Compositions and methods for treating monogenic neurodevelopmental disorders

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 haploid insufficiency, such as the Ferron-Mecanum Syndrome (Phelan-McDermid Syndrome).

Description

[0001] This application claims priority to AU 2022901557 filed on June 7, 2022, AU2022902778 filed on September 26, 2022, and AU 2022903040 filed on October 17, 2022, the entire contents of each of which are incorporated herein by reference. Technical Field

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

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

[0004] Phelan-McDermid syndrome is characterized by varying degrees of intellectual disability, neonatal hypotonia, absent to severely delayed speech development, moderate to profound developmental delay, motor regression and mild dysmorphic features. Approximately 14%-70% of affected individuals develop mild to severe epileptic seizures. Other complications include renal abnormalities, gastrointestinal problems, reduced sweating and risk of overheating, lack of pain perception, arachnoid cysts or other comorbid neuropsychiatric disorders. Children are usually diagnosed in early childhood, usually 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). Therefore, there is a need to provide effective compositions and methods for treating such conditions. Summary of the invention

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

[0006] While not wishing to be bound by theory, SHANK3 haploinsufficiency due to loss-of-function gene mutations (including nonsense, missense, and frameshift mutations) as well as partial or complete gene deletions can result in insufficient protein production.

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

[0008] Thus, in one aspect, provided herein is an antisense oligonucleotide that is incorporated within the targeted portion of:

[0009] (i) 5' untranslated region (UTR) of SHANK3 mRNA;

[0010] (ii) the 5' proximal noncoding region (PNCR) of SHANK3 pre-mRNA; or

[0011] (iii) 3′UTR of SHANK3 mRNA;

[0012] Binding of the antisense oligonucleotide within the targeted portion of the mRNA or pre-mRNA in the mammalian cell thereby results in an increase in the level of SHANK3 protein in the mammalian cell.

[0013] In a related aspect, provided herein is a vector for expressing an antisense RNA (AR) in mammalian neurons, wherein the AR is bound within a targeted portion of:

[0014] (i) 5'UTR of SHANK3 mRNA;

[0015] (ii) 5' PNCR of SHANK3 pre-mRNA; or

[0016] (iii) 3′UTR of SHANK3 mRNA;

[0017] Thus, AR is combined in the targeted portion of RNA in mammalian cells so that the level of SHANK3 protein in the mammalian cells is increased. In some examples, the vector includes a neuron-selective promoter for driving AR expression in mammalian neurons. In some examples, the neuron-selective promoter is selective for expression in a neuron type selected from a list consisting of: cortical glutamatergic neurons, cortical GABA 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, a non-viral vector is provided as a composition comprising a transfection agent. In other examples, the vector is a viral vector. In some examples, when the vector is a viral vector, the viral vector is a recombinant virus selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, and anellovirus.

[0018] In some examples, the nucleotide sequence of the ASO or AR corresponds to any of the following: 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 of any of the ASOs, vectors, or compositions, the binding of the ASO or AR is carried out within the targeted portion of the 5'UTR corresponding to SEQ ID NO: 1. In other examples of any of the foregoing methods, ASOs, vectors, or compositions, the binding of the ASO or AR is carried out within the targeted portion of the 5'PNCR corresponding to SEQ ID NO: 3. In other examples of any of the foregoing methods, ASOs, vectors, or compositions, binding of the ASO or AR is performed within the targeted portion of the 3'UTR corresponding to SEQ ID NO:2.

[0019] 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 moiety over the length of the ASO or AR. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of the following: SEQ ID NO: 5-622, 4175-4181 or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR corresponds to any one of the following: SEQ ID NO: 559, 606 or 4178-4181. In other examples, the nucleotide sequence of the antisense oligonucleotide or AR corresponds to any one of the following: SEQ ID NO: 1935-4168, 4182, 4183, 12646-12654 or 12664-12671. In some examples, the nucleotide sequence of the ASO or AR corresponds to any of the following: SEQ ID NO: 1935-1937 or 2849. In other examples, the nucleotide sequence of the ASO or AR corresponds to any of the following: SEQ ID NO: 623-1934, 4169-4174, 12645, 12655-12663 or 12688. In some examples, the nucleotide sequence of the ASO or AR corresponds to any of the following: SEQ ID NO: 1847, 1852, 1934, 12661-12663 or 12688.

[0020] In some instances, any of the aforementioned ASOs includes backbone modifications. In some instances, backbone modifications include phosphorothioate bonds or diaminophosphorothioate bonds. In other instances, ASOs include diaminophosphorothioate morpholinos, locked nucleic acids, peptide nucleic acids or 2'-O-modifications, such as 2'-O-methyl, 2'-fluoro or 2'-O-methoxyethyl moieties. In some instances, ASOs include at least one modified sugar moiety. In other instances, each sugar moiety of ASOs is a modified sugar moiety. In some instances, ASOs include 2'-O-methoxyethyl moieties. In other instances, each nucleotide of ASOs includes 2'-O-methoxyethyl moieties.

[0021] In some examples of any of the aforementioned ASOs or vectors, the length of 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, or 26 to 30 nucleotides. In some examples of the ASO being 17 to 30 nucleotides in length, the ASO includes one or more phosphorodiamidate morpholino moieties.

[0022] In some instances, any of the aforementioned ASOs is connected to a functional part. In some instances, the functional part includes a delivery part. In some instances, the delivery part is selected from the group consisting of lipids, polyethers, peptides, carbohydrates, receptor binding domains (RBDs) and antibodies. In some instances where ASOs include a delivery part, the delivery part includes a cell penetrating peptide (CPP). In some instances, the delivery part includes N-acetylgalactosamine (GalNAc) or a polysaccharide part. In some instances, the delivery part includes a fatty acid or a lipid part. In some embodiments, the fatty acid chain length is about C8 to C20. In other instances, the functional part includes a stabilizing part. In some instances, the functional part is covalently linked to the ASO. In other instances, the functional part is non-covalently linked to the ASO. In some instances, the functional part is connected to the 5' end of the ASO. In other instances, the functional part is connected to the 3' end of the ASO. In some instances, any of the aforementioned ASOs also includes a delivery nanocarrier, wherein the nanocarrier is compounded with the ASO. In some instances, the delivery nanocarrier is selected from the group consisting of: lipid complexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In some instances, the delivery nanocarrier comprises lipid nanoparticles (LNPs) encapsulating the ASO.

[0023] In a related aspect, provided herein is a pharmaceutical composition comprising any of the aforementioned ASOs, a carrier or a composition, and a pharmaceutically acceptable excipient.

[0024] In a further related aspect, 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 instances, the condition to be treated is Phelan-McDermid syndrome, autism spectrum disorder, schizophrenia, or intellectual disability. In some instances, the condition to be treated is Phelan-McDermid syndrome. In some instances, the subject to be treated is a human subject.

[0025] In further aspects, provided herein is the use of any of the aforementioned antisense oligonucleotides, vectors or compositions for the preparation of a medicament for preventing or treating a condition associated with SHANK3 haploinsufficiency.

[0026] In some examples of the aforementioned methods of treatment or use, the level of SHANK3 protein in at least a plurality of cells of the subject is increased in cells in vitro or ex vivo (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 the level of SHANK3 protein is increased from about 1.1 fold to about 5 fold in the cells of the subject, compared to the level in the absence of the pharmaceutical composition.

[0027] On the other hand, a genetically modified cell is provided herein, which comprises any of the aforementioned ASO or vector. In some instances, the genetically modified cell is a mammalian cell. In some instances, the genetically modified mammalian cell is a human cell. In some instances, the genetically modified mammalian cell is a neuron or a neural progenitor cell. In some instances, the genetically modified mammalian cell is a neuron selected from the group consisting of: cortical glutamatergic neurons, cortical GABAergic neurons, hippocampal glutamatergic neurons, and striatal inhibitory neurons. In some instances, the genetically modified mammalian cell is from a cell line. In some instances, the cell line is a human induced pluripotent stem cell (hiPSC) system or a cell line derived from a neuron.

[0028] It is also known that SHANK3 mRNA is affected by alternative splicing, which may have an impact on the overall level of typical SHANK3 mRNA and protein levels. The term "alternative splicing" refers to a process in which an exon or a portion of an exon of a gene or an intron or a portion of an intron can be included in or excluded from the final mRNA transcript. Mature atypical mRNA transcripts may be non-productive because frameshifting may induce degradation of the transcript through a nonsense-mediated decay pathway. In other cases, translation of atypical mRNA can produce truncated but non-functional proteins. Alternative splicing of SHANK3 precursor RNA transcripts can downregulate overall SHANK3 mRNA and protein expression.

[0029] Introns are removed from mature transcripts by a large RNA-protein complex called the spliceosome, which coordinates complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and a large number of proteins. The spliceosome assembles on each intron in an orderly manner, beginning with recognition of the 5' splice site (5'ss) by the U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, which involves binding of the U2 auxiliary factor (U2AF) to the 3'ss region to facilitate U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65-kD subunit (U2AF65) encoded by U2AF2 that binds to the polypyrimidine tract (PPT) and a 35-kD subunit (U2AF35) encoded by U2AF1 that 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 inhibit splice site recognition, called intronic or exonic splicing enhancers or silencers. These elements enable the true splice sites to be distinguished from cryptic or pseudo splice sites, which have the same sequence motifs as the true sites but are an order of magnitude more numerous than the true sites in the genomes of higher eukaryotes.

[0030] Thus, in various aspects, provided herein is a method for increasing the amount of functional SHANK3 protein in a mammalian cell expressing SHANK3 pre-mRNA, the method comprising contacting the cell with an antisense oligonucleotide or antisense RNA that binds to a targeted portion of the SHANK3 pre-mRNA to modulate the splicing of the SHANK3 mRNA derived therefrom, thereby increasing the level of SHANK3 mRNA encoding a full-length functional SHANK3 protein in the mammalian cell.

[0031] In a further aspect, provided herein is an antisense oligonucleotide that binds to a targeted portion of a SHANK3 pre-mRNA to modulate the splicing of the SHANK3 mRNA, thereby increasing the level of the SHANK3 mRNA encoding a full-length functional SHANK3.

[0032] In a related aspect, a vector for expressing an antisense RNA (AR) in mammalian neurons is provided herein, wherein the AR binds to the targeted portion of the SHANK3 pre-mRNA to regulate the splicing of the SHANK3 mRNA, thereby increasing the level of the SHANK3 mRNA encoding the full-length functional SHANK3. In some examples, the nucleotide sequence of the ASO or AR for regulating splicing corresponds to any one of the following: SEQ ID NO: 4187-12693 or 12695-3953. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 - Exemplary SHANK3 PMO collections induce variable SHANK3 protein upregulation

[0034] The normalized change fold of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression was shown relative to the level of transfected control cells (no ASO transfection control). SH-SY5Y cells were transfected with 25 μM and 50 μM PMO 4-10 (SEQ ID NO: 4169, 4170, 1934 and 4171-4174, respectively) and 19-37 (SEQ ID NO: 4184-4186, 4175, 559, 4176, 4177, 598, 4178-4183, 606, 1935, 1936, 2849 and 1937, respectively). SHANK3 protein (190 kDa) was normalized relative to the total amount of loaded protein. Data are expressed as mean values ​​(n = 3-6 biological replicates, 1-2 technical replicates per experiment).

[0035] Figure 2 - Exemplary SHANK3 PMO subset induces robust upregulation of SHANK3 protein

[0036] Normalized fold change of SHANK3 protein expression was assessed by Western blot. SHANK3 protein expression is shown relative to the level of transfected control cells (no ASO transfection control). SH-SY5Y cells transfected with 25 μM and 50 μM PMO 6 (SEQ ID NO: 1934), 33-35 (SEQ ID NO: 606, 1935 and 1936, respectively), 37 (SEQ ID NO: 1937) showed SHANK3 protein upregulation relative to transfected control SH-SY5Y cells (no ASO transfection control). Data are expressed as mean ± SEM (n = 3-6 biological replicates, 1-2 technical replicates per experiment). Statistical significance was calculated as a two-way unpaired t-test between treated cells and transfected control cells (no ASO transfection control); *: p ≤ 0.05, **: p ≤ 0.01.

[0037] Figure 3 - Screening of PMOs targeting 5'UTR, 3'UTR or retained introns of SHANK3 in human neuroblastoma cell lines

[0038] According to the manufacturer's instructions, SH-SY5Y cells were transfected with 25 μM and 50 μM of 5'UTR, 3'UTR or PMO retaining introns targeting SHANK3 by electroporation (NEON, Thermo Fisher Scientific). Total protein was harvested (96 hours) from transfected cells using 15% SDS lysis buffer. SHANK3 protein (190 kDa) expression was analyzed by Western blotting in TBST containing 5% BSA using 1:10 000 rabbit-SHANK3 polyclonal antibody (Bethyl Laboratory, BLA304178A-T). Experimental controls were identified as TFC (transfection control, no ASO), UTC (untreated control), + control (rat cerebellum lysate positive control) and NTC (untargeted negative control (Smn1 and scrambled PMO)). Housekeeping proteins used for normalization included vinculin (116 kDa; 1:200 mouse-monoclonal antibody in TBST containing 5% BSA, Sigma Aldrich, V9131) and Beclin1 (60 kDa; 1:2 000 rabbit-polyclonal antibody in TBST containing 5% BSA, Cell Signaling Technology, S3495). Blots are representative of n=2-5 biological replicates. The corresponding SEQ ID NOs through (A) and (B) are indicated above each lane.

[0039] Figure 4 - Exemplary SHANK3 PMO set induces variable SHANK3 protein upregulation by targeting the 5'UTR and putative extended 5'UTR of SHANK3 transcripts

[0040] SH-SY5Y cells were transfected with 25 μM and 50 μM PMOs targeting the 5'UTR of SHANK3 and putative extended 5'UTR by electroporation (NEON, Thermo Fisher Scientific). Normalized fold change of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression is shown relative to the level of transfected control cells (no PMO, zap). Data are presented as mean ± SEM (n = 2-5 biological replicates). ASOs (except positive controls) were identified by their SEQ ID NO and are listed in 5'-3' target site order as follows: Fig.14 Schematically shown in . Control treatments were identified as NTC (non-targeted control PMO), TFC (transfection control) and UTC (untreated control). SHANK3 protein (190 kDa) was normalized to the total amount of protein loaded.

[0041] Figure 5- Exemplary SHANK3 PMO set induces variable SHANK3 protein upregulation by targeting the 3'UTR of SHANK3 transcripts

[0042] SH-SY5Y cells were transfected with 25 μM and 50 μM of PMOs targeting the 3'UTR of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). Normalized fold change of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression is shown relative to the level of transfected control cells (no PMO, zap). Data are presented as mean ± SEM (n = 2-5 biological replicates). ASOs (except positive controls) were identified by their SEQ ID NO and are listed in 5'-3' target site order as follows: Fig.17 Schematically shown in . Control treatments were identified as NTC (non-targeted control PMO), TFC (transfection control) and UTC (untreated control). SHANK3 protein (190 kDa) was normalized to the total amount of protein loaded.

[0043] Figure 6 - An exemplary SHANK3 PMO subset induces robust upregulation of SHANK3 protein by targeting the 5' and 3' UTRs of the SHANK3 transcript

[0044] SH-SY5Y cells were transfected with 25 μM and 50 μM PMOs targeting the 5' and 3' UTRs of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). Normalized fold change of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression is shown relative to the level of transfected control cells (no PMO, zap). Data are presented as mean ± SEM (n = 2-5 biological replicates). ASOs (except positive controls) were identified by their SEQ ID NOs and are listed in 5'-3' target site order as follows: Fig.14 and 17 As shown, and control treatments are identified as NTC (non-targeted control PMO), TFC (transfection control), and UTC (untreated control). SHANK3 protein (190 kDa) was normalized to the total amount of protein loaded.

[0045] Figure 7 - Exemplary SHANK3 PMO collection induces upregulation of alternative SHANK3 proteins by modulating splicing of SHANK3 transcripts

[0046] SH-SY5Y cells were transfected with 25 μM and 50 μM of PMOs that modulate alternative splicing of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). Normalized fold change of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression is shown relative to the level of transfected control cells (no PMO, zap). Data are presented as mean ± SEM (n = 2-5 biological replicates). ASOs (except positive controls) were identified by their SEQ ID NO and are listed in 5'-3' target site order as follows: Fig.15 and 16 Control treatments were identified as NTC (non-targeted control PMO), TFC (transfection control) and UTC (untreated control). SHANK3 protein (190 kDa) was normalized to the total amount of protein loaded.

[0047] Figure 8 - Exemplary SHANK3 PMO subset induces robust upregulation of SHANK3 protein by modulating splicing of SHANK3 transcripts

[0048] SH-SY5Y cells were transfected with 25 μM and 50 μM of PMOs that modulate alternative splicing of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). Normalized fold change of SHANK3 protein expression was assessed by Western blotting. SHANK3 protein expression is shown relative to the level of transfected control cells (no PMO, zap). Data are presented as mean ± SEM (n = 2-5 biological replicates). ASOs (except positive controls) were identified by their SEQ ID NO and are listed in 5'-3' target site order as follows: Fig.15 and 16 Control treatments were identified as NTC (non-targeted control PMO), TFC (transfection control), and UTC (untreated control). SHANK3 protein (190 kDa) was normalized to the total amount of protein loaded.

[0049] Fig. 9 - Screening of MOEs targeting the 5'UTR or 3'UTR of SHANK3 in human neuroblastoma cell lines

[0050] According to the manufacturer's instructions, a group of MOEs targeting SHANK3's 5'UTR, 3'UTR or retained introns were screened in SH-SY5Y cells by electroporation (NEON, Thermo Fisher Scientific). Protein was harvested (96 hours) using 15% SDS protein lysis buffer. SHANK3 (190 kDa) expression was evaluated by Western blotting using 1:10,000 rabbit-SHANK3 polyclonal antibody (Bethyl Laboratories, BLA304-178A-T) in TBST buffer containing 5% BSA. Beclin1 (60 kDa) and focal adhesion protein (116 kDa) were used as housekeeping proteins and evaluated using 1:2,000 rabbit-Beclin1 monoclonal antibody in TBST containing 5% BSA (Cell Signaling Technology, S3495) and 1:200 mouse-focular adhesion protein monoclonal antibody in TBST containing 5% BSA (Sigma Aldrich, V9131). Control treatments were identified as NTC (non-targeted control PMO), TFC (transfection control), UTC (untreated control), and rat cerebellum lysate positive control (+control). The SEQ ID NO for each MOE is indicated above each lane. Blots are representative of n=1-3 biological replicates.

[0051] Fig.10 - Exemplary SHANK3 MOE set induces variable SHANK3 protein upregulation by targeting the 5'UTR and putative extended 5'UTR of SHANK3 transcripts

[0052] SH-SY5Y cells were transfected with 2.5 μM, 5.0 μM and / or 25 μM MOE designed to target the 5'UTR or putative extended 5'UTR of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). SHANK3 protein levels were assessed by Western blotting, and expression was normalized for housekeeping (HK) protein Beclin1. SHANK3 expression was measured relative to the change fold of transfection control (TFC; no ASO). Relative to TFC, 5 μM (SEQ ID NO: 3510, 299, 301, 302, 305, 306, 309 and 318) and 25 μM (SEQ ID NO: 293, 305, 306, 307, 308, 311, 313 and 315) MOE subsets induced protein upregulation ≥ 1.5 times. Data are presented as mean ± SEM (n = 1-3 biological replicates, 1-2 technical replicates per experiment).

[0053] Fig.11 - Exemplary SHANK3 MOE subsets induce variable upregulation of SHANK3 protein by targeting the 3'UTR of SHANK3 transcripts

[0054] SH-SY5Y cells were transfected with a set of MOEs targeting the 3'UTR of SHANK3 by electroporation (NEON, Thermo Fisher Scientific). The normalized change fold of SHANK3 protein expression (at 96 hours) was assessed by Western blotting. SHANK3 protein expression in transfected and untransfected (UTC) cells was shown relative to transfection control (TFC; no ASO). MOEs were tested at concentrations of 2.5 μM, 5.0 μM or 25 μM, although not all concentrations of each MOE were tested. Among the sequences tested, one MOE (SEQ ID NO: 1193) induced the upregulation of SHANK3 protein at both lower (5.0 μM) and higher (25 μM) concentrations relative to TFC. MOE subsets (SEQ ID NO: 797, 1194 and 1195) induced SHANK3 upregulation only when used at 25 μM. Data are presented as mean ± SEM (n = 1-3 biological replicates, 1-2 technical replicates per experiment).

[0055] Fig.12 -Through lipofectamine TM Screening for an expanded panel of SHANK3 MOEs targeting both the 5'UTR and 3'UTR

[0056] Lipofectamine was used according to the manufacturer's instructions. TM3000 (Life Technologies) screened a panel of MOEs targeting 5'UTR or 3'UTR in SH-SY5Y cells to facilitate transfection. Total protein was harvested (96 hours) from transfected cells and untreated controls (UTC) using 15% SDS protein lysis buffer, and SHANK3 expression was assessed by Western blotting. (A) SHANK3 expression was normalized to housekeeping (HK) proteins, and the fold change in its expression was measured for the transfection control (TFC; no ASO). On average, none of the screened MOEs induced ≥1.5-fold upregulation of SHANK3 relative to TFC. Data are presented as mean ± SEM (n = 2-4 biological replicates, 1-2 technical replicates per experiment). The corresponding SEQ ID NO for each MOE tested is indicated under each figure set. (B) Representative Western blots were performed using 1:10000 rabbit anti-SHANK3 polyclonal antibody (Bethyl Laboratories, A304-178A-T) in TBST containing 5% BSA to detect SHANK3. HK protein was measured using 1:2000 rabbit anti-Beclin1 monoclonal antibody in TBST containing 5% BSA (Cell Signaling Technology, S3495) and 1:200 mouse anti-vinculin monoclonal antibody in TBST containing 5% BSA (Sigma-Aldrich, V9131). The corresponding SEQ ID NO for each MOE tested is indicated on each lane.

[0057] Fig.13 - Exemplary SHANK3 MOE subset induces robust upregulation of SHANK3 protein by targeting both 5' and 3' UTRs of SHANK3 transcripts

[0058] The ability of a subset of MOEs targeting the 5' and 3' UTRs of SHANK3 to upregulate SHANK3 protein expression in SH-SY5Y cells was tested. Total protein was harvested (96 h) from transfected cells and untreated controls (UTC) using 15% SDS protein lysis buffer. SHANK3 expression was assessed by Western blotting and total protein levels were determined (Revert TM700 total protein staining, LI-COR) for normalization. Transfection control (TFC; no ASO) is used to calculate the change fold of SHANK3 expression. Relative to TFC, MOE subsets (SEQ ID NO: 3510, 299, 301, 302, 304, 305, 309, 318 and 1193) cause upregulation at 5.0 μM. The selected MOE is also tested at 25 μM and shows that SHANK3 expression is further increased by ≥ 1.5 times relative to TFC (SEQ ID NO: 293, 305-308, 311, 313, 315, 316, 797 and 1195). Data are represented by mean ± SEM (n = 1-2 biological replicates, 1-2 technical replicates per experiment).

[0059] Fig.14 -PMO 5'UTR map

[0060] Binding sites of PMOs targeting putative extended 5'UTRs and typical 5'UTRs of SHANK3 transcripts. Gray rectangles above uppercase sequences (ACGT) represent exons or exon fragments; dashed lines above lowercase sequences (agct) represent introns or intron fragments. Ellipses (...) indicate that a portion of an exon or intron sequence has been omitted from the figure. =Write the span of the missing sequence between the two shown sequences (e.g., 1-1152 bp- in an intron) when necessary. Black bars indicate target sites of PMOs, which are marked with corresponding SEQ IDs; gray bars indicate target sites of a single PMO (SEQID 598) bridging exon 3 and exon 4, where asterisks indicate adjacent positions of two target site fragments in mature spliced ​​mRNAs. SHANK3 exon / intron boundaries and sequences are derived from the ENSEMBL transcript reference sequence ENST00000262795.6.

[0061] Fig.15 - PMO Retained Intron Map (Page 1 of 2)

[0062] Binding sites of PMOs targeting exons of the mature SHANK3 transcript. Gray rectangles above uppercase sequences (ACGT) indicate exons or exon fragments; dashed lines above lowercase sequences (agct) indicate introns or intron fragments. Ellipses (…) indicate that a portion of an exon or intron sequence has been omitted from the figure. Black bars indicate target sites of PMOs whose sequences correspond to the labeled SEQ IDs. SHANK3 exon / intron boundaries and sequences are derived from the ENSEMBL transcript reference sequence ENST00000262795.6.

[0063] Fig.16 - PMO preserved intron map (page 2 of 2)

[0064] Binding sites of PMOs targeting exons of mature SHANK3 transcripts. Grey rectangles above uppercase sequences (ACGT) represent exons or exon fragments, and dashed lines above lowercase sequences (agct) represent introns or intron fragments. Ellipses (…) indicate that a portion of an exon or intron sequence has been omitted from the figure. Black bars indicate target sites of PMOs, whose sequences correspond to the labeled SEQ IDs. SHANK3 exon / intron boundaries and sequences are derived from the ENSEMBL transcript reference sequence ENST00000262795.6.

[0065] Fig.17 -PMO 3'UTR map

[0066] Binding sites for PMOs targeting the 3'UTR of the SHANK3 transcript. The gray rectangle above the uppercase sequence (ACGT) represents a fragment of exon 23. The ellipsis (...) indicates that a portion of the sequence has been omitted from the figure and indicates the span of the missing sequence (i.e., 1128 bp). The black bar represents the target site of a given PMO, which is marked with the corresponding SEQ ID. The SHANK3 exon boundaries and sequence are derived from the ENSEMBL transcript reference sequence ENST00000262795.6.

[0067] Fig.18 -MOE 5'UTR and 3'UTR maps

[0068] Binding sites of MOE targeting the 5'UTR (top) and 3'UTR (middle, bottom) of the mature SHANK3 transcript. The gray rectangle above the uppercase sequence (ACGT) represents an exon or exon fragment, where the vertical black line indicates the exon-exon junction. The ellipsis (...) indicates that a portion of the exon or intron sequence is omitted in the figure. If necessary, the span of the missing sequence is also indicated. The black bar represents the target site of each MOE, which is marked with the corresponding SEQ ID. The SHANK3 exon / intron boundaries and numbering are derived from the GenBank transcript reference sequence NM_001372044.2.

[0069] Fig.19 - Exemplary SHANK3 PMO subset induces robust transcript regulation of SHANK3

[0070] SH-SY5Y cells were transfected with a subset of PMOs designed to target the 5'UTR or putative extended 5'UTR of SHANK3, showing ≥1.5-fold upregulation of SHANK3 protein as assessed by Western blot. SH-SY5Y cells were transfected with 25 μM and 50 μM PMOs by electroporation (NEON, Thermo Fisher Scientific) following the manufacturer's instructions. Cells were harvested (96 h) and quantified using MagMAXTM -96 total RNA isolation kit (AM1830, Thermo Fisher Scientific). Target engagement was determined by the percentage of regulation of altered SHANK3 transcripts relative to TFC (transfection control) and UTC (untreated control), as indicated by RT-PCR gel bands (LabChip GXII, PerkinElmer). Data are presented as mean ± SEM (n = 3 biological replicates). DETAILED DESCRIPTION

[0071] Overview

[0072] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter are to be considered to encompass both one and multiple (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise. 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 a single as well as two or more; reference to "a" includes a single as well as two or more; reference to "the" includes a single as well as two or more, etc.

[0073] Unless specifically stated otherwise, each example of the present disclosure described herein applies mutatis mutandis to every other example.

[0074] Those skilled in the art will appreciate that, in addition to the disclosure specifically described, changes and modifications may be made to the disclosure herein. It should be understood that the disclosure includes all such changes and modifications. The disclosure also includes all steps, features, compositions and compounds mentioned or indicated individually or collectively in this specification, as well as any and all combinations of any two or more of the steps or features.

[0075] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0076] Unless otherwise indicated, the present disclosure is performed without undue experimentation using conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such techniques are described and explained throughout the literature in sources such as Perbal 1984, Sambrook et al., 2001, Brown (ed.) 1991, Glover and Hames (eds.) 1995 and 1996, Ausubel et al., including all updates to date, Coligan et al. (eds.) (including all updates to date), Maniatis et al. 1982, Gait (ed.) 1984, Hames and Higgins (eds.) 1984, Freshney (ed.) 1986.

[0077] The term "and / or", such as "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0078] Unless otherwise stated, the term "about" refers to + / -20%, more preferably + / -10% of the specified value. For the avoidance of doubt, the term "about" followed by a specified value should be interpreted as also covering the exact specified value itself (e.g., "about 10" also covers exactly 10).

[0079] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of stated elements, integers or steps or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps or groups of elements, integers or steps.

[0080] As used herein, the term "antisense oligonucleotide," "antisense oligomer," or "ASO" encompasses oligonucleotides and any other oligomeric molecule comprising a nucleobase capable of hybridizing to a complementary sequence on a target RNA transcript, including but not limited to those that do not comprise a sugar moiety, such as in the case of peptide nucleic acids (PNAs). Preferably, the ASO is one that is resistant to nuclease cleavage or degradation.

[0081] As used herein, the phrase "bound to the targeted portion" or "bound within the targeted portion" with respect to an ASO or AR refers to specific hybridization between an ASO or AR nucleotide sequence and a target nucleotide sequence complementary to the range set forth herein. In some instances, specific hybridization occurs when hybridization occurs under highly stringent conditions under in vitro conditions. "High stringency conditions" means that under such in vitro conditions, an ASO or AR hybridizes to a target sequence in an amount that is detectably stronger than non-specific hybridization. Then, high stringency conditions are conditions that distinguish a polynucleotide having an exact complementary sequence or a polynucleotide containing only a few dispersed mismatches from a random sequence that happens to have a few cells (e.g., 1-5 bases) that match the probe. Such complementary cells are more easily melted than full-length complements of 12-17 or more bases, and moderately stringent hybridization makes them easily distinguishable. In one example, high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1M NaCl or an equivalent, at a temperature of about 50-70°C. The skilled artisan will appreciate that under in vivo conditions, the specificity of hybridization between an ASO or AR and its target sequence is defined based on the level of complementarity between the ASO or AR and its target sequence to which it hybridizes within the cell.

[0082] The term "peptide" is intended to include compounds consisting of amino acid residues connected by amide bonds. Peptides can be natural or non-natural, ribosomally translated or synthetically derived. Typically, a peptide is composed of 2 to 200 amino acids. For example, the length of a peptide can be 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 range. A peptide can include 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 consist of.

[0083] Peptides as referred to herein include "inverso" peptides, in which all L-amino acids are replaced by 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.

[0084] The peptide may include amino acids in both L-form and / or D-form. For example, both L-form and D-form may be used for different amino acids in the same peptide sequence. In some instances, the amino acids in the peptide sequence are in L-form, such as natural amino acids. In some instances, the amino acids in the peptide sequence are a combination of L-form and D-form. In addition, the peptide may include unusual but naturally occurring amino acids, including but not limited to hydroxyproline (Hyp), β-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 homologous amino acids, N-methyl amino acids, α-methyl amino acids, β (homologous) amino acids, γ amino acids, and N-substituted glycine. The peptide may be a linear peptide or a cyclic peptide.

[0085] The term "protein" should be considered to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, the 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.

[0086] The amino acid sequence identity percentage relative to a given amino acid sequence is defined as the percentage of the amino acid residue in the candidate sequence identical to the amino acid residue in the reference sequence after the alignment sequence and when necessary introducing a gap to achieve the maximum sequence identity percentage and without considering any conservative substitution as a part of sequence identity. Amino acid sequence identity can be determined using the EMBOSS pairwise alignment algorithm tool that can be obtained from the European Bioinformatics Institute (The European Bioinformatics Institute, EMBL-EBI) as a part of the European Molecular Biology Laboratory (the European Molecular Biology Laboratory). This tool can be obtained on the website located at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Using default settings, the default settings include gap opening: 10.0 and gap extension 0.5. Default matrix "Blosum62" is used for amino acid sequences and default matrix. "Nucleic acid sequence identity" percentage (%) or percentage relative to the nucleotide sequences disclosed herein is defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage. Alignment for the purpose of determining the nucleic acid sequence identity percentage can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST or ALIGN. The skilled person can readily determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequence.

[0087] The term "cell penetrating peptide" (CPP) refers to a peptide that can pass through a cell membrane. In one example, a CPP is able to translocate across a mammalian cell membrane and enter a cell. In another example, a CPP can guide a conjugate to a desired subcellular compartment. Thus, a CPP can guide or promote the penetration of a molecule of interest through a phospholipid, mitochondrial, endosome, lysosome, vesicle, or nuclear membrane. A CPP can translocate across a membrane or alternatively partially degrade when its amino acid sequence is complete and intact.

[0088] CPP can direct the molecule of interest (such as ASO disclosed herein) from outside the cell through the plasma membrane and into the cytoplasm or desired subcellular compartment. Alternatively or in addition, CPP can direct the molecule of interest across the blood-brain barrier, through the mucosal barrier, the blood-retinal barrier, the skin barrier, the gastrointestinal barrier and / or the lung barrier.

[0089] The term "peptide ligand" or "receptor binding domain" refers to a peptide that can bind to a membrane surface receptor to enable the peptide to translocate across the cell membrane. In one example, the peptide ligand can achieve translocation across the cell membrane by natural endocytosis of the targeted receptor. In another example, the peptide ligand can utilize a complementary mechanism of translocation across the cell membrane, including the use of a conjugated CPP. In one example, the peptide ligand is able to translocate across the mammalian cell membrane and enter the cell. In another example, the peptide ligand can guide the conjugate to a desired subcellular compartment. Therefore, the peptide ligand can guide or promote the cellular uptake of the molecule of interest through phospholipids, mitochondria, endosomes, lysosomes, vesicles or nuclear membranes. The peptide ligand can be translocated across the membrane or alternatively partially degraded when its amino acid sequence is complete and intact.

[0090] The peptide ligand, through its binding to the target receptor, can guide the molecule of interest (such as the ASO disclosed herein) from outside the cell through the plasma membrane and into the cytoplasm or a desired subcellular compartment. Alternatively or in addition, the peptide ligand, through its binding to the target receptor, can guide the molecule of interest through relevant biological barriers, such as the blood-brain barrier, the mucosal barrier, the blood-retinal barrier, the skin barrier, the gastrointestinal barrier and / or the lung barrier.

[0091] Compositions for increasing SHANK3 protein levels

[0092] Secondary structures and upstream open reading frames in the 5'UTR of SHANK3 mRNA reduce translation efficiency and transcript stability, thereby limiting protein production. In addition, the SHANK3 5'UTR includes 3 exons, and exon 2 contains sequences that may 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, thereby removing elements that reduce translation efficiency. Further, microRNAs (miRNAs) often bind to complementary RNA sequences in the 3' untranslated region (3'UTR) and regulate gene expression by stimulating mRNA degradation or translation inhibition. Both of these mechanisms lead to reduced gene expression. Despite this "inefficiency", in the genetic background of two functional (wild-type) alleles, the levels of productive RNA transcripts and translation produce sufficient levels of functional protein for a given gene. However, in the case of rare monogenic diseases, the loss of one functional allele (e.g., SHANK3 allele) may lead to haploinsufficiency and related diseases.

[0093] Although not wishing to be bound by theory, it is believed that ASOs targeting sequences within the 5'UTR of SHANK3 mRNA can enhance translation, for example, by reducing / disrupting the formation of secondary structures that would otherwise reduce translation efficiency. ASOs targeting the 5'PNCR can enhance the translation efficiency of SHANK3 transcripts. Further, it is believed that antisense sequences that are at least partially complementary to the binding sites of miRNAs located within the 3'UTR of SHANK3 mRNA will hybridize to the 3'UTR and sterically hinder ("mask") these miRNAs from accessing their binding sites, thereby increasing the level of SHANK3 mRNA and ultimately allowing increased translation of SHANK3 protein.

[0094] Thus, disclosed herein is an ASO that binds within the 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;

[0095] Thus, incorporation of the antisense oligonucleotide into the targeted moiety in a mammalian cell results in an increase in the level of SHANK3 protein in the mammalian cell.

[0096] For reference, the nucleotide sequence of a typical human SHANK3 pre-mRNA transcript ("SHANK3-201") is provided herein as SEQ ID NO: 4. The nucleotide sequence of the 5'UTR of a typical human SHANK3 mRNA transcript is provided herein as SEQ ID NO: 1. The nucleotide sequence of the 5'PNCR of a typical SHANK3 pre-mRNA is provided herein as SEQ ID NO: 3. The nucleotide sequence of the 3'UTR of a typical human SHANK3 mRNA is provided herein as SEQ ID NO: 2 (Appendix).

[0097] Antisense Oligonucleotides (ASO) and Antisense RNA (AR)

[0098] In some examples of the compositions and methods described herein, ASO and AR have sequences that are completely or almost completely complementary to the target sequence in their length. ASO and AR are designed so that they bind (hybridize) to the target RNA sequence (e.g., the targeted portion of the mRNA transcript) and keep hybridization under physiological conditions. Where possible, selecting suitable sequences for ASO and AR generally avoids similar nucleic acid sequences in other (i.e., off-target) positions in genomes or cell mRNA or miRNA, so that the possibility that ASO or AR will hybridize at these sites is limited. In some examples, ASO disclosed herein can be used to weaken the formation of SHANK3 mRNA secondary structure, particularly in the 5'UTR region that interferes with translation. In other examples, ASO disclosed herein binds to a target region in the 5'PNCR of SHANK3 pre-mRNA (e.g., in an intron sequence, or partially in an intron sequence and partially in a flanking exon sequence). In other examples, the ASOs disclosed herein mask miRNA access to its target binding site in the SHANK3 3'UTR, thereby reducing the level of miRNA-dependent SHANK3 mRNA instability. Thus, the ASOs disclosed herein result in a net increase in the level of canonical SHANK3 mRNA and, therefore, the level of functional SHANK3 protein.

[0099] In some examples, the ASO or AR "specifically hybridizes" or is "specific" to a target nucleic acid or a targeted portion of a SHANK3 mRNA 5'UTR or 3'UTR or a SHANK3 pre-mRNA 5'PNCR. m It is the temperature at which 50% of the target sequence hybridizes to the complementary oligonucleotide.

[0100] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the ASO and AR sequences are "complementary" to their target sequences. According to the generally accepted base pairing rules, complementarity can be quantified according to the ratio of bases in the relative chains that are expected to form hydrogen bonds with each other (e.g., percentage). The nucleotide sequence of an ASO or AR does not need to be 100% complementary to the nucleotide sequence of the target nucleic acid with which it hybridizes. In certain examples, the nucleotide sequence of an ASO or AR in the composition disclosed herein can be 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, 18 of the 20 nucleotides of the ASO or AR sequence are complementary to the target region and will therefore specifically hybridize to the ASO or AR will represent 90% complementarity. In such examples, the remaining non-complementary nucleotides of the ASO or AR can be gathered together or interspersed with complementary nucleotides, and need not be continuous. The complementarity of the ASO or AR sequence to the target nucleotide sequence (expressed as "complementarity percentage" with its target sequence; or "identity percentage" with its reverse complementary sequence) can be routinely determined using algorithms known in the art, such as BLAST programs (Basic Local Alignment Search Tool) and PowerBLAST programs (Altschul et al., 1990, "Journal of Molecular Biology (J.Mol.Biol.)", 215: 403-410; Zhang et al., 1997, "Genome Res. (Genome Res.)", 7: 649-656) as exemplified.

[0101] In some examples, the ASO or AR does not hybridize to all nucleotides in the target sequence, and the nucleotide positions to which it hybridizes may be continuous or discontinuous. The ASO or AR may hybridize to one or more fragments of the SHANK3 mRNA 5'UTR or 3'UTR; to one or more fragments of the SHANK3 mRNA 5'PNCR, such that no intermediate or adjacent fragments are involved in the hybridization event (e.g., a loop structure or a hairpin structure may be formed).

[0102] In some examples, the ASOs or ARs disclosed herein are complementary to the targeted portion of the canonical SHANK3 mRNA 5'UTR sequence corresponding to SEQ ID NO: 1. In other examples, the ASOs or ARs disclosed herein are complementary to the targeted portion of the canonical SHANK3 pre-mRNA 5'PNCR sequence corresponding to SEQ ID NO: 3. In other examples, the ASOs or ARs disclosed herein are complementary to the targeted portion of the canonical SHANK3 mRNA 3'UTR sequence corresponding to SEQ ID NO: 2.

[0103] 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 comprises the following sequence: (a) having at least about 40% to about 60% identity to the nucleotide sequence of the ASO or AR sequence disclosed herein, for example, 45%, 48%, 50%, 52%, 55%, 58% identity or about 40% to 60% another sequence identity to the entire length of any ASO or AR sequence disclosed herein; and (b) comprising a continuous sequence of at least 8 bases to 16 bases, which is 100% identical to the continuous sequence of at least 8 to 16 bases in any of the ASO or AR sequences disclosed herein, for example, 100% sequence identity to the continuous 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases or 16 bases of the ASO or AR sequence disclosed herein. The ASO or AR used in the compositions described herein can have any length suitable for specific hybridization with the target sequence. In some instances, the nucleotide sequence of ASO or AR consists of 8 to 50 nucleotides. For example, the length of 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 some instances, the nucleotide sequence of ASO or AR consists of 8 to 50 nucleotides. For example, the length of 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 some examples, the ASO consists of more than 50 nucleotides but no more than 100 nucleotides in length.In some examples, the length of 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, 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, 1 2 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 In some instances, the length of the ASO or AR is 17 nucleotides. In other instances, the length of the ASO or AR is 20 nucleotides. In some instances, the length of the nucleotide sequence of the ASO or AR nucleotide is 25 nucleotides.

[0104] In other examples, the ASO or AR comprises at least 10 consecutive nucleotides of the ASO or AR sequence described herein. In some examples, the ASO or AR comprises at least 10 consecutive nucleotides (subsequences) from each of two or more ASO or AR sequences described herein, wherein the two or more subsequences are discontinuous in the SHANK3 mRNA sequence.

[0105] In some examples of each occurrence of "G" in the ASO or AR sequences disclosed herein, "G" is guanosine or inosine. In some examples of each occurrence of "T" in the ASO or AR sequences disclosed herein, "T" is any of the following: thymidine, inosine, uracil, or an isomer or modified form of uracil (e.g., pseudouridine or N1-methyl-pseudouridine). In some examples of 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).

[0106] In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one 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 the sequence of any one of SEQ ID NO: 5-622, 4175-4181, or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one of the following: SEQ ID NO: 5-188, 191-622, 4175-4181 or 4184-4186. In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one of the following: SEQ ID NO: 559, 606 or 4178-4181. In other examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one of the following: SEQ ID NO: 1935-4168, 4182, 4183, 12646-12654 or 12664-12671. In some examples, the nucleotide sequence of the ASO or AR comprises the sequence of any one of the following: 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 the sequence of any one of the following: 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 any one of the following: SEQ ID NO: 1934.

[0107] In some examples, the nucleotide sequence of the ASO or AR consists of the nucleotide sequence of any of the following: SEQ ID NO: 5-4186, 12646-12654, or 12664- 12671. The sequences of the aforementioned SEQ ID NOs are provided in Tables 4 and 5 in the Appendix.

[0108] ASO chemistry and modifications

[0109] The ASO used in the compositions described herein may include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination thereof. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or with modified backbones. In some examples, all nucleotides of ASO are modified nucleotides. Chemical modifications of ASOs or ASO components compatible with the compositions and methods described herein are known in the art, such as disclosed in, for example, 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 Review Drug Discovery (Nature Rev. Drug Disc.), 19: 673-694.

[0110] The one or more nucleotides of the ASO can be any naturally occurring, unmodified nucleobase (e.g., adenine, guanine, cytosine, thymine, uracil, and inosine) or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with 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.

[0111] ASO includes a "backbone" structure, which refers to the connection between the nucleotides / monomers of ASO. In naturally occurring oligonucleotides, the backbone includes a 3'-5' phosphodiester bond connecting the sugar moieties of adjacent nucleotides. The types of backbone bonds applicable to ASOs described herein include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphorodiamidate, phosphoroselenoate, phosphorodiselenoate, thioanilide phosphate (phosphoroanilothioate), anilide phosphate, phosphoramidate, etc. In some examples, the backbone modification is a phosphorothioate bond. In other examples, the backbone modification is a phosphorodiamidate bond. See, for example, Roberts et al., supra; and Agrawal (2021), "Biomedicines", 9: 503. In some examples, the backbone structure of ASO does not contain a phosphorus-based bond, but contains, for example, a peptide bond in a peptide nucleic acid (PNA) or a linking group including carbamates, amides, and straight and cyclic hydrocarbon groups.

[0112] In some instances, the stereochemistry at each phosphorus internucleotide bond in the phosphorus internucleotide bond of the ASO skeleton is random. In other instances, the stereochemistry at each phosphorus internucleotide bond in the phosphorus internucleotide bond of the ASO skeleton is controlled and not random. For example, U.S. Patent No. 9,605,019 describes a method for independently selecting the chiral handedness at each phosphorus atom in an oligonucleotide. In some instances, the ASO used in the compositions and methods provided herein (including but not limited to the ASO whose sequence is disclosed as SEQ ID NO:5-39535 in this article) is an ASO with a non-random phosphodiester internucleotide bond. In some instances, the compositions or compositions used in the methods disclosed herein include pure diastereoisomer ASO. In other examples, the composition comprises an ASO 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% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0113] In some examples, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus internucleotide linkage. In some examples, the ASO used in the compositions and methods disclosed herein comprises 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 about 100% Rp.

[0114] In some instances, ASO as described herein contains sugar moieties including ribose or deoxyribose or modified sugar moieties or sugar analogs, including morpholine ring. Suitable examples of modified sugar moieties include but are not limited to 2' substitutions, such as 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'-guanidine, 2'-O-guanidineethyl, carbamate modified sugar and dicyclo modified sugar. In some instances, sugar moiety modifications are selected from 2'-O-Me, 2'F and 2'MOE. In other instances, sugar moiety modifications are additional bridge bonds, such as in locked nucleic acids (LNA). In some instances, sugar analogs contain morpholine rings, such as diaminophosphorothioate morpholino (PMO). In some instances, the sugar moiety comprises a ribofuranosyl or 2'deoxyribofuranosyl modification. In some instances, the sugar moiety comprises a 2'4'-restricted 2'-O-methoxyethyl (cMOE) modification. In some instances, the sugar moiety comprises a cEt 2',4'-restricted 2'-O ethyl BNA modification. In other instances, the sugar moiety comprises a tricyclic DNA (tcDNA) modification. In some instances, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some instances, the sugar moiety comprises a 2'-O-(2-N-methylcarbamoylethyl) (MCE). Modifications are known in the art, as exemplified in Jarver et al., 2014, Nucleic Acid Therapeutics, 24(1): 37-47.

[0115] In some examples, each constituent nucleotide of the ASO is modified in the same manner, for example, each bond of the backbone of the ASO comprises a phosphorothioate bond, or each ribose moiety comprises a 2'-O-methyl modification. In other examples, a combination of different modifications is used, for example, an ASO comprising a combination of a phosphorodiamidate bond and a sugar moiety comprising a morpholine ring (morpholino).

[0116] In some instances, the ASO comprises one or more backbone modifications. In some instances, the ASO comprises one or more sugar moiety modifications. In some instances, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some instances, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some instances, the ASO comprises a peptide nucleic acid (PNA).

[0117] In some preferred embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO).

[0118] Those skilled in the art will appreciate that an ASO can be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. In some instances, the ASO is modified to change one or more properties. For example, such modifications may: enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation caused by cellular nucleases (e.g., RNase H); improve uptake of the ASO into cells and / or specific subcellular compartments; alter the pharmacokinetics or pharmacodynamics of the ASO; and / or regulate the half-life of the ASO in vivo.

[0119] In some examples, the ASO comprises one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides, which have been shown to confer significantly enhanced resistance to nuclease degradation and increased bioavailability to the ASO.

[0120] Methods for synthesizing and chemically modifying ASOs and synthesizing ASO conjugates are well known in the art, and such ASOs are commercially available.

[0121] In some examples, the compositions (e.g., pharmaceutical compositions) provided herein include two or more ASOs having different chemistry but complementary to the same targeted portion of SHANK3 mRNA 5'UTR or 3'UTR or SHANK3 pre-mRNA 5'PNCR. In other examples, the compositions include two or more ASOs complementary to different targeted portions of 5'UTR, 3'UTR or 5'PNCR.

[0122] In some instances, the compositions disclosed herein include an ASO linked to a functional moiety. In some instances, the functional moiety is a delivery moiety, a targeted moiety, a detection moiety, a stabilizing moiety, or a therapeutic moiety. In some instances, the functional moiety includes a delivery moiety or a targeted moiety. In some instances, the functional moiety includes a stabilizing moiety. In some preferred instances, the functional moiety is a delivery moiety.

[0123] Suitable delivery moieties include, but are not limited to, lipids, polyethers, peptides, carbohydrates, polysaccharides, receptor binding domains (RBDs), and antibodies.

[0124] In some instances, the delivery moiety includes a cell penetrating peptide (CPP). Suitable examples of CPP are described in, for example, PCT / AU2020 / 051397. In some instances, the amino acid sequence of CPP comprises or consists of: RRSRTARAGRPGRNSSRPSAPR (SEQ ID NO: 12694). In other instances, the delivery moiety includes RBD.

[0125] 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), polysaccharides and mannose. In one example, the carbohydrate delivery moiety comprises a GalNac or polysaccharide moiety.

[0126] In other examples, the delivery moiety includes a lipid. Examples of suitable lipids as delivery moieties include, but are not limited to, cholesterol moieties, cholesteryl moieties, and aliphatic lipids. In some examples, the delivery moiety includes a fatty acid or lipid moiety. In some embodiments, the fatty acid chain length is about C8 to C20. Examples of suitable fatty acid moieties and their conjugation to oligonucleotides are found in, for example, International Patent Publication WO 2019232255 and Prakash et al., (2019).

[0127] In further examples, the delivery moiety comprises an antibody, as described, for example, in Dugal-Tessier et al., (2021).

[0128] Suitable examples of stabilizing moieties include, but are not limited to, polyethylene glycol (PEG), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and poly(2-oxazoline)s (POx).

[0129] In some instances, where the ASO is linked to a functional moiety, the functional moiety is covalently linked to the ASO. In other instances, the functional moiety is non-covalently linked to the ASO.

[0130] The functional moiety can be attached to one or more of any nucleotide in the ASO at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, for example, using a linker. The linker can include a divalent or trivalent branched linker. In some instances, the functional moiety is attached to the 5' end of the ASO. In other instances, the functional moiety is attached to the 3' end of the ASO.

[0131] In some instances, the composition comprising any of the ASOs disclosed herein further comprises a delivery nanocarrier compounded with the ASO. In some instances, the delivery nanocarrier is selected from lipid complexes, liposomes, exosomes, inorganic nanoparticles, and DNA nanostructures. In other instances, the delivery nanocarrier comprises lipid nanoparticles encapsulating the ASO. Various delivery ASO-nanocarrier complex forms are known in the art, as summarized in, for example, Roberts et al., supra.

[0132] Vector expressing SHANK3 antisense RNA (AR)

[0133] In some examples, provided herein is a vector for expressing an antisense RNA (AR) in mammalian neurons or other cell types, wherein the AR is bound within a targeted portion of: (i) 5'UTR of SHANK3 mRNA; (ii) 5'PNCR of SHANK3 pre-mRNA; or (iii) 3'UTR of SHANK3 mRNA; thereby binding the AR within the targeted portion in mammalian cells to increase the level of SHANK3 protein in the mammalian cells.

[0134] In some instances, the promoter used in the expression vector is a neuron-selective promoter for driving AR expression in mammalian cells. In some instances, the neuronal 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.

[0135] In some, the promoter is an inducible promoter, for example, a ligand-regulated transactor (such as tet-induced rtTA) can be induced, which allows titration of AR transcription in target mammalian cells. In some examples, the promoter driving AR expression is U6 or other Pol III promoters, which are particularly suitable for transcribing short RNA sequences, such as AR sequences disclosed herein. In some examples, the expression vector utilizes a hybrid promoter system, for example, a U6 promoter system regulated by Tet-O as described in the following literature: Lin et al. (2004), "FEBS Letters", 577 (2004) 376-380. In some examples where both cell type specificity and inducibility of an AR expression vector are desired, a two-part expression system is used, in which the expression of a ligand-regulated transactor is driven by a cell type selective promoter, and the expression of the AR disclosed herein is driven by a promoter regulated by a ligand-regulated transactor.

[0136] In some examples, the expression vector used in the compositions disclosed herein is a non-viral expression vector, for example, a plasmid vector, a minicircular DNA vector, a linear amplicon expression cassette, and the like.

[0137] In some examples, 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 Strasbourg, France) SA); TurboFect in vivo transfection reagent (Thermo Fisher Scientific) and cationic derivatives of polyisoprenylated alcohol (PTAI) as described in Rak et al., (2016).

[0138] In other examples, the expression vector to be used is a viral vector, ie, a non-replicating recombinant virus suitable for expressing the AR disclosed herein.

[0139] Preferably, the recombinant virus used to express 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 designing, producing, and using recombinant DNA viruses of this type are well established in the art, as exemplified by Fukazawa et al. (2010), International J of Mol. Med, 25(1), 3-10, and “Gene Therapy Protocols” for adenovirus; “Adeno-associated viruses: methods and protocols” for AAV; Cody et al. (2013), Journal of Genetic Syndromes & Gene Therapy, 4(1), 126, and “Herpes simplex virus: methods and protocols” for HSV; “Gene Therapy Protocols Vol. 1: Generation and in vivo use of gene transfer vectors”; and Merten et al. (2016), Molecular Therapy–Methods & Clinical Development, 3, 16017, and Emeagi et al. (2013), Current Molecular Medicine, 1, 16017 for lentiviruses. Medicine)》13(4),602-625. In some preferred embodiments, the viral vector is a recombinant AAV.

[0140] Genetically modified cells

[0141] 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 vector disclosed herein). In other examples, the genetically modified cells are genetically modified mammalian cells that have been transfected with any of ASO or non-viral AR expression vectors or transduced with any of the viral AR expression vectors disclosed herein. In some examples, the genetically modified mammalian cells are in vitro, for example, as a cultured cell colony. In other examples, the genetically modified mammalian cells are in vivo, for example, in mice. In some examples, the genetically modified mammalian cells are human cells.

[0142] In some examples, the genetically modified mammalian cell is a neuron or a neural progenitor cell. Suitable examples of neurons include, but are not limited to, cortical glutamatergic neurons, cortical GABA 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 cells (hESC) lines). Methods for obtaining a variety of different neuronal cell types are known in the art, as reviewed in, for example, Alia et al., (2019), Fitzgerald et al., (2020) and Kim et al. (2014). In other examples, the genetically modified mammalian cell is derived from a cell line. In some examples, the cell line is a pluripotent stem cell line (e.g., hiPSC or hESC) or a neuronal cell line. Suitable neuronal cell lines or neuronal stem cell lines include, but are not limited to, SH-SY5Y, NTera, CTX0E16, ReNcell VM, ReNcellCx. In some preferred examples, the genetically modified mammalian cell endogenously expresses SHANK3.

[0143] Genetically modified cells disclosed herein can be genetically modified by any of a number of methods and strategies known in the art (e.g., transient transfection, stable transfection, and viral transduction). In some instances, transfection with ASOs or non-viral vectors is performed by nuclear transfection. In other instances, cell transfection is performed by lipofection.

[0144] Pharmaceutical composition

[0145] Also provided herein is a pharmaceutical composition comprising any of the aforementioned ASOs, non-viral expression vectors, and viral expression vectors disclosed herein, and formulated together with at least one pharmaceutically acceptable excipient (including carriers, fillers, preservatives, adjuvants, solubilizers, and / or diluents).

[0146] Pharmaceutical compositions containing any of the ASO or expression vector compositions described herein for use in the methods disclosed herein can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the disclosed literature. In some examples, a pharmaceutical composition for treating a subject comprises a therapeutically effective amount of any ASO or expression vector disclosed herein.

[0147] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without 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 (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., 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, dodecylsulfate, 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, sodium p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed when appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0148] In some instances, the pharmaceutical composition is formulated into any of many possible dosage routes or forms, including but not limited to intravenous administration, intrathecal administration, magna administration, tablets, capsules, gel capsules, liquid syrups and soft gels. In some instances, the composition is formulated into a suspension in an aqueous, non-aqueous or mixed medium. The aqueous suspension may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain a stabilizer. In some instances, the pharmaceutical formulations disclosed herein are provided in the form of solutions, emulsions, microemulsions, foams or formulations containing liposomes (e.g., cationic or non-cationic liposomes).

[0149] In some instances, the pharmaceutical formulation comprising any of the ASOs or expression vectors described herein may include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients suitable and known to the skilled person. In some instances, when the pharmaceutical composition includes liposomes, such liposomes may also include spatially stable liposomes, such as liposomes including one or more specialized lipids. These specialized lipids produce liposomes with enhanced circulation life. In some instances, spatially stable liposomes include one or more glycolipids or are derivatized with one or more hydrophilic polymers (such as PEG moieties). In some instances, surfactants are included in the pharmaceutical formulation.

[0150] In some examples, the pharmaceutical composition also includes a penetration enhancer to enhance the delivery of the ASO or non-viral expression vector, for example to aid diffusion across the cell membrane and / or enhance the permeability of lipophilic drugs. In some examples, the penetration enhancer includes a surfactant, a fatty acid, a bile salt, or a chelating agent.

[0151] In some examples, where administration is by a systemic route, such as intravenous, the method further comprises a step of promoting translocation of any of the ASOs or vectors described herein across the blood-brain barrier (BBB) ​​into the CNS and particularly into the brain. In some examples, the BBB is transiently disrupted, for example by administering one or more antibodies that disrupt the binding of spindle protein-1 to Unc5B, as described in Boyé et al., (2022).

[0152] In some examples, the pharmaceutical composition comprises an ASO or non-viral vector at a dose in the range of about 0.01 mg / kg to 20 mg / kg (e.g., 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 dose in the range of about 0.01 mg / kg to 20 mg / kg. In some examples, when an ASO disclosed herein is administered directly to the CNS or brain, for example by intraventricular administration, the total dose is in the range of about 50 mg to about 500 mg, for example 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 dose of about 50 mg to about 500 mg. Assuming the average human brain volume is about 1200 cm 3 , which corresponds to a dose range of approximately 0.050 mg / cm 3 The brain volume is about 0.42mg / cm 3 of brain volume.

[0153] In some examples, the pharmaceutical composition comprises multiple ASO or AR expression vectors. In some examples, the pharmaceutical composition comprises another drug or therapeutic agent suitable for treating a subject with SHANK3 haploinsufficiency in addition to the ASO or AR expression vector.

[0154] method

[0155] As described herein, many conditions (e.g., Phelan-McDermid syndrome) are associated with insufficient functional SHANK3 levels. Therefore, the methods described herein include methods for preventing or treating conditions 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 is used to prepare a drug for treating conditions 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.

[0156] Also provided herein is a method for increasing the amount of functional SHANK3 protein in a mammalian cell expressing SHANK3 mRNA, the method comprising contacting the cell with any of the ASOs or expression vectors disclosed herein.

[0157] In some examples, administration of any of the ASOs, AR expression vectors, or pharmaceutical compositions disclosed herein to a subject or contacting it with cells in vitro or ex vivo increases the level of SHANK3 protein in cells (e.g., neurons) in vitro or ex vivo by about 1.1 to about 5 times, for example, 1.2 times, 1.3 times, 1.5 times, 1.7 times, 2 times, 2.2 times, 2.5 times, 2.7 times, 3 times, 3.3 times, 3.5 times, 4 times, 4.3 times, 4.5 times, 4.7 times, or the level of SHANK3 protein in a subject or in vitro or ex vivo cells increases from about 1.1 times to about 5 times.

[0158] Suitable routes of administration for treatment with a composition, pharmaceutical composition, or medicament disclosed herein include, but are not limited to, intravenous, intraarterial, intraparenchymal, intraventricular, intracisternal, intrathecal, intravenous, intraarterial, subcutaneous, and topical administration.

[0159] As will be appreciated by those skilled in the art, the methods of treatment disclosed herein include administering the compositions and pharmaceutical compositions disclosed herein to a subject (e.g., a human subject) in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of one or more symptoms and / or clinical markers associated with SHANK3 haploinsufficiency in a particular disease or health condition that the ASO, non-viral or viral expression vector disclosed herein is administered in an amount sufficient to alleviate to a certain extent. In some instances, the "effective amount" for therapeutic use is the amount of one of the aforementioned agents required to provide a clinically significant reduction in disease symptoms to prevent disease symptoms without excessive adverse side effects. Examples of suitable symptoms alleviated by the methods of treatment provided herein include, but are not limited to, epileptic seizures, anxiety, repetitive behaviors, learning and memory defects, and impaired social skills. Techniques such as dose escalation studies can be used to determine the appropriate "effective amount" in any individual case. The term "therapeutically effective amount" includes, for example, a preventive effective amount. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to changes in the metabolism of the compound, any of the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation including, but not limited to, dose escalation clinical trials. When more than one therapeutic agent is used in combination, a "therapeutically effective amount" of each therapeutic agent may refer to an amount of the therapeutic agent that has a therapeutic effect when used alone, or may refer to a reduced amount that has a therapeutic effect due to use in combination with one or more additional therapeutic agents.

[0160] Combination therapy

[0161] Pharmaceutical compositions comprising any of the ASOs or AR expression vectors disclosed herein can also be used in combination with other agents of therapeutic value to treat conditions associated with SHANK3 haploinsufficiency. Typically, other agents do not necessarily have to be administered in the same pharmaceutical composition, and due to different physical and chemical properties, may preferably be administered by different routes. Where possible, determining the mode of administration and the rationality of administration in the same pharmaceutical composition is entirely within the knowledge of a skilled clinician. Initial administration can be performed according to established protocols known in the art, and then based on the observed effects, a skilled clinician can modify the dosage, mode of administration, and time of administration.

[0162] Depending on the stage and progression of the condition associated with SHANK3 haploinsufficiency to be treated, the condition of the patient, and the choice of the specific therapeutic agent used, compositions and pharmaceutical compositions comprising an ASO and / or expression vector and an additional therapeutic agent may be administered concurrently (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen) or sequentially. Determining the order of administration of each therapeutic agent and the number of administration repetitions during the treatment regimen is well within the knowledge of the skilled physician after evaluating the disease being treated and the condition of the patient.

[0163] It is known to those skilled in the art that when drugs are used for treatment combination, the therapeutically effective dose can vary. Methods for experimentally determining the therapeutically effective dose of drugs and other medicaments for combination treatment regimens are described in the literature. For example, the use of metronomic administration has been widely described in the literature, that is, more frequent, lower dosages are provided to minimize toxic and side effects. Combination therapy further includes periodic treatments that start and stop at different times to help the clinical management of patients.

[0164] For combination therapy, dosages of the co-administered therapeutic agents will of course vary depending on the type of adjuvant, ASO or expression vector employed and the stage of the disease in the patient being treated.

[0165] The pharmaceutical composition comprising the ASO, AR or expression vector constituting the combination therapy disclosed herein and an additional therapeutic agent can be a combined dosage form or a separate dosage form intended for substantially simultaneous administration. The pharmaceutical composition constituting the combination therapy can also be administered sequentially, wherein any therapeutic agent is administered by a regimen requiring two-step administration. The two-step administration regimen may require sequential administration of active agents or separate administration of active agents. Depending on the characteristics of each agent, such as the efficacy, solubility, bioavailability, plasma half-life and kinetic characteristics of the agent, the time period between multiple administration steps can range from a few minutes to a few hours. The diurnal changes of various physiological parameters can also be evaluated to determine the optimal dosage interval.

[0166] 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.

[0167] Examples

[0168] Example 1: Identification of SHANK3 target sequences

[0169] The annotation and expression transcripts that can generate the canonical transcripts of the SHANK3 gene were identified by sequence alignment of all SHANK3 protein coding and NMD transcripts described in Gencode v38. A 25-mer and 17-mer ASO sequence "micro-walk" was performed in increments of 3 bases on the 5'PNCR of the SHANK3 canonical pre-RNA transcript and the 5'UTR and 3'UTR of the SHANK3 canonical mRNA transcript (ENST00000262795.6). The resulting ASO sequences corresponded to SEQ ID NOs: 5-4186, 12646-12671, and 12688 provided in Table 4 (Appendix).

[0170] Example 2: Measuring SHANK3 protein upregulation by PMOs targeting SHANK3 mRNA 5'UTR or 3'UTR in neuronal cell lines

[0171] A subset of the ASO sequences identified above were synthesized as phosphorodiamidate morpholino oligonucleotides (PMO 4-37) targeting the canonical SHANK3 mRNA 5'UTR, 5'PNCR and 3'UTR regions. The PMOs, corresponding SEQ ID NOs 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 an electroporation system (Thermo Fisher Scientific) and incubated for 96 hours. At this point, total protein was extracted using 15% SDS complete lysis reagent, and mouse anti-SHANK3 monoclonal antibody (Merck, catalog number: SAB520004) at a dilution of 1:1000 in TBST buffer containing 5% BSA was used, followed by western blotting using goat anti-mouse IgG H&L antibody (Abcam, catalog number: ab216776) to assess SHANK3 protein levels. The expression level of SHANK3 protein was compared with cells without PMO transfection (untreated), controls treated with transfection conditions, and controls treated with scrambled / non-targeted control sequences. The average signal from SHANK3 after imaging analysis was normalized to the average signal of total protein and 'housekeeping gene' proteins (e.g., focal adhesion protein, beclin, SRSF4)

[0172]

[0173]

[0174] like Figure 1 , 4As shown in Figures 5 and 6, some of the PMOs tested induced an increase in SHANK3 protein levels relative to the control, while other PMOs failed to induce any changes in SHANK3 protein levels. Figure 2 and 6 As shown, a subset of PMOs increased SHANK3 protein levels from approximately 50% above control to more than fourfold.

[0175] Example 3: Measurement of SHANK3 protein upregulation by MOE oligonucleotides targeting SHANK3 mRNA 5'UTR or 3'UTR in neuronal cell lines

[0176] A subset of the ASO sequences identified above were synthesized as 2'-O-(2-methoxyethyl) (MOE) modified oligonucleotides that also contained fully phosphorothioated backbones targeting the canonical SHANK3 mRNA 5'UTR, 5'PNCR, and 3'UTR regions ("MOE" 1-6, 13-60). The numbered MOEs, corresponding SEQ ID NOs, and target regions are shown in Table 2 below. Antisense MOE was electroporated into SH-SY5Y neuronal cell line cultures at concentrations of 2.5 μM and 5.0 μM using an electroporation system (Thermo Fisher Scientific) 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 assessed by western blotting using a mouse anti-SHANK3 monoclonal antibody (Merck, catalog number: SAB520004) at a dilution of 1:1000 in TBST buffer containing 5% BSA, followed by a goat anti-mouse IgG H&L antibody (Abcam, catalog number: ab216776). The expression level of SHANK3 protein was compared with cells without MOE transfection (untreated), controls treated with transfection conditions, and controls treated with scrambled / non-targeted control sequences. The average signal from SHANK3 after imaging analysis was normalized to the average signal of total protein and 'housekeeping gene' proteins (e.g., focal adhesion protein, beclin, SRSF4)

[0177]

[0178]

[0179] like Fig.10 , 11 As shown in Figures 1 and 2, some of the tested MOEs induced an increase in SHANK3 protein levels relative to the control, while other MOEs failed to induce any changes in SHANK3 protein levels. Fig.13 As shown, a subset of MOE increased SHANK3 protein levels from approximately 50% above control to more than fourfold.

[0180] Example 4: Identification of SHANK3 target intron / exon sequences

[0181] Annotated and expressed transcripts that can produce typical transcripts of the SHANK3 gene were identified by sequence alignment of all SHANK3 protein coding and NMD transcripts described in Gencode v38. A 25-mer ASO sequence "microwalk" was performed on the sequence of introns 7, 17, 21 and exon 21 of the ENST00000262795.6 pre-mRNA transcript in 5 bp increments and was designed to target intron splicing enhancer motifs to mediate the exclusion of retained introns or portions thereof and produce productive SHANK3 mRNA transcripts. The resulting ASO sequences correspond to SEQ ID NOs: 4187-12644.

[0182] Example 5: Measuring SHANK3 protein upregulation by PMO targeting SHANK3 pre-mRNA in neuronal cell lines

[0183] The 25-nucleotide ASO sequences identified above were synthesized as phosphorodiamidate morpholino oligonucleotides (PMOs). Electroporation system (Thermo Fisher Scientific) electroporates antisense PMOs targeting intron 7, 18 or 21 (as described in Example 1) into the original SH-SY5Y neuronal cell line culture at a concentration of 25 μM and 50 μM and incubates for 96 hours. At this time, 15% SDS complete lysis reagent is used to extract total protein, and a mouse anti-SHANK3 monoclonal antibody (Merck, catalog number: SAB520004) with a dilution of 1:1000 is used in TBST buffer containing 5% BSA, followed by Western blotting to assess the level of SHANK3 protein using goat anti-mouse IgG H&L antibody (Abcam, catalog number: ab216776). The expression level of SHANK3 protein is compared with cells (UT) without PMO transfection.

[0184]

[0185]

[0186] like Figure 7 and 8 As shown, some of the tested PMOs induced an increase in SHANK3 protein levels relative to the control, while other PMOs failed to induce any changes in SHANK3 protein levels.

[0187] It will be appreciated by those skilled in the art that, without departing from the spirit or scope of the invention as broadly described, various changes and / or modifications may be made to the invention as shown in the specific examples. Therefore, the present invention examples should be considered to be illustrative and not restrictive in all aspects.

[0188] All publications cited herein are hereby incorporated by reference in their entirety. Where a URL or other such identifier or address is mentioned, it is understood that such identifiers may change, and particular information on the Internet may be added or deleted, but equivalent information may be found by searching the Internet. Reference hereto evidences the availability and public dissemination of such information.

[0189] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. This should not be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0190] References

[0191] Alia et al. (2019), Frontiers in Neuroscience, 13:684.

[0192] Boyé et al., (2022), Nature Communications, 13:1169.doi.org / 10.1038 / s41467-022-28785-9.

[0193] Dugal-Tessier et al., (2021), J Clin Med., 10(4):838.

[0194] Fitzgerald et al. (2020), Stem Cell, 38(11): 1375-1386.

[0195] Kim et al. (2014), Frontiers in Neuroscience, 8:109.

[0196] Prakash et al. (2019), Nucleic Acids Research, 47(12): 6029-6044.

[0197] Rak et al., (2016), J Gene Med, 18(11-12): 331-342.

[0198] Appendix: Sequence and SEQ ID NO

[0199] SEQ ID NO:1

[0200] SHANK3 mRNA typical transcript 5'UTR sequence (SHANK3-201 ENST00000262795.7)

[0201]

[0202] SEQ ID NO:2

[0203] SHANK3 mRNA typical transcript 3'UTR sequence (SHANK3-201 ENST00000262795.7)

[0204]

[0205] SEQ ID NO:3

[0206] SHANK3 5' PNCR of SHANK3 precursor RNA (SHANK3-201 ENST00000262795.7)

[0207]

[0208] SEQ ID NO:4

[0209] SHANK3 pre-mRNA typical sequence (SHANK3-201 ENST00000262795.7)

[0210]

[0211] Table 4: Exemplary ASO or AR sequences targeting SHANK3 mRNA 5'UTR or 3'UTR or SHANK3 5'PNCR

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313] Table 5: Exemplary PMO and MOE oligonucleotide sequences targeting SHANK3 mRNA 5'UTR or 3'UTR or SHANK3 5'PNCR

[0314]

[0315]

[0316] SEQ ID NO:12694

[0317] CPP amino acid sequence (artificial / synthetic)

[0318] RRSRTARAGRPGRNSSRPSAPR

[0319] The sequence table of the present application is provided in five separate attached .xml format files, and the entire contents of each file are incorporated herein by reference. The sequence table .xml file name (automatically generated by the WIPO sequence program), the sequence number range, the corresponding applicant-assigned SEQ ID NO range for each of these files, and the first sequence in each file are summarized in Table 6 below.

[0320]

[0321] If any differences are identified between (i) the sequence in the accompanying sequence listing file, the number corresponding to the SEQ ID NO, or the corresponding title / name provided in the accompanying sequence listing file; and (ii) the sequence and SEQ ID NO disclosed in this specification and the appendices thereto, the sequence and SEQ ID NO (and SEQ ID NO range) provided in this specification and the appendices are deemed to be correct.

Claims

1. An antisense oligonucleotide that binds within the targeted portion of: (i) 5' untranslated region (UTR) of SHANK3 mRNA; (ii) the 5' proximal noncoding region (PNCR) of SHANK3 pre-mRNA; or (iii) 3′UTR of SHANK3 mRNA; Incorporation of the antisense oligonucleotide into the targeted moiety in a mammalian cell thereby results in an increase in the level of SHANK3 protein in the mammalian cell.

2. A vector for expressing an antisense RNA (AR) in mammalian neurons, wherein the AR is bound within a targeted portion of: (i) 5' untranslated region (UTR) of SHANK3 mRNA; (ii) the 5' proximal noncoding region (PNCR) of SHANK3 pre-mRNA; or (iii) 3′UTR of SHANK3 mRNA; Thus, binding of the AR within the targeted moiety in a mammalian cell results in an increase in the level of SHANK3 protein in the mammalian cell.

3. The vector of 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 neurons of the mammal.

5. The vector of 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 any one of claims 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 of claim 9, wherein the composition further comprises a transfection agent.

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

12. The method or antisense oligonucleotide of claim 11, wherein the antisense oligonucleotide comprises a backbone modification comprising a phosphorothioate bond or a phosphorodiamidate bond.

13. The antisense oligonucleotide of claim 11 or claim 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 of 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 of claim 16, wherein each nucleotide of the antisense oligonucleotide comprises a 2'-O-methoxyethyl moiety.

18. The antisense oligonucleotide according to any one of claims 1 or 11 to 17, or the vector according to any one of claims 2 to 9, or the composition according to claim 10, wherein the nucleotide sequence of the antisense oligonucleotide or 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 of 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 of claim 21, wherein the functional moiety comprises a delivery moiety.

23. The antisense oligonucleotide of claim 22, wherein the delivery moiety is selected from the group consisting of a lipid, a polyether, a peptide, a carbohydrate, a receptor binding domain (RBD), and an antibody.

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

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

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

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

28. The antisense oligonucleotide of claim 21, wherein the functional portion comprises a stabilizing portion.

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

34. The antisense oligonucleotide of any one of claims 1 or 11 to 33, the vector of any one of claims 2 to 9, or the composition of claim 10, wherein the nucleotide sequence of the ASO or the AR corresponds to any one of the following: 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.

35. 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, wherein the binding is carried out within the targeted portion of the 5'UTR corresponding to SEQ ID NO:

1.

36. 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, wherein the binding is carried out within the targeted portion of the 5' PNCR corresponding to SEQ ID NO:

3.

37. 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, wherein the binding is carried out within the targeted portion of the 3'UTR corresponding to SEQ ID NO:

2.

38. The antisense oligonucleotide, vector or composition according to claim 35, wherein the nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of the following: SEQ ID NO: 5-622, 4175-4181 or 4184-4186.

39. The antisense oligonucleotide, vector or composition of claim 35, wherein the nucleotide sequence of the antisense oligonucleotide or the AR corresponds to any one of the following: SEQ ID NO: 559, 606 or 4178-4181.

40. The antisense oligonucleotide, vector or composition according to claim 36, wherein the nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of the following: SEQ ID NO: 1935-4168, 4182, 4183, 12646-12654 or 12664-12671.

41. The antisense oligonucleotide, vector or composition of claim 36, wherein the nucleotide sequence of the antisense oligonucleotide or the AR corresponds to any one of SEQ ID NO: 1935-1937, 2849, 2858, 2864, 2874, 3510, 12647, 12648 or 12664-12671.

42. The antisense oligonucleotide, vector or composition according to claim 37, wherein the nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of the following: SEQ ID NO: 623-1934, 4169-4174, 12645, 12655-12663 or 12688.

43. The antisense oligonucleotide, vector or composition according to claim 37, wherein the nucleotide sequence of the antisense oligonucleotide or the antisense RNA corresponds to any one of the following: SEQ ID NO: 1847, 1852, 1934, 12661-12663 or 12688.

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

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

46. ​​The antisense oligonucleotide of claim 44, wherein the delivery nanocarrier comprises a lipid nanoparticle (LNP) encapsulating the antisense oligonucleotide.

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 of claim 47.

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

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

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

52. Use of an antisense oligonucleotide, vector or composition according to any one of claims 1 to 46 for the preparation of a medicament for preventing or treating a condition associated with SHANK3 haploinsufficiency.

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

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 of claim 54, wherein the genetically modified cell is a mammalian cell.

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

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

58. The genetically modified mammalian neuron of claim 57, wherein the genetically modified mammalian cell 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 of claim 55 or claim 56, wherein the genetically modified mammalian cell is from a cell line.

60. The genetically modified mammalian cell of claim 59, wherein the cell line is a hiPSC cell line or a neuron-derived cell line.

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