Method for identifying and employing target site blocking oligonucleotides in 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. Herein are described methods of identifying target site binding (TSB) oligonucleotides that hybridize to the 3' untranslated region of the MEF2C mRNA and thereby block the binding of inhibitory non-coding RNAs. Such TSB oligonucleotides are therefore useful in treating MCHS.

Description

[0001]DESCRIPTION METHOD FOR IDENTIFYING AND EMPLOYING TARGET SITE BLOCKING OLIGONUCLEOTIDES IN THE TREATMENT OF MEF2C HAPLOINSUFFICIENCY SYNDROME ^^ ^ ^ PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 595,442, filed November 2, 2023, the entire contents of which are hereby incorporated by reference. ^^^ ^ STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 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. REFERENCE TO A SEQUENCE LISTING 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 October 31, 2024, is named MESCP0141WO_ST26.xml and is 66,620 bytes in size. BACKGROUND 1. Field of the Disclosure ^^^ The present disclosure relates generally to the fields of medicine, genetics, and molecular biology. More particularly, the disclosure relates to method of identifying target site blocking (TSB) oligonucleotides and their use in treating MEF2C haploinsufficiency syndrome. ^ ^^^ 2. Background 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 is commonly associated with severe language 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 5q14.3 (FIG. 1), 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 of 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. To date, there are more than 300 patients worldwide with MCHS, and with the increasing affordability and accessibility of genetic sequencing technology, the number of individuals with documented or suspected MCHS are 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 society and could lay the groundwork for ^^^ treating numerous single-gene disorders, such as Fragile X syndrome, Rett syndrome, CHAMP1 haploinsufficiency, and many, many others.^ ^ 4888-9547-9283, v. 2 SUMMARY Thus, in accordance with the present disclosure, a method of identifying a target site blocking (TSB) oligonucleotide that selectively regulates MEF2C expression comprising (a) providing a non-coding RNA that inhibits expression of MEF2C by hybridizing to a 3’ ^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ (b) designing a candidate oligonucleotide ^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^!"^^^^^^^^^^^^^^^^^^^^^^^^ (c) contacting said candidate oligonucleotide with a first cell expressing MEF2C and said non-^^^^^^^ ^^^^^ ^^^ (d) comparing the expression of MEF2C in said first cell as compared to a control cell expressing MEF2C and said non-coding RNA in the absence of said candidate oligonucleotide, wherein ^^^ an increase in expression of MEF2C in said first cell as compared to said control cell identifies said candidate oligonucleotide as a TSB oligonucleotide. The non-coding RNA may be a micro-RNA. The candidate oligonucleotide may be 15- 50 nucleotides in length. The non-coding RNA is a mouse or human non-coding RNA. The first cell may be a mouse cell or human cell, such as a wild-type neuronal cell, a neural ^^^ progenitor cell, a wild-type microglial cell, a MEF2C Haploinsufficiency syndrome neuronal cell, or a MEF2C haploinsufficiency syndrome microglial cell. The candidate oligonucleotide may be a non-natural / modified oligonucleotide, such as where said candidate oligonucleotide contains one or more non-natural / modified nucleotides that increase stability. The method may further comprise identifying the binding site of said non-coding RNA, ^^^ such as by measuring expression in a second cell that comprises a reporter construct comprising a promoter operationally linked to a MEF2C coding sequence, a luciferase coding region or a green fluorescent protein sequence fused to a wild-type or mutated 3’ UTR from MEF2C, wherein said cell is contacted with said non-coding RNA. The second cell may be a mouse or human cell, such as a mouse brain cell or a human SH-SY5Y cell. The candidate ^^^ oligonucleotide may be designed to bind to the identified binding site. The method may also further comprise performing one of more control reactions, such as assessing the effect of said candidate oligonucleotide on non-MEF2C protein expression, or generating and testing point mutants of said TSB oligonucleotide. The method may further comprise testing an identified TSB oligonucleotide in any in ^^^ vivo model, such as a mouse model, such as a mouse model for MEF2C Haploinsufficiency syndrome. Two, 3, 4 or more identified TSB oligonucleotides may be tested in the same mouse. The method may further comprise modifying said identified TSB oligonucleotide to increase activity and / or stability and / or to decrease an off-target effect. The candidate oligonucleotide 4888-9547-9283, v. 2 may comprise one or more non-natural modifications, such as a backbone modification or a sugar modification. Also provided is a target site blocking (TSB) oligonucleotide that selectively regulates MEF2C expression identified according to the method as described herein, or a target site ^^ blocking (TSB) oligonucleotide consisting essentially or consisting of 15-30 nucleotides, 19- 25 nucleotides, 18-30 nucleotides, 19-30 nucleotides, 20-30 nucleotides, 20-50 nucleotides, or 10-50 nucleotides, that hybridizes to a 3’ untranslated region of a MEF2C mRNA, and that selectively regulates MEF2C expression identified, wherein said TSB oligonucleotide comprises one or more non-natural / modified nucleotides, such as a backbone modification or ^^^ sugar modification. The TSB oligonucleotide may be completely complementary over its full length to a portion of the 3’ untranslated region of a MEF2C mRNA, such as comprising the sequence AACUGAC. The TSB oligonucleotide may comprise 0, 1, 2, 3, 4 or 5 mismatches over its full length as compared to a portion of the 3’ untranslated region of a MEF2C mRNA but retains complete complementarity to AACUGAC. The TSB oligonucleotide may comprise ^^^ the sequence: 5’-TGTCAGTTGACCCAATAGATTGC-3’ (SEQ ID NO: 1), 5’-AATGGATGTCAGTTGACCCAA-3’ (SEQ ID NO: 2), 5’-UGUCAGUUGACCCAAUAGAUUGC-3’ (SEQ ID NO: 3), 5’-AAUGGAUGUCAGUUGACCCAA-3’ (SEQ ID NO: 4), ^^^ 5’ -TTATTTCCAGCAGGAGAAACT-3’ (SEQ ID NO: 6), 5’ -CAATTTGAGGTATGAACAG-3’ (SEQ ID NO: 8), 5’ -ATTAAACTGAGGTATGAAT-3’ (SEQ ID NO: 9), 5’ -CCACATCTTTGGTTAAAAC-3’ (SEQ ID NO: 10), 5’ -TGTTAATTTCACAGATTTT-3’ (SEQ ID NO: 11), ^^^ 5’ -CCGCTTTTGGCAAATGTTT-3’ (SEQ ID NO: 12), 5’ -TTTCTTATGGCACTCACTT-3’ (SEQ ID NO: 13), 5’ -TATTTCCAGCAGGAGAAAC-3’ (SEQ ID NO: 14), 5’ -ATGTTAATTTCACAGATTTTT-3’ (SEQ ID NO: 16), 5’ -GCCGCTTTTGGCAAATGTTTC-3’ (SEQ ID NO: 17), ^^^ 5’ -CTTTCTTATGGCACTCACTTA-3’ (SEQ ID NO: 18), 5’ -ACGGCAGATGGCACAAATGAG-3’ (SEQ ID NO: 19), 5’ -CAAUUUGAGGUAUGAACAG-3’ (SEQ ID NO: 20), 5’ -AUUAAACUGAGGUAUGAAU-3’ (SEQ ID NO: 21), 5’ -CCACAUCUUUGGUUAAAAC-3’ (SEQ ID NO: 22), 4888-9547-9283, v. 2 5’ -UGUUAAUUUCACAGAUUUU-3’ (SEQ ID NO: 23), 5’ -CCGCUUUUGGCAAAUGUUU-3’ (SEQ ID NO: 24), 5’ -UUUCUUAUGGCACUCACUU-3’ (SEQ ID NO: 25), 5’ -UAUUUCCAGCAGGAGAAAC-3’ (SEQ ID NO: 26), ^^ 5’ -AUGUUAAUUUCACAGAUUUUU-3’ (SEQ ID NO: 27), 5’ -GCCGCUUUUGGCAAAUGUUUC-3’ (SEQ ID NO: 28), 5’ -CUUUCUUAUGGCACUCACUUA-3’ (SEQ ID NO: 29), 5’ -ACGGCAGAUGGCACAAAUGAG-3’ (SEQ ID NO: 30), or 5’ -UUAUUUCCAGCAGGAGAAACU-3’ (SEQ ID NO: 31). ^^^ The TSB may also be complementary to and bind a target sequence of: GTCAGTT (SEQ ID NO: 34), GAGGTA (SEQ ID NO: 35), GAGGTA (SEQ ID NO: 36), CTTTGGT (SEQ ID NO: 37), ^^^ TTCACAGA (SEQ ID NO: 38), TTGGCAA (SEQ ID NO: 39), ATGGCAC (SEQ ID NO: 40), CAGCAGG (SEQ ID NO: 41), TTCACAGA (SEQ ID NO: 42), ^^^ TTGGCAA (SEQ ID NO: 43), ATGGCAC (SEQ ID NO: 44), ATGGCAC (SEQ ID NO: 45) or CAGCAGG (SEQ ID NO: 46). In another embodiment, there is provided a pharmaceutical formulation comprising (a) ^^^ one or more target site blocking (TSB) oligonucleotides consisting essentially or consisting of about 18-50 nucleotides, that hybridize(s) to a 3’ untranslated region of a MEF2C mRNA, and that selectively regulates MEF2C expression identified, optionally where said TSB oligonucleotide comprises one or more non-^^^^^^^#^^^^^^^^^ ^^^^^^^^^^^^^ ^^^ (b) a pharmaceutically acceptable buffer, diluent or excipient. ^^^ In still another embodiment, there is provided a method of treating a subject having MEF2C haploinsufficiency syndrome comprising providing a target site blocking (TSB) oligonucleotide as described here or a pharmaceutical formulation comprising the TSB oligonucleotide. The method may further comprise treating said subject with said TSB oligonucleotide or said pharmaceutical formulation a second time. The TSB oligonucleotide or 4888-9547-9283, v. 2 pharmaceutical formulation may be administered intracerebroventricularly or intrathecally. The TSB oligonucleotide may be delivered using a modified nanocarrier or exosome capable of binding to and crossing the blood-brain barrier. The TSB oligonucleotide may comprise the sequence: ^^ 5’-TGTCAGTTGACCCAATAGATTGC-3’ (SEQ ID NO: 1), 5’-AATGGATGTCAGTTGACCCAA-3’ (SEQ ID NO: 2), 5’-UGUCAGUUGACCCAAUAGAUUGC-3’ (SEQ ID NO: 3), 5’-AAUGGAUGUCAGUUGACCCAA-3’ (SEQ ID NO: 4), 5’ -TTATTTCCAGCAGGAGAAACT-3’ (SEQ ID NO: 6), ^^^ 5’ -CAATTTGAGGTATGAACAG-3’ (SEQ ID NO: 8), 5’ -ATTAAACTGAGGTATGAAT-3’ (SEQ ID NO: 9), 5’ -CCACATCTTTGGTTAAAAC-3’ (SEQ ID NO: 10), 5’ -TGTTAATTTCACAGATTTT-3’ (SEQ ID NO: 11), 5’ -CCGCTTTTGGCAAATGTTT-3’ (SEQ ID NO: 12), ^^^ 5’ -TTTCTTATGGCACTCACTT-3’ (SEQ ID NO: 13), 5’ -TATTTCCAGCAGGAGAAAC-3’ (SEQ ID NO: 14), 5’ -ATGTTAATTTCACAGATTTTT-3’ (SEQ ID NO: 16), 5’ -GCCGCTTTTGGCAAATGTTTC-3’ (SEQ ID NO: 17), 5’ -CTTTCTTATGGCACTCACTTA-3’ (SEQ ID NO: 18), ^^^ 5’ -ACGGCAGATGGCACAAATGAG-3’ (SEQ ID NO: 19), 5’ -CAAUUUGAGGUAUGAACAG-3’ (SEQ ID NO: 20), 5’ -AUUAAACUGAGGUAUGAAU-3’ (SEQ ID NO: 21), 5’ -CCACAUCUUUGGUUAAAAC-3’ (SEQ ID NO: 22), 5’ -UGUUAAUUUCACAGAUUUU-3’ (SEQ ID NO: 23), ^^^ 5’ -CCGCUUUUGGCAAAUGUUU-3’ (SEQ ID NO: 24), 5’ -UUUCUUAUGGCACUCACUU-3’ (SEQ ID NO: 25), 5’ -UAUUUCCAGCAGGAGAAAC-3’ (SEQ ID NO: 26), 5’ -AUGUUAAUUUCACAGAUUUUU-3’ (SEQ ID NO: 27), 5’ -GCCGCUUUUGGCAAAUGUUUC-3’ (SEQ ID NO: 28), ^^^ 5’ -CUUUCUUAUGGCACUCACUUA-3’ (SEQ ID NO: 29), 5’ -ACGGCAGAUGGCACAAAUGAG-3’ (SEQ ID NO: 30), or 5’ -UUAUUUCCAGCAGGAGAAACU-3’ (SEQ ID NO: 31). The TSB may also be complementary to and bind a target sequence of: GTCAGTT (SEQ ID NO: 34), 4888-9547-9283, v. 2 GAGGTA (SEQ ID NO: 35), GAGGTA (SEQ ID NO: 36), CTTTGGT (SEQ ID NO: 37), TTCACAGA (SEQ ID NO: 38), ^^ TTGGCAA (SEQ ID NO: 39), ATGGCAC (SEQ ID NO: 40), CAGCAGG (SEQ ID NO: 41), TTCACAGA (SEQ ID NO: 42), TTGGCAA (SEQ ID NO: 43), ^^^ ATGGCAC (SEQ ID NO: 44), ATGGCAC (SEQ ID NO: 45) or CAGCAGG (SEQ ID NO: 46). The TSB may be oligonucleotide provided by administering an expression construct encoding and expressing the TSB oligonucleotide, such as a viral construct, such as an adeno-associated ^^^ viral construct. 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. ^^^ 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. 4888-9547-9283, v. 2 BRIEF DESCRIPTION OF THE DRAWINGS 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. FIG.1. MEF2C haploinsufficiency caused by mutations / deletions that reduce the levels of functioning MEF2C. FIG.2. MEF2C haploinsufficiency in mice (Mef2c+ / -) produces a variety of behavioral ^^^ and brain circuit phenotypes, and our follow-up studies have shown that MEF2C plays an important role in multiple brain cells, but predominantly in neurons. FIG. 3. TSB 223 (5’-TGTCAGTTGACCCAATAGATTGC-3’^ SEQ ID NO: 1) tested for increase in relative MEF2C protein levels in various concentrations versus a sequence scrambled oligonucleotide in a human neuroblastoma cell line, SH-SY5Y. ^^^ FIG. 4. Site-specific binding of a microRNA (miRNA) to the MEF2C mRNA 3’ untranslated region suppresses translation of MEF2C protein. FIG.5. Assay design in human cells to identify MEF2C mRNA 3’UTR target sites for regulatory miRNAs. FIG. 6. Schematic of how an effective target site blocking (TSB) oligonucleotide ^^^ inhibits miRNA-mediated suppression of MEF2C protein production. FIG.7. Schematic of the human MEF2C 3' UTR showing potential microRNA binding sites. Highlighted are the sites successfully targeted. Image generated using TargetScan (world- wide-web at targetscan.org). FIG. 8. (Left) Table showing TSBs (SEQ ID NOS: 8-14 and 16-20) tested for effects ^^^ on MEF2C levels in HEK 293 cells, along with the corresponding statistical data. (Right) Same data in graph form. FIG.9. (Left) TSB214 (SEQ ID NO: 6) effect on MEF2C levels. (Right) Combination of 2 TSBs versus individual ones and observed a ceiling effect on MEF2C levels (in cells). This is beneficial because MEF2C is dose-sensitive and overexpression can be detrimental. ^^^ FIG.10. Testing of TSB223 (SEQ ID NO: 1) in human iPSC-derived Neural Progenitor Cells (NPCs) showing an increase in MEF2C levels. FIG.11. Neonatal mice were injected with 16 micrograms of either TSB 223 (SEQ ID NO: 1) or its scramble sequence at postnatal day 0 (P0). Cortical tissue (outlined in the brain 4888-9547-9283, v. 2 schematic below right) was collected 10 days later to assess MEF2C expression via western blot. FIG.12. Ten days after the injection protocol shown in FIG.11 was performed, MEF2C protein levels increased in the mice that received the TSB. This was confirmed by normalizing ^^ against two different housekeeping genes (GAPDH and beta-actin), both of which showed an increase. 4888-9547-9283, v. 2 DETAILED DESCRIPTION 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. ^^^ Messenger RNAs (mRNAs) encode proteins that are synthesized based on the mRNA encoding information. However, others are RNAs are non-coding and can function to regulate the amount of protein produced from mRNAs (FIG. 4). One such non-coding RNA class is termed microRNAs (miRNA or miR). These miRNAs bind to specific mRNAs and often reduce the ability of that mRNA to produce a protein. The inventors here propose a therapeutic ^^^ approach that involves targeting miRNA silencing pathways to increase the levels of total MEF2C protein in cells. In short, they proposed protecting the MEF2C mRNA from miRNA repression by blocking the binding site for miRNAs with modified short, synthesized RNAs (i.e., target site blocking oligonucleotides or TSBs). The first step is to identify the relevant miRNAs and their binding sites on the MEF2C mRNA. ^^^ Multiple microRNAs likely regulate the expression of human MEF2C, and the inventors have identified several optimal miRNA candidates that are expressed in both human and mouse brains (FIG. 5). The next step is to identify the specific target sites on the MEF2C mRNA where these miRNA’s bind and limit MEF2C expression. Identified miRNAs that regulate MEF2C expression and will then be targeted using TSB oligonucleotides, leading to a ^^^ viable therapy for MCHS. These and other aspects of the disclosure are described below. ^ I. MEF2C and MCHS 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 5q14.3 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. Multiple isoforms have been identified. Several post 4888-9547-9283, v. 2 translational modifications have been identified including phosphorylation on serine-59 and serine-396, sumoylation on lysine-391, acetylation on lysine-4 and proteolytic cleavage. 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.^ 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. Only about 300 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. 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 (Mef2c+ / -), 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 (FIG.2). 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 4888-9547-9283, v. 2 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. ^^ 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. ^ II. Target Site Blocking (TSB) Oligonucleotides ^^^ In one aspect of the disclosure, there are provided oligonucleotides designed to target and protect a site in the 3’ untranslated region of the MEF2C mRNA. In a particular embodiment, the target sequence is AACUGAC. The TSBs may comprise DNA bases, RNA bases, or non- natural bases. The oligonucleotides will therefore be 10-50, 10-40, 10-30, 10-25, 10-22, 15-50, 15-40, 15-30, 15-25, 15-22, 20-50, 20-40, 20-30, 19-25, 20-25, 20-22, 21, 22, 23, 24, 25, 26,^^^ 27, 28, 29, or 30 nucleotides in length and contain the core sequences 5’-GTCAGTT-3’ or 5’- GUCAGUU-3’. The regions of the TSBs outside these core sequences may contain 0, 1 or more mismatches as compared to the corresponding MEF2C mRNA sequences, such as 1, 2, 3, 4 or 5 mismatches. Specific oligonucleotides include 5’- TGTCAGTTGACCCAATAGATTGC-3’ (SEQ ID NO: 1), 5’-^^^ AATGGATGTCAGTTGACCCAA-3’ (SEQ ID NO: 2), 5’- UGUCAGUUGACCCAAUAGAUUGC-3’ (SEQ ID NO: 3), 5’- AAUGGAUGUCAGUUGACCCAA-3’ (SEQ ID NO: 4), 5’ - TTATTTCCAGCAGGAGAAACT-3’ (SEQ ID NO: 6), 5’ -CAATTTGAGGTATGAACAG-3’ (SEQ ID NO: 8), 5’ -ATTAAACTGAGGTATGAAT-3’ (SEQ ID NO: 9), 5’ - ^^^ CCACATCTTTGGTTAAAAC-3’ (SEQ ID NO: 10), 5’ -TGTTAATTTCACAGATTTT-3’ (SEQ ID NO: 11), 5’ -CCGCTTTTGGCAAATGTTT-3’ (SEQ ID NO: 12), 5’ - TTTCTTATGGCACTCACTT-3’ (SEQ ID NO: 13), 5’ -TATTTCCAGCAGGAGAAAC-3’ (SEQ ID NO: 14), 5’ -ATGTTAATTTCACAGATTTTT-3’ (SEQ ID NO: 16), 5’ - GCCGCTTTTGGCAAATGTTTC-3’ (SEQ ID NO: 17), 5’ -^^^ CTTTCTTATGGCACTCACTTA-3’ (SEQ ID NO: 18), 5’ - ACGGCAGATGGCACAAATGAG-3’ (SEQ ID NO: 19), 5’ - CAAUUUGAGGUAUGAACAG-3’ (SEQ ID NO: 20), 5’ -AUUAAACUGAGGUAUGAAU- 3’ (SEQ ID NO: 21), 5’ -CCACAUCUUUGGUUAAAAC-3’ (SEQ ID NO: 22), 5’ - UGUUAAUUUCACAGAUUUU-3’ (SEQ ID NO: 23), 5’ -CCGCUUUUGGCAAAUGUUU- 4888-9547-9283, v. 2 3’ (SEQ ID NO: 24), 5’ -UUUCUUAUGGCACUCACUU-3’ (SEQ ID NO: 25), 5’ - UAUUUCCAGCAGGAGAAAC-3’ (SEQ ID NO: 26), 5’ - AUGUUAAUUUCACAGAUUUUU-3’ (SEQ ID NO: 27), 5’ - GCCGCUUUUGGCAAAUGUUUC-3’ (SEQ ID NO: 28), 5’ - ^^ CUUUCUUAUGGCACUCACUUA-3’ (SEQ ID NO: 29), 5’ - ACGGCAGAUGGCACAAAUGAG-3’ (SEQ ID NO: 30), and 5’ - UUAUUUCCAGCAGGAGAAACU-3’ (SEQ ID NO: 31). The TSB may also be complementary to and bind a target sequence of GTCAGTT (SEQ ID NO: 34), GAGGTA (SEQ ID NO: 35), GAGGTA (SEQ ID NO: 36), CTTTGGT (SEQ ID NO: 37), TTCACAGA (SEQ ^^^ ID NO: 38), TTGGCAA (SEQ ID NO: 39), ATGGCAC (SEQ ID NO: 40), CAGCAGG (SEQ ID NO: 41), TTCACAGA (SEQ ID NO: 42), TTGGCAA (SEQ ID NO: 43), ATGGCAC (SEQ ID NO: 44), ATGGCAC (SEQ ID NO: 45), or CAGCAGG (SEQ ID NO: 46). In certain embodiments, oligonucleotides provided herein may comprise one or more modifications to a nucleobase, sugar, and / or internucleoside linkage, and as such is a modified ^^^ oligonucleotide. A modified nucleobase, sugar, and / or internucleoside linkage may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases. In certain embodiments, a 2’-modified nucleoside comprises a 2’-substituent group ^^^ selected from F, OCF3,O-CH3(also referred to as “2’-OMe”), OCH2CH2OCH3(also referred to as “2’-O-methoxyethyl” or “2’-MOE”), 2'-O(CH2)2SCH3, O-(CH2)2-O- N(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3. Another non-natural modified oligonucleotide is a locked nucleic acid (LNA), often referred to as inaccessible RNA. The ribose moiety of an LNA nucleotide is modified with an ^^^ extra bridge connecting the 2$ and 4$ carbons. The bridge “locks” the ribose in the 3$-endo structural conformation, which is often found in the A-form of DNA or RNA. LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. Such oligomers are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the thermal stability (melting ^^^ temperature) of oligonucleotides (Kaur et al., 2006). Two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, methylenephosphonates, vinylphosphonates, phosphonoacetates, thiophosphonoacetates, 4888-9547-9283, v. 2 phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, amide, triazole, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--^^^ ^^^^%^^^^ ^--O--Si(H)2--O--^^^ ^^^^ ^&^'- ^^dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of ^^^ preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art. ^ III. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising TSB ^^^ oligonucleotides. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. 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. The term “carrier” refers to ^^^ a diluent, 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. In certain embodiments, the oligonucleotide 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 oligonucleotide 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 or as an intravenous infusion. The lyophilized drug 4888-9547-9283, v. 2 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. 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. 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, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. ^^^ The TSB oligonucleotide may also be 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. The AAV vector may be replication-defective or conditionally replication defective and / or may be a recombinant AAV vector. The AAV vector ^^^ may comprise a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11) Generally, the ingredients of 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 4888-9547-9283, v. 2 for injection or saline can be provided so that the ingredients may be mixed prior to administration. ^ IV. Kits ^^ In still further embodiments, the present disclosure concerns kits for use with the methods described herein. TSB oligonucleotides are included in the kit. The immunodetection kits will thus comprise, in suitable container means, one or more TSB oligonucleotides. The kits may further comprise a suitably aliquoted composition of the TSB oligonucleotide(s), whether in dry (e.g., lyophilized) or in aqueous form. ^^^ 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 TSB oligonucleotide(s) may be placed, or preferably, suitably aliquoted. Such containers / devices for mixing, diluting and administering the TSB oligonucleotides. Directions for mixing, diluting and administering the TSB oligonucleotides may also be included. ^^^ The kits of the present disclosure will also typically include a means for containing the TSB oligonucleotide(s) 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. ^ ^^^ V. Examples 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 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. ^ Example 1 ^^^ To determine the miRNA target sequence on MEF2C mRNA, the inventors will use a “reporter gene” assay (i.e., MEF2C (e.g., overexpressed), firefly luciferase or a green fluorescent protein (GFP) gene combined with the 3’ untranslated regions (UTR) of the MEF2C mRNA sequence) to confirm the ability of the miRNA to regulate the MEF2C mRNA translation (FIG.6). The luciferase gene encodes an enzyme that produces light when a specific 4888-9547-9283, v. 2 chemical substrate is added, and the levels of this enzyme, like MEF2C itself, are regulated by the MEF2C mRNA sequence attached to it. Similarly, expression of GFP would allow the inventors to monitor cellular fluorescence to assess the influence of miRNAs and TSBs. The inventors will introduce a reporter gene into mouse (e.g., Neuro2A cells) human cells (e.g., ^^ SH-SY5Y) together with the candidate FIG. 5. Process for miRNA(s) that are synthesized by a commercial company. If the selecting miRNA binds to the target sequence and is functional, it will reduce the expression of the luciferase or GFP protein, resulting in lower levels of activity it the test. The inventors can then use site-directed mutagenesis in the targeted MEF2C mRNA region, and this should disrupt the ability of the miRNA to reduce assay activity.^^^ After identifying the MEF2C mRNA target site(s) that are sensitive to miRNA- mediated suppression of protein synthesis, the inventors will next design and synthesize one or more TSBs (locked RNAs or 2’MOE with phosphorothioate backbone) that are exactly complementary to the identified miRNA-sensitive sequence(s) on MEF2C mRNA (FIG. 7). The candidate TSBs will be produced. The TSBs are modified RNAs to increase their stability ^^^ and usefulness as a therapeutic. The inventors will then test the effectiveness of the TSBs to increase MEF2C levels. This will be accomplished by introducing a range of different TSB amounts into cultured human cells, and the inventors will assess levels of MEF2C using protein immunoblotting. For successful TSBs, they will next test them in wild-type and MCHS mouse neurons or microglia (brain immune cells that express MEF2C) in culture (i.e., growing in a ^^^ dish). For TSBs that increase MEF2C expression, the inventors could potentially combine two or more together to get a larger increase in MEF2C levels. The inventors will measure the precise levels of MEF2C protein using highly specific antibodies that only recognize MEF2C. The inventors will select the TSB, or combination of TSBs, that increase the expression of the non-mutated MEF2C protein to levels comparable to wild-type cells. To assess the specificity ^^^ of this approach, the inventors will use a range of controls and additional experiments, including testing the levels of non-MEF2C proteins, testing mutant forms of the TSBs that don’t precisely match the MEF2C mRNA, etc. To evaluate in vivo efficacy, the inventors will use the lead TSB candidate(s) that work well in cells and administer them via intracerebroventricular (ICV) injections (similar to ^^^ intrathecal injections in humans) at multiple postnatal timepoints in the MCHS mouse. They will first analyze the brain tissues for expression levels to confirm their ability to increase MEF2C levels. Second, if increases are observed, then the inventors will perform similar studies with the MCHS mice to test for rescue of MCHS-like behaviors previously observed (e.g., social preference deficits, hyperactivity, ultrasonic vocalizations, altered 4888-9547-9283, v. 2 approach / avoidance, repetitive behavior, high pain tolerance). They plan to introduce the TSBs at four key ages in the mice (i.e., postnatal days 0, 14, 28, or 60). These ages roughly correspond in humans to third trimester to young adulthood, and these ages will provide insight and preclinical proof-of-principle into a potential therapeutic window for TSBs in human MCHS. ^^ TSB safety and toxicity measures will also be evaluated, including weight monitoring, cytokine production, and blood, liver, and kidney function tests. The inventors’ laboratory has extensive experience in all of the described approaches, including molecular biology, cell culture, in vivo injections, and mouse behavior testing. When a TSB approach shows promise in mice, the inventors will optimize the target ^^^ site blocking oligonucleotides before clinical use and determine the most appropriate method of administration. They plan to collaborate with companies that manufacture similar compounds, as there are already FDA-approved medications that use the same technology, but working through slightly different mechanisms. Thus, the safety and efficacy of RNA-based TSB compounds have already been tested and approved for treating other neurological diseases ^^^ in humans. The TSBs would likely be administered via intrathecal delivery, which is routine procedure in most hospitals worldwide. Similar RNA-based medications have a half-life of up to ~200 days in people, and as such, they require re-administration every ~6-12 months. One benefit of this non-permanent approach is that emergence of adverse indications can be ^^^ addressed by treatment discontinuation or adjustment of the dosing amount in subsequent treatments. In addition, targeted delivery is less of an issue because even if the target site blocking oligonucleotides end up in a cell that does not express MEF2C, they will not have an effect, thereby minimizing the risk of aberrant expression or other off-target effects. The inventors have identified at least eight potential Target Site Blocking (TSB) ^^^ oligonucleotides targeting several microRNA binding sites (FIG. 7) capable of regulating MEF2C protein levels. They demonstrated increased levels in the human embryonic kidney cell line HEK-293 (FIG.8) using two different lengths (19 and 21 bases) with distinct chemical modifications (phosphorothioate and phosphodiester bonds), both proving effective to varying extents. ^^^ Additionally, the inventors explored combining TSB oligonucleotides versus individual ones in the SY5Y neuronal cell line to assess potential additive or synergistic effects. Results indicate that each TSB oligonucleotide alone may be sufficient to achieve a ceiling effect in protein level increase, which is beneficial in therapeutic applications due to the dose-sensitive nature of MEF2C. This precision is important as MEF2C overexpression can be detrimental, 4888-9547-9283, v. 2 and at least one MCHS patient has been diagnosed with a full gene duplication. Similar duplication conditions, such as MeCP2 Duplication Syndrome, further underscore the importance of therapeutic modalities with controlled protein regulation. ^^ Furthermore, TSB 223 was tested in iPSC-derived human Neural Progenitor Cells (NPCs), resulting in successful upregulation of MEF2C protein expression (Figure 10). In vivo, the injection of TSB 223 into the cortex of neonatal mice showed a 62% increase in MEF2C levels compared to a scramble sequence control (FIG.11). ^ ^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 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. 4888-9547-9283, v. 2 VI. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. ^ Kaur et al., Biochemistry, 45:7347-55, 2006.

Claims

WHAT IS CLAIMED:

1. A method of identifying a target site blocking (TSB) oligonucleotide that selectively regulates MEF2C expression comprising: (a) providing a non-coding RNA that inhibits expression of MEF2C by hybridizing ^^^^^!"^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ (b) designing a candidate oligonucleotide that is complementary to said 3’ UTR of ^^^^^^^^^^^^^^^^ (c) contacting said candidate oligonucleotide with a first cell expressing MEF2C and said non-^^^^^^^^^^^^^^^ (d) comparing the expression of MEF2C in said first cell as compared to a control cell expressing MEF2C and said non-coding RNA in the absence of said candidate oligonucleotide, wherein an increase in expression of MEF2C in said first cell as compared to said control cell identifies said candidate oligonucleotide as a TSB oligonucleotide.

2. The method of claim 1, wherein said non-coding RNA is a micro-RNA.

3. The method of claim 1 or claim 2, wherein said candidate oligonucleotide is 15-50 nucleotides in length, 18-50 nucleotides in length, 19-50 nucleotides in length, 20-50 nucleotides in length, 19-25 nucleotides in length, or 20-30 nucleotides in length.

4. The method of any one of claims 1-3, wherein said non-coding RNA is a mouse or human non-coding RNA.

5. The method of any one of claims 1-4, wherein step a) further comprises identifying the binding site of said non-coding RNA.

6. The method of claim 5, wherein identifying comprises measuring luciferase expression in a second cell that comprises a reporter construct comprising a promoter operationally linked to a MEF2C coding sequence, a luciferase coding sequence or green fluorescent protein coding sequence fused to a wild-type or mutated 3’ UTR from MEF2C, wherein said cell is contacted with said non-coding RNA.

7. The method of claim 6, wherein said second cell is a mouse or human cell, such as a mouse brain cell or a human SH-SY5Y cell.4888-9547-9283, v.

28. The method of any one of claims 5-7, wherein said candidate oligonucleotide is designed to bind to the identified binding site.

9. The method of any one of claims 1-8, wherein the first cell is a mouse cell or human cell, such as a wild-type neuronal cell, a neural progenitor cell, a wild-type microglial cell, a MEF2C Haploinsufficiency syndrome neuronal cell, or a MEF2C haploinsufficiency syndrome microglial cell.

10. The method of any one of claim 1-9, further comprising performing one of more control reactions, such as assessing the effect of said candidate oligonucleotide on non-MEF2C protein expression, or generating and testing point mutants of said TSB oligonucleotide.

11. The method of any one of claims 1-10, wherein said candidate oligonucleotide is a non- natural / modified oligonucleotide, such as where said candidate oligonucleotide contains one or more non-natural / modified nucleotides that increase stability.

12. The method of any one of claims 1-11, further comprising testing an identified TSB oligonucleotide in any in vivo model, such as a mouse model, such as a mouse model for MEF2C Haploinsufficiency syndrome.

13. The method of claim 12, wherein 2, 3, 4 or more identified TSB oligonucleotides are tested in the same mouse.

14. The method of any one of claims 1-13, further comprising modifying said identified TSB oligonucleotide to increase activity and / or stability and / or to decrease an off-target effect.

15. The method of claims 1-14, wherein the candidate oligonucleotide comprises one or more non-natural modifications, such as a backbone modification or a sugar modification.

16. A target site blocking (TSB) oligonucleotide that selectively regulates MEF2C expression identified according to the method of any one of claims 1-15.

17. A target site blocking (TSB) oligonucleotide consisting essentially or consisting of 18- 30 nucleotides, that hybridizes to a 3’ untranslated region of a MEF2C mRNA, and that selectively regulates MEF2C expression identified, wherein said TSB oligonucleotide comprises one or more non-natural / modified nucleotides, such as a backbone modification or sugar modification.4888-9547-9283, v.

218. The TSB oligonucleotide of claim 17, wherein said TSB oligonucleotide is completely complementary over its full length to a portion of the 3’ untranslated region of a MEF2C mRNA.

19. The TSB oligonucleotide of claim 18, wherein said portion comprises the sequence AACUGAC.

20. The TSB oligonucleotide of claim 17, wherein the TSB oligonucleotide comprises the sequence: 5’-TGTCAGTTGACCCAATAGATTGC-3’ (SEQ ID NO: 1), 5’-AATGGATGTCAGTTGACCCAA-3’ (SEQ ID NO: 2), 5’-UGUCAGUUGACCCAAUAGAUUGC-3’ (SEQ ID NO: 3), 5’-AAUGGAUGUCAGUUGACCCAA-3’ (SEQ ID NO: 4), 5’ -TTATTTCCAGCAGGAGAAACT-3’ (SEQ ID NO: 6), 5’ -CAATTTGAGGTATGAACAG-3’ (SEQ ID NO: 8), 5’ -ATTAAACTGAGGTATGAAT-3’ (SEQ ID NO: 9), 5’ -CCACATCTTTGGTTAAAAC-3’ (SEQ ID NO: 10), 5’ -TGTTAATTTCACAGATTTT-3’ (SEQ ID NO: 11), 5’ -CCGCTTTTGGCAAATGTTT-3’ (SEQ ID NO: 12), 5’ -TTTCTTATGGCACTCACTT-3’ (SEQ ID NO: 13), 5’ -TATTTCCAGCAGGAGAAAC-3’ (SEQ ID NO: 14), 5’ -ATGTTAATTTCACAGATTTTT-3’ (SEQ ID NO: 16), 5’ -GCCGCTTTTGGCAAATGTTTC-3’ (SEQ ID NO: 17), 5’ -CTTTCTTATGGCACTCACTTA-3’ (SEQ ID NO: 18), 5’ -ACGGCAGATGGCACAAATGAG-3’ (SEQ ID NO: 19), 5’ -CAAUUUGAGGUAUGAACAG-3’ (SEQ ID NO: 20), 5’ -AUUAAACUGAGGUAUGAAU-3’ (SEQ ID NO: 21), 5’ -CCACAUCUUUGGUUAAAAC-3’ (SEQ ID NO: 22), 5’ -UGUUAAUUUCACAGAUUUU-3’ (SEQ ID NO: 23), 5’ -CCGCUUUUGGCAAAUGUUU-3’ (SEQ ID NO: 24), 5’ -UUUCUUAUGGCACUCACUU-3’ (SEQ ID NO: 25), 5’ -UAUUUCCAGCAGGAGAAAC-3’ (SEQ ID NO: 26), 5’ -AUGUUAAUUUCACAGAUUUUU-3’ (SEQ ID NO: 27), 5’ -GCCGCUUUUGGCAAAUGUUUC-3’ (SEQ ID NO: 28), 5’ -CUUUCUUAUGGCACUCACUUA-3’ (SEQ ID NO: 29),4888-9547-9283, v. 25’ -ACGGCAGAUGGCACAAAUGAG-3’ (SEQ ID NO: 30), or 5’ -UUAUUUCCAGCAGGAGAAACU-3’ (SEQ ID NO: 31)^ or is complementary and binds a target sequence of: GTCAGTT (SEQ ID NO: 34), GAGGTA (SEQ ID NO: 35), GAGGTA (SEQ ID NO: 36), CTTTGGT (SEQ ID NO: 37), TTCACAGA (SEQ ID NO: 38), TTGGCAA (SEQ ID NO: 39), ATGGCAC (SEQ ID NO: 40), CAGCAGG (SEQ ID NO: 41), TTCACAGA (SEQ ID NO: 42), TTGGCAA (SEQ ID NO: 43), ATGGCAC (SEQ ID NO: 44), ATGGCAC (SEQ ID NO: 45) or CAGCAGG (SEQ ID NO: 46) ^ 21. A pharmaceutical formulation comprising: (a) one or more target site blocking (TSB) oligonucleotides consisting essentially or consisting of about 18-50 nucleotides, that hybridize(s) to a 3’ untranslated region of a MEF2C mRNA, and that selectively regulates MEF2C expression identified, optionally where said TSB oligonucleotide comprises one or more non-natural / modified ^^^^^^^^^^^^^^^^ (b) a pharmaceutically acceptable buffer, diluent or excipient.

22. A method of treating a subject having MEF2C haploinsufficiency syndrome comprising providing a target site blocking (TSB) oligonucleotide of any one of claims 16-19 or a pharmaceutical formulation of claim 20 to said subject.

23. The method of claim 22, further comprising treating said subject with said TSB oligonucleotide or said pharmaceutical formulation a second time.

24. The method of claim 22 or claim 23, wherein said TSB oligonucleotide or said pharmaceutical formulation is administered intracerebroventricularly or intrathecally.4888-9547-9283, v.

225. The method of any one of claims 22-24, wherein said TSB oligonucleotide is delivered using a modified nanocarrier or exosome capable of binding to and crossing the blood- brain barrier.

26. The method of any one of claims 22-25, wherein the TSB oligonucleotide comprises the sequence: 5’-TGTCAGTTGACCCAATAGATTGC-3’ (SEQ ID NO: 1), 5’-AATGGATGTCAGTTGACCCAA-3’ (SEQ ID NO: 2), 5’-UGUCAGUUGACCCAAUAGAUUGC-3’ (SEQ ID NO: 3), 5’-AAUGGAUGUCAGUUGACCCAA-3’ (SEQ ID NO: 4), 5’ -TTATTTCCAGCAGGAGAAACT-3’ (SEQ ID NO: 6), 5’ -CAATTTGAGGTATGAACAG-3’ (SEQ ID NO: 8), 5’ -ATTAAACTGAGGTATGAAT-3’ (SEQ ID NO: 9), 5’ -CCACATCTTTGGTTAAAAC-3’ (SEQ ID NO: 10), 5’ -TGTTAATTTCACAGATTTT-3’ (SEQ ID NO: 11), 5’ -CCGCTTTTGGCAAATGTTT-3’ (SEQ ID NO: 12), 5’ -TTTCTTATGGCACTCACTT-3’ (SEQ ID NO: 13), 5’ -TATTTCCAGCAGGAGAAAC-3’ (SEQ ID NO: 14), 5’ -ATGTTAATTTCACAGATTTTT-3’ (SEQ ID NO: 16), 5’ -GCCGCTTTTGGCAAATGTTTC-3’ (SEQ ID NO: 17), 5’ -CTTTCTTATGGCACTCACTTA-3’ (SEQ ID NO: 18), 5’ -ACGGCAGATGGCACAAATGAG-3’ (SEQ ID NO: 19), 5’ -CAAUUUGAGGUAUGAACAG-3’ (SEQ ID NO: 20), 5’ -AUUAAACUGAGGUAUGAAU-3’ (SEQ ID NO: 21), 5’ -CCACAUCUUUGGUUAAAAC-3’ (SEQ ID NO: 22), 5’ -UGUUAAUUUCACAGAUUUU-3’ (SEQ ID NO: 23), 5’ -CCGCUUUUGGCAAAUGUUU-3’ (SEQ ID NO: 24), 5’ -UUUCUUAUGGCACUCACUU-3’ (SEQ ID NO: 25), 5’ -UAUUUCCAGCAGGAGAAAC-3’ (SEQ ID NO: 26), 5’ -AUGUUAAUUUCACAGAUUUUU-3’ (SEQ ID NO: 27), 5’ -GCCGCUUUUGGCAAAUGUUUC-3’ (SEQ ID NO: 28), 5’ -CUUUCUUAUGGCACUCACUUA-3’ (SEQ ID NO: 29), 5’ -ACGGCAGAUGGCACAAAUGAG-3’ (SEQ ID NO: 30), or 5’ -UUAUUUCCAGCAGGAGAAACU-3’ (SEQ ID NO: 31)^4888-9547-9283, v. 2or the TSB is complementary to and binds a target sequence of: GTCAGTT (SEQ ID NO: 34), GAGGTA (SEQ ID NO: 35), GAGGTA (SEQ ID NO: 36), CTTTGGT (SEQ ID NO: 37), TTCACAGA (SEQ ID NO: 38), TTGGCAA (SEQ ID NO: 39), ATGGCAC (SEQ ID NO: 40), CAGCAGG (SEQ ID NO: 41), TTCACAGA (SEQ ID NO: 42), TTGGCAA (SEQ ID NO: 43), ATGGCAC (SEQ ID NO: 44), ATGGCAC (SEQ ID NO: 45), or CAGCAGG (SEQ ID NO: 46). ^ 27. The method of any one of claims 22-26, wherein the TSB is oligonucleotide provided by administering an expression construct encoding and expressing the TSB oligonucleotide.

28. The method of claim 26, wherein the expression construct is a viral construct.

29. The method of claim 28, wherein the viral construct is an adeno-associated viral construct. ^ ^4888-9547-9283, v. 2

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