Antisense oligonucleotides that increase Shank3 expression

Abstract

The present invention relates to an oligonucleotide that specifically binds to a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1. The present invention also relates to said oligonucleotide for use in medicine and for use in the treatment and / or prevention of Shank3 deficiency, preferably for use in the treatment and / or prevention of Phelan-McDermid syndrome. The present invention also relates to expression constructs, host cells, and methods related thereto.

Description

Technical Field

[0001] The present invention relates to oligonucleotides that specifically bind to a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1. The present invention also relates to said oligonucleotides for use in medicine and for use in the treatment and / or prevention of Shank3 deficiency, preferably for use in the treatment and / or prevention of Phelan-McDermid syndrome. The present invention also relates to expression constructs, host cells, and methods related thereto.

Background Art

[0002] Reduced expression of the Shank3 gene has been associated with diseases such as autism (Wang et al. (2019), Mol Psychiatry; doi.org / 10.1038 / s41380-018-0324-x) and Phelan-McDermid syndrome (PMDS). PMDS is a syndrome caused by deletions or mutations on chromosome 22, and thus affected patients have only one functional copy of the Shank3 gene. Symptoms associated with PMDS are specifically global developmental delay, intellectual disability, and speech, motor, and gait abnormalities (Phelan and McDermott, 2011). Notably, the Shank3 gene product has been found to stabilize synapses (Roussignol et al. (2005), J Neurosci 25(14):3560).

[0003] Currently available treatments for PMDS are primarily symptomatic, involving the administration of insulin and neurotrophic factors such as lithium, risperidone, or IGF-I, resulting in improvement of a single symptom (e.g., Darville et al., 2016). Increased Shank3 levels have been shown to lead to improved molecular properties and behavioral changes in diseased cells, both in vitro and in mouse models (Mei et al., 2016). The Shank3 gene has been found to be regulated by several miRNAs that reduce its gene expression (Choi et al., 2015).

[0004] Antisense oligonucleotides have been used, at least experimentally, to treat a variety of diseases, particularly neurodegenerative diseases. For example, the onset of spinocerebellar ataxia type 2 could be delayed in a mouse model by administration of antisense oligonucleotides (Scoles et al., 2017). Similarly, antisense oligonucleotides that bind to the 3' sequence of the translation initiation reduce the translation of α-4 integrin mRNA and suppress lymphocyte and monocyte migration in multiple sclerosis (Evers et al., 2015). Furthermore, antisense oligonucleotides have been investigated for the treatment of, for example, Usher syndrome type IIa, USH2A-asymptomatic retinal degeneration, and Pompe disease (US 10,131,910, WO 2015 / 190922 A1, DE 1998 122954, WO 2003 / 025144 A2). WO 2017 / 106382 A1 discloses the use of antisense oligonucleotides to enhance the production of correctly spliced ​​mRNA expressed from genes associated with central nervous system disorders, such as Shank3. [Overview of the Initiative]

[0005] Nevertheless, in the field of technology, there is still a need for means and methods to improve the treatment of Shank3 deficiency disorders.

[0006] This challenge is addressed by oligonucleotides, expression constructs, host cells, and methods comprising elements of the independent claims. Advantageous embodiments that can be realized individually or in any combination are described in the dependent claims.

[0007] Therefore, the present invention relates to an oligonucleotide that specifically binds to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1: Transfection of human CTRL and PMDS iPSCs with 50 bp antisense oligonucleotides. SHANK3 protein levels in the 110 kDa isoform after transfection were detected by Western blotting and normalized against SHANK3 levels in scrambled transfected iPSCs. SHANK3 levels (A / C) in transfected CTRL1 / PMDS2 iPSCs detected by Western blot (B / D). Some AONs increase SHANK3 expression, while others decrease its expression compared to scrambled transfected samples. [Figure 2] Figure 2: Transfection of iPSCs and motor neurons using the most effective AON obtained from screening. SHANK3 protein levels after transfection were detected by Western blotting and standardized against the SHANK3 levels of transfected iPSCs using scrambling. SHANK3 levels in transfected CTRL1 / PMDS2 iPSCs detected by Western blot (B) (A), and SHANK3 levels in transfected CTRL1 / PMDS2 motor neurons detected by Western blot (D) (C). The bar graph represents overall SHANK3 expression as three consecutive mean + mean standard error (SEM) (one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***). [Figure 3]Figure 3: SHANK3 expression in iPSCs transfected with 18bp AON prepared from the most effective 50bp AON. Post-transfection SHANK3 protein levels were detected by Western blotting and standardized for transfected iPSCs using scrambling. SHANK3 levels detected by Western blotting in transfected CTRL1 / PMDS2 iPSCs. The bar graph represents overall SHANK3 expression as the mean + standard error of the mean (SEM) of three consecutive bars (one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***). [Figure 4] Figure 4: SHANK3 levels in motor neurons after transfection with promising 18bp AON. d42 motor neurons derived from CTRL1 and PMDS2 were transfected, and SHANK3 levels were measured by Western blotting. The bar graph represents overall SHANK3 expression as three consecutive mean + mean standard error (SEM) (one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***). [Figure 5] Figure 5: SHANK3 expression in NPC motor neurons transfected with the most effective 18bp AON. The bar graph represents overall SHANK3 expression as the mean + standard error of the mean (SEM) of three consecutive bars (one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***). [Figure 6] Figure 6: SHANK3 expression detected by ICC in transfected motor neurons. NPC motor neurons stained with DAPI (nucleus), SHANK3, biotin, and NEFH (neurofilament). Merged (overlapping) and SHANK3 of all channels are shown. Bar graphs represent mean + SEM (one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***). Scale bars are 20 μm. [Figure 7]Figure 7: SHANK3 protein levels in transfected NPC motor neurons. Protein levels detected by Western blotting. Expression was normalized for scrambling and all isoforms were pooled. Bar graphs represent mean + SEM (n=3, one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***, p=0.001:****). [Figure 8] Figure 8: SHANK3 expression detected by ICC in transfected NPC motor neurons. NPC motor neurons stained with DAPI (nucleus), SHANK3, biotin, and MAP2 (neurofilament). Quantification of nuclear SHANK3 levels. Bar graphs represent mean values. Scale bar is 20 μm. [Figure 9-1] Figure 9: SHANK3 expression detected by ICC in transfected motor neurons. (A) Motor neurons stained with DAPI, SHANK3, biotin, and MAP2. Scale bar is 10 μm. [Figure 9-2] Figure 9: Expression of SHANK3 detected by ICC in transfected motor neurons. (B), (C) Quantification of nuclear SHANK3 levels. (B) shows standardized SHANK3 levels detected in each cell line, and (C) shows pooled data for controls and PMDS patients. Bar graphs represent mean + SEM (n=3, one-way ANOVA, p<0.05:*, p=0.01:**, p=0.001:***, p=0.0001:****). [Figure 10] Figure 10: Transfection of human CTRL and PMDS iPSCs with shifted 18 bp antisense oligonucleotides. SHANK3 protein levels in the 300 kDa isoform after transfection were detected by Western blotting and normalized to SHANK3 levels in untreated iPSCs. (A), (B) SHANK3 levels in transfected CTRL1 (A) or PMDS2 (B) iPSCs detected by Western blotting. [Modes for carrying out the invention]

[0009] In general, the terms used herein are given their usual and idiomatic meanings to those skilled in the art and should not be limited to any special or customized meanings unless otherwise indicated. The terms “have,” “comprise,” or “include,” as used below, or any grammatical variations thereof, are used in a non-exclusive sense. Thus, these terms may refer to both situations in which there are no other features in the entity described in this context besides the feature introduced by these terms, and situations in which there are one or more other features. For example, the expressions “A has B,” “A comprises B,” and “A includes B” may refer to both situations in which there are no other elements in A besides B (i.e., A consists exclusively of B), and situations in which there are one or more other elements in entity A besides B, such as element C, elements C and D, or other elements. Furthermore, as will be understood by those skilled in the art, the expressions “comprising a” and “comprising an” (“including (indefinite article + singular noun)”) preferably mean “including one or more,” that is, “including at least one.” Similarly, the expression “determining an X” means determining one X, but also means determining two or more X, for example, two X, three X, or four X. Also, the term “plurality” refers to a number of referent items, with at least two in one embodiment, at least three in another embodiment, at least four in yet another embodiment, and at least five in yet another embodiment.

[0010] Furthermore, the terms “preferably,” “more preferably,” “most preferably,” “in detail,” “more specifically,” “specifically,” “more specifically,” or similar terms used below are used in relation to any feature without limiting any further possibilities. Thus, any feature introduced by these terms is any feature and is not intended to limit the scope of the claims in any way. As will be recognized to those skilled in the art, the present invention may be carried out by using alternative features. Similarly, any feature introduced by “in one embodiment” or similar expressions is intended to be any feature without limiting any further embodiments of the present invention, without limiting any scope of the present invention, and without limiting any possibility of combining such introduced features with any other features or non-exclusive features of the present invention.

[0011] As used herein, the term “standard conditions” refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., preferably a temperature of 25°C and an absolute pressure of 100 kPa, unless otherwise specified; preferably, the standard conditions also include pH 7. Furthermore, the term “about” refers to a stated value with a technical precision generally accepted in the art, unless otherwise specified, preferably within ±20%, more preferably ±10%, and most preferably ±5% of the stated value. Furthermore, the expression “essentially” indicates that there are no deviations that would affect the indicated results or use, i.e., any possible deviations would not cause the indicated results to deviate by more than ±20%, more preferably more than ±10%, and most preferably more than ±5%. Thus, “essentially consisting of” means that it contains the specified components but excludes other components, except for components present as impurities, components present as a result of processes used to provide the specified components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase “essentially consisting of” includes any known acceptable additives, pharmaceutical additives, diluents, carriers, etc. Preferably, a composition consisting of essentially a set of components contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of non-specific components (multiple components are possible). With respect to nucleic acid sequences, the expression "essentially identical" indicates a percentage identity of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%. Naturally, the term "essentially identical" includes 100% identity. The same applies to the expression "essentially complementary." Unless otherwise specified, the letters representing amino acids and nucleotides are those of WIPO standard ST.25.

[0012] As used herein, the term “polynucleotide” refers to a polynucleotide comprising the nucleic acid sequence shown in SEQ ID NO: 1, fragments thereof, and / or variants thereof, as defined below. Preferably, the polynucleotide comprises at least 10, more preferably at least 12, and even more preferably at least 14 consecutive bases from nucleotides 100 to 300 of SEQ ID NO: 1, preferably nucleotides 50 to 600 of SEQ ID NO: 1, more preferably nucleotides 1 to 1000 of SEQ ID NO: 1, but most preferably comprising the nucleotides of SEQ ID NO: 1. Preferably, the polynucleotide is a nucleic acid molecule comprising the nucleic acid sequence of Genbank acceptance number NM_0013720044.1, preferably a nucleic acid molecule consisting of this nucleic acid sequence, i.e., a gene or RNA encoding the Shank3 polypeptide, and preferably an mRNA encoding the Shank3 polypeptide. Preferably, the encoded Shank3 polypeptide is a human Shank3 polypeptide, and more preferably has Genbank acceptance number NP_001358973.1. As will be understood by those skilled in the art, several isoforms of the Shank3 protein exist, and therefore, the encoding mRNAs are all preferably included as polynucleotides as described herein. Naturally, polypeptides having the amino acid sequences detailed above may also be encoded by two or more polynucleotides due to the degeneracy of the genetic code. Furthermore, the term polynucleotide encompasses variants of the particular polynucleotides described above. Such variants may represent orthologues, paralogs, or other homologs of a given polynucleotide. A polynucleotide variant preferably comprises a nucleic acid sequence whose sequence is obtained from the particular nucleic acid sequence described above by at least one nucleotide substitution, addition, and / or deletion, provided that the polynucleotide is preferably contained in the gene encoding the Shank3 polypeptide, or in RNA transcribed therefrom, preferably mRNA.The variants also include polynucleotides containing nucleic acid sequences that can hybridize with the specific nucleic acid sequences described above, preferably under stringent hybridization conditions. Such stringent conditions are known to those skilled in the art and are described in standard textbooks. A preferred example of stringent hybridization conditions is hybridization in 6 × sodium chloride / sodium citrate (=SSC) at about 45°C, followed by one or more washing steps in 0.2 × SSC and 0.1% SDS at 50–65°C. As is well known to those skilled in the art, these hybridization conditions vary depending on the type of nucleic acid, and there are differences in buffer temperature and concentration, for example, when organic solvents are present. For example, under “standard hybridization conditions,” the temperature varies depending on the type of nucleic acid, and is between 42°C and 58°C in aqueous buffer at a concentration of 0.1 × ~ 5 × SSC (pH 7.2). If an organic solvent, such as 50% formamide, is present in the buffer, the temperature under standard conditions is about 42°C. The hybridization conditions for DNA:DNA hybrids are preferably, for example, 0.1 × SSC and 20°C to 45°C, but more preferably 30°C to 45°C. The hybridization conditions for DNA:RNA hybrids are preferably, for example, 0.1 × SSC and 30°C to 55°C, but more preferably 45°C to 55°C. The above hybridization temperatures are determined, for example, for nucleic acids with a length of about 100 bp (= base pairs) and a G+C content of 50% in the absence of formamide. Those skilled in the art will know how to determine the required hybridization conditions by referring to textbooks. Alternatively, polynucleotide variants can be obtained by PCR-based techniques, such as amplification based on mixed oligonucleotide primers of DNA, i.e., by using degenerate primers for the conserved domains of the polypeptide of the present invention. The conserved domains of the polypeptide of the present invention can be identified by sequence comparison of the nucleic acid sequence of the polynucleotide of the present invention or the amino acid sequence of the polypeptide identified above. Appropriate PCR conditions are well known in the art.Mammalian DNA or cDNA, particularly human DNA, can be used as a template. Furthermore, variants also include polynucleotides containing nucleic acid sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid sequences detailed above. In addition, polynucleotides containing nucleic acid sequences encoding amino acid sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequences shown above. The identity percentage value is preferably calculated over the entire amino acid or nucleic acid sequence region. To compare different sequences, those skilled in the art can utilize a range of programs based on various algorithms. In this regard, the Needleman and Wunsch, or Smith and Waterman algorithms, provide particularly reliable results. To perform sequence alignment, the PileUp program (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the Gap and BestFit programs (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)), and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))) should be used, as these are part of the GCG software packet (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)).The sequence identity values ​​expressed as percentages (%) above should preferably be determined using the GAP program across the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000, and Average Mismatch: 0.000, and these settings shall always be used as the standard settings for sequence alignment unless otherwise specified. Polynucleotides containing fragments of any of the above nucleic acid sequences are also preferably included as polynucleotides. Fragments as used herein may also preferably contain at least 50, at least 100, at least 250, or at least 500 consecutive nucleotides of any one of the above nucleic acid sequences. Polynucleotides consist of or contain the above nucleic acid sequences in essence. Therefore, polynucleotides may contain additional nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode a fusion protein such that one partner of the fusion protein is a polypeptide encoded by the above nucleic acid sequences. The polynucleotides are preferably provided as isolated polynucleotides (i.e., polynucleotides isolated from their natural background) or as genetically modified polynucleotides. The polynucleotides are preferably DNA, including cDNA, or RNA, but preferably mRNA. The term encompasses single-stranded and double-stranded polynucleotides, and preferably refers to single-stranded polynucleotides. In addition, chemically modified polynucleotides are also preferably included, which include naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified ones such as biotinylated polynucleotides. Preferably, the polynucleotides have a length of 10 bp to 5 Mb, more preferably 10 bp to 100 kb, even more preferably 50 bp to 20 kb, and most preferably 100 bp to 10 kb.

[0013] The term “oligonucleotide” as used herein will be understood by those skilled in the art. The term preferably relates to nucleic acid molecules containing 10 to 100 nucleic acid bases, more preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 nucleic acid bases. Oligonucleotides as referred herein have the activity to bind to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1, and the binding preferably refers to specific binding, more specifically specific binding under standard conditions, and more preferably specific binding under stringent conditions as described in another section herein; also preferably, the binding relates to in vivo binding, i.e., binding under conditions within a living cell. Preferably, oligonucleotides contain, and more preferably consist of, a sequence of at least 10, preferably at least 12, more preferably at least 14 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000, more preferably nucleotides 50 to 600, and most preferably nucleotides 100 to 300. More preferably, the oligonucleotide comprises 10 to 100, preferably 12 to 50, more preferably 14 to 25, most preferably about 18 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000, more preferably nucleotides 50 to 600, and most preferably nucleotides 100 to 300. In a preferred embodiment, the oligonucleotide comprises, more preferably, at least 10, preferably at least 12, more preferably at least 14 consecutive bases that are essentially complementary to nucleotides 140 to 200 of SEQ ID NO: 1. Even more preferably, the oligonucleotide comprises 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000, more preferably nucleotides 50 to 600, and most preferably nucleotides 100 to 300.Most preferably, the oligonucleotide comprises, and more preferably consists of, a sequence of 10 to 100, preferably 12 to 50, more preferably 14 to 25, most preferably about 18 consecutive bases, which are essentially identical to SEQ ID NO: 2, more preferably SEQ ID NO: 3. In addition, the term oligonucleotide further encompasses variants of the indicated specific oligonucleotide. Oligonucleotide variants preferably comprise nucleic acid sequences whose sequence is obtained from the above-mentioned specific nucleic acid sequence by substitution, addition and / or deletion of at least one nucleotide, and the variant oligonucleotide still possesses the specified binding properties. Furthermore, oligonucleotides comprising nucleic acid sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the above-mentioned specific nucleic acid sequence. The percentage identity value is preferably calculated over the entire nucleic acid sequence region, as specified above for the polynucleotide described herein. As will be understood by those skilled in the art, the parameters of the determination algorithm can be specifically corrected for short sequence comparisons. Oligonucleotides consist essentially of or contain the above nucleic acid sequences; therefore, oligonucleotides may contain additional nucleic acid sequences. However, more preferably, oligonucleotides consist of the above nucleic acid sequences. Oligonucleotides are preferably provided as isolated oligonucleotides (i.e., oligonucleotides isolated from their native background) or as genetically modified oligonucleotides. Oligonucleotides are preferably DNA including cDNA, or RNA, but more preferably DNA. The term encompasses single-stranded and double-stranded oligonucleotides, and preferably relates to single-stranded molecules. In addition, chemically modified polynucleotides are also preferably included, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified ones such as biotinylated polynucleotides.Preferably, the oligonucleotide is modified to reduce its rate of degradation within cells; suitable modifications are known in the art and specifically include replacing one or more nucleotides, preferably at least two, more preferably at least three, with phosphorothioate nucleotides. Preferably, the oligonucleotide comprises, preferably consists of, any one nucleic acid sequence of SEQ ID NOs: 4-17, more preferably consisting of such nucleic acid sequences, but more preferably comprising, preferably consisting of, any one nucleic acid sequence of SEQ ID NOs: 12-17, more preferably 14-17, most preferably 15 or 17, and preferably consisting of such nucleic acid sequences. Also preferably, the oligonucleotide is included in a composition, preferably a pharmaceutical composition. In a preferred embodiment, the oligonucleotide comprises, preferably consisting of, any one nucleic acid sequence of SEQ ID NOs: 4-13 or 15-17, or consisting of the nucleic acid sequence of SEQ ID NO: 14. In a preferred embodiment, the oligonucleotide comprises, and more preferably consists of, one of the following nucleic acid sequences: SEQ ID NOs: 12-17 and 65, 70, 71, 75, 81, 83, 87-89; more preferably SEQ ID NOs: 14-17 and one of the following: 65, 70, 71, 75, 81, 83, 87-89; and most preferably one of the following nucleic acid sequences: SEQ ID NOs: 15, 17, 65, 71, 75, 81, 83, 87, 88, or 89.

[0014] As used herein, the term “composition” refers to any composition comprising the indicated component(s). Preferably, the composition is a pharmaceutical composition.

[0015] As used herein, the term “pharmaceutical composition” refers to a composition comprising the compounds of the present invention and, optionally, one or more pharmaceutically acceptable carriers, wherein the components of the pharmaceutical composition are provided in a pharmaceutically acceptable form, i.e., in a form that is not harmful to the recipient. Specifically, the compounds of the present invention can be formulated as pharmaceutically acceptable salts or solutions. Preferably acceptable salts include acetates, methyl esters, HCl, sulfates, and chlorides. The pharmaceutical composition is preferably administered topically or systemically, more preferably systemically. Suitable conventional routes of administration for drug administration are oral, intravenous, subcutaneous, or parenteral administration, as well as inhalation. Preferably, administration is intracerebral, epidural, and / or intraventricular. However, depending on the properties and mode of action of the compounds, the pharmaceutical composition may also be administered by other routes. Furthermore, the compounds can be administered in combination with other drugs as a common pharmaceutical composition, or as separate pharmaceutical compositions as specified elsewhere herein, where the separate pharmaceutical compositions may be provided in the form of a kit of parts. Preferably, the combined compound formulation is a sustained-release formulation of one or more of the compounds.

[0016] The compounds are preferably administered in conventional dosage forms, prepared by combining the drug with a standard pharmaceutical carrier according to conventional procedures. These procedures may include mixing, granulation and compression, or dissolution of components to suit the desired preparation. Naturally, the form and properties of a pharmaceutically acceptable carrier or diluent depend on the amount of active ingredient to be combined, the route of administration, and other well-known variables. The carrier(s) must be acceptable in the sense that they are compatible with the other components of the formulation and are not harmful to the recipient. Pharmaceutical carriers used can be, for example, solids, gels, or liquids. Examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic (acacia), magnesium stearate, and stearic acid. Exemplary liquid carriers include phosphate-buffered saline, syrups, oils such as peanut oil and olive oil, water, emulsions, various types of wetting agents, and sterile solutions. Similarly, the carrier or diluent may contain time retarders well known to those skilled in the art, such as glyceryl monostearate or glyceryl distearate, either alone or in combination with wax. Suitable carriers include those described above and others well known in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The diluent(s) are selected so as not to affect the compound or the biological activity of those compounds. Examples of such diluents are distilled water, saline, Ringer's solution, glucose solution, and Hanks' solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, reactive oxygen species scavengers, etc.

[0017] A therapeutically effective dose means the amount of compound used in the pharmaceutical composition of the present invention that prevents, improves, or treats the symptoms associated with the diseases or conditions referred to herein. The therapeutic effect and toxicity of such compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, such as determining the ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose ratio of therapeutic effect to toxic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. The administration plan is determined by the attending physician and other clinical factors; preferably, according to one of the methods described above. As is well known in the medical field, the dose for any one patient is determined by many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general condition, and other drugs being administered simultaneously. The progress can be monitored by periodic evaluation. Typical doses can be in the range of, for example, 1 μg to 1500 mg; however, doses below or above this exemplary range are conceivable, especially considering the factors described above. In general, regimens for the regular administration of pharmaceutical compositions should be in the range of 100 ng to 100 mg units per day. Even when the regimen is a continuous infusion, the range should be 100 ng to 100 mg units per minute per kg of body weight. Preferably, the continuous-release formulation of each drug is infused at intervals of once a week to once every two months, or more. The progress can be monitored by periodic evaluations. Preferred doses and concentrations of the compounds of the present invention are described in a separate section of this specification.

[0018] The pharmaceutical compositions and formulations described herein are preferably administered at least once, for example in the case of sustained release formulations, to treat or ameliorate or prevent the diseases or medical conditions described herein. However, the pharmaceutical compositions may be administered more than once, for example, they can be administered from once to four times a day for an unlimited number of days. Also, compounds with short clearance times can be applied as an infusion into the bloodstream to supply an effective amount throughout the body during a long treatment period.

[0019] Individual pharmaceutical compositions are prepared by methods well known in the pharmaceutical art and contain the above at least one active compound, either in a mixture with a pharmaceutically acceptable carrier or diluent or otherwise accompanied by them. To manufacture those individual pharmaceutical compositions, the active compound(s) are usually mixed with a carrier or diluent or enclosed or encapsulated in capsules, sachets, cachets, paper, or other suitable containers or vehicles. The resulting formulations will be employed in the method of administration in the form of tablets, capsules, suppositories, solutions, suspensions, etc. The recommended dosage shall be as indicated in the prescription or instructions for use so that dosage adjustment can be made in advance according to the recipient to be considered.

[0020] Advantageously, in the studies underlying the present invention, it has been found that the oligonucleotides of the present invention can increase the amount of Shank3 protein present in cells compared to the control. Without wishing to be bound by theory, it is presumed that the oligonucleotides prevent the degradation of Shank3 mRNA and thus increase the amount of protein produced from each mRNA molecule.

[0021] The above definitions shall apply mutatis mutandis hereinafter. Also, the additional definitions and explanations made hereinafter shall also apply mutatis mutandis to all embodiments described herein.

[0022] The present invention also relates to the nucleotides of the present invention for use in pharmaceuticals; and oligonucleotides of the present invention for use in the treatment and / or prevention of Shank3 deficiency, preferably for use in the treatment and / or prevention of Phelan-McDermid syndrome. The present invention also relates to pharmaceutical compositions comprising the oligonucleotides of the present invention, preferably for use in pharmaceuticals, preferably for use in the treatment and / or prevention of Shank3 deficiency, preferably for use in the treatment and / or prevention of Phelan-McDermid syndrome. The present invention also relates to the use of oligonucleotides of the present invention for producing pharmaceutical compositions, specifically pharmaceutical compositions for the treatment and / or prevention of Shank3 deficiency, preferably pharmaceutical compositions for the treatment and / or prevention of Phelan-McDermid syndrome.

[0023] The term “treatment” means a significant improvement in the disease or disorder described herein, or the symptoms associated therewith. As used herein, such treatment also includes the complete recovery of health related to the disease or disorder described herein. It should be understood that the treatments used in the present invention may not be effective for all subjects to be treated. However, this term preferably requires that a statistically significant portion of subjects suffering from the disease or disorder described herein can be successfully treated. Whether a portion is statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, p-values, Student's t-tests, and Mann-Whitney U tests. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferred p-values ​​are 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment is effective for at least 20%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the subjects in a given cohort or population. As will be obvious to those skilled in the art, the effectiveness of the treatment depends on various factors, including, for example, the stage and severity of the disease. Also obviously, the term treatment includes measures that prevent and / or delay the progression of the disease or disorder described herein.

[0024] The term “prevention” refers to maintaining health in a subject with respect to the disease or disorder described herein for a certain period of time. Naturally, the said period depends on the amount of the drug compound administered and the individual factors of the subject, which are discussed in other sections of this specification. Naturally, prevention may not be effective for all subjects treated with the compounds of the present invention. However, this term preferably requires that a statistically significant portion of a cohort or population of subjects be effectively prevented from developing the disease or disorder described herein, or its associated symptoms. Preferably, in this context, a cohort or population of subjects who would normally, i.e., without the preventive treatment according to the present invention, develop the disease or disorder described herein is assumed. Whether a portion is statistically significant can be readily determined by those skilled in the art using the various well-known statistical evaluation tools described separately herein. As used herein, the term prevention preferably includes gene therapy treatments, as described in other sections of this specification. Thus, subjects receiving preventive treatment may not have any symptoms of the disease, but may be identified, for example, by genetic analysis, as being at high risk of developing the disease or disorder described herein.

[0025] Those skilled in the art know that the term “Shank3 deficiency” relates to any disease or disorder caused by or exacerbated by a decrease in Shank3 expression in the cells of a subject, which results in morphological and neurological symptoms. Preferably, Shank3 deficiency is synaptic degeneration, particularly synaptic degeneration involving the loss of excitatory synapses; therefore, preferably, Shank3 deficiency is Alzheimer’s disease, amyotrophic lateral sclerosis, and / or stroke, particularly cerebral stroke. Preferably, Shank3 deficiency is Phelan-McDermid syndrome, also known as 22q13 deletion syndrome. The symptoms of Phelan-McDermid syndrome are known in the art, most commonly general developmental delay, intellectual disability, and language, motor, and gait abnormalities. Diagnosis of Phelan-McDermid syndrome is usually established by a combination of genetic, cognitive, and behavioral assessments. Shank3 deficiency can also be determined by measuring Shank3 expression in the subject's cells, for example, by the method described in the examples herein.

[0026] As used herein, the term “subject” refers to a vertebrate. Preferably, the subject is a mammal, more preferably a mouse, rat, cat, dog, hamster, guinea pig, sheep, goat, pig, cattle, or horse. Even more preferably, the subject is a primate. Most preferably, the subject is a human. Preferably, the subject is known to have, be at high risk of having, or suspect to have, Shank3 deficiency, particularly Phelan-McDermid syndrome.

[0027] Preferably, treatment and / or prevention of Shank3 deficiency comprises contacting a subject with the oligonucleotides described herein; more preferably, treatment and / or prevention of Shank3 deficiency comprises contacting the neurons of the subject with the oligonucleotides; also preferably, treatment and / or prevention comprises contacting the subject, preferably the neurons, with a plurality of non-identical oligonucleotides and / or with one or more constructs that induce the expression of the oligonucleotides described below.

[0028] The present invention also relates to an expression construct, which is preferably contained in an expression vector and encodes an oligonucleotide of the present invention.

[0029] As used herein, the term “expression construct” refers to a nucleic acid molecule encoding an oligonucleotide described herein, comprising at least one nucleic acid sequence for expressing the oligonucleotide in a host cell. Preferably, when the expression construct is DNA, the nucleic acid sequence for expressing the oligonucleotide is preferably a promoter, more preferably an RNA polymerase promoter. Those skilled in the art can select an appropriate promoter depending on the purpose; preferably, the promoter is an inductive promoter, more preferably a constitutive promoter. It is also preferable that the promoter is a cell-type specific promoter, particularly a neuron-specific promoter. More preferably, when the expression construct is RNA, the expression construct comprises at least one reverse transcriptase initiation site, i.e., preferably, the above-mentioned DNA oligonucleotide is expressed from the RNA expression construct in a host cell containing reverse transcriptase. Therefore, preferably, the expression construct further preferably comprises an expressible reverse transcriptase gene.

[0030] The present invention further relates to host cells, preferably neuronal host cells, comprising oligonucleotides and / or expression constructs described herein.

[0031] As used herein, the term “host cell” refers to a eukaryotic cell, preferably a vertebrate cell, more preferably a mammalian cell, and most preferably a human cell. Preferably, the host cell is a neuronal host cell. Preferably, the host cell comprises at least one polynucleotide polymerase, preferably a reverse transcriptase and / or RNA polymerase. More preferably, the polynucleotide polymerase initiates polymerization at the site of the nucleic acid sequence contained in the expression construct, preferably at the reverse transcriptase start site and / or promoter site.

[0032] The present invention also relates to a kit comprising oligonucleotides and administration means as described herein.

[0033] As used herein, the term “kit” refers to a collection of the above-mentioned compounds, means, or reagents, which may or may not be packaged together. The components of the kit may consist of separate vials (i.e., as kits of separate parts), or they may be supplied in a single vial. Furthermore, naturally, the kits of the present invention should preferably be used to carry out the methods described in other sections herein. Preferably, it is assumed that all components are provided ready for immediate use to carry out the above methods. Furthermore, the kit preferably includes instructions for use to carry out the above methods. These instructions may be provided in the form of a user manual, either in paper or electronic format. In addition, this manual may include instructions for administration and / or dosage instructions for carrying out the above methods using the kits of the present invention. As can be understood from the above, preferably, the description of a kit containing oligonucleotides relates to a kit containing the corresponding expression construct, with necessary modifications.

[0034] Preferably, the kit comprises the oligonucleotides described herein and an administration means. The administration means is any means suitable for administering the oligonucleotides and / or expression constructs to a subject. Therefore, preferably, the administration means is any means configured and / or suitable for introducing the oligonucleotides and / or expression constructs into the body of a subject and / or into the host cells of the subject. The administration means may include a delivery unit for administering the compound or composition and a storage unit for storing the compound or composition until administration. However, the means of the present invention may also appear as separate devices in such embodiments, and preferably may be packaged together in the kit. A preferred administration means is one that can be applied without the special knowledge of a specialist. Preferably, the administration means is a syringe, more preferably a syringe with a needle, containing the compound or composition of the present invention. In another preferred embodiment, the administration means is an intravenous infusion (IV) device containing the compound or composition. In yet another preferred embodiment, the administration means is an inhaler containing the compound of the present invention, more preferably the compound is formulated for administration as an aerosol. Preferably, the administration means is a chemical reagent that induces or improves the uptake of oligonucleotides and / or expression constructs into host cells. Therefore, the administration means may be a transfection reagent. Suitable reagents are known in the art.

[0035] The present invention also relates to a device comprising an oligonucleotide and / or an expression construct as described herein.

[0036] The device may be one of the above-described administration means; however, the device may also be a device configured to carry out one of the in vitro methods of the present invention, specifically a pipetting device and / or incubation device configured to carry out a method for determining whether a subject suffering from Shank3 deficiency is suitable for treatment with the following oligonucleotides.

[0037] The present invention also relates to a method for increasing Shank3 expression in host cells, the method comprising contacting the host cells with an oligonucleotide described herein to increase Shank3 expression.

[0038] The method for increasing Shank3 expression according to the present invention is preferably an in vitro method. Furthermore, the method may include additional steps in addition to the steps expressed above. For example, the additional steps may relate to providing oligonucleotides or incubation after contact with the host cells, etc. Furthermore, one or more of the above steps may be performed by an automated apparatus.

[0039] The present invention also relates to a method for treating Shank3 deficiency in a subject, the method comprising contacting the subject with an oligonucleotide described herein, thereby treating Shank3 deficiency.

[0040] The present invention further relates to a method for determining whether a subject suffering from Shank3 deficiency is suitable for treatment with oligonucleotides described herein, the method comprising: a) The step of bringing the cell sample of the subject into contact with the oligonucleotide; b) A step of measuring the amount of Shank3 polypeptide in the cell sample; c) A step of comparing the amount of Shank3 polypeptide measured in step b) to a reference; and d) A step to determine whether a subject suffering from Shank3 deficiency is suitable for treatment, based on the results of the comparison step c).

[0041] A method for determining whether a subject suffering from Shank3 deficiency is suitable for the treatment of the present invention is preferably an in vitro method. Furthermore, the method may include additional steps in addition to those explicitly stated above. For example, the additional steps may relate to providing a cell sample for step a) and / or incubating the cells of step a) before step b), for example, to increase Shank3 expression. Preferably, the method further includes a1) providing a control cell sample that has not been in contact with the oligonucleotide, and b1) measuring the amount of Shank3 polypeptide in the control cell sample, and step c) comparing the amount of Shank3 polypeptide measured in step b) with the amount of Shank3 polypeptide measured in step b1); preferably, in such a case, if the amount of Shank3 measured in step b) is higher, preferably significantly higher, than the amount of Shank3 measured in step b1), then the subject suffering from Shank3 deficiency is confirmed to be suitable for treatment. Furthermore, one or more of the above steps may be performed by an automated apparatus.

[0042] As used herein, the term “cell sample” refers to a sample containing intact cells, preferably isolated cells, from the tissue or organs of a subject. Cultured cell samples are also included as samples. Methods for obtaining cell samples are known in the art and include, specifically, biopsy, excision, and blood collection. Preferably, the cell sample is a sample containing blood cells, but a sample containing nerve cells is also preferred.

[0043] The term “criteria” is known to those skilled in the art to refer to an identifier that can confirm whether a subject suffering from Shank3 deficiency is suitable for treatment with the oligonucleotides described herein. Such an identifier may be a target value for Shank3 concentration or a target value for relative amounts in host cells. Preferably, the criterion is a value such as a quantitative index, more preferably a threshold, a range such as a range of values, a score, or any other value or range that is deemed appropriate by those skilled in the art. Preferably, the criterion is determined based on (i) a control cell sample of the subject that has not been exposed to the oligonucleotide; (ii) a population of seemingly healthy subjects; (iii) a population of subjects found not to have Shank3 deficiency; (iv) a population of subjects found to have Shank3 deficiency. Preferably, the criterion is obtained from a subject or group of subjects suffering from Shank3 deficiency; in that case, if the criterion is, for example, a threshold, then a subject suffering from Shank3 deficiency suitable for the specified treatment is identified if the amount of Shank3 measured in step b) is higher than the criterion. Preferably, the criteria are obtained from subjects or groups of subjects who have been found not to have Shank3 deficiency; in that case, if the criteria is, for example, a threshold, subjects with Shank3 deficiency who are suitable for the specified treatment are identified if the amount of Shank3 measured in step b) is the same as or higher than the criteria; in the latter case, the criteria may also be based on a population of seemingly healthy subjects.Therefore, preferably, the criteria are: (I) an amount of Shank3 based on any one of (i) to (iii) above, where an amount that is essentially increased or higher than the criterion indicates a subject suitable for the treatment; (II) an amount of Shank3 based on (iv) above, where an amount that is essentially the same as or lower than the criterion indicates a subject unsuitable for the treatment; (III) a reference range determined based on any one of (i) to (iv) above, or any combination of (i) to (iv) above, preferably determined based on any one of (i) to (iii) above and (iv) above, where an amount within the reference range indicates a subject suitable for the treatment; and (IV) a score calculated including any one of (i) to (iv) above.

[0044] Based on the above, the following embodiments are particularly conceivable.

[0045] Embodiment 1: An oligonucleotide that specifically binds to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1, preferably nucleotides 1 to 1000 of SEQ ID NO: 1, more preferably nucleotides 50 to 600 of SEQ ID NO: 1, and most preferably nucleotides 100 to 300 of SEQ ID NO: 1.

[0046] Embodiment 2: The oligonucleotide according to Embodiment 1, wherein the oligonucleotide comprises at least 10, preferably at least 12, more preferably at least 14 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000 of SEQ ID NO: 1, more preferably nucleotides 50 to 600 of SEQ ID NO: 1, and most preferably nucleotides 100 to 300 of SEQ ID NO: 1.

[0047] Embodiment 3: The oligonucleotide according to Embodiment 1 or 2, wherein the oligonucleotide comprises 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000, more preferably nucleotides 50 to 600, and most preferably nucleotides 100 to 300 of SEQ ID NO: 1.

[0048] Embodiment 4: The oligonucleotide according to any one of Embodiments 1 to 3, wherein the oligonucleotide consists of 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000, more preferably nucleotides 50 to 600, and most preferably nucleotides 100 to 300 of SEQ ID NO: 1.

[0049] Embodiment 5: The oligonucleotide according to any one of Embodiments 1 to 4, wherein the oligonucleotide contains 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases, which are essentially identical to SEQ ID NO: 2, more preferably SEQ ID NO: 3.

[0050] Embodiment 6: The oligonucleotide according to any one of Embodiments 1 to 5, wherein the oligonucleotide consists of 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases, which are essentially identical to SEQ ID NO: 2.

[0051] Embodiment 7: The oligonucleotide according to any one of Embodiments 1 to 6, wherein the oligonucleotide consists of 10 to 100 nucleotides, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 nucleotides.

[0052] Embodiment 8: The oligonucleotide according to any one of Embodiments 1 to 7, wherein the oligonucleotide comprises one of the nucleic acid sequences of SEQ ID NOs: 4 to 17, preferably SEQ ID NOs: 12 to 17, and preferably consists of the said nucleic acid sequence.

[0053] Embodiment 9: The oligonucleotide according to any one of Embodiments 1 to 8, wherein the oligonucleotide comprises one of SEQ ID NOs: 14 to 17, preferably SEQ ID NO: 15 or 17, and preferably consists of the said nucleic acid sequence.

[0054] Embodiment 10: The oligonucleotide according to any one of Embodiments 1 to 9, wherein the oligonucleotide comprises a deoxynucleotide, preferably a DNA oligonucleotide, and / or a phosphorothioate-type nucleotide.

[0055] Embodiment 11: The oligonucleotide according to any one of Embodiments 1 to 10, wherein the oligonucleotide is bound to at least one of the membrane transport signal and the nuclear localization signal, preferably covalently.

[0056] Embodiment 12: An oligonucleotide according to any one of Embodiments 1 to 11, which is contained in a composition, preferably a pharmaceutical composition.

[0057] Embodiment 13: An oligonucleotide according to any one of Embodiments 1 to 12 for use in pharmaceuticals.

[0058] Embodiment 14: An oligonucleotide according to any one of Embodiments 1 to 12, for use in the treatment and / or prevention of Shank3 deficiency, preferably for use in the treatment and / or prevention of Phelan-McDermid syndrome.

[0059] Embodiment 15: The oligonucleotide for use according to Embodiment 14, wherein the use comprises bringing the neurons of a subject into contact with the oligonucleotide.

[0060] Embodiment 16: The oligonucleotide for use according to Embodiment 14 or 15, wherein the use comprises contacting the neurons of a subject suffering from Shank3 deficiency with a construct that induces the expression of the oligonucleotide within the neurons, preferably the expression construct described in Embodiment 19.

[0061] Embodiment 17: An oligonucleotide for use according to Embodiment 15 or 16, wherein the use comprises contacting the neuron with a plurality of non-identical oligonucleotides as described in any one of Embodiments 1 to 12.

[0062] Embodiment 18: A pharmaceutical composition comprising an oligonucleotide according to any one of Embodiments 1 to 12.

[0063] Embodiment 19: An expression construct, preferably contained in an expression vector, which encodes an oligonucleotide described in any one of Embodiments 1 to 12.

[0064] Embodiment 20: A host cell, preferably a neuronal host cell, comprising an oligonucleotide according to any one of Embodiments 1 to 12 and / or an expression construct according to Embodiment 19.

[0065] Embodiment 21: A kit comprising an oligonucleotide according to any one of Embodiments 1 to 12, and / or an expression construct according to Embodiment 19; and means for administering them.

[0066] Embodiment 22: A device comprising an oligonucleotide according to any one of Embodiments 1 to 12, and / or an oligonucleotide according to Embodiment 19, and / or an expression construct.

[0067] Embodiment 23: A method for increasing Shank3 expression in host cells, comprising contacting the host cells with an oligonucleotide described in any one of Embodiments 1 to 12, thereby increasing Shank3 expression.

[0068] Embodiment 24: The method according to Embodiment 23, wherein the method is an in vitro method.

[0069] Embodiment 25: A method for treating Shank3 deficiency in a subject, the method comprising contacting the subject with an oligonucleotide described in any one of Embodiments 1 to 12, thereby treating Shank3 deficiency.

[0070] Embodiment 26: The method according to Embodiment 25, wherein the neurons of the subject are brought into contact with the oligonucleotide.

[0071] Embodiment 27: A method for determining whether a subject suffering from Shank3 deficiency is suitable for treatment with an oligonucleotide described in any one of Embodiments 1 to 12, wherein the method is a) The step of bringing the cell sample of the subject into contact with the oligonucleotide; b) A step of measuring the amount of Shank3 polypeptide in the cell sample; c) A step of comparing the amount of Shank3 polypeptide measured in step b) to a reference; and d) A step to determine whether a subject suffering from Shank3 deficiency is suitable for treatment, based on the results of the comparison step c). The method, including the method described above.

[0072] Embodiment 28: The above criteria are, (i) A control cell sample of the subject that has not been in contact with the oligonucleotide; (ii) A group of subjects who appear healthy; (iii) A group of subjects who have been found not to have Shank3 deficiency; (iv) A group of subjects who have been found to have Shank3 deficiency The method according to embodiment 27, determined based on the following.

[0073] Embodiment 29: The above criteria are, (I) A quantity of Shank3 based on any one of (i) to (iii) of Embodiment 28, in which case an amount that is essentially increased or higher than the criterion indicates a subject suitable for the treatment; (II) An amount of Shank3 based on Embodiment 28(iv), in which case an amount essentially the same as or lower than the aforementioned standard indicates a subject unsuitable for the treatment; (III) A reference range determined based on any one of Embodiments 28(i) to (iv) or any combination of Embodiments 28(i) to (iv), preferably based on any one of Embodiments 28(i) to (iii) and Embodiment 28(iv), wherein the amount within the reference range indicates a subject suitable for the treatment; (IV) A score calculated including any one of (i) to (iv) of Embodiment 28, The method described in Embodiment 28.

[0074] Embodiment 30: Use of an oligonucleotide according to any one of Embodiments 1 to 12 for manufacturing a drug for treating and / or preventing Shank3 deficiency.

[0075] All documents cited herein are incorporated herein by reference, in their entirety and in any way specifically referred to herein. The present invention includes, for example, the following embodiments: [1] An oligonucleotide specifically binding to a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1 for use in the treatment and / or prevention of Shank3 deficiency, wherein the oligonucleotide comprises a deoxynucleotide, and the oligonucleotide comprises at least 10 consecutive bases that are essentially complementary to SEQ ID NO: 1, preferably nucleotides 1 to 1000 of SEQ ID NO: 1, more preferably nucleotides 50 to 600 of SEQ ID NO: 1, and most preferably nucleotides 100 to 300 of SEQ ID NO: 1. [2] The oligonucleotide for use according to [1], wherein the Shank3 deficiency is Phelan-McDermid syndrome. [3] The oligonucleotide according to [1] or [2], wherein the oligonucleotide comprises 10 to 100 consecutive bases that are essentially complementary to SEQ ID NO: 1. [4] The oligonucleotide according to any one of [1] to [3], wherein the oligonucleotide comprises 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 consecutive bases that are essentially identical to SEQ ID NO: 2, preferably SEQ ID NO: 3. [5] The oligonucleotide according to any one of [1] to [4], wherein the oligonucleotide consists of 10 to 100, preferably 12 to 50, more preferably 14 to 25, and most preferably about 18 nucleotides. [6] The oligonucleotide according to any one of [1] to [5], wherein the oligonucleotide comprises the nucleic acid sequence described in any one of SEQ ID NOs: 4 to 17, and preferably consists of that nucleic acid sequence. [7] The oligonucleotide according to any one of [1] to [6], wherein the oligonucleotide comprises the nucleic acid sequence described in SEQ ID NO: 15 or 17, and preferably consists of that nucleic acid sequence. [8] The oligonucleotide according to any one of [1] to [7], wherein the oligonucleotide is a DNA oligonucleotide. [9] Oligonucleotides according to any one of [1] to [8], which are contained in a composition, preferably a pharmaceutical composition.

[10] An oligonucleotide that specifically binds to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1, wherein the oligonucleotide contains a deoxynucleotide, and the oligonucleotide contains one nucleic acid sequence from SEQ ID NOs: 4 to 13 or SEQ ID NOs: 15 to 17, preferably consisting of that nucleic acid sequence, or consisting of the nucleic acid sequence of SEQ ID NO: 14.

[11] Oligonucleotides as described in

[10] for use in pharmaceuticals.

[12] An expression construct encoding an oligonucleotide according to any of [1] to [9] for use in the treatment and / or prevention of Shank3 deficiency, preferably comprising an expression vector.

[13] Host cells, preferably neuronal host cells, for use in the treatment and / or prevention of Shank3 deficiency, comprising oligonucleotides described in any of [1] to [9] and / or the expression construct described in

[12] .

[14] A method for determining whether a subject suffering from Shank3 deficiency is suitable for treatment with an oligonucleotide described in any of [1] to [9], wherein the method is a) The step of bringing the cell sample of the subject into contact with the oligonucleotide; b) A step of measuring the amount of Shank3 polypeptide in the cell sample; c) A step of comparing the amount of Shank3 polypeptide measured in step b) to a baseline; and d) A step of determining whether a subject suffering from Shank3 deficiency is suitable for treatment based on the results of comparison step c). The method, including the method described above.

[15] The above criteria (i) A control cell sample of the subject that has not been in contact with the oligonucleotide; (ii) A group of subjects who appear healthy; (iii) A group of subjects who have been found not to have Shank3 deficiency; (iv) A group of subjects who have been found to have Shank3 deficiency The method described in

[14] , which is determined based on

[14] . A kit comprising the oligonucleotide described in

[16]

[10] and means for administering the same.

[17] A method for increasing Shank3 expression in host cells, comprising contacting the host cells with an oligonucleotide described in any of [1] to

[12] , thereby increasing Shank3 expression.

[0076] The following embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way.

[0077] [Example 1] method All reagents used, their manufacturers, and catalog numbers can be found in Table 1.

[0078] [Table 1] JPEG0007911969000002.jpg150165

[0079] [Example 2] iPSC and differentiation Human induced pluripotent stem cells (iPSCs) were generated from hair follicles extracted from PMDS patients and healthy controls using a protocol previously established by Linta et al. (2012), Stem Cells and Development, 21(6):965, to reprogram keratinocytes into iPSCs. Two healthy control cell lines and three cell lines generated from PMDS patients were used. Their deletion sizes, clinical phenotypes, and detailed information can be found in Table 2.

[0080] [Table 2-1]

[0081] Human iPS cells were cultured in mTeSR1 medium on Matrigel-coated plates in a feeder-free system in a 37°C, 5% CO2 incubator under a reduced oxygen concentration (5% O2) atmosphere. All cell culture procedures were performed under sterile conditions. Spontaneously differentiated cells were mechanically removed daily using a pipette tip, followed by a washing step with DMEM-F12 to remove differentiated or dead cells. After washing, 1.5 mL of fresh mTeSR1 was added. When the cells reached approximately 80% confluence or large colony size, they were split using Dispase in a ratio of 1:3 to 1:8 and transferred to new 6-well plates.

[0082] Motor neurons were generated using two different protocols. Motor neurons directly generated from iPSCs according to the protocol published in Hu & Zhang (2009), Nature Protocols 4(9):1295, are referred to as mn (Method 1), while motor neurons generated from neural progenitor cells (NPCs) are referred to as NPC-mn (Method 2). To differentiate human iPSCs into mn for immunocytochemistry, the protocol published in Catanese and colleagues (2019), Autophagy 15(10):1719 was followed.

[0083] At least six wells of densely grown iPSCs were used to initiate differentiation to mn for Western blot analysis. For the next 28 days, the cells were treated as described in the scheme previously published by Hu & Zhang (2009, loc. cit.).

[0084] Method 1: Day 1: Creation of embryoid bodies Human iPSCs were detached from 10 mL of hESC medium (77% DMEM / F12, 20% KO-SR, 1% NEAA, 1% Antibiotic-Antimycotic, 1% β-mercaptoethanol) and transferred to a low-adhesion flask. 10 μM Rho-related kinase (ROCK) inhibitor was added during the first 48 hours. Embryoid bodies (EBs) formed after 1 day. The embryoid bodies were allowed to settle at the bottom of the flask, and a portion of the medium was replaced daily by replacing 5–7 mL of the medium with fresh ESC medium.

[0085] Days 4-8: Induction of neuronal differentiation All subsequent differentiation media were based on mn basic medium (96% DMEM / F12, 2% Hormone Mix, 1% NEAA, 1% Antibiotic-Antimycotic, 0.00004% heparin (50 mg / mL)). After cell sedimentation, 7 mL of the old medium was removed and replaced with Diff. 1 medium (mn basic medium, 1:1000 GDNF, BDNF, IGF-1 and Vitamin C, 1:10000 cAMP), which contains brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and insulin-like growth factor 1 (IGF1) along with vitamin C (Vitamin C) and cyclic adenosine monophosphate (cAMP). A portion of the medium was replaced daily.

[0086] Day 8: Cell plating and formation of nerve cell rosettes One 12-well plate was coated with laminin (1:50 in mn basal medium, 1 hour, 37°C). EB was transferred to a Falcon tube and centrifuged at 100g for 2 minutes. The supernatant was discarded, and EB was carefully resuspended in 6 mL of Diff. 1 medium. 0.5 mL of the EB suspension was transferred to each well of the 12-well plate. After 24 hours, 0.5 mL of Diff. 1 medium was added to each well.

[0087] Days 10-14: Induction of caudalization The culture medium was changed to Diff. 2 medium containing retinoic acid (RA) (mn basic medium, 1:1000 GDNF, BDNF, IGF-1, and Vitamin C, 1:10000 cAMP, and RA) to induce caudalization. The medium was changed three times a week.

[0088] Day 15: Induction of floating and ventralization The culture medium was removed, and the neuronal rosettes were washed off the plate using Diff. 3 medium containing palmorfamine (PU) (mn basic medium, 1:1000 GDNF, BDNF, IGF-1 and Vitamin C, 1:10000 cAMP, RA and PU, 1:50 B27) to induce ventralization. These were then transferred to low-adhesion flasks and placed in suspension culture to form neuronal spheres. The medium was changed three times a week.

[0089] Day 28: Seeding of nerve cell spheres and maturation of motor neurons. First, the ibidiμ dishes were coated with poly-L-ornithine (PLO) at 37°C for 2 hours to enhance surface hydrophobicity. Trace amounts of toxic PLO residue were removed by washing three times with PBS + MgCl2 / + CaCl2, and then coated with laminin as previously described. After removing the laminin, 1.5 mL of Diff. 4 medium (mn basic medium, 1:1000 GDNF, BDNF, IGF-1 and Vitamin C, 1:10000 cAMP, and PU, 1:20000 RA, 1:50 B27) was added to each μ dish. The neuronal cell sphere suspension was transferred from a low-adhesion flask to a 15 mL tube and centrifuged at 100 g for 2 minutes. The supernatant was discarded, and the neuronal cell spheres were incubated with 2 mL of TrypLE in a 37°C water bath for 2 minutes to reduce their size. The reaction was stopped by adding DMEM-F12, and the cells were centrifuged again, discarding the supernatant to remove residual enzymes. The neuronal cell spheres were resuspended in 500 μL of Diff.4 medium per dish and evenly dispensed between coated dishes. 1 mL of medium was carefully replaced weekly. Mature motor neurons were obtained 42 days after the final plating.

[0090] Method 2: To differentiate human iPSCs into NPCs and then into NPC-mn cells, the following protocol was used, as previously reported by Reinhardt et al. (2013), PLoS One 8(11):10.1371 / annotation / 6a917a2e-df4a-4ad9-99bb-6aa7218b833e. The NPC basal medium was a 1:1 mixture of DMEM-F12 and Neurobasal containing 1% Anti-Anti, and all media used were based on this basal medium.

[0091] Days 0-1, hESC medium Human iPSCs were detached as described above, resuspended in hESC medium (10 μM SB-431542, 1 μM Dorsomorphin, 3 μM CHIR99021, 0.5 μM PU), and further cultured in a low-adhesion flask. The medium was changed daily.

[0092] Days 2-3, NPC d2-3 medium The old culture medium was completely removed and replaced with NPC d2-3 medium (1:200 N2, 1:100 B27, 10 μM SB-431542, 1 μM Dorsomorphin, 3 μM CHIR99021, 0.5 μM PU), and the cells were further cultured in a low-adhesion flask.

[0093] Days 4-5, NPC d4-X+3 medium EB was further cultured in a low-adhesion flask, and the culture medium was replaced with NPC d4-X+3 medium (1:200 N2, 1:100 B27, 3 μM CHIR99021, 0.5 μM PU, 150 μM ascorbic acid).

[0094] Day 6, NPC d4-X+3 medium EBs were completely seeded in a 12-well plate coated with Matrigel (with reduced growth factors). Depending on the size of the EBs, 6 to 15 EBs were seeded in each well. On day 6, 0.5 mL of medium was needed for each seeding well. Therefore, the plate was coated with 500 μL of Matrigel (1:100 in Knockout-DMEM) with reduced growth factors. On day 7, 0.25 mL of medium was added to each well. After 1 day, the medium was not completely removed because the EBs were not strongly adhered to the plate. After 5 subculturings, the culture did not contain non-NPCs.

[0095] NPC d4-X+3 medium, used from days 8 to 10 and up to day X (X = the last split before differentiation begins). For the first split, EB cells exhibiting neuroepithelial proliferation were selected and split in a 1:6 ratio between two suitable wells on a coated 12-well plate. The EB cells were digested with pre-warmed Accutase (37°C, 15 mins), 2 mL of DMEM-F12 was added to stop the enzymatic reaction, and the cells were carefully resuspended using a pipette until the EB cells were no longer visible. The turbid single-cell suspension was transferred to a reaction tube and centrifuged at 100 g for 2 minutes. The supernatant was discarded, and the pellet was resuspended in NPC medium containing supplements. 0.75 mL of cell suspension was added to each well. At this point, the NPC can be frozen, but to initiate differentiation, the NPC must be passaged three times without freezing, and then split as described below at the "final split before differentiation".

[0096] All other splits were performed in a 1:6 split ratio when the cells reached at least 90% confluence. The cells were treated with pre-warmed Accutase as described above and seeded into new 12-well plates. In the final pre-differentiation split, the cells were again treated with Accutase, centrifuged, and resuspended in 1 mL of NPC medium in a reaction tube. Approximately 50,000 cells were used per well, which corresponds to approximately 20,000 cells / cm³. 2 This corresponds to [the following]. To count the number of cells, the suspension was diluted 1:100 with DMEM-F12 and counted using a Neubauer Counting Chamber. The following formula was used to calculate the number of cells: Cells / mL = Number of cells × Dilution factor × 10 4

[0097] Days X to X+3, NPC d4-X+3 medium The cells were further cultured in 12-well plates, and the culture medium was completely changed every two days. If a large number of dead cells were observed, a washing step was performed before adding new medium.

[0098] Days X+4 to X+5, NPC dX+4 / 5 medium The old culture medium was aspirated and replaced with NPC dX+4 / 5 medium (1:200 N2, 1:100 B27, 1 μM PU).

[0099] Day X+6 to Day X+13, NPC dX+6 medium The culture medium was completely replaced with NPC dX+6 medium (1:200 N2, 1:100 B27, 1 μM PU, 1 μM RA), and a complete medium change was performed every two days (Monday, Wednesday, and Friday). If a large number of dead cells were observed, the cells were washed with DMEM-F12.

[0100] Day X+14 - End, NPC Mature Medium The culture medium was changed to NPC maturation medium (1:200 N2, 1:100 B27, 10 ng / μL BDNF and GDNF, 1 μM cAMP), and the cells were cultured for 1 day. On day 15, the cells were divided, counted, and seeded to a cell density of 50,000 cells per well. Splitting was performed on a 12-well plate. Half of the medium was removed and replaced with fresh medium twice a week. After 1 month, mature NPC-mn cells were obtained.

[0101] How to differentiate into mn for ICC: To differentiate human iPSCs into mn for ICC, the following protocol was used, as previously reported by Catanese and colleagues (2019). EBs were generated in hESC medium containing additives (7.7 mL DMEM / F12 + GlutaMAX, 2 mL KO-SR, 100 μL Anti-Anti, 100 μL NEAA, 100 μL mercaptoethanol), plated, and then maintained in Neurobasal medium containing additives (480 mL DMEM / F12 + GlutaMAX, 10 mL Hormone Mix, 5 mL Anti-Anti, 5 mL NEAA, 20 μL heparin).

[0102] Day 0 Cells were detached with Dispase (3 minutes), cultured in suspension in an ultra-low adhesion flask T75, and allowed to form EBs in New hESC medium (10 mL hESC medium, 100 μL B27 (without Vitamin A), 50 μL N2, 3 μM CHIR99021, 10 μM SB-431542, and 200 μM Vitamin C, 10 μL of 1 μM Dorsomorphin, and 1 μL of 1 μM Purmorphamine).

[0103] Day 1 Complete removal of old hESC medium and addition of new hESC medium.

[0104] Day 3 The culture medium was replaced with New Diff medium (10 mL Neurobasal, 100 μL B27 (without Vitamin A), 50 μL N2, 3 μM CHIR99021, 10 μM SB-431542, 10 μM BDNF, 10 μM GDNF, 10 μM IGF1, and 200 μM Vitamin C, respectively, along with 5 μL of 1 μM Dorsomorphin, 1 μL of 1 μM Purmorphamine, 1 μL of 500 μM cAMP, and 1 μL of 1 μM retinoic acid).

[0105] Day 5 + Day 7 Remove 5 mL of old medium and add 5 mL of fresh New Diff medium.

[0106] Day 8 Cells were counted (40,000 cells / ibidi) and plated (coated with Growth Factor Reduced Matrigel, 37°C, 2 hours). MN was then cultured for 28 days, followed by transfection, fixation, and staining.

[0107] [Example 3] General method: Transfection using Lipofectamine® For each approach, two reaction tubes were prepared. 50 μL of DMEM-F12 was pipetted into each tube. 0.71% LF3000 was added to one tube, and 7.2% P3000 reagent and 1 μg AON were added to the other tube. Transfection of iPSCs and NPC-mn cells was performed in 12-well plates, with mn cells transfected in microdishes. The two reaction tubes were mixed, homogenized, and incubated at room temperature for 10 minutes. This transfection mix was then dispensed onto cells. After incubation with the transfection mix for 24 hours, the culture medium was changed as usual.

[0108] immunocytochemistry Cells were fixed using 4% paraformaldehyde (PFA) containing 0.1% sucrose. Cell permeabilization was performed at room temperature for 10 minutes using 0.2% Triton in PBS + CaCl2 / +MgCl2. Nonspecific binding was blocked for 1 hour using a blocking solution (5% FBS, 10% goat serum in PBS + CaCl2 / +MgCl2). Subsequently, the primary antibody was diluted with the blocking solution and incubated overnight at 4°C. After removing the antibody solution, the cells were treated with Alexa-labeled secondary antibody for 1 hour after three washing steps of 5 minutes each with PBS + CaCl2 / +MgCl2. The procedure was completed with four washing steps with PBS + CaCl2 / +MgCl2 (1 min, 5 min, 5 min, 20 min), one washing step with PBS - CaCl2 / -MgCl2 (5 min), and a short washing step (30 seconds) with Millipore water. After these cleaning steps, the liquid was removed and the specimens were mounted using ProLong® Gold Antifade Mountant containing DAPI, which required drying in the dark for at least 4 hours before microscopic observation.

[0109] Western blot Cell lysis was performed using RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1 mM sodium orthovanadate, 1% NP-40, protease and phosphatase inhibitors). Cells were detached and transferred to a reaction tube, where they were lysed on ice for 15 minutes. Subsequently, the mixture was mechanically homogenized 10 times using a 1 mL syringe with a hollow needle (20 μm diameter) and incubated for a further 15 minutes. After incubation, the sample was sonicated 10 times and centrifuged at 13000 rpm at 4°C for 10 minutes. The supernatant containing protein was transferred to a new reaction tube, and the pellet containing cell fragments was discarded. The generated lysate was subjected to a Bradford assay to calculate the protein concentration, and the volume per 10 μg protein concentration was determined. The sample was diluted with 4 × loading buffer and water, and Western blotting was performed. Proteins were separated using 8% SDS-page and transferred to polyvinylidene fluoride membranes using the Trans-Blot® turbotransfer system. The membranes were blocked with blocking solution (5% BSA, 0.1% Tween-20) for 1–3 hours. The primary antibody was then left overnight on a shaker at 4°C. After washing with 0.2% TBS-T, the horseradish peroxidase-labeled secondary antibody was incubated at RT for 1 hour, and any remaining antibody was removed by washing again with TBS-T. Images were acquired using GelCapture software and analyzed using GelAnalyzer software.

[0110] Data analysis and statistics Statistical analysis and graphing were performed using GraphPad Prism 6. First, the data for all groups were examined for normal distribution using the Shapiro-Wilk test, and then for equal variances using the Levene test. Since more than two groups were compared, analysis of variance (ANOVA) was performed on data that followed a normal distribution and showed equal variances to detect whether there was a significant difference between their means. If a significant difference was found in the ANOVA, Tukey's post-hoc test was used to determine which groups had the significant difference. For data that did not follow a normal distribution, the Kruskal-Wallis test was followed by a post-hoc test to detect significant differences between the group means.

[0111] The data are presented as bar graphs representing the mean and standard error (SEM) of the mean for each group. Statistical significance is indicated by asterisks according to the calculated probability (p-value). If p < 0.05, the data was considered statistically significant and represented by one asterisk (*). If p < 0.01, significance was represented by two asterisks (**), and if p < 0.001, significance was represented by three asterisks (***) above the bar graph and was considered statistically very significant.

[0112] [Example 4] 50 bp antisense oligonucleotide (AON).

[0113] A 50bp AON, an analog of the 3' end of the human SHANK3 gene, was constructed (Table 2). Scrambled AONs were then constructed by randomizing the base sequence according to the base ratio of the 50bp AON. AON1 could not be constructed. All other AONs were used in a screening method using iPSCs. The screening results are shown in Figures 1 and 2.

[0114] [Example 5] 18bp antisense oligonucleotide.

[0115] The most effective 50 bp AON was split into 18 bp AONs, each containing the end of the previous AON or the beginning of the next AON (Table 3). Scrambled AONs were constructed with randomized base sequences according to the proportion of bases in the 50 bp AON. * indicates the position of the PTO backbone, and each AON was tagged with biotin at the beginning and end. The results are shown in Figures 3-8, 9, and 10, and it is clear that the 18 bp AONs increase the amount of Shank3 protein present in cells transfected with them.

[0116] [Table 2-2] TIFF0007911969000005.tif80169

[0117] Table 3

[0118] Table 4 TIFF0007911969000008.tif177166

[0119] References: TIFF0007911969000009.tif52162TIFF0007911969000010.tif73165

Claims

1. An oligonucleotide that specifically binds to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1, for use in the treatment and / or prevention of Shank3 deficiency, The oligonucleotide comprises a deoxynucleotide, The oligonucleotide contains 14 to 25 consecutive bases that are essentially complementary to nucleotides 100 to 300 of SEQ ID NO: 1, The oligonucleotide comprises the nucleic acid sequence described in SEQ ID NO: 15 or 17. The aforementioned oligonucleotide.

2. The oligonucleotide for use according to claim 1, wherein the Shank3 deficiency is Phelan-McDermid syndrome.

3. The oligonucleotide according to claim 1 or 2, wherein the oligonucleotide comprises the nucleic acid sequence described in SEQ ID NO: 15 or 17.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide is a DNA oligonucleotide.

5. Oligonucleotides according to any one of claims 1 to 4, which are contained in a composition, preferably a pharmaceutical composition.

6. An oligonucleotide that specifically binds to a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 1, The oligonucleotide contains a deoxynucleotide, The oligonucleotide contains 14 to 25 consecutive bases that are essentially complementary to nucleotides 100 to 300 of SEQ ID NO: 1, The oligonucleotide comprises the nucleic acid sequence of sequence number 15 or 17.

7. The oligonucleotide according to claim 6, wherein the oligonucleotide comprises the nucleic acid sequence described in SEQ ID NO: 15 or 17.

8. The oligonucleotide according to claim 6 or 7 for use in pharmaceuticals.

9. An expression construct encoding an oligonucleotide according to any one of claims 1 to 5, for use in the treatment and / or prevention of Shank3 deficiency, preferably contained in an expression vector.

10. A host cell, preferably a neuronal host cell, for use in the treatment and / or prevention of Shank3 deficiency, comprising an oligonucleotide according to any one of claims 1 to 5 and / or an expression construct according to claim 9.

11. A method for determining whether a subject suffering from Shank3 deficiency is suitable for treatment with an oligonucleotide according to any one of claims 1 to 5, wherein the method is: a) The step of bringing the cell sample of the subject into contact with the oligonucleotide; b) A step of measuring the amount of Shank3 polypeptide in the cell sample; c) A step of comparing the amount of Shank3 polypeptide measured in step b) to a reference; and d) A step to determine whether a subject suffering from Shank3 deficiency is suitable for treatment, based on the results of the comparison step c). The method, including the method described above.

12. The aforementioned standards (i) A control cell sample of the subject that has not been in contact with the oligonucleotide; (ii) A group of subjects who appear healthy; (iii) A group of subjects who have been found not to have Shank3 deficiency; (iv) A group of subjects who have been found to have Shank3 deficiency The method according to claim 11, determined based on the following:

13. A kit comprising an oligonucleotide according to claim 6 or 7, and means for administering the same.

14. An in vitro method for increasing Shank3 expression in host cells, comprising contacting the host cells with an oligonucleotide according to any one of claims 1 to 9, thereby increasing Shank3 expression.

15. Use of an oligonucleotide according to any one of claims 1 to 9 for the manufacture of a medicament for use in a method for increasing Shank3 expression in host cells, wherein the method comprises contacting the host cells with the oligonucleotide to increase Shank3 expression.

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