RNA modulating oligonucleotides with improved characteristics for treatment of neuromuscular disorders

Abstract

To provide an antisense oligonucleotide for preventing, delaying, and / or treating a human cis-element repeat instability-associated genetic disorder.SOLUTION: A medicament comprises, as an active ingredient, an oligonucleotide, wherein the oligonucleotide comprises 2'-O-methyl RNA nucleotide residues, has a backbone with at least one phosphate moiety being replaced by a phosphorothioate moiety, and comprises or consists of a repetitive nucleotide unit (XYG)m, wherein m is an integer from 4 to 12, each X is C or 5-methylcytosine, each Y is U or 5-methyluracil, and at least one X is 5-methylcytosine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to human genetics, more specifically to the field of neuromuscular disorders. The present invention particularly relates to the use of antisense oligonucleotides (AONs) having improved characteristics that enhance clinical applicability as further defined herein. Background of the Invention

[0002] Neuromuscular diseases are characterized by muscle dysfunction due to either muscle or nerve pathologies (myopathies and neuropathies). Neuropathies are characterized by neurodegeneration and disorders of innervation, leading to problems with movement, spasticity or paralysis. Examples include Huntington's disease (HD), several types of spinocerebellar ataxia (SCA), Friedreich's ataxia (FA), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A subset of neuropathies results from cis-element repeat instability. For example, HD results from a triplet (CAG) n repeat expansion in exon 1 of the HTT gene. The expansion of these repeats results in an elongation of the polyglutamine stretch at the N-terminus of the 348 kDa cytoplasmic huntingtin protein. Huntingtin has a characteristic sequence of 6-29 glutamine amino acid residues in its normal form, and the mutant huntingtin that causes the disease has more than 38 residues. The continuous expression of mutant huntingtin molecules in neurons ultimately leads to the formation of large protein deposits that cause cell death, particularly in the frontal lobe and basal ganglia (mainly in the caudate nucleus). The severity of the disease generally correlates with the number of extra residues. AONs that specifically target the expanded CAG repeat (e.g., PS57(CUG)7 as 2'-O-methyl phosphorothioate RNA (SEQ ID NO: 1) (Evers et al.)) can be applied to effectively reduce mutant huntingtin transcript and (toxic) protein levels in cells derived from HD patients. For the treatment of neuropathies, systemically administered AONs need to cross the blood-brain barrier. Therefore, there is a need for optimization of oligochemistry that enables and / or demonstrates improved brain delivery.

[0003] Myopathies include hereditary muscular dystrophies characterized by progressive weakness and degeneration of skeletal, cardiac, and / or smooth muscle. Examples of myopathies are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), and myotonic dystrophy type 2 (DM2). Both DM1 and DM2 are caused by cis-element repeat instability. DM1 is due to trinucleotide (CTG) n repeat expansions in the 3' untranslated region of exon 15 in the DMPK gene, and DM2 is due to tetranucleotide (CCTG) n repeat expansions in the DM2 / ZNF9 gene. Also, AONs (e.g., PS58, a 2'-O-methyl phosphorothioate RNA for (CAG)7 for DM1 (Mulders et al.)) that specifically target expanded repeats have been shown to efficiently induce specific degradation of (toxic) expanded repeat transcripts. In contrast to DMD, where gene deletion is associated with increased permeability of the muscle fiber membrane to small compounds such as AONs, for most other myopathies, enhanced AON distribution to and uptake by muscle tissue are essential to obtain a therapeutic effect. Therefore, there is also a need to optimize oligochemistry that enables and / or demonstrates improved muscle delivery.

[0004] The specific characteristics of the selected chemistries at least partially affect the delivery of AONs to the target transcript: route of administration, in vivo stability, biodistribution, tissue distribution, and cellular uptake and transport. Further optimization of oligonucleotide chemistries is also thought to reduce the cost of the product by enhancing binding affinity and stability, enhancing activity, improving safety, and / or shortening length or improving synthesis and / or purification procedures. A number of chemical modifications have become generally and / or commercially available to the research community (e.g., 2'-O-methyl RNA and 5-substituted pyrimidines and 2,6-diaminopurine), but most others still require substantial synthetic efforts to obtain. In particular, promising preliminary results have been obtained using the 2'-O-methyl phosphorothioate RNA described herein that contains modifications to pyrimidine and purine bases.

[0005] In conclusion, there is a need for AONs with further improved characteristics to enhance the therapeutic applicability of AONs for treating human cis - element repeat instability - related genetic disorders exemplified herein. Description of the Invention

[0006] Oligonucleotide: In a first aspect, the present invention provides an oligonucleotide for use as a medicament for treating human cis - element repeat instability - related genetic disorders, which comprises 2'-O - methyl RNA nucleotide residues, has a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety, and contains one or more 5 - methylpyrimidine and / or one or more 2,6 - diamino purine bases, or an oligonucleotide for use as a medicament for treating human cis - element repeat instability - related genetic disorders, which consists of 2'-O - methyl RNA nucleotide residues, has a backbone in which all phosphate moieties are replaced by phosphorothioate moieties, and contains one or more 5 - methylpyrimidine and / or one or more 2,6 - diamino purine bases.

[0007] In the present invention, the term "backbone" is used to indicate a chain in which ribose rings and internucleoside linkages are alternately linked to which nucleobases are attached. The term "linkage" is used for the connection between two ribose units (i.e., "internucleoside linkage") and is generally a phosphate moiety. Thus, an oligonucleotide having 10 nucleotides can have 9 linkages that link 10 ribose units together. There can also be one or more terminal linkages present on one or both sides of the oligonucleotide that connect to only one nucleotide. The terms "linkage" and "internucleoside linkage" shall also denote such pendant linkages. At least one linkage in the backbone of the oligonucleotide of the present invention consists of a phosphorothioate moiety that links two ribose units. Thus, at least one naturally occurring 3'-to-5' phosphodiester moiety present in RNA is replaced by a phosphorothioate moiety.

[0008] In the present invention, "a" in each of the following expressions means "at least one": 2'-O-methyl RNA nucleotide residue, 2'-O-methyl RNA residue, phosphorothioate moiety, 2'-O-methyl phosphorothioate RNA residue, 5-methylpyrimidine base, 5-methylcytosine base, 5-methyluracil base, thymine base, 2,6-diaminopurine base.

[0009] Preferably, the oligonucleotide of the present invention is an oligonucleotide having a length of less than 37 nucleotides. The oligonucleotide can have 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Such an oligonucleotide can also be identified as an oligonucleotide having 12 to 36 nucleotides.

[0010] Accordingly, the oligonucleotides of the present invention having a backbone that includes 2'-O-methyl RNA nucleotide residues and in which at least one phosphate moiety is replaced by a phosphorothioate moiety contain less than 37 nucleotides (i.e., contain 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides), and contain 5-methylpyrimidine and / or 2,6-diaminopurine bases.

[0011] Accordingly, the oligonucleotides of the present invention consisting of 2'-O-methyl RNA nucleotide residues and having a backbone in which all phosphate moieties are replaced by phosphorothioates contain less than 34 nucleotides (i.e., contain 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides), and contain 5-methylpyrimidine and / or 2,6-diaminopurine bases.

[0012] In a preferred embodiment, the oligonucleotide of the present invention comprises, or consists of, 2'-O-methyl phosphorothioate RNA nucleotide residues. Such oligonucleotides contain 2'-O-methyl RNA residues that are connected to the next nucleotide in the sequence through phosphorothioate linkages. The next nucleotide may or may not be another 2'-O-methyl phosphorothioate RNA nucleotide residue. Alternatively, such oligonucleotides consist of 2'-O-methyl phosphorothioate RNA nucleotide residues in which all nucleotides contain a 2'-O-methyl moiety and a phosphorothioate moiety. Preferably, such oligonucleotides consist of 2'-O-methyl phosphorothioate RNA nucleotide residues. Such chemistries are known to those skilled in the art. Throughout this specification, oligonucleotides containing 2'-O-methyl RNA residues and phosphorothioate linkages may be replaced by oligonucleotides containing 2'-O-methyl phosphorothioate RNA nucleotide residues or oligonucleotides containing 2'-O-methyl phosphorothioate RNA residues. Throughout this specification, oligonucleotides consisting of 2'-O-methyl RNA residues linked or connected by phosphorothioate linkages or oligonucleotides consisting of 2'-O-methyl phosphorothioate RNA nucleotide residues may be replaced by oligonucleotides consisting of 2'-O-methyl phosphorothioate RNA.

[0013] Also, the oligonucleotide of the present invention comprises at least one base modification that increases the binding affinity to the target strand and / or raises the melting temperature of the duplex formed between the oligonucleotide and its target and / or reduces the immunostimulatory effect and / or increases the in vivo stability and / or improves the in vivo distribution and / or tissue distribution and / or cellular uptake and transport.

[0014] In one embodiment, the oligonucleotides of the present invention contain 5-methylpyrimidine and / or 2,6-diaminopurine bases. The 5-methylpyrimidine base is selected from 5-methylcytosine and / or 5-methyluracil and / or thymine, and thymine is identical to 5-methyluracil. When the oligonucleotides of the present invention have two or more such base modifications, the base modifications can be the same. For example, all such modified bases in the oligonucleotide can be 5-methylcytosine. Alternatively, the base modifications can be combinations of various base modifications. For example, the oligonucleotide can have one or more 5-methylcytosines and one or more 5-methyluracils.

[0015] In a preferred embodiment, the oligonucleotide of the present invention (i.e., an oligonucleotide comprising 2'-O-methyl RNA nucleotide residues, having a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety, and comprising one or more 5-methylpyrimidines and / or one or more 2,6-diaminopurine bases, or consisting of 2'-O-methyl RNA nucleotide residues, having a backbone in which all phosphate moieties are replaced by phosphorothioate moieties, and comprising one or more 5-methylpyrimidines and / or one or more 2,6-diaminopurine bases) does not contain 2'-deoxy-2'-fluoronucleotides (i.e., 2'-deoxy-2'-fluoro-adenosine, -guanosine, -uridine and / or -cytidine). Such oligonucleotides containing 2'-fluoro (2'-F) nucleotides have been shown to be able to recruit interleukin enhancer-binding factors 2 and 3 (ILF2 / 3) and thus induce exon skipping in a targeted mRNA precursor (Rigo F et al., International Publication No. WO 2011 / 097614). In the present invention, the oligonucleotide used preferably does not recruit such factors and / or the oligonucleotide of the present invention does not form a heteroduplex with RNA specifically recognized by ILF2 / 3. The mechanism of action of the oligonucleotide of the present invention is presumed to be different from that of oligonucleotides containing 2'-F nucleotides, and the oligonucleotide of the present invention is expected to mainly induce specific degradation of (toxic) expanded repeat transcripts.

[0016] "Thymine" and "5-methyluracil" may be interchangeable throughout this specification. Similarly, 2,6-diaminopurine is identical to 2-aminoadenine, and these terms may be interchangeable throughout this specification.

[0017] As used herein, the term "base modification" or "modified base" refers to the modification of a base that naturally occurs in RNA (i.e., a pyrimidine or purine base) or the de novo synthesis of a base. This de novo synthesized base can be recognized as "modified" by comparison with an existing base.

[0018] The oligonucleotides of the present invention containing 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine bases are each such that at least one of the cytosine nucleobases of the oligonucleotide is modified by substitution of a proton at the 5-position of the pyrimidine ring with a methyl group (i.e., 5-methylcytosine) and / or at least one of the uracil nucleobases of the oligonucleotide is modified by substitution of a proton at the 5-position of the pyrimidine ring with a methyl group (i.e., 5-methyluracil) and / or at least one of the adenine nucleobases of the oligonucleotide is modified by substitution of a proton at the 2-position with an amino group (i.e., 2,6-diaminopurine). In the present invention, the expression "substitution of a proton at the 5-position of the pyrimidine ring with a methyl group" is replaced by the expression "substitution with 5-methylpyrimidine of the pyrimidine", and the pyrimidine can refer to only uracil, only cytosine or both. Similarly, in the present invention, the expression "substitution of a proton at the 2-position of adenine with an amino group" can be replaced by the expression "substitution with 2,6-diaminopurine of adenine". When the oligonucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or more cytosines, uracils and / or adenines, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more cytosines, uracils and / or adenines are each modified in this way. Preferably, all cytosines, uracils and / or adenines are modified in this way or are each replaced by 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine. It goes without saying that the present invention is applicable only to oligonucleotides that each contain at least one cytosine, uracil or adenine in their sequences.

[0019] The inventors have found that the presence of 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine in the oligonucleotides of the present invention has a favorable effect on at least one of the parameters of said oligonucleotides or an improvement in at least one parameter. Here, the parameters can include, as described below, the binding affinity and / or kinetics, silencing activity, in vivo stability, (in tissue) distribution, cellular uptake and / or transport, and / or immunogenicity of said oligonucleotides.

[0020] Binding affinity and kinetics depend on the thermodynamic properties of the AON. These are at least partially determined by the melting temperature (Tm; for single-stranded RNA, calculated using the basic Tm and neighboring model, for example, Oligo Calculator (http: / / www.unc.edu / ~cail / biotool / oligo / index.html or http: / / eu.idtdna.com / analyzer / Applications / OligoAnalyzer / )), and / or by the free energy of the oligonucleotide-target exon complex (using RNA Structure version 4.5 or RNA mfold version 3.5). When Tm increases, exon skipping activity typically increases, but when Tm is too high, the AON is predicted to be less sequence-specific. The acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to indicate a preferred range for each of these parameters.

[0021] The activity of the oligonucleotides of the present invention is, as described hereinafter, to inhibit the formation of mutant proteins and / or to suppress, reduce or decrease the amount of transcripts of disease-related or disease-causing or mutant containing an extended or unstable number of repeats in the cells of a patient, in the tissues of a patient and / or in a patient. The oligonucleotides of the present invention comprising or consisting of 2'-O-methyl phosphorothioate RNA and 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine bases are expected to be able to more efficiently suppress, reduce or decrease the amount of said transcripts than oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA but not containing 5-methylcytosine, not containing 5-methyluracil and not containing 2,6-diaminopurine bases. This difference in efficiency can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%. The reduction or decrease can be evaluated by Northern blotting or (semi-)quantitative RT-PCR for transcript levels (preferably performed in the experimental part), or by Western blotting for protein levels. The oligonucleotides of the present invention can first be tested in cell lines such as patient-derived fibroblasts, as described in Example 1.

[0022] Biodistribution and biostability are preferably at least partially determined by the validated hybridization ligation assay of Yu et al., 2002. In one embodiment, plasma or homogenized tissue samples are incubated with specific capture oligonucleotide probes. After separation, the DIG-labeled oligonucleotide is ligated to the complex and detection is performed using an anti-DIG antibody-conjugated peroxidase. Non-compartmental pharmacokinetic analysis is performed using the WINNONLIN software package (Model 200, Version 5.2, Pharsight, Mountainview, CA). The level of AON (μg) per 1 mL of plasma or 1 mg of tissue is the area under the curve (AUC), peak concentration (C max ), time to peak concentration (T max ), terminal phase half-life, and absorption lag time (t lag ) are monitored over time to evaluate. Such preferred assays are disclosed in the experimental part.

[0023] AONs can stimulate the innate immune response by activating toll-like receptors (TLRs) including TLR9 and TLR7 (Krieg et al., 1995). Activation of TLR9 typically occurs by mimicking bacterial DNA that activates the innate immune system through TLR9-mediated cytokine release due to the presence of unmethylated CG sequences present in oligodeoxynucleotides (ODNs). However, 2'-O-methyl modification has been shown to significantly reduce such possible effects. TLR7 has been described as recognizing uracil repeats in RNA (Diebold et al., 2006).

[0024] Activation of TLR9 and TLR7 gives rise to a coordinated immune response involving innate immunity (macrophages, dendritic cells (DCs), NK cells) (Krieg et al., 1995; Krieg, 2000). Several chemo- and cytokines such as IP-10, TNFα, IL-6, MCP-1 and IFNα (Wagner, 1999; Popovic et al., 2006) are associated with this process. Inflammatory cytokines attract additional defensive cells such as T and B cells from the blood. The levels of these cytokines can be investigated by in vitro assays. Briefly, whole human blood is incubated with increasing concentrations of AON, and then the cytokine levels are determined by standard commercially available ELISA kits. A decrease in immunogenicity is preferably corresponding to a detectable decrease in the concentration of at least one of the above cytokines by comparison of the corresponding cytokine concentrations in assays in cells treated with oligonucleotides containing at least one 5-methylcytosine and / or 5-methyluracil, and / or 2,6-diaminopurine (comparison with cells treated with the corresponding oligonucleotides without 5-methylcytosine, 5-methyluracil or 2,6-diaminopurine).

[0025] Accordingly, the preferred oligonucleotides of the invention have improved parameters such as acceptable or decreased immunogenicity and / or better in vivo distribution and / or acceptable or improved RNA binding kinetics and / or thermodynamic properties compared to the corresponding oligonucleotides consisting of 2'-O-methyl phosphorothioate RNA that do not contain 5-methylcytosine, 5-methyluracil and 2,6-diaminopurine. Each of these parameters can be known to those skilled in the art or preferably evaluated using the assays described herein.

[0026] Other chemistries and modifications of the oligonucleotides of the invention are defined below. These additional chemistries and modifications may be present in combination with the chemistries already defined for said oligonucleotides, namely the presence of 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine and oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA.

[0027] Preferred oligonucleotides of the invention comprise or consist of an RNA molecule or a modified RNA molecule. In a preferred embodiment, the oligonucleotide is single-stranded. However, one of ordinary skill in the art will understand that a single-stranded oligonucleotide may form internal double-stranded structures. However, in the present invention, this oligonucleotide is still referred to as a single-stranded oligonucleotide. Single-stranded oligonucleotides have several advantages compared to double-stranded siRNA oligonucleotides: (i) its synthesis is expected to be simpler than that of two complementary siRNA strands; (ii) there are a wider range of chemical modifications that can enhance cellular uptake, better (physiological) stability and reduce potential general harmful effects; (iii) siRNAs have a higher potential for non-specific effects (including off-target genes) and excessive pharmacology (e.g., lower controllability of efficacy and selectivity by treatment regimens or dosages), and (iv) siRNAs do not act much in the nucleus and cannot target introns.

[0028] In addition to the above modifications, the oligonucleotides of the invention may include further modifications such as various nucleic acid nucleotide residues or nucleotides as described below. Various nucleic acid nucleotide residues can be used to generate the oligonucleotides of the invention. The oligonucleotide may have at least one backbone modification (internucleoside linkage and / or sugar modification) and / or at least one base modification compared to an RNA-based oligonucleotide.

[0029] Base modifications include hypoxanthine (e.g., inosine), orotic acid, agmatidine, lysidine, pseudouracil, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, super G, super A and N4-ethylcytosine, or derivatives thereof; N 2 -cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP) and N 2 -propyl-2-aminopurine (Pr-AP), or modified versions of natural purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine, thymine) such as derivatives thereof; and degenerate or universal bases such as 2,6-difluorotoluene or abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives (azaribose) where the ring oxygen is replaced by nitrogen). Examples of derivatives of super A, super G and super T can be found in U.S. Patent No. 6,683,173 (Epoch Biosciences), which is hereby incorporated by reference in its entirety. cPent-G, cPent-AP and Pr-AP have been shown to reduce the immunostimulatory effect when incorporated into siRNA (Peacock H. et al.).

[0030] In one embodiment, the oligonucleotide of the present invention contains an abasic site or an abasic monomer. In the present invention, such a monomer may also be referred to as an abasic site or an abasic monomer. An abasic monomer or abasic site is a nucleotide residue or component that lacks a nucleobase as compared to the corresponding nucleotide residue containing a nucleobase. Thus, in the present invention, an abasic monomer is a component part of an oligonucleotide (lacking a nucleobase). Such an abasic monomer may be present at, linked to, conjugated to, or bound to a free end of the oligonucleotide.

[0031] In a more preferred embodiment, the oligonucleotide of the present invention contains 1 to 10 or more abasic monomers. Thus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more abasic monomers may be present in the oligonucleotide of the present invention.

[0032] The abasic monomer may be of any type known and conceivable to those skilled in the art. Non-limiting examples thereof are shown below.

[0033]

Chemical formula

[0034] In the formula, R1 and R2 are independently H, an oligonucleotide, or another abasic site (provided that the case where both R1 and R2 are H and the case where both R1 and R2 are oligonucleotides are excluded). The abasic monomer can be bound to one or both of the ends of the oligonucleotide defined above. It should be noted that an oligonucleotide bound to one or two abasic sites or abasic monomers may contain less than 12 nucleotides. In this regard, the oligonucleotide of the present invention may contain at least 12 nucleotides (optionally containing one or more abasic sites or abasic monomers at one or both of its ends).

[0035] In the Sequence Listing, the oligonucleotides of the present invention containing abasic monomers can be represented by their nucleotide or base sequences. Abasic monomers are not represented because they are considered to be linked or bound or conjugated to the free ends of the oligonucleotides. This is the case for the base sequences of SEQ ID NOs: 107 and 108. Table 2 provides the full-length sequences of preferred oligonucleotides containing SEQ ID NO: 107 or 108. Such oligonucleotides contain SEQ ID NO: 107 or 108 and four abasic monomers at the 3'-end of SEQ ID NO: 107 or 108. SEQ ID NOs: 220 and 221 correspond to SEQ ID NOs: 107 and 108 and further contain four additional abasic monomers at the 3'-end of the oligonucleotides.

[0036] When an abasic monomer is present within the base sequence of an oligonucleotide, the abasic monomer is shown in the Sequence Listing as part of the sequence of the oligonucleotide, such as SEQ ID NOs: 210 and 213.

[0037] In Tables 1 and 2, abasic monomers are indicated using the letter Q.

[0038] The oligonucleotides of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 or 36 base modifications, depending on their length. The present invention also encompasses introducing more than one different base modification into the oligonucleotide.

[0039] "Sugar modification" refers to the presence of modified versions of the ribosyl moiety (i.e., the furanosyl moiety) that naturally occurs in RNA, such as bicyclic sugars, tetrahydropyrans, morpholinos, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars. Examples of suitable sugar modifications include, but are not limited to, 2'-O-alkyl or 2'-O-(substituted)alkyl (e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propynyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl)), 2'-deoxy (DNA), 2'-O-(haloalkoxy)methyl (Arai K. et al.) (e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM)), 2'-O-alkoxycarbonyl (e.g., 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME)), 2'-halo (e.g., 2'-F, FANA (2'-F arabinosyl nucleic acid)), etc. of 2'-O-modified RNA nucleotide residues; carbocyclic sugar and azasugar modifications; 3'-O-alkyl (e.g., 3'-O-methyl, 3'-O-butyryl, 3'-O-propynyl); 5'-alkyl (e.g., 5'-methyl); and derivatives thereof.

[0040] Another sugar modification is "bridged" or "bicyclic" nucleic acid (BNA), e.g., locked nucleic acid (LNA), xyl-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), BNA NC[N-Me] (as described in Chem. Commun. 2007, 3765, which is incorporated herein by reference in its entirety), tricyclic DNA (tcDNA), unlocked nucleic acid (UNA), 5'-methyl-substituted BNA (as described in U.S. Patent Application No. 13 / 530218, which is incorporated herein by reference in its entirety), cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA), morpholino (e.g., those in PMO, PMOPlus, PMO-X), and derivatives thereof, preferably locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclic DNA (tcDNA), cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA), morpholino (e.g., those in PMO, PMOPlus, PMO-X), and derivatives thereof. A preferred tcDNA is tc-PS-DNA (tricyclic DNA containing phosphorothioate nucleoside internucleotide linkages). The oligonucleotides of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 or 36 sugar modifications depending on their length. The introduction of more than one different sugar modification into the oligonucleotide is also encompassed by the present invention. In one embodiment, the oligonucleotides described herein contain or consist of LNA or a derivative thereof. BNA derivatives are described, for example, in International Publication No. WO 2011 / 097641, which is incorporated herein by reference in its entirety. In a further preferred embodiment, the oligonucleotides of the present invention are completely 2'-O-methyl modified.Examples of PMO-X are described in WO 2011 / 150408, which is hereby incorporated by reference in its entirety.

[0041] In a preferred embodiment, the oligonucleotides of the invention, apart from the essential 2'-O-methyl sugar modification, comprise at least one other sugar modification selected from 2'-O-methyl, 2'-O-(2-methoxy)ethyl, morpholino, bridged nucleotides or BNA, or the oligonucleotide comprises both bridged nucleotides and 2'-deoxy modified nucleotides (BNA / DNA mixmer). More preferably, the oligonucleotides of the invention are modified over their entire length with a sugar modification selected from 2'-O-methyl, 2'-O-(2-methoxy)ethyl, morpholino, bridged nucleic acid (BNA) or BNA / DNA mixmer.

[0042] In a more preferred embodiment, the oligonucleotides of the invention are fully 2'-O-methyl modified, preferably fully 2'-O-methyl phosphorothioate modified.

[0043] "Skeleton modification" indicates the presence of a modified version of the ribosyl moiety described above ("sugar modification") and / or the presence of a modified version of the phosphodiester that occurs naturally in RNA ("internucleoside linkage modification"). Examples of internucleoside linkage modifications compatible with the present invention are phosphorothioate (PS), chirally pure phosphorothioate, phosphorodithioate (PS2), phosphonoacetic acid (PACE), phosphonoacetamide (PACA), thiophosphonoacetic acid, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylphosphonothioate, methylphosphate, methylphosphorothioate, ethylphosphate, ethylphosphorothioate, boranophosphate, boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, methylboranophosphonate, methylboranophosphonothioate, and derivatives thereof. Another modification is phosphoramidite, phosphoramidate, N3’→P5’ phosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamic acid, dimethylenesulfoxide, sulfonic acid, triazole, oxalyl, carbamate, methyleneimino (MMI), thioacetamide nucleic acid (TANA), and derivatives thereof. The oligonucleotides of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 or 35 skeleton modifications, depending on their length. The introduction of more than one different skeleton modification into the oligonucleotide is also encompassed by the present invention.

[0044] The oligonucleotides of the present invention contain at least one phosphorothioate modification. In a more preferred embodiment, the oligonucleotides of the present invention are fully phosphorothioate modified.

[0045] Other chemical modifications of the oligonucleotides of the present invention include peptide-based nucleic acids (PNA), boron cluster-modified PNA, pyrrolidine-based oxy-peptide nucleic acids (POPNA), glycol or glycerol-based nucleic acids (GNA), trehalose-based nucleic acids (TNA), acyclic threoninol-based nucleic acids (aTNA), morpholino-based oligonucleotides (PMO, PPMO, PMO-X), cationic morpholino-based oligomers (PMOPlus), oligonucleotides having integrated bases and a backbone (ONIB), pyrrolidine amide oligonucleotides (POM), and derivatives thereof.

[0046] In another embodiment, the oligonucleotide comprises a peptide nucleic acid and / or a morpholino phosphorodiamidate, or a derivative thereof.

[0047] Thus, preferred oligonucleotides according to one aspect of the present invention are (a) at least one base modification selected from 5-methylpyrimidine and 2,6-diaminopurine, and / or (b) at least one sugar modification that is 2'-O-methyl, and / or (c) at least one backbone modification that is phosphorothioate and include.

[0048] Thus, the preferred oligonucleotides according to this aspect of the present invention contain base modification (a), do not contain sugar modification (b), and do not contain backbone modification (c). Another preferred oligonucleotide according to this aspect of the present invention contains sugar modification (b), does not contain base modification (a), and does not contain backbone modification (c). Another preferred oligonucleotide according to this aspect of the present invention contains backbone modification (c), does not contain base modification (a), and does not contain sugar modification (b). Also, it is understood that oligonucleotides having none of the above modifications are covered by the present invention in the same way as oligonucleotides containing two of the above (i.e., (a) and (b), (a) and (c), and / or (b) and (c)) or all three of the modifications (a), (b), and (c). In another preferred embodiment, any oligonucleotide described in the previous paragraph is (a) at least one (additional) base modification selected from 2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, and / or (b) at least one (additional) sugar modification selected from 2'-O-methyl, 2'-O-(2-methoxy)ethyl, 2'-deoxy (DNA), morpholino, bridged nucleotide or BNA, or the oligonucleotide contains both a bridged nucleotide and a 2'-deoxy modified nucleotide (BNA / DNA mixmer), and / or (c) at least one (additional) backbone modification selected from (another) phosphorothioate or phosphordiamidate may contain.

[0049] In another preferred embodiment, the oligonucleotide of the present invention is modified over its entire length with one or more of the same modifications selected from (a) one of the base modifications, and / or (b) one of the sugar modifications, and / or (c) one of the backbone modifications.

[0050] The emergence of nucleic acid mimicking technology has made it possible to generate molecules that do not need to be identical to nucleic acids themselves in whole, but have similar, preferably the same hybridization characteristics. Such functional equivalents are, of course, also suitable for use in the present invention.

[0051] Those skilled in the art understand that each sugar, base and / or backbone may not be similarly modified. Several different modified sugars, bases and / or backbones can be combined in one oligonucleotide of the present invention.

[0052] Those skilled in the art also recognize that there are numerous synthetic derivatives of oligonucleotides.

[0053] Since RNA / RNA duplexes are very stable, the oligonucleotide preferably contains RNA. Preferably, the RNA oligonucleotide contains modifications that provide RNA with additional properties such as resistance to endonucleases, exonucleases and RNaseH, increased additional hybridization strength, increased stability (e.g., in body fluids), increased or decreased mobility, increased activity, reduced toxicity, increased intracellular transport, tissue specificity, etc. Also, mRNA complexed with the oligonucleotide of the present invention is preferably not sensitive to RNaseH cleavage. Preferred modifications are as described above. Oligonucleotides that at least partially contain naturally occurring DNA nucleotides are useful for inducing the degradation of DNA-RNA hybrid molecules in cells by RNaseH activity (EC.3.1.26.4).

[0054] RNA-like synthetic ribonucleotides containing naturally occurring RNA ribonucleotides or oligonucleotides are included herein and act as enzyme-dependent antisense through the RNA interference or silencing (RNAi / siRNA) pathway, which is involved in the degradation of target RNA by the RNA-induced silencing complex (RISC) following target RNA recognition through sense-antisense strand pairing to form double-stranded RNA-RNA hybrids.

[0055] Alternatively or additionally, the oligonucleotide can bind to the target sequence of the RNA transcript and interfere with the processing or expression of the precursor RNA or messenger RNA (steric blocking, RNaseH-independent process), particularly RNA splicing and exon skipping (but not limited to these), by preventing processes such as translation or blocking of splice donor or splice acceptor sites. Further, the oligonucleotide can inhibit the binding of proteins, nuclear factors, etc. by steric hindrance, and / or interfere with the proper spatial folding of the target RNA, and / or bind itself to the protein that originally binds to the target RNA, and / or have other effects on the target RNA, thereby contributing to the destabilization of the target RNA, preferably the mRNA precursor, and / or contributing to the reduction in the amount of disease-causing or toxic transcripts and / or proteins in the HD-like diseases described below.

[0056] As used herein, an oligonucleotide may include nucleotides having a chemical substitution (RNaseH resistance) that confers intracellular stability at at least one of its 5' or 3' ends, and may include fewer than 9, more preferably fewer than 6, consecutive (RNaseH sensitive) deoxyribose nucleotides in other portions of its sequence. The other portion of the sequence is preferably the central portion of the sequence. Such oligonucleotides are referred to as gapmers. Gapmers are extensively described in WO 2007 / 089611. Gapmers are designed to enable the recruitment and / or activation of RNaseH. Without being bound by theory, RNaseH is thought to be recruited and / or activated through binding to the central region of a gapmer made of deoxyribose. The oligonucleotides of the present invention, which are preferably substantially RNaseH-independent or RNaseH-independent, are designed to have a central region that is substantially unable or unable to recruit and / or activate RNaseH. In a preferred embodiment, the other portion of the sequence of the oligonucleotide, more preferably the central portion thereof, contains fewer than 9, 8, 7, 6, 5, 4, 3, 2, 1 deoxyribose or no deoxyribose. Thus, the oligonucleotides of the present invention are preferably partially or completely replaced as described above. "Partially replaced" means that the oligonucleotide contains at least some nucleotides that have been replaced, preferably at least 50% of the nucleotides have been replaced, or at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% have been replaced. 100% replacement of the nucleotides corresponds to "completely replaced".

[0057] Accordingly, the present invention provides an oligonucleotide comprising 2'-O-methyl phosphorothioate RNA residues or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylpyrimidine and / or 2,6-diaminopurine bases. This oligonucleotide consists of 2'-O-methyl RNA residues connected through a phosphorothioate backbone, and it is most preferred that all of its cytosines and / or all of its uracils and / or all of its adenines are independently replaced by 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine, respectively. Accordingly, the oligonucleotide of the present invention is one in which at least one, preferably all, cytosines are replaced by 5-methylcytosine, one in which at least one, preferably all, cytosines are replaced by 5-methylcytosine and at least one, preferably all, uracils are replaced by 5-methyluracil, one in which at least one, preferably all, cytosines are replaced by 5-methylcytosine and at least one, preferably all, adenines are replaced by 2,6-diaminopurine, one in which at least one, preferably all, cytosines are replaced by 5-methylcytosine, at least one, preferably all, uracils are replaced by 5-methyluracil, and at least one, preferably all, adenines are replaced by 2,6-diaminopurine, one in which at least one, preferably all, uracils are replaced by 5-methyluracil, one in which at least one, preferably all, uracils are replaced by 5-methyluracil and at least one, preferably all, adenines are replaced by 2,6-diaminopurine, or one in which at least one, preferably all, adenines are replaced by 2,6-diaminopurine and can be such.

[0058] The oligonucleotides of the present invention are for use as a drug for preventing, delaying and / or treating human cis-element repeat instability-related genetic disorders preferably exemplified herein. In the present specification, the human cis-element repeat instability-related genetic disorder is preferably a neuromuscular disorder. Preferably, the oligonucleotide is for use in therapeutic RNA regulation. Thus, the oligonucleotides of the present invention may be described as antisense oligonucleotides (AONs). An antisense oligonucleotide is an oligonucleotide that binds (or is capable of binding), targets, hybridizes (or is capable of hybridizing) and / or is reverse complementary to a specific sequence of a transcript of a gene known to be associated with or involved in human cis-element repeat instability-related genetic neuromuscular disorders.

[0059] According to the present invention, an antisense oligonucleotide comprising or consisting of 2'-O-methyl RNA nucleotide residues and having a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety and further comprising at least one of 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine is (CAG) n , (GCG) n , (CGG) n , (GAA) n , (GCC) n , (CCG) n , (AUUCU) n、 (GGGGCC) n or (CCUG) n and is represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is capable of binding), hybridizes (or is capable of hybridizing), targets and / or is reverse complementary to a repeat element in an RNA transcript having the selected repeat nucleotide unit as a repeat nucleotide unit. The oligonucleotide is preferably a single-stranded oligonucleotide.

[0060] It is understood that the oligonucleotides of the invention hybridize (or are capable of hybridizing), bind (or are capable of binding), target and / or are reverse complementary to the repetitive elements present in the above-described RNA transcript, but it cannot be excluded that such oligonucleotides may also interfere with, bind (or be capable of binding) to, or hybridize (or be capable of hybridizing) to the corresponding DNA from which this RNA transcript is derived.

[0061] As used herein, a repeat or repetitive element or repetitive sequence or repetitive stretch is a repetitive unit or repetitive nucleotide unit or repeat nucleotide unit comprising a trinucleotide repeat unit or a 4, 5 or 6 nucleotide repeat unit in a transcribed gene sequence in the genome of a subject, including a human subject, of at least 3, 4, 5, 10, 100, 1000 or more repeats (e.g., (CAG) n , (GCG) n , (CGG) n , (GAA) n , (GCC) n , (CCG) n , (AUUCU) n , (GGGGCC) n , (CCUG) n ). Thus, n is an integer and can be at least 3, 4, 5, 10, 100, 1000 or more. The present invention is not limited to the exemplified repeat nucleotide units. Other repeat nucleotide units can be found at http: / / neuromuscular.wustl.edu / mother / dnarep.htm. In most patients, the above-described "pure" repeats or repetitive elements or repetitive sequences or repetitive stretches (e.g., (CAG) n , (GCG) n , (CGG) n , (GAA) n , (GCC) n , (CCG) n , (AUUCU) n , (GGGGCC) n , (CCUG) n) is present in the transcribed gene sequences in the patient's genome. However, in some patients, for example, in the above-mentioned repeats or repetitive elements or repetitive sequences or repetitive stretches, there may be at least 1, 2 or 3 nucleotides that do not match the nucleotides of the repeat or repetitive element or repetitive sequence or repetitive stretch. In such cases, the repeat or repetitive element or repetitive sequence or repetitive stretch is not considered "pure" or is considered "mutated" (Braida C. et al.), and this is also encompassed by the present invention.

[0062] Therefore, the oligonucleotides of the present invention do not necessarily have to be 100% reverse complementary to the targeted repeat. Usually, the oligonucleotides of the present invention can be at least 90%, 95%, 97%, 99% or 100% reverse complementary to the targeted repeat.

[0063] In one embodiment, the antisense oligonucleotide comprises or consists of 2'-O-methyl phosphorothioate RNA, contains 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine, and (CAG) in the transcript nA nucleotide sequence comprising or consisting of an array that binds to (or is capable of binding to), hybridizes to (or is capable of hybridizing to), targets, and / or is reverse complementary, represented by the nucleotide sequence, for treating, delaying, remitting, and / or preventing human genetic diseases Huntington's disease (HD), spinocerebellar ataxia type 1, 2, 3, 6, 7, 12, or 17 (SCA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal and bulbar muscular atrophy (SBMA), and / or dentatorubral-pallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in transcripts of the HTT (SEQ ID NO: 80), ATXN1 (SEQ ID NO: 81), ATXN2 (SEQ ID NO: 82), ATXN3 (SEQ ID NO: 83), CACNA1A (SEQ ID NO: 84), ATXN7 (SEQ ID NO: 85), PPP2R2B (SEQ ID NO: 86), TBP (SEQ ID NO: 87), AR (SEQ ID NO: 88), or ATN1 (SEQ ID NO: 89) genes. These genes are preferably of human origin. In this embodiment, the oligonucleotide comprises or consists of 2'-O-methyl phosphorothioate RNA, contains 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine, and binds to (or is capable of binding to), hybridizes to (or is capable of hybridizing to), targets, and / or is reverse complementary to the above-mentioned (CAG) n A nucleotide sequence comprising or consisting of an array that binds to (or is capable of binding to), hybridizes to (or is capable of hybridizing to), targets, and / or is reverse complementary, represented by the nucleotide sequence, having (CUG) as a repeating nucleotide unit m . (CUG) m In (CUG), m is preferably an integer of 4, 5, 6, 7, 8, 9, 10, 11, 12. In a preferred embodiment, m is 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12

[0064] Note that for ALS and FTD, it is known that at least two different repeats in at least two different transcripts may be involved in, cause, or be associated with the disease. One is identified in the previous paragraph (i.e., (CAG) in the ATXN2 transcript) n ). The other one is (GGGGCC) in the C9ORF72 transcript nIt is later identified as a repeat or tract. This means that for each of these two diseases, it can be envisioned that the specific degradation of the corresponding (toxic) extended repeat transcript can be specifically induced using any one of these two different oligonucleotides of the present invention.

[0065] Throughout this specification, oligonucleotides that are identified as being complementary to the reverse of the above-described repeat, binding (able to bind), hybridizing (able to hybridize), or targeting, and having or containing repetitive nucleotide units, can have any length including 12 to 36 nucleotides. Taking CUG as an example of the repetitive nucleotide unit contained within the oligonucleotide, any oligonucleotide containing UGC or GCU as the repetitive nucleotide unit is also encompassed by the present invention. Depending on the length of the oligonucleotide (for example, 12 to 36 nucleotides), a given repetitive nucleotide unit may not terminate at the 5' and / or 3' side of the oligonucleotide. Each of the oligonucleotides is encompassed by the present invention.

[0066] Alternatively, taking an oligonucleotide having CUG as the repetitive nucleotide unit as an example, it is H-(P) p -(CUG) m -(Q) q-H [wherein m is an integer defined above.] can be represented by. The presence of each of P and Q is, individually, a nucleotide such as the above-described deoxy base monomer or A, C, G, U or an analog or equivalent thereof, and p and q are each individually an integer, preferably an integer from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to 100. Therefore, p and q are each individually an integer from 0 to 100, preferably an integer from 0 to 20, more preferably an integer from 0 to 10, more preferably an integer from 0 to 6, and even more preferably an integer from 0 to 3. Therefore, when p is 0, P does not exist, and when q is 0, Q does not exist. Those skilled in the art will understand that regardless of the amount and nature of the nucleotides present in the oligonucleotide, the oligonucleotide always starts and ends with a hydrogen atom (H).

[0067] In the present specification, (CUG) m It is understood that it can be replaced by any repeating nucleotide unit within the scope of the present invention. Therefore, a preferred oligonucleotide of the present invention is H-(P) p -(R) r -(Q) q -H [wherein (R) r is a repeating nucleotide unit within the scope of the present invention, and P, Q, p and q are as defined above.] can be represented by.

[0068] In the present invention, a "nucleotide analog" or "equivalent" is understood as a nucleotide containing at least one modification with respect to nucleotides naturally present in RNA such as A, C, G and U. Such modifications can be internucleoside linkage modifications and / or sugar modifications and / or base modifications, as described and exemplified above.

[0069] Again, when taking an oligonucleotide having CUG as a repeating nucleotide unit, it is understood that the repeating sequence can start with any of C, U or G. Therefore, in a preferred embodiment, p is not 0, and (P) p is (P') p’UG or (P’) p” G [wherein each P’ is individually a deprotected base site or nucleotide (e.g., A, C, G, U, or an analog or equivalent thereof), p’ is p - 2, and p” is p - 1.] is represented thereby. Such oligonucleotides are H-(P’) p’ UG-(CUG) m -(Q) q -H, or H-(P’) p” G-(CUG) m -(Q) q -H can be represented as

[0070] Similarly, in a preferred embodiment, q is not 0, and (Q) q is CU(Q’) q’ or C(Q’) q” [wherein each Q’ is individually a deprotected base site or nucleotide (e.g., A, C, G, U, or an analog or equivalent thereof), q’ is q - 2, and q” is q - 1.] is represented thereby. Such oligonucleotides are H-(P) p -(CUG) m -CU(Q’) q’ -H, or H-(P) p -(CUG) m -C(Q’) q” -H can be represented as

[0071] In another preferred embodiment, neither p nor q is 0, and both (P) p and (Q) q are each represented by (P’) p’ UG or (P’) p” G and CU(Q’) q’ or C(Q’) q” [wherein P’, Q’, p’, p”, q’, and q” are as defined above.] respectively. Such oligonucleotides are H-(P’) p’ UG-(CUG) m -CU(Q’) q’-H, H-(P’) p” G-(CUG) m -CU(Q’) q’ -H, H-(P’) p’ UG-(CUG) m -C(Q’) q” -H, or H-(P’) p” G-(CUG) m -C(Q’) q” -H can be represented as

[0072] It is understood that p’, p”, q’ and q” may not be negative integers. Thus, (P) p is (P’) p’ UG or (P’) p” represented by G, p is at least 1 or at least 2 respectively, and (Q) q is CU(Q’) q’ or C(Q’) q” represented by, q is at least 1 or at least 2 respectively.

[0073] It is understood that all that has been said herein regarding the CUG repeat unit can be extended to any repeat unit within the scope of the present invention.

[0074] In a preferred embodiment, an oligonucleotide identified as binding (or capable of binding), hybridizing (or capable of hybridizing) or targeting, which is reverse complementary to the (CAG) n repeat, comprises or consists of repeating nucleotide units (XYG) m [wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine and / or at least one Y is 5-methyluracil. m is an integer.] and has a length of 12 to 36 nucleotides. In this embodiment, m can be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value of m is 7.

[0075] Therefore, more preferred oligonucleotides are the repeating nucleotide unit (XYG) m [wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine, and / or at least one Y is 5-methyluracil. m is an integer from 4 to 12.](SEQ ID NOs: 2-12) and comprises or consists of.

[0076] Even more preferred oligonucleotides are the repeating nucleotide unit (XYG) m [wherein each X is 5-methylcytosine, and / or each Y is 5-methyluracil. m is an integer from 4 to 12.](SEQ ID NOs: 2-12) and comprises or consists of.

[0077] Therefore, even more preferred oligonucleotides are the repeating nucleotide units (XYG)5, (XYG)6 or (XYG)7, (XYG)8 or (XYG)9 [wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine, and / or at least one Y is 5-methyluracil.]. More preferred is (XYG)7 [wherein each X is C or 5-methylcytosine, and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine, and / or at least one Y is 5-methyluracil.](SEQ ID NO: 7) and comprises or consists of oligonucleotides.

[0078] Even more preferred oligonucleotides are the repeating nucleotide unit (XYG)7 [wherein each X is 5-methylcytosine and each Y is uracil (SEQ ID NO: 2), or each X is cytosine and each Y is 5-methyluracil (SEQ ID NO: 3).] and comprises or consists of. Even more preferred oligonucleotides comprise SEQ ID NO: 2 or 3 and have a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides.

[0079] The repeating nucleotide unit (XYG) mThe most preferred oligonucleotide sequences comprising or consisting of are set forth in Table 2 as SEQ ID NOs: 90 to 118.

[0080] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the base sequences of SEQ ID NOs: 90 to 106 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the base sequences of SEQ ID NOs: 90 to 106, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NOs: 90 to 106, and have a length of 21 nucleotides.

[0081] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the base sequences of SEQ ID NOs: 107 or 108 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the base sequences of SEQ ID NOs: 107 or 108, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NOs: 107 or 108, and have a length of 21 nucleotides.

[0082] The most preferred oligonucleotide consists of 2'-O-methyl phosphorothioate RNA, has a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 107 or 108, has a length of 21 nucleotides, and additionally contains 4 abasic monomers at one end thereof, preferably at the 3' end. The most preferred oligonucleotide is represented by the nucleotide sequence consisting of SEQ ID NO: 220 or 221.

[0083] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 109 or 110 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. More preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 109 or 110, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotide consists of 2'-O-methyl phosphorothioate RNA, has a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 109 or 110, and has a length of 24 nucleotides.

[0084] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 111 or 112 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. More preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 111 or 112, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotide consists of 2'-O-methyl phosphorothioate RNA, has a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 111 or 112, and has a length of 27 nucleotides.

[0085] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 113 or 114 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 113 or 114, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 113 or 114, and have a length of 30 nucleotides.

[0086] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 115 or 116 and have a length of 33 to 36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 115 or 116, and have a length of 33 to 36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 115 or 116, and have a length of 33 nucleotides.

[0087] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 117 or 118 and have a length of 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 117 or 118, and have a length of 36 nucleotides.

[0088] In another embodiment, an antisense oligonucleotide comprising or consisting of 2'-O-methyl phosphorothioate RNA and comprising 5-methylcytosine binds to a (GCG) n repeat in a transcript, hybridizes to (or is capable of hybridizing to), targets, and / or is reverse complementary to a nucleotide sequence comprising or consisting of a sequence represented by a nucleotide sequence which binds to (or is capable of binding to), hybridizes to (or is capable of hybridizing to), and is particularly useful for the treatment, delay, remission and / or prevention of infantile spasm syndrome, cleidocranial dysplasia, blepharophimosis, hand-foot-genital disease, polysyndactyly, oculopharyngeal muscular dystrophy and / or holoprosencephaly caused by repeat expansion in the human hereditary diseases: ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM / PABP2, TCFBR1 or ZIC2 genes. These genes are preferably of human origin.

[0089] In a preferred embodiment, an oligonucleotide that binds to, hybridizes to (or is capable of hybridizing to), targets, and is identified as being reverse complementary to a (GCG) n repeat consists of or comprises repeating nucleotide units (XGX) m [wherein each X is C or 5-methylcytosine; at least one X is 5-methylcytosine; m is an integer.], and has a length of 12 to 36 nucleotides. In this embodiment, m can be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value of m is 7.

[0090] Accordingly, a more preferred oligonucleotide consists of or comprises repeating nucleotide units (XGX) m [wherein at least one X is 5-methylcytosine; m is an integer from 4 to 12] (SEQ ID NOs: 13-21). An even more preferred oligonucleotide consists of or comprises repeating nucleotide units (XGX) m [wherein each X is 5-methylcytosine; m is an integer from 4 to 12] (SEQ ID NOs: 13-21).

[0091] Accordingly, more preferred oligonucleotides comprise or consist of the repeating nucleotide unit (XGX)7 [wherein at least one X is 5-methylcytosine.](SEQ ID NO: 16). Even more preferred oligonucleotides comprise or consist of the repeating nucleotide unit (XGX)7 [wherein each X is 5-methylcytosine.](SEQ ID NO: 16).

[0092] Repeating nucleotide unit (XGX) m The most preferred oligonucleotide sequences comprising or consisting of the repeating nucleotide unit (XGX) are set forth in Table 2 as SEQ ID NOS: 119 to 132.

[0093] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the base sequences of SEQ ID NO: 119 or 120 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the base sequences of SEQ ID NO: 119 or 120, and have a length of 16 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NO: 119 or 120, and have a length of 12 nucleotides.

[0094] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 121 or 122 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 90 to 106, and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 121 or 122, and have a length of 15 nucleotides.

[0095] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 123 or 124 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 123 or 124, and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 123 or 124, and have a length of 18 nucleotides.

[0096] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 125 or 126 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 125 or 126, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 125 or 126, and have a length of 21 nucleotides.

[0097] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 127 or 128 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 127 or 128, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 127 or 128, and have a length of 24 nucleotides.

[0098] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 129 or 130 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 129 or 130, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 129 or 130, and have a length of 27 nucleotides.

[0099] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 131 or 132 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 131 or 132, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 131 or 132, and have a length of 30 nucleotides.

[0100] In another embodiment, oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylcytosine are (CGG) in the transcript nIt is represented by a nucleotide sequence comprising or consisting of an array that binds to (or can bind to), targets, hybridizes to (or can hybridize to) and / or is reverse complementary to the repeat, and is particularly useful for the treatment, delay, remission and / or prevention of human fragile X syndrome caused by repeat expansion in the FMR1 gene. These genes are preferably of human origin.

[0101] In a preferred embodiment, (CGG) n Oligonucleotides identified as being reverse complementary to, binding to (or capable of binding to), hybridizing to (or capable of hybridizing to) or targeting the repeat are repeat nucleotide units (XXG) m [wherein each X is C or 5-methylcytosine. At least one X is 5-methylcytosine.] and has a length of 12 to 36 nucleotides.

[0102] m is an integer. In this embodiment, m can be 4, 5, 6, 7, 8, 9, 10, 11, 12. The preferred value of m is 7.

[0103] Therefore, a more preferred oligonucleotide is a repeat nucleotide unit (XXG) m [wherein each X is C or 5-methylcytosine. At least one X is 5-methylcytosine. m is an integer from 4 to 12.](SEQ ID NOs: 22-30) and comprises or consists of.

[0104] An even more preferred oligonucleotide is a repeat nucleotide unit (XXG) m [wherein each X is 5-methylcytosine. m is an integer from 4 to 12.](SEQ ID NOs: 22-30) and comprises or consists of.

[0105] Therefore, an even more preferred oligonucleotide is a repeat nucleotide unit (XXG)7 [wherein each X is C or 5-methylcytosine. At least one X is 5-methylcytosine.](SEQ ID NO: 25) and comprises or consists of.

[0106] More preferred oligonucleotides contain or consist of the repeating nucleotide unit (XXG)7 [wherein each X is 5-methylcytosine.] (SEQ ID NO: 25).

[0107] Repeating nucleotide unit (XXG) m The most preferred oligonucleotide sequences containing or consisting of the repeating nucleotide unit (XXG) are set forth in Table 2 as SEQ ID NOs: 133 to 146.

[0108] Preferred oligonucleotides containing or consisting of 2'-O-methyl phosphorothioate RNA contain one of the base sequences of SEQ ID NO: 133 or 134 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the base sequences of SEQ ID NO: 133 or 134, and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NO: 133 or 134, and have a length of 12 nucleotides.

[0109] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 135 or 136 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 135 or 136, and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 135 or 136, and have a length of 15 nucleotides.

[0110] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 137 or 138 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 137 or 138, and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 137 or 138, and have a length of 18 nucleotides.

[0111] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 139 or 140 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 139 or 140, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 139 or 140, and have a length of 21 nucleotides.

[0112] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 141 or 142 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 141 or 142, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 141 or 142, and have a length of 24 nucleotides.

[0113] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 143 or 144 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 143 or 144, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of base numbers 143 or 144, and have a length of 27 nucleotides.

[0114] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 145 or 146 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 145 or 146, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 145 or 146, and have a length of 30 nucleotides.

[0115] In another embodiment, oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylcytosine and / or 5-methyluracil are represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is capable of binding), targets, hybridizes (or is capable of hybridizing) to the (GAA) n repeat in the transcript and is particularly useful for the treatment, delay and / or prevention of the human genetic disorder Friedreich's ataxia.

[0116] In a preferred embodiment, (GAA) n Oligonucleotides that are identified as binding (or capable of binding), hybridizing (or capable of hybridizing), or targeting and are reverse complementary to the repeat are the repeat nucleotide unit (YYX) m [wherein each X is C or 5-methylcytosine and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine and / or at least one Y is 5-methyluracil.] and contain or consist of and have a length of 12 to 36 nucleotides.

[0117] m is an integer. In this embodiment, m can be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value of m is 7.

[0118] Therefore, a more preferred oligonucleotide is the repeat nucleotide unit (YYX) m [wherein each X is C or 5-methylcytosine and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine and / or at least one Y is 5-methyluracil. m is an integer from 4 to 12.](SEQ ID NOs: 31-39) and contain or consist of.

[0119] An even more preferred oligonucleotide is the repeat nucleotide unit (YYX) m [wherein each X is 5-methylcytosine and / or each Y is 5-methyluracil. m is an integer from 4 to 12.](SEQ ID NOs: 31-39) and contain or consist of.

[0120] Therefore, an even more preferred oligonucleotide is the repeat nucleotide unit (YYX)7 [wherein each X is C or 5-methylcytosine and each Y is U or 5-methyluracil. At least one X is 5-methylcytosine and / or at least one Y is 5-methyluracil.](SEQ ID NO: 34) and contain or consist of.

[0121] More preferred oligonucleotides comprise or consist of the repeating nucleotide unit (YYX)7 [wherein each X is 5-methylcytosine and / or each Y is 5-methyluracil.] (SEQ ID NO: 34).

[0122] The most preferred oligonucleotide sequences comprising or consisting of the repeating nucleotide unit (XYG) m are set forth in Table 2 as SEQ ID NOS: 147 to 167.

[0123] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the base sequences of SEQ ID NO: 147 or 148 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the base sequences of SEQ ID NO: 147 or 148, and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NO: 147 or 148, and have a length of 12 nucleotides.

[0124] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 149 or 150, and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 149 or 150, and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 149 or 150, and have a length of 15 nucleotides.

[0125] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 151 or 152, and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 151 or 152, and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 151 or 152, and have a length of 18 nucleotides.

[0126] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NOs: 153 to 157 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NOs: 153 to 157, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NOs: 153 to 157, and have a length of 21 nucleotides.

[0127] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 158 or 159 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 158 or 159, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 158 or 159, and have a length of 24 nucleotides.

[0128] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 160 or 161, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 160 or 161, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 160 or 161, and have a length of 27 nucleotides.

[0129] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 162 or 163, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 162 or 163, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 162 or 163, and have a length of 30 nucleotides.

[0130] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 164 or 165 and have a length of 33 to 36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the nucleotide sequences of SEQ ID NO: 164 or 165, and have a length of 33 to 36 nucleotides, more preferably 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 164 or 165, and have a length of 33 nucleotides.

[0131] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the nucleotide sequences of SEQ ID NO: 166 or 167 and have a length of 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 166 or 167, and have a length of 36 nucleotides.

[0132] In another embodiment, antisense oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylcytosine are (CCG) in the transcript n or (GCC) n represented by a nucleotide sequence that binds (or is capable of binding), hybridizes (or is capable of hybridizing), targets and / or is reverse complementary to the repeat, and is particularly useful for the treatment, delay, remission and / or prevention of the human hereditary disorder fragile XE mental retardation caused by repeat expansion in the FMR2 gene. These genes are preferably of human origin.

[0133] In a preferred embodiment, (CCG) nAn oligonucleotide that is identified as being complementary to the repeat in the reverse orientation, binding (or capable of binding), hybridizing (or capable of hybridizing), or targeting, comprises or consists of the repeating nucleotide unit (XGG) m or (GGX) m [wherein each X is C or 5-methylcytosine. m is an integer.], and has a length of 12 to 36 nucleotides. In this embodiment, m can be 4, 5, 6, 7, 8, 9, 10, 11, 12. A preferred value of m is 7.

[0134] Therefore, a more preferred oligonucleotide comprises or consists of the repeating nucleotide unit (XGG) m or (GGX) m [wherein each X is C or 5-methylcytosine. m is an integer from 4 to 12.](SEQ ID NOs: 49 to 57 or SEQ ID NOs: 40 to 48).

[0135] An even more preferred oligonucleotide comprises or consists of the repeating nucleotide unit (XGG) m or (GGX) m [wherein each X is 5-methylcytosine. m is an integer from 4 to 12.](SEQ ID NOs: 49 to 57 or SEQ ID NOs: 40 to 48).

[0136] Therefore, an even more preferred oligonucleotide comprises or consists of the repeating nucleotide unit (XGG)7 or (GGX)7 [wherein each X is C or 5-methylcytosine.](SEQ ID NO: 52 or SEQ ID NO: 43).

[0137] An even more preferred oligonucleotide comprises or consists of the repeating nucleotide unit (XGG)7 or (GGX)7 [wherein each X is 5-methylcytosine.](SEQ ID NO: 52 or SEQ ID NO: 43).

[0138] The most preferred oligonucleotide sequence that comprises or consists of the repeating nucleotide unit (GGX) m is set forth in Table 2 as SEQ ID NOs: 168 to 177.

[0139] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 168 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 168 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 168 and have a length of 12 nucleotides.

[0140] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 169 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 169 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 169 and have a length of 15 nucleotides.

[0141] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 170 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 170 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 170 and have a length of 18 nucleotides.

[0142] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NOs: 171 to 174 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NOs: 171 to 174 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NOs: 171 to 174 and have a length of 21 nucleotides.

[0143] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 175 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 175, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 175, and have a length of 24 nucleotides.

[0144] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 176 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 176, and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 176, and have a length of 27 nucleotides.

[0145] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 177 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 177, and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 177, and have a length of 30 nucleotides.

[0146] Repeated nucleotide unit (XGG) m The most preferred oligonucleotide sequences comprising or consisting of are set forth in Table 2 as SEQ ID NOs: 178-184.

[0147] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 178 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 178, and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 178, and have a length of 12 nucleotides.

[0148] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 179 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 179 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 179 and have a length of 15 nucleotides.

[0149] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 180 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 180 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 180 and have a length of 18 nucleotides.

[0150] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 181 and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 181, and have a length of 21 to 36 nucleotides, more preferably 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 181, and have a length of 21 nucleotides.

[0151] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 182 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 182, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 182, and have a length of 24 nucleotides.

[0152] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 183 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 183 and have a length of 27 to 36 nucleotides, more preferably 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 183 and have a length of 27 nucleotides.

[0153] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 184 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 184 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 1,84 and have a length of 30 nucleotides.

[0154] In another embodiment, oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylcytosine and / or 2,6-diaminopurine are represented by a nucleotide sequence comprising or consisting of a sequence that hybridizes to (or can hybridize to), binds to (or can bind to), targets and / or is reverse complementary to the (CCUG) repeat in the transcript and is particularly useful for the treatment, delay and / or prevention of type 2 myotonic dystrophy (DM2), a human hereditary disorder caused by a repeat expansion in the DM2 / ZNF9 gene. These genes are preferably of human origin. n ​

[0155] In a preferred embodiment, (CCUG) n Oligonucleotides that are identified as binding (or capable of binding), hybridizing (or capable of hybridizing), or targeting and are reverse complementary to the repeat are the repeating nucleotide unit (XZGG) m [wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine. At least one X is 5-methylcytosine, and / or at least one Z is 2,6-diaminopurine.] and comprises or consists of, and has a length of 12 to 36 nucleotides.

[0156] m is an integer. In this embodiment, m can be 3, 4, 5, 6, 7, 8, 9. A preferred value of m is 5.

[0157] Therefore, a more preferred oligonucleotide is the repeating nucleotide unit (XZGG) m [wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine. At least one X is 5-methyl-cytosine, and / or at least one A is 2,6-diaminopurine. m is an integer from 3 to 9.](SEQ ID NOs: 63-69) and comprises or consists of.

[0158] An even more preferred oligonucleotide is the repeating nucleotide unit (XZGG) m [wherein each X is 5-methylcytosine, and / or each Z is 2,6-diaminopurine. m is an integer from 3 to 9.](SEQ ID NOs: 63-69) and comprises or consists of.

[0159] Therefore, an even more preferred oligonucleotide is the repeating nucleotide unit (XZGG)5 [wherein each X is C or 5-methylcytosine, and each Z is A or 2,6-diaminopurine. At least one X is 5-methylcytosine, and / or at least one Z is 2,6-diaminopurine.](SEQ ID NO: 65) and comprises or consists of.

[0160] More preferred oligonucleotides comprise or consist of the repeating nucleotide unit (XZGG)5 [wherein each X is 5-methyl-cytosine and / or each Z is 2,6-diaminopurine.](SEQ ID NO: 65).

[0161] The repeating nucleotide unit (XZGG) m The most preferred oligonucleotide sequences comprising or consisting of the repeating nucleotide unit (XZGG) are set forth in Table 2 as SEQ ID NOS: 193 to 208.

[0162] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA include one of the base sequences of SEQ ID NO: 193 or 194 and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. More preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, include one of the base sequences of SEQ ID NO: 193 or 194, and have a length of 12 to 36 nucleotides, more preferably 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a base sequence consisting of one of the base sequences of SEQ ID NO: 193 or 194, and have a length of 12 nucleotides.

[0163] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 195 or 196 and have a length of 16 to 36 nucleotides, more preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 195 or 196, and have a length of 16 to 36 nucleotides, more preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 195 or 196, and have a length of 16 nucleotides.

[0164] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 197 - 200 and have a length of 20 to 36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 197 - 200, and have a length of 20 to 36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 197 - 200, and have a length of 20 nucleotides.

[0165] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 201 or 202 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 201 or 202, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 201 or 202, and have a length of 24 nucleotides.

[0166] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 203 or 204 and have a length of 28 to 36 nucleotides, more preferably 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 203 or 204, and have a length of 28 to 36 nucleotides, more preferably 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 203 or 204, and have a length of 28 nucleotides.

[0167] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 205 or 206 and have a length of 32 to 36 nucleotides, more preferably 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 205 or 206, and have a length of 32 to 36 nucleotides, more preferably 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 205 or 206, and have a length of 32 nucleotides.

[0168] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 207 or 208 and have a length of 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 207 or 208, and have a length of 36 nucleotides.

[0169] In another embodiment, oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methyluracil and / or 2,6-diaminopurine are represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is capable of binding), hybridizes (or is capable of hybridizing), targets and / or is reverse complementary to the (AUUCU) n repeat in an intron and are particularly useful for the treatment, delay, remission and / or prevention of type 10 human hereditary disorder spinocerebellar ataxia (SCA10). This gene is preferably of human origin.

[0170] In a preferred embodiment, an oligonucleotide identified as binding (or capable of binding), hybridizing (or capable of hybridizing) or targeting and being reverse complementary to the (AUUCU) n repeat is the repetitive nucleotide unit (ZGZZY)m [Wherein each Y is U or 5-methyluracil, and each Z is A or 2,6-diaminopurine. At least one Y is 5-methyluracil, and / or at least one Z is 2,6-diaminopurine.] It contains or consists of, and has a length of 12 to 36 nucleotides.

[0171] m is an integer. In this embodiment, m can be 3, 4, 5, 6, 7. The preferred value of m is 4.

[0172] Therefore, a more preferred oligonucleotide is the repeating nucleotide unit (ZGZZY) m [Wherein each Y is U or 5-methyluracil, and each Z is A or 2,6-diaminopurine. At least one Y is 5-methyluracil, and / or at least one Z is 2,6-diaminopurine. m is an integer from 3 to 7.](SEQ ID NOs: 58 to 62) It contains or consists of.

[0173] An even more preferred oligonucleotide is the repeating nucleotide unit (ZGZZY) m [Wherein each Y is 5-methyluracil, and / or each Z is 2,6-diaminopurine. m is an integer from 3 to 7.](SEQ ID NOs: 58 to 62) It contains or consists of.

[0174] Therefore, an even more preferred oligonucleotide is the repeating nucleotide unit (ZGZZY)4 [Wherein each Y is C or 5-methyluracil, and each Z is A or 2,6-diaminopurine. At least one Y is 5-methyluracil, and / or at least one Z is 2,6-diaminopurine.](SEQ ID NO: 59) It contains or consists of.

[0175] An even more preferred oligonucleotide is the repeating nucleotide unit (ZGZZY)4 [Wherein each Y is 5-methyluracil, and / or each Z is 2,6-diaminopurine.](SEQ ID NO: 59) It contains or consists of.

[0176] Repeating nucleotide unit (ZGZZY) mThe most preferred oligonucleotide sequences comprising or consisting of are set forth in Table 2 as SEQ ID NOs: 185 to 192.

[0177] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 185 and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 185, and have a length of 15 to 36 nucleotides, more preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 185, and have a length of 15 nucleotides.

[0178] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NOs: 186 to 189 and have a length of 20 to 36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NOs: 186 to 189, and have a length of 20 to 36 nucleotides, more preferably 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NOs: 186 to 189, and have a length of 20 nucleotides.

[0179] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 190 and have a length of 25 to 36 nucleotides, more preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 190 and have a length of 25 to 36 nucleotides, more preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 190 and have a length of 25 nucleotides.

[0180] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 191 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 191 and have a length of 30 to 36 nucleotides, more preferably 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 191 and have a length of 30 nucleotides.

[0181] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 192 and have a length of 35 to 36 nucleotides, more preferably 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 192, and have a length of 35 to 36 nucleotides, more preferably 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 192, and have a length of 35 nucleotides.

[0182] In another embodiment, oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA and containing 5-methylcytosine and / or abasic monomers and / or inosine are present in the C9ORF72 human transcript (GGGGCC) n represented by a nucleotide sequence comprising or consisting of a sequence that binds (or is capable of binding), hybridizes (or is capable of hybridizing), targets and / or is reverse complementary to the (GGGGCC) repeat and is particularly useful for the treatment, delay, remission and / or prevention of the human hereditary disorder amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). These genes are preferably of human origin.

[0183] In a preferred embodiment, (GGGGCC) n Oligonucleotides identified as being reverse complementary to, binding (or capable of binding), hybridizing (or capable of hybridizing) or targeting the (GGGGCC) repeat are the repetitive nucleotide units (GGXUXX) m , (GGXQXX) m , (GGXIXX) m or (GGCCUC) m[Wherein each X is C or 5-methylcytosine, each Q is a deoxyribonucleotide monomer, and each I is inosine. At least one X is 5-methylcytosine. m is an integer.] comprising or consisting of, having a length of 17 to 36 nucleotides. In this embodiment, m can be 3, 4, 5, 6, or 7. A preferred value of m is 3 or 4.

[0184] More preferably, the oligonucleotide comprises or consists of the repetitive nucleotide unit sequence numbers 216 to 219 described in Table 1. Repetitive nucleotide unit (GGXUXX) m , (GGXQXX) m , (GGXIXX) m or (GGCCUC) m A more preferred oligonucleotide sequence comprising or consisting of is described in Table 2 as SEQ ID NOs: 209 to 215.

[0185] A preferred oligonucleotide comprising or consisting of 2'-O-methyl phosphorothioate RNA contains one of the base sequences of SEQ ID NO: 209 or 211 and has a length of 17 to 36 nucleotides, more preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. A more preferred oligonucleotide consists of 2'-O-methyl phosphorothioate RNA, contains one of the base sequences of SEQ ID NO: 209 or 211, and has a length of 17 to 36 nucleotides, more preferably 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. The most preferred oligonucleotide consists of 2'-O-methyl phosphorothioate RNA, has a base sequence consisting of one of the base sequences of SEQ ID NO: 209 or 211, and has a length of 17 or 18 nucleotides.

[0186] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 210 and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 210, and have a length of 18 to 36 nucleotides, more preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 210, and have a length of 18 nucleotides.

[0187] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain one of the nucleotide sequences of SEQ ID NO: 212 or 215 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain one of the nucleotide sequences of SEQ ID NO: 212 or 215, and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of one of the nucleotide sequences of SEQ ID NO: 212 or 215, and have a length of 24 nucleotides.

[0188] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 213 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 213 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 213 and have a length of 24 nucleotides.

[0189] Preferred oligonucleotides comprising or consisting of 2'-O-methyl phosphorothioate RNA contain the nucleotide sequence of SEQ ID NO: 214 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Even more preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, contain the nucleotide sequence of SEQ ID NO: 214 and have a length of 24 to 36 nucleotides, more preferably 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. Most preferred oligonucleotides consist of 2'-O-methyl phosphorothioate RNA, have a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 214 and have a length of 24 nucleotides.

[0190] In one embodiment, the oligonucleotide preferably comprises or consists of 2'-O-methyl phosphorothioate RNA, contains 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine bases, and is represented by a nucleotide sequence comprising or consisting of at least 12 to 36 consecutive nucleotides, and the oligonucleotide targets, hybridizes (or is capable of hybridizing), binds (or is capable of binding) and / or is reverse complementary to the aforementioned repeat. More preferably, the nucleotide sequence comprises or consists of at least 12 to 36 nucleotides, still more preferably 15 to 24, and most preferably 20 or 21 nucleotides. The length of the oligonucleotide can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 nucleotides. The oligonucleotide is reverse complementary to, and / or capable of hybridizing to, and / or capable of targeting, and / or capable of binding to a repeat in the coding region of the transcript, preferably polyglutamine (CAG) n in the coding tract and / or capable of hybridizing and / or capable of targeting and / or capable of binding. Also, the oligonucleotide can be reverse complementary to, and / or capable of hybridizing to, and / or capable of targeting, and / or capable of binding to a non-coding region present in the precursor RNA molecule, such as the 5' or 3' untranslated region or intron sequence.

[0191] In the present invention, the expression "is capable of" can be replaced with "(is) able to".

[0192] In a second aspect, the present invention relates to an oligonucleotide comprising one or more abasic sites at one or both ends. Preferably 1 to 10, more preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10, and most preferably 4 abasic sites are present at one or both ends of the oligonucleotide. The one or more abasic sites can be present at both or only one of the free ends (5' and 3') of the oligonucleotide. The oligonucleotide according to this aspect of the present invention has the aforementioned (CAG) n , (GCG) n, (CGG) n , (GAA) n , (GCC) n , (CCG) n , (AUUCU) n , (GGGGCC) n or (CCUG) n Preferably represented by a nucleotide or base sequence that binds to (or is capable of binding to), hybridizes to (or is capable of hybridizing to), targets, and / or is antisense to a repetitive element in an RNA transcript selected from. Said oligonucleotide is preferably a single-stranded oligonucleotide and may further contain one or more base modifications, sugar modifications, and / or backbone modifications such as 5-methyl-C, 5-methyl-U, 2,6-diaminopurine, 2'-O-methyl, phosphorothioate, and combinations thereof, as discussed herein. It is understood that these modifications are not mandatory in this aspect of the invention.

[0193] An oligonucleotide according to this aspect of the invention containing one or more abasic sites at one or both ends has improved parameters compared to an oligonucleotide that does not contain such abasic sites. Here, as previously explained herein in connection with the improved parameters of the oligonucleotide of the first aspect, the parameters may include the binding affinity and / or kinetics, silencing activity, allelic selectivity, biological stability, (in tissue) distribution, cellular uptake and / or transport, and / or immunogenicity of said oligonucleotide. Each assay and definition provided herein with respect to the improved parameters of the oligonucleotide of the first aspect is also retained for the oligonucleotide of the second aspect.

[0194] Hereinafter, consisting of or containing 2'-O-methyl phosphorothioate RNA, containing 5-methylcytosine and / or 5-methyluracil bases, represented by a nucleotide or base sequence containing (CUG) m and thus, (CAG) nThe present invention will be further described by taking as an example oligonucleotides that bind to (or can bind to), hybridize to (or can hybridize to), target, and / or are reverse complementary. Similar parameters defined for such oligonucleotides are included within the scope of the present invention and can be defined by those skilled in the art for other oligonucleotides that bind to (or can bind to), hybridize to (or can hybridize to), target, and / or are reverse complementary to other repeats described herein. Also, other or similar symptoms can be identified by those skilled in the art for other diseases described herein.

[0195] In a preferred embodiment, the oligonucleotides designed herein can delay and / or cure and / or treat and / or prevent and / or remit human genetic disorders such as Huntington's disease (HD), spinocerebellar ataxia (SCA) type 1, 2, 3, 6, 7, 12 or 17, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal muscular atrophy (SBMA), and / or dentatorubral-pallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in the transcripts of the HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes, if the oligonucleotide can reduce or decrease the amount of (toxic) transcripts of the disease alleles of the HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes in the cells of a patient, in the tissues of a patient, and / or in a patient. In one embodiment, the HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 gene is a human gene.

[0196] In the case of HD, the expanded CAG repeat region is present in exon 1 of the HTT gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 38 to 180 repeat units containing the CAG trinucleotide in the transcriptional sequence of the HTT gene.

[0197] In the case of SCA1, the expanded CAG repeat region is present in exon 8 of the ATXN1 gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 41 to 83 repeat units containing the CAG trinucleotide in the transcriptional sequence of the ATXN1 gene.

[0198] In the case of SCA2, the expanded CAG repeat region is present in exon 1 of the ATXN2 gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 32 to 200 repeat units containing the CAG trinucleotide in the transcriptional sequence of the ATXN2 gene.

[0199] In the case of SCA3, the expanded CAG repeat region is present in exon 8 of the ATXN3 gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 52 to 86 repeat units containing the CAG trinucleotide in the transcriptional sequence of the ATXN3 gene.

[0200] In the case of SCA6, the expanded CAG repeat region is present in exon 47 of the CACNA1A gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 20 to 33 repeat units containing the CAG trinucleotide in the transcriptional sequence of the CACNA1A gene.

[0201] In the case of SCA7, the expanded CAG repeat region is present in exon 3 of the ATXN7 gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 36 to 460 repeat units containing the CAG trinucleotide in the transcriptional sequence of the ATXN7 gene.

[0202] In the case of SCA12, the expanded CAG repeat region may be present in the 5’ untranslated region (UTR), intron, or open reading frame of the PPP2R2B gene in the patient's genome. As used herein, the expanded CAG repeat region may be defined as including a continuous repeat of 66 to 78 repeat units containing the CAG trinucleotide in the transcribed sequence of the PPP2R2B gene.

[0203] In the case of SCA17, the expanded CAG repeat region is present in exon 3 of the TBP gene in the patient's genome. As used herein, the expanded CAG repeat region may be defined as including a continuous repeat of 45 to 66 repeat units containing the CAG trinucleotide in the transcribed sequence of the TBP gene.

[0204] In the case of ALS or FTD, the expanded CAG repeat region is present in exon 1 of the ATXN2 gene in the patient's genome. As used herein, the expanded CAG repeat region may be defined as including a continuous repeat of 27 to 33 repeat units containing the CAG trinucleotide in the transcribed sequence of the ATXN2 gene.

[0205] In the case of ALS or FTD, the expanded GGGGCC repeat region is present in the first intron of the C9ORF72 gene in the patient's genome. As used herein, the expanded GGGGCC repeat region may be defined as including a continuous repeat of more than 30 repeat units containing the GGGGCC hexanucleotide in the transcribed sequence of the C9ORF72 gene.

[0206] In the case of SBMA, the expanded CAG repeat region is present in exon 1 of the AR gene in the patient's genome. As used herein, the expanded CAG repeat region may be defined as including a continuous repeat of 40 repeat units containing the CAG trinucleotide in the transcribed sequence of the AR gene.

[0207] In the case of DRPLA, the expanded CAG repeat region is present in exon 5 of the ATN1 gene in the patient's genome. As used herein, the expanded CAG repeat region can be defined as including a continuous repeat of 49 to 88 repeat units containing the CAG trinucleotide in the transcriptional sequence of the ATN1 gene.

[0208] Throughout this specification, the term CAG repeat is (CAG) n is replaced by, and vice versa, where n is 6 to 29 if the repeat is present in exon 1 of the HTT transcript of a healthy individual, 6 to 39 if the repeat is present in exon 8 of the ATXN1 gene of a healthy individual, less than 31 if the repeat is present in exon 1 of the ATXN2 gene of a healthy individual, 12 to 40 if the repeat is present in exon 8 of the ATXN3 gene of a healthy individual, less than 18 if the repeat is present in exon 47 of the CACNA1A gene of a healthy individual, 4 to 17 if the repeat is present in exon 3 of the ATXN7 gene of a healthy individual, 7 to 28 if the repeat is present in the 5'UTR of the PPP2R2B gene of a healthy individual, 25 to 42 if the repeat is present in exon 3 of the TBP gene of a healthy individual, 13 to 31 if the repeat is present in exon 1 of the AR gene of a healthy individual, 12 to 40 if the repeat is present in exon 8 of the ATXN3 gene of a healthy individual, or 6 to 35 if the repeat is present in exon 5 of the ATN1 gene of a healthy individual, and can be an integer.

[0209] This preferably means that the oligonucleotide of the invention reduces a detectable amount of a disease-related or disease-causing or mutant transcript containing an extended or unstable number of CAG repeats in the cells of a patient, in the tissues of a patient and / or in a patient. Alternatively or additionally, the oligonucleotide may reduce the translation of the mutant transcript and thus the amount of mutant (toxic) protein. The reduction or decrease in the amount of the extended CAG repeat transcript can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the amount of the extended CAG repeat transcript before treatment. Another parameter can be a decrease in the amount of the (CAG) n transcript or the mutant transcript. This can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the amount of the transcript detected at the start of treatment.

[0210] The reduction or decrease can be evaluated by Northern blotting or Q-RT-PCR (preferably performed in the experimental part). The oligonucleotide of the invention can first be tested in a cell line as described in Example 1 of the experimental part.

[0211] Alternatively, or in combination with the foregoing preferred embodiments, the oligonucleotides designed herein can alleviate one or more symptoms and / or features and / or improve parameters associated with or linked to Huntington's disease (HD), spinocerebellar ataxia type 1, 2, 3, 6, 7, 12 or 17 (SCA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal muscular atrophy (SBMA), and / or dentatorubral-pallidoluysian atrophy (DRPLA) in an individual, when the oligonucleotide is capable of alleviating one or more symptoms and / or features and / or improving parameters associated with or linked to Huntington's disease (HD), spinocerebellar ataxia type 1, 2, 3, 6, 7, 12 or 17 (SCA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal muscular atrophy (SBMA), and / or dentatorubral-pallidoluysian atrophy (DRPLA) caused by CAG repeat expansions in transcripts of the HTT, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, AR or ATN1 genes. The oligonucleotide can delay and / or cure and / or treat and / or prevent and / or remit human hereditary disorders such as Huntington's disease (HD), spinocerebellar ataxia type 1, 2, 3, 6, 7, 12 or 17 (SCA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), X-linked spinal muscular atrophy (SBMA), and / or dentatorubral-pallidoluysian atrophy (DRPLA) if the parameters are improved or the symptoms or features are reduced after at least one week, one month, six months, one year or more of treatment using the dosage of the oligonucleotide of the invention described herein. The oligonucleotide can improve the parameter or reduce the symptom or feature if it can be said that the parameter has improved or the symptom or feature has been reduced after treatment with the dosage of the oligonucleotide of the invention described herein for at least one week, one month, six months, one year or more.

[0212] Here, improvement can mean that the parameter has changed significantly towards the value of the parameter for a healthy individual and / or towards the value of the parameter corresponding to the value of the parameter in the same individual at the start of treatment.

[0213] Here, reduction or alleviation can mean that the symptom or feature has changed significantly towards the absence of the symptom or feature characteristic of a healthy individual and / or towards the change in the symptom or feature corresponding to the state of the same individual at the start of treatment.

[0214] Here, the symptoms of Huntington's disease are chorea-like movements, progressive dementia, and psychiatric findings (such as depression and psychosis). Chorea-like movements consist of involuntary, rapid, irregular, spasm-like motor movements that include facial contractions or risings and contractions of distal sites, and later, more general forms (which may be gait disturbances) (Ropper and Brown, 2005). Each of these symptoms can be evaluated by a physician using known and described methods. Preferred methods are monitoring of total function (TFC), validated criteria for the three main symptom areas of HD measured by validated assessment criteria, or progression of symptoms. These areas are, in particular, progression of motor signs, progression of neuropsychiatric symptoms, and progression of cognitive decline. Thus, another preferred criterion is the Unified HD Rating Scale (UHDRS; Huntington Study Group (Kieburtz K. et al., 1996; 11: 136-142)).

[0215] Huntington's disease (HD), spinocerebellar ataxia type 1, 2, 3, 6, 7 or 17 (SCA), X-linked spinal muscular atrophy (SBMA), and dentatorubral-pallidoluysian atrophy (DRPLA) all result from CAG triplet repeat expansions in the coding regions of genes. Although the disease-causing proteins in these disorders are different, in each case the resulting stretch of glutamines causes a toxic gain of function of the protein, which leads to neurodegeneration. Protein aggregates are seen in the nuclei and cytoplasm of cells, suggesting that protein misfolding is a common feature of these disorders. Thus, common favorable parameters can be the (mutant) protein levels that can be determined by Western blot analysis (Evers et al.) or the presence of protein aggregates in the nucleus and / or cytoplasm that can be monitored by in situ hybridization. Improvement of HD parameters can be a decrease in the detection of the amount of protein aggregates. Such a decrease can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to the amount of protein aggregates before the start of treatment.

[0216] With respect to HD, various other proteins have been found to coexist with htt aggregates, namely, the TATA box binding protein (TBP), the CREB binding protein (CBP), and several molecular chaperones (Huang et al.; Muchowski et al.; Roon-Mom et al.; Steffan et al.). Also, numerous intracellular processes affected (e.g., deregulation of transcription, mitochondrial dysfunction, vesicular transport disorders) have been identified in HD, whereby alternative parameters for HD can be provided (Bauer et al., 2009; Ross et al.). Improvement of each of these possible alternative HD parameters (i.e., the TATA box binding protein (TBP), the CREB binding protein (CBP), and several molecular chaperones) can be considered an improvement of the protein aggregates described above.

[0217] Composition: In a second aspect, there is provided a composition comprising the oligonucleotide described in the section headed "Oligonucleotide". This composition preferably comprises or consists of or consists essentially of the above oligonucleotide.

[0218] As described in the first aspect of the invention with respect to ALS and FTD, it is known that at least two different repeats in at least two different transcripts may be involved in, cause or be associated with the disease. All preferred characteristics associated with each of these oligonucleotides are disclosed in the section entitled "Oligonucleotide".

[0219] In a preferred embodiment, the composition is for use as a medicament. Accordingly, the composition is a pharmaceutical composition. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, diluent and / or excipient. In a preferred embodiment, the composition of the present invention comprises the compounds described herein and optionally further comprises a pharmaceutically acceptable formulation, filler, preservative, solubilizer, carrier, diluent, excipient, salt, adjuvant and / or solvent. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, diluents, salts, adjuvants, solvents and / or excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition, Baltimore, MD: Lippincott Williams & Wilkins, 2000. The compounds described in the present invention have at least one ionizable group. The ionizable group can be a base or an acid and can be charged or neutral. The ionizable group may exist as an ion pair with a suitable counterion carrying an opposite charge. Examples of cationic counterions are sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium and tetraalkylammonium. Examples of anionic counterions are chloride, bromide, iodide, lactate, mesylate, acetate, trifluoroacetate, dichloroacetate and citrate. Examples of counterions are described [e.g., Kumar L. et al., 2008 (which is hereby incorporated by reference in its entirety)].

[0220] The pharmaceutical composition can be further formulated to further assist in enhancing the stability, solubility, absorbability, bioavailability, pharmacokinetics and cellular uptake of the compound, in particular, formulations containing excipients capable of forming complexes, nanoparticles, microparticles, nanotubes, nanogels, hydrogels, poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes and / or liposomes. Examples of nanoparticles include polymeric nanoparticles, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, protein nanoparticles and peptide nanoparticles.

[0221] The preferred composition further comprises at least one excipient that may further assist in promoting the targeting and / or delivery of the composition and / or the oligonucleotide to and / or into muscle and / or brain tissue and / or nerve tissue and / or cells. The cells can be muscle or nerve cells.

[0222] Many of these excipients are known in the art (see, e.g., Bruno, 2011) and can be classified as the first type of excipient. Examples of the first type of excipient include polymers (e.g., polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivatives, butyl cyanoacrylate (PBCA), hexyl cyanoacrylate (PHCA), poly(lactic-co-glycolic acid) (PLGA), polyamines (e.g., spermine, spermidine, putrescine, cadaverine), chitosan, poly(amidoamine) (PAMAM), poly(esteramine), polyvinyl ether, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cyclodextrin, hyaluronic acid, colominic acid, and their derivatives), dendrimers (e.g., poly(amidoamine)), lipids {e.g., 1,2-dioleoyl-3-dimethylammonium propane (DODAP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholine derivatives [e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)], lysophosphatidylcholine derivatives [e.g., 1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-LysoPC)], sphingomyelin, 2-{3-[bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecylcarbamoylmethylacetamide (RPR209120), phosphoglycerol derivatives [e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol sodium salt (DPPG-Na)], phosphatidic acid derivatives [1,2-distearoyl-sn-glycero-3-phosphatidic acid sodium salt (DSPA)], phosphatidylethanolamine derivatives [e.g., dioleoyl-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE)], N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), 1,3 - dioleoyloxy - 2 - (6 - carboxy - spermidyl - propylamide (DOSPER), (1,2 - dimyristyloxypropyl - 3 - dimethylhydroxyethylammonium (DMRIE), N1 - cholesteryloxycarbonyl - 3,7 - diazanonan - 1,9 - diamine (CDAN), dimethyldioctadecylammonium bromide (DDAB), 1 - palmitoyl - 2 - oleoyl - sn - glycerol - 3 - phosphocholine (POPC), b - L - arginyl - 2,3 - L - diaminopropionic acid - N - palmitoyl - N - oleyl - amide trihydrochloride (AtuFECT01), N,N - dimethyl - 3 - aminopropane derivatives [e.g., 1,2 - distearoyloxy - N,N - dimethyl - 3 - aminopropane (DSDMA), 1,2 - dioleyloxy - N,N - dimethyl - 3 - aminopropane (DoDMA), 1,2 - dilinoleyloxy - N,N - 3 - dimethylaminopropane (DLinDMA), 2,2 - dilinoleyl - 4 - dimethylaminomethyl [1,3] - dioxolane (DLin - K - DMA)], phosphatidylserine derivatives [1,2 - dioleoyl - sn - glycero - 3 - phospho - L - serine sodium salt (DOPS)], cholesterol}, proteins (e.g., albumin, gelatin, atelocollagen), and peptides (e.g., protamine, PepFects, NickFects, polyarginine, polylysine, CADY, MPG) are included.,

[0223] Another preferred composition may include at least one excipient classified as a second type of excipient. The second type of excipient may include or may contain a conjugate group described herein for enhancing the targeting and / or delivery of the compositions and / or oligonucleotides of the present invention to and / or into tissues and / or cells such as, for example, muscle or nerve tissue or cells. Any type of excipient may be mixed together in one single composition described herein.,

[0224] One of ordinary skill in the art can select, mix, and / or adapt the above or other alternative excipients and delivery systems for formulating and delivering the compounds for use in the present invention.

[0225] Such pharmaceutical compositions of the invention can be administered to animals, preferably mammals, at effective concentrations at a set time. A more preferred mammal is a human. The oligonucleotides or compositions described herein for use in accordance with the present invention can be suitable for direct administration in vivo to cells, tissues, and / or organs of an individual suffering from or at risk of developing the diseases or conditions described herein, and can be administered directly in vivo, ex vivo, or in vitro. Administration can be via systemic and / or parenteral routes, such as intravenous, subcutaneous, intraventricular, intrathecal, intramuscular, intranasal, enteral, intravitreal, intracerebral, epidural, or oral routes.

[0226] Preferably, such pharmaceutical compositions of the invention can be encapsulated in the form of an emulsion, suspension, pill, tablet, capsule, or soft gel for oral delivery, or in the form of an aerosol or dry powder for delivery to the airway and lungs.

[0227] In one embodiment, the oligonucleotide of the present invention may be used in combination with another compound that is already known to be used for the treatment of the disease. Such other compounds can be used to slow the progression of the disease, reduce abnormal behavior or movement, reduce muscle tissue inflammation, improve the function, integrity and / or survival of muscle fibers and / or nerves, and / or improve, increase or restore cardiac function. Examples include, but are not limited to, steroids, preferably (glucocorticoid) corticosteroids, ACE inhibitors (preferably perindopril), type 1 angiotensin II receptor blockers (preferably losartan), tumor necrosis factor-alpha (TNFα) inhibitors, TGFβ inhibitors (preferably decorin), human recombinant biglycan, mIGF-1 sources, myostatin inhibitors, mannose-6-phosphate, dantrolene, halofuginone, antioxidants, ion channel inhibitors, protease inhibitors, phosphodiesterase inhibitors (preferably PDE5 inhibitors such as sildenafil or tadalafil, and / or PDE10A inhibitors and / or MP-10), L-arginine, dopamine blockers, amantadine, tetrabenazine, coenzyme Q10, antidepressants, antipsychotics, antiepileptic drugs, mood stabilizers (generally omega 3-fatty acids), creatine monohydrate, KMO inhibitors (kynurenine monooxygenase) such as CHDI246 or HDAC4 inhibitors such as PBT2. Such combined use may be sequential use, and each component may be administered in a separate composition. Alternatively, each component may be used together in a single composition.

[0228] Use: In a further aspect, there is provided the use of the composition or oligonucleotide described in the previous section for use as part of a medicament or treatment, or an application in which the oligonucleotide confers its activity intracellularly.

[0229] Preferably, the oligonucleotide or composition of the present invention is for use as part of a drug or therapy for preventing, delaying, curing, remitting and / or treating human cis-element repeat instability-related genetic disorders. The human cis-element repeat instability-related genetic disorder is preferably a neuromuscular genetic disorder, more preferably the aforementioned one.

[0230] Method: In a further aspect, there is provided a method for preventing, treating, curing, remitting and / or delaying the condition or disease described in the previous section in an individual, the cells, tissues or organs of said individual. The method comprises the step of administering the oligonucleotide or composition of the present invention to said individual or subject in need thereof.

[0231] In the method according to the present invention, the oligonucleotide or composition described herein may be suitable for administration in vivo to the cells, tissues and / or organs of an individual suffering from or at risk of developing any of the diseases described herein, and may be administered in vivo, ex vivo or in vitro. The individual or subject in need is preferably a mammal, more preferably a human.

[0232] In a further aspect, there is provided a method for diagnosis, in which the oligonucleotide of the present invention is provided together with a radioactive label or a fluorescent label. In this method, the oligonucleotide of the present invention can be used as an in situ probe for detecting foci (RNA / protein aggregates resulting from repeat expansion) in a sample derived from a subject. Said sample contains cells derived from said subject.

[0233] In one embodiment, in the method of the present invention, the concentration of the oligonucleotide or composition is from 0.01 nM to 1 μM. More preferably, the concentration used is from 0.05 to 500 nM or from 0.1 to 500 nM or from 0.02 to 500 nM or from 0.05 to 500 nM, and even more preferably from 1 to 200 nM.

[0234] The dosage range of the oligonucleotide or composition according to the present invention is preferably designed based on an escalating dose study in a clinical trial (in vivo use) where strict protocol requirements exist. The oligonucleotides described herein can be used at dosages in the range of 0.01 to 200 mg / kg or 0.05 to 100 mg / kg or 0.1 to 50 mg / kg or 0.1 to 20 mg / kg, preferably 0.5 to 10 mg / kg.

[0235] The dosage range of the oligonucleotide or composition of the present invention is 100 to 300 μg / week, a total of 8 to 12 injections, or 150 to 250 μg / week, a total of 9 to 11 injections, or 200 μg / week, a total of 11 injections, or 10 to 350 μg / day, for 2 weeks, or 50 to 250 μg / day, for 2 weeks, or 100 to 200 μg / day, for 2 weeks, or 20 to 80 μg / day, for 2 weeks, or 200 to 320 μg / day, for 2 weeks, or 320 μg / day, for 2 weeks, or 30 μg / day, for 2 weeks and can also be used at dosages that are

[0236] The above-mentioned concentration or dosage ranges of the oligonucleotide or composition are preferred concentrations or dosages for in vitro or ex vivo use. Those skilled in the art will understand that depending on the identity of the oligonucleotide used, the target cells to be treated, the gene target and its expression level, the medium used, as well as the transfection and incubation conditions, the concentration or dosage of the oligonucleotide used can be further varied and may need to be further optimized.

[0237] In the specification, the verb "comprising" and its conjugations are used in their non-limiting sense to mean that the items following the term are included, without excluding items not specifically recited. The verb "comprising" is, unless otherwise specified, synonymous with the verb "having". Also, the verb "consisting of" may be replaced by "consisting essentially of", meaning that the oligonucleotides or compositions described herein may contain additional components other than those specifically defined, provided that such additional components do not change the unique features of the invention. Further, references to an element by the indefinite article "a" or "an" do not exclude the possibility that more than one element is present, unless it is clear from the context that there is one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0238] Each embodiment described herein may be combined together, unless otherwise specified. All patents and references cited herein are hereby incorporated by reference in their entirety.

[0239] Definitions: Throughout this specification, the terms "binding", "targeting", "hybridizing" may be used interchangeably when used in connection with an antisense oligonucleotide that is reverse complementary to a portion of the mRNA precursor described herein. In the present invention, "hybridizing" or "binding" is used under physiological conditions, unless otherwise specified, in cells, preferably human cells.

[0240] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., antisense compounds and their target nucleic acids). Without being limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding (which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotide bases (nucleobases). For example, the natural base adenine is a nucleobase that is complementary to the natural nucleobases thymine, 5-methyluracil and uracil with which it forms hydrogen bonds. The natural base guanine is a nucleobase that is complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur under various circumstances. In particular, hybridization of the oligonucleotides of the invention with the targeted mRNA precursors can occur under various environments. Similarly, binding of the oligonucleotides of the invention to the targeted mRNA precursors can occur under various environments. Preferably, said hybridization or said binding is evaluated under physiological conditions in cells, more preferably in human cells. The oligonucleotides of the invention are said to preferably be able to bind, or be bindable, or be able to hybridize, or be hybridizable when said binding or hybridization occurs under physiological conditions in cells, preferably in human cells.

[0241] As used herein, "nucleotide" refers to a nucleoside further comprising a modified or unmodified phosphate-linked group or a non-phosphate nucleoside-to-nucleoside linkage.

[0242] As used herein, "nucleotide analog" or "nucleotide equivalent" refers to a nucleotide that contains at least one modification with respect to a nucleotide that occurs naturally in RNA, such as A, C, G and U. Such modifications can be internucleoside linkage modifications and / or sugar modifications and / or base modifications.

[0243] As used herein, "monomer" refers to a precursor in the synthesis of an oligomeric or polymeric compound. Also, monomer units or residues in such oligomeric or polymeric compounds are included in the term "monomer". Thus, "monomer" and "nucleotide residue" may be used interchangeably throughout the specification. In the present invention, the monomer is preferably a nucleotide. Preferred monomers incorporated into the oligonucleotides of the present invention are nucleotides containing 2'-O-methyl substitution, phosphorothioate internucleoside linkages and 5-methylpyrimidine and / or 2,6-diaminopurine nucleobases.

[0244] As used herein, "nucleobase" refers to the heterocyclic base portion of a nucleoside. The nucleobases may be naturally occurring or modified, and thus include, but are not limited to, adenine, cytosine, guanine, uracil, thymine, and analogs thereof (e.g., 5-methyl-cytosine). In certain embodiments, the nucleobase may include a group of atoms that can hydrogen bond to any atom or an atom of another nucleic acid base.

[0245] As used herein, "T m " means the melting temperature, which is the temperature at which the two strands of a double-stranded nucleic acid separate. T m is often used as a measure of the stability of a double-stranded or the binding affinity of an antisense compound for a complementary RNA molecule.

[0246] As used herein, "2'-modification" or "2'-substitution" refers to a nucleoside containing a pentose sugar with a substituent at the 2'-position other than H or OH. 2'-modified nucleosides include, but are not limited to, bicyclic nucleosides in which a bridge connecting two carbon atoms of the sugar ring connects the 2'-carbon and another carbon of the sugar ring, and allyl, amino, azide, thio, O-allyl, O-C1-C 10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O-N(R m )(R n ) or O-CH2-C(=O)-N(R m )(Rn ), wherein R m and R n are each independently H or a substituted or unsubstituted C1-C 10 alkyl. Nucleosides having non-bridged 2'-substituents such as] are included. The 2'-modified nucleoside may further contain other modifications at other positions of the sugar and / or the nucleobase, for example.

[0247] As used herein, "2'-O-Me", "2'-OMe", "2'-OCH3" or "2'-O-methyl" each refers to a nucleoside containing a sugar having an -OCH3 group at the 2'-position of the sugar ring.

[0248] As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-O-methoxyethyl" each refers to a nucleoside containing a sugar having an -OCH2CH2OCH3 group at the 2'-position of the sugar ring.

[0249] As used herein, the term "adenine analog" means a chemically modified purine nucleobase that can form a base pair with either thymine or uracil of the complementary strand of RNA or DNA when incorporated into an oligomer. Such base pairs are preferably Watson-Crick base pairs, but analogs and minor deviations thereof are also considered acceptable in the present invention.

[0250] As used herein, the term "uracil analog" means a chemically modified pyrimidine nucleobase that can form a base pair with adenine of the complementary strand of RNA or DNA when incorporated into an oligomer. Such base pairs are preferably Watson-Crick base pairs, but analogs and minor deviations thereof are also considered acceptable in the present invention.

[0251] As used herein, the term "thymine analog" means a chemically modified pyrimidine nucleobase that can form a base pair with adenine of the complementary strand of RNA or DNA when incorporated into an oligomer. Such base pairs are preferably Watson-Crick base pairs, but analogs and minor deviations thereof are also considered acceptable in the present invention.

[0252] As used herein, the term "cytosine analog" means a chemically modified pyrimidine nucleobase that can form a base pair with guanine of the complementary strand of RNA or DNA when incorporated into an oligomer. For example, a cytosine analog can be 5-methylcytosine. Such base pairs are preferably Watson-Crick base pairs, but analogs and minor deviations thereof are also considered acceptable in the present invention.

[0253] As used herein, the term "guanine analog" means a chemically modified purine nucleobase that can form a base pair with cytosine of the complementary strand of RNA or DNA when incorporated into an oligomer. Such base pairs are preferably Watson-Crick base pairs, but analogs and minor deviations thereof are also considered acceptable in the present invention.

[0254] As used herein, the term "guanosine" refers to a nucleoside or sugar-modified nucleoside containing guanine or a guanine analog nucleobase.

[0255] As used herein, the term "uridine" refers to a nucleoside or sugar-modified nucleoside containing uracil or a uracil analog nucleobase.

[0256] As used herein, the term "thymidine" refers to a nucleoside or sugar-modified nucleoside containing thymine or a thymine analog nucleobase.

[0257] As used herein, the term "cytidine" refers to a nucleoside or sugar-modified nucleoside containing cytosine or a cytosine analog nucleobase.

[0258] As used herein, the term "adenosine" refers to a nucleoside or a sugar-modified nucleoside that contains an adenine or an adenine analog nucleobase.

[0259] As used herein, "oligonucleotide" refers to a compound that contains a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides are modified. In certain embodiments, the oligonucleotide contains one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0260] As used herein, "internucleoside linkage" refers to a covalent bond between adjacent nucleosides. The internucleoside linkage can be a naturally occurring internucleoside linkage, i.e., a 3' to 5' phosphodiester linkage, or a modified internucleoside linkage.

[0261] As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0262] As used herein, "backbone" refers to an alternating chain of sugar moieties and internucleoside linkages that occur in an oligonucleotide. The oligonucleotides of the invention contain at least one phosphorodithioate internucleoside linkage, but it should be understood that additional backbone modifications such as sugar modifications and / or internucleoside linkage modifications may be present in the backbone.

[0263] As used herein, "oligomeric compound" refers to a polymeric structure that contains two or more basic structures. In certain embodiments, the oligomeric compound is an oligonucleotide. In certain embodiments, the oligomeric compound is a single-stranded oligonucleotide. In certain embodiments, the oligomeric compound is a double-stranded structure that contains two oligonucleotides. In certain embodiments, the oligomeric compound is a single-stranded or double-stranded oligonucleotide that contains one or more conjugate groups and / or terminal groups.

[0264] As used herein, "conjugate" refers to an atom or group of atoms attached to an oligonucleotide or oligomeric compound. Generally, conjugate groups modify one or more properties of the compound to which the conjugate group is attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are commonly used in the chemical arts and are attached to a parent compound, such as an oligomeric compound, either directly or via an optional linking moiety or linker group. In certain embodiments, conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocolesterol, cholanic acid moieties, folic acid, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluorescein, rhodamine, coumarin, and dyes. In certain embodiments, the conjugate is a terminal group. In certain embodiments, the conjugate is attached to an internal nucleoside of a 3' or 5' terminal nucleoside or oligonucleotide.

[0265] As used herein, "conjugate linking group" refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linker groups or bifunctional linking moieties such as those known in the art are contemplated for use in accordance with the present invention.

[0266] As used herein, "antisense compound" refers to an oligomeric compound, at least a portion of which is at least partially complementary or at least partially oriented to a target nucleic acid to which the oligomeric compound hybridizes to modulate the activity, processing, or expression of the target nucleic acid.

[0267] As used herein, "expression" refers to the process by which a gene ultimately gives rise to a protein. Expression includes, but is not limited to, transcription, splicing, post-transcriptional modification, and translation.

[0268] As used herein, "antisense oligonucleotide" refers to an antisense compound that is an oligonucleotide.

[0269] As used herein, "antisense activity" refers to any detectable and / or measurable activity resulting from hybridization of an antisense compound to its target nucleic acid. In certain embodiments, such activity can be an increase or decrease in the amount of nucleic acid or protein. In certain embodiments, such activity can be a change in the ratio of splice variants of nucleic acid or protein. Detection and / or measurement of antisense activity can be direct or indirect. In certain embodiments, antisense activity is evaluated by observing a change in phenotype in a cell or animal.

[0270] As used herein, "target nucleic acid" refers to any nucleic acid molecule whose expression, amount or activity can be regulated by an antisense compound. In certain embodiments, the target nucleic acid is DNA or RNA. In certain embodiments, the target RNA is miRNA, mRNA, mRNA precursor, non-coding RNA or natural antisense transcript. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene), the expression of which is associated with a particular disorder or disease state.

[0271] As used herein, "target mRNA" refers to a preselected RNA molecule that encodes a protein.

[0272] As used herein, "targeting" or "to target" refers to the association of an antisense compound with a particular target nucleic acid molecule or a particular region of nucleotides within the target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions. Here, "sufficient reverse complementarity" can be at least 90%, 95%, 97%, 99% or 100% reverse complementarity to the targeted nucleic acid molecule.

[0273] As used herein, "target site" refers to the region of a target nucleic acid to which an antisense compound binds. In certain embodiments, the target site is at least partially within the 3' untranslated region of an RNA molecule. In certain embodiments, the target site is at least partially within the 5' untranslated region of an RNA molecule. In certain embodiments, the target site is at least partially within the coding region of an RNA molecule. In certain embodiments, the target site is at least partially within an exon of an RNA molecule. In certain embodiments, the target site is at least partially within an intron of an RNA molecule. In certain embodiments, the target site is at least partially within a miRNA target site of an RNA molecule. In certain embodiments, the target site is at least partially within a repetitive region of an RNA molecule.

[0274] As used herein, "target protein" refers to a protein whose expression is regulated by an antisense compound. In certain embodiments, the target protein is encoded by a target nucleic acid. In certain embodiments, the expression of the target protein is otherwise affected by the target nucleic acid.

[0275] As used herein, "complementarity" with respect to nucleobases refers to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobases refer to the nucleobases of an antisense compound that can base pair with the nucleobases of its target nucleic acid. For example, if the nucleobase at a particular position of an antisense compound can hydrogen bond with the nucleobase at a particular position of a target nucleic acid, the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is considered to be complementary in that nucleobase pair. Nucleobases containing certain modifications can still be nucleobase complementary because they can maintain the ability to pair with the corresponding nucleobase.

[0276] As used herein, "non-complementary" with respect to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise do not support hybridization.

[0277] As used herein, with respect to a linked nucleoside, oligonucleotide or nucleic acid, "complementary" refers to the ability of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid by nucleobase complementarity. In certain embodiments, an antisense compound and its target are complementary to each other if a sufficient number of corresponding positions within each molecule are occupied by nucleobases that can bind to each other to allow for a stable association between the antisense compound and the target. Those skilled in the art recognize that the inclusion of mismatches can be possible without excluding the ability of the oligomeric compound to maintain the association. Thus, antisense compounds are described herein that can contain up to about 20% nucleotides that are mismatched (i.e., not nucleobases complementary to the corresponding nucleotides of the target). Preferably, the antisense compound contains 15% or less, more preferably 10% or less, and most preferably 5% or less mismatches or no mismatches. The remaining nucleobases are either complementary nucleobases or nucleobases that do not disrupt hybridization (e.g., universal bases). Those skilled in the art will recognize that the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary or reverse complementary to the target nucleic acid.

[0278] As used herein, "modulation" refers to a perturbation of the amount or quality of a function or activity as compared to the function or activity prior to modulation. For example, modulation can include any change in gene expression, either an increase (stimulation or induction) or a decrease (inhibition or reduction). As a further example, modulation of expression can include perturbing the splice site selection of mRNA precursor processing that results in a change in the amount of a particular splice variant present as compared to unperturbed conditions. As a further example, modulation includes perturbing the translation of a protein.

[0279] As used herein, "motif" refers to the pattern of modification in an oligomeric compound or a region thereof. A motif can be defined by modifications in specific nucleosides and / or specific linking groups of the oligomeric compound.

[0280] As used herein, "same modification" refers to modifications to naturally occurring molecules that are the same as each other, including the absence of modification. Thus, for example, two unmodified DNA nucleosides have the "same modification" even though the DNA nucleosides are not modified.

[0281] As used herein, "type of modification" with respect to a nucleoside or a "type" of nucleoside refers to the modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0282] As used herein, "pharmaceutically acceptable salt" refers to a salt of an active compound that retains the desired biological activity of the active compound and does not impart undesirable toxic effects to the salt.

[0283] As used herein, the term "independently" means that each occurrence of a repeating variable within the claimed oligonucleotide is selected independently of the others. For example, each repeating variable can be selected such that (i) each of the repeating variables is the same, (ii) two or more are the same, or (iii) each of the repeating variables can be different.

[0284] General chemical definitions: As used herein, "alkyl" refers to a saturated straight-chain or branched hydrocarbon substituent or radical typically containing up to 24 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, etc. An alkyl group typically has from 1 to 24 carbon atoms, more typically from 1 to 12 carbon atoms (C1-C 12It contains an alkyl group, and 1 to 6 carbon atoms (C1-C6 alkyl) are more preferred. In this specification, the term "lower alkyl" includes 1 to 6 carbon atoms (C1-C6 alkyl). In this specification, the alkyl group may optionally contain one or more additional substituents.

[0285] In this specification, "alkenyl" typically refers to a straight-chain or branched-chain hydrocarbon radical or substituent containing up to 24 carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, dienes such as ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, 1,3-butadienyl, etc. The alkenyl group typically contains 2 to 24 carbon atoms, more typically 2 to 12 carbon atoms, and 2 to 6 carbon atoms are more preferred. In this specification, the alkenyl group may optionally contain one or more additional substituents.

[0286] In this specification, "alkynyl" typically refers to a straight-chain or branched-chain hydrocarbon radical or substituent containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, etc. The alkynyl group typically contains 2 to 24 carbon atoms, more typically 2 to 12 carbon atoms, and 2 to 6 carbon atoms are more preferred. In this specification, the alkynyl group may optionally contain one or more additional substituents.

[0287] In this specification, "aminoalkyl" refers to an amino-substituted alkyl radical or substituent. This term has an amino substituent at any position, and the aminoalkyl group is attached to the parent molecule via its alkyl moiety, meaning it contains a C1-C 12 alkyl group. The alkyl and / or amino portions of the aminoalkyl group may optionally be further substituted by additional substituents.

[0288] As used herein, "aliphatic" refers to a straight-chain or branched hydrocarbon radical or substituent typically containing up to 24 carbon atoms, with saturation between any two carbon atoms being a single, double, or triple bond. Aliphatic groups preferably contain 1 to 24 carbon atoms, more typically 1 to 12 carbon atoms, and even more preferably 1 to 6 carbon atoms. The straight-chain or branched chain of the aliphatic group may be interrupted by one or more heteroatoms including nitrogen, oxygen, sulfur, and phosphorus. Such aliphatic groups interrupted by heteroatoms include, but are not limited to, polyalkoxys such as polyalkylene glycols, polyamines, and polyimines. As used herein, aliphatic groups may optionally include further substituents.

[0289] As used herein, "alicyclic" or "alicyclic" refers to a cyclic radical or substituent in which the ring system is aliphatic. The ring system may include one or more rings in which at least one ring is aliphatic. Preferred alicyclic moieties include rings having 5 to 9 carbon atoms in the ring. As used herein, alicyclic groups may optionally include further substituents.

[0290] As used herein, "alkoxy" refers to a radical or substituent containing an alkyl group and an oxygen atom, wherein the alkoxy group is bonded to the parent molecule through its oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. As used herein, alkoxy groups may optionally include further substituents.

[0291] As used herein, "halo", "halide", and "halogen" refer to an atom, radical, or substituent selected from fluorine, chlorine, bromine, and iodine.

[0292] As used herein, "aryl" and "aromatic" refer to radicals or substituents that include mono- or polycyclic carbocyclic systems having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Preferred aryl ring systems have 5 to 20 carbon atoms in one or more rings. As used herein, an aryl group may optionally include further substituents.

[0293] As used herein, "aralkyl" and "arylalkyl" refer to radicals or substituents that include alkyl groups and aryl groups in which the aralkyl or arylalkyl group is attached to the parent molecule through its alkyl moiety. Examples include, but are not limited to, benzyl, phenethyl, and the like. As used herein, an aralkyl group may optionally include further substituents attached to the alkyl portion, the aryl portion, or both portions that form the radical or substituent.

[0294] As used herein, "heterocyclyl" refers to a radical or substituent that includes at least one heteroatom and includes unsaturated, partially saturated, or fully saturated mono- or polycyclic ring systems, thereby including heteroaryl groups. Heterocyclyl also means that one or more of the fused rings contain at least one heteroatom and the other rings may contain one or more heteroatoms or optionally may not contain heteroatoms and include fused ring system moieties. Heterocyclic groups typically include at least one atom selected from sulfur, nitrogen, or oxygen. Examples of heterocyclic groups include [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and the like. As used herein, a heterocyclic group may optionally include further substituents.

[0295] As used herein, "heteroaryl" and "heteroaromatic ring" refer to a radical or substituent that includes a mono- or polycyclic aromatic ring, ring system, or fused ring system in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl also means including a fused ring system in which one or more of the fused rings contain no heteroatoms. A heteroaryl group typically contains one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. A heteroaryl radical or substituent may be bonded directly to the parent molecule or via a linking moiety such as an aliphatic group or a heteroatom. As used herein, a heteroaryl group may optionally include further substituents.

[0296] As used herein, "heteroarylalkyl" refers to a radical or substituent that includes a heteroaryl group and an alkyl moiety, wherein the heteroarylalkyl group is bonded to the parent molecule through its alkyl moiety. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, naphthyridinylpropyl, and the like. As used herein, a heteroarylalkyl group may optionally include further substituents on one or both of the heteroaryl or alkyl moieties.

[0297] As used herein, "mono- or polycyclic" refers to any ring system, such as a monocyclic or polycyclic system having fused or linked rings, and means including single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic ring and heteroarylalkyl. Such single and polycyclic structures may contain rings having uniform or varying degrees of saturation, including fully saturated, partially saturated or fully unsaturated rings. Each ring may contain ring atoms selected from C, N, O and S such that heterocyclic rings and rings containing only C ring atoms are formed. Heterocyclic rings and all carbon rings may be present in a mixed motif such as benzimidazole where one ring of the fused ring system has only carbon ring atoms and the other ring has two nitrogen atoms. The mono- or polycyclic structure may be further substituted, for example, by substituents such as phthalimide having two oxo groups (=O) attached to one of the rings. In another aspect, the mono- or polycyclic structure may be attached to the parent molecule directly through a ring atom or through a substituent or a bifunctional linking moiety.

[0298] As used herein, "acyl" refers to a radical or substituent containing a carbonyl moiety (C=O or -C(O)-) and a further substituent X, where the acyl group is attached to the parent molecule through its carbonyl moiety. Thus, an acyl group is formally obtained by removal of a hydroxyl group from an organic acid and has the general formula -C(O)-X [wherein X is typically an aliphatic group, an alicyclic group or an aromatic group]. The term "acyl" also means including a heteroacyl radical or substituent having the general formula -Y(O) n -X [wherein X is as defined above and Y(O) n is typically a sulfonyl, sulfinyl or phosphate]. Examples of acyl groups include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, etc. As used herein, an acyl group may optionally contain further substituents.

[0299] As used herein, "substituent" includes a group that is typically added to another substituent or a parent compound to enhance a desired property or impart a desired effect. The substituent may or may not be protected and may be attached to one or more available sites within the parent compound. The substituent may also be further substituted by other substituents and may be attached to the parent compound directly or via a linking group such as an alkyl or hydrocarbyl group. As used herein, "hydrocarbyl" refers to any group containing C, O, and H. Linear, branched, and cyclic groups having any degree of saturation are included. Such hydrocarbyl groups may contain one or more heteroatoms selected from N, O, and S and may be further substituted by one or more substituents.

[0300] Unless otherwise specified, the terms "substituted" or "optionally substituted" refer to the (optional) presence of any of the following substituents: halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)R aa ), carboxyl (-C(O)O-R aa ), aliphatic group, alicyclic group, alkoxy, substituted oxo (-O-R aa ), aryl, aralkyl, heterocycle, heteroaryl, heteroarylalkyl, amino (-NR bb R cc ), imino (=NR bb ), amide (-C(O)NR bb R cc or -N(R bb )C(O)R aa ), azide (-N3), nitro (-NO2), cyano (-CN), carbamide (-OC(O)NR bb R cc or -N(R bb )C(O)OR aa ), ureido (-N(R bb )C(O)NR bb R cc ), thioureido (-N(R bb )C(S)NR bb R cc ), guanidinyl (-N(R bb)C(=NR bb )NR bb R cc )、 amidinyl (-C(=NR bb )NR bb R cc or -N(R bb )C(NR bb )R aa )、 thiol (-SR bb )、 sulfinyl (-S(O)R bb )、 sulfonyl (-S(O)2R bb )、 sulfonamidyl (-S(O)2NR bb R cc or -N(R bb )S(O)2R bb )、 and conjugate groups. As used herein, R aa 、 R bb and R cc are each independently H, an optionally linked chemical functional group, or a further substituent, and preferably, but not limited to, are selected from the group consisting of H, alkyl, alkenyl, alkynyl, aliphatic group, alkoxy, acyl, aryl, aralkyl, heteroaryl, cycloaliphatic group, heterocycle, and heteroarylalkyl. The selected substituents within the compounds described herein are present recursively.

[0301] Here, a "recursive substituent" means that a substituent can cite another example of itself. Due to the recursive nature of such substituents, theoretically, there can be a large number in any given claim. Those skilled in the fields of pharmaceutical chemistry and organic chemistry understand that the total number of such substituents is reasonably limited by the desired properties of the intended compound. Such properties include, for example, physical properties (e.g., molecular weight, solubility, log P), applicability (e.g., activity against a target), and practical properties (e.g., ease of synthesis).

[0302] Recursive substituents are an intended aspect of the present invention. Those skilled in the fields of pharmaceuticals and organic chemistry understand the diversity of such substituents. To the extent that recursive substituents are present in the claims of the present invention, the total number is determined as described above.

[0303] In this specification, in a range indicating the number of a specific unit, zero (0) means that the unit may not exist. For example, an oligomeric compound containing 0 to 2 regions of a specific motif means that the oligomeric compound may contain one or two such regions having the specific motif or may not have any region having the specific motif. When an internal part of a molecule does not exist, the parts adjacent to the non-existent part are directly bonded to each other. Similarly, in this specification, the term "none" indicates that a specific feature does not exist.

[0304] In this specification, "analog" or "derivative" means either a compound or a moiety that is structurally similar but different from the parent compound in elemental composition regardless of the method by which the compound is made. For example, an analog or derivative compound is not required to be made from a parent compound such as a chemical starting material.

[0305] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

Brief Description of Drawings

[0306]

Figure 1

Figure 2

[0307] Table 1: General structure of AONs [X=C or 5-methylcytosine; Y=U or 5-methyluracil; Z=A or 2,6-diaminopurine; I=inosine; Q=abasic monomer] All AONs containing SEQ ID NOs: 4-69 or 216-219 contain at least one base modification selected from 5-methylcytosine, 5-methyluracil, and 2,6-diaminopurine. [Table 1-1] [Table 1-2] [Table 1-3]

[0308] Table 2: General structure of AONs All AONs were 2'-O-methyl phosphorothioate AONs. C is 5-methylcytosine, U is 5-methyluracil, A is 2,6-diaminopurine, I is inosine, and Q is an abasic monomer.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0309] [Example 1] Introduction: Specific features of the selected antisense oligonucleotide (AON) chemistries are that they can at least partially enhance binding affinity and stability, enhance activity, improve safety, and / or reduce the cost of the product by shortening the length or improving the synthesis and / or purification procedures. This example describes a comparative analysis of the activity of AONs designed to target the elongation (CAG) n repeats in HTT transcripts in HD fibroblasts in vitro, and includes AONs having either 5-methylcytosine (XYG)7 [where X is 5-methylcytosine and Y = U.](also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2)) or 5-methyluracil (XYG)7 [where X = C and Y = 5-methyluracil.](also represented as SEQ ID NO: 97 (derived from SEQ ID NO: 3)).

[0310] Materials and Methods: Cell Culture: Patient-derived HD fibroblasts (GM04022) (purchased from Coriell Cell Repositories, Camden, USA) were cultured in minimum essential medium (MEM) (Gibco Invitrogen, Carlsbad, USA) containing 15% heat-inactivated fetal bovine serum (FBS) (Clontech, Palo Alto, USA), 1% Glutamax (Gibco), and 100 U / ml penicillin / streptomycin (P / S) (Gibco) at 37°C and 5% CO2.

[0311] Oligonucleotides: The AONs were fully 2'-O-methyl phosphorothioate modified and were the following. PS659, (XYG)7 [wherein X is 5-methylcytosine and Y = U.](also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2)); and PS661, (XYG)7 [wherein X = C and Y = 5-methyluracil.](also represented as SEQ ID NO: 97 (derived from SEQ ID NO: 3)).

[0312] Transfection: Cells were transfected with AON complexed with PEI (2 μL per 1 μg of AON in 0.15 M NaCl). The AON-PEI complex was added to the cells in MEM medium containing 5% FBS such that the final AON concentration was 0.5 - 200 nM. Fresh medium was replenished after 4 hours, and RNA was isolated after 24 hours.

[0313] RNA Isolation: RNA was isolated from cultured cells using the Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol.

[0314] RT-PCR and Lab-on-a-chip Analysis: Approximately 200 ng of RNA was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 μL. PCR was performed with primers for HTT (across the CAG repeat) and β-actin. The PCR program started with an initial denaturation at 95 °C for 4 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 45 seconds, and then a final extension step at 72 °C for 7 minutes. Lab-on-a-Chip was performed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using the Agilent DNA 1000 kit (Agilent DNA 1000 kit). Expression levels were normalized to β-actin levels and to transcript levels without transfection. The following primers were used. HTT forward: 5’-ATGGCGACCCTGGAAAAGCTGAT-3’ (SEQ ID NO: 70) HTT reverse: 5’-TGAGGCAGCAGCGGCTG-3’ (SEQ ID NO: 71) β-actin forward: 5’-GGACTTCGAGCAAGAGATGG-3’ (SEQ ID NO: 72) β-actin reverse: 5’-AGCACTGTGTTGGCGTACAG-3’ (SEQ ID NO: 73)

[0315] Results: Both PS659 (SEQ ID NO: 90 derived from SEQ ID NO: 2) and PS661 (SEQ ID NO: 97 derived from SEQ ID NO: 3) were highly effective and dose-dependently reduced HTT transcripts in HD fibroblasts (Fig. 1a and 1b). Both AONs also showed selectivity for alleles with extended CAG repeats. PS659 (SEQ ID NO: 90 derived from SEQ ID NO: 2) showed a higher effect at a lower concentration (highest effect at 5 nM) (1a) compared to PS661 (SEQ ID NO: 97 derived from SEQ ID NO: 3) (highest effect at 20 nM) (1b) and was more allele-specific.

[0316] [Example 2] Introduction: PS659(XYG)7 [wherein X is 5-methylcytosine and Y = U.](Also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2).) was selected from in vitro tests as the most efficient and safe candidate. The activity in a transgenic HD rat model after a series of intracerebroventricular direct injections is described in this example.

[0317] Materials and Methods: Animals: Transgenic HD rats carry a truncated huntingtin cDNA fragment with 51 CAG repeats under the control of the native rat huntingtin promoter. The expressed gene product is approximately 75 kDa and corresponds to 22% of full-length huntingtin (cDNA positions 324 - 2321, amino acid positions 1 - 709 / 825 (corresponding to exons 1 - 16)), and is under the control of 886 bp of the rat huntingtin promoter (von Horsten S. et al.). All animal experiments were approved by the Institutional Animal Care and Use Committees of the Maastricht University, Maastricht.

[0318] Oligonucleotides: PS659(XYG)7 [wherein X is 5-methylcytosine and Y = U.](Also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2).) is a fully 2'-O-methyl phosphorothioate modified AON.

[0319] In Vivo Treatment: Transgenic HD rats were given 15 intracerebroventricular injections with a final dose of 200 μg of PS659 (also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2).) over a period of 18 weeks. Control HD rats were administered vehicle only. The rats were sacrificed 1 week after the final injection.

[0320] RNA Isolation: Brain tissue-derived RNA was isolated using RNA-Bee reagent (Tel Test, Inc). Briefly, RNA-Bee (50 mg of tissue per 1 mL of RNA-Bee) was added, and the tissue sample was homogenized in a MagNA Lyser green bead tube (Roche) by homogenizing with a MagNA Lyser instrument (Roche). The lysate was transferred to a new tube, chloroform (SIGMA) was added (0.2 mL per 1 mL of RNA-Bee), mixed, incubated on ice for 5 minutes, and centrifuged at 13,000 rpm at 4 °C for 15 minutes. The upper aqueous phase was recovered, an equal volume of isopropanol (SIGMA) was added, and incubation and centrifugation (13,000 rpm, 4 °C, 15 minutes) were performed at 4 °C for 1 hour. The RNA precipitate was washed with 70% (v / v) ethanol (BioSolve), air-dried, and dissolved in MilliQ.

[0321] Quantitative RT-PCR analysis: Approximately 200 ng was subjected to cDNA synthesis with random hexamers using the SuperScript first-strand synthesis system (Invitrogen) in a total volume of 20 μL. Subsequently, 3 μL of a 1 / 40 dilution preparation of the cDNA was used for quantitative PCR analysis by standard procedures in the presence of iQ (trademark) SYBR (registered trademark) Green Supermix (Bio-Rad). Quantitative PCR primers were designed based on the NCBI database sequence information. The identity of the product was confirmed by DNA sequencing. Signals for Rab2 and YWHAZ were used for normalization. The following primers were used. Rat Htt-F: 5’-CGCCGCCTCCTCAGCTTC-3’ (SEQ ID NO: 74) Rat Htt-R: 5’-GAGAGTTCCTTCTTTGGTCGGTGC-3’ (SEQ ID NO: 75) Rab2-F: 5’-TGGGAAACAGATAAAACTCCAGA-3’ (SEQ ID NO: 76) Rab2-R: 5’-AATATGACCTTGTGATAGAACGAAAG-3’(SEQ ID NO: 77) YWHAZ-F: 5’-AAATGAGCTGGTGCAGAAGG-3’(SEQ ID NO: 78) YWHAZ-R: 5’-GGCTGCCATGTCATCGTAT-3’(SEQ ID NO: 79)

[0322] Results: PS659 (also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2)) decreased transgenic Htt transcript levels in the cortex (Fig. 2a), hippocampus (Fig. 2b), olfactory bulb (Fig. 2c), and thalamus (Fig. 3d) compared to saline-treated rats. These results indicate that PS659 (also represented as SEQ ID NO: 90 (derived from SEQ ID NO: 2)) is effective in vivo after intracerebroventricular injection.

[0323] Preferred embodiments of the present invention are as follows. [1] An oligonucleotide for use as a drug for preventing, delaying, and / or treating human cis-element repeat instability-related genetic disorders, comprising 2'-O-methyl RNA nucleotide residues, having a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety, comprising one or more 5-methylpyrimidines and / or one or more 2,6-diaminopurine bases, (CAG) n , (GCG) n , (CGG) n , (GAA) n , (GCC) n , (CCG) n , (AUUCU) n , (GGGGCC) n and (CCUG) n and capable of hybridizing to a repetitive element having a repetitive nucleotide unit selected from the group consisting of Oligonucleotide. [2] Consisting of 2'-O-methyl RNA nucleotide residues, having a backbone in which all phosphate moieties are replaced by phosphorothioate moieties, the oligonucleotide according to [1]. [3] (CAG) n which can hybridize to the repeat, the repeating nucleotide unit (XYG) m comprising or consisting of, where m is an integer from 4 to 12, each X is C or 5-methylcytosine, each Y is U or 5-methyluracil, at least one X is 5-methylcytosine, and / or at least one Y is 5-methyluracil, the oligonucleotide according to [1] or [2]. [4] The oligonucleotide according to [3], wherein each X is 5-methylcytosine, and / or each Y is 5-methyluracil. [5] m is 7, and preferably, the oligonucleotide comprises or consists of the repeating nucleotide unit (XYG)7 [wherein each X is 5-methylcytosine and each Y is uracil (SEQ ID NO: 2). Alternatively, each X is cytosine and each Y is 5-methyluracil (SEQ ID NO: 3).], the oligonucleotide according to [3] or [4]. [6] The oligonucleotide according to [5], wherein the base sequence of the oligonucleotide comprises or consists of any one of the base sequences of SEQ ID NOs: 90 to 118. [7] Consisting of 2'-O-methyl RNA nucleotide residues, having a backbone in which all phosphate moieties are replaced by phosphorothioate moieties, having the base sequence consisting of SEQ ID NO: 90, oligonucleotide. [8] The oligonucleotide according to any one of [1] to [6], comprising 5-methylcytosine and / or 5-methyluracil bases. [9] The oligonucleotide according to any one of [1] to [6] and [8], comprising 2,6-diaminopurine bases.

[10] An oligonucleotide as described in any one of [1] to [6], [8], and [9], having improved parameters as compared to the corresponding oligonucleotide that does not contain 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine, wherein the corresponding oligonucleotide contains 2'-O-methyl RNA nucleotide residues, and has a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety, oligonucleotide.

[11] An oligonucleotide as described in any one of [1] to [6] and [8] to

[10] , having a length of 12 to 36 nucleotides.

[12] An oligonucleotide as described in any one of [1] to

[11] , which is a single-stranded oligonucleotide.

[13] A composition comprising an oligonucleotide as described in any one of [1] to

[12] .

[14] A composition as described in

[13] , further comprising at least one excipient that can further assist in enhancing the targeting and / or delivery of the above composition and / or the above oligonucleotide to and / or into tissues and / or cells.

[15] A method for the prevention, treatment, and / or delay of human cis-element repeat instability-related genetic disorders, wherein the prevention, treatment, and / or delay is effected by administering an oligonucleotide as described in any one of [1] to

[12] or a composition as described in

[13] or

[14] to a subject in need thereof. References

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Claims

**Claim 1** A medicament for preventing, delaying and / or treating a human cis - element repeat instability - related genetic disorder caused by CAG repeat expansion, comprising an oligonucleotide as an active ingredient, wherein the oligonucleotide contains 2'-O-methyl RNA nucleotide residues, has a backbone in which at least one phosphate moiety is replaced by a phosphorothioate moiety, Repeating nucleotide unit (XYG) ｍ comprising or consisting of m is an integer from 4 to 12, each X is C or 5 - methylcytosine, each Y is U or 5 - methyluracil, and at least one X is 5 - methylcytosine, a medicament.

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