Compositions and methods for the treatment of a genetic neurodevelopmental disorder

Abstract

Described herein are antisense oligonucleotides, vectors, and related compositions and methods for increasing the endogenous expression of the SHANK3 protein, and their use for conditions associated with SHANK3 haploinsufficiency such as Phelan-McDermid syndrome.

Description

Technical Field

[0001] This application claims priority from AU2022 / 901557, filed on 7 June 2022, AU2022 / 902778, filed on 26 September 2022, and AU2022 / 903040, filed on 17 October 2022, the entire contents of each of which are incorporated herein by reference.

[0002] The present disclosure generally relates to oligonucleotides for treating conditions associated with mutations in the SHANK3 gene, and related compositions and methods.

Background Art

[0003] SHANK3 is a widely expressed scaffold protein rich in postsynaptic density of excitatory synapses in the brain. SHANK3 recruits ionotropic and metabotropic glutamate receptors (AMPA, NMDA, mGluR) and stabilizes them up to the postsynaptic density. SHANK3 gene mutations / deletions and SHANK3 haploinsufficiency are the cause of Phelan-McDermid syndrome, a rare hereditary neurodevelopmental disorder, and are involved in 0.5% - 1% of autism spectrum disorder (ASD) diagnoses, 2% of intellectual disability diagnoses, and 0.6% - 2.16% of schizophrenia diagnoses.

[0004] Phelan-McDermid syndrome is characterized by varying degrees of intellectual disability, neonatal hypotonia, absent to severely delayed language development, moderate to severe developmental delay, motor regression, and mild dysmorphism. Approximately 14 - 70% of affected individuals develop mild to severe seizures. Other complications include kidney abnormalities, gastrointestinal problems, risk of decreased sweating and hyperthermia, lack of pain perception, arachnoid cysts, or other co-existing neuropsychiatric disorders. Children are typically diagnosed in early childhood, often due to significant delays in reaching early developmental milestones. There is currently no effective treatment for conditions caused by SHANK3 haploinsufficiency such as Phelan-McDermid syndrome. Thus, there is a continuing need to provide effective compositions and methods for treating such conditions.

Summary of the Invention

[0005] The SHANK3 gene contains 22 exons spanning 58 kb of genomic DNA on the end of chromosome 22 (the 22q13 region), and its main protein product is a 1,607 amino acid polypeptide. There are at least 6 known isoforms that are temporally and spatially specific in synapses and have different functions. SHANK3 contains 5 protein-protein interaction domains, and each isoform contains a different combination of these 5 domains.

[0006] Without wishing to be bound by theory, SHANK3 haploinsufficiency resulting from loss of functional gene mutations, including nonsense, missense, and frameshift mutations, as well as partial or complete gene deletions, results in insufficient protein production.

[0007] The present disclosure provides antisense oligonucleotides (ASOs), antisense RNA (AR) expression vectors, and related compositions and methods for increasing SHANK3 protein levels by modulating the translation efficiency or stability of SHANK3 mRNA to increase the level of SHANK3 mRNA encoding a functional SHANK3 isoform. Methods for treating conditions associated with SHANK3 haploinsufficiency are also disclosed.

[0008] Accordingly, in one aspect, provided herein is (i) an antisense oligonucleotide that binds within a targeted portion of the 5' untranslated region (UTR) of SHANK3 mRNA, (ii) the 5' proximal non-coding region (PNCR) of SHANK3 pre-mRNA, or (iii) the 3' UTR of SHANK3 mRNA, wherein binding of the antisense oligonucleotide within the targeted portion results in an increased level of SHANK3 protein in mammalian cells.

[0009] In a related aspect, provided herein is (i) an antisense RNA (AR) that binds within a targeted portion of the 5' UTR of SHANK3 mRNA, (ii) the 5' PNCR of SHANK3 pre-mRNA, or (iii) the 3' UTR of SHANK3 mRNA, for expression in mammalian neurons, A vector in which the binding of AR within the targeting portion of RNA in mammalian cells results in increased levels of SHANK3 protein in mammalian cells. In some examples, the vector includes a neuron-selective promoter for driving the expression of AR in mammalian neurons. In some examples, the neuron-selective promoter is selective for expression in a neuron type selected from the list consisting of cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons. In some examples, the vector includes an inducible promoter. In some examples, the vector is a non-viral vector. In some examples, the non-viral vector is provided as a composition comprising a transfection agent. In other examples, the vector is a viral vector. In some examples, the vector is a viral vector and the viral vector is a recombinant virus selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and anellovirus.

[0010] In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 293, 299, 301, 302, 304-309, 311, 313, 315, 318, 606, 797, 1193, 1195, 1847, 1934-1937, 2858, 2874, 3510, 12644, 12666, 12669, 12671, 12688, or 12690. In some examples of any of the ASO, vector, or composition, the binding of the ASO or AR is within the targeting portion of the 5'UTR corresponding to SEQ ID NO: 1. In other examples of any of the aforementioned methods, ASO, vector, or composition, the binding of the ASO or AR is within the targeting portion of the 5'PNCR corresponding to SEQ ID NO: 3. In other examples of any of the aforementioned methods, ASO, vector, or composition, the binding of the ASO or AR is within the targeting portion of the 3'UTR corresponding to SEQ ID NO: 2.

[0011] In some examples, the nucleotide sequence of the ASO or AR is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeting portion over the length of the ASO or AR. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 5-622, 4175-4181, or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 559, 606, or 4178-4181. In other examples, the nucleotide sequence of the antisense oligonucleotide or AR corresponds to any one of SEQ ID NOs: 1935-4168, 4182, 4183, 12646-12654, or 12664-12671. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 1935-1937, or 2849. In other examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 623-1934, 4169-4174, 12645, 12655-12663, or 12688. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 1847, 1852, 1934, 12661-12663, or 12688.

[0012] In some examples, any of the aforementioned ASOs includes a backbone modification. In some examples, the backbone modification includes a phosphorothioate bond or a phosphorodiamidate bond. In other examples, the ASO includes a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, or a 2'-O-modification such as a 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some examples, the ASO includes at least one modified sugar moiety. In other examples, each sugar moiety in the ASO is a modified sugar moiety. In some examples, the ASO includes a 2'-O-methoxyethyl moiety. In other examples, each nucleotide of the ASO includes a 2'-O-methoxyethyl moiety.

[0013] In some examples of any of the foregoing ASOs or vectors, the nucleotide sequence of the ASO or AR is 10 to 50 nucleotides, 15 to 40 nucleotides, 17 to 30 nucleotides, 18 to 40 nucleotides, 17 to 25 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 22 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides in length. In some examples where the ASO is 17 to 30 nucleotides in length, the ASO contains one or more phosphorodiamidate morpholino moieties.

[0014] In some examples, any of the foregoing ASOs is linked to a functional moiety. In some examples, the functional moiety includes a delivery moiety. In some examples, the delivery moiety is selected from the group consisting of lipids, polyethers, peptides, carbohydrates, receptor binding domains (RBDs), and antibodies. In some examples, the ASO includes a delivery moiety, and the delivery moiety includes a cell-penetrating peptide (CPP). In some examples, the delivery moiety includes N-acetylgalactosamine (GalNAc) or a glycan moiety. In some examples, the delivery moiety includes a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is about C8 - C20. In other examples, the functional moiety includes a stabilizing moiety. In some examples, the functional moiety is covalently linked to the ASO. In other examples, the functional moiety is non-covalently linked to the ASO. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO. In some examples of any of the foregoing ASOs, a delivery nanocarrier is also included, and the nanocarrier is complexed with the ASO. In some examples, the delivery nanocarrier is selected from the group consisting of lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In some examples, the delivery nanocarrier includes lipid nanoparticles (LNPs) that encapsulate the ASO.

[0015] In related aspects, provided herein is a pharmaceutical composition comprising any one of the aforementioned ASO, vector, or composition, and a pharmaceutically acceptable excipient.

[0016] In further related aspects, provided herein is a method for preventing or treating a condition associated with SHANK3 haploinsufficiency, the method comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned pharmaceutical composition. In some examples, the condition being treated is Phelan-McDermid syndrome, autism spectrum disorder, schizophrenia, or intellectual disability. In some examples, the condition being treated is Phelan-McDermid syndrome. In some examples, the subject being treated is a human subject.

[0017] In a further aspect, provided herein is the use of any one of the aforementioned antisense oligonucleotides, vectors, or compositions in the manufacture of a medicament for the prevention or treatment of a condition associated with SHANK3 haploinsufficiency.

[0018] In some examples of the aforementioned treatment method or use, the level of SHANK3 protein in at least a plurality of cells in the subject is increased in vitro or ex vivo, in cells (e.g., neurons), by about 1.1 to about 5-fold, e.g., 1.2-fold, 1.3-fold, 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold, 2.7-fold, 3-fold, 3.3-fold, 3.5-fold, 4-fold, 4.3-fold, 4.5-fold, 4.7-fold compared to the level in the absence of the pharmaceutical composition, or is another increase in the SHANK3 protein level of about 1.1-fold to about 5-fold in the cells in the subject.

[0019] In yet another aspect, provided herein is a genetically modified cell comprising any of the foregoing ASOs or vectors. In some examples, the genetically modified cell is a mammalian cell. In some examples, the genetically modified mammalian cell is a human cell. In some examples, the genetically modified mammalian cell is a neuron or a neural progenitor cell. In some examples, the genetically modified mammalian cell is a neuron selected from the group consisting of a cortical glutamatergic neuron, a cortical GABAergic neuron, a hippocampal glutamatergic neuron, and a striatal inhibitory neuron. In some examples, the genetically modified mammalian cell is derived from a cell line. In some examples, the cell line is a human induced pluripotent stem cell (hiPSC) line, or a cell line derived from a neuron.

[0020] SHANK3 mRNA is also known to be a target of alternative splicing, which can affect the overall levels of standard SHANK3 mRNA and protein. The term "alternative splicing" refers to a process by which exons, or parts of exons of a gene, or introns or parts of introns can be included in or excluded from the final mRNA transcript. Mature non-standard mRNA transcripts can be non-productive due to frameshifts that can induce degradation of the transcript via the nonsense-mediated decay pathway. In other cases, translation of non-standard mRNA is truncated but can produce a non-functional protein. Alternative splicing of SHANK3 pre-RNA transcripts can downregulate overall SHANK3 mRNA and protein expression.

[0021] Introns are removed from the mature transcript by a large RNA-protein complex called the spliceosome, which coordinates complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome is assembled on each intron in an ordered manner that begins with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, which facilitates binding of U2 auxiliary factor (U2AF) to the 3'ss region and binding of U2 to the branch point sequence (BPS). U2AF is a stable heterodimer composed of the 65kD subunit encoded by U2AF2 (U2AF65), which binds to the polypyrimidine tract (PPT), and the 35kD subunit encoded by U2AF1 (U2AF35), which interacts with the highly conserved AG dinucleotide at the 3'ss and stabilizes U2AF65 binding. In addition to the BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures that activate or suppress splice site recognition, known as splicing enhancers or silencers of introns or exons. These elements enable the true splice sites to be distinguished from potential or pseudo-splice sites that have the same sequence motifs as the genuine sites but are present in the genomes of higher eukaryotes in numbers that exceed them by an order of magnitude or more.

[0022] Accordingly, in different aspects, provided herein is a method for increasing the amount of functional SHANK3 protein in mammalian cells that express SHANK3 pre-mRNA, the method comprising contacting the cells with an antisense oligonucleotide or antisense RNA that binds to a targeted portion of the SHANK3 pre-mRNA to modulate splicing of the resulting SHANK3 mRNA, whereby the level of SHANK3 mRNA encoding full-length functional SHANK3 protein is increased in the mammalian cells.

[0023] In a further aspect, provided herein is an antisense oligonucleotide that binds to a targeted portion of SHANK3 pre-mRNA and modulates splicing of SHANK3 mRNA, whereby the level of SHANK3 mRNA encoding full-length functional SHANK3 is increased.

[0024] In a related aspect, provided herein is a vector for expression in mammalian neurons of an antisense RNA (AR) that binds within a targeted portion of SHANK3 pre-mRNA and modulates splicing of SHANK3 mRNA, whereby the level of SHANK3 mRNA encoding full-length functional SHANK3 is increased. In some examples, the nucleotide sequence of the ASO or AR used to modulate splicing corresponds to any one of SEQ ID NOs: 4187-12693 or 12695-3953. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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DETAILED DESCRIPTION OF THE INVENTION

[0026] General Throughout this specification, unless specifically stated otherwise or the context otherwise requires, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to include one and more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter. Thus, as used herein, the singular forms “a,” “an,” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes not only a single one, but also two or more, reference to “an” includes not only a single one, but also two or more, reference to “the” includes not only a single one, but also two or more, and so forth.

[0027] Each example of the present disclosure described herein is to be applied mutatis mutandis to each and all other examples, unless otherwise specified.

[0028] Those skilled in the art will understand that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, as well as any and all combinations, or any two or more of the above steps or features.

[0029] The present disclosure should not be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure, as described herein.

[0030] The present disclosure is implemented without undue experimentation using conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid-phase peptide synthesis, and immunology, unless otherwise indicated. Such techniques are described and explained in the literature cited, such as Perbal 1984, Sambrook et al., 2001, Brown (editor) 1991, Glover and Hames (editors) 1995 and 1996, Ausubel et al. including all updates to date, Coligan et al. (editors) (including all updates to date), Maniatis et al. 1982, Gait (editor) 1984, Hames and Higgins (editors) 1984, Freshney (editor) 1986, etc.

[0031] The term "and / or", e.g., "X and / or Y", is to be understood to mean either "X and Y" or "X or Y", and is to be construed as providing explicit support for both meanings or either meaning.

[0032] The term "about", unless the contrary meaning is indicated, refers to + / - 20% of the specified value, more preferably + / - 10%. To avoid doubt, the term "about" before a specified value should be construed to include the exact specified value itself (e.g., "about 10" includes exactly 10).

[0033] Throughout this specification, the term "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0034] The terms "antisense oligonucleotide", "antisense oligomer" or "ASO", as used herein, include, but are not limited to, oligonucleotides and any other oligomeric molecules containing nucleobases capable of hybridizing to a complementary sequence on a target RNA transcript, including those that do not contain a sugar moiety, such as in the case of peptide nucleic acids (PNAs). Preferably, the ASO is an ASO that is resistant to nuclease cleavage or degradation.

[0035] As used herein, with respect to an ASO or an AR, the phrases “binds to the targeting moiety” or “binds within the targeting moiety” refer to specific hybridization between the ASO or AR nucleotide sequence and a target nucleotide sequence that is complementary within the ranges set forth herein. In some examples, specific hybridization occurs under ex vivo conditions when hybridization occurs under high stringency conditions. “High stringency conditions” means that an ASO or an AR hybridizes to a target sequence in a detectably stronger amount than non-specific hybridization under such ex vivo conditions. Thus, high stringency conditions are conditions that distinguish polynucleotides having exact complementary sequences, or conditions that contain only a few scattered mismatches from random sequences that fortuitously have a few small regions (e.g., 1-5 bases) that match the probe. Such small regions of complementarity are more readily melted than a full-length complement of 12-17 or more bases, and moderate stringency hybridization readily distinguishes them. In one example, high stringency conditions include, for example, low salt and / or high temperature conditions provided by about 0.02-0.1 M NaCl or an equivalent at a temperature of about 50-70 °C. Those skilled in the art will understand that under in vivo conditions, the specificity of hybridization between an ASO or an AR and its target sequence is defined in terms of the level of complementarity between the ASO or an AR and the target sequence that hybridizes intracellularly.

[0036] The term "peptide" is intended to include compounds composed of amino acid residues linked by amide bonds. Peptides may be natural or non-natural, ribosomally translated, or synthetically derived. Typically, peptides consist of 2 to 200 amino acids. For example, a peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within the above ranges. A peptide may contain less than about 150 amino acids, or less than about 125 amino acids, or less than about 100 amino acids, or less than about 90 amino acids, or less than about 80 amino acids, or less than about 70 amino acids, or less than about 60 amino acids, or less than about 50 amino acids, or may consist of them.

[0037] As referred to herein, peptides include "inverso" peptides in which all L-amino acids are replaced by the corresponding D-amino acids, and "retro-inverso" peptides in which the amino acid sequence is reversed and all L-amino acids are replaced by D-amino acids.

[0038] The peptide may contain both L - type and / or D - type amino acids. For example, both L - type and D - type can be used for different amino acids within the same peptide sequence. In some examples, the amino acids within the peptide sequence are of the L - type, such as natural amino acids. In some examples, the amino acids within the peptide sequence are a combination of L - type and D - type. Further, the peptide may contain non - conventional but naturally occurring amino acids including, but not limited to, hydroxyproline (Hyp), beta - alanine, citrulline (Cit), ornithine (Orn), norleucine (Nle), 3 - nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr). The peptide may also incorporate non - natural amino acids including, but not limited to, homo - amino acids, N - methyl amino acids, alpha - methyl amino acids, beta(homo)amino acids, gamma amino acids, and N - substituted glycines. The peptide may be a linear peptide or a cyclic peptide.

[0039] The term "protein" must be interpreted to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., polypeptide complex) linked to each other by covalent or non - covalent bonds. For example, a series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non - covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0040] The percentage of amino acid sequence identity to a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Amino acid sequence identity can be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is available on the website at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings including Gap Open: 10.0 and Gap Extend 0.5 are utilized. The default matrix “Blosum62” is used for the amino acid sequence and the default matrix. The “nucleic acid sequence identity” percentage (%) or percentage for a nucleotide sequence disclosed herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining nucleic acid sequence identity percentage can be achieved in various ways known in the art using publicly available computer software such as BLAST or ALIGN, for example. One of ordinary skill in the art can readily determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0041] The term "cell-penetrating peptide" (CPP) refers to a peptide that can cross the cell membrane. In one example, a CPP can move across a mammalian cell membrane and enter the cell. In another example, a CPP can direct a conjugate to a desired subcellular compartment. Thus, a CPP can direct or facilitate the penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A CPP can cross the membrane with its amino acid sequence intact or alternatively in a partially degraded state.

[0042] A CPP can direct a molecule of interest, such as an ASO disclosed herein, from outside the cell, across the plasma membrane, and into the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, a CPP can direct a molecule of interest across the blood-brain, trans-mucosal, blood-retinal, skin, gastrointestinal, and / or pulmonary barriers.

[0043] The term "peptide ligand" or "receptor-binding domain" refers to a peptide that can bind to a membrane surface receptor to enable the movement of a peptide across the cell membrane. In one example, a peptide ligand can enable movement across the cell membrane via the natural endocytosis of a target receptor. In another example, a peptide ligand can utilize a complementary mechanism of movement across the cell membrane, including by utilizing a conjugated CPP. In one example, a peptide ligand can move across a mammalian cell membrane and enter the cell. In another example, a peptide ligand can direct a conjugate to a desired subcellular compartment. Thus, a peptide ligand can direct or facilitate the cellular uptake of a molecule of interest across a phospholipid, mitochondrial, endosomal, lysosomal, vesicular, or nuclear membrane. A peptide ligand can cross the membrane with its amino acid sequence intact or alternatively in a partially degraded state.

[0044] Peptide ligands that act through binding to a target receptor can direct a molecule of interest, such as an ASO disclosed herein, from outside the cell, through the plasma membrane, and towards the cytoplasm or a desired subcellular compartment. Alternatively, or in addition, peptide ligands that act through binding to a target receptor can direct a molecule of interest through relevant biological barriers such as, for example, the blood-brain, transmucosal, blood-retinal, skin, gastrointestinal, and / or pulmonary portals.

[0045] Compositions for increasing SHANK3 protein levels The secondary structure and upstream open reading frame in the 5’UTR of SHANK3 mRNA can reduce translation efficiency and transcript stability, thereby limiting protein production. In addition, the SHANK3 5’UTR contains three exons, and exon 2 contains sequences that can affect translation efficiency, ultimately affecting the level of SHANK3 protein. Exclusion of SHANK3 exon 2 during splicing of the 5’PNCR shortens the resulting 5’UTR and removes elements that reduce translation efficiency. Furthermore, microRNAs (miRNAs) typically bind to complementary RNA sequences within the 3’untranslated region (3’UTR) and regulate gene expression by stimulating either mRNA degradation or translational repression. Both classes of mechanisms result in decreased gene expression. Despite such “inefficiencies,” in the genetic background of two functional (wild-type) alleles, the levels of productive RNA transcripts and translation result in sufficient levels of functional protein for a given gene. However, in the case of rare monogenic diseases, the loss of one functional allele, for example, the SHANK3 allele, can result in haploinsufficiency and related diseases.

[0046] Although not wishing to be bound by theory, it is thought that ASOs targeting sequences within the 5’UTR of SHANK3 mRNA can enhance translation, for example, by reducing / destroying the formation of secondary structures that in other cases reduce translation efficiency. ASOs targeting the 5’PNCR can enhance the translation efficiency of SHANK3 transcripts. Further, antisense sequences that are at least partially complementary to the binding sites for miRNAs located within the 3’UTR of SHANK3 mRNA hybridize to the 3’UTR, sterically hindering (“masking”) their access to those binding sites of these miRNAs, thereby resulting in increased levels of SHANK3 mRNA and ultimately enabling increased translation of SHANK3 protein.

[0047] Accordingly, disclosed herein are ASOs that bind within a targeted portion of (i) the 5’UTR of SHANK3 mRNA, (ii) the 5’PNCR of SHANK3 pre-mRNA, or (iii) the 3’UTR of SHANK3 mRNA, wherein binding of the antisense oligonucleotide within the targeted portion in mammalian cells results in increased levels of SHANK3 protein in mammalian cells.

[0048] For reference, the nucleotide sequence of a standard human SHANK3 pre-mRNA transcript (“SHANK3-201”) is provided herein as SEQ ID NO: 4. The nucleotide sequence of the 5’UTR of a standard human SHANK3 mRNA transcript is provided herein as SEQ ID NO: 1. The nucleotide sequence of the 5’PNCR of a standard SHANK3 pre-mRNA is provided herein as SEQ ID NO: 3. The nucleotide sequence of the 3’UTR of a standard human SHANK3 mRNA is provided herein as SEQ ID NO: 2 (Appendix).

[0049] Antisense oligonucleotides (ASOs) and antisense RNAs (AR) In some examples of the compositions and methods described herein, the ASOs and ARs have sequences that are completely or nearly completely complementary to the target sequence over their length. The ASOs and ARs are designed to bind (hybridize) to a target RNA sequence (e.g., the targeted portion of an mRNA transcript) and remain hybridized under physiological conditions. Selection of a suitable sequence for the ASOs and ARs generally avoids similar nucleic acid sequences at other (i.e., off-target) positions within the genome or in cellular mRNA or miRNA, such that the likelihood that the ASO or AR hybridizes at such sites is limited. In some examples, the ASOs disclosed herein are useful for attenuating the formation of SHANK3 mRNA secondary structure in the 5’UTR region, particularly for interfering with translation. In other examples, the ASOs disclosed herein bind to targeted regions within the 5’PNCR of SHANK3 pre-mRNA, such as within an intron sequence, or partially within an intron sequence and partially within an adjacent exon sequence. In other examples, the ASOs disclosed herein mask the access of miRNAs to their target binding sites within the SHANK3 3’UTR, thereby reducing the level of miRNA-dependent SHANK3 mRNA destabilization. Thus, the ASOs disclosed herein result in a net increase in the level of canonical SHANK3 mRNA and, as a result, a net increase in the level of functional SHANK3 protein.

[0050] In some examples, the ASO or AR “specifically hybridizes” to, or is “specific” for, the target nucleic acid or the targeted portion of the SHANK3 mRNA 5’UTR or 3’UTR, or the SHANK3 pre-mRNA 5’PNCR. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0051] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, ASO and AR sequences are "complementary" to their target sequences. Complementarity can be quantified in terms of the proportion (e.g., percentage) of bases in the opposing strand that are expected to form hydrogen bonds with each other according to the generally accepted base pairing rules. The nucleotide sequence of an ASO or AR does not need to be 100% complementary to the nucleotide sequence of its target nucleic acid for hybridization to occur. In certain examples, the nucleotide sequence of an ASO or AR in the compositions disclosed herein is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementary to the nucleotide sequence of the targeted portion of the RNA transcript over the length of the ASO or AR nucleotide sequence. For example, an ASO or AR in which 18 out of 20 nucleotides of the ASO or AR sequence are complementary to the target region and will thus hybridize specifically will exhibit 90 percent complementarity. In such examples, the remaining non-complementary nucleotides of the ASO or AR can be clustered together with the complementary nucleotides or be scattered and do not need to be contiguous. The complementarity of an ASO or AR sequence to a target nucleotide sequence (expressed as the "percent complementarity" to its target sequence or the "percent identity" to its reverse complement sequence) can be routinely determined using algorithms known in the art, as exemplified by the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul, et al., 1990, J. Mol. Biol., 215:403-410, Zhang et al., 1997, Genome Res., 7:649-656).

[0052] In some examples, the ASO or AR does not hybridize to all nucleotides within the target sequence, and the nucleotide positions that hybridize may be adjacent or non - adjacent. The ASO or AR can hybridize across one or more segments of the SHANK3 mRNA 5’UTR or 3’UTR, or one or more segments of the SHANK3 mRNA 5’ PNCR, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure can form).

[0053] In some examples, the ASO or AR disclosed herein is complementary to a targeted portion of the standard SHANK3 mRNA 5’UTR sequence corresponding to SEQ ID NO: 1. In other examples, the ASO or AR disclosed herein is complementary to a targeted portion of the standard SHANK3 pre - mRNA 5’PNCR sequence corresponding to SEQ ID NO: 3. In other examples, the ASO or AR disclosed herein is complementary to a targeted portion of the standard SHANK3 mRNA 3’UTR sequence corresponding to SEQ ID NO: 2.

[0054] In some examples, the nucleotide sequence of the ASO or AR is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeted portion of the SHANK3 mRNA over the length of the ASO or AR. In some examples, the nucleotide sequence of the ASO or AR has (a) at least about 40% identity to about 60% identity to the nucleotide sequence of the ASO or AR sequence disclosed herein, e.g., 45% identity, 48% identity, 50% identity, 52% identity, 55% identity, 58% identity, or another sequence identity of at least about 40% identity to about 60% identity to the full length of any ASO or AR sequence disclosed herein, and (b) a continuous sequence of at least 8 to 16 bases that is 100% identical to a continuous sequence of at least 8 to 16 bases in any one of the ASO or AR sequences disclosed herein, e.g., a continuous sequence of 9, 10, 11, 12, 13, 14, 15, or 16 bases in the ASO or AR sequence disclosed herein that is 100% sequence identical. The ASO or AR for use in the compositions described herein can be of any length suitable for specific hybridization to the target sequence. In some examples, the nucleotide sequence of the ASO or AR consists of 8 to 50 nucleotides. For example, the ASO or AR sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleotides in length. In some examples, the nucleotide sequence of the ASO or AR consists of 8 to 50 nucleotides. For example, the ASO or AR sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleotides in length. In some examples, the ASO is more than 50 nucleotides but consists of a length of 100 nucleotides or less.In some examples, the ASO or AR nucleotide sequence is 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 35 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 9 to 50 nucleotides, 9 to 40 nucleotides, 9 to 35 nucleotides, 9 to 30 nucleotides, 9 to 25 nucleotides, 9 to 20 nucleotides, 9 to 15 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, 10 to 15 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 11 to 20 nucleotides, 11 to 15 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 35 nucleotides, 12 to 30 nucleotides, 12 to 25 nucleotides, 12 to 20 nucleotides, 12 to 15 nucleotides, 13 to 50 nucleotides, 13 to 40 nucleotides, 13 to 35 nucleotides, 13 to 30 nucleotides, 13 to 25 nucleotides, 13 to 20 nucleotides, 14 to 50 nucleotides, 14 to 40 nucleotides, 14 to 35 nucleotides, 14 to 30 nucleotides, 14 to 25 nucleotides, 14 to 20 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 15 to 20 nucleotides, 17 to 30 nucleotides, 17 to 25 nucleotides, 17 to 20 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 20 to 25 nucleotides, 25 to 50 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 25 to 30 nucleotides in length. In some examples, the ASO or AR is 17 nucleotides in length. In other examples, the ASO or AR is 20 nucleotides in length.In some examples, the nucleotide sequence of the ASO or AR nucleotide is 25 nucleotides in length.

[0055] In other examples, the ASO or AR comprises at least 10 contiguous nucleotides of the ASO or AR sequences described herein. In some examples, the ASO or AR comprises at least 10 contiguous nucleotides (subsequences) from each of two or more of the ASO or AR sequences described herein, and the two or more subsequences are not contiguous within the SHANK3 mRNA sequence.

[0056] In some examples for each occurrence of "G" in the ASO or AR sequences disclosed herein, "G" is guanosine or inosine. In some examples for each occurrence of "T" in the ASO or AR sequences disclosed herein, "T" is any one of thymidine, inosine, uracil, or an isomer or modified form of uracil (e.g., pseudouridine or N1-methyl-pseudouridine). In some examples for each occurrence of "C" in the ASO or AR sequences disclosed herein, C is cytosine or a modified form of cytosine (e.g., 5'-methylcytosine).

[0057] In some examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 293, 299, 301, 302, 304-309, 311, 313, 315, 318, 606, 797, 1193, 1195, 1847, 1934-1937, 2858, 2874, 3510, 12644, 12666, 12669, 12671, 12688, or 12690. In some examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 5-622, 4175-4181, or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 5-188, 191-622, 4175-4181, or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 559, 606, or 4178-4181. In other examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 1935-4168, 4182, 4183, 12646-12654, or 12664-12671. In some examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 1935-1937, 2849, 2858, 2864, 2874, 3510, 12647, 12648, or 12664-12671. In other examples, the nucleotide sequence of the ASO or AR comprises any one of the sequences of SEQ ID NO: 623-1934, 4169-4174, 12645, 12655-12663, or 12688. In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of SEQ ID NO: 1934.

[0058] In some examples, the nucleotide sequence of the ASO or AR consists of any one of the nucleotide sequences of SEQ ID NO: 5-4186, 12646-12654, or 12664-12671. The sequences for the foregoing SEQ ID NOs are provided in Tables 4 and 5 of the Appendix.

[0059] ASO Chemistry and Modifications The ASOs used in the compositions described herein can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination thereof. The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having a modified or substituted sugar moiety and / or a modified backbone. In some instances, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the compositions and methods described herein are known in the art, such as those disclosed in U.S. Patent No. 8,258,109, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Roberts et al., 2020, Nature Rev. Drug Disc., 19:673-694.

[0060] One or more nucleotides of the ASO can be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, uracil, and inosine, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase to be able to hydrogen bond to a nucleobase present on the target RNA transcript. Examples of suitable modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0061] An ASO contains a "backbone" structure that refers to the connection between nucleotides / monomers of the ASO. In naturally occurring oligonucleotides, the backbone contains 3'-5' phosphodiester linkages that connect the sugar moieties of adjacent nucleotides. Suitable types of backbone linkages for the ASOs described herein include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphorodiamidate, phosphorosenoate, phosphorodiselenoate, phosphoranilidate, phosphoramidate, and the like. In some examples, the backbone modification is a phosphorothioate linkage. In other examples, the backbone modification is a phosphorodiamidate linkage. See, for example, Roberts et al. (supra), and Agrawal (2021), Biomedicines, 9:503. In some examples, the backbone structure of the ASO does not contain phosphorus-based linkages, but rather contains peptide bonds, for example, in peptide nucleic acid (PNA), or in linkers containing carbamate, amide, and linear and cyclic hydrocarbon groups.

[0062] In some examples, the stereochemistry at each internucleotide linkage of the ASO backbone is random. In other examples, the stereochemistry at each internucleotide linkage of the ASO backbone is controlled and not random. For example, U.S. Patent No. 9,605,019 describes a method for independently selecting the chirality of the chirality at each phosphorus atom in an oligonucleotide. In some examples, the ASOs used in the compositions and methods provided herein include, but are not limited to, the sequences of the ASOs disclosed herein as SEQ ID NOs: 5-39535, and are ASOs having non-random phosphodiester internucleotide linkages. In some examples, the compositions or compositions used in the methods disclosed herein include pure diastereomeric ASOs. In other examples, the composition includes ASOs having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% - about 100%, about 91% - about 100%, about 92% - about 100%, about 93% - about 100%, about 94% - about 100%, about 95% - about 100%, about 96% - about 100%, about 97% - about 100%, about 98% - about 100%, or about 99% - about 100%.

[0063] In some examples, the ASO has a non-random mixture of Rp and Sp configurations at its internucleotide linkages. In some examples, the ASOs used in the compositions and methods disclosed herein include about 5 - 100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp, or include about 100% Rp.

[0064] In some examples, the ASOs described herein include a sugar moiety that includes ribose or deoxyribose, or a modified sugar moiety or sugar analog, that includes a morpholine ring. Suitable examples of modified sugar moieties include 2'-O-modifications, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminoethyl, 2'-F, N3'->P5' phosphoramidate, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars, such as 2'-substitutions, but are not limited thereto. In some examples, the sugar moiety modification is selected from among 2'-O-Me, 2'-F, and 2'-MOE. In other examples, the sugar moiety modification is an extra cross-link, such as a locked nucleic acid (LNA). In some examples, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some examples, the sugar moiety includes a ribofuranosyl or 2'-deoxyribofuranosyl modification. In some examples, the sugar moiety includes a 2',4'-constrained 2'-O-methyloxyethyl (cMOE) modification. In some examples, the sugar moiety includes a cEt 2',4'-constrained 2'-O-ethyl BNA modification. In other examples, the sugar moiety includes a tricyclic DNA (tcDNA) modification. In some examples, the sugar moiety includes an ethylene nucleic acid (ENA) modification. In some examples, the sugar moiety includes 2'-O-(2-N-methylcarbamoylethyl) (MCE). Modifications are known in the art as exemplified by Jarver, et al., 2014, Nucleic Acid Therapeutics, 24(1):37-47.

[0065] In some examples, each constituent nucleotide of the ASO is modified in the same manner, e.g., all linkages of the ASO backbone include phosphorothioate linkages, or each ribose sugar moiety includes a 2'-O-methyl modification. In other examples, combinations of different modifications, such as ASOs that include a combination of phosphoramidate linkages that include a morpholine ring (morpholino) and sugar moieties, are used.

[0066] In some examples, the ASO includes one or more backbone modifications. In some examples, the ASO includes one or more sugar moiety modifications. In some examples, the ASO includes one or more backbone modifications and one or more sugar moiety modifications. In some examples, the ASO includes a 2’MOE modification and a phosphorothioate backbone. In some examples, the ASO includes a peptide nucleic acid (PNA). In some preferred examples, the ASO includes a phosphorodiamidate morpholino (PMO).

[0067] One of ordinary skill in the art will understand that the ASO can be modified to achieve the desired properties or activities of the ASO or to reduce the undesired properties or activities of the ASO. In some examples, the ASO is modified to alter one or more properties. For example, such modifications can enhance the binding affinity to the target sequence on the pre-mRNA transcript, reduce binding to any non-target sequences, reduce degradation by cellular nucleases (e.g., RNase H), improve the uptake of the ASO into cells and / or specific subcellular compartments, modify the pharmacokinetics or pharmacodynamics of the ASO, and / or regulate the half-life of the ASO in vivo.

[0068] In some examples, the ASO includes one or more 2’-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides, which have been shown to significantly enhance the resistance of the ASO to nuclease degradation and increase bioavailability.

[0069] Methods for the synthesis and chemical modification of ASOs, as well as for the synthesis of ASO conjugates, are well known in the art and such ASOs are commercially available.

[0070] In some examples, the compositions provided herein (e.g., pharmaceutical compositions) include two or more ASOs that have different chemistries but are complementary to the same targeting portion of the SHANK3 mRNA 5’UTR or 3’UTR, or the SHANK3 pre-mRNA 5’PNCR. In other examples, the composition includes two or more ASOs that are complementary to different targeting portions of the 5’UTR, 3’UTR, or 5’PNCR.

[0071] In some examples, the compositions disclosed herein include an ASO linked to a functional moiety. In some examples, the functional moiety is a delivery moiety, a targeting moiety, a detection moiety, a stabilizing moiety, or a therapeutic moiety. In some examples, the functional moiety includes a delivery moiety or a targeting moiety. In some examples, the functional moiety includes a stabilizing moiety. In some preferred examples, the functional moiety is a delivery moiety.

[0072] Suitable delivery moieties include, but are not limited to, lipids, polyethers, peptides, carbohydrates, glycans, receptor binding domains (RBDs), and antibodies.

[0073] In some examples, the delivery moiety includes a cell-penetrating peptide (CPP). Suitable examples of CPPs are described, for example, in PCT / AU2020 / 051397. In some examples, the amino acid sequence of the CPP includes or consists of RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 12694). In other examples, the delivery moiety includes an RBD.

[0074] In other examples, the delivery moiety includes a carbohydrate. In some examples, the carbohydrate delivery moiety is selected from N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), glycans, and mannose. In one example, the carbohydrate delivery moiety includes GalNac or a glycan moiety.

[0075] In other examples, the delivery moiety includes a lipid. Examples of suitable lipids as the delivery moiety include, but are not limited to, cholesterol moieties, cholesteryl moieties, and aliphatic lipids. In some examples, the delivery moiety includes a fatty acid or a lipid moiety. In some embodiments, the fatty acid chain length is about C8-C20. Examples of suitable fatty acid moieties and their conjugation to oligonucleotides can be found, for example, in International Patent Publication No. WO 2019 / 232255 and Prakash et al., (2019).

[0076] In a further example, the delivery moiety includes an antibody as described, for example, in Dugal-Tessier et al., (2021).

[0077] Suitable examples of the stabilizing moiety include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and poly(2-oxazoline) (POx).

[0078] In some examples, when the ASO is linked to the functional moiety, the functional moiety is covalently linked to the ASO. In other examples, the functional moiety is non-covalently linked to the ASO.

[0079] Functional moieties are understood in the art and can be linked to one or more of any of the nucleotides in the ASO at any of several positions on the sugar, base, or phosphate group, for example, using a linker as described in the literature. The linker can include a divalent or trivalent branched-chain linker. In some examples, the functional moiety is linked to the 5' end of the ASO. In other examples, the functional moiety is linked to the 3' end of the ASO.

[0080] In some examples, a composition comprising any of the ASOs disclosed herein also comprises a delivery nanocarrier complexed with the ASO. In some examples, the delivery nanocarrier is selected from lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In other examples, the delivery nanocarrier comprises lipid nanoparticles encapsulating the ASO. Various delivery ASO-nanocarrier complex formats are known in the art, for example, as outlined in Roberts et al. (supra).

[0081] Vector for expression of SHANK3 antisense RNA (AR) In some examples, provided herein is a vector for expression of an antisense RNA (AR) that binds within a targeting portion of (i) the 5’UTR of SHANK3 mRNA, (ii) the 5’PNCR of SHANK3 pre-mRNA, or (iii) the 3’UTR of SHANK3 mRNA, in mammalian neurons or other cell types, whereby binding of the AR within the targeting portion in mammalian cells results in increased levels of SHANK3 protein in mammalian cells.

[0082] In some examples, the promoter used in the expression vector is a neuron-type selective promoter for driving expression of the AR in mammalian cells. In some examples, the neuron cell-type selective promoter is selective for expression in neurons selected from the list consisting of cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons.

[0083] In some cases, the promoter is an inducible promoter, which is induced by a ligand-regulated transactivator, such as tet-inducible rtTA, and enables titration of AR transcription in target mammalian cells. In some examples, the promoter driving AR expression is a U6 or other Pol III promoter, which is particularly suitable for the transcription of short RNA sequences such as the AR sequences disclosed herein. In some examples, the expression vector utilizes a hybrid promoter system, such as the Tet-O-regulated U6 promoter system described in Lin et al. (2004), FEBS Letters, 577(2004)376-380. In some examples, when both cell-type specificity and inducibility of the AR expression vector are desired, a two-part expression system is used in which the expression of the ligand-regulated transactivator is driven by a cell-type selective promoter and the expression of the AR disclosed herein is driven by a promoter regulated by the ligand-regulated transactivator.

[0084] In some examples, the expression vector used in the compositions disclosed herein is a non-viral expression vector, such as a plasmid vector, a minicircle DNA vector, a linear amplicon expression cassette, etc.

[0085] In some cases, the composition containing the non-viral expression virus further comprises a transfection agent. Exemplary transfection agents for transfection include, but are not limited to, jet-PEI® (available from Polyplus-transfection® SA, Strasbourg, France), TurboFect in vivo Transfection Reagent (ThermoFisher), and cationic derivatives of polyisoprenoid alcohols (PTAI) such as those described in Rak et al., (2016).

[0086] In other examples, the expression vector used is a viral vector, i.e., a non-replicating recombinant virus suitable for the expression of AR disclosed herein.

[0087] Preferably, the recombinant virus for the expression of SHANK3 AR is a DNA virus. Suitable types of DNA viruses include adeno-associated virus (AAV), adenovirus, lentivirus, herpes simplex virus (HSV), and anellovirus. Methods for the design, production, and use of such types of recombinant DNA viruses are well-established in the art as exemplified for adenovirus by Fukazawa et al., (2010), International J of Mol.Med, 25(1), 3-10, and “Gene Therapy Protocols”, for AAV by “Adeno-Associated Virus: Methods and Protocols”, for HSV by Cody et al(2013), Journal of Genetic Syndromes & Gene Therapy, 4(1), 126, and “Herpes Simplex Virus: Methods and Protocols”, for lentivirus by “Gene Therapy Protocols Vol.1: Production and In Vivo Applications of Gene Transfer Vectors”, and Merten et al.(2016), Molecular Therapy-Methods & Clinical Development, 3, 16017, and Emeagi et al.(2013), Current Molecular Medicine 13(4), 602-625. In some preferred examples, the viral vector is recombinant AAV.

[0088] Genetically modified cells Genetically modified cells are also provided herein. In some examples, the genetically modified cells are genetically modified bacterial cells (e.g., recombinant E. coli for amplifying the AR expression vectors disclosed herein). In other examples, the genetically modified cells are genetically modified mammalian cells transfected with either an ASO or a non-viral AR expression vector, or transduced with any of the viral AR expression vectors disclosed herein. In some examples, the genetically modified mammalian cells are ex vivo, e.g., as a cultured cell population. In other examples, the genetically modified mammalian cells are in vivo, e.g., in a mouse. In some examples, the genetically modified mammalian cells are human cells.

[0089] In some embodiments, the genetically modified mammalian cells are neurons or neural progenitor cells. Suitable examples of neurons include, but are not limited to, cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons. In some examples, such primary cell types can be obtained by differentiation of human pluripotent stem cell lines, e.g., hiPSC lines or human embryonic stem cell (hESC) lines. Methods for obtaining various different neuronal cell types are known in the art, as outlined, for example, in Alia et al., (2019), Fitzgerald et al., (2020) and Kim et al. (2014). In other examples, the genetically modified mammalian cells are derived from cell lines. In some examples, the cell lines are pluripotent stem cell lines (e.g., hiPSC or hESC) or neuronal cell lines. Suitable neuronal or neural stem cell lines include, but are not limited to, SH-SY5Y, NTera, CTX0E16, ReNcell VM, ReNcell Cx. In some preferred examples, the genetically modified mammalian cells endogenously express SHANK3.

[0090] The genetically modified cells disclosed herein can be genetically modified by any of several methods and strategies known in the art, such as transient transfection, stable transfection, and viral transduction. In some examples, transfection by ASO or non-viral vectors is carried out by nucleofection. In other examples, transfection of cells is by lipofection.

[0091] Pharmaceutical composition Also provided herein are pharmaceutical compositions comprising any of the foregoing ASOs, non-viral expression vectors, and viral expression vectors disclosed herein, formulated with at least a pharmaceutically acceptable excipient including a carrier, filler, preservative, adjuvant, solubilizer and / or diluent.

[0092] For use in the methods disclosed herein, pharmaceutical compositions containing any of the ASO or expression vector compositions described herein are well known in the pharmaceutical industry and can be prepared according to conventional techniques described in the published literature. In some examples, a pharmaceutical composition for treating a subject comprises a therapeutically effective amount of any ASO or expression vector disclosed herein.

[0093] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without producing excessive toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylic acids, sulfuric acid, phosphoric acid, nitric acid, lower alkylsulfonic acids, and arylsulfonic acids.

[0094] In some examples, the pharmaceutical composition is formulated into any of several possible routes of administration or dosage forms, including but not limited to intravenous administration, intrathecal administration, tablets, capsules, gel capsules, liquid syrups, and soft gels. In some examples, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. An aqueous suspension may further contain substances that increase the viscosity of the suspension, such as, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer. In some examples, the pharmaceutical formulations disclosed herein are provided in forms including, but not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0095] In some examples, a pharmaceutical formulation comprising any of the ASOs or expression vectors described herein may be appropriate and may contain one or more permeation enhancers, carriers, excipients, or other active or inactive ingredients as known to those skilled in the art. In some examples, when the pharmaceutical composition contains liposomes, such liposomes may also include sterically stabilized liposomes, e.g., liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with an extended circulation lifetime. In some examples, sterically stabilized liposomes contain one or more glycolipids or are derivatized with one or more hydrophilic polymers, e.g., PEG moieties. In some examples, a surfactant is included in the pharmaceutical formulation.

[0096] In some examples, the pharmaceutical composition also includes a permeation enhancer to enhance the delivery of the ASO or non-viral expression vector, e.g., to assist in diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In some examples, the permeation enhancer includes a surfactant, fatty acid, bile salt, or chelating agent.

[0097] In some examples, when administration is via a systemic route, such as an intravenous route, the method also includes the step of facilitating the movement of any of the ASOs or vectors described herein across the blood-brain barrier (BBB) into the CNS, particularly the brain. In some examples, the BBB is transiently disrupted by administration of one or more antibodies that disrupt the binding of Netrin-1 to Unc5B, as described, for example, in Boye et al., (2022).

[0098] In some examples, the pharmaceutical composition comprises an ASO or non-viral vector at a dosage in the range of about 0.01 mg / kg to 20 mg / kg, such as 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, or another dosage in the range of about 0.01 mg / kg to 20 mg / kg. In some examples, when the ASO disclosed herein is administered directly to the CNS or brain, for example, by intracerebroventricular administration, the total dosage is in the range of about 50 mg to about 500 mg, such as 60 mg, 70 mg, 80 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 220 mg, 250 mg, 270 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg, or another dosage in the range of about 50 mg to about 500 mg. This dosage range corresponds to approximately 0.050 mg / cm 3 of average human brain volume, and is approximately 0.050 mg / cm 3 to about 0.42 mg / cm 3 of brain volume.

[0099] In some examples, the pharmaceutical composition comprises multiple ASOs or AR expression vectors. In some examples, the pharmaceutical composition comprises, in addition to the ASO or AR expression vector, another drug or therapeutic agent suitable for the treatment of a subject suffering from SHANK3 haploinsufficiency.

[0100] Method As described herein, several conditions (e.g., Phelan-McDermid syndrome) are associated with insufficient levels of functional SHANK3. Accordingly, the methods described herein include methods for preventing or treating a condition associated with SHANK3 haploinsufficiency by administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising any of the ASOs or expression vectors disclosed herein. Similarly, in some instances, any of the ASOs or AR expression vectors disclosed herein are used in the manufacture of a medicament for treating a condition associated with SHANK3 haploinsufficiency. In some instances, the conditions associated with SHANK3 haploinsufficiency that are prevented or treated by the methods or compositions disclosed herein are Phelan-McDermid syndrome, autism spectrum disorder, schizophrenia, or intellectual disability. In some preferred instances, the condition is Phelan-McDermid syndrome.

[0101] Methods are also provided herein for increasing the amount of functional SHANK3 protein in mammalian cells expressing SHANK3 mRNA, the method comprising contacting the cells with any of the ASOs or expression vectors disclosed herein.

[0102] In some instances, administration of any of the ASOs, AR expression vectors, or pharmaceutical compositions disclosed herein to a subject, or contact thereof with cells, in vitro or ex vivo increases the level of SHANK3 protein in the cells (e.g., neurons) by about 1.1- to about 5-fold, e.g., 1.2-fold, 1.3-fold, 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold, 2.7-fold, 3-fold, 3.3-fold, 3.5-fold, 4-fold, 4.3-fold, 4.5-fold, 4.7-fold, or another increase in SHANK3 protein level of about 1.1- to about 5-fold in the subject's cells or in vitro or in vivo.

[0103] Suitable routes of administration for the compositions, pharmaceutical compositions, or treatments by the medicaments disclosed herein include, but are not limited to, intravenous, intraarterial, intrasubstantial, intracerebroventricular, intracisternal, intrathecal, intravenous, intraarterial, subcutaneous, and topical.

[0104] As will be appreciated by those skilled in the art, the treatment methods disclosed herein involve administering to a subject (e.g., a human subject) a therapeutically effective amount of the compositions and pharmaceutical compositions disclosed herein. The terms "effective amount" or "therapeutically effective amount" as used herein refer to an amount of the disclosed ASO, non-viral or viral expression vector administered such that a sufficient amount alleviates to some extent one or more of the symptoms and / or clinical indicators associated with SHANK3 haploinsufficiency in a particular disease or health condition. In some instances, the "effective amount" for therapeutic use is the amount of one of the aforementioned agents necessary to bring about a clinically significant decrease in disease symptoms to prevent disease symptoms without undue adverse side effects. Examples of suitable symptoms alleviated by the treatment methods provided herein include, but are not limited to, seizures, anxiety, repetitive behaviors, learning and memory impairments, and social impairments. The appropriate "effective amount" in any individual case can be determined using techniques such as dose escalation studies. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. The "effective amount" or "therapeutically effective amount" can vary depending on the subject, due to factors such as any age, weight, general condition, condition being treated, severity of the condition being treated, and variability in the metabolism of the compound as determined by the prescribing physician. By way of mere example, the therapeutically effective amount can be determined by routine experimentation, including but not limited to dose escalation clinical trials. When two or more therapeutic agents are used in combination, the "therapeutically effective amount" of each therapeutic agent can refer to the amount of the therapeutic agent that would be therapeutically effective when used alone, or to a decreased amount that is therapeutically effective in combination with one or more additional therapeutic agents.

[0105] Combination therapy A pharmaceutical composition comprising any of the ASO or AR expression vectors disclosed herein can also be used in combination with other therapeutically valuable agents in the treatment of conditions associated with SHANK3 haploinsufficiency. Generally, the other agents need not necessarily be administered within the same pharmaceutical composition and, due to their different physical and chemical properties, can preferably be administered by different routes. If possible, the determination of the mode of administration and suitability for administration within the same pharmaceutical composition is well within the knowledge of those skilled in the art. The initial administration can be carried out according to established protocols known in the art, and then, based on the observed effects, the dosage, mode of administration and time of administration can be changed by a skilled clinician.

[0106] Compositions and pharmaceutical compositions comprising an ASO and / or expression vector, and additional therapeutic agents, can be administered concurrently (e.g., simultaneously, essentially simultaneously, or within the same treatment protocol), or sequentially, depending on the stage and progression of the SHANK3 haploinsufficiency-related condition being treated, the condition of the patient, and the selection of the particular therapeutic agent being used. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during the treatment protocol, are well within the knowledge of a skilled physician following evaluation of the disease being treated and the condition of the patient.

[0107] It is known to those skilled in the art that the therapeutically effective dosage can vary when the drug is used in a combination therapy. Methods for experimentally determining the therapeutically effective dosages of drugs and other agents for use in combination therapy regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses to minimize toxic side effects, is widely described in the literature. Combination therapy further includes periodic treatments that are started and stopped at various times to assist in the clinical management of the patient.

[0108] In the case of combination therapy, the dosages of the co-administered therapeutic agents will of course vary depending on the type of co-drug utilized, the ASO or expression vector, and the stage of the patient being treated.

[0109] The pharmaceutical compositions comprising an ASO, an AR, or an expression vector, and additional therapeutic agents constituting the combination therapies disclosed herein may be in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical compositions constituting the combination therapies may be administered continuously while any of the therapeutic agents is being administered according to a regimen that requires two-step administration. This two-step administration regimen may require sequential administration of the active agent or spaced administration of separate active agents. The period between multiple administration steps may range from minutes to hours depending on the characteristics of each pharmaceutical, such as the efficacy, solubility, bioavailability, plasma half-life, and kinetic profile of the pharmaceutical. Also, circadian variations in various physiological parameters may be evaluated to determine the optimal dosing interval.

[0110] Examples of suitable therapeutic agents for co-administration with the compositions or pharmaceutical compositions disclosed herein include, but are not limited to, growth hormone, insulin-like growth factor-1, risperidone, lumateperone, sodium valproate, lithium, and D-serine.

Example

[0111] Example 1: Identification of SHANK3 Target Sequences Identification of annotated and expressed transcripts capable of generating the canonical transcript of the SHANK3 gene was performed by sequence alignment of all SHANK3 protein-coding and NMD transcripts described in Gencode v38. 25mer and 17mer ASO sequences “micro-walked” every three bases across the 5’ PNCR of the canonical pre-RNA transcript of SHANK3 and across the 5’ UTR and 3’ UTR of the canonical mRNA transcript of SHANK3 (ENST00000262795.6). The resulting ASO sequences correspond to SEQ ID NOs: 5-4186, 12646-12671, and 12688 provided in Table 4 (Appendix).

[0112] Example 2: Measurement of upregulation of SHANK3 protein by PMO targeting SHANK3 mRNA 5’UTR or 3’UTR in neuronal cell lines A subset of the ASO sequences identified above was synthesized as phosphorodiamidate morpholino oligonucleotides (PMO4 - 37) targeting the standard SHANK3 mRNA 5’UTR, 5’PNCR, and 3’UTR regions. The PMOs, corresponding SEQ ID numbers, and target regions are shown in Table 1 below. Antisense PMOs were electroporated into SH - SY5Y neuronal cell line cultures at concentrations of 25 μM and 50 μM using the NEON® electroporation system (ThermoFisher) and incubated for 96 hours. At this point, total protein was extracted using 15% SDS complete lysis reagent, and the level of SHANK3 protein was evaluated by Western blot using a 1:1000 dilution in 5% BSA in TBST buffer with a mouse anti - SHANK3 monoclonal antibody (Merck, catalog number SAB520004), followed by a goat anti - mouse IgG H&L antibody (Abcam, catalog number ab216776). The expression level of SHANK3 protein was compared to cells not transfected with PMO (untreated), a control treated under transfection conditions, and a scrambled / non - targeting control sequence. The average signal from SHANK3 after image analysis was normalized to the average signals of total protein and “housekeeping gene” proteins (e.g., vinculin, beclin, SRSF4).

Table 1 - 1

Table 1 - 2

[0113] As shown in FIGS. 1, 4, and 5, some of the tested PMOs induced increased SHANK3 protein levels compared to the control, while other PMOs were unable to induce changes in SHANK3 protein levels. As shown in FIGS. 2 and 6, a subset of PMOs increased SHANK3 protein levels from about 50% higher than the control to more than four-fold.

[0114] Example 3: Measurement of SHANK3 Protein Upregulation by MOE Oligonucleotides Targeting SHANK3 mRNA 5’UTR or 3’UTR in Neuronal Cell Lines A subset of the ASO sequences identified above was synthesized as 2’-O-(2-methoxyethyl) (MOE)-modified oligonucleotides, which also contained a fully phosphorothioated backbone (「MOE」1 - 6, 13 - 60) targeting the standard SHANK3 mRNA 5’UTR, 5’PNCR, and 3’UTR regions. The numbered MOEs, corresponding SEQ ID NOs, and target regions are shown in Table 2 below. Antisense MOEs were electroporated into SH-SY5Y neuronal cell line cultures at concentrations of 2.5 μM and 5.0 μM using the NEON® electroporation system (ThermoFisher) and incubated for 96 hours. At this point, total protein was extracted using 15% SDS complete lysis reagent, and the level of SHANK3 protein was evaluated by Western blot using a 1:1000 dilution in 5% BSA in TBST buffer with a mouse anti-SHANK3 monoclonal antibody (Merck, catalog number SAB520004), followed by a goat anti-mouse IgG H&L antibody (Abcam, catalog number ab216776). The expression level of SHANK3 protein was compared to cells not transfected with MOE (untreated), a control treated under transfection conditions, and a scrambled / non-targeting control sequence. The average signal from SHANK3 after image analysis was normalized to the average signal of total protein and 「housekeeping gene」 proteins (e.g., vinculin, beclin, SRSF4).

Table 2-1

Table 2-2

[0115] As shown in FIGS. 10, 11, and 12, some of the tested MOEs induced increased SHANK3 protein levels compared to the control, while other MOEs were unable to induce changes in SHANK3 protein levels. As shown in FIG. 13, a subset of MOEs increased SHANK3 protein levels from about 50% higher than the control to more than four-fold.

[0116] Example 4: Identification of SHANK3 Target Intron / Exon Sequences Identification of annotated and expressed transcripts capable of generating the canonical transcript of the SHANK3 gene was performed by sequence alignment of all SHANK3 protein-coding and NMD transcripts described in Gencode v38. A 25-mer ASO sequence “micro-walk” was performed at 5-bp increments of distance across the sequences of introns 7, 17, 21, and exon 21 of the ENST00000262795.6 pre-mRNA transcript, designed to mediate exclusion of retained introns or portions thereof and target intron splice enhancer motifs to generate productive SHANK3 mRNA transcripts. The resulting ASO sequences correspond to SEQ ID NOs: 4187 to 12644.

[0117] Example 5: Measurement of SHANK3 Protein Upregulation by PMOs Targeting SHANK3 Pre-mRNA in Neuronal Cell Lines The 25-nucleotide length of the ASO sequence identified above is synthesized as a phosphorodiamidate morpholino oligonucleotide (PMO). Antisense PMOs targeting intron 7, 18, or 21 (as described in Example 1) are electroporated into naive SH-SY5Y neuronal cell line cultures at concentrations of 25 μM and 50 μM using a NEON® electroporation system (ThermoFisher) and incubated for 96 hours. At this point, total protein is extracted using 15% SDS complete lysis reagent, and the level of SHANK3 protein is evaluated by Western blot using a mouse anti-SHANK3 monoclonal antibody (Merck, catalog number SAB520004) at a 1:1000 dilution in 5% BSA in TBST buffer, followed by a goat anti-mouse IgG H&L antibody (Abcam, catalog number ab216776). The expression level of the SHANK3 protein is compared to non-PMO transfected cells (UT). [Table 3]

[0118] As shown in Figures 7 and 8, some of the PMOs tested induced increased SHANK3 protein levels compared to the control, while other PMOs were unable to induce a change in SHANK3 protein levels.

[0119] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, this embodiment is to be considered in all respects as illustrative and not restrictive.

[0120] All publications cited in this specification are hereby incorporated by reference in their entirety. When reference is made to a URL or other such identifier or address, such identifier may change and specific information on the Internet may come and go, but it is understood that equivalent information can be found by searching the Internet. Such reference attests to the availability and public dissemination of such information.

[0121] Any description of documents, acts, materials, devices, articles, etc. contained in this specification is for the sole purpose of providing context for the present invention. It is not to be regarded as an admission that any or all of these matters formed part of the basis of the prior art or were common general knowledge in the field related to the present invention as they existed prior to the priority date of each claim of this application.

[0122] References Alia et al.,(2019),Frontiers in Neuroscience,13:684. Boye et al.,(2022),Nature Communications,13:1169.doi.org / 10.1038 / s41467-022-28785-9. Dugal-Tessier et al.,(2021),J Clin Med.,10(4):838. Fitzgerald et al.,(2020),Stem Cells,38(11):1375-1386. Kim et al.,(2014),Frontiers in Neuroscience,8:109. Prakash et al.,(2019),Nucleic Acids Research,47(12):6029-6044. Rak et al.,(2016),J Gene Med,18(11-12):331-342.

[0123] Appendix: Sequences and Sequence Numbers Array number 1 SHANK3 mRNA standard transcript 5'UTR sequence (SHANK3-201 ENST00000262795.7) Accession number 2 SHANK3 mRNA canonical transcript 3’UTR sequence (SHANK3-201 ENST00000262795.7) Accession number 3 SHANK3 5’PNCR of SHANK3 pre-RNA (SHANK3-201 ENST00000262795.7) Array number 4 SHANK3 pre-mRNA canonical sequence (SHANK3-201 ENST00000262795.7)

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

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Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

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Table 4-28

Table 4-29

Table 4-30

Table 4-31

Table 4-32

Table 4-33

Table 4-34

Table 4-35

Table 4-36

Table 4-37

Table 4-38

Table 4-39

Table 4-40

Table 4-41

Table 4-42

Table 4-43

Table 4-44

Table 4-45

Table 4-46

Table 4-47

Table 4-48

Table 4-49

Table 4-50

Table 4-51

Table 4-52

Table 4-53

Table 4-54

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Table 4-56

Table 4-57

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Table 4-59

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Table 4-61

Table 4-62

Table 4-63

Table 4-64

Table 4-65

Table 4-66

Table 4-67

Table 4-68

Table 4-69

Table 4-70

Table 4-71

Table 4-72

Table 4-73

Table 4-74

Table 4-75

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Table 4-77

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Table 4-79

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Table 4-83

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Table 4-85

Table 4-86

Table 4-87

Table 4-88

Table 4-89

Table 4-90

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Table 4-95

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Table 4-99

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Table 4-101

Table 4-102

Table 4-103

Table 5

[0124] Sequence number 12694 CPP amino acid sequence (artificial / synthetic) RRSRTARAGRPGRNSSRPSAPR

[0125] The sequence listing of this application is provided in five separate attached.xml format files, the entire contents of each of which are incorporated herein by reference. The arrays listing the.xml file names, the sequence numbering ranges (WIPO Sequence Program autogenerated), the corresponding applicant-assigned sequence numbering ranges for each of these, and the first sequence within each file, are summarized in Table 6 below.

Table 6

[0126] If any discrepancies are identified between (i) the sequences, numberings corresponding to the sequence numbers, or corresponding headings / names provided in the attached sequence listing files, and (ii) the sequences and sequence numbers disclosed in this specification and its appendices, the sequences and sequence numbers (and sequence number ranges) provided in this specification and appendices shall be accepted as accurate.

Claims

1. An antisense oligonucleotide that binds within a targeting portion of (i) the 5′ untranslated region (UTR) of SHANK3 mRNA, (ii) the 5′ proximal non-coding region (PNCR) of SHANK3 pre-mRNA, or (iii) the 3′ UTR of SHANK3 mRNA, wherein binding of the antisense oligonucleotide within the targeting portion in mammalian cells results in an increased level of SHANK3 protein in the mammalian cells.

2. A vector for expression of an antisense RNA (AR) that binds within a targeting portion of (i) the 5′ untranslated region (UTR) of SHANK3 mRNA, (ii) the 5′ proximal non-coding region (PNCR) of SHANK3 pre-mRNA, or (iii) the 3′ UTR of SHANK3 mRNA, in mammalian neurons, wherein binding of the AR within the targeting portion in mammalian cells results in an increased level of SHANK3 protein in the mammalian cells.

3. The vector according to claim 2, wherein the mammalian cell is a neuron.

4. The vector according to claim 3, wherein the vector comprises a neuron-selective promoter for driving expression of the antisense RNA in the mammalian neuron.

5. The vector according to claim 4, wherein the neuron-selective promoter is selective for expression in a neuron type selected from the list consisting of cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons.

6. The vector according to any one of claims 2 to 5, wherein the vector comprises an inducible promoter.

7. The vector according to any one of claims 2 to 6, wherein the vector is a viral vector.

8. The vector according to claim 7, wherein the viral vector is a recombinant virus selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and anellovirus.

9. The vector according to any one of claims 2 to 8, wherein the vector is a non-viral vector.

10. A composition comprising the non-viral vector according to claim 9, wherein the composition further comprises a transfection agent.

11. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide comprises a backbone modification.

12. The method or antisense oligonucleotide according to claim 11, wherein the antisense oligonucleotide comprises a backbone modification including a phosphorothioate bond or a phosphorodiamidate bond.

13. The antisense oligonucleotide according to claim 11 or 12, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-fluoro, or a 2'-O-methoxyethyl moiety.

14. The antisense oligonucleotide according to any one of claims 11 to 13, wherein the antisense oligonucleotide comprises at least one modified sugar moiety.

15. The antisense oligonucleotide according to claim 14, wherein each sugar moiety in the antisense oligonucleotide is a modified sugar moiety.

16. The antisense oligonucleotide according to any one of claims 11 to 15, wherein the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

17. The antisense oligonucleotide according to claim 16, wherein each nucleotide of the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

18. The antisense oligonucleotide according to claim 1 or any one of claims 11 to 17, or the vector according to any one of claims 2 to 9, or the composition according to claim 10, wherein the antisense oligonucleotide or the nucleotide sequence of the AR consists of 10 to 50 nucleotides, 15 to 40 nucleotides, 18 to 40 nucleotides, 17 to 25 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 22 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, or 26 to 30 nucleotides.

19. The antisense oligonucleotide according to claim 18, wherein the nucleotide sequence of the antisense oligonucleotide or the AR consists of 17 to 30 nucleotides.

20. The antisense oligonucleotide according to claim 19, wherein the antisense oligonucleotide comprises one or more phosphorodiamidate morpholino moieties.

21. The antisense oligonucleotide according to any one of claims 1 or 11 to 20, wherein the antisense oligonucleotide is linked to a functional moiety.

22. The antisense oligonucleotide according to claim 21, wherein the functional moiety comprises a delivery moiety.

23. The antisense oligonucleotide according to claim 22, wherein the delivery moiety is selected from the group consisting of lipids, polyethers, peptides, carbohydrates, receptor binding domains (RBDs), and antibodies.

24. The antisense oligonucleotide according to claim 22 or 23, wherein the delivery moiety comprises a cell-penetrating peptide (CPP).

25. The antisense oligonucleotide according to claim 22 or 23, wherein the delivery moiety comprises an N-acetylgalactosamine (GalNAc) moiety or a glycan moiety.

26. The antisense oligonucleotide according to claim 22 or 23, wherein the delivery moiety comprises a fatty acid or a lipid moiety.

27. The antisense oligonucleotide according to claim 26, wherein the chain length of the fatty acid is about C8 - C20.

28. The antisense oligonucleotide according to claim 21, wherein the functional moiety comprises a stabilizing moiety.

29. The antisense oligonucleotide according to any one of claims 21 to 28, wherein the functional moiety is covalently linked to the antisense oligonucleotide.

30. The antisense oligonucleotide according to any one of claims 21 to 28, wherein the functional moiety is non-covalently linked to the antisense oligonucleotide.

31. The antisense oligonucleotide according to any one of claims 21 to 30, wherein the functional moiety is linked to the 5'-end of the antisense oligonucleotide.

32. The antisense oligonucleotide according to any one of claims 21 to 30, wherein the functional moiety is linked to the 3'-end of the antisense oligonucleotide.

33. The nucleotide sequence of the antisense oligonucleotide or the AR is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the nucleotide sequence of the targeting portion over the length of the antisense oligonucleotide or the AR. The antisense oligonucleotide, vector, or composition according to any one of claims 1 to 32.

34. The nucleotide sequence of the ASO or AR corresponds to any one of SEQ ID NOs: 293, 299, 301, 302, 304 - 309, 311, 313, 315, 318, 606, 797, 1193, 1195, 1847, 1934 - 1937, 2858, 2874, 3510, 12644, 12666, 12669, 12671, 12688, or 12690. The antisense oligonucleotide according to any one of claims 1 or 11 to 33, the vector according to any one of claims 2 to 9, or the composition according to claim 10.

35. The binding is within the targeting portion of the 5'UTR corresponding to SEQ ID NO:

1. The antisense oligonucleotide according to any one of claims 1 or 11 to 33, the vector according to any one of claims 2 to 9, or the composition according to claim 10.

36. The binding is within the targeting portion of the 5'PNCR corresponding to SEQ ID NO:

3. The antisense oligonucleotide according to any one of claims 1 or 11 to 33, the vector according to any one of claims 2 to 9, or the composition according to claim 10.

37. The binding is within the targeting portion of the 3'UTR corresponding to SEQ ID NO:

2. The antisense oligonucleotide according to any one of claims 1 or 11 to 33, the vector according to any one of claims 2 to 9, or the composition according to claim 10.

38. The nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of SEQ ID NOs: 5 - 622, 4175 - 4181, or 4184 - 4186. The antisense oligonucleotide, vector, or composition according to claim 35.

39. The antisense oligonucleotide or the nucleotide sequence of the AR corresponds to any one of SEQ ID NOs: 559, 606, or 4178 to 4181, the antisense oligonucleotide, vector, or composition according to claim 35.

40. The nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of SEQ ID NOs: 1935 to 4168, 4182, 4183, 12646 to 12654, or 12664 to 12671, the antisense oligonucleotide, vector, or composition according to claim 36.

41. The nucleotide sequence of the antisense oligonucleotide or the AR corresponds to any one of SEQ ID NOs: 1935 to 1937, 2849, 2858, 2864, 2874, 3510, 12647, 12648, or 12664 to 12671, the antisense oligonucleotide, vector, or composition according to claim 36.

42. The nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of SEQ ID NOs: 623 to 1934, 4169 to 4174, 12645, 12655 to 12663, or 12688, the antisense oligonucleotide, vector, or composition according to claim 37.

43. The nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of SEQ ID NOs: 1847, 1852, 1934, 12661 to 12663, or 12688, the antisense oligonucleotide, vector, or composition according to claim 37.

44. Further comprising a delivery nanocarrier, wherein the nanocarrier is complexed with the antisense oligonucleotide, the antisense oligonucleotide according to any one of claims 1 or 11 to 43.

45. The delivery nanocarrier is selected from the group consisting of lipoplexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures, the antisense oligonucleotide according to claim 44.

46. The delivery nanocarrier comprises lipid nanoparticles (LNP) encapsulating the antisense oligonucleotide, the antisense oligonucleotide according to claim 44.

47. A pharmaceutical composition comprising the antisense oligonucleotide, vector, or composition according to any one of claims 1 to 46 and a pharmaceutically acceptable excipient.

48. A method for preventing or treating a condition associated with SHANK3 haploinsufficiency, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 47.

49. The method according to claim 48, wherein the condition is Phelan-McDermid syndrome, autism spectrum disorder, schizophrenia, or intellectual disability.

50. The method according to claim 48 or 49, wherein the condition is Phelan-McDermid syndrome.

51. The method according to claim 48 or 50, wherein the subject is a human subject.

52. Use of the antisense oligonucleotide, vector, or composition according to any one of claims 1 to 46 in the manufacture of a medicament for the prevention or treatment of a condition associated with SHANK3 haploinsufficiency.

53. The level of SHANK3 protein in at least a plurality of cells in the subject is increased intracellularly by about 1.1 to about 5-fold, for example, 1.2-fold, 1.3-fold, 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold, 2.7-fold, 3-fold, 3.3-fold, 3.5-fold, 4-fold, 4.3-fold, 4.5-fold, 4.7-fold compared to the level in the absence of the pharmaceutical composition, or another increase in the SHANK3 protein level of about 1.1-fold to about 5-fold, the method according to any one of claims 48 to 51, or the use according to claim 52.

54. A genetically modified cell comprising the antisense oligonucleotide or vector according to any one of claims 1 to 46.

55. The genetically modified cell according to claim 54, wherein the genetically modified cell is a mammalian cell.

56. The genetically modified mammalian cell according to claim 55, wherein the genetically modified mammalian cell is a human cell.

57. The genetically modified mammalian cell according to claim 55 or 56, wherein the genetically modified mammalian cell is a neuron or a neural progenitor cell.

58. The genetically modified mammalian neuron according to claim 57, wherein the genetically modified mammalian neuron is a neuron selected from the group consisting of cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons.

59. The genetically modified mammalian cell according to claim 55 or 56, wherein the genetically modified mammalian cell is derived from a cell line.

60. The genetically modified mammalian cell according to claim 59, wherein the cell line is a hiPSC cell line or a cell line derived from neurons.

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