Guanosine analog for use in therapeutic polynucleotides

Abstract

The present invention relates to a polynucleotide comprising at least one phosphorothioate internucleoside linkage and at least one guanosine analog comprising a guanine nucleobase analog selected from the group consisting of Formula (I) and Formula (II). Polynucleotides comprising such guanosine analogs exhibit relatively reduced neurotoxicity compared to polynucleotides comprising natural guanosine. TIFF2023529457000035.tif34128

Description

[Technical Field]

[0001] Field of Invention This invention relates to antisense oligonucleotides or polynucleotides such as siRNA or shRNA used as pharmaceutical active ingredients. More specifically, this invention relates to guanosine analogs for use with such polynucleotides. [Background technology]

[0002] background Currently, there is considerable interest in the development of polynucleotides, such as antisense oligonucleotides (ASOs) or siRNA therapies, to treat neurological disorders. Such polynucleotides can be administered, for example, intrathecally. However, the discovery of new polynucleotide therapies has been hampered by the finding that a significant proportion of polynucleotides induce neurotoxicity in animal studies (see, for example, International Publication No. 2016 / 126995). Mice administered some polynucleotides showed signs of acute neurotoxicity within 30 minutes to 1 hour after administration, indicating that the toxicity is unlikely to be due to hybridization events.

[0003] International Publication No. 2016 / 127000 reports the use of a calcium oscillation assay to identify nucleic acid molecules, such as antisense oligonucleotides, that are likely to induce acute neurotoxicity in vivo, and a method for selecting polynucleotides with acceptable in vivo neurotoxicity by calculating the number obtained by subtracting the number of guanosine nucleotides or their analogues from the number of cytosine nucleotides or their analogues and dividing by the total length of the polynucleotide.

[0004] Seela et al., Chim. Acta 1988, 71, 1191-1198, describe the use of 6-chloro-7-deazapurine and 7-deaza-6-(methylthio)purine as general candidates for glycosylation of pyrrolo[2,3-d]pyrimidine.

[0005] Seela, F., and Becher, G., Chemical Communications (1998), (18), 2017-2018, describe the introduction of 7-halogenated 7-deazapurine (pyrrolo-[2,3-d]pyrimidine A) into oligonucleotides. The purpose of this introduction into DNA is presumably to act as a reporter group, cleavage agent, or residue useful for sequencing by mass spectrometry or atomic force microscopy. Kutyavin et al., NAR2002, Vol. 30, pp. 4952-4959, disclose that 8-aza-7-deazaguanine (pyrazolo[3,4-d]pyrimidine, PPG) reduces guanine self-association of guanine-rich oligodeoxyribonucleotides, and that guanine substitution of PPG enhances the affinity, specificity, sensitivity, and predictiveness of guanine-rich DNA probes.

[0006] Hara et al., J Org Chem, reported that LNA-7-deazaguanine and LNA-8-aza-7-deazaguanine-modified phosphodiester oligonucleotides, which were found to have lower binding affinity than natural DNA, as well as LNA-7-deazaguanine, effectively suppress aggregation even in guanine-rich sequences.

[0007] A brief overview of this literature indicates that the problem of neurotoxicity observed when certain polynucleotides are administered to the central nervous system remains largely unresolved.

[0008] Object of the invention The inventors were surprised to find that the proportion of natural, i.e., unmodified guanosine nucleic acid bases within a polynucleotide sequence directly correlates with the potential for polynucleotides such as antisense oligonucleotides or siRNAs to be neurotoxic. This invention provides guanosine analogs for use in therapeutic oligonucleotides, thereby offering reduced neurotoxicity. [Overview of the project]

[0009] This invention is based on the finding that the proportion of natural, i.e., unmodified guanosine (G) nucleic acid bases in a polynucleotide sequence is directly correlated with the potential for polynucleotides, such as antisense oligonucleotides or siRNAs, to be neurotoxic. Having identified unmodified G nucleic acid bases as a trigger for neurotoxicity, the inventors screened numerous G analogs and identified certain G analogs that reduce or mitigate the neurotoxicity of polynucleotides, exemplified by reducing the neurotoxicity of phosphorothioate polynucleotides such as antisense oligonucleotides or siRNAs when used in place of unmodified G. In particular, the inventors identified that substitution of unmodified G nucleic acid bases with 8-aza-7-deazaguanine (PPG) or 8-oxo-deoxyguanosine (8-oxo-dG) bases in phosphorothioate antisense oligonucleotides or siRNAs mitigates neurotoxicity in in vivo and in vitro neurotoxicity assays.

[0010] Furthermore, the inventors identified that the incorporation of PPG bases into polynucleotides can reduce polynucleotide binding affinity, and that this depends heavily on the sequence configuration of the incorporated PPG bases. Using these observations, the inventors identified short double- and triple-stranded motifs associated with maintained effective binding affinity. Alternatively, binding affinity can be compensated for by the incorporation of further high-affinity nucleosides, such as LNAs, into oligonucleotides. [Invention 1001] - at least one phosphorothioate nucleoside interbonding, -below: TIFF0007899100000001.tif34128 A guanosine analog comprising at least one guanine analog selected from the group consisting of and Polynucleotides containing these nucleotides. [Invention 1002] A single-stranded polynucleotide according to the present invention 1001. [Invention 1003] The polynucleotide of the present invention 1002, which is an antisense oligonucleotide. [Invention 1004] A double-stranded polynucleotide according to the present invention 1001. [Invention 1005] A polynucleotide according to the present invention 1004, which is siRNA or shRNA. [Invention 1006] A polynucleotide of the present invention 1001, further comprising one or more 2'-sugar-modified nucleosides. [Invention 1007] The polynucleotide of the present invention 1002 or 1003, wherein the 2'-sugar-modified nucleoside is independently selected from the group consisting of locked nucleic acids and 2'-sugar-substituted nucleosides. [Invention 1008] One or more of the aforementioned 2' sugar-modified nucleosides TIFF0007899100000002.tif165150 A locked nucleic acid selected from the group consisting of, where B is a natural or modified nucleic acid base, and Z is an internucleoside bond to an adjacent nucleoside or 5' terminal group, Z * The polynucleotide of the present invention 1006 or 1007, wherein is an internucleoside bond to an adjacent nucleoside or a 3' terminal group. [Invention 1009] One or more 2' sugar-modified nucleosides, TIFF0007899100000003.tif64128 A polynucleotide selected from the group consisting of either 1006 or 1007 of the present invention. [Invention 1010] The guanosine analog is TIFF0007899100000004.tif49128 (In the formula, R is either H or OH) TIFF0007899100000005.tif103128TIFF0007899100000006.tif163128TIFF0007899100000007.tif58128 A polynucleotide selected from the group consisting of any of the present invention 1001 to 1009. [Invention 1011] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ia). [Invention 1012] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ia1). [Invention 1013] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ia2). [Invention 1014] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ib). [Invention 1015] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIa). [Invention 1016] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIa1). [Invention 1017] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIa2). [Invention 1018] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIb). [Invention 1019] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ic). [Invention 1020] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIc). [Invention 1021] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Id). [Invention 1022] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IId). [Invention 1023] The polynucleotide of the present invention 1010, wherein the guanosine analog is (Ie). [Invention 1024] The polynucleotide of the present invention 1010, wherein the guanosine analog is (IIe). [Invention 1025] A polynucleotide according to any one of the present invention 1002-1003 or 1006-1020, wherein the antisense oligonucleotide is a gapmer. [Invention 1026] A polynucleotide according to any one of the present invention 1002-1003 or 1006-1021, comprising at least one further nucleoside having a modified ribose, wherein the ribose modification is selected from the group consisting of locked nucleic acids or 2' modifications. [Invention 1027] The polynucleotide according to any of the present invention 1025 to 1026, wherein the guanosine analog is located in the gap region of the gapmer and is of formula Ia or formula IIb, where R is H. [Invention 1028] A polynucleotide according to any of the present invention 1025 to 1027, wherein the guanosine analog is not present in the flank of the gapmer. [Invention 1029] A polynucleotide of any of the present inventions 1001 to 1020, comprising one guanosine analog of any of the present inventions 1001 to 1028. [Invention 1030] A polynucleotide of any of the inventions 1001 to 1029, comprising two guanosine analogs of any of the inventions 1001 to 1024. [Invention 1031] A polynucleotide of any of the present inventions 1001 to 1030, comprising three guanosine analogs of any of the present inventions 1001 to 1024. [Invention 1032] A polynucleotide according to any of the present invention 1001 to 1031, which does not contain natural guanosine. [Invention 1033] The aforementioned polynucleotide is CTCAacttg オキソ ctttaAT(sequence_number_4); CTCAtacttg N ctttaAT(sequence number 5); CTCAtacttg PPG ctttaAT(sequence number 6); CTAcatctcatactTgC(Sequence ID 9); CTAcatctcatactTg PPG C(sequence code 10); CTAcatctcatactTg オキソ C(sequence code 11); CTAcatctcatactTg N C(sequence number 13); ACAg オキソ g オキソ attag オキソ ttCTA(sequence number 15); and ACAg PPG g PPG attag PPG ttCTA (SEQ ID NO: 16) Selected from the group consisting of, uppercase letters in these sequences indicate nucleosides with LNA-modified ribose, all LNA Cs are 5-methylcytosine, and lowercase letters in these sequences indicate DNA. g PPG It is 7-deaza-8-aza-deoxyguanosine, g N It is 8-amino-dG, g オキソ It is 8-oxo-deoxyguanosine. A polynucleotide according to any of the present invention 1001 to 1028. [Invention 1034] A polynucleotide according to any of the invention 1001 to 1033 for use as a pharmaceutical. [Invention 1035] The polynucleotide of the present invention 1034 is for administration to the central nervous system or for treating CNS disorders selected from the group consisting of amyotrophic lateral sclerosis (ALS), Angelman syndrome, Alzheimer's disease, aneurysms, back pain, Bell's palsy, congenital defects of the brain and spinal cord, brain injury, brain tumors, cerebral palsy, chronic fatigue syndrome, concussions, dementia, cervical and lumbar disc diseases, vertigo, epilepsy, Guillain-Barré syndrome, headaches and migraines, multiple sclerosis, muscular dystrophy, neuralgia, neuropathy, neuromuscular and related diseases, Parkinson's disease, psychiatric symptoms (severe depression, obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury, spinal deformities and disorders, spinal tumors, stroke and vertigo. [Invention 1036] A polynucleotide of the present invention 1035, intended for administration via intrathecal injection. [Invention 1037] Any of the polynucleotides 1001-1034 of this invention for use as a pharmaceutical to treat medical conditions in which the regulation of Ube3A is beneficial, for example, for treating angelman. [Invention 1038] Any of the polynucleotides 1001-1034 of the present invention for use as a pharmaceutical for treating medical conditions in which the regulation of ATXN2 is beneficial. [Invention 1039] Any of the polynucleotides 1001-1034 of the present invention for use as pharmaceuticals to treat medical conditions in which the regulation of ATXN3 is beneficial. [Invention 1040] A polynucleotide of any of the present invention 1001 to 1038, used as a pharmaceutical product where reduced neurotoxicity is required. [Invention 1041] A method for synthesizing a polynucleotide with reduced toxicity according to any of Invention 1001 to 1034, comprising coupling a nucleotide monomer such as a phosphoramidite to a further nucleotide or oligonucleotide, wherein the nucleotide monomer comprises a guanosine analog according to any of Invention 1001 to 1024. [Invention 1042] A method for selecting a polynucleotide that is less toxic than a reference polynucleotide, wherein the polynucleotide is one of the present invention 1001 to 1024, and the less neurotoxic polynucleotide differs from the reference polynucleotide in that it contains at least one guanosine analog, wherein the reference polynucleotide and the less neurotoxic antisense oligonucleotide have the same nucleotide sequence and contain at least one guanosine. [Invention 1043] The use of compounds containing guanosine analogs selected from the group consisting of (Ia), (Ib), (Ic), (Id), (Ie), (IIa), (IIb), (IIc), (IId), and (IIe) in the production of polynucleotides. [Invention 1044] A method for upregulating Ube3a expression in target cells expressing Ube3a-ATS, comprising administering an effective amount of any polynucleotide of the present invention 1001 to 1024 that targets Ube3a-ATS to the cells. [Invention 1045] The method of the present invention 1038, which is an in vivo or in vitro method. [Invention 1046] A method for treating or preventing neurological disorders in subjects such as human beings who are suffering from or are at risk of suffering from neurological disorders, the method comprising administering a therapeutically effective amount or a preventively effective amount of any polynucleotide of the present invention 1001 to 1024 for the purpose of preventing or alleviating the neurological disorders.

[0011] Sequence List The sequence listing submitted with this application is incorporated herein by reference. The antisense oligonucleotide sequence motifs listed in the sequence listing are shown as DNA sequences. [Brief explanation of the drawing]

[0012] [Figure 1] A graph showing the relief of acute neurotoxicity using guanine analogs in the test according to Example 3. [Modes for carrying out the invention]

[0013] definition Oligonucleotides As used herein, the term “oligonucleotide” is defined as is generally understood by those skilled in the art to be a molecule containing two or more covalently bonded nucleosides. Such covalently bonded nucleosides may also be called nucleic acid molecules or oligomers. Oligonucleotides are typically prepared in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, the sequence or order of the nucleic acid base portions of the covalently bonded nucleotide or nucleoside, or their modification, is referred to. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides, such as 2'-sugar modified nucleosides. The oligonucleotides of the present invention may contain one or more modified nucleoside bonds, such as one or more phosphorothioate nucleoside bonds.

[0014] Antisense oligonucleotides As used herein, the term “antisense oligonucleotide” is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid. Antisense oligonucleotides are not inherently double-stranded and are therefore neither siRNA nor shRNA. Preferably, the antisense oligonucleotides of the present invention are single-stranded. It is understood that single-stranded oligonucleotides of the present invention can form hairpin or intermolecular double-stranded structures (double strands between two molecules of the same oligonucleotide) as long as the degree of internal or mutual self-complementarity over the entire length of the oligonucleotide is less than 50%.

[0015] In some embodiments, the single-stranded antisense oligonucleotide of the present invention may not contain an unmodified RNA nucleoside.

[0016] Advantageously, the oligonucleotide of the present invention comprises one or more modified nucleosides or nucleotides, such as 2'-sugar modified nucleosides. Furthermore, it is advantageous that the unmodified nucleosides are DNA nucleosides.

[0017] Sequential nucleotide sequence The term “continuous nucleotide sequence” refers to a region of an antisense oligonucleotide that is complementary to the target nucleic acid. This term is used herein interchangeably with the terms “continuous nucleic acid base sequence” and “oligonucleotide motif sequence.” In some embodiments, all nucleotides of the oligonucleotide constitute a continuous nucleotide sequence. In some embodiments, the oligonucleotide may include a continuous nucleotide sequence, such as an FG-F' gapmer region, and optionally include a nucleotide linker region that can be used to attach further nucleotides, such as functional groups (e.g., conjugate groups), to the continuous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the nucleic acid base sequence of the antisense oligonucleotide is a continuous nucleotide sequence.

[0018] Nucleotides and nucleosides Nucleotides and nucleosides are the constituent units of oligonucleotides and polynucleotides, and for the purposes of this invention, include both naturally occurring and non-naturally occurring nucleotides and nucleosides. Nucleotides, such as DNA nucleotides and RNA nucleotides, naturally consist of a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides may also be interchangeably referred to as “units” or “monomers.”

[0019] Modified nucleoside As used herein, the terms “modified nucleoside” or “nucleoside modification” refer to a nucleoside modified compared to an equivalent DNA or RNA nucleoside by introducing one or more modifications to a sugar moiety or (nucleic acid) base moiety. Advantageously, a modified nucleoside of one or more antisense oligonucleotides of the present invention includes a modified sugar moiety. The term “modified nucleoside” may also be used interchangeably with the terms “nucleoside analog” or modified “unit” or modified “monomer.” A nucleoside having an unmodified DNA or RNA sugar moiety is referred to herein as a DNA or RNA nucleoside. A nucleoside having modifications to the base region of a DNA or RNA nucleoside is still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing.

[0020] Guanosine analog As used herein, the terms “guanine analog” or “G analog” refer to nucleosides or nucleotides containing the nucleic acid base 8-oxo-guanine (8-oxo-G) and / or 7-deaza-8-aza-guanine (PPG) of the following formula: TIFF0007899100000008.tif49128 (where R is H or OH).

[0021] 8-Oxo-deoxyguanosine (8-oxo-dG) and / or 7-deaza-8-aza-deoxyguanosine (PPG) can also be expressed in more detail as follows: TIFF0007899100000009.tif49128 or TIFF0007899100000010.tif49128

[0022] It should be understood that this also includes guanosine analogs that have sugar modifications, such as the following: TIFF0007899100000011.tif49128TIFF0007899100000012.tif210111TIFF0007899100000013.tif58128

[0023] Parts (Ia) and (IIa) can be obtained using phosphoramidites commercially available from Glen Research (Sterling, Virginia).

[0024] Parts (IIb) and (IIc) can be obtained according to the synthetic routes described by Hara et al., J.Org.Chem.2017, 82, 25-36 and Blade et al., J.Org.Chem.2015, 80, 5337-5343.

[0025] Parts (Ib), (Ic), (Id), and (Ie) can be obtained from the corresponding guanosine nucleosides, as described by Kannan et al., J. Org. Chem. 2011, 76, 720-723.

[0026] Parts (IId) and (IIe) can be obtained from the corresponding pentofuranocylloride intermediates as described by Seela et al., Nucleosides & Nucleotides (1989), 8(5-6), 789-792 and Seela et al., Helvetica Chimica Acta (1986), 69(7), 1602-1613.

[0027] Inter-modified nucleoside bonding The term “modified nucleoside bond” is defined as is commonly understood by those skilled in the art, as a bond other than a phosphodiester (PO) bond that covalently bonds two nucleosides to each other. Accordingly, the oligonucleotides of the present invention may contain one or more modified nucleoside bonds, such as one or more phosphorothioate nucleoside bonds or one or more phosphorodithioate nucleoside bonds.

[0028] In some embodiments, at least 50% of the internucleoside bonds of the oligonucleotide or its sequence of nucleotides are phosphorothioates, for example, at least 60%, for example at least 70%, for example at least 75%, for example at least 80%, or for example at least 90% of the internucleoside bonds of the oligonucleotide or its sequence of nucleotides are phosphorothioates. In some embodiments, all of the internucleoside bonds of the oligonucleotide or its sequence of nucleotides are phosphorothioates.

[0029] In some advantageous embodiments, all nucleoside-to-nucleoside bonds in the continuous nucleotide sequence of the oligonucleotide are phosphorothioates, or all nucleoside-to-nucleoside bonds of the oligonucleotide are phosphorothioate bonds.

[0030] As disclosed in European Patent No. 2742135, it is recognized that antisense oligonucleotides may include other nucleoside bonds (other than phosphodiesters, phosphorothioates, and phosphorodithioates), such as alkylphosphonate / methylphosphonate nucleoside bonds, which, according to European Patent No. 2742135, may be resistant, for example, within the gap region of another DNA phosphorothioate.

[0031] Nucleic acid bases The term "nucleic acid base" includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of this invention, the term "nucleic acid base" may differ from naturally occurring nucleic acid bases, but also includes modified nucleic acid bases that are functional in nucleic acid hybridization. In this context, "nucleic acid base" refers to both naturally occurring nucleic acid bases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, and variants that do not exist in nature. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

[0032] In some embodiments, the nucleic acid base moiety is modified by replacing a purine or pyrimidine with a nucleic acid base selected from modified purines or pyrimidines, such as substituted purines or pyrimidines, such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolocytosine, 5-propynylcytosine, 5-propynyluracil, 5-bromouracil, 5-thiazolouracil, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0033] The nucleic acid base portion may be represented by a letter code for each corresponding nucleic acid base, for example, A, T, G, C, or U, where each letter may optionally contain a modified nucleic acid base with equivalent function. For example, in the illustrated oligonucleotide, the nucleic acid base portion is selected from A, T, G, C, and 5-methylcytosine. Optionally, for the LNA gapmer, a 5-methylcytosine LNA nucleoside may be used.

[0034] Modified oligonucleotides The term "modified oligonucleotide" refers to an oligonucleotide containing one or more sugar-modified nucleosides and / or intermodified nucleoside bonds. The term "chimeric oligonucleotide" is a term used in the literature to describe oligonucleotides containing sugar-modified nucleosides and DNA nucleosides. The antisense oligonucleotides of the present invention are advantageously chimeric oligonucleotides.

[0035] Complementarity The term "complementarity" describes the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may also contain nucleosides with modified nucleic acid bases; for example, 5-methylcytosine is often used in place of cytosine, the guanosine analog described herein. Therefore, the term complementarity will be understood to encompass Watson-Crick base pairing between unmodified and modified nucleic acid bases (see, e.g., Hirao et al., (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0036] As used herein, the term "% complementary" refers to the percentage of nucleotides in a sequence of nucleic acid molecules (e.g., oligonucleotides) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across the sequence. Therefore, the percentage of complementarity is calculated by counting the number of complementary (from Watson-Crick base pairs) nucleic acid bases between two sequences (when the oligonucleotide sequence is aligned from the target sequence 5'-3' and 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form base pairs) are called mismatches. Insertions and deletions are not permitted in the calculation of the % complementarity of a sequence of nucleotides. In determining complementarity, it will be understood that chemical modifications of nucleic acid bases are disregarded as long as the nucleic acid base retains its functional ability to form Watson-Crick base pairs (for example, 5'-methylcytosine and the guanosine analogs described herein are considered identical to cytosine and guanosine, respectively, for the purposes of calculating % identity).

[0037] The term "perfectly complementary" refers to 100% complementarity.

[0038] identity As used herein, the term “identity” refers to the percentage of nucleotides in a sequence of nucleic acid molecules (e.g., oligonucleotides) that are identical to a reference sequence (e.g., a sequence motif) across the sequence. Therefore, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleic acid bases between two sequences (in the sequence of the compound of the present invention and the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches × 100) / length of the aligned region (e.g., sequence of nucleotides). Insertions and deletions are not permitted in the calculation of the percentage of identity of a sequence of nucleotides. It will be understood that, in determining identity, chemical modifications of nucleic acid bases are ignored as long as the nucleic acid base retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for the purposes of calculating % identity).

[0039] Hybridization As used herein, the terms “hybridize” or “to hybridize” should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a double helix by forming hydrogen bonds between base pairs on opposing strands. The affinity of the bond between the two nucleic acid strands is the strength of the hybridization. This is often defined as the melting temperature (T) at which half of the oligonucleotide forms a double helix with the target nucleic acid. m It is represented by ). Under physiological conditions, T m It is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515~537). The Gibbs free energy ΔG° at standard conditions more accurately represents the binding affinity, where ΔG° = -RTln(K d The dissociation constant of the reaction (K) is determined by the equation (where R is the gas constant and T is the absolute temperature). d) is associated with this. Therefore, a very low ΔG° for the reaction between oligonucleotides and target nucleic acids reflects strong hybridization between oligonucleotides and target nucleic acids. ΔG° is the energy associated with a reaction at an aqueous solution concentration of 1 M, pH 7, and temperature of 37°C. Hybridization of oligonucleotides with target nucleic acids is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by using isothermal titration calorimetry (ITC) methods, as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will know that commercially available instruments are available for ΔG° measurement. ΔG° can be numerically estimated using the nearest-neighbor model described in SantaLucia, 1998, Proc Natl Acad Sci USA.95:1460~1465, and appropriately obtained thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211~11216 and McTigue et al., 2004, Biochemistry 43:5388~5405. To ensure the possibility of modulating the intended nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal for oligonucleotides of 10 to 30 nucleotide lengths. In some embodiments, the degree or intensity of hybridization is measured by the Gibbs free energy ΔG° at standard conditions. Oligonucleotides can hybridize to target nucleic acids with estimated ΔG° values ​​in the range of less than -10 kcal for oligonucleotides of 8 to 30 nucleotides in length, e.g., less than -15 kcal, e.g., less than -20 kcal, and e.g., less than -25 kcal. In some embodiments, oligonucleotides hybridize to target nucleic acids with estimated ΔG° values ​​of -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or -16 to -27 kcal, e.g., -18 to -25 kcal.

[0040] target nucleic acid According to the present invention, the target nucleic acid may be a nucleic acid, RNA, mRNA, or premRNA, mature mRNA, or cDNA sequence that encodes a human gene. When the oligonucleotide of the present invention is used for research or diagnostic purposes, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0041] target sequence As used herein, the term “target sequence” refers to a sequence of nucleotides present in a target nucleic acid, comprising a nucleic acid base sequence complementary to the antisense oligonucleotide of the present invention. In some embodiments, the target sequence comprises a region on the target nucleic acid having a nucleic acid base sequence complementary to the continuous nucleotide sequence of the antisense oligonucleotide of the present invention. This region of the target nucleic acid may interchangeably be called a target nucleotide sequence, target sequence, or target region. In some embodiments, the target sequence may be longer than the complementary sequence of a single antisense oligonucleotide and represent, for example, a preferred region of the target nucleic acid that can be targeted by some antisense oligonucleotides of the present invention.

[0042] In some embodiments, the antisense oligonucleotide or its sequence of nucleotides according to the present invention is complementary, for example, perfectly complementary, to the target sequence.

[0043] The antisense oligonucleotides of the present invention include a sequence of nucleotides that are complementary to and hybridize with a target nucleic acid, such as a target sequence described herein.

[0044] The target sequences to which antisense oligonucleotides are complementary generally contain a sequence of at least 10 consecutive nucleic acid bases. The sequence of consecutive nucleotides is 10 to 30 nucleotides long, e.g., 12 to 30, e.g., 14 to 20, e.g., 15 to 18 consecutive nucleotides long, e.g., 15, 16, 17 consecutive nucleotides long.

[0045] In some embodiments, the antisense oligonucleotide of the present invention is completely complementary to the target sequence along the entire length of the antisense oligonucleotide.

[0046] target cell As used herein, the term “target cell” refers to a cell expressing a target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell may be a mammalian cell, e.g., a rodent cell, e.g., a mouse cell or a rat cell, or a primate cell, e.g., a monkey cell or a human cell.

[0047] Typically, target cells express target mRNA, such as target pre-mRNA or target mature mRNA. For experimental evaluation, target cells expressing nucleic acids containing the target sequence may be used.

[0048] The antisense oligonucleotides of the present invention can typically inhibit the expression of target nucleic acids in cells expressing the target nucleic acid (target cells), either in vivo or in vitro.

[0049] The continuous sequence of nucleic acid bases of the antisense oligonucleotide of the present invention, measured over the length of the antisense oligonucleotide, is complementary, e.g., perfectly complementary, to the target nucleic acid, except for optionally a nucleotide-based linker region that can conjugate the antisense oligonucleotide to any functional group such as a conjugate, or other non-complementary terminal nucleotides (e.g., region D' or D''). The target nucleic acid may be, for example, messenger RNA such as mature mRNA or premRNA encoding a given protein.

[0050] Naturally occurring variants The term "naturally occurring variant" refers to variants of a gene or transcript, and allele variants, that originate from the same locus as the target nucleic acid but may differ due to, for example, degeneracy of the genetic code resulting from multiple codons encoding the same amino acid, or due to alternative splicing of premRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs). Therefore, based on the presence of sufficiently complementary sequences for the oligonucleotide, the oligonucleotides of the present invention may target the target nucleic acid and its naturally occurring variants.

[0051] In some embodiments, naturally occurring variants have at least 95%, for example, at least 98% or at least 99% homology to the mammalian target nucleic acid.

[0052] Inhibition of expression As used herein, the term “inhibition of expression” should be understood as a general term for the ability of an oligonucleotide to inhibit the amount or activity of a given protein in a target cell. Inhibition of activity may be determined by measuring the level of target pre-mRNA or target mRNA, or by measuring the level of the target gene or target gene activity in the cell. Thus, inhibition of expression may be determined in vitro or in vivo. Inhibition of target expression may also be determined by measuring activity or protein levels.

[0053] Typically, inhibition of expression is determined by comparing the inhibition of activity by administering an effective amount of antisense oligonucleotide to target cells and comparing the level to a reference level (control experiment) obtained from target cells without administration of the antisense oligonucleotide, or a known reference level (e.g., the expression level before administration of an effective amount of antisense oligonucleotide, or a predetermined or other known expression level).

[0054] For example, the control experiment may involve animals or humans, or target cells, treated with a saline composition or a reference oligonucleotide (often a scrambled control).

[0055] The terms inhibition or suppression can also be said to downregulate, reduce, suppress, decrease, or lower the expression of a given gene A.

[0056] Inhibition of expression can occur, for example, by degradation of premRNA or mRNA (e.g., using RNaseH-mobilizing oligonucleotides, e.g., gapmers).

[0057] High affinity modified nucleoside A "high affinity modified nucleoside" is a modified nucleotide that, when incorporated into an antisense oligonucleotide, enhances the affinity of the antisense oligonucleotide to its complementary target, as measured, for example, by its melting temperature (Tm). The high affinity modified nucleosides of the present invention preferably result in an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0058] sugar modification The antisense oligonucleotides of the present invention may contain one or more nucleosides having a modified sugar moiety, that is, a modification of the sugar moiety compared to the ribose sugar moiety found in DNA and RNA.

[0059] Numerous nucleosides having modifications to the ribose sugar moiety have been fabricated primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance. In some embodiments of the present invention, the term “sugar modification” means “ribose modification” or “modified ribose” or “ribose modification.”

[0060] Such modifications include those in which the ribose ring structure is modified by replacing it with, for example, a hexose ring (HNA), or a bicyclic ring (LNA) typically having a biradical bridge between the C2 and C4 carbons on the ribose ring, or an unbonded ribose ring typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (International Publication No. 2011 / 017521) or tricyclic nucleic acids (International Publication No. 2013 / 154798). Modified nucleosides also include those in which the sugar moiety is replaced with a non-sugar moiety, for example, a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0061] Sugar modifications also include modifications made by changing substituents on the ribose ring to non-hydrogen groups or naturally occurring 2'-OH groups in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' positions.

[0062] 2' sugar-modified nucleoside 2'-sugar-modified nucleosides, also known as 2'-sugar-modified nucleosides, are nucleosides that have a substituent other than H or -OH at the 2' position (2'-substituted nucleosides), or nucleosides that contain a 2'-bonded biradical that can form a bridge between the 2' carbon and the second carbon in the ribose ring, such as LNA (2'-4' biradical bridged) nucleosides.

[0063] In fact, the development of 2'-sugar-substituted nucleosides has attracted considerable attention, and many 2'-substituted nucleosides have been found to possess beneficial properties when incorporated into antisense oligonucleotides. For example, 2'-modified sugars can impart enhanced binding affinity and / or increased nuclease resistance to antisense oligonucleotides. Examples of 2'-substituted nucleosides include 2'-O-alkyl-RNA / DNA, 2'-O-methyl-RNA / DNA, 2'-alkoxy-RNA / DNA, 2'-O-methoxyethyl-RNA / DNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA / DNA, and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, as well as Deleavey and Damha, Chemistry and Biology 2012, 19, 937. The following are some examples of 2' substitution-modified nucleosides. TIFF0007899100000014.tif68128

[0064] In relation to the present invention, the 2'-substituted sugar-modified nucleoside does not contain a 2'-crosslinked nucleoside such as LNA.

[0065] Locked nucleic acid nucleosides (LNA nucleosides) An "LNA nucleoside" is a 2'-modified nucleoside containing a biradical (also called a "2'-4' bridge") that links the C2' and C4' of the ribose sugar ring of the nucleoside, thereby restricting or fixing the conformation of the ribose ring. These nucleosides are also referred to in the literature as cross-linked nucleic acids or bicyclic nucleic acids (BNAs). Fixation of the ribose conformation is associated with improved hybridization affinity (double-strand stabilization) when LNAs are incorporated into antisense oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the antisense oligonucleotide / complementary double-strand.

[0066] Non-restrictive, exemplary LNA nucleosides are described in International Publication No. 99 / 014226, International Publication No. 00 / 66604, International Publication No. 98 / 039352, International Publication No. 2004 / 046160, International Publication No. 00 / 047599, International Publication No. 2007 / 134181, International Publication No. 2010 / 077578, International Publication No. 2010 / 036698, and International Publication No. 2007 / 090071. This information is disclosed in Lett, International Publication No. 2009 / 006478, International Publication No. 2011 / 156202, International Publication No. 2008 / 154401, International Publication No. 2009 / 067647, International Publication No. 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al., J. Org. Chem. 2010, Vol. 75(5), pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0067] Further non-limiting, exemplary LNA nucleosides are disclosed in Scheme 1. TIFF0007899100000015.tif159137(wherein B is a natural or non-natural (modified) nucleic acid base, Z is an internucleoside bond to an adjacent nucleoside or 5' terminal group, Z * (This is an internucleoside bond to an adjacent nucleoside or 3' terminal group).

[0068] Unless otherwise specified, it should be recognized that LNA nucleosides can be β-D or α-L stereoisoforms.

[0069] Exemplary nucleosides with HELM annotations DNA nucleoside TIFF0007899100000016.tif44160β-D-oxy-LNA nucleoside TIFF0007899100000017.tif461602'-O-methylnucleoside TIFF0007899100000018.tif46159

[0070] Exemplary phosphorothioate nucleoside interbonding with HELM annotation TIFF0007899100000019.tif26128

[0071] The dotted lines represent covalent bonds between each nucleoside and the 5' or 3' phosphorothioate nucleoside bond. In the 5'-terminal nucleoside, the 5' dotted line represents a bond to the hydrogen atom (forming a 5'-terminal -OH group). In the 3'-terminal nucleoside, the 3' dotted line represents a bond to the hydrogen atom (forming a 3'-terminal -OH group).

[0072] RNaseH activation and recruitment The RNaseH activity of an antisense oligonucleotide refers to its ability to recruit RNaseH when double-stranded with a complementary RNA molecule. International Publication No. 01 / 23613 provides an in vitro method for determining RNaseH activity that can be used to determine the ability to recruit RNaseH. Typically, an antisense oligonucleotide is considered capable of recruiting RNase H if it has an initial rate measured at at least 5% of the initial rate determined using the methodology provided in Examples 91-95 of International Publication No. 01 / 23613 (incorporated herein by reference), e.g., at least 10% or more than 20% of the initial rate measured at pmol / l / min. For use in determining RNase H activity, recombinant human RNase H1 is available from Creative Biomart® (recombinant human RNase H1 fused with a His tag expressed in E. coli).

[0073] Gapmar The antisense oligonucleotide or its sequence of nucleotides according to the present invention may also be a gapmer, and may also be called a gapmer antisense oligonucleotide or gapmer design. Gapmers are typically used to inhibit target nucleic acids via RNaseH-mediated degradation. A gapmer comprises at least three distinct structural regions, 5'-flank, gap, and 3'-flank, FG-F', in a 5'->3' orientation. The "gap" region (G) contains an extension of sequence DNA nucleotides that allows the gapmer to recruit RNaseH. The gap region is flanked by a 5' flanking region (F) containing one or more glycosylated nucleosides, preferably high-affinity glycosylated nucleosides, and a 3' flanking region (F') containing one or more glycosylated nucleosides, preferably high-affinity glycosylated nucleosides. One or more glycosylated nucleosides in regions F and F' enhance the affinity of the gapmer to the target nucleic acid (i.e., they are affinity-enhancing glycosylated nucleosides). In some embodiments, one or more glycosylated nucleosides in regions F and F' are 2' glycosylated nucleosides, such as high-affinity 2' glycosylation nucleosides, which are independently selected from, for example, LNA and 2'-MOE.

[0074] In gapmer design, the 5' and 3' nucleosides in the gap region are DNA nucleosides, located adjacent to the glycosylated nucleosides in the 5'(F) or 3'(F') region, respectively. A flank can be further defined by having at least one glycosylated nucleoside at the end furthest from the gap region, i.e., the 5' end of a 5' flank and the 3' end of a 3' flank.

[0075] Region FG-F' forms a continuous nucleotide sequence. The antisense oligonucleotide or its continuous nucleotide sequence according to the present invention may include a gapmer region of formula FG-F'.

[0076] The total length of the gapmer design FG-F' can be, for example, 12-32 nucleosides, 13-24, 14-22 nucleosides, 14-17, or 16-18 nucleosides.

[0077] For example, the gapmer oligonucleotide of the present invention can be represented by the following formula: F 1~8 -G 5~16 -F' 1~8 ,for example F 1~8 -G 7~16 -F' 2~8 However, the total length of the gapmer region FG-F' is at least 12, for example, at least 14 nucleotides long.

[0078] In one aspect of the present invention, an antisense oligonucleotide or a sequence of nucleotides thereof consists of or comprises a gapmer of formula 5'-FG-F'-3' (wherein regions F and F' independently contain or consist of 1 to 8 nucleosides, of which 1 to 4 are 2'-sugar modified and define the 5' and 3' ends of the F and F' regions, and G is a region of 6 to 16 nucleosides capable of recruiting RNase H).

[0079] Regions F, G, and F' are further defined below and can be incorporated into the FG-F' formula.

[0080] LNA gapmer An LNA gapmer is a gapmer in which either or both of regions F and F' contain or consist of an LNA nucleoside. A β-D-oxy gapmer is a gapmer in which either or both of regions F and F' contain or consist of a β-D-oxy LNA nucleoside.

[0081] In some embodiments, the LNA gapmer is given by formula: [LNA] 1~5 -[Area G]-[LNA] 1~5(In the formula, region G is a region of a continuous DNA nucleoside capable of recruiting RNaseH, or contains such a region.)

[0082] MOE Gap Marker A MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, the MOE gapmer is designed [MOE] 1~8 -[Area G] 5~16 -[MOE] 1~8 For example, [MOE] 2~7 -[Area G] 6~14 -[MOE] 2~7 For example, [MOE] 3~6 -[Area G] 8~12 -[MOE] 3~6 (In the formula, region G is as defined in the definition of a gapmer). MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0083] Mixed wing gap mar A mixed wing gapmer is an LNA gapmer in which one or both of regions F and F' contain a 2'-substituted nucleoside, such as a MOE nucleoside, independently selected from the group consisting of 2'-substituted nucleosides, e.g., 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments in which at least one of regions F and F', or both regions F and F', contain at least one LNA nucleoside, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments in which at least one of regions F and F', or both regions F and F', contain at least two LNA nucleosides, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further contain one or more DNA nucleosides.

[0084] Alternating Frank Gap Marker The flanking regions may contain both LNA nucleosides and DNA nucleosides, and are called "alternating flanks" because they contain an alternating motif of LNA-DNA-LNA nucleosides. Gapmers containing such alternating flanks are called "alternating flank gapmers." Thus, an "alternating flank gapmer" is an LNA gapmer oligonucleotide in which at least one flank (F or F') contains DNA in addition to an LNA nucleoside. In some embodiments, at least one of regions F or F', or both regions F and F', contains both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking regions F or F', or both F and F', contain at least three nucleosides, and the nucleosides on the furthest 5' and 3' ends of regions F and / or F' are LNA nucleosides.

[0085] Region D' or D'' within an antisense oligonucleotide In some embodiments, the antisense oligonucleotides of the present invention may comprise, or consist of, a sequential nucleotide sequence of oligonucleotides complementary to the target nucleic acid, e.g., a gapmer region FG-F', and further 5' and / or 3' nucleosides. The further 5' and / or 3' nucleosides may be fully complementary to the target nucleic acid or not. Such further 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0086] The addition of region D' or D'' may be used to link a continuous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used for linking, the continuous nucleotide sequence with the conjugate moiety may act as a bio-cleavable linker. Alternatively, it may be used to provide exonuclease protection or to facilitate synthesis or manufacture.

[0087] Regions D' and D'' can be bonded to the 5' end of region F or the 3' end of region F', respectively, to generate the following designs of formula D'-FG-F', FG-F'-D'', or D'-FG-F'-D''. In this case, FG-F' is the gapmer portion of the antisense oligonucleotide, and region D' or D'' constitutes a separate portion of the antisense oligonucleotide.

[0088] Region D' or D'' independently contains or consists of 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides, but are, for example, DNA or RNA or their base-modified versions. Region D' or D'' can act as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, further 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or D'' are disclosed in International Publication 2014 / 076195, which includes, as an example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in International Publication WO2015 / 113922, which are used to link multiple antisense constructs (e.g., gapmer regions) within a single antisense oligonucleotide.

[0089] In one embodiment, the antisense oligonucleotide of the present invention comprises regions D' and / or D'' in addition to the continuous nucleotide sequence constituting the gapmer.

[0090] In some embodiments, the antisense oligonucleotide of the present invention may be represented by the following formula: FG-F'; especially F 1~8 -G 5~16 -F' 2~8 D'-FG-F', especially D' 1~3 -F 1~8 -G 5~16 -F' 2~8 FG-F'-D'', especially F 1~8 -G 5~16 -F' 2~8 -D'' 1~3 D'-FG-F'-D'', especially D' 1~3-F 1~8 -G 5~16 -F' 2~8 -D'' 1~3 .

[0091] In some embodiments, the internucleoside bond located between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside bond located between region F' and region D'' is a phosphodiester bond.

[0092] Conjugate As used herein, the term "conjugate" refers to an antisense oligonucleotide covalently bonded to a non-nucleotide portion (the conjugate portion or region C or a third region). The conjugate portion may optionally be covalently bonded to the antisense oligonucleotide via a linker group such as region D' or D''.

[0093] Antisense oligonucleotide conjugates and their synthesis have also been reported in comprehensive reviews: Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, edited by STCrooke, Chapter 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0094] In some embodiments, the non-nucleotide portion (conjugate portion) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, active pharmaceutical ingredients, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0095] Linker A linker or conjugate is a connection between two atoms that, via one or more covalent bonds, attaches one target chemical group or segment to another target chemical group or segment. A conjugate portion can be attached to an antisense oligonucleotide directly or via a linking portion (e.g., a linker or tether). The linker plays a role in covalently attaching a third region, e.g., the conjugate portion (region C), to a first region, e.g., an antisense oligonucleotide or sequential nucleotide sequence complementary to the target nucleic acid (region A).

[0096] In some embodiments of the present invention, the conjugate or antisense oligonucleotide conjugate of the present invention may optionally include a linker region (second region or region B and / or region Y) located between an antisense oligonucleotide or sequential nucleotide sequence (region A or first region) complementary to the target nucleic acid and the conjugate portion (region C or third region).

[0097] Region B refers to biocleavable linkers containing or consisting of physiologically unstable bonds that can be cleaved under conditions normally found in mammals, or conditions similar to those found therein. Conditions under which physiologically unstable linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidation or reduction conditions or oxidizing or reducing agents, and salt concentration, which are found in or similar to those found in mammalian cells. Intracellular mammalian conditions also include the presence of enzyme activity normally present in mammalian cells, such as proteases or hydrolases or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker contains 1 to 5 nucleosides, such as a DNA nucleoside containing at least two consecutive phosphodiester bonds. Biocleavable linkers containing phosphodiesters are described in detail in International Publication No. 2014 / 076195.

[0098] Region Y refers to a linker that is not necessarily biocleavable but primarily serves to covalently bond the conjugate portion (region C or third region) to the antisense oligonucleotide (region A or first region). The region Y linker may contain a chain structure or oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The antisense oligonucleotide conjugate of the present invention can be constructed from the following local elements AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group containing a C6-C12 aminoalkyl group. In some embodiments, the linker (region Y) is a C6 aminoalkyl group. In some embodiments, the linker is NA.

[0099] siRNA A “small interfering RNA” or “small interfering RNA” or siRNA is an RNA double-stranded molecule of nucleotides that targets a gene of interest. “RNA double-stranded” refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA “targets” a gene in that the nucleotide sequence of the double-stranded portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA double-stranded molecule is less than 30 nucleotides. In some embodiments, the double-stranded molecule may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides long. In some embodiments, the double-stranded molecule is 19–25 nucleotides long. The RNA double-stranded portion of siRNA may be part of a hairpin structure. In addition to the double-stranded portion, the hairpin structure may include a loop portion positioned between the two sequences forming the double-stranded molecule. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides long. The hairpin structure may also include a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides long.

[0100] As used herein, “shRNA molecule” includes conventional stem-loop shRNAs that form precursor miRNAs (pre-miRNAs). “shRNA” also includes microRNA-embedded shRNAs (miRNA-based shRNAs), where the guide and passenger strands of a miRNA double-strand are incorporated into an existing (or native) miRNA or a modified or synthetic (designed) miRNA. Upon transcription, conventional shRNAs form a structure very similar to primary miRNAs (pre-miRNAs) or native pre-miRNAs. Pre-miRNAs are then processed into pre-miRNAs by Drosha and its cofactors. Therefore, the term “shRNA” includes both pre-miRNA (shRNA-mir) molecules and pre-miRNA molecules.

[0101] A “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides known or expected to form a double-stranded or double-stranded (stem) structure, primarily linked on one side by a region of single-stranded nucleotides (loop portion). The terms “hairpin” and “folded” structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are used in accordance with their known meanings in the art. As is known in the art, secondary structures do not require precise base pairing. Therefore, the stem may contain one or more base mismatches or bulges, or the base pairing may be precise, i.e., without any mismatches.

[0102] The term "RNAi expression construct" or "RNAi construct" is a general term encompassing nucleic acid preparations designed to achieve RNA interference effects. RNAi expression constructs include RNAi molecules that can be cleaved in vivo to form siRNA or mature shRNA. For example, an RNAi construct is an expression vector capable of producing siRNA or mature shRNA in vivo. Non-limiting examples of vectors that may be used in accordance with the present invention are described herein, for example, in Section 4.6. Exemplary methods for constructing and delivering long or short RNAi constructs can be found, for example, in International Publication No. 01 / 68836 and International Publication No. 01 / 75164.

[0103] siRNA can be encoded by a nucleic acid sequence, which may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a minimal synthetic polyadenylation signal.

[0104] treatment As used herein, the term “treatment” refers to both the treatment of an existing disease (e.g., any disease or disorder referred to herein) and the prevention, or prophylaxis, of a disease. Therefore, it will be recognized that, in some embodiments, the treatments referred to herein may be prophylactic.

[0105] Detailed description of the invention In the first aspect, the present invention is - at least one phosphorothioate nucleoside interbonding, -below: At least one guanosine analog containing a guanine analog selected from the group consisting of TIFF0007899100000020.tif34128 and Regarding polynucleotides, including those mentioned above.

[0106] Polynucleotides can be single-stranded, such as antisense oligonucleotides.

[0107] Polynucleotides can be double-stranded, such as siRNA or shRNA.

[0108] A polynucleotide can contain one or more 2'-sugar-modified nucleosides. Such 2'-sugar-modified nucleosides can be independently selected from the group consisting of locked nucleic acids and 2'-sugar-substituted nucleosides.

[0109] 2' sugar-modified nucleosides are TIFF0007899100000021.tif102151TIFF0007899100000022.tif65150(wherein B is a natural or modified nucleic acid base, Z is an internucleoside bond to an adjacent nucleoside or 5' terminal group, Z * (This is an internucleoside bond to an adjacent nucleoside or 3' terminal group.) It may be a locked nucleic acid selected from the group consisting of the following.

[0110] 2' sugar-modified nucleosides are The group consisting of TIFF0007899100000023.tif68128 can be selected.

[0111] Guanosine analogs are TIFF0007899100000024.tif49128 (wherein R is H or OH) The group can be selected from TIFF0007899100000025.tif157128 and TIFF0007899100000026.tif165128.

[0112] In one embodiment of the present invention, the guanosine analog is (Ia).

[0113] In one embodiment of the present invention, the guanosine analog is (Ib).

[0114] In one embodiment of the present invention, the guanosine analog is (IIa).

[0115] In one embodiment of the present invention, the guanosine analog is (IIb).

[0116] In one embodiment of the present invention, the guanosine analog is (Ic).

[0117] In one embodiment of the present invention, the guanosine analog is (IIc).

[0118] In one embodiment of the present invention, the guanosine analog is (Id).

[0119] In one embodiment of the present invention, the guanosine analog is (IId).

[0120] In one embodiment of the present invention, the guanosine analog is (Ie).

[0121] In one embodiment of the present invention, the guanosine analog is (IIe).

[0122] According to the present invention, the antisense oligonucleotide can be a gapmer.

[0123] According to the present invention, a polynucleotide may comprise at least one further nucleoside having a modified ribose, the ribose modification being selected from the group consisting of locked nucleic acids or other 2' modifications.

[0124] In one embodiment of the present invention, a guanosine analog is located in the gap region of the gapmer and is of formula Ia or formula IIb, where R is H.

[0125] In one embodiment of the present invention, the guanosine analog is absent in the gapmer flank.

[0126] In one embodiment of the present invention, the polynucleotide contains one guanosine analog.

[0127] In one embodiment of the present invention, the polynucleotide contains two guanosine analogs.

[0128] In one embodiment of the present invention, the polynucleotide contains three guanosine analogs.

[0129] In one embodiment of the present invention, the polynucleotide does not contain natural guanosine.

[0130] In one embodiment of the present invention, a polynucleotide is CTCAacttg オキソ ctttaAT(sequence_number_4); CTCAtacttg N ctttaAT(sequence number 5); CTCAtacttg PPG ctttaAT(sequence number 6); CTAcatctcatactTgC(Sequence ID 9); CTAcatctcatactTg PPG C(sequence code 10); CTAcatctcatactTg オキソ C(sequence code 11); CTAcatctcatactTg N C(sequence number 13); ACAg オキソ g オキソ attag オキソ ttCTA(sequence number 15); and ACAg PPG g PPG attag PPG ttCTA (SEQ ID NO: 16) (Uppercase letters in these sequences indicate nucleosides with LNA-modified ribose, all LNA Cs are 5-methylcytosine, and lowercase letters in these sequences indicate DNA.) g PPG It is 7-deaza-8-aza-deoxyguanosine, g N It is 8-amino-dG, g オキソ (This is 8-oxo-deoxyguanosine) This is an antisense selected from the group consisting of the following:

[0131] In one embodiment of the present invention, the antisense oligonucleotide is CTCAacttg オキソ This is ctttaAT (sequence number 4).

[0132] In one embodiment of the present invention, the antisense oligonucleotide is CTCAtacttg N This is ctttaAT (sequence number 5).

[0133] In one embodiment of the present invention, the antisense oligonucleotide is CTCAtacttg PPG This is ctttaAT (sequence number 6).

[0134] In one embodiment of the present invention, the antisense oligonucleotide is CTAcatctcatactTgC (SEQ ID NO: 9).

[0135] In one embodiment of the present invention, the antisense oligonucleotide is CTAcatctcatactTg PPG This is C (sequence number 10).

[0136] In one embodiment of the present invention, the antisense oligonucleotide is CTAcatctcatactTg オキソ This is C (sequence number 11).

[0137] In one embodiment of the present invention, the antisense oligonucleotide is CTAcatctcatactTg N This is C (sequence number 13).

[0138] In one embodiment of the present invention, the antisense oligonucleotide is ACAg オキソ g オキソ attag オキソ This is ttCTA (sequence number 15).

[0139] In one embodiment of the present invention, the antisense oligonucleotide is ACAg PPG g PPG attag PPG ttCTA (SEQ ID NO: 16) (In the above sequences, uppercase letters indicate nucleosides with LNA-modified ribose, all LNA Cs are 5-methylcytosine, and lowercase letters in these sequences indicate DNA.) g PPG It is 7-deaza-8-aza-deoxyguanosine, g N It is 8-amino-dG, g オキソ (This is 8-oxo-deoxyguanosine) That is the case.

[0140] The present invention also relates to polynucleotides according to the present invention for use as pharmaceuticals. These may be used for administration to the central nervous system or to treat CNS disorders selected from the group consisting of amyotrophic lateral sclerosis (ALS), Angelman syndrome, Alzheimer's disease, aneurysms, back pain, Bell's palsy, congenital defects of the brain and spinal cord, brain injury, brain tumors, cerebral palsy, chronic fatigue syndrome, concussions, dementia, cervical and lumbar disc diseases, vertigo, epilepsy, Guillain-Barré syndrome, headaches and migraines, multiple sclerosis, muscular dystrophy, neuralgia, neuropathy, neuromuscular and related disorders, Parkinson's disease, psychiatric symptoms (severe depression, obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury, spinal deformities and disorders, spinal tumors, stroke and vertigo.

[0141] The polynucleotide according to the present invention can be administered via intrathecal injection.

[0142] The polynucleotides according to the present invention can be used as pharmaceuticals to treat medical conditions in which the regulation of Ube3A is beneficial, for example, to treat angelman.

[0143] The polynucleotides according to the present invention can be used as pharmaceuticals to treat medical conditions in which the regulation of ATXN2 is beneficial.

[0144] The polynucleotides according to the present invention can be used as pharmaceuticals to treat medical conditions in which the regulation of ATXN3 is beneficial.

[0145] The polynucleotides of the present invention can be used as pharmaceuticals in which reduced neurotoxicity is required.

[0146] The present invention also relates to a method for synthesizing a polynucleotide of the present invention with reduced toxicity, comprising coupling a nucleotide monomer such as a phosphoramidite to a further nucleotide or oligonucleotide, wherein the nucleotide monomer comprises a guanosine analog as defined above.

[0147] The present invention also relates to a method for selecting a polynucleotide having lower toxicity than a reference polynucleotide, wherein the polynucleotide contains at least one phosphorothioate nucleoside linkage, and the less neurotoxic polynucleotide contains at least one guanosine analogue as compared to the reference polynucleotide, and the reference polynucleotide and the less neurotoxic antisense oligonucleotide have the same nucleotide sequence and contain at least one guanosine.

[0148] The present invention also relates to the use of a compound containing a guanosine analogue selected from the group consisting of (Ia), (Ib), (Ic), (Id), (Ie), (IIa), (IIb), (IIc), (IId) and (IIe) in the manufacture of oligonucleotide antisense or shRNA or siRNA.

[0149] The present invention also relates to a conjugate comprising a polynucleotide according to the invention and at least one conjugate moiety covalently bound to said polynucleotide.

[0150] The present invention also relates to a pharmaceutically acceptable salt of a polynucleotide according to the invention or a conjugate as defined above.

[0151] In some embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

[0152] A pharmaceutical composition comprising an antisense oligonucleotide according to the invention, or a conjugate according to the invention, or a pharmaceutically acceptable salt according to the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0153] The present invention also relates to a method for upregulating Ube3a expression in target cells expressing Ube3a-ATS, comprising administering an effective amount of an antisense oligonucleotide, a conjugate, or a pharmaceutically acceptable salt thereof, that targets Ube3a-ATS according to the present invention to the cells.

[0154] In some embodiments, the method is an in vivo or in vitro method.

[0155] The present invention also relates to a method for treating or preventing neurological disorders in subjects such as humans who are suffering from or are at risk of suffering from neurological disorders, the method comprising administering a therapeutically effective or preventively effective amount of a polynucleotide, a conjugate, or a pharmaceutically acceptable salt thereof according to the present invention for purposes such as preventing or mitigating neurological disorders.

[0156] Antisense oligonucleotides containing at least one phosphorothioate nucleoside linkage. It should be understood that the antisense oligonucleotides of the present invention may contain two or more phosphorothioates. Thus, the oligonucleotides of the present invention may contain one or more modified nucleoside links, such as one or more phosphorothioate nucleoside links or one or more phosphorothioate nucleoside links. In some embodiments, at least 50% of the nucleoside links of the oligonucleotide or its sequence of nucleotides are phosphorothioates, for example, at least 60%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, or e.g., at least 90% of the nucleoside links of the oligonucleotide or its sequence of nucleotides are phosphorothioates. In some embodiments, all of the nucleoside links of the oligonucleotide or its sequence of nucleotides are phosphorothioates.

[0157] In one embodiment, locked nucleic acid TIFF0007899100000027.tif171152(where B is a natural or modified nucleobase, Z is a internucleoside linkage to an adjacent nucleoside or 5'-terminal group, and Z * is an internucleoside linkage to an adjacent nucleoside or 3'-terminal group). is selected from the group consisting of.

[0158] In one embodiment of the antisense of the present invention, the 2'-modification can be selected from the group consisting of TIFF0007899100000028.tif68128.

[0159] In one embodiment of the present invention, the antisense oligonucleotide can include at least one additional nucleoside having a modified ribose, and the ribose modification is selected from the group consisting of locked nucleic acid or 2'-modification.

[0160] In one embodiment of the present invention, the antisense oligonucleotide is a gapmer as defined above.

[0161] In some embodiments, the antisense oligonucleotide of the present invention can be represented by the following formula: F-G-F'; particularly F 1~8 -G 5~16 -F' 2~8 D'-F-G-F', particularly D' 1~3 -F 1~8 -G 5~16 -F' 2~8 F-G-F'-D'', particularly F 1~8 -G 5~16 -F' 2~8 -D'' 1~3 D'-F-G-F'-D'', particularly D' 1~3 -F 1~8 -G 5~16 -F' 2~8 -D'' 1~3 (where F is a flank, and G is a gap).

[0162] In some embodiments, the internucleoside linkage located between region D’ and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage located between region F’ and region D’’ is a phosphodiester linkage.

[0163] In the antisense oligonucleotides of the present invention, the guanosine analog may be within the gap of the gapmer. In some embodiments according to the present invention, the guanosine analog is not in the flank of the gapmer. [[ID=*6]]

[0164] The antisense oligonucleotides of the present invention can contain one guanosine analog. The antisense oligonucleotides of the present invention can contain two guanosine analogs. The antisense oligonucleotides of the present invention can contain three guanosine analogs.

[0165] In some embodiments of the present invention, the antisense oligonucleotide does not contain natural guanosine.

[0166] The antisense oligonucleotides of the present invention provide relatively low neurotoxicity compared to conventional antisense oligonucleotides and thus can be used as a medicine. This can be administered to the central nervous system, for example, via intrathecal injection.

[0167] The antisense oligonucleotides according to the present invention can be used as a medicine for treating medical conditions where the regulation of Ube3A is beneficial, such as Angelman syndrome.

[0168] The antisense oligonucleotides according to the present invention can be used as a medicine for treating medical conditions where the regulation of ATXN2 is beneficial, such as spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS).

[0169] The antisense oligonucleotides according to the present invention can be used as pharmaceuticals to treat medical conditions in which modulation of ATXN3 is beneficial, such as spinocerebellar ataxia type 3 (SCA3).

[0170] The antisense oligonucleotides according to the present invention can be used as pharmaceuticals to treat medical conditions where a reduction in the neurotoxicity of the drug is required.

[0171] The present invention further relates to a method for designing an antisense oligonucleotide of the present invention that is less neurotoxic than a reference compound, wherein the reference compound and the less neurotoxic antisense oligonucleotide have the same nucleotide sequence and contain at least one guanosine, and the less neurotoxic antisense oligonucleotide contains at least one guanosine analog compared to the reference compound.

[0172] The present invention further relates to the use of guanosine analogs selected from the group consisting of (Ia), (Ib), (Ic), (Id), (Ie), (IIa), (IIb), (IIc), (IId), and (IIe) in the production of oligonucleotides.

[0173] The present invention also relates to a conjugate comprising an antisense oligonucleotide and at least one conjugate moiety covalently bonded to the antisense oligonucleotide.

[0174] The present invention further relates to pharmaceutically acceptable salts or conjugates of the antisense oligonucleotides of the present invention. In some embodiments, the non-nucleotide portion (conjugate portion) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, active pharmaceutical ingredients, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0175] The antisense oligonucleotides of the present invention may be in the form of pharmaceutically acceptable salts, such as sodium salts or potassium salts.

[0176] The present invention also relates to a pharmaceutical composition comprising an antisense oligonucleotide or conjugate or pharmaceutically acceptable salt of the present invention and one or more pharmaceutically acceptable diluents, solvents, carriers, salts and / or adjuvants.

[0177] The present invention also relates to a method for inhibiting Ube3a expression in target cells expressing Ube3a, comprising administering an effective amount of the antisense oligonucleotide, conjugate, or pharmaceutically acceptable salt thereof of the present invention to said cells. The present invention may be an in vivo or in vitro method.

[0178] The present invention further relates to a method for treating or preventing neurological disorders in subjects such as human beings who are or may be affected by neurological disorders, comprising administering a therapeutically effective or preventively effective amount of the antisense oligonucleotide, conjugate, or pharmaceutically acceptable salt of the present invention for purposes such as preventing or mitigating neurological disorders.

[0179] Antisense oligonucleotides In some embodiments, the antisense oligonucleotides of the present invention can be modulated by inhibiting or downregulating the expression of a target. Preferably, such modulation results in an inhibition of expression of at least 20% compared to the normal expression level of the target, more preferably at least 30%, at least 40%, and at least 50% compared to the normal expression level of the target. In some embodiments, the antisense oligonucleotides of the present invention may inhibit the expression level of target mRNA by at least 60% or 70% in vitro after application of 0.031 μM, 0.1 μM, and 0.4 μM antisense oligonucleotides to target cells. In some embodiments, the antisense oligonucleotides of the present invention may inhibit the expression level of a target gene by at least 50% in vitro after application of 0.031 μM, 0.1 μM, and 0.4 μM oligonucleotides to target cells. Preferably, the examples provide assays that can be used to measure target RNA or protein inhibition. Target modulation is caused by hybridization between the sequential nucleotide sequence of the antisense oligonucleotide and the target nucleic acid. In some embodiments, the antisense oligonucleotides of the present invention include a mismatch between the antisense oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to exhibit the desired regulation of target gene expression. The decrease in binding affinity resulting from the mismatch can be favorably compensated by increasing the number of nucleotides in the antisense oligonucleotide and / or the number of modified nucleosides that can increase binding affinity to the target, such as an increase in the number of 2' sugar-modified nucleosides containing LNAs present in the antisense oligonucleotide sequence.

[0180] One aspect of the present invention relates to an antisense oligonucleotide comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length having at least 90% complementarity to a target premRNA or mRNA or a transcript variant derived therefrom.

[0181] In some embodiments, the antisense oligonucleotide comprises a continuous sequence of 10 to 30 nucleotides in length, which is at least 90% complementary, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, or 100% complementary, to a region of the target nucleic acid or target sequence.

[0182] The antisense oligonucleotide or its sequence of nucleotides according to the present invention is advantageous when it is perfectly complementary (100% complementary) to the region of the target nucleic acid, or, in some embodiments, when there may be one or two mismatches between the antisense oligonucleotide and the target nucleic acid.

[0183] In some embodiments, the antisense oligonucleotide comprises a sequence of nucleotides that is completely (or 100%) complementary to the region of the target nucleic acid.

[0184] The antisense oligonucleotides of the present invention comprise a continuous nucleotide sequence that is complementary to or hybridizes with a region of a target nucleic acid, such as a target sequence as described herein.

[0185] The target nucleic acid sequences to which therapeutic antisense oligonucleotides are complementary or hybridize generally contain an extension of at least 10 consecutive nucleic acid bases. The consecutive nucleotide sequences are 12 to 70 nucleotides, e.g., 12 to 50, e.g., 13 to 30, e.g., 14 to 25, e.g., 14 to 20 consecutive nucleotides.

[0186] In some embodiments, the antisense oligonucleotide or its sequence of nucleotides of the present invention comprises or consists of 10 to 30 nucleotides in length, for example 12 to 25, for example 11 to 22, for example 12 to 20, for example 14 to 18 or 14 to 16 sequence of nucleotides.

[0187] In some embodiments, the antisense oligonucleotide or its sequence of nucleotides contains or consists of 22 or fewer nucleotides, e.g., 20 or fewer nucleotides, e.g., 18 or fewer nucleotides, e.g., 14, 15, 16, or 17 nucleotides. It should be understood that any range provided herein includes the endpoint of the range. Therefore, when an antisense oligonucleotide is described as containing 10 to 30 nucleotides, it includes both 10 and 30 nucleotides.

[0188] In some embodiments, the continuous nucleotide sequence includes or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 continuous nucleotide lengths.

[0189] In some embodiments, the continuous nucleotide sequence includes or consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 continuous nucleotide lengths.

[0190] In a favorable embodiment, the antisense oligonucleotide comprises one or more glycosylated nucleosides, for example, one or more 2'-glycosylated nucleosides, for example, one or more 2'-glycosylated nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. It is advantageous that one or more of the modified nucleosides are locked nucleic acid (LNA).

[0191] In some embodiments, the continuous nucleotide sequence includes an LNA nucleoside.

[0192] In some embodiments, the continuous nucleotide sequence includes LNA nucleosides and DNA nucleosides.

[0193] In some embodiments, the continuous nucleotide sequence includes LNA nucleosides, DNA nucleosides, and 2'-O-methylRNA nucleosides.

[0194] In some embodiments, the continuous nucleotide sequence comprises LNA nucleosides, DNA nucleosides, and 2'-O-methylRNA nucleosides, and the internucleoside bonds between each nucleoside in the continuous nucleotide linkage are phosphorothioate internucleoside bonds.

[0195] In some embodiments, the continuous nucleotide sequence comprises LNA nucleosides, DNA nucleosides, and 2'-O-methylRNA nucleosides, and the internucleoside bonds between each nucleoside in the continuous nucleotide linkage are phosphorothioate internucleoside bonds.

[0196] In some embodiments, the continuous nucleotide sequence comprises a 2'-O-methoxyethyl (2'MOE) nucleoside.

[0197] In some embodiments, the continuous nucleotide sequence comprises a 2'-O-methoxyethyl (2'MOE) nucleoside and a DNA nucleoside.

[0198] Advantageously, the 3'-most nucleoside of the antisense oligonucleotide or its sequence of nucleotides is a 2'-sugar-modified nucleoside.

[0199] Advantageously, the antisense oligonucleotide contains at least one modified internucleoside bond, such as a phosphorothioate or phosphorodithioate.

[0200] In some embodiments, at least one nucleoside bond in a continuous nucleotide sequence is a phosphorothioate nucleoside bond.

[0201] In some embodiments, at least one nucleoside bond in a sequence of nucleotides is a phosphorodithioate nucleoside bond.

[0202] In some embodiments, at least one nucleoside bond in a continuous nucleotide sequence is a phosphodiester nucleoside bond.

[0203] In some embodiments, all nucleoside bonds within a continuous nucleotide sequence are phosphorothioate nucleoside bonds.

[0204] In some embodiments, at least 75% of the nucleoside-to-nucleoside bonds within an antisense oligonucleotide or its sequence of nucleotides are phosphorothioate-nucleoside bonds.

[0205] In some embodiments, all nucleoside-to-nucleoside bonds within an antisense oligonucleotide or its sequence of nucleotides are phosphorothioate nucleoside bonds.

[0206] In advantageous embodiments of the present invention, the antisense oligonucleotide of the present invention can recruit RNase H, for example, RNase H1. In some embodiments, the antisense oligonucleotide of the present invention or its sequence of nucleotides is a gapmer.

[0207] In some embodiments, the antisense oligonucleotide or its sequence of nucleotides consists of or includes a gapmer of formula 5'-FG-F'-3'.

[0208] In some embodiments, region G consists of 6 to 16 DNA nucleosides.

[0209] In some embodiments, regions F and F' each contain at least one LNA nucleoside.

[0210] Pharmaceutically acceptable salts In a further embodiment, the present invention provides pharmaceutically acceptable salts of antisense oligonucleotides or their conjugates, such as pharmaceutically acceptable sodium salts, ammonium salts, or potassium salts.

[0211] Manufacturing method In further embodiments, the present invention provides a method for producing an antisense oligonucleotide of the present invention, comprising reacting nucleotide units to form a covalently linked sequence of nucleotide units contained within the antisense oligonucleotide. Preferably, this method uses phosphoramidite chemistry (see, for example, Caruthers et al., 1987, Methods in Enzymology, Vol. 154, pp. 287-313). In further embodiments, this method further comprises reacting a sequence of nucleotides with a conjugate moiety (ligand) to covalently bond the conjugate moiety to the antisense oligonucleotide. In further embodiments, a method is provided for producing a composition of the present invention, comprising mixing the antisense oligonucleotide or conjugated antisense oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0212] Pharmaceutical composition In further embodiments, the present invention provides pharmaceutical compositions comprising any of the aforementioned antisense oligonucleotides and / or antisense oligonucleotide conjugates or salts thereof, and a pharmaceutically acceptable diluent, carrier, salt, and / or adjuvant. The pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS), and the pharmaceutically acceptable salt includes, but is not limited to, sodium salts and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate-buffered saline or sterile sodium carbonate buffer.

[0213] In some embodiments, the antisense oligonucleotide of the present invention is in the form of a solution in a pharmaceutically acceptable diluent, such as PBS or sodium carbonate buffer. In some embodiments, the antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt thereof, is in a solid form, such as a powder, such as a lyophilized powder. In some embodiments, the antisense oligonucleotide may be pre-formulated in solution, or in some embodiments, it may be in the form of a dry powder (e.g., lyophilized powder) that can be dissolved in a pharmaceutically acceptable diluent before administration.

[0214] Appropriately, for example, antisense oligonucleotides can be dissolved at concentrations of 0.1 to 100 mg / ml, for example, 1 to 10 mg / a pharmaceutically acceptable diluent.

[0215] In some embodiments, the oligonucleotides of the present invention are formulated in unit doses of 0.5 to 100 mg, for example, 1 mg to 50 mg or 2 to 25 mg.

[0216] In some embodiments, the antisense oligonucleotide is used at a concentration of 50–300 μM in a pharmaceutically acceptable diluent.

[0217] The antisense oligonucleotides or antisense oligonucleotide conjugates of the present invention may be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but are not limited to, the route of administration, the severity of the disease, or the dose administered.

[0218] Pharmaceutical compositions, such as solutions, may be sterilized by conventional sterilization techniques or filtered through a sterile filter. The resulting solutions may be packaged for immediate use or lyophilized, and the lyophilized preparations may be combined with a sterile aqueous carrier before administration. The pH of the preparations will typically be 3 to 11, more preferably 5 to 9 or 6 to 8, most preferably 7 to 8, for example, 7 to 7.5. The resulting solid-form compositions may be packaged in multiple single-dose units, each containing a fixed amount of the above-mentioned drug or group of drugs, such as in sealed packages of tablets or capsules. The solid-form compositions may also be packaged in flexible-volume containers, such as squeezeable tubes designed for topically administered creams or ointments.

[0219] In some embodiments, the antisense oligonucleotide or antisense oligonucleotide conjugate of the present invention is a prodrug. In particular with respect to antisense oligonucleotide conjugates, when the prodrug is delivered to the site of action, for example, a target cell, the conjugate portion is cleaved from the antisense oligonucleotide.

[0220] Purpose The antisense oligonucleotides of the present invention can be used, for example, as research reagents for diagnosis, treatment, and prevention.

[0221] In research, such oligonucleotides can be used to specifically regulate protein synthesis in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating the functional analysis of targets or the evaluation of their usefulness as targets for therapeutic interventions. Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting the protein-producing gene or mRNA modulator.

[0222] When the antisense oligonucleotides of the present invention are used for research or diagnostic purposes, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0223] The present invention provides an in vivo or in vitro method for regulating gene expression in target cells expressing a target protein, comprising administering an effective amount of the antisense oligonucleotide of the present invention to the cells.

[0224] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells forming part of mammalian tissue. In preferred embodiments, the target cells are located in the central nervous system.

[0225] therapeutic use The antisense oligonucleotides of the present invention, or the antisense oligonucleotide conjugates, salts, or pharmaceutical compositions of the present invention, may be administered to animals or humans for the prevention or treatment of neurological disorders.

[0226] Neurological disorders that can be treated with the antisense oligonucleotides of the present invention, or the antisense oligonucleotide conjugates, salts, or pharmaceutical compositions of the present invention, may include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, aneurysms, back pain, Bell's palsy, congenital defects of the brain and spinal cord, brain injury, brain tumors, cerebral palsy, chronic fatigue syndrome, concussions, dementia, cervical and lumbar disc diseases, vertigo, epilepsy, Guillain-Barré syndrome, headaches and migraines, multiple sclerosis, muscular dystrophy, neuralgia, neuropathy, neuromuscular and related disorders, Parkinson's disease, psychiatric symptoms (severe depression, obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury, spinal deformities and disorders, spinal tumors, strokes, and vertigo.

[0227] The present invention provides antisense oligonucleotides, antisense oligonucleotide conjugates, compositions, or salts for use in preventing or treating the above-mentioned neurological disorders.

[0228] The present invention further relates to the use of the antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition of the present invention for manufacturing a pharmacopoeia for treating or preventing the above-mentioned neurological disorders.

[0229] The present invention provides antisense oligonucleotides, antisense oligonucleotide conjugates, pharmaceutical compositions, or salts for use as pharmaceuticals.

[0230] The present invention provides the use of antisense oligonucleotides, antisense oligonucleotide conjugates, pharmaceutical compositions, or salts of the present invention for the manufacture of pharmaceuticals.

[0231] The present invention provides antisense oligonucleotides, antisense oligonucleotide conjugates, pharmaceutical compositions, or salts for use in preventing or treating the above-mentioned neurological disorders.

[0232] The present invention further relates to the use of the antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition of the present invention for manufacturing a pharmacopoeia for treating or preventing the above-mentioned neurological disorders.

[0233] Treatment method The present invention provides a method for treating or preventing neurological disorders in subjects such as humans who are suffering from or are likely to suffer from the above-mentioned neurological disorders, comprising administering a therapeutically effective amount or a preventively effective amount of the antisense oligonucleotide, antisense oligonucleotide conjugate or pharmaceutical composition of the present invention to subjects suffering from or susceptible to the above-mentioned neurological disorders.

[0234] For example, the treatment method may be for subjects suffering from an indication selected from the group of neurological disorders described above.

[0235] The method of the present invention may be for treating the above-mentioned neurological disorders.

[0236] The method of the present invention is preferably used to treat or prevent the above-mentioned neurological disorders.

[0237] Administration The antisense oligonucleotides, antisense oligonucleotide conjugates, or pharmaceutical compositions of the present invention may be administered via parenteral administration.

[0238] In some embodiments, the route of administration is subcutaneous or intravenous.

[0239] In some embodiments, the route of administration is selected from the group consisting of intravenous, subcutaneous, intramuscular, intracerebral, epidural, intraventricular, intraocular, intrathecal, and transvertebral foramen administration.

[0240] In some embodiments, the antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition of the present invention is targeted to the brain, i.e., delivered to the brain.

[0241] In some advantageous embodiments, administration is via intrathecal administration, epidural administration, or transforaminal administration.

[0242] Advantageously, the antisense oligonucleotide, antisense oligonucleotide conjugate, or pharmaceutical composition of the present invention is administered intrathecally.

[0243] The present invention also provides the use of the antisense oligonucleotide or antisense oligonucleotide conjugate of the present invention, for example, the pharmaceutical salt or composition of the present invention, for the manufacture of a pharmacopoeia for the prevention or treatment of neurological disorders, wherein the pharmacopoeia is in a dosage form for intrathecal administration.

[0244] The present invention also provides a use of antisense oligonucleotides or antisense oligonucleotide conjugates described for manufacturing a pharmacopoeci for the manufacture of

[0245] The present invention also provides antisense oligonucleotides or antisense oligonucleotide conjugates, such as pharmaceutical salts or compositions of the present invention, for use as pharmaceuticals for preventing or treating neurological disorders, wherein the pharmaceutical is in a dosage form for intrathecal administration.

[0246] The present invention also provides an antisense oligonucleotide or antisense oligonucleotide conjugate for use as a pharmaceutical for preventing or treating neurological disorders, wherein the pharmaceutical is in a dosage form for intrathecal administration.

[0247] Combination therapy In some embodiments, the antisense oligonucleotides, antisense oligonucleotide conjugates, or pharmaceutical compositions of the present invention are used in combination therapy with another therapeutic agent, which may, for example, be a standard treatment for the diseases or disorders described above. In some embodiments, the compounds of the present invention are used in combination with low-molecular-weight analgesics, which may be administered concurrently with or independently of the compounds or compositions of the present invention. The advantage of combination therapy of the compounds of the present invention and low-molecular-weight analgesics is that while low-molecular-weight analgesics typically have a short duration of action (several hours to several days) and a rapid onset of neuropathic activity, the compounds of the present invention have a delayed onset of activity (typically several days or even a week or more) but a long duration of action (several weeks to several months, e.g., 2+, 3+, or 4 months or more). [Examples]

[0248] Example A: Synthesis of oligomer compounds Single-stranded oligonucleotides were synthesized using standard phosphoramidite chemistry. Unmodified DNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). LNA phosphoramidites were manufactured in-house (LNA phosphoramidites are also commercially available, for example, from Merck KGaA). 8-amino-dG, 8-oxo-dG, 7-deaza-8-aza-dG (PPG), and 2'-deoxyinosine phosphoramidites were purchased from Glen Research (Sterling, Virginia).

[0249] Oligonucleotides were synthesized on a 20 μmol scale on NittoPhase HL UnyLinker350 supports (Kinovate, Oceanside, California) on MerMade12 (LGC BioAutomation, Irving, Texas). After synthesis, the oligonucleotides were cleaved from the supports overnight at 65°C using aqueous ammonia. The oligonucleotides were purified by ion exchange on SuperQ-5PW gels (Tosoh Bioscience, Griesheim, Germany) using a gradient of 10 mM NaOH buffer and 2 M NaCl, and desalted using a Millipore membrane. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse-phase and electrospray ionization-mass spectrometry).

[0250] Example 1: In vivo study of alkyl groups containing guanosine analogs in mice In this example, gapmer antisense alkyl groups containing natural guanosine or guanosine analogs were tested for their neurotoxicity in the following acute neurotoxicity assay.

[0251] Nine groups of C57BL / 6 black mice, each consisting of six mice, were injected with a single dose of 100 μg of antisense oligonucleotide via intracerebroventricular injection.

[0252] Group 1 mice were injected with physiological saline. Group 2 mice were injected with SEQ ID NO: 1 (control). Groups 3-9 mice were injected with SEQ ID NOs: 2-8, respectively.

[0253] The behavior of mice after injection was observed and reported at 30 minutes, 1 hour, 24 hours, and 14 days according to the following behavioral scoring categories (0-4): A. "Hyperactivity, stereotyped behavior, and home cage behavior" B. "Reduced arousal, exploration, and responsiveness" C. "Motor coordination and strength" D. "Posture, appearance, and breathing" E. "Tremor, hyperactivity, convulsions"

[0254] Typically, high neurotoxicity is observed within 30 minutes of injection as ataxia, convulsions, and seizures.

[0255] (Table 1) The capital letters in these sequences indicate nucleosides with LNA-modified ribose. All LNA Cs are 5-methylcytosine. Lowercase letters in these sequences indicate DNA. Na: No abnormalities I: Inosine nucleoside The following modified guanosine nucleosides were used in these sequences: TIFF0007899100000029.tif54128 g PPG :7-deaza-8-aza-deoxyguanosine TIFF0007899100000030.tif103165g N :8-amino-dG: TIFF0007899100000031.tif55128g オキソ :8-Oxo-deoxyguanosine

[0256] This test shows the following: - The sequence without g (SEQ ID NO: 1, control) does not show neurotoxicity. - Sequences containing two or three g (sequences 7 and 8) exhibit relatively higher neurotoxicity compared to sequences containing one g (sequence 2). - Sequences containing g (SEQ ID NO: 2) exhibit neurotoxicity. - Sequences containing I (SEQ ID NO: 3) exhibit neurotoxicity.

[0257] Conclusion: - Many g in the sequence: relatively high neurotoxicity - Modified g in the sequence exhibits relatively lower neurotoxicity compared to sequences containing unmodified g. - g in the array オキソ and g PPG It exhibits the lowest relatively low neurotoxicity.

[0258] Acute neurotoxicity in groups 7 and 8 leads to euthanasia. The 'g' in the name indicates toxicity, suggesting that many 'g's are more toxic, while modified 'g's are less toxic.

[0259] Example 2 Following the procedure of Example 1, the gapmer compounds listed in Table 2 were tested in eight groups of six C57BL / 6 mice. The results are shown in Table 2.

[0260] (Table 2) TIFF0007899100000032.tif75168 The capital letters in these sequences indicate nucleosides with LNA-modified ribose. All LNA Cs are 5-methylcytosine. Lowercase letters in these sequences indicate DNA. Na: No abnormalities I: Inosine nucleoside The following modified guanosine nucleosides were used in these sequences: g PPG :7-deaza-8-aza-deoxyguanosine g N :8-amino-dG g オキソ :8-Oxo-deoxyguanosine

[0261] This test demonstrates that sequences without guanosine do not exhibit neurotoxicity; please refer to sequence (SEQ ID NO: 1, control). PPG The sequence containing (SEQ ID NO: 10) exhibits far less neurotoxicity compared to the same sequence containing natural guanosine (SEQ ID NO: 9). オキソ The sequence containing (SEQ ID NO: 11) does not exhibit neurotoxicity. This is because natural guanosine (SEQ ID NO: 9) and g PPG It can be compared to the same sequence containing (SEQ ID NO: 10). The sequence containing I (SEQ ID NO: 12) exhibits relatively higher neurotoxicity than the same sequence containing guanosine. N The sequence containing (SEQ ID NO: 13) exhibits relatively higher neurotoxicity than the same sequence containing guanosine.

[0262] Conclusion: The inventors were surprised to find that the proportion of natural, i.e., unmodified guanosine nucleic acid bases within a polynucleotide sequence directly correlates with the potential for polynucleotides, such as antisense oligonucleotides, to be neurotoxic. Such neurotoxicity indicates that it is acute and lethal. Guanosine analog g PPG and g オキソ Substitution of natural guanosine by [this method] reduced neurotoxicity.

[0263] Example 3 Following the procedure of Example 1, the gapmer compounds listed in Table 2 were tested in nine groups of six C57BL / 6 mice. The results are shown in Table 3.

[0264] (Table 3) TIFF0007899100000033.tif124164 Uppercase letters in these sequences indicate nucleosides with LNA-modified ribose. All LNA Cs are 5-methylcytosine. Lowercase letters in these sequences indicate DNA. Na: No abnormalities I: Inosine nucleoside The following modified guanosine nucleosides were used in these sequences: g PPG :7-deaza-8-aza-deoxyguanosine g N :8-amino-dG g オキソ :8-Oxo-deoxyguanosine

[0265] This study revealed that the same nucleotide sequence (SEQ ID NO: 14) containing three natural guanosines in the gap, tested at 50 μg and 100 μg, showed a dose-dependent increase in neurotoxicity.

[0266] It has the same nucleotide sequence as the gapmer of SEQ ID NO: 14, but it has three guanosine analogs (g オキソ When a sample containing (SEQ ID NO: 15) was tested at 50 μg and 100 μg, it showed significantly lower neurotoxicity compared to the gapmer of SEQ ID NO: 14. Interestingly, there was no increase in neurotoxicity even when the dose was increased from 50 μg to 100 μg.

[0267] It has the same nucleotide sequence as the gapmer of SEQ ID NO: 14, but it has three guanosine analogs (g PPG When a sample containing (SEQ ID NO: 16) was tested at 50 μg and 100 μg, it showed significantly lower neurotoxicity compared to the gapmer of SEQ ID NO: 14. Interestingly, there was no increase in neurotoxicity even when the dose was increased from 50 μg to 100 μg.

Claims

1. - At least one phosphorothioate nucleoside interbonding, -below: A guanosine analog comprising at least one guanine analog selected from the group consisting of and A polynucleotide that is siRNA or shRNA, containing the above.

2. - At least one phosphorothioate nucleoside interbonding, -below: A guanosine analog comprising at least one guanine analog selected from the group consisting of and A polynucleotide which is an antisense oligonucleotide, A polynucleotide in which the antisense oligonucleotide is a gapmer.

3. The polynucleotide according to claim 2, comprising at least one further nucleoside having a modified ribose, wherein the modified ribose is selected from the group consisting of locked nucleic acids or 2' modifications.

4. The guanosine analog is located in the gap region of the gapmer, and the following formula Ia or formula IIb: It is, The polynucleotide according to claim 2 or 3, wherein R is H in the formula.

5. The polynucleotide according to any one of claims 2 to 4, wherein the guanosine analog is not present in the flank of the gapmer.

6. The polynucleotide according to claim 1 or 2, further comprising one or more 2'-sugar-modified nucleosides.

7. The polynucleotide according to claim 6, wherein the 2'-sugar-modified nucleoside is independently selected from the group consisting of locked nucleic acids and 2'-sugar-substituted nucleosides.

8. One or more of the 2' sugar-modified nucleosides are A locked nucleic acid selected from the group consisting of, where B is a natural or modified nucleic acid base, and Z is an internucleoside bond to an adjacent nucleoside or 5' terminal group, Z ＊ The polynucleotide according to claim 6 or 7, wherein is an internucleoside bond to an adjacent nucleoside or a 3' terminal group.

9. One or more 2'-sugar-modified nucleosides, A polynucleotide according to claim 6 or 7, selected from the group consisting of the following.

10. The guanosine analog is (In the formula, R is either H or OH) A polynucleotide according to any one of claims 1 to 3 and 6 to 9, selected from the group consisting of the following.

11. The polynucleotide according to claim 10, wherein the guanosine analog is (Ia).

12. The polynucleotide according to claim 10, wherein the guanosine analog is (Ia1).

13. The polynucleotide according to claim 10, wherein the guanosine analog is (Ia2).

14. The polynucleotide according to claim 10, wherein the guanosine analog is (Ib).

15. The polynucleotide according to claim 10, wherein the guanosine analog is (IIa).

16. The polynucleotide according to claim 10, wherein the guanosine analog is (IIa1).

17. The polynucleotide according to claim 10, wherein the guanosine analog is (IIa2).

18. The polynucleotide according to claim 10, wherein the guanosine analog is (IIb).

19. The polynucleotide according to claim 10, wherein the guanosine analog is (Ic).

20. The polynucleotide according to claim 10, wherein the guanosine analog is (IIc).

21. The polynucleotide according to claim 10, wherein the guanosine analog is (Id).

22. The polynucleotide according to claim 10, wherein the guanosine analog is (IId).

23. The polynucleotide according to claim 10, wherein the guanosine analog is (Ie).

24. The polynucleotide according to claim 10, wherein the guanosine analog is (IIe).

25. A polynucleotide according to any one of claims 1 to 24, comprising one guanosine analog according to any one of claims 1 to 24.

26. A polynucleotide according to any one of claims 1 to 24, comprising two guanosine analogs according to any one of claims 1 to 24.

27. A polynucleotide according to any one of claims 1 to 24, comprising three guanosine analogs according to any one of claims 1 to 24.

28. A polynucleotide according to any one of claims 1 to 27, which does not contain natural guanosine.

29. The aforementioned polynucleotide is CTCAacttg オキソ ctttaAT (SEQ ID NO: 4); CTCAtacttg ＰＰＧ ctttaAT (SEQ ID NO: 6); CTAcatctcatactTg ＰＰＧ C (Sequence ID 10); CTAcatctcatactTg オキソ C (Sequence ID 11); ACAg オキソ g オキソ tag オキソ ttCTA (SEQ ID NO: 15); and ACA g ＰＰＧ g ＰＰＧ attag ＰＰＧ ttCTA (SEQ ID NO: 16) Selected from the group consisting of, uppercase letters in these sequences indicate nucleosides with LNA-modified ribose, all LNA Cs are 5-methylcytosine, and lowercase letters in these sequences indicate DNA. g ＰＰＧ is 7-deaza-8-aza-deoxyguanosine, and g オキソ It is 8-oxo-deoxyguanosine. The polynucleotide according to claim 2.

30. A pharmaceutical product comprising a polynucleotide according to any one of claims 1 to 29.

31. The pharmacopoeia according to claim 30, for administration to the central nervous system, or for treating CNS disorders selected from the group consisting of amyotrophic lateral sclerosis (ALS), Angelman syndrome, Alzheimer's disease, aneurysms, back pain, Bell's palsy, congenital defects of the brain and spinal cord, brain injury, brain tumors, cerebral palsy, chronic fatigue syndrome, concussions, dementia, cervical and lumbar disc diseases, vertigo, epilepsy, Guillain-Barré syndrome, headaches and migraines, multiple sclerosis, muscular dystrophy, neuralgia, neuropathy, neuromuscular and related diseases, Parkinson's disease, psychiatric symptoms (severe depression, obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury, spinal deformities and disorders, spinal tumors, stroke and vertigo.

32. The pharmaceutical product according to claim 31, for administration via intrathecal injection.

33. A pharmaceutical product comprising a polynucleotide according to any one of claims 1 to 29 for treating a medical condition in which the regulation of Ube3A is beneficial, for example, for treating angelman.

34. A pharmaceutical product comprising a polynucleotide according to any one of claims 1 to 29 for treating a medical condition in which the regulation of ATXN2 is beneficial.

35. A pharmaceutical product comprising a polynucleotide according to any one of claims 1 to 29 for treating a medical condition in which the regulation of ATXN3 is beneficial.

36. A pharmaceutical product comprising a polynucleotide according to any one of claims 1 to 29, which is used as a pharmaceutical product for which a reduction in neurotoxicity is required.

37. A method for synthesizing a polynucleotide with reduced toxicity according to any one of claims 1 to 29, comprising coupling a nucleotide monomer such as a phosphoramidite to a further nucleotide or oligonucleotide, wherein the nucleotide monomer comprises a guanosine analog according to any one of claims 1 to 24.

38. A method for selecting a polynucleotide having lower neurotoxicity than a reference polynucleotide, wherein the polynucleotide is described in any one of claims 1 to 29, and the less neurotoxic polynucleotide and the less neurotoxic antisense oligonucleotide have the same nucleotide sequence and contain at least one guanosine, with the difference being that the less neurotoxic polynucleotide contains at least one guanosine analog compared to the reference polynucleotide.

39. The use of a compound containing a guanosine analog selected from the group consisting of (Ia), (Ib), (Ic), (Id), (Ie), (IIa), (IIb), (IIc), (IId), and (IIe) as defined in claim 10, in the production of a polynucleotide according to any one of claims 1 to 29.

40. A pharmaceutical composition for use in a method for upregulating Ube3a expression in target cells expressing Ube3a-ATS, comprising an effective amount of a polynucleotide according to any one of claims 1 to 29, which targets Ube3a-ATS.

41. The pharmaceutical composition according to claim 34, wherein the treatment of the aforementioned medical symptoms is performed by an in vivo or in vitro method.

42. A pharmaceutical composition for treating or preventing neurological disorders in subjects such as humans who are suffering from or are at risk of suffering from neurological disorders, comprising a therapeutically effective amount or a preventively effective amount of the polynucleotide described in any one of claims 1 to 29.

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