neuron expression), a Navi.8 promoter (neuron expression), a Navl.9 promoter (neuron expression), or an Advillin promoter (neuron expression).

[0064] Non-limiting examples of neuron-specific promoters include: human synapsin- 1 (SYN- 1) promoter, calcium-calmodulin dependent protein kinase IIA (CaMKIIA) promoter, tubulin

[0065] -11-

[0066] 4900-4314-0443, v. 1 alpha 1 promoter, neuron- specific enolase (NSE) promoter, platelet derived growth factor beta chain promoter (PDGFB), TRPV1 promoter, Navl.7 promoter, Navi.8 promoter, Navl.9 promoter, Advillin promoter, the Drosophila single minded homolog 1 (SIM1) promoter, oxytocin (OXT) promoter, Agouti-related peptide (AgRP) promoter, protein kinase C-delta (PKC-delta) promoter or ghrelin promoter.

[0067] Neuronal-specific promoters may be used to enable precise expression in targeted neuronal populations. One of the most widely used neuronal-specific promoters is the synapsin I promoter, which drives gene expression in almost all neurons. The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between murine and human.

[0068] Another notable example is the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, which is particularly active in excitatory neurons in the forebrain.

[0069] Recent advancements have introduced activity -dependent promoters, such as the E- SARE (enhanced synaptic activity-responsive element), which allow for the labeling of neurons based on their activity levels.

[0070] III. Delivery of MEF2C Coding Regions

[0071] A. Formulation and Administration

[0072] The present disclosure provides pharmaceutical compositions comprising expression vectors. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. The term “treat” and its grammatical equivalents used herein generally refer to the use of a composition or method to reduce, eliminate, or prevent symptoms of a disease and includes achieving a therapeutic benefit and / or a prophylactic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder or condition being treated. A prophylactic benefit of the treatment includes reducing the risk of a condition, retarding the progress of a condition, or decreasing the likelihood of occurrence of a condition. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In a specific embodiment, the term “effective amount” means an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or attenuate particular symptoms. The term “carrier” refers to a diluent,

[0073] -12-

[0074] 4900-4314-0443, v. 1 excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0075] In certain embodiments, the nucleic acid molecule or pharmaceutical composition will be prepared in a suitable diluent, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified nucleic acid molecule or pharmaceutical composition is subsequently reconstituted with a suitable diluent, e.g., aqueous solution, such as water or physiologically compatible buffers such as saline solution, Hanks’s solution, or Ringer’s solution. The reconstituted product is administered as a subcutaneous injection, a bilateral transverse sinus injection, an intracerebroventricular injection, or as an intravenous infusion. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal.

[0076] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0077] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium,

[0078] -13-

[0079] 4900-4314-0443, v. 1 ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine , 2-ethylamino ethanol, histidine, procaine, and the like.

[0080] Generally, ingredients of the compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0081] The composition may be administered to a subject by various routes. Non-limiting examples of methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, transdermal administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, and intraneural administration. In some cases, administration can involve injection of a liquid formulation of the composition. In other cases, administration can involve oral delivery of a solid formulation of the composition. In a particular case, a composition is administered by oral administration (e.g., a pill, tablet, capsule and the like). In some cases, the oral composition can be administered with food. In another particular case, a composition is administered by intrathecal injection (i.e., into the subarachnoid space of the spinal cord) for delivery to the cerebrospinal fluid (CSF) of the subject. In another particular case, a ligand is administered topically (e.g., dermal patch, cream, lotion, ointment and the like).

[0082] B. Expression Vectors

[0083] The MEF2C is delivered via expression from an expression vector. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a retroviral vector, a lentiviral vector, a pox viral vector, a herpesviral vector, an adenoviral vector or an adeno- associated viral (AAV) vector.

[0084] Adeno-associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family. To date, numerous serologically distinct AAV s have been identified, and more than a dozen have been isolated from humans or primates. AAV is distinct from other members of this family by its dependence upon a helper virus for replication. AAV genomes can exist in an extrachromosomal state without integrating into host cellular genomes; possess a broad host

[0085] -14-

[0086] 4900-4314-0443, v. 1 range; transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes. AAV viral particles are heat stable; resistant to solvents, detergents, changes in pH, and temperature; and can be column purified and / or concentrated on CsCl gradients or by other means. The AAV genome comprises a singlestranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The approximately 4.7 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. The ends of the genome are short-inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as the origin of viral DNA replication.

[0087] An AAV “genome” refers to a recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. An AAV particle often comprises an AAV genome packaged with AAV capsid proteins. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the AAV vector genome does not include the portion of the “plasmid” that does not correspond to the vector genome sequence of the recombinant plasmid. This non vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is needed for plasmid propagation and production but is not itself packaged or encapsulated into viral particles. Thus, an AAV vector “genome” refers to nucleic acid that is packaged or encapsulated by AAV capsid proteins.

[0088] The AAV virion (particle) is a non-enveloped, icosahedral particle approximately 25 nm in diameter that comprises an AAV capsid. The AAV particle comprises an icosahedral symmetry comprised of three related capsid proteins, VP1 , VP2 and VP3, which interact together to form the capsid. The genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1 :10 respectively, but may be in varied ratios, and are all derived from the right-hand ORE The VP1 , VP2 and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1 which is translated from an alternatively spliced message results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any singlestranded progeny DNA or infectious particles. In certain embodiments, the genome of an AAV particle encodes one, two or all three VP1, VP2 and VP3 polypeptides.

[0089] The left ORF often encodes the non- structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated

[0090] -15-

[0091] 4900-4314-0443, v. 1 with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68 / 78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities. Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. In certain embodiments the genome of an AAV (e.g. , an rAAV) encodes some or all of the Rep proteins. In certain embodiments the genome of an AAV e.g., an rAAV) does not encode the Rep proteins. In certain embodiments one or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a polypeptide.

[0092] The ends of the AAV genome comprise short-inverted terminal repeats (ITR) which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Accordingly, the genome of an AAV comprises one or more (e.g., a pair of) ITR sequences that flank a single stranded viral DNA genome. The ITR sequences often have a length of about 145 bases each. Within the ITR region, two elements have been described which are believed to be central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration.

[0093] The term “recombinant,” as a modifier of vector, such as recombinant viral, e.g., lentivirus or parvovirus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e. , engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted within the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into a vector, with or without 5', 3' and / or intron regions that the gene is normally associated within the viral genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission.

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[0095] 4900-4314-0443, v. 1 A recombinant viral “vector” is derived from the wild-type genome of a virus by using molecular methods to remove part of the wild-type genome from the virus, and replacing it with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A “recombinant” viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome, since part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”

[0096] In certain embodiments, an AAV (e.g., a rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., a rAAV) comprises a pair of ITRs that flank (i.e., are at each 5' and 3' end) of a nucleic acid sequence that at least encodes a polypeptide having function or activity.

[0097] An AAV vector (e.g., rAAV vector) can be packaged and is referred to herein as an “AAV particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle can also be referred to as a “rAAV particle.” In certain embodiments, an AAV particle is a rAAV particle, A rAAV particle often comprises a rAAV vector, or a portion thereof. A rAAV particle can be one or more rAAV particles (e.g. , a plurality of AAV particles). rAAV particles typically comprise proteins that encapsulate or package the rAAV vector genome (e.g., capsid proteins). It is noted that reference to a rAAV vector can also be used to reference a rAAV particle.

[0098] Any suitable AAV particle (e.g., rAAV particle) can be used for a method or use herein. A rAAV particle, and / or genome comprised therein, can be derived from any suitable serotype or strain of AAV. A rAAV particle, and / or genome comprised therein, can be derived from two or more serotypes or strains of AAV. Accordingly, a rAAV can comprise proteins and / or nucleic acids, or portions thereof, of any serotype or strain of AAV, wherein the AAV particle is suitable for infection and / or transduction of a mammalian cell. Non-limiting examples of AAV serotypes include serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), and 12 (AAV12). The AAV vector may be replication-defective or conditionally replication defective and / or may be a recombinant AAV vector.

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[0100] 4900-4314-0443, v. 1 In certain embodiments a plurality of rAAV particles comprises particles of, or derived from, the same strain or serotype (or subgroup or variant). In certain embodiments a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g. , of different serotypes and / or strains).

[0101] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants e.g. , due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0102] In certain embodiments, a rAAV vector based upon a first serotype genome corresponds to the serotype of one or more of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g. , ITRs) that comprises the AAV vector genome corresponds to the serotype of a capsid that comprises the rAAV particle.

[0103] In certain embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence having at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV 12 particle. In particular embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence having at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 serotype. In certain embodiments, a method herein comprises use, administration or delivery of an rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV 10, rAAVl 1, or rAAV 12.

[0104] Recombinant AAVs (rAAV s) may be relatively safe (do not integrate into the genome and produce limited immunogenicity), may confer long-lasting (years to decades) transgene expression, and they may be engineered to selectively enter and express in brain neurons or

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[0106] 4900-4314-0443, v. 1 immune cells. rAAVs may be efficacious in nondividing tissues, such as neurons (Kaplitt et al., 1994; Mandel et al., 1997).

[0107] AAV capsids may be designed to alter tropism, targeting specificity, and antigenicity (Castle et al., 2016). In the brain, AAVs 1, 2, 5, 7, 8, 9, and rh.10 may exhibit strong neuronal tropism (Bartlett et al., 1998; Burger et al., 2004; Cearley & Wolfe, 2006; Passini et al., 2003); however, AAVs 1, 2, 5, 6, and 8 also may transduce astroglia (Davidson et al., 2000; Hutson et al., 2012; Klein et al., 2008). AAVs 2 and 4 may display weaker and restricted expression in neuronal populations (Burger et al., 2004; Davidson et al., 2000; Klein et al., 2006; Reimsnider et al., 2007; Sondhi et al., 2007; Taymans et al., 2007; Vite et al., 2003). Conversely, AAVs 1, 9, and rh.10 may exhibit strong and widespread neuronal transduction (Burger et al., 2004; Cearley & Wolfe, 2006; Issa et al., 2023; Klein et al., 2008; Li et al.. 2006; Sondhi et al., 2007). AAV9 may be an appealing serotype for gene therapy, as they may robustly transduce neurons and cross the blood brain barrier. AAV9 may be more readily delivered to the brain compared to other serotypes (Cearley & Wolfe, 2006; Foust et al., 2009). In mice, intravenous administration of AAV9 may be found to deliver virus to the central nervous system in a dose- responsive manner.

[0108] In some embodiments, high-titer AAV9-hSYN- Mef2c (single-stranded) are packaged into the self-complementary form (scAAV). Self-complementary AAVs may bypass the need for dsDNA conversion by packaging both strands as a single molecule. A limiting factor of the efficiency of rAAV vectors may be complementary strand synthesis (McCarty et al., 2001). A drawback to the scAAV vector may be that the DNA construct may be about half (-2500 bp) the capacity of a typical AAV. scAAV may be 5-140-fold more efficient at transducing virus than rAAV (McCarty et al., 2001). In sc AAVs, the transgenic cassette itself may be expressed as an inverted repeat with 3 ’ terminal repeated that have been deleted or mutated, and may be between two 5’ terminal repeats (Raj et al., 2011). Once within a cell, the inverted repeats may pair up and create a double-stranded form that may be transcriptionally active. scAAV genomes may be more stable and may circularize once in the cell compared to AAVs (Raj et al., 2011).

[0109] Suitable host cells for producing transduction competent AAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells that can be, or have been, used as recipients of a heterologous rAAV vectors. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. In certain embodiments a modified human embryonic kidney cell line (e.g., HEK293), which is transformed with adenovirus type-5 DNA fragments and expresses the adenoviral El a and Elb genes is used to generate recombinant

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[0111] 4900-4314-0443, v. 1 AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform in which to produce rAAV particles. Methods of generating high titer AAV particles capable of transducing mammalian cells are known in the art.

[0112] In certain embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. A number of other vectors are known which encode Rep and / or Cap expression products.

[0113] IV. Kits

[0114] In still further embodiments, the present disclosure concerns kits for use with the methods described herein. Expression vectors are included in the kit. The kits will thus comprise, in suitable container means, one or more expression vectors. The kits may further comprise a suitably aliquoted composition of the vectors, whether in dry (e.g., lyophilized) or in aqueous form.

[0115] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the vectors may be placed, or preferably, suitably aliquoted. Such containers / devices for mixing, diluting and administering the vectors. Directions for mixing, diluting and administering the vectors may also be included.

[0116] The kits of the present disclosure will also typically include a means for containing the vectors and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0117] V. Examples

[0118] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many

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[0120] 4900-4314-0443, v. 1 changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0121] Example 1: Materials and Methods

[0122] Neonatal Transverse Sinus Injections (nTSI). The pups were anesthetized using hypothermia. The inventors injected 4 pL of virus to the bilateral transverse sinuses (2 LIL in each sinus) at postnatal days 0-1 using a glass micropipette attached to Nanoject II Injector and stereotax. The inventors used sterilized fine scissors to make a small V-shaped cut (~2mm) in the skin above the right or left transverse sinus (dorsal surface of the mouse head). The pups were positioned such that the site of injection was perpendicular to the pipette. The inventors placed the glass capillary atop the transverse sinus and took note of the dorsal / ventral coordinates. Gentle downward pressure was applied to break through the skull, and the inventors retracted the capillary so that the tip was below the skull’s surface (-300-400 um). The inventors injected the virus mixed with Fast Green FCF dye (Sigma Aldrich, F7252; .2 pL dye for 30 pL of virus) at a rate of 23 nL per second and observed spreading of the dye. Following a 5 second delay, the inventors retracted the pipette and moved the skin back over the cut. The inventors applied a small amount of VetBond glue to close the incision. The inventors then stimulated the pup by warming it in hands and then placing the pup on a heating pad. After injecting the whole litter, the pups were returned to their home cage and gently rubbed with bedding to prevent rejection by the mother. The inventor perfused the pups transcardially at various developmental time points.

[0123] Neonatal Intracerebroventricular Injections (ICV). Postnatal day 0 - 1 pups were anesthetized using hypothermia and placed on an administration platform surrounded by dry ice to maintain a subthermal environment. The administration platform was placed on the stereotaxic frame (David Kopf Instruments) and a glass capillary pipette loaded with virus was maneuvered to the position above the lateral ventricle. For administration, the needle is lowered into the right ventricle and 1 pL of virus is delivered at a flow rate of 50 nL per second using a Nanoject III (Drummond Scientific). After a one minute thirty second delay, the needle was retracted halfway, followed by a one-minute delay before full retraction of the needle from the site of injection. The pup was then recovered on a warming pad positioned in left lateral recumbency before being returned to the mother.

[0124] Immunohistochemistry (IHC). In some preparations, the brains were dropped fixed in 4% paraformaldehyde (PFA) overnight, at 4°C then transferred to 30% sucrose in IX PBS

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[0126] 4900-4314-0443, v. 1 and sodium azide; in other preparations, the mouse underwent cardio perfusion prior to collection of the brain. Briefly, mice were injected intraperitoneally with ketamine (12 mg / mL) / xylazine (1.6 mg / mL) and placed into a staging box. The rib cage was cut to expose the beating heart. The right atrium was punctured using scissors, and a 26G needle attached to a 20 mL syringe was inserted into the left ventricle. Cold IX PBS (phosphate buffered saline) was slowly ejected. In another syringe with a 26G needle, 20 mL of cold paraformaldehyde (PFA) was slowly ejected into the left ventricle. The mouse was decapitated and the brain was carefully excised. Brains were post-fixed in 4% PFA for 24 hours. The brains were then transferred to 30% sucrose in IX PBS and sodium azide for cryopreservation. The brains were sectioned at 40 pL using a sliding microtome and stored in IX PBS with 0.02% sodium azide. Brain sections were blocked in TritonX-100, BSA, normal donkey serum, and normal goat serum, and immunostained with primary antibodies at 4°C overnight. Brain sections were incubated with secondary antibody for 2 hours at room temperature. Brain sections were mounted with Prolong Gold Antifade Mountant (ThermoFisher, #P36930). The sections were imaged using the departmental Leica Stellaris 5 confocal microscope. All confocal settings were kept the same for every image within each experimental group. The confocal images were analyzed using Cell Counter in FIJI or the Surfaces Workflow in Imaris.

[0127] 4900-4314-0443, v. 1 Table 1. Antibodies

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[0129] 4900-4314-0443, v. 1 Example 2: Neonatal Transverse Sinus Injection is a Robust Method for Transgene Delivery

[0130] While vector delivery methods that leverage the circulatory system have been characterized previously, direct access to the brain’s venous system has been less investigated (Gessler et al., 2019; Hamodi et al., 2020; Wasala et al., 2019). Neonatal transverse sinus injection (nTSI) yields effective (broad and robust labeling of cells) and efficient easily accessible and requires smaller volumes for widespread expression) method for delivering transgenes to the neonatal circulatory system. Tn preliminary studies, the inventors found that infusion of the rAAV expressing a reporter gene (enhanced green fluorescent protein, EGFP) under control of a neuronal promoter (human Synapsin) produced EGFP protein expression in 45-50% of cortical neurons in a wildtype mouse brain (FIG. 1 and FIG. 2), which could be sufficient for therapeutic effects. nTSI can be used for proof-of-concept studies to test whether increasing MEF2C levels in neurons of the mouse brain is sufficient to restore neurotypical behavior and physiology in Mef2c global heterozygous mice.

[0131] Example 3: AAV9-hSyn-EGFP Infects Cortical Layers Unevenly

[0132] To characterize infection patterns of AAV9-hSyn-EGFP using nTSI, two wildtype animals were injected at P0-1 and euthanized at 3-5 weeks of age. Using confocal microscopy, the inventors imaged the somatosensory cortex (SSC) and quantified the number of virally infected neurons in the 1) superficial and 2) deeper layers (FIG. 2). Given that deeper layers of the cortex have increased cellular density (Keller et al., 2018), the inventors used a proportion — GFP+and NeuN+co-localized cells over NeuN+cells — to assess infection pattern in the cortex. The inventors found that the deeper layers were preferentially infected over the superficial layers, possibly due to blood vessel distribution patterns.

[0133] Moreover, the inventors confirmed that this virus targeted inhibitory neurons known to express MEF2C under the synapsin promoter. The inventors observed co-localization of PV and GFP in the PFC and SSC (FIG. 3). Moreover, given MEF2C’s expression in cerebellar Purkinje neurons, the inventors assessed whether this cell population was targeted. Using morphology and GAD67 (pan-inhibitory neuron marker) staining, the inventors confirmed GFP transduction in Purkinje cells of the cerebellum (FIG. 4).

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[0135] 4900-4314-0443, v. 1 Example 4: Development of Mef2c Constructs for Brain Delivery

[0136] The inventors previously found that MEF2C exists as two main isoforms in the brain and multiple developmental timepoints (FIGS. 12A-D). Due to alternative splicing of Mef2c mRNA, a small polypeptide, called the gamma domain, is either included in or absent from MEF2C protein. In 2021, two different constructs that could increase levels of MEF2C in the brain were generated (FIG. 5). To determine whether increasing levels of MEF2C in the brain is sufficient to restore abnormal phenotypes observed in Mef2c global heterozygous mice, the inventors performed proof-of-concept experiments and used nTSI to deliver AAV9-hSyn- EGFP (control virus), AAV9-hSyn-Mef2c-+G, and AAV9-hSyn-Mef2c to the venous system of P0-1 mice. The inventor’s initial studies with rAAVhSyn- MEF2C showed detectable expression of vector in the cortex; however, the number of infected cells was somewhat low and optimization of the virus should improve the infection rate.

[0137] In some embodiments, a pAAV vector with a human synapsin promoter plus mouse MEF2C open-reading-frame (alpha2 = beta domain + gamma domain) with a 3X Flag epitope tag + P2A cleavage site + mCherry ORF and hGH polyA sequence was used (SEQ ID NO: 1). The complete nucleic acid sequence of SEQ ID NO: 1 is described in Table 2. The complete sequence map of SEQ ID NO: 1 is illustrated in FIG. 6.

[0138] An exemplary, but non-limiting sequence of a human MEF2C open-reading frame that may be used is GenBank Gene ID: 4208. An exemplary, but non- limiting human MEF2C amino acid sequence is described in Table 2 as SEQ ID NO: 2. An exemplary, but non-limiting human MEF2C mRNA transcript is described in Table 2 as SEQ ID NO: 3. Other exemplary human MEF2C sequences that may be used are GenBank Accessions: NM_001364330.2, NM_001364353.2, NM_001308002.3, NM_001193350.2, NM_001364333.2,

[0139] NM_001364331.2, NM_002397.5, NM_001364329.2, or NM_001364344.2. An exemplary non-limiting sequence of a P2A cleavage site used is described in Table 2 as SEQ ID NO: 4.

[0140] In some embodiments, MEF2C may be generated by alternative splicing, for example, with two splice acceptor sites in one of the exons, and when the second acceptor site is chosen, then it skips the gamma domain coding region. An illustrative example of a splicing event generating MEF2C, in an embodiment, is depicted in FIGS. 7A-D. In some embodiments, a truncated version of the human intron leading up to the gamma-included exon may be used. The inventors have found that a 200 nucleotide mini-intron comprising the first 100 nucleotides and the last 100 nucleotides of human MEF2C intron 10 is sufficient to allow for expression of

[0141] -25-

[0142] 4900-4314-0443, v. 1 human MEF2C protein containing or lacking the gamma domain sequences (dotted line in FIG. 8).

[0143] In some embodiments, a rAAV vector with a human synapsin promoter plus human MEF2C open-reading-frame (alpha2 = beta domain + gamma domain) with a 3X Flag epitope tag + hGH polyA sequence and AAV inverted terminal repeat was used (SEQ ID NO: 5) in preclinical proof-of-concept studies to confirm MEF2C expression. The complete nucleic acid sequence of SEQ ID NO: 5 is described in Table 3. The complete sequence map of SEQ ID NO: 5 is illustrated in FIG. 8. Given the homology of the MEF2C sequence in humans and mice, the 3X FLAG epitope in the preclinical vector construct is fused to the MEF2C protein and used as a reporter to indicate vector-derived expression. An alternative embodiment of the vector may not contain the 3X FLAG epitope and would be used for the purposes of clinical evaluations of safety and efficacy (SEQ ID NO: 6). The inventors have demonstrated improved transduction efficiency relative to AAV9-hSyn-MEF2C following ICV administration of AAV.PHP.Eb-hSyn-MEF2C-FLAG to PO-1 pups (FIG 9). Vector-derived ME2C expression, as indicated by the presence of FLAG (filled arrowheads, FIG 9 E-H), was observed to colocalize with endogenous expression of MEF2C protein (open arrows, FIG 9 E-H) in brain slices from the somatosensory cortex and prefrontal cortex.

[0144] Given the previously observed performance of the pAAV vector AAV9-hSyn-MEF2C, the inventors performed quantitative analysis of the transduction efficiency observed using AAV.PHP.Eb-hSyn-MEF2C-FLAG (FIG 10). In the somatosensory cortex, the inventors observed a 30% increase in the overall MEF2C expression level detected in neuronal cells expressing both MEF2C and FLAG, relative to the cells expressing MEF2C alone (FIG 10 C). In this region of the brain, approximately 54% of the cells analyzed (n=1026) were found to demonstrate vector-mediated expression of MEF2C and FLAG (FIG 10 D), indicating successful transduction. In the prefrontal cortex, a 62% increase in overall MEF2C expression level was detected in transduced neuronal cells (MEF2C+ FLAG+), relative to the cells expressing MEF2C alone (FIG 10 G). In this brain region, approximately 60% of the neuronal cells analyzed (n=1023) demonstrated vector- mediated expression of MEF2C and FLAG (FIG 10 H).

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[0146] 4900-4314-0443, v. 1 Table 2. Mef2C and P2A Construct Sequences

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[0148] 4900-4314-0443, v. 1

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[0150] 4900-4314-0443, v. 1

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[0152] 4900-4314-0443, v. 1

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[0154] 4900-4314-0443, v. 1

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[0156] 4900-4314-0443, v. 1 Table 3. Mef2C Alternative Splicing Construct Sequences

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[0158] 4900-4314-0443, v. 1

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[0160] 4900-4314-0443, v. 1

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[0162] 4900-4314-0443, v. 1

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[0164] 4900-4314-0443, v. 1 All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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[0166] 4900-4314-0443, v. 1

Claims

WHAT IS CLAIMED:

1. A nucleic acid molecule comprising an expression cassette comprising a MEF2C isoform coding sequence and a neuronal specific promoter that is operably linked to the MEF2C isoform coding sequence.

2. The nucleic acid molecule of claim 1 , wherein the MEF2C isoform coding sequence does not comprise a gamma-domain, optionally wherein the MEF2C isoform coding sequence comprises a miniature intron that permits alternative splicing to both exclude the gammadomain.

3. The nucleic acid molecule of claim 1, wherein the MEF2C isoform coding sequence comprises a gamma-domain, optionally wherein the MEF2C isoform coding sequence comprises a miniature intron that permits alternative splicing to include the gamma-domain.

4. The nucleic acid molecule of claim 1 , wherein the MEF2C isoform coding sequence does not comprise an alpha 1 domain.

5. The nucleic acid molecule of claim 1, wherein the MEF2C isoform coding sequence does comprise an alphal domain.

6. The nucleic acid molecule of claim 1, wherein the MEF2C isoform coding sequence does not comprise a beta domain.

7. The nucleic acid molecule of claim 1 , wherein the MEF2C isoform coding sequence does comprise a beta domain.

8. The nucleic acid molecule of claim 1, wherein the MEF2C isoform coding sequence does not comprise an alpha2 domain.

9. The nucleic acid molecule of claim 1 , wherein the MEF2C isoform coding sequence does comprise an alpha2 domain.

10. The nucleic acid molecule of claim 1, wherein the neuronal specific promoter is a synapsin promoter (hSyn).

11. The nucleic acid molecule of claim 1 , wherein the MEF2C isoform coding sequence is at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 3.

12. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid molecule of any one of claims 1-11.-37-4900-4314-0443, v.

113. A self-complementary form adeno-associated virus (sc AAV) vector comprising the nucleic acid molecule of any one of claims 1-11.

14. A pharmaceutical composition for treating MEF2C haploinsufficiency syndrome in a patient in need thereof, the pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 -11.

15. A pharmaceutical composition for treating MEF2C haploinsufficiency syndrome in a patient in need thereof, the pharmaceutical composition comprising the recombinant adeno- associated virus (rAAV) vector of claim 12 or 13.

16. A method of treating MEF2C haploinsufficiency syndrome in a patient in need thereof comprising administering an effective amount of the pharmaceutical composition of claim 14 or 15 to a patient.

17. A method of inducing transgenic expression of MEF2C in a patient comprising administering an effective amount of the pharmaceutical composition of claim 14 or 15 to a patient.

18. The method of claim 16, wherein the treating comprises improving one or more symptoms selected from the group consisting of autism spectrum disorder, intellectual disability, speech disability, hyperactivity, epilepsy, attention-deficit / hyperactivity disorder, and schizophrenia.

19. The method of claim 16 or claim 17, wherein administering comprises injection of the pharmaceutical composition to bilateral transverse sinus regions.

20. The method of claim 16 or claim 17, wherein the patient is between 0 and 5, 0 and 10, 0 and 15, 0 and 20, or 0 and 25 years of age.

21. The method of claim 16 or claim 17, wherein the pharmaceutical composition is administered in combination with a second MEF2C haploinsufficiency syndrome treatment.

22. The method of claim 16 or claim 17, wherein administering comprises oral, intravenous, subcutaneous, intrathecal, or topical administration.

23. The recombinant adeno-associated virus (rAAV) vector of claim 12 wherein the rAAV vector is derived from serotype AAV9.

24. The self-complementary form adeno-associated virus (scAAV) vector of claim 13 wherein the scAAV vector is derived from serotype AAV9.-38-4900-4314-0443, v. 1

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