Antisense oligonucleotides for treating pitt hopkin syndrome

Abstract

The present disclosure provides an antisense oligonucleotide that specifically targets the TCF4 gene, particularly at TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS-2, and method of use thereof.

Description

ANTISENSE OLIGONUCLEOTIDES FOR TREATING PITT HOPKIN SYNDROMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 574,784 filed April 4, 2024, which is hereby incorporated by reference herein in its entirety.BACKGROUND

[0002] Pitt-Hopkins syndrome (PTHS) is a rare genetic disorder characterized by developmental delay, epilepsy, distinctive facial features, and possible intermittent hyperventilation followed by apnea. PTHS is reported to be caused by an insufficient expression of the TCF4 gene.

[0003] This disorder is due to a haploinsufficiency of the transcription factor 4 (TCF4) gene which is located on the long arm of chromosome 18 (18q21.2). The mutational spectrum appears to be 40% point mutations, 30% small deletions / insertions and 30% deletions. All appear to be de novo mutations.

[0004] Currently there is no specific treatment for this condition, and people with PTHS use behavioral and training approaches to manage the condition.SUMMARY

[0005] According to one aspect, the present disclosure provides a method for increasing the expression of the transcription factor 4 (TCF4) gene in a cell. The method comprises introducing to the cell an antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS- 2.

[0006] In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene that overlaps with any one of SEQ ID NOS: 1-15. In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides with any one of SEQ ID NO: 1-15. In some embodiments, the antisenseoligonucleotide is single-stranded. In some embodiments, the antisense oligonucleotide is chemically modified. In some embodiments, the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16-30. In some embodiments, the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 16-30. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 31-45. In some embodiments, the cell is a human cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is a human patient having a genetic defect in the TCF4 gene leading to reduced expression of the TCF4 protein as compared to a healthy human subject. In some embodiments, the cell is a human patient suffering from a Pitt Hopkins syndrome. In some embodiments, the introduction is via intrathecal injection.

[0007] According to another aspect, the present disclosure provides a method of treating a disease or condition mediated by TCF4 in a patient in need thereof. The method comprises administering an antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS-2.

[0008] In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps with any one of SEQ ID NO: 1-15. In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleotides with any one of SEQ ID NOs:l-15. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the antisense oligonucleotide is chemically modified. In the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16-30. In some embodiments, the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 16-30. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 31-45. In some embodiments, the disease or condition is a genetic defect in the TCF4 geneleading to reduced expression of the TCF4 protein as compared to a healthy human subject. In some embodiments, the disease or condition is a Pitt Hopkins syndrome. In some embodiments, the administration is via intrathecal injection.

[0009] According to another aspect, the present disclosure provides an antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS-2.

[0010] In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps with any one of SEQ ID NO: 1-15. In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleotides with any one of SEQ ID NO: 1-15. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the antisense oligonucleotide is chemically modified. In some embodiments, the oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16- 30. In some embodiments, the oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides. In some embodiments, the oligonucleotide comprises or consists of any one of SEQ ID NOs: 16-30. In some embodiments, the oligonucleotide comprises or consists of any one of SEQ ID NOs: 31 -45.

[0011] According to another aspect, the present disclosure provides a pharmaceutical composition comprising the oligonucleotide of the preceding paragraphs.BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 is a drawing showing a map of the TCF4 genomic locus.

[0013] FIG. 2 is a graph showing TCF4 mRNA levels upon treatment of antisense oligonucleotides (ASO) in human neural progenitor cells (hNPC).

[0014] FIG. 3 is a graph showing EdU incorporation in healthy control and Pitt Hopkin Syndrome neural progenitor cells (hNPC).

[0015] FIG. 4 is a graph showing EdU incorporation in Pitt Hopkin Syndrome human neural progenitor cells (hNPC) after transduction with antisense oligonucleotides (ASO).DETAILED DESCRIPTIONDefinitions

[0016] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0017] As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.

[0018] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.

[0019] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.

[0020] The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. Likewise, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope- binding) or structure (e.g., salt-bridge) of the reference sequence.

[0021] Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40°C in about 10 x SSC or a solution of equivalent ionic strength / temperature. A moderate stringency hybridization is typically performed at about 50°C in about 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60°C in about 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the ail. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+normally found in a cell.

[0022] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotideis RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “polymorphism” refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a “polymorphic region of a gene”. A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0023] The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double- stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form.

[0024] The term “antisense oligonucleotides (ASO)” refer to short oligonucleotides that can bind to RNA or DNA in a target- specific manner and that may modify protein expressions. ASOs can be single- or double-stranded oligonucleotides and it may have been chemically modified to increase stability, improve the target affinity and bioavailability, and enhance cellular uptake. ASOs can bind to their target RNA transcript or genomic region in a sequence- specific manner via Watson-Crick base pairing. They can thus be designed to target distinct sequences or specificgenetic variants. Through this complementary binding, the molecules can alter protein expression by either knocking down or blocking antisense transcripts or increasing or modifying promoter activity, ultimately leading to restoration of gene expression.

[0025] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of Pitt-Hopkins syndrome. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0026] By ‘ ‘subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0027] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of a composition of the present disclosure.

[0028] As used herein, “double- stranded ribonucleic acid (dsRNA)”, as used herein, refers to an oligoribonucleotide or polyribonucleotide, modified or unmodified, and fragments or portions thereof, of genomic or synthetic origin or derived from the expression of a vector, which may be partly or fully double-stranded and which may be blunt-ended or contain a 5'- and / or 3'- overhang, and also may be of a hairpin form comprising a single oligoribonucleotide which folds back upon itself to give a double- stranded region.

[0029] As used herein “siRNA” denotes short interfering RNAs and refers to short double stranded ribonucleic acids useful for RNAi.

[0030] As used herein “inhibition” of gene expression means the reduction of the expression of said gene (such as an antisense transcript) by at least 10%, 33%, 50%, 90%, 95% or 99%.

[0031] In accordance with the present disclosure, the ribonucleic acid used for inhibition will have at least a partially double- stranded character, but may also be totally double- stranded. The RNA can be a single strand that is self-complementary or may comprise two or more separate complementary strands.

[0032] In one aspect, provided in accordance with the present disclosure are short double- stranded RNAs, also termed siRNAs, having a length of 10 to 50 nucleotides, preferably 15 to 25 nucleotides. For example, dsRNA's may be composed of oligoribonucleotides having a duplex length of 17 to 21 ribonucleotides. The oligoribonucleotides may have a duplex length of 19 ribonucleotides.

[0033] The efficiency, i.e. the degree of inhibition of the target gene (e.g., antisense transcript), is dependent on a number of different factors including the specificity of the oligonucleotide for its target sequence. In this context, specificity means homology, i.e. sequence identity between the oligonucleotide (or sequence complementary thereof) and the target sequence. It is understood by a person skilled in the ail that 100% sequence identity is not required in order to achieve significant inhibition. Normally, at least 75% sequence identity is sufficient in order to inhibit expression of the target nucleic acid. Preferred is a sequence identity of at least 80%, more preferred is a sequence identity of at least 90%. Most preferred is a sequence identity of at least 95% between the oligonucleotide and the target sequence. The best is clearly 100%. In order to target only the desired target antisense transcript, the oligonucleotide reagent should have 100% homology to the target antisense transcript and at least 2 mismatched nucleotides to all other genes present in the cell or organism. Methods to analyze and identify oligonucleotides with sufficient sequence identity in order to effectively inhibit expression of a specific target sequence are known in the art. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University ofWisconsin Genetic Computing Group). Another factor affecting the efficiency of the oligonucleotide reagent is the target region of the target transcript. The region of a target transcript effective for inhibition by the oligonucleotide reagent may be determined by experimentation.. For instance, transfection assays as described in Elbashir et al. (2001) may be performed for this purpose. A number of other suitable assays and methods exist in the art which are well known to a person skilled in the art.

[0034] The oligonucleotide according to the present disclosure may also contain modified nucleotide residues. As anyone having skill in the art of drug development would readily understand, ASOs and siRNAs can exist in various formats as described in Tolen et al. 2002, Nucl. Acids Res. 30, 1757-1766; Elbashir S. M. et al, 2001 EMBO J., 20, 6877-6888; February 2002, 521, 195-199; Current Biology 2001, 11, 1776-1780; Nature Biotech. 2002, 19, 497-500; Nature Biotech. 2002, 19, 505-508; Nucleic Acids Research 2002, 20, 1757-1766; Science 2002, 296, 5567, 550-553; Methods (San Diego, Calif., United States) 2002, 26(2), 199-213.

[0035] In the case of dsRNA, the dsRNA may be blunt ended or ligated at or on at least one end with either loops composed of ribonucleotides or deoxyribonucleotides or a chemical synthetic linker (WO00 / 44895). In a preferred embodiment, the ribonucleic acid contains 3'-end nucleotide overhangs on the antisense strand and / or the sense strands of the dsRNA of at least one ribonucleotide or deoxyribonucleotide, or modified nucleotide. Preferred are overhangs with 1, 2, 3 or 4 nucleotides. The overhangs may contain both ribonucleotide(s) and deoxyribonucleotide(s) which in addition may contain modified sugar moieties. The overhang may be of any sequence, but in a preferred embodiment, the overhang is complementary to the target mRNA strand. In another preferred embodiment the overhang contains at least one UU group or dTdT group. In another preferred embodiment, the overhang on the antisense strand has the penultimate overhanging nucleotide complementary to the antisense target. Preferably, such an overhang is a 2-nucleotides overhang.

[0036] In another embodiment, the extreme 3 '-position of the oligonucleotide is a hydroxyl group. Additionally, the 5'-end may be a hydroxyl or phosphate group.

[0037] The sugar moieties may be unmodified or modified. Preferred modified sugar moieties of oligonucleotides comprise one of the following at the 2' position: F; O — , S — , or N-alkyl; O — ,S — , or N-alkenyl; O — , S — or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nNR2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes an alkoxyalkoxy group, such as 2'-methoxyethoxy (2'-O — CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'- MOE). A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2'-DMAOE, 2'-methoxy (2'-OCH3), 2 '-aminopropoxy (2'-OCH2CH2CH2NH2). A further preferred modification of this category is the bicyclic class of modifications known collectively as LNAs (Locked Nucleic Acids) as described in Rajwanshi et al., Angew. Chem. Int. Ed. 2000, 39, 1656-1659. One of skill in the art may use conventional methods to create such modified sugar structures. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,700,920 and 5,969,116 each of which is incorporated by reference herein in its entirety.

[0038] The intemucleoside linkage of the dsRNA may be the “normal” 3' to 5' phosphodiester linkage or contain at least one chemically modified linkage. In a preferred embodiment, the at least one overhanging nucleotides contains one or more modified linkages, whereas the double- stranded part of the oligonucleotide contains phosphodiester intemucleoside linkages. Preferred modified linkages include but are not limited to, for example, those disclosed in U.S. Pat. Nos. 3,687,808; 4,469,863 and 5,625050; each of which is incorporated by reference herein in its entirety. In a preferred embodiment the linkages are phosphorothioates, chiral phosphorothioates or phosphorodithioates. Techniques for the synthesis of compounds containing oligonucleotides with modified linkages as described above may be achieved using conventional methodologies, and are familiar to one of skill in the art.

[0039] The oligoribonucleotides may be prepared by chemical synthesis (Micura R., Angcwandtc Chcmic, International Edition (2002), 41(13), 2265-2268) on commercially available or homemade oligonucleotide synthesizers using a number of different chemistries that are well known in the art. The oligonucleotide may also be prepared by in vitro transcription of a suitable template using for instance a commercially available kit such as the Silencer™ siRNA construction kit by Ambion. Alternatively, the oligoribonucleotides may be synthesized by transcription of siRNA's intracellularly from plasmids through both transient or stable transfection (Paddison P J et al., 2002, Genes and Development 16, 948-958, Paul et al., 2002, Nat. Biotech 29, 505-508).

[0040] The effect of oligonucleotide, such as ASO or dsRNA, on gene expression will typically result in expression of the target transcript being inhibited by at least 10%, 33%, 50%, 90%, 95% or 99% when compared to a cell not treated according to the present disclosure. Lower doses of administered material, lower concentrations of the oligonucleotide in the cell and / or longer times after administration of the oligonucleotide may result in inhibition at a lower level and / or in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). However, it is within the skill of the art to adapt conditions to provide the desired result. Quantitation of gene expression can be established by assessing the amount of the targeted gene product in the cell. For example, any transcript transcribed from the target gene may be detected with a hybridization probe, or RT-PCR based methodologies, or translated polypeptide may be detected with an antibody raised against the encoded polypeptide.

[0041] In embodiments, the oligonucleotide is delivered in accordance with the present application by intrathecal injection (i.e. injection into the spinal fluid which bathes the brain and spinal cord tissue). Intrathecal injection into the spinal fluid can be performed as a bolus injection or via minipumps which can be implanted beneath the skin, providing a regular and constant delivery into the spinal fluid. The circulation of the spinal fluid from the choroid plexus, where it is produced, down around the spinal cord and dorsal root ganglia and subsequently up past the cerebellum and over the cortex to the arachnoid granulations, where the fluid can exit the CNS, that, depending upon size, stability, and solubility of the compounds injected, molecules delivered intrathecally could hit targets throughout the entire CNS.Antisense oligonucleotides (ASO)

[0042] According to one aspect, the present disclosure provides an antisense oligonucleotide (ASO) targeting transcription factor 4 (TCF4) gene. The ASO may target any suitable sequence in the TCF4 gene, so that it can increase the expression of the transcription factor (TCF4) gene. In some embodiments, the ASO targets one or more of TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, the TCF4 antisense transcript AS-2, or a portion thereof. In some embodiments, the ASO targets TCF4 promoter TSS-1 or a portion thereof. In some embodiments, the ASO targets TCF4 promoter TSS-2 or a portion thereof. In some embodiments, the ASO targets antisense transcript AS-1 or a portion thereof. In some embodiments, the ASO targets antisense transcript AS-2 or a portion thereof.

[0043] In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps with any one of SEQ ID NOS: 1-15. In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides with any one of SEQ ID NOS: 1-15. In some embodiments, the antisense oligonucleotide specifically targets a sequence in the TCF4 gene that overlaps for at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% with any one of SEQ ID NOS: 1 -15. In some embodiments, the antisense oligonucleotide specifically targets a sequence comprising any one of SEQ ID NOS: 1-15. In some embodiments, the antisense oligonucleotide specifically targets any one of SEQ ID NOS: 1-15.

[0044] In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the antisense oligonucleotide is chemically modified. In some embodiments, the antisense oligonucleotide includes one or more non-canonical nucleotides. In some embodiments, the antisense oligonucleotide includes one or more chemically modified nucleotides. In some embodiments, the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16- 30. In embodiments, such antisense oligonucleotide is complementary to the TCF4 gene or antisense transcript, but comprises at least a portion of any one of SEQ ID NOS: 16-30. In some embodiments, the antisense oligonucleotide has sequence identity of at least 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% with any one of SEQ ID Nos: 16-30. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NO: 16-30. In some embodiments, the antisense oligonucleotide has a sequence of any one of SEQ ID NO: 16-30. In some embodiments, the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides, and comprises a nucleotide sequence of any one of SEQ ID NOS: 16-30 or a portion thereof as described above.

[0045] In some embodiments, the antisense oligonucleotide has one or more chemically modified nucleotides. In some embodiments, the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 31-45. In some embodiments, the antisense oligonucleotide has sequence identity of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% with any one of SEQ ID Nos: 31-45. In some embodiments, the antisense oligonucleotide comprises or consists of any one of SEQ ID NO: 31-45. In some embodiments, the antisense oligonucleotide has a sequence of any one of SEQ ID NO: 31-45. In some embodiments, the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides.

[0046] In embodiments, the present disclosure provides a compound comprising an oligonucleotide that is 10 to 30 linked nucleotides in length, and the oligonucleotide comprises at least 8, 10, 12, or 15 contiguous nucleobases of any one of SEQ ID NOs: 16 to 30.

[0047] In embodiments targeting antisense transcripts for degradation, the oligonucleotide has a stretch of at least 6 DNA nucleotides sufficient to recruit RNaseH. RNaseH is a non-sequence- specific endonuclease enzyme that catalyzes the cleavage of RNA in a hybridized RNA / DNA substrate. In some embodiments, the oligonucleotide of the present disclosure is a “gapmer,” that is, the oligonucleotides contains a central block of deoxynucleotides (also referred to herein as “DNA nucleotides”). As used herein, the term “DNA nucleotide” refers to a nucleotide that is not an RNA nucleotide. DNA nucleotides typically have a 2' H, but may alternatively have various 2' chemical modifications, including 2'-halo and 2'-lower alkyl (e.g., C1-4). In someembodiments, the 2' chemical modifications of DNA nucleotides are independently selected from 2'-Fluoro, 2'-Mcthyl, and 2'-Ethyl.

[0048] In embodiments, the oligonucleotide is a gapmer having a 5' and a 3' segment, each of the 5' and 3' segments being from 2 to 6 nucleotides, and where the 5' and 3' segments do not contain DNA nucleotides. In embodiments, one or more nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents, optionally where all of the nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents. In embodiments, the 2'-O substituents are selected from 2'-O alkyl (e.g., 2'-O methyl, 2'-O ethyl), 2'-O methoxyethyl (MOE), and a bridged nucleotide having a 2' to 4' bridge. In embodiments, the bridged nucleotide has a methylene bridge (LNA) or a constrained ethyl bridge (cEt).

[0049] In embodiments, the oligonucleotide does not recruit RNaseH, and therefore does not induce degradation of the transcript. Instead, the oligonucleotide sterically blocks binding of an antisense transcript to its target, without inducing degradation of the antisense transcript. In such embodiments, the oligonucleotide does not contain a stretch of more than four consecutive DNA nucleotides and maybe consist only of RNA nucleotides or nucleotide mimics (such as morpholino or thiomorpholino).

[0050] In some embodiments, the oligonucleotide comprises one or more locked or bi-cyclic nucleotides, e.g., bridging the 2’ and 4’ positions (“a bridged nucleotide”). Locked nucleic acid (LNA) or “locked nucleotides” are described, for example, in U.S. Pat. Nos. 6,268,490;6,316,198; 6,403,566; 6,770,748; 6,998,484; 6,670,461; and 7,034,133, all of which are hereby incorporated by reference in their entireties. LNAs are modified nucleotides that contain a bridge between the 2' and 4' carbons of the sugar moiety resulting in a “locked” conformation, and / or bicyclic structure. Other suitable locked nucleotides that can be incorporated in the oligonucleotides of this disclosure include those described in U.S. Pat. Nos. 6,403,566 and 6,833,361, both of which are hereby incorporated by reference in their entireties. In exemplary embodiments, the locked nucleotides are independently selected from a 2' to 4' methylene bridge and a constrained ethyl (cEt) bridge (see US Patent Nos. 7,399,845 and 7,569,686, which are hereby incorporated by reference in their entireties).

[0051] In embodiments, the oligonucleotide has a modified polynucleotide backbone or modified intcmuclcotidc linkages. The term “intcrnuclcotidc linkage” refers to the linkage between two adjacent nucleosides in a polynucleotide molecule. Naturally, the internucleotide linkage is a phosphodiester bond that forms between two oxygen atoms of the phosphate group and an oxygen atom of the sugar (either at 3' or 5' position) to form two ester bonds bridging between the two adjacent nucleosides. Modification of the intemucleotide linkage may provide different characteristics, including but not limited to enhanced stability. For example, phosphorothioate or phosphorodithioate linkages increase the resistance of the intemucleotide linkage to nucleases. Another example is phosphoacetate linkage (PACE), which improves transfection characteristics and enhances nuclease resistance. Internucleotide linkages and oligonucleotide backbone modifications which may be employed in the oligonucleotides of the present description include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodiamidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages.

[0052] In some embodiments, the oligonucleotide comprises one or more phosphorothioate or phosphorodithioate intemucleotide linkages. These bonds substitute a sulfur atom for a non- bridging oxygen in the phosphate backbone of the oligonucleotide, and can be effective for reducing nuclease digestion. In some embodiments, phosphorothioate or phosphorodithioate bonds can be introduced between the last three to five nucleotides at the 5'- and / or 3'-end of the oligonucleotide to inhibit exonuclease degradation. In some embodiments, the oligonucleotides have a combination of phosphodiester and phosphorothioate linkages. In some embodiments, the oligonucleotides contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten phosphorothioate intemucleotide linkages. In some embodiments, the oligonucleotides comprise substantially alternating phosphodiester and phosphorothioate intemucleotide linkages. In some embodiments, the oligonucleotides are fully phosphorothioate linked (i.e., all intemucleotide linkages are phosphorothioate).

[0053] In some embodiments, particularly where RNaseH recruitment is not desired, the oligonucleotides have a morpholino backbone. Morpholino oligonucleotides do not trigger the degradation of their target RNA molecules, and can be effective for steric blocking of a target RNA sequence. Morpholino oligonucleotides and their synthesis are disclosed generally in US Patent No. 11,028,386, US Patent No. 10,947,533, and US Patent No. 10,927,378, each of which is hereby incorporated by reference in its entirety. In some embodiments, the oligonucleotides comprise thiomorpholino nucleotides and / or other substituted or modified nucleotides such as those described, for example, in International Patent Application Publication No.WO / 2019 / 060522 and International Patent Application Publication No. WO / 2018 / 057430, each of which is hereby incorporated by reference in its entirety. For example, Langner et al. describe methods for synthesizing oligonucleotide analogs dubbed thiophosphoramidate morpholino oligonucleotides (TMOs) which incorporate morpholino nucleosides and phosphoro thioate linkages (“Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras.” JACS 142.38 (2020): 16240-16253; see also Dumbovic, Gabrijela, et al. “Nuclear compartmentalization of TERT mRNA and TUG1 IncRNA is driven by intron retention.” Nature communications 12.1 (2021): 1-19; both of which are hereby incorporated by reference in their entireties). Thus, the oligonucleotides described herein may comprise full or partial TMO-modified nucleotides, or may comprise chimeras of TMO-modified nucleotides and unmodified nucleotides and / or other nucleotides comprising different modifications (e.g., LNAs).

[0054] In various embodiments, the oligonucleotide may contain one or more modified bases. For example, in some embodiments, cytosine (as shown in sequences of Tables 1-4) is replaced with 5-methylcytosine, which may enhance base pairing. Other modified bases (particularly of cytosine or guanine) can be employed to reduce immunogenicity, where needed.Compositions and Formulations

[0055] The present disclosure provides compositions and / or formulations including the antisense oligonucleotides (ASO) described herein. In some embodiments, compositions or formulations may be formulated for intrathecal administration. In some embodiments, the compositions or formulations, for example for intrathecal administration, may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0056] The pharmaceutical compositions may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. In embodiments, the oligonucleotide of the disclosure is packages in lipid nano-particles as known in the art. The pharmaceutical formulations may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with carriers.

[0057] The compositions of the present disclosure may be formulated into any of many appropriate dosage forms. The compositions of the present disclosure may for instance be formulated as suspensions in aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0058] The pharmaceutical compositions suitable for use described herein include compositions wherein the active ingredients arc contained in an effective amount to achieve the intended purpose, for example methods described herein. The determination of an effective dose is well within the capability of those skilled in the art.Methods

[0059] In one aspect, the antisense oligonucleotides (ASO) described herein or the pharmaceutical composition or formulation including the ASOs may be used for a method for increasing the expression of the transcription factor 4 (TCF4) gene in a cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is from a human patient having a genetic defect in the TCF4 gene leading to reduced expression of the TCF4 protein as compared to a healthy human subject. In some embodiments, the cell is from a human patient suffering from a Pitt Hopkins syndrome.

[0060] In one aspect, the antisense oligonucleotides (ASO) described herein or the pharmaceutical composition or formulation including the ASOs may be used for a method of treating a disease or condition mediated by TCF4 in a patient in need thereof. The ASOs may increase the expression of TCF4 gene, thereby treating or alleviating the symptom of the disease or condition caused by defects in TCF4, leading to reduced expression of the TCF4 protein. In some embodiments, the disease or condition is a genetic defect in the TCF4 gene leading to reduced expression of the TCF4 protein as compared to a healthy human subject. In some embodiments, the disease or condition is a Pitt Hopkins syndrome. In some embodiments, the administration is done via intrathecal injection. Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, parenteral administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.Dose

[0061] A therapeutically effective dose refers to that amount of active ingredient in accordance with the present disclosure. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD5O / ED5O. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0062] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance / response to therapy. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.

[0063] The disclosure is further described by reference to the following examples. These examples are provided for illustration purposes and are not intended to be limiting.EXAMPLESExample 1

[0064] In this example, specific antisense oligonucleotides (ASO) useful for treating Pitt Hopkins syndrome were designed. Pitt Hopkins syndrome is reported to be caused by the loss of function of one of the TCF4 allele. The ASO oligonucleotides can degrade a negative regulator of TCF4 and as consequence the transcribed mRNA and expressed protein of TCF4 will increase, mitigating the effect of Pitt Hopkins syndrome. ASOs, as single stranded oligonucleotides, are small (e.g., 18-20 base pairs) and can traffic easily through the plasma membrane, including without packaging material.

[0065] We have identified several regions of TCF4 DNA that can be targeted by non-coding small RNA molecules to induce TCF4 upregulation. In this example, we have developed single stranded oligonucleotides to target TCF4 DNA regions with the goal of increasing TCF4 expression. The selected target regions of TCF4 locus are shown in FIG. 1. In FIG. 1, main TCF transcript isoforms are shown. Also shown are TCF4 antisense 1 (TCF4AS-1) and 2 (TCF4AS-2) and a long non-coding RNA (AC022031.2) in which overlap with the transcription start site 1 (TSS1) and 2 (TSS2) are designated. We have selected the two antisense mRNA TCF4AS-1 and TCF4AS-2 to be possible regulators of TCF4, along with regions of the transcription start site 1 and 2 (TSSl and TSS2).

[0066] We developed two types of ASO targeting the same sequence. Oligonucleotides that were partially modified were referred to as “ASO”, and the fully modified oligonucleotides to achieve better stability were referred to as “SBO”. In Table 1 are reported the list of ASOs generated and used.Table 1Methods

[0067] Transfection Procedure: Neural Progenitor cells (hNPC) were differentiated from induced Pluripotent Stem Cells following standard differentiation protocol. 20nM ASO was transfected into hNPC using BioT transfection reagents. 24 hours post-transduction, media was replaced, and cell collected 48 hours post transduction.

[0068] Semi-quantitative qPCR: RNA was extracted using Takara RNA extraction plus kit. cDNA was obtained using Prime script RNA synthesis kit (Takara), and qPCR was performed using Taqman master mix and Applied Biosystem quant studio pro instrument. TCF4 mRNA level were normalized on GAPDH level (used as internal control).Results

[0069] Upon establishing the protocol and ASO concentration to achieve greater than 80% transduction, we have tested all the ASO RNA (for a total of 42 ASO) in two independent experiments. Specifically, we performed a screening of 42 ASOs to identify whether they were capable to induce TCF4 expression. Using methods described herein, ASO were transfected into hNPC and RNA extracted at 48 hours post-transduction. qPCR was performed using TCF4 specific Taqman probes.

[0070] In FIG. 2, the impact of ASO on the induction of endogenous TCF4 mRNA levels in hNPC, normalized on GAPDH level (used as internal control), is shown. As shown in FIG. 2, at least the following ASO successfully induced TCF4 upregulation: ID 19 target TCF4 TSS1; ID 24, 25, 30, 31 target TCF4 TSS2; ID 36 targets TCF4-AS1; ID 46, 48 and 51 target TCF4-AS2. As the listed ASOs increased mRNA levels of TCF4, and they are capable of increasing TCF4 endogenous levels.Example 2

[0071] It was reported that neural progenitor cells (NPCs) derived from subjects having Pitt Hopkins Syndrome exhibit a defect in proliferation. Papes F et al. Nat Commun 13, 2387 (2022).

[0072] The neural progenitor cells (NPCs) derived from subjects having Pitt Hopkins Syndrome (Pitt Hopkins NPCs) were obtained from induced pluripotent stem cells (iPSC) and differentiated following the embryoid body (EB) protocol from Stem Cell Technologies Inc. In theexperiments, NPC were treated with 20mM 5-ethynyl-2'-deoxyuridine (EdU), which incorporated for 4 hours. The EdU incorporation is indicative of cell proliferation because EdU is incorporated only when cells undergo division (proliferation) because it is integrated into DNA during replication. As shown in FIG. 3, Pitt Hopkins NPCs exhibited approximately half the EdU incorporation over a four-hour period compared to controls (healthy cells). Accordingly, Pitt Hopkins NPCs showed lower level of proliferation compared to control.

[0073] Because antisense oligonucleotides (ASOs) induce TCF4 upregulation as shown in Example 1, it was contemplated that such increase in TCF4 expression can also enhance NPC proliferation, thereby demonstrating biological activity in the cells. The Pitt Hopkins NPCs were treated for two days with 40nM ASOs capable of inducing TCF4 expression, listed in Table 1 above. On Day 3, the cells were treated with 20mM 5-ethynyl-2'-deoxyuridine (EdU) and 4-hour incorporation was analyzed, to evaluate whether ASO treatment affected their proliferation capacity. The EdU incorporation level is shown in FIG. 4 (*p<0.05). As shown in FIG. 4, ASO IDs 19, 30, 31, 33, 36, 46, 48, and 51 exhibited increased EdU incorporation, thus the ASOs significantly increased the proliferation of Pitt Hopkins NPCs compared to NCI (Negative Control). These results provide evidence that ASO treatment can correct a biological defect in Pitt Hopkins NPCs and demonstrate its efficacy in one aspect of their function.Example 3

[0074] ASO transfection is performed without packaging reagents (BioT) to assess ASO uptake efficiency. To determine whether ASOs can traverse the cell membrane, fluorescently labeled ASO 31 is used. Various ASO concentrations (ranging from 20-200 nM) are directly applied to healthy control (HC) and Pitt Hopkins (PH) NPCs. Cells are harvested and analyzed via flow cytometry to measure transduction efficiency, expressed as the percentage of fluorescent cells over the total cell population, at multiple time points (2-5 days post-treatment). Additionally, the timing of ASO administration are varied to control transduction rates.Example 4

[0075] TCF4 isoforms affected by treatment with ASOs described herein are identified via Deep RNA Sequencing. Pitt Hopkins (PH) and healthy control (HC) iPSC-derived NPCs are furtherdifferentiated into forebrain neurons (FBNs) following Stem Cell Technologies Inc. protocols. Both NPCs and FBNs arc treated with the ASOs, after which cells arc harvested, RNA arc extracted, and samples are subject to PAC-BIO sequencing. Baseline TCF4 expression is established using untreated HC cells and compared to PH cells in both NPC and FBN stages. From the sequencing, isoform abundance and type are analyzed to assess the impact of different ASO treatments.Example 5

[0076] PH and HC iPSC-derived NPCs are differentiated into forebrain neurons (FBNs) following Stem Cell Technologies Inc. protocols. At the start of differentiation, ASO treatment is initiated with ASOs administered every two days throughout the two-week differentiation period. At the end of differentiation, FBNs are fixed and immunostained for MAP2, a neuron- specific cytoskeletal protein, and TRIM46, an axon-expressed protein. DAPI is used to label cell nuclei. Using confocal microscopy, six different fields per sample are imaged and soma diameter (ImageJ), axon length (TRIM46-positive neurites), and dendrite length (TRIM46-negative neurites) are quantified.* * *

[0077] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and valuations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0078] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference

Claims

CLAIMSWhat is claimed is:

1. A method for increasing the expression of the transcription factor 4 (TCF4) gene in a cell, comprising introducing to the cell an antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS-2.

2. The method of claim 1, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene that overlaps with any one of SEQ ID NO: 1-15.

3. The method of claim 1, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides with any one of SEQ ID NO: 1-15.

4. The method of any preceding claim, wherein the antisense oligonucleotide is single- stranded.

5. The method of any preceding claim, wherein the antisense oligonucleotide is chemically modified.

6. The method of any preceding claim, wherein the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16-30.

7. The method of claim 6, wherein the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides.

8. The method of claim 6, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 16-30.

9. The method of claim 6, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 31-45.

10. The method of any preceding claim, wherein the cell is a human cell.

11. The method of any preceding claim, wherein the cell is in vivo.

12. The method of claim 11, wherein the cell is a human patient having a genetic defect in the TCF4 gene leading to reduced expression of the TCF4 protein as compared to a healthy human subject.

13. The method of claim 12, wherein the cell is a human patient suffering from a Pitt Hopkins syndrome.

14. The method of any one of claims 11-13, wherein the introduction is via intrathecal injection.

15. A method of treating a disease or condition mediated by TCF4 in a patient in need thereof, the method comprising administering an antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS- 1, or the TCF4 antisense transcript AS-2.

16. The method of claim 15, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps with any one of SEQ ID NO:1- 15.

17. The method of claim 15 or 16, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides with any one of SEQ ID NO: 1-15.

18. The method of any one of claims 15-17, wherein the antisense oligonucleotide is single- stranded.

19. The method of any one of claims 15-18, wherein the antisense oligonucleotide is chemically modified.

20. The method of any one of claims 15-19, wherein the antisense oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16-30.

21. The method of claim 20, wherein the antisense oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides.

22. The method of claim 20, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 16-30.

23. The method of claim 20, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 31-45.

24. The method of any one of claims 15-23, wherein the disease or condition is a genetic defect in the TCF4 gene leading to reduced expression of the TCF4 protein as compared to a healthy human subject.

25. The method of claim 24, wherein the disease or condition is a Pitt Hopkins syndrome.

26. The method of any one of claims 15-25, wherein the administration is via intrathecal injection.

27. An antisense oligonucleotide that specifically targets the TCF4 promoter TSS-1, the TCF4 promoter TSS-2, the TCF4 antisense transcript AS-1, or the TCF4 antisense transcript AS- 2.

28. The oligonucleotide of claim 27, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps with any one of SEQ IDNO:1-15.

29. The oligonucleotide of claim 27, wherein the antisense oligonucleotide specifically targets a sequence in the TCF4 gene or antisense transcript that overlaps for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides with any one of SEQ ID NO:1-15.

30. The oligonucleotide of any one of claims 27-29, wherein the antisense oligonucleotide is single- stranded.

31. The oligonucleotide of any one of claims 27-30, wherein the antisense oligonucleotide is chemically modified.

32. The oligonucleotide of any one of claims 27-31, wherein the oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of any one of SEQ ID NOs: 16-30.

33. The oligonucleotide of claim 32, wherein the oligonucleotide is of a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, or less than 30, 35, 40, 45 of 55 nucleotides.

34. The oligonucleotide of claim 32, wherein the oligonucleotide comprises any one of SEQ ID NOs: 16-30.

35. The oligonucleotide of claim 32, wherein the oligonucleotide comprises any one of SEQ ID NOs: 31-45.

36. A pharmaceutical composition comprising the oligonucleotide of any of claims 27-35.

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