Oligonucleotides comprising inter-trithiophosphate nucleoside bonds

Abstract

The present invention relates to oligonucleotides comprising a trithiophosphate inter-nucleoside bond, in particular to an oligonucleotide comprising at least one trithiophosphate inter-nucleoside bond of formula (I) as defined in the description and in the claims. # imgabs0 #. The oligonucleotide disclosed by the invention can be used as a medicine.

Description

[0001] The applicant submitted a PCT application PCT / EP2019 / 070331 entitled “Oligonucleotides containing trithiophosphate internucleoside bonds” on July 29, 2019. The PCT application entered the Chinese national phase on December 8, 2020 with application number 201980038240.1. This application is a divisional application of the Chinese application. Technical Field

[0002] The present invention relates to an oligonucleotide comprising at least one phosphorothioate internucleoside bond of formula (I)

[0003]

[0004] One of the two bridging sulfur atoms is bound to a DNA nucleoside or an RNA nucleoside (A 1 ) and the other is connected to the 3' carbon atom of another nucleoside (A 2 ), and wherein R is hydrogen or a phosphate protecting group. Background Art

[0005] Short synthetic nucleic acids show great potential as therapeutic agents. Since the first proof-of-concept studies in the late 1970s, which demonstrated that unmodified deoxynucleotides could inhibit virus production in vitro (PC Zamecnik, ML Stephenson, Proc. Nat. Acad. Sci. USA 1978, 75, 280-284), significant progress has been made through chemical modification. Due to the sensitivity to nucleic acid degradation, stabilization of the phosphodiester backbone of nucleic acids is an obvious starting point for chemical optimization of therapeutic oligonucleotides. Therefore, various phosphate modifications have been studied, including phosphorothioates (PS) (F. Eckstein, Antisense and Nucleic Acid Drug Development 2009, 10, 117-121), phosphorodithioates (e.g., WT Weisler, MH Caruthers, J. Org. Chem 1996, 61, 4272-4281), boron phosphoroates (e.g., JS Summers, BR Shaw, Curr. Med. Chem. 2001, 8, 1147-1155), (thio) phosphoramidates (e.g., S. Gryaznov, T. Skorski, C. Cucco, M. Nieborowska-Skorska, CY Chiu, D. Lloyd, J. Chen, M. Koziolkiewicz, B. Calabretta, Nucleic Acids Res.1996,24,1508-1514) and methylphosphonates (e.g. P.S. Sarin, S. Agrawal, M.P. Civeira, J. Goodchild, T. Ikeuchi, P.C. Zamecnik, Proc. Nat. Acad. Sci. USA 1988,20,7448-7451). Among these phosphate analogs, the most successful modification is undoubtedly the thiophosphate, in which one of the non-bridging phosphate oxygen atoms is replaced by sulfur. Due to its high stability to nucleases and its pharmacokinetic advantages, thiophosphate oligonucleotides have become the first generation of oligonucleotide therapeutic drugs and have paved the way for a new generation of modification methods such as locked nucleic acids (LNA) or 2'-O-(2-methoxyethyl)-oligoribonucleotides (2'-MOE).

[0006] The present invention relates to (thio)phosphorothioate modifications, wherein trithiophosphate bonds are incorporated into oligonucleotide sequences. In such trithiophosphates, not only one non-bridging phosphate oxygen atom is substituted by sulfur, but also two bridging oxygen atoms at the 3' and 5' positions of adjacent ribose sugar rings are substituted by sulfur.

[0007] We have surprisingly found that a single modification according to the invention results in oligonucleotides with improved activity both in vitro and in vivo compared to the unmodified phosphorothioate analogue. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Shown are ApoB mRNA levels and intracellular oligonucleotide concentrations (distribution) in primary rat hepatocytes after 24 h and 72 h of incubation at a treatment concentration of 1 μM.

[0009] Figure 2 Shown are ApoB mRNA levels in liver and kidney of C57BL / 6JbomTac female mice treated with a single 1 mg / kg dose of an oligonucleotide carrying a modification according to the invention and sacrificed on day 7, compared with the results of saline and unmodified controls.

[0010] Figure 3 Shown are serum cholesterol levels measured on days 0, 3 and 7 in C57BL / 6JbomTac female mice treated with a single 1 mg / kg dose of an oligonucleotide carrying a modification according to the invention, and compared with the results of saline and unmodified controls.

[0011] Figure 4 The figure shows the oligonucleotide tissue content in the liver, kidney and heart of C57BL / 6JbomTac female mice treated with a single dose of 1 mg / kg of the oligonucleotide carrying the modification according to the present invention and sacrificed on the 7th day, and the comparison with the results after receiving unmodified control treatment. DETAILED DESCRIPTION

[0012] In the present specification, the term "alkyl" refers alone or in combination to a straight or branched chain alkyl group having 1 to 8 carbon atoms, in particular a straight or branched chain alkyl group having 1 to 6 carbon atoms and more particularly a straight or branched chain alkyl group having 1 to 4 carbon atoms. Examples of straight and branched C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, isomeric amyl, isohexyl, isoheptyl and isooctyl, in particular methyl, ethyl, propyl, butyl and amyl. Specific examples of alkyl groups are methyl, ethyl and propyl.

[0013] The term "cycloalkyl" alone or in combination refers to a cycloalkyl ring having 3 to 8 carbon atoms, particularly a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, more particularly cyclopropyl and cyclobutyl. A specific example of "cycloalkyl" is cyclopropyl.

[0014] The term "alkoxy" alone or in combination refers to a radical of the formula alkyl-O-, wherein the term "alkyl" has the meaning given previously, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. Specific "alkoxy" are methoxy and ethoxy. Methoxyethoxy is a specific example of "alkoxyalkoxy".

[0015] The term "oxy" alone or in combination refers to an -O- group.

[0016] The term "alkenyl", alone or in combination, refers to a straight-chain or branched hydrocarbon residue comprising an olefinic bond and up to 8, preferably up to 6, particularly preferably up to 4 carbon atoms. Examples of alkenyl are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl and isobutenyl.

[0017] The term "alkynyl" alone or in combination refers to a straight-chain or branched hydrocarbon residue comprising a triple bond and up to 8, in particular 2, carbon atoms.

[0018] The term "halogen" or "halogen" refers alone or in combination to fluorine, chlorine, bromine or iodine and particularly to fluorine, chlorine or bromine, more particularly to fluorine. In combination with another group, the term "halogen" refers to the group substituted with at least one halogen, especially with one to five halogens, especially one to four halogens (i.e. one, two, three or four halogens).

[0019] The term "haloalkyl" alone or in combination refers to an alkyl group substituted with at least one halogen, especially with one to five halogens, particularly one to three halogens. Examples of haloalkyl groups include monofluoro, difluoro or trifluoromethyl, ethyl or propyl, such as 3,3,3-trifluoropropyl, 2-difluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl or trifluoromethyl. Fluoromethyl, difluoromethyl and trifluoromethyl are specific "haloalkyl groups".

[0020] The term "halocycloalkyl" alone or in combination refers to a cycloalkyl as defined above which is substituted with at least one halogen, especially with one to five halogens, particularly one to three halogens. Specific examples of "halocycloalkyl" are halocyclopropyls, especially fluorocyclopropyls, difluorocyclopropyls and trifluorocyclopropyls.

[0021] The terms "hydroxy" and "hydroxyl," alone or in combination, refer to a -OH group.

[0022] The terms "sulfhydryl" and "mercapto," alone or in combination, refer to a -SH group.

[0023] The term "carbonyl" alone or in combination refers to a -C(O)- group.

[0024] The term "carboxy" or "carboxy", alone or in combination, refers to a -COOH group.

[0025] The term "amino" alone or in combination refers to a primary amino group (-NH2), a secondary amino group (-NH-) or a tertiary amino group (-N-).

[0026] The term "alkylamino" alone or in combination refers to an amino group as defined above substituted by one or two alkyl groups as defined above.

[0027] The term "sulfonyl" alone or in combination means a -SO2 group.

[0028] The term "sulfinyl" alone or in combination refers to a -SO- group.

[0029] The term "sulfanyl," alone or in combination, refers to a -S- group.

[0030] The term "cyano" alone or in combination refers to a -CN group.

[0031] The term "azido" alone or in combination refers to a -N3 group.

[0032] The term "nitro", alone or in combination, refers to a NO2 group.

[0033] The term "formyl" alone or in combination refers to a -C(O)H group.

[0034] The term "carbamyl", alone or in combination, refers to a -C(O)NH2 group.

[0035] The term "urea" alone or in combination refers to a -NH-C(O)-NH2 group.

[0036] The term "aryl", alone or in combination, refers to a monovalent aromatic carbocyclic mono- or bicyclic ring system containing 6 to 10 carbon ring atoms, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of aryl include phenyl and naphthyl, especially phenyl.

[0037] The term "heteroaryl", alone or in combination, refers to a monovalent aromatic heterocyclic mono- or bicyclic ring system of 5 to 12 ring atoms, said system comprising 1, 2, 3 or 4 heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, carbazolyl, or acridinyl.

[0038] The term "heterocyclyl" alone or in combination refers to a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system of 4 to 12, in particular 4 to 9 ring atoms, comprising 1, 2, 3 or 4 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of monocyclic saturated heterocyclyl are azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl or oxazepanyl. Examples of bicyclic saturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl or 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocycloalkyl are dihydrofuranyl, imidazolinyl, dihydrooxazolyl, tetrahydropyridinyl or dihydropyranyl.

[0039] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and characteristics of free alkali or free acid and are biologically or otherwise desirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid (particularly hydrochloric acid) and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methylsulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins). The oligonucleotides of the present invention may also exist in the form of zwitterions. Particularly preferred pharmaceutically acceptable salts of the present invention are sodium salts, lithium salts, potassium salts and trialkylammonium salts.

[0040] The term "protecting group" alone or in combination refers to a group that selectively blocks a reactive site in a multifunctional compound so that a chemical reaction can be selectively performed at another unprotected reactive site. The protecting group can be removed. Exemplary protecting groups are amino protecting groups, carboxyl protecting groups, or hydroxyl protecting groups.

[0041] A "phosphate protecting group" is a protecting group for a phosphate group. Examples of phosphate protecting groups are 2-cyanoethyl and methyl. A specific example of a phosphate protecting group is 2-cyanoethyl.

[0042] "Hydroxy protecting groups" are protecting groups for hydroxyl groups and are also used to protect thiol groups. Examples of hydroxy protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (or tris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silyl ether (e.g., trimethylsilyl imidazole (TMS), tert-butyldimethylsilyl (TBDMS), triisobutylsiloxymethyl (TOM), and triisopropylsilyl (TIPS) ether), methyl ether, and ethoxyethyl ether (EE). Specific examples of hydroxyl protecting groups are DMT and TMT, especially DMT.

[0043] A "sulfhydryl protecting group" is a protecting group for a sulfhydryl group. Examples of the sulfhydryl protecting group are those of the "hydroxyl protecting group".

[0044] If one of the starting materials or compounds of the invention contains one or more functional groups that are unstable or reactive under the reaction conditions of one or more reaction steps, suitable protecting groups may be introduced prior to the key steps using methods well known in the art (as described in, for example, "Protective Groups in Organic Chemistry" by TW Greene and PGM Wuts, 3rd edition, 1999, Wiley, New York). Such protecting groups may be removed at a later stage of the synthesis using standard methods described in the literature. Examples of protecting groups are tert-butyloxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), carbobenzyl (Cbz), and p-methoxybenzyloxycarbonyl (Moz).

[0045] The compounds described herein may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers such as racemates, diastereomeric mixtures, diastereomeric racemates or mixtures of diastereomeric racemates, and the like.

[0046] Oligonucleotides

[0047] As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides as commonly understood by the skilled artisan. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually prepared in a laboratory by solid phase chemical synthesis and then purified. When referring to the sequence of an oligonucleotide, reference is made to the core base portion of the covalently linked nucleotides or nucleosides or their modified sequence or order. The oligonucleotide of the present invention is artificial, and is chemically synthesized, and is generally purified or separated. The oligonucleotide of the present invention may comprise one or more modified nucleosides or nucleotides.

[0048] Antisense Oligonucleotides

[0049] As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing with a target nucleic acid, particularly with a continuous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and are therefore not siRNA or shRNA. Preferably, the antisense oligonucleotides of the present invention are single-stranded. It should be understood that the single-stranded oligonucleotides of the present invention can form a hairpin or an intermolecular duplex structure (a duplex between two molecules of the same oligonucleotide), as long as the degree of self-complementarity within or between them is less than 50% across the total length of the oligonucleotide.

[0050] Continuous nucleotide sequence

[0051] The term "continuous nucleotide sequence" refers to an oligonucleotide region that is complementary to a target nucleic acid. This term is used interchangeably with the term "continuous core base sequence" and the term "oligonucleotide motif sequence" herein. In certain embodiments, all nucleotides of an oligonucleotide constitute a continuous nucleotide sequence. In certain embodiments, an oligonucleotide comprises a continuous nucleotide sequence such as a FG-F' gap polymer region, and may optionally comprise other nucleotides, such as a nucleotide joint region that can be used to join a functional group to a continuous nucleotide sequence. The nucleotide joint region may or may not be complementary to a target nucleic acid.

[0052] Nucleotides

[0053] Nucleotide is the component of oligonucleotide and polynucleotide, and for the purpose of the present invention, comprises naturally occurring and non-naturally occurring nucleotide.In fact, nucleotide such as DNA nucleotide and RNA nucleotide comprises ribose moiety, core base moiety and one or more phosphate groups (not present in nucleoside).Nucleoside and nucleotide can also be referred to as " unit " or " monomer " interchangeably.

[0054] Modified Nucleosides

[0055] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by introducing one or more modifications of a sugar moiety or (core) base moiety compared to an equivalent DNA or RNA nucleoside. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside can also be used interchangeably with the term "nucleoside analog" or modified "unit" or modified "monomer" in this article. Nucleosides with unmodified DNA sugar moieties or nucleosides with RNA sugar moieties are referred to as DNA or RNA nucleosides in this article. If Watson Crick base pairing is allowed, the nucleosides modified in the DNA nucleobase region or RNA nucleobase region are still usually referred to as DNA or RNA.

[0056] Modified internucleoside bonds

[0057] The term "modified internucleoside bond" is defined as a bond other than the phosphodiester (PO) bond that covalently links two nucleosides together as generally understood by the technician. Therefore, the oligonucleotide of the present invention may include a modified internucleoside bond. In certain embodiments, the modified internucleoside bond increases the nuclease resistance of the oligonucleotide compared to the phosphodiester bonding. For naturally occurring oligonucleotides, the internucleoside bond includes a phosphate group that produces a phosphodiester bond between adjacent nucleosides. The modified internucleoside bond is particularly useful for stabilizing oligonucleotides for in vivo use, and can be used in the DNA nucleoside or RNA nucleoside region (e.g., inside the gap region of a gap polymer oligonucleotide) in the oligonucleotide of the present invention and in the modified nucleoside region (e.g., region F and region F') to protect from nuclease shearing.

[0058] In one embodiment, the oligonucleotide comprises one or more internucleoside bonds modified by natural phosphodiester, such as one or more modified internucleoside bonds, which are, for example, more resistant to nuclease attacks. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (such as snake venom phosphodiesterase (SVPD)), which is well known in the art. The internucleoside bond that can enhance the nuclease resistance of the oligonucleotide is called an anti-nuclease internucleoside bond. In certain embodiments, at least 50% of the internucleoside bonds in an oligonucleotide or its continuous nucleotide sequence are modified, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% of the oligonucleotide or its continuous nucleotide sequence The internucleoside bond is an anti-nuclease internucleoside bond. In certain embodiments, all internucleoside bonds of an oligonucleotide or its continuous nucleotide sequence are anti-nuclease internucleoside bonds. It should be appreciated that in some embodiments, the nucleoside to which the oligonucleotide of the present invention is connected to a non-nucleotide functional group such as a conjugate can be a phosphodiester.

[0059] A preferred modified internucleoside linkage for use in the oligonucleotides of the invention is phosphorothioate.

[0060] Phosphorothioate internucleoside bonds are particularly beneficial due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In certain embodiments, at least 50% of the internucleoside bonds in an oligonucleotide or its continuous nucleotide sequence are phosphorothioates, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% of the internucleoside bonds in an oligonucleotide or its continuous nucleotide sequence are phosphorothioates. In certain embodiments, except for the trithiophosphate internucleoside bonds, all internucleoside bonds of an oligonucleotide or its continuous nucleotide sequence are phosphorothioates. In certain embodiments, except for the trithiophosphate bonds, the oligonucleotide of the present invention also comprises a thiophosphate internucleoside bond and at least one phosphodiester bond such as 2,3 or 4 phosphodiester bonds. In a gap polymer oligonucleotide, when present, the phosphodiester bond is suitably not located between the continuous DNA nucleosides in the gap region G.

[0061] Anti-nuclease bonds, such as phosphorothioate bonds, are particularly useful in regions of the oligonucleotide that are capable of recruiting nucleases when forming a duplex with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate bonds can also be used in non-nuclease recruitment regions and / or affinity enhancement regions, such as regions F and F' of a gapmer. In some embodiments, a gapmer oligonucleotide can contain one or more phosphodiester bonds in region F or F' or in both regions F and F', wherein the internucleoside bonds in region G can be entirely phosphorothioate.

[0062] Advantageously, all internucleoside bonds in the contiguous nucleotide sequence of the oligonucleotide or all internucleoside bonds of the oligonucleotide are phosphorothioate bonds.

[0063] It will be appreciated that, as disclosed in EP 2 742 135, antisense oligonucleotides may comprise other internucleoside linkages (besides phosphodiester and phosphorothioate), for example alkylphosphonate / methylphosphonate internucleosides, which according to EP 2 742 135 may for example otherwise be tolerated in the spacer region of DNA phosphorothioate.

[0064] Stereo-random phosphorothioate bond

[0065] Phosphorothioate bonds are phosphate bonds between nucleosides in which one of the non-bridging oxygens has been replaced by sulfur. The replacement of one of the non-bridging oxygens by sulfur introduces a chiral center and thereby inside a single phosphorothioate oligonucleotide, each phosphorothioate nucleoside bond will be in S (Sp) or R (Rp) stereoisomerism. This type of nucleoside bond is referred to as a "chiral nucleoside bond". In contrast, phosphodiester nucleoside bonds are achiral because they have two non-terminal oxygen atoms.

[0066] The nomenclature for the chirality of stereocenters is determined according to the standard Cahn-Ingold-Prelog rules (CIP priority rules) first published in the following literature: Cahn, RS; Ingold, CK; Prelog, V. (1966) "Specification of Molecular Chirality", Angewandte Chemie International Edition 5(4):385–415, doi:10.1002 / anie.196603851.

[0067] In standard oligonucleotide synthesis, the stereoselectivity of the coupling and subsequent sulfurization is not controlled. For this reason, the stereochemistry of each phosphorothioate internucleoside bond is randomly either Sp or Rp, and thus phosphorothioate oligonucleotides produced by traditional oligonucleotide synthesis can actually be in up to 2X There are 2 different phosphorothioate diastereomers, where X is the number of phosphorothioate internucleoside bonds. Such oligonucleotides are referred to herein as stereo-random phosphorothioate oligonucleotides and do not contain any stereo-defined internucleoside bonds. Thus, stereo-random phosphorothioate oligonucleotides are mixtures of diastereomers derived from non-stereochemically defined synthesis. In this context, a mixture is defined as having up to 2 X Different phosphorothioate diastereoisomers.

[0068] Stereospecific internucleoside bonds

[0069] A stereospecific internucleoside bond is a chiral internucleoside bond having a diastereomeric excess in one of its two diastereoisomeric forms, Rp or Sp.

[0070] It will be appreciated that stereoselective oligonucleotide synthesis methods used in the art typically provide at least about 90% or at least about 95% diastereoselectivity at each chiral internucleoside bond, and thus up to about 10%, such as about 5%, of the oligonucleotide molecules may have diastereomeric alternative forms.

[0071] In some embodiments, the diastereomeric ratio of each stereospecific chiral internucleoside bond is at least about 90: 10. In some embodiments, the diastereomeric ratio of each chiral internucleoside bond is at least about 95:5.

[0072] A stereospecific phosphorothioate bond is a specific example of a stereospecific internucleoside bond.

[0073] Stereospecific phosphorothioate bond

[0074] A stereospecific phosphorothioate bond is one that has a diastereomeric excess in one of its two diastereoisomeric forms, Rp or Sp.

[0075] The Rp and Sp configurations of phosphorothioate internucleoside bonds are shown below

[0076]

[0077] The 3'R group represents the 3' position of the adjacent nucleoside (5' nucleoside), and the 5'R group represents the 5' position of the adjacent nucleoside (3' nucleoside).

[0078] Herein, an Rp internucleoside linkage may also be represented as srP, and an Sp internucleoside linkage may be represented as ssP.

[0079] In a particular embodiment, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 90:10 or at least 95:5.

[0080] In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 97: 3. In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 98: 2. In some embodiments, the diastereomeric ratio of each stereospecific phosphorothioate bond is at least about 99: 1.

[0081] In some embodiments, the stereospecific internucleoside linkages have the same diastereomeric form (Rp or Sp) in at least 97%, such as at least 98%, such as at least 99%, or (substantially) all of the oligonucleotide molecules present in a population of oligonucleotide molecules.

[0082] The diastereomeric purity can be measured in a model system with only an achiral skeleton (i.e., a phosphodiester). The diastereomeric purity of each monomer can be measured by, for example, coupling a monomer with a stereospecific internucleoside bond to the following model system "5't-po-t-po-t-po 3'". The result of the measurement will be given subsequently: 5'DMTr-t-srp-t-po-t-po-t-po 3' or 5'DMTr-t-ssp-t-po-t-po-t-po 3' that can be separated using HPLC. The diastereomeric purity is determined by integrating the UV signals from two possible diastereomers and obtaining the ratio of these diastereomers (e.g., 98:2, 99:1, or>99:1).

[0083] It will be appreciated that the diastereomeric purity of a particular single diastereomer (single stereospecific oligonucleotide molecule) will vary with the coupling selectivity of the stereocenter defined at each internucleoside position and the number of stereospecific internucleoside bonds to be introduced. By way of example, if the coupling selectivity at each position is 97%, the resulting purity of a stereospecific oligonucleotide having 15 stereospecific internucleoside bonds would be 0.97. 15 , i.e. 63% of the desired diastereomer compared to 37% of the other diastereomer. The purity of a defined diastereomer can be improved after synthesis by purification, for example by HPLC, such as ion exchange chromatography or reverse phase chromatography.

[0084] In some embodiments, stereospecific oligonucleotides refer to a population of oligonucleotides in which at least about 40%, such as at least about 50%, of the population of oligonucleotides are of the desired diastereomer.

[0085] In other words, in some embodiments, stereospecific oligonucleotides refer to a population of oligonucleotides wherein at least about 40%, such as at least about 50%, of the population of oligonucleotides consists of a desired (specific) stereospecific internucleoside linkage motif (also referred to as a stereospecific motif).

[0086] For stereospecific oligonucleotides containing both stereorandom and stereospecific internucleoside stereocenters, the purity of the stereospecific oligonucleotide is determined by reference to the % of the oligonucleotide population that retains the desired stereospecific internucleoside linkage motif, ignoring the stereorandom linkages in the calculation.

[0087] Nucleobase

[0088] The term core base includes 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 the present invention, the term core base also encompasses modified core bases, which may be different from naturally occurring core bases, but work during nucleic acid hybridization. In this context, "core base" refers to naturally occurring core bases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring variants. Such variants are, for example, described 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.

[0089] In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-mercapto-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazole-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0090] The nucleobase moiety may be represented by the letter code for each corresponding nucleobase, such as A, T, G, C or U, wherein each letter may optionally include a modified nucleobase having an equivalent function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides may be used.

[0091] Modified oligonucleotides

[0092] The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar modified nucleosides and / or modified internucleoside linkages and / or the presence of conjugate moieties. The term chimeric "oligonucleotide" is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.

[0093] Stereospecific oligonucleotides

[0094] A stereospecific oligonucleotide is an oligonucleotide in which at least one internucleoside linkage is a stereospecific internucleoside linkage.

[0095] A stereospecific phosphorothioate oligonucleotide is an oligonucleotide in which at least one internucleoside linkage is a stereospecific phosphorothioate internucleoside linkage.

[0096] Complementarity

[0097] The term "complementarity" describes the ability of the Watson-Crick base pairing of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It should be understood that oligonucleotides may include nucleosides with modified nucleobases, for example, 5-methylcytosine is often used instead of cytosine, and therefore, the term complementarity encompasses Watson Crick base pairing between unmodified and modified nucleobases (see, for example, 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).

[0098] As used herein, the term "% complementary" refers to the ratio of nucleotides within a continuous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) wherein at a given position, the nucleotide is complementary to (i.e., forms a Watson Crick base pair with) a continuous nucleotide sequence at a given position of a separate nucleic acid molecule (e.g., a target nucleic acid). The percentage is calculated by counting the number of aligned bases that form a pair between the two sequences (when aligned from 3'-5' to the target sequence 5'-3' and the oligonucleotide sequence), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such comparisons, a nucleobase / nucleotide that is not aligned (forming a base pair) is referred to as a mismatch. Preferably, insertions and deletions are not allowed when calculating the complementarity % of a continuous nucleotide sequence.

[0099] The term "fully complementary" refers to 100% complementarity.

[0100] consistency

[0101] As used herein, the term "consistency" refers to the number of nucleotides in a continuous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) as a percentage, wherein at a given position, the nucleotide is identical to a continuous nucleotide sequence at a given position of a separate nucleic acid molecule (e.g., a target nucleic acid) (i.e., in terms of its ability to form Watson Crick base pairs with complementary nucleosides). The percentage is calculated in the following manner: count the number of identical aligned bases between the two sequences, divide by the total number of nucleotides in the oligonucleotide and multiply by 100. Consistency percentage = (match × 100) / length of the alignment region. Preferably, insertions and deletions are not allowed when calculating the complementarity % of continuous nucleotide sequences.

[0102] Hybridization

[0103] As used herein, the term "hybridization" should be understood as the formation of hydrogen bonds between base pairs on opposite strands to form two nucleic acid chains (e.g., an oligonucleotide and a target nucleic acid) in a duplex. The affinity of the binding between the two nucleic acid chains is the strength of the hybridization. The melting temperature (T m ), the melting temperature (Tm) is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions, T m It is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515–537). The standard state Gibbs free energy ΔG° is a more accurate expression of binding affinity and is related to the dissociation constant (K d ) multiplied by ΔG° = -RTln(K d), where R is the gas constant and T is the absolute temperature. Therefore, the very low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with the reaction in which the water concentration is 1M, the pH is 7 and the temperature is 37°C. The hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, and for a spontaneous reaction, ΔG° is less than zero. For example, ΔG° can be measured experimentally using the isothermal titration calorimetry (ITC) method as described in Hansen et al. (1965, Chem. Comm. 36–38) and Holdgate et al. (2005, Drug Discov Today). Those skilled in the art will know that commercial equipment can be used to measure ΔG°. ΔG can also be estimated numerically by using the nearest neighbor model as described by SantaLucia (1998, Proc Natl Acad Sci USA.95:1460–1465), appropriately using the thermodynamic parameters described by Sugimoto et al. (1995, Biochemistry 34:11211–11216) and McTigue et al. (2004, Biochemistry 43:5388–5405). To obtain the possibility of regulating its expected nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize with the target nucleic acid at a ΔG° estimate of less than -10 kcal for oligonucleotides of 10-30 nucleotides in length. In some embodiments, the degree or intensity of hybridization is measured according to the standard state Gibbs free energy ΔG°. The oligonucleotide can hybridize with the target nucleic acid at a ΔG° estimate of less than 10 kcal, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal for oligonucleotides of 8-30 nucleotides in length. In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated AG° of -10 kcal to -60 kcal, such as -12 kcal to -40 kcal, such as -15 kcal to -30 kcal, or -16 kcal to -27 kcal, such as -18 kcal to -25 kcal.

[0104] Sugar modification

[0105] The oligomers of the invention may comprise one or more nucleosides having a modified sugar moiety (ie, a modified sugar moiety) when compared to the ribose sugar moiety present in DNA and RNA.

[0106] A number of nucleosides with modifications of the ribose sugar moiety have been generated, primarily with the goal of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance.

[0107] Such modifications include the following modifications, wherein the ribose ring structure is modified, for example, by replacing with a hexose ring (HNA) or a bicyclic ring (LNA) generally having a diradical bridge between C2 and C4 carbons on the ribose ring or a non-connected ribose ring (e.g., UNA) generally lacking a bond between C2 carbon and C3 carbon. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, for example, in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0108] Sugar modifications also include modifications made by changing substituents on the ribose ring to groups other than hydrogen or the 2'-OH group naturally present in DNA and RNA nucleosides. Sugar modifications may, for example, introduce substituents at the 2', 3', 4' or 5' positions.

[0109] 2' Sugar-Modified Nucleosides.

[0110] A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or contains a 2' linked diradical capable of forming a bridge between the 2' carbon and the second carbon atom in the ribose ring, such as an LNA (2'-4' diradical bridged) nucleoside.

[0111] In fact, people have spent a lot of effort to develop 2' substituted nucleosides, and found that many 2' substituted nucleosides have beneficial properties after being incorporated into oligonucleotides. For example, 2' modified sugars can provide enhanced binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA nucleosides. Other examples can be found, for example: Freier and Altmann, Nucl. Acid Res., 1997, 25, 4429-4443; Uhlmann, Curr. Opinion in Drug Development, 2000, 3 (2), 293-213; and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are schematic diagrams of some 2' substituted modified nucleosides.

[0112]

[0113] With respect to the present invention, 2' substitution does not include 2' bridged molecules such as LNA.

[0114] Locked Nucleic Acid Nucleosides (LNA Nucleosides)

[0115] An "LNA nucleoside" is a 2'-modified nucleoside comprising a double radical of C2' and C4' linking the ribose ring of the nucleoside (also referred to as a "2'-4' bridge"), which constrains or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). When LNA is incorporated into an oligonucleotide complementary to an RNA or DNA molecule, the locking of the ribose conformation is associated with an enhanced affinity for hybridization (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0116] Non-limiting exemplary LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 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).

[0117] The 2'-4' bridge contains 2 to 4 bridging atoms and is in particular of the formula -XY-, wherein X is attached to C4' and Y is attached to C2',

[0118] in

[0119] X is oxygen, sulfur, -CR a R b -、-C(R a )=C(R b )-、-C(=CR a R b )-、-C(R a )=N-、-Si(R a )2-、-SO2-、-NR a -;-O-NR a -、-NR a -O-, -C(=J)-, Se, -O-NR a -、-NR a -

[0120] CR a R b -、-N(R a )-O-or-O-CR a R b -;

[0121] Y is oxygen, sulfur, -(CR a R b ) n -、-CR a R b -O-CR a R b -、-C(R a )=C(R b )-、-C(R a )=N-、

[0122] -Si(R a )2-、-SO2-、-NR a -, -C(=J)-, Se, -O-NR a -、-NR a -CR a R b -、

[0123] -N(R a )-O-or-O-CR a R b -;

[0124] Provided that -XY- is not -OO-, Si(R a )2-Si(R a )2-、-SO2-SO2-、-C(R a )=C(R b )-

[0125] C(R a )=C(R b )、-C(R a )=NC(R a )=N-、-C(R a )=NC(R a )=C(R b ),

[0126] -C(R a )=C(R b )-C(R a )=N- or -Se-Se-;

[0127] J is oxygen, sulfur, =CH2 or =N(R a );

[0128] R a and R b independently selected from hydrogen, halogen, hydroxyl, cyano, sulfhydryl, alkyl, substituted alkane

[0129] alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclic, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, ureido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, alkylthiosulfide, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC(=X a )R c 、-OC(=X a )NR c R d and-

[0130] NR e C(=X a )NR c R d ;

[0131] or two R a and R b together form an optionally substituted methylene group;

[0132] or two R a and R b Together with the carbon atom to which they are attached, they form a cycloalkyl or halocycloalkane

[0133] The radical contains only one carbon atom of -XY-;

[0134] wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy and substituted methylene are alkyl, alkenyl, alkynyl and methylene substituted with 1 to 3 substituents independently selected from halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, methyl

[0135] acyl, heterocyclyl, aryl and heteroaryl;

[0136] X a is oxygen, sulfur or -NR c ;

[0137] R c , R d and R e are independently selected from hydrogen and alkyl; and

[0138] n is 1, 2 or 3.

[0139] In another specific embodiment of the present invention, X is oxygen, sulfur, -NR a -、-CR a R b -or-C(=CR a R b )-, in particular oxygen, sulfur, -NH-, -CH2- or -C(=CH2)-, more in particular oxygen.

[0140] In another specific embodiment of the present invention, Y is -CR a R b -、-CR a R b -CR a R b -or-CR a R b- CR a R b- CR a R b -, in particular -CH2-CHCH3-, -CHCH3-CH2-, -CH2-CH2- or -CH2-CH2-CH2-.

[0141] In a specific embodiment of the present invention, -XY- is -O-(CR a R b ) n -、-S-CR a R b -、-N(R a )CR a R b -、-CR a R b -CR a R b -、-O-CR a R b -O-CR a R b -、-CR a R b -O-CR a R b -、-C(=CR a R b )-CR a R b -、-N(R a )CR a R b -、-ON(R a )-CR a R b -or-N(R a)-O-CR a R b -.

[0142] In a specific embodiment of the present invention, R a and R b Independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl and alkoxyalkyl, in particular hydrogen, halogen, alkyl and alkoxyalkyl.

[0143] In another specific embodiment of the present invention, R a and R b Independently selected from the group consisting of: hydrogen, fluorine, hydroxyl, methyl and -CH2-O-CH3, in particular hydrogen, fluorine, methyl and -CH2-O-CH3.

[0144] Advantageously, the R of -XY- a and R b One of them is as defined above and the other is hydrogen.

[0145] In another specific embodiment of the present invention, R a is hydrogen or alkyl, in particular hydrogen or methyl.

[0146] In another specific embodiment of the present invention, R b is hydrogen or alkyl, in particular hydrogen or methyl.

[0147] In a specific embodiment of the present invention, R a and R b One or both of them are hydrogen.

[0148] In a specific embodiment of the present invention, R a and R b Only one of them is hydrogen.

[0149] In a specific embodiment of the present invention, R a and R b One of them is methyl and the other is hydrogen.

[0150] In a specific embodiment of the present invention, R a and R b Both are methyl groups.

[0151] In a specific embodiment of the present invention, -XY- is -O-CH2-, -S-CH2-, -S-CH(CH3)-, -NH-CH2-, -O-CH2CH2-, -O-CH(CH2-O-CH3)-, -O-CH(CH2CH3)-, -O-CH(CH3)-, -O-CH 2-O-CH2-, -O-CH2-O-CH2-, -CH2-O-CH2-, -C(=CH2)CH2-, -C(=CH2)CH(CH3)-, -N(OCH3)CH2- or -N(CH3)CH2-;

[0152] In a specific embodiment of the present invention, -XY- is -O-CR a R b -'R a and R b Independently selected from the group consisting of hydrogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl and -CH2-O-CH3.

[0153] In a specific embodiment, -XY- is -O-CH2- or -O-CH(CH3)-, specifically -O-CH2-.

[0154] The 2'-4' bridge can be located below the plane of the ribose ring (β-D-configuration), or above the plane of the ring (α-L-configuration), as shown in formula (A) and formula (B), respectively.

[0155] The LNA nucleosides according to the invention are in particular of formula (B1) or (B2)

[0156]

[0157] in

[0158] W is oxygen, sulfur, -N(R a )-or-CR a R b -, in particular oxygen;

[0159] B is a nucleobase or a modified nucleobase;

[0160] Z is an internucleoside bond to an adjacent nucleoside or a 5' terminal group;

[0161] Z* is an internucleoside bond to an adjacent nucleoside or a 3' terminal group;

[0162] R 1 , R 2 , R 3 , R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, haloalkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkoxyalkyl, azido, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, and aryl; and

[0163] X, Y, R a and R b As defined above.

[0164] In one embodiment, in the definition of -XY-, R a is hydrogen or alkyl, in particular hydrogen or methyl. In another specific embodiment, in the definition of -XY-, R b is hydrogen or alkyl, in particular hydrogen or methyl. In another specific embodiment, in the definition of -XY-, R a and R b In one embodiment, in the definition of -XY-, R a and R b In one embodiment, in the definition of -XY-, R a and R b In one embodiment, in the definition of -XY-, R a and R b Both are methyl groups.

[0165] In yet another specific embodiment, in the definition of X, R a is hydrogen or alkyl, in particular hydrogen or methyl. In another specific embodiment, in the definition of X, R b is hydrogen or alkyl, in particular hydrogen or methyl. In a specific embodiment, in the definition of X, R a and R b In one embodiment, in the definition of X, R a and R b In one embodiment, in the definition of X, R a and R b One of them is methyl and the other is hydrogen. In one embodiment, in the definition of X, R a and R b Both are methyl groups.

[0166] In yet another specific embodiment, in the definition of Y, R a is hydrogen or alkyl, in particular hydrogen or methyl. In another specific embodiment, in the definition of Y, R b is hydrogen or alkyl, in particular hydrogen or methyl. In a specific embodiment, in the definition of Y, R a and R b In one embodiment, in the definition of Y, R a and R b In one embodiment, in the definition of Y, R a and R b One of them is methyl and the other is hydrogen. In one embodiment, in the definition of Y, Ra and R b Both are methyl groups.

[0167] In a specific embodiment of the present invention, R 1 , R 2 , R 3 , R 5 and R 5* Independently selected from hydrogen and alkyl, in particular hydrogen and methyl.

[0168] In yet another particularly advantageous embodiment of the present invention, R 1 , R 2 , R 3 , R 5 and R 5* Both are hydrogen.

[0169] In another specific embodiment of the present invention, R 1 , R 2 , R 3 At the same time, both are hydrogen, R 5 and R 5* One of is hydrogen and the other is as defined above, particularly is alkyl, more particularly is methyl.

[0170] In a specific embodiment of the present invention, R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, alkoxyalkyl and azido, in particular selected from hydrogen, fluorine, methyl, methoxyethyl and azido. In particular, in an advantageous embodiment of the present invention, R 5 and R 5* one of them is hydrogen and the other is alkyl, especially methyl, halogen, especially fluorine, alkoxyalkyl, especially methoxyethyl or azido; or R 5 and R 5* At the same time, all are hydrogen or halogen, in particular, all are hydrogen or fluorine. In such specific embodiments, W may advantageously be oxygen, and -XY- is advantageously -O-CH2-.

[0171] In a specific embodiment of the present invention, -XY- is -O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352 and WO 2004 / 046160, all of which are hereby incorporated by reference, and include beta-D-oxy LNA nucleosides and alpha-L-oxy LNA nucleosides generally known in the art.

[0172] In another specific embodiment of the present invention, -XY- is -S-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such thio-LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are hereby incorporated by reference.

[0173] In another specific embodiment of the present invention, -XY- is -NH-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such amino LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are hereby incorporated by reference.

[0174] In another specific embodiment of the present invention, -XY- is -O-CH2CH2- or -OCH2CH2CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such LNA nucleosides are disclosed in WO 00 / 047599 and Morita et al. (Bioorganic & Med. Chem. Lett. 12, 73-76), which are hereby incorporated by reference, and include 2'-0-4'C-ethylene bridged nucleic acids (ENA) generally known in the art.

[0175] In another specific embodiment of the present invention, -XY- is -O-CH2-, W is oxygen, R 1 , R 2 , R 3 At the same time, both are hydrogen, R 5 and R 5* One of the 5'-substituted LNA nucleosides is hydrogen and the other is not hydrogen, such as an alkyl group (eg, methyl). Such 5'-substituted LNA nucleosides are disclosed in WO 2007 / 134181, which is hereby incorporated by reference.

[0176] In another specific embodiment of the present invention, -XY- is -O-CR a R b -, where R a and R bOne or both of them are not hydrogen, in particular alkyl such as methyl, W is oxygen, R 1 , R 2 , R 3 At the same time, both are hydrogen, R 5 and R 5* One of the is hydrogen and the other is not hydrogen, in particular is an alkyl group (eg methyl). Such doubly modified LNA nucleosides are disclosed in WO 2010 / 077578, which is hereby incorporated herein by reference.

[0177] In another specific embodiment of the present invention, -XY- is -O-CHR a -, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such 6' substituted LNA nucleosides are disclosed in WO 2010 / 036698 and WO 2007 / 090071, which are hereby incorporated by reference. In such 6' substituted LNA nucleosides, R a In particular C1-C6 alkyl such as methyl.

[0178] In another specific embodiment of the present invention, -XY- is -O-CH(CH2-O-CH3)- ("2'O-methoxyethyl bicyclic nucleic acid", Seth et al., J. Org. Chem. 2010, Vol. 75, No. 5, pp. 1569-81).

[0179] In another specific embodiment of the present invention, -XY- is -O-CH(CH2CH3)-;

[0180] In another specific embodiment of the present invention, -XY- is -O-CH(CH2-O-CH3)-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* At the same time, both are hydrogen. In the art, such LNA nucleosides are also known as cyclic MOE (cMOE) and are disclosed in WO2007 / 090071.

[0181] In another specific embodiment of the present invention, -XY- is -O-CH(CH3)- ("2'O-ethyl bicyclic nucleic acid", Seth et al., J. Org. Chem. 2010, Vol. 75, No. 5, pp. 1569-81).

[0182] In another specific embodiment of the present invention, -XY- is -O-CH 2-O—CH 2 — (Seth et al., J. Org. Chem 2010, supra).

[0183] In another specific embodiment of the present invention, -XY- is -O-CH(CH3)-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* Such 6'-methyl LNA nucleosides are also referred to in the art as cET nucleosides and may be (S)-cET or (R)-cET diastereomers as disclosed in WO 2007 / 090071 (β-D) and WO 2010 / 036698 (α-L), both of which are hereby incorporated by reference.

[0184] In another specific embodiment of the present invention, -XY- is -O-CR a R b -, where R a and R b are not hydrogen, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* In one embodiment, R a and R b At the same time both are alkyl, in particular at the same time both are methyl. Such 6'-disubstituted LNA nucleosides are disclosed in WO 2009 / 006478, which is hereby incorporated herein by reference.

[0185] In another specific embodiment of the present invention, -XY- is -S-CHR a -, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* and are both hydrogen. Such 6'-substituted thio-LNA nucleosides are disclosed in WO 2011 / 156202, which is hereby incorporated by reference herein. In a specific embodiment of such 6'-substituted thio-LNA, R a is an alkyl group, especially a methyl group.

[0186] In a specific embodiment of the present invention, -XY- is -C(=CH2)C(R a R b )-、-C(=CHF)C(R a R b )-or-C(=CF2)C(R a Rb )-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* At the same time, they are all hydrogen. a and R b R is advantageously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluorine and methoxymethyl. a and R b In particular, both are hydrogen or methyl, or R a and R b One of the is hydrogen and the other is methyl. Such vinyl carbon LNA nucleosides are disclosed in WO 2008 / 154401 and WO 2009 / 067647, which are hereby incorporated by reference herein.

[0187] In a specific embodiment of the present invention, -XY- is -N(OR a )-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* In one embodiment, R a Such LNA nucleosides are also known as N-substituted LNAs and are disclosed in WO 2008 / 150729, which is hereby incorporated by reference herein.

[0188] In a specific embodiment of the present invention, -XY- is -ON(R a )-、-N(R a )-O-、-NR a -CR a R b -CR a R b -or-NR a -CR a R b -, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* At the same time, they are all hydrogen. a and R b Advantageously, R is independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluorine and methoxymethyl. a is an alkyl group such as a methyl group, R bis hydrogen or methyl, in particular hydrogen (Seth et al., J. Org. Chem 2010, supra).

[0189] In a specific embodiment of the present invention, -XY- is -ON(CH3)- (Seth et al., J. Org. Chem 2010, supra).

[0190] In a specific embodiment of the present invention, R 5 and R 5* In another specific embodiment of the present invention, R 5 and R 5* One of is hydrogen and the other is an alkyl group such as methyl. In such embodiments, R 1 , R 2 and R 3 may be especially hydrogen, and -XY- may be especially -O-CH2- or -O-CHC(R a )3- such as -O-CH(CH3)-.

[0191] In a specific embodiment of the present invention, -XY- is -CR a R b -O-CR a R b - such as -CH2-O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* In this embodiment, R a It may be particularly an alkyl group such as a methyl group, R b is hydrogen or methyl, in particular hydrogen. Such LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO 2013 / 036868, which is hereby incorporated herein by reference.

[0192] In a specific embodiment of the present invention, -XY- is -O-CR a R b -O-CR a R b - such as -O-CH2-O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5* At the same time, they are all hydrogen. a and R b Advantageously, R is independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluorine and methoxymethyl.a It may be particularly an alkyl group such as a methyl group, R b is hydrogen or methyl, in particular hydrogen. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuok et al., Nucleic Acids Research 2009, 37(4), 1225-1238, which is hereby incorporated herein by reference.

[0193] It will be appreciated that, unless otherwise stated, LNA nucleosides may be in either the β-D or α-L stereoisomeric form.

[0194] Specific examples of LNA nucleosides of the invention are shown in Scheme 1 (wherein B is as defined above).

[0195] Solution 1

[0196]

[0197]

[0198]

[0199] Particular LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy-LNA such as (S)-6'-methyl-β-D-oxy-LNA ((S)-cET) and ENA.

[0200] RNase H activity and recruitment

[0201] The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when forming a duplex with a complementary RNA molecule. WO01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. An oligonucleotide is generally considered to be able to recruit RNase H if the following occurs: when a complementary target nucleic acid sequence is provided, it has an initial rate, as measured in pmol / l / min, that is at least 5% such as at least 10% or more than 20% of the initial rate determined using the following oligonucleotide and using the method provided in Examples 91-95 of WO01 / 23613 (hereby incorporated herein by reference), wherein the oligonucleotide has the same base sequence as the oligonucleotide to be modified, but contains only DNA monomers, and all monomers of the oligonucleotide are phosphorothioate bonds. For determining RNase H activity, recombinant human RNase H1 can be obtained from Lubio Science GmbH (Lucerne, Switzerland).

[0202] Gapmer

[0203] The antisense oligonucleotide of the present invention or its continuous nucleotide sequence can be a gapmer. Antisense gapmers are commonly used to inhibit target nucleic acids by degradation mediated by ribonuclease H. Gapmer oligonucleotides contain at least three unique structural regions, 5'-flank, gap and 3'-flank, which are FG-F' in the '5->3' direction. The "gap" region (G) contains a section of continuous DNA nucleotides that enable the oligonucleotide to recruit ribonuclease H. The gap region is flanked by a 5' flank region (F) containing one or more sugar-modified nucleosides, advantageously sugar-modified high-affinity nucleosides, and a 3' flank region (F') containing one or more sugar-modified nucleosides, advantageously sugar-modified high-affinity nucleosides. One or more sugar-modified nucleosides in region F and region F' enhance the affinity of the oligonucleotide to the target nucleic acid (i.e., sugar-modified nucleosides that enhance affinity). In some embodiments, one or more sugar-modified nucleosides in region F and region F' are 2' sugar-modified nucleosides such as high affinity 2' sugar modifications, such as independently selected from LNA and 2'-MOE.

[0204] In the gapmer design, the 5' and 3' most terminal nucleosides of the gap region are DNA nucleosides, and sugar-modified nucleosides are present adjacent to the 5' (F) region or the 3' (F') region, respectively. These flanking regions can be further defined by having at least one sugar-modified nucleoside at the end farthest from the gap region (i.e., at the 5' end of the 5' flanking region and at the 3' end of the 3' flanking region).

[0205] The region FG-F' forms a continuous nucleotide sequence. The antisense oligonucleotide of the present invention or its continuous nucleotide sequence may comprise a gapmer region of the formula FG-F'.

[0206] The overall length of the gapmer design FG-F' can be, for example, 12 to 32 nucleosides, such as 13 to 24 nucleosides, such as 14 to 22 nucleosides, such as 14 to 17 nucleosides, such as 16 to 18 nucleosides.

[0207] By way of example, the gapmer oligonucleotide of the invention may be represented by the following formula:

[0208] F 1-8 -G 5-16 -F' 1-8 , such as

[0209] F 1-8 -G 7-16 -F' 2-8

[0210] Provided that the overall length of the gapmer region FG-F' is at least 12, such as at least 14, nucleotides.

[0211] Regions F, G and F' are further defined below and may be incorporated into the formula FG-F'.

[0212] Gapmer-Region G

[0213] The region G (gap region) of the gap polymer is a nucleoside region that enables oligonucleotides to recruit ribonuclease H such as human ribonuclease H1, which is generally a DNA nucleoside. RNase H is a cellular enzyme that recognizes duplexes between DNA and RNA and enzymatically cleaves RNA molecules. Suitable gap polymers can have a gap region (G) of at least 5 or 6 continuous DNA nucleosides, such as 5-16 continuous DNA nucleosides, such as 6-15 continuous DNA nucleosides, such as 7-14 continuous DNA nucleosides, such as 8-12 continuous DNA nucleotides, such as 8-12 continuous DNA nucleosides. In certain embodiments, the gap region G can be composed of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 continuous DNA nucleosides. In some instances, the cytosine (C) DNA in the gap region can be methylated, and such residues are labeled as 5-methyl-cytosine ( me C or e instead of c). If the cg dinucleotide is present in the gap, methylation of the cytosine DNA in the gap is beneficial to reduce potential toxicity, and this modification has no significant effect on the efficacy of the oligonucleotide.

[0214] In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside bonds in the gap are phosphorothioate bonds.

[0215] Although conventional gapmers have a DNA gap region, there are numerous examples of modified nucleosides that allow for the recruitment of ribonuclease H when the modified nucleosides are used inside the gap region. Modified nucleosides that are capable of recruiting ribonuclease H when the modified nucleosides are incorporated inside the gap region have been reported, including, for example, α-L-LNA, C4' alkylated DNA (as described in PCT / EP2009 / 050349 and Vester et al. (Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300), both of which are incorporated herein by reference), nucleosides derived from arabinose such as ANA and 2'F-ANA (Mangosr et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al. (Mol. Biosyst., 2009, 10, 1039), which is incorporated herein by reference). UNA is a non-locked nucleic acid, generally in which the bond between C2 and C3 of the ribose has been removed, forming a non-locked "sugar" residue. The modified nucleosides used in such gapmers can be nucleosides that adopt a 2' internal (DNA-like) structure when introduced into the gap region, i.e., modifications that allow the recruitment of RNase H. In some embodiments, the DNA gap region (G) described herein may optionally contain 1 to 3 sugar-modified nucleosides that adopt a 2' internal (DNA-like) structure when introduced into the gap region.

[0216] Area G - "Gap Busters"

[0217] Alternatively, there are numerous reports of inserting modified nucleosides that confer a 3' endo conformation into the gap region of a gapmer while retaining some RNase H activity. Such gapmers with a gap region comprising one or more 3' endo modified nucleosides are referred to as "gap-breakers" or "gap-disrupted" gapmers, see, for example, WO2013 / 022984. Gap-breaker oligonucleotides retain sufficient DNA nucleoside regions inside the gap region to allow recruitment of RNase H. The ability of gap-breaker oligonucleotides to recruit RNase H is generally sequence-specific or even compound-specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses "gap-breaker" oligonucleotides that recruit RNase H, which in some cases provide more specific target RNA cleavage. The modified nucleosides used inside the gap region of the gap breaker oligonucleotide can be, for example, modified nucleosides that confer a 3' endo conformation such as 2'-O-methyl (OMe) or 2'-O-MOE (MOE) nucleosides or β-D LNA nucleosides (the bridge between C2' and C4' of the ribose sugar ring of the nucleoside is in the β conformation) such as β-D-oxy LNA or ScET nucleosides.

[0218] As with the gapmer comprising region G described above, the gap region of the gapbreaker gapmer or the gap-blocking gapmer has a DNA nucleoside at the 5' end of the gap (adjacent to the 3' nucleoside of region F) and a DNA nucleoside at the 3' end of the gap (adjacent to the 5' nucleoside of region F'). The gapmer comprising the gap-blocking gap generally retains a region of at least 3 or 4 consecutive DNA nucleosides at the 5' end or 3' end of the gap region.

[0219] Exemplary designs of gap breaker oligonucleotides include

[0220] F 1-8 -[D 3-4 -E1-D 3-4 ]-F' 1-8

[0221] F 1-8 -[D 1-4 -E1-D 3-4 ]-F' 1-8

[0222] F 1-8 -[D 3-4 -E1-D 1-4 ]-F' 1-8

[0223] The region G is in brackets [D n -Er -D m ], D is a contiguous DNA nucleoside sequence, E is a modified nucleoside (a gap breaker or a gap-blocking nucleoside), and F and F' are flanking regions as defined herein, and provided that the overall length of the gapmer region FG-F' is at least 12, such as at least 14, nucleotides.

[0224] In some embodiments, the region G of the gapmer that interferes with the gap comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be continuous or may be optionally interspersed with one or more modified nucleosides, provided that the gap region G is capable of mediating RNase H recruitment.

[0225] Gapmer-flanking regions F and F'

[0226] Region F is immediately adjacent to the 5' DNA nucleoside of region G. The 3'-most nucleoside of region F is a sugar-modified nucleoside, such as a high affinity sugar-modified nucleoside, for example a 2'-substituted nucleoside such as a MOE nucleoside or a LNA nucleoside.

[0227] Region F' is immediately adjacent to the 3' DNA nucleoside of region G. The 5' most terminal nucleoside of region F' is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2' substituted nucleoside such as a MOE nucleoside or a LNA nucleoside.

[0228] The length of region F is 1-8 consecutive nucleotides, such as 2-6, such as 3-4 consecutive nucleotides. Advantageously, the 5'-terminal nucleoside of region F is a sugar-modified nucleoside. In some embodiments, the two 5'-terminal nucleosides of region F are sugar-modified nucleosides. In some embodiments, the 5'-terminal nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-terminal nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-terminal nucleosides of region F are 2'-substituted nucleosides such as two 3'MOE nucleosides. In some embodiments, the 5'-terminal nucleosides of region F are 2'-substituted nucleosides such as MOE nucleosides.

[0229] The length of region F' is 2-8 consecutive nucleotides, such as 3-6 such as 4-5 consecutive nucleotides. Advantageously, in an embodiment, the 3'-most nucleoside of region F' is a sugar-modified nucleoside. In some embodiments, the two 3'-most nucleosides of region F' are sugar-modified nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are 2'-substituted nucleosides such as two 3'MOE nucleosides. In some embodiments, the 3'-most nucleosides of region F' are 2'-substituted nucleosides such as MOE nucleosides.

[0230] It should be noted that when region F and / or region F' has a length of 1, it is advantageously an LNA nucleoside.

[0231] In some embodiments, regions F and F' independently consist of or comprise a continuous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F may be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0232] In some embodiments, region F and region F' independently comprise LNA and 2' substituted modified nucleosides (mixed flank design).

[0233] In some embodiments, region F and region F' consist of only one type of sugar-modified nucleosides such as only MOE or only β-D-oxy LNA or only ScET. Such designs are also referred to as unified flank designs or unified gapmer designs.

[0234] In some embodiments, all nucleosides in region F or region F' or F and F' are LNA nucleosides, such as independently selected from β-D-oxy LNA, ENA or ScET nucleosides. In some embodiments, region F consists of 1-5, such as 2-4, such as 3-4, such as 1, 2, 3, 4 or 5 consecutive LNA nucleosides. In some embodiments, all nucleosides in region F and region F' are β-D-oxy LNA nucleosides.

[0235] In some embodiments, all nucleosides of region F or region F' or F and F' are 2' substituted nucleosides such as OMe or MOE nucleosides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7 or 8 consecutive OMe or MOE nucleosides. In some embodiments, only one of the flanking regions may consist of 2' substituted nucleosides such as OMe or MOE nucleosides. In some embodiments, the 5' (F) flanking region consists of 2' substituted nucleosides such as OMe or MOE nucleosides, and the 3' (F') flanking region comprises at least one LNA nucleoside such as β-D-oxy LNA nucleosides or cET nucleosides. In some embodiments, the 3' (F') flanking region consists of 2' substituted nucleosides such as OMe or MOE nucleosides, and the 5' (F) flanking region comprises at least one LNA nucleoside such as β-D-oxy LNA nucleosides or cET nucleosides.

[0236] In some embodiments, all modified nucleosides of region F and region F' are LNA nucleosides, such as independently selected from β-D-oxy LNA, ENA or ScET nucleosides, wherein region F or region F' or F and F' may optionally comprise DNA nucleosides (alternating flanks, see the definitions of these regions for more information). In some embodiments, all modified nucleosides of region F and region F' are β-D-oxy LNA nucleosides, wherein region F or region F' or F and F' may optionally comprise DNA nucleosides (alternating flanks, see the definitions of these regions for more information).

[0237] In some embodiments, the 5'-most nucleoside and the 3'-most nucleoside of region F and region F' are LNA nucleosides such as β-D-oxy LNA nucleosides or ScET nucleosides.

[0238] In some embodiments, the internucleoside bond between region F and region G is a phosphorothioate internucleoside bond. In some embodiments, the internucleoside bond between region F' and region G is a phosphorothioate internucleoside bond. In some embodiments, the internucleoside bond between nucleosides in region F or F' and between F and F' is a phosphorothioate internucleoside bond.

[0239] Other gapmer designs are disclosed in WO 2004 / 046160, WO 2007 / 146511 and WO 2008 / 113832, which are hereby incorporated by reference.

[0240] LNA gapmer

[0241] An LNA gapmer is one in which either or both of region F and region F' comprises or consists of an LNA nucleoside. A β-D-oxy gapmer is one in which either or both of region F and region F' comprises or consists of a β-D-oxy LNA nucleoside.

[0242] In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[Region G]-[LNA] 1-5 , wherein region G is as defined in the definition of gapmer region G.

[0243] MOE gapmer

[0244] A MOE gapmer is a gapmer in which region F and region F' consist of MOE nucleosides. In some embodiments, the MOE gapmer has the following design: [MOE] 1-8 -[Region G]-[MOE] 1-8 , such as [MOE] 2-7 -[Area G] 5-16 -[MOE] 2-7 , such as [MOE] 3-6 -[Region G]-[MOE] 3-6 , wherein region G is as defined in the definition of gapmer. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

[0245] Hybrid Flank Gapmer

[0246] The hybrid flanking gapmer is an LNA gapmer wherein one or both of regions F and F' comprise 2'-substituted nucleosides, such as 2'-substituted nucleosides independently selected from the group consisting of 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 such as MOE nucleosides. In some embodiments, wherein at least one of regions F and F' or both regions F and F' comprise 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, wherein at least one of regions F and F' or both regions F and F' comprise 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 region F and region F' may further comprise one or more DNA nucleosides.

[0247] Hybrid flank gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are hereby incorporated by reference.

[0248] Alternating flank gapmers

[0249] The flanking regions may comprise both LNA nucleosides and DNA nucleosides and are referred to as "alternating flankings" because they comprise an alternating motif of LNA-DNA-LNA nucleosides. Gapmers comprising such alternating flankings are referred to as "alternating flanking gapmers". Thus, an "alternating flanking gapmer" is an LNA gapmer oligonucleotide in which at least one flank (F or F') comprises DNA in addition to LNA nucleosides. In some embodiments, at least one of region F or region F' or both region F and region F' comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking regions F or F' or both F and F' comprise at least three nucleosides, wherein the 5' and 3' most terminal nucleosides of the F and / or F' regions are LNA nucleosides.

[0250] Alternating flanking LNA gapmers are disclosed in WO 2016 / 127002.

[0251] The alternating flanking regions may comprise up to 3 consecutive DNA nucleosides, such as 1 to 2 or 1 or 2 or 3 consecutive DNA nucleosides.

[0252] The alternating flanks may be labeled as a series of integers representing the number of LNA nucleosides (L) followed by the number of DNA nucleosides (D), e.g.

[0253] [L] 1-3 -[D] 1-4 -[L] 1-3

[0254] [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2

[0255] In oligonucleotide design, these will often be represented as numbers such that 2-2-1 represents 5'[L]2-[D]2-[L]3' and 1-1-1-1-1 represents 5'[L]-[D]-[L]-[D]-[L]3'. The length of the flanks (region F and region F') in oligonucleotides with alternating flanks may independently be 3 to 10 nucleosides, such as 4 to 8 nucleosides, such as 5 to 6 nucleosides, such as 4, 5, 6 or 7 modified nucleosides. In some embodiments, only one of the flanks in the gapmer oligonucleotide has alternation, while the other is composed of LNA nucleotides. It may be advantageous to have at least two LNA nucleosides at the 3' end of the 3' flank (F') to confer additional exonuclease resistance. Some examples of oligonucleotides with alternating flanks are:

[0256] [L]1-5 -[D] 1-4 -[L] 1-3 -[G] 5-16 -[L] 2-6

[0257] [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2 -[G] 5-16 -[L] 1-2 -[D] 1-3 -[L] 2-4

[0258] [L] 1-5 -[G] 5-16 -[L]-[D]-[L]-[D]-[L]2

[0259] Provided that the overall length of the gapmer is at least 12, such as at least 14, nucleotides.

[0260] Region D' or D" in the oligonucleotide

[0261] In some embodiments, the oligonucleotides of the invention may comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to the target nucleic acid (such as a gapmer FG-F') and other 5' and / or 3' nucleosides. Other 5' and / or 3' nucleosides may or may not be completely complementary to the target nucleic acid. Such other 5' and / or 3' nucleosides herein may be referred to as regions D' and D".

[0262] To link a contiguous nucleotide sequence such as a gapmer to a conjugate moiety or another functional group, an additional region D' or D" may be used. When used for ligation, the contiguous nucleotide sequence with the conjugate moiety may act as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or facilitate synthesis or manufacturing.

[0263] Regions D' and D" can be linked to the 5' end of region F or the 3' end of region F', respectively, to produce designs having the formula: D'-FG-F', FG-F'-D" or

[0264] D'-FG-F'-D". In this example, FG-F' is the gapmer portion of the oligonucleotide and the regions D' or D" constitute independent portions of the oligonucleotide.

[0265] Region D' or D" may independently comprise or consist 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, such as DNA or RNA or base-modified forms of these. The D' or D" region may act as a nuclease-labile biocleavable linker (see definition of linker). In some embodiments, the additional 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 WO 2014 / 076195, which include, by way of example, phosphodiester-linked DNA dinucleotides. WO 2015 / 113922 discloses the use of biocleavable linkers in multi-oligonucleotide constructs, wherein they are used to connect multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0266] In one embodiment, in addition to the continuous nucleotide sequence constituting the gapmer, the oligonucleotide of the present invention further comprises a region D' and / or D".

[0267] In some embodiments, the oligonucleotides of the invention may be represented by the following formula:

[0268] FG-F', especially F 1-8 -G 5-16 -F' 2-8

[0269] D'-FG-F', especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8

[0270] FG-F'-D", especially F 1-8 -G 5-16 -F' 2-8 -D" 1-3

[0271] D'-FG-F'-D", especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8 -D" 1-3

[0272] In some embodiments, the internucleoside bond between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside bond between region F' and region D" is a phosphodiester bond.

[0273] Full polymer

[0274] In some embodiments, all nucleosides of an oligonucleotide or a contiguous nucleotide sequence thereof are sugar-modified nucleosides. Such oligonucleotides are referred to herein as full polymers.

[0275] In some embodiments, all sugar-modified nucleosides of the holopolymer comprise the same sugar modification, for example, they may all be LNA nucleosides, or may all be 2'O-MOE nucleosides. In some embodiments, the sugar-modified nucleosides of the holopolymer may be independently selected from LNA nucleosides and 2' substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises LNA nucleosides and 2' substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises an LNA nucleoside and a 2'-O-MOE nucleoside. In some embodiments, the oligonucleotide comprises a (S)cET LNA nucleoside and a 2'-O-MOE nucleoside. In some embodiments, each nucleoside unit of the oligonucleotide is a 2'-substituted nucleoside. In some embodiments, each nucleoside unit of the oligonucleotide is a 2'-O-MOE nucleoside.

[0276] In some embodiments, all nucleosides of the oligonucleotide or its contiguous nucleotide sequence are LNA nucleosides, such as β-D-oxy-LNA nucleosides and / or (S) cET nucleosides. In some embodiments, the length of such LNA full polymer oligonucleotides is between 7 and 12 nucleosides (see, e.g., WO 2009 / 043353). Such short complete LNA oligonucleotides are particularly effective in inhibiting microRNAs.

[0277] Several full-polymer compounds are very effective as therapeutic oligomers, especially when targeting microRNAs (anti-miRs) or as splice-switching oligomers (SSOs).

[0278] In some embodiments, the full polymer comprises or consists of at least one XYX or YXY sequence motif such as a repeating sequence XYX or YXY, wherein X is LNA and Y is an alternative (i.e., non-LNA) nucleotide analog, such as a 2'-OMe RNA unit and a 2'-fluoro DNA unit. In some embodiments, the above sequence motif may be, for example, XXY, XYX, YXY, or YYX.

[0279] In some embodiments, the full polymer may comprise or consist of a contiguous nucleotide sequence of between 7 and 24 nucleotides, such as 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0280] In some embodiments, the continuous nucleotide sequence of the full polymer comprises at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as 95%, such as 100% LNA units. For complete LNA compounds, it is advantageous that their length is less than 12, such as 7-10 nucleotides.

[0281] The remaining units may be selected from the non-LNA nucleotide analogues mentioned herein, such as those selected from the group consisting of 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, PNA units, HNA units, INA units and 2'MOE RNA units or 2'-OMe RNA units and 2'-fluoro DNA units.

[0282] Polymer

[0283] The term "mixed polymer" refers to an oligomer comprising both DNA nucleosides and sugar-modified nucleosides, wherein there are insufficient continuous DNA nucleosides of length to recruit ribonuclease H. Suitable mixed polymers may contain up to 3 or up to 4 continuous DNA nucleosides. In certain embodiments, the mixed polymer or its continuous nucleotide sequence comprises alternating sugar-modified nucleoside regions and DNA nucleoside regions. Oligonucleotides that do not recruit ribonuclease H can be prepared by incorporating alternating regions of sugar-modified nucleosides that form an RNA-like (3' internal) conformation with a short DNA nucleoside region when incorporated into an oligonucleotide. Advantageously, the sugar-modified nucleosides are sugar-modified nucleosides that enhance affinity.

[0284] Oligonucleotide polymers are often used to provide occupancy-based regulation of target genes such as splicing regulators or microRNA inhibitors.

[0285] In some embodiments, the sugar-modified nucleosides in the mixed polymer or contiguous nucleotide sequence thereof comprise or are all LNA nucleosides, such as (S)cET or β-D-oxy LNA nucleosides.

[0286] In some embodiments, all sugar-modified nucleosides of the mixed polymer contain the same sugar modification, for example, they can all be LNA nucleosides, or can all be 2'O-MOE nucleosides. In some embodiments, the sugar-modified nucleosides of the mixed polymer can be independently selected from LNA nucleosides and 2' substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of the following items: 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide contains LNA nucleosides and 2' substituted nucleosides, such as 2' substituted nucleosides selected from the group consisting of the following items: 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises an LNA nucleoside and a 2'-O-MOE nucleoside. In some embodiments, the oligonucleotide comprises a (S)cET LNA nucleoside and a 2'-O-MOE nucleoside.

[0287] In some embodiments, the mixed polymer or its contiguous nucleotide sequence comprises only LNA and DNA nucleosides, such LNA mixed polymer oligonucleotides may be, for example, between 8 and 24 nucleosides in length (see, for example, WO2007112754, which discloses LNA anti-miR inhibitors of microRNAs).

[0288] Various mixed-polymer compounds are very effective as therapeutic oligomers, especially when targeting microRNAs (anti-miRs) or as splice-switching oligomers (SSOs).

[0289] In some embodiments, the polymer comprises the following motif

[0290] …[L]m[D]n[L]m[D]n[L]m…or

[0291] …[L]m[D]n[L]m[D]n[L]m[D]n[L]m…or

[0292] …[L]m[D]n[L]m[D]n[L]m[D]n[L]m[D]n[L]m…or

[0293] …[L]m[D]n[L]m[D]n[L]m[D]n[L]m[D]n[L]m[D]n[L]m…

[0294] Wherein L represents a sugar-modified nucleoside such as LNA or a 2'substituted nucleoside (e.g., 2'-O-MOE), D represents a DNA nucleoside, and wherein each m is independently selected from 1-6, and each n is independently selected from 1, 2, 3, and 4 such as 1-3. In some embodiments, each L is an LNA nucleoside. In some embodiments, at least one L is an LNA nucleoside, and at least one L is a 2'-O-MOE nucleoside. In some embodiments, each L is independently selected from: LNA and 2'-O-MOE nucleosides.

[0295] In some embodiments, the mixed polymer may comprise or consist of a contiguous nucleotide sequence of between 10 and 24 nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0296] In some embodiments, the contiguous nucleotide sequence of the mixed polymer comprises at least 30%, such as at least 40%, such as at least 50%, LNA units.

[0297] In some embodiments, the mixed polymer comprises or consists of a contiguous nucleotide sequence of a nucleotide analog and a repeating pattern of naturally occurring nucleotides or a nucleotide analog of one type and a nucleotide analog of a second type. The repeating pattern may be, for example, that every second or every third nucleotide is a nucleotide analog such as LNA, and the remaining nucleotides are naturally occurring nucleotides such as DNA, or may be a 2' substituted nucleotide analog, such as 2'MOE of a 2' fluoro analog as mentioned herein, or in some embodiments, selected from the group of nucleotide analogs mentioned herein. It should be appreciated that a repeating pattern of nucleotide analogs such as LNA units may be combined with nucleotide analogs at fixed positions (e.g., 5' or 3' ends).

[0298] In some embodiments, the first nucleotide of the oligomer, counting from the 3' end, is a nucleotide analog, such as an LNA nucleotide or a 2'-O-MOE nucleoside.

[0299] In some embodiments, which may be the same or different, the second nucleotide of the oligomer, counting from the 3' end, is a nucleotide analog, such as an LNA nucleotide or a 2'-O-MOE nucleoside.

[0300] In some embodiments, which may be the same or different, the 5' end of the oligomer is a nucleotide analog, such as an LNA nucleotide or a 2'-O-MOE nucleoside.

[0301] In some embodiments, the mixed polymer comprises at least one region comprising at least two consecutive nucleotide analogue units such as at least two consecutive LNA units.

[0302] In some embodiments, the mixed polymer comprises at least one region comprising at least three consecutive nucleotide analogue units such as at least three consecutive LNA units.

[0303] Conjugate

[0304] The term "conjugate" as used herein refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

[0305] The conjugation of the oligonucleotide of the present invention to one or more non-nucleotide parts can improve the pharmacological characteristics of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In certain embodiments, the conjugate part regulates or enhances the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of the oligonucleotide. In particular, the conjugate can target the oligonucleotide to a specific organ, tissue or cell type, and thus enhance the effectiveness of the oligonucleotide in such an organ, tissue or cell type. At the same time, the conjugate can play a role in reducing the activity of the oligonucleotide in non-target cell types, tissues or organs (e.g., off-target activity or activity in non-target cell types, tissues or organs).

[0306] WO 93 / 07883 and WO 2013 / 033230 provide suitable conjugate moieties, which are hereby incorporated by reference herein. Other suitable conjugate moieties are those capable of binding to asialoglycoprotein receptors (ASGPR). In particular, trivalent N-acetylgalactosamine conjugate moieties are suitable for ASGPR binding, see, for example, WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620 (hereby incorporated by reference herein). Such conjugates are used to enhance the uptake of oligonucleotides by the liver while reducing their content in the kidney, thereby increasing the liver / kidney ratio of the conjugated oligonucleotides compared to the unconjugated form of the same oligonucleotide.

[0307] Oligonucleotide conjugates and their synthesis have also been reported in the following literature: Manoharan's review, cited in "Antisense Drug Technology, Principles, Strategies, and Applications", edited by ST Crooke, Chapter 16, Marcel Dekker, Inc., 2001; and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, both of which are incorporated herein by reference in their entirety.

[0308] In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a sugar, a cell surface receptor ligand, a drug, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0309] Connectors

[0310] A bonding or joint is a connection between two atoms that links a target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a joint or tether). The joint serves to covalently link a third region, such as a conjugate moiety (region C), to a first region, such as an oligonucleotide or a continuous nucleotide sequence (region A) complementary to the target nucleic acid.

[0311] In some embodiments of the present invention, the conjugate or oligonucleotide conjugate of the present invention may optionally include a linker region (second region or region B and / or region Y) located between the oligonucleotide or continuous nucleotide sequence (region A or first region) complementary to the target nucleic acid and the conjugate portion (region C or third region).

[0312] Region B refers to a biodegradable joint comprising or consisting of a physiologically unstable bond, which is cleavable under conditions commonly encountered in mammals or under conditions similar thereto. The conditions under which the physiologically unstable joint undergoes chemical transformation (e.g., cleavage) include chemical conditions, such as pH, temperature, oxidation or reduction conditions or reagents, and salt concentrations encountered in mammalian cells or salt concentrations similar thereto. Conditions within mammalian cells also include enzyme activities commonly present in mammalian cells, such as enzyme activities from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment, the biodegradable joint is sensitive to S1 nuclease cleavage. In a preferred embodiment, the joint sensitive to nucleases comprises 1 to 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably comprises 2 to 6 nucleosides, most preferably comprises 2 to 4 linked nucleosides, the nucleosides comprising at least two continuous phosphodiester bonds, such as at least 3 or 4 or 5 continuous phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Biocleavable linkers comprising phosphodiesters are described in detail in WO 2014 / 076195 (incorporated herein by reference).

[0313] Region Y refers to a linker that is not necessarily biodegradable but is primarily used to covalently link the conjugate moiety (region C or the third region) to the oligonucleotide (region A or the first region). The region Y linker may comprise a chain structure or oligomer of repeating units such as ethylene glycol units, amino acid units or aminoalkyl groups. The oligonucleotide conjugates of the present invention may be composed of the following regional elements AC, ABC, ABYC, AYBC or AYC. In certain embodiments, the linker (region Y) is an aminoalkyl group such as a C2-C36 aminoalkyl group, including, for example, a C6 to C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

[0314] Therefore, the oligonucleotide according to the present invention has the formula (II)

[0315]

[0316] Among them (A 1 ) and (A 2 ) as defined above.

[0317] Therefore, the present invention particularly relates to:

[0318] According to the oligonucleotide of the present invention, the nucleoside (A 1 ) is a DNA nucleoside;

[0319] According to the oligonucleotide of the present invention, the nucleoside (A 2 ) is a DNA nucleoside, an RNA nucleoside or a sugar-modified nucleoside;

[0320] According to the oligonucleotide of the present invention, the nucleoside (A 2 ) is a DNA nucleoside or a sugar-modified nucleoside;

[0321] According to the oligonucleotide of the present invention, the sugar-modified nucleoside is a 2' sugar-modified nucleoside;

[0322] According to the oligonucleotide of the present invention, the nucleoside (A 2 ) is: 2'-alkoxy-RNA, in particular 2'-methoxy-RNA; 2'-alkoxyalkoxy-RNA, in particular 2'-methoxyethoxy-RNA; 2'-amino-DNA; 2'-fluoro-RNA; or 2'-fluoro-ANA;

[0323] According to the oligonucleotide of the present invention, the nucleoside (A 2 ) is an LNA nucleoside;

[0324] According to the oligonucleotide of the present invention, the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA and ENA, in particular β-D-oxy LNA;

[0325] According to the oligonucleotide of the present invention, the nucleoside (A 1 ) and (A 2 ) is 2'-alkoxyalkoxy-RNA;

[0326] According to the oligonucleotide of the present invention, the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA;

[0327] According to the oligonucleotide of the present invention, the nucleoside (A 1 ) and (A 2 ) are all DNA nucleosides;

[0328] The oligonucleotide according to the present invention comprises other internucleoside bonds selected from phosphodiester internucleoside bonds, phosphorothioate internucleoside bonds and trithiophosphate internucleoside bonds of formula (I) as defined above;

[0329] The oligonucleotide according to the present invention comprises other internucleoside bonds selected from phosphorothioate internucleoside bonds and trithioate internucleoside bonds of formula (I) as defined above;

[0330] The oligonucleotide according to the invention comprises between 1 and 15, particularly between 1 and 5, more particularly 1, 2, 3, 4 or 5 trithiophosphate internucleoside bonds of formula (I) as defined above;

[0331] According to the oligonucleotide of the present invention, the internucleoside bonds are all phosphorothioate internucleoside bonds of the formula -P(=S)(OR)O2-, wherein R is as defined above;

[0332] The oligonucleotide according to the present invention comprises other nucleosides selected from DNA nucleosides, RNA nucleosides and sugar-modified nucleosides;

[0333] According to the oligonucleotide of the present invention, one or more nucleosides are nucleobase-modified nucleosides;

[0334] According to the oligonucleotide of the present invention, the oligonucleotide is an antisense oligonucleotide, siRNA, microRNA mimic or ribozyme;

[0335] According to the oligonucleotide of the present invention, the oligonucleotide is an antisense gapmer oligonucleotide;

[0336] According to the oligonucleotide of the present invention, the trithiophosphate internucleoside bond of formula (I) is in the gap region of the gapmer oligonucleotide;

[0337] According to the oligonucleotide of the present invention, the gapmer oligonucleotide is an LNA gapmer, a mixed flank gapmer or a 2'-substituted gapmer, in particular a 2'-O-methoxyethyl gapmer;

[0338] According to the gapmer oligonucleotide of the present invention, wherein the gapmer oligonucleotide comprises a continuous nucleotide sequence of the formula 5'-FG-F'-3', wherein G is a region having 5 to 18 nucleosides capable of recruiting ribonuclease H, and the region G has flanking regions F and F' on the 5' and 3' sides, respectively, wherein regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, wherein the nucleosides of region F adjacent to region G are 2'-sugar-modified nucleosides and wherein the nucleosides of region F' adjacent to region G are 2'-sugar-modified nucleosides;

[0339] The gapmer oligonucleotide according to the present invention, wherein the at least one phosphorothioate internucleoside bond of formula (I) as defined above is positioned between adjacent nucleosides of region G or between region G and region F';

[0340] According to the oligonucleotide of the present invention, the oligonucleotide is an antisense oligonucleotide mixed polymer or full polymer, in particular a splicing-switching oligonucleotide or a microRNA inhibitor oligonucleotide;

[0341] A pharmaceutically acceptable salt of the oligonucleotide according to the present invention, in particular a sodium salt or a potassium salt;

[0342] A conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt according to the present invention and at least one conjugate moiety covalently linked to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety;

[0343] A pharmaceutical composition comprising an oligonucleotide according to the present invention, a pharmaceutically acceptable salt or conjugate and a therapeutically inert carrier;

[0344] The oligonucleotide, pharmaceutically acceptable salt or conjugate according to the present invention is used as a therapeutically active substance;

[0345] The oligonucleotide, pharmaceutically acceptable salt or conjugate according to the present invention is used for treating or preventing heart disease or blood disease;

[0346] Use of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to the present invention for preparing a drug for treating or preventing heart disease or blood disease;

[0347] Use of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to the present invention in treating or preventing heart disease or blood disease;

[0348] A method for treating or preventing heart disease or blood disease, comprising administering an effective amount of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to the present invention to a patient in need thereof;

[0349] A method for inhibiting a target RNA (particularly human mRNA or viral RNA) in a cell, comprising administering an oligonucleotide according to the invention to a cell expressing the target RNA;

[0350] A method for regulating or inhibiting a target RNA (particularly a human mRNA or a viral RNA) in a cell in vitro, comprising administering an oligonucleotide or a gapmer oligonucleotide according to the invention to a cell expressing the target RNA;

[0351] A method for producing an oligonucleotide according to the present invention comprises the following steps:

[0352] (a) coupling a 3'S-modified nucleoside phosphoramidite to the terminal 5' sulfur atom of a 5'S-modified nucleoside or oligonucleotide to generate a dithiophosphite triester intermediate;

[0353] (b) sulfur-oxidizing the dithiophosphite triester intermediate obtained in step a); and

[0354] (c) optionally further extending the oligonucleotide;

[0355] According to the method of the present invention, the 5'S-modified nucleoside or oligonucleotide of step (a) is attached to a solid support;

[0356] The method according to the present invention further comprises cleaving the oligonucleotide from the solid support; and

[0357] An oligonucleotide produced according to the method of the present invention.

[0358] In certain embodiments, compared with the oligonucleotides connected by corresponding complete phosphorothioate, the oligonucleotides of the present invention have higher activity in regulating its target nucleic acid. In certain embodiments, the invention provides oligonucleotides with enhanced activity, enhanced efficacy, enhanced specific activity or enhanced cellular uptake. In certain embodiments, the invention provides oligonucleotides with external or internal action duration of change such as extended external or internal action duration. In certain embodiments, in the cell expressing the target nucleic acid, higher activity of regulating target nucleic acid is recorded in vitro or in vivo.

[0359] In some embodiments, the oligonucleotides of the present invention have changed pharmacological properties, such as decreased toxicity, such as decreased nephrotoxicity, decreased hepatotoxicity or decreased immunostimulation. Hepatotoxicity can be determined, for example, in vivo or by using the in vitro assay disclosed in WO 2017 / 067970 (incorporated herein by reference). Nephrotoxicity can be determined, for example, in vivo or by using the in vitro assay disclosed in PCT / EP2017 / 064770 (incorporated herein by reference). In some embodiments, the oligonucleotides of the present invention include 5'CG 3' dinucleotides, such as DNA 5'CG 3' dinucleotides, wherein the internucleoside bond between C and G is a trithiophosphate internucleoside bond with formula (I) defined above.

[0360] In some embodiments, the oligonucleotides of the invention have improved nuclease resistance, such as improved biological stability in serum. In some embodiments, the 3' terminal nucleoside of the oligonucleotides of the invention has an A or G base, such as a 3' terminal LNA-A or LNA-G nucleoside. Suitably, according to formula (I) defined above, the internucleoside bond between the two 3' terminal nucleosides of the oligonucleotide may be a trithioate internucleoside bond.

[0361] In some embodiments, the oligonucleotides of the invention have enhanced bioavailability. In some embodiments, the oligonucleotides of the invention have higher blood exposure, such as longer blood retention time.

[0362] The oligonucleotide according to the present invention can be prepared, for example, according to the following scheme.

[0363] Phosphoramidite method can be utilized to introduce the phosphorothioate bond containing sulfur atom in non-bridging position and 3' and 5' position of adjacent furanose ring into oligonucleotide by solid phase synthesis.Use controlled pore glass column (CPG) equipped with universal joint to synthesize.On this type of solid support, oligonucleotide is usually built in 3' to 5' direction by sequential circulation, and this sequential circulation comprises the nucleoside phosphoramidite structural unit of coupling 5'O-DMT protection, and then carries out (thio) oxidation, end-blocking and deprotection of DMT group.For the introduction of phosphorothioate as described in the present patent application, the 5'-deoxy-5'-thiol phosphoramidite structural unit of DMT protection is coupled to the free 5'-hydroxyl group of the oligonucleotide chain of solid support combination.After (thio) oxidation and end-blocking, the intermediate deprotection of gained is to release 5'-thiol group.

[0364] Solution 1

[0365]

[0366] This deprotection is challenging and is best performed by repeated (greater than 15 times) application of standard deprotection conditions (typically 3-5% dichloroacetic acid or trichloroacetic acid in dichloromethane) or using higher acid concentrations (e.g., up to 10% trichloroacetic acid in dichloromethane) or strong acids (e.g., 5% trifluoroacetic acid in dichloromethane), preferably with an appropriate cation scavenger (typically 20% triethylsilane, 5% 4-methoxythiophenol, or a mixture of the two).

[0367] Solution 2

[0368]

[0369] The free 5'thiol group thus obtained is then coupled with a suitable 5'O-DMT protected 3'-deoxy-3'-thiol phosphoramidite building block. Due to the low reactivity of such building blocks, a typical procedure includes 12 couplings using 4 equivalents and a longer reaction time (7.5 min). The resulting dithiophosphite intermediate can then be sulfur-oxidized using a suitable reagent (e.g., 3-amino-1,2,4-dithiazole-5-thione) to provide the desired trithiophosphate bond.

[0370] Solution 3

[0371]

[0372] In the above scenario:

[0373] R 2a and R 4a together form -XY- as defined above; or

[0374] R 4a is hydrogen, and R 2a is selected from alkoxy (especially methoxy), halogen (especially fluorine), alkoxyalkoxy (especially methoxyethoxy), alkenyloxy (especially allyloxy) and aminoalkoxy (especially aminoethoxy);

[0375] R 2b and R 4b together form -XY- as defined above; or

[0376] R 2b and R 4b both are hydrogen; or

[0377] R 4b is hydrogen, and R 2b is selected from alkoxy (especially methoxy), halogen (especially fluorine), alkoxyalkoxy (especially methoxyethoxy), alkenyloxy (especially allyloxy) and aminoalkoxy (especially aminoethoxy);

[0378] R 3 is dialkylamino or pyrrolidinyl;

[0379] R 5 is a hydroxyl or sulfhydryl protecting group; and

[0380] R is as defined above.

[0381] In one aspect, the present application provides the following embodiments:

[0382] 1. An oligonucleotide comprising at least one phosphorothioate internucleoside bond of formula (I)

[0383]

[0384] One of the two bridging sulfur atoms is bound to a DNA nucleoside or an RNA nucleoside (A 1 ) and the other is connected to the 3' carbon atom of another nucleoside (A 2 ), and wherein R is hydrogen or a phosphate protecting group.

[0385] 2. The oligonucleotide according to embodiment 1, characterized in that the nucleoside (A 1 ) are DNA nucleosides.

[0386] 3. The oligonucleotide according to embodiment 1 or 2, characterized in that the nucleoside (A 2 ) is a DNA nucleoside, an RNA nucleoside or a sugar-modified nucleoside.

[0387] 4. The oligonucleotide according to any one of embodiments 1 to 3, characterized in that the nucleoside (A 2 ) is a DNA nucleoside or a sugar-modified nucleoside.

[0388] 5. The oligonucleotide according to embodiment 3 or 4, characterized in that the sugar-modified nucleoside is a 2' sugar-modified nucleoside.

[0389] 6. An oligonucleotide according to any one of embodiments 1 to 5, characterized in that the nucleoside (A 2 ) is: 2'-alkoxy-RNA, in particular 2'-methoxy-RNA; 2'-alkoxyalkoxy-RNA, in particular 2'-methoxyethoxy-RNA; 2'-amino-DNA; 2'-fluoro-RNA; or 2'-fluoro-ANA.

[0390] 7. An oligonucleotide according to any one of embodiments 1 to 5, characterized in that the nucleoside (A 2 ) is an LNA nucleoside.

[0391] 8. The oligonucleotide according to embodiment 7, characterized in that the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA and ENA, in particular β-D-oxy LNA.

[0392] 9. The oligonucleotide according to embodiment 1, characterized in that the nucleoside (A 1 ) and nucleosides (A 2 ) is 2'-alkoxyalkoxy-RNA.

[0393] 10. The oligonucleotide according to embodiment 9, wherein the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA.

[0394] 11. An oligonucleotide according to any one of embodiments 1 to 4, characterized in that the nucleoside (A 1 ) and nucleosides (A 2 ) Both are DNA nucleosides.

[0395] 12. The oligonucleotide according to any one of embodiments 1 to 11, comprising other internucleoside bonds selected from phosphodiester internucleoside bonds, phosphorothioate internucleoside bonds and trithiophosphate internucleoside bonds of formula (I) as defined in embodiment 1.

[0396] 13. The oligonucleotide according to any one of embodiments 1 to 12, comprising further internucleoside linkages selected from phosphorothioate internucleoside linkages and trithiophosphate internucleoside linkages of formula (I) as defined in embodiment 1.

[0397] 14. The oligonucleotide according to any one of embodiments 1 to 13, comprising between 1 and 15, particularly between 1 and 5, more particularly 1, 2, 3, 4 or 5 trithioate phosphoroside internucleoside bonds of formula (I) as defined in embodiment 1.

[0398] 15. An oligonucleotide according to any one of embodiments 1 to 14, characterized in that the other internucleoside bonds are all phosphorothioate internucleoside bonds of the formula -P(=S)(OR)O2-, wherein R is as defined in embodiment 1.

[0399] 16. The oligonucleotide according to any one of embodiments 1 to 15, comprising further nucleosides selected from DNA nucleosides, RNA nucleosides and sugar-modified nucleosides.

[0400] 17. The oligonucleotide according to any one of embodiments 1 to 16, wherein one or more nucleosides are nucleobase-modified nucleosides.

[0401] 18. The oligonucleotide according to any one of embodiments 1 to 17, characterized in that the oligonucleotide is an antisense oligonucleotide, siRNA, microRNA mimic or ribozyme.

[0402] 19. The oligonucleotide according to any one of embodiments 1 to 18, characterized in that the oligonucleotide is an antisense gapmer oligonucleotide.

[0403] 20. The oligonucleotide of embodiment 19, wherein the trithiophosphate internucleoside linkage of formula (I) is in the gap region of the gapmer oligonucleotide.

[0404] 21. An oligonucleotide according to embodiment 19 or 20, characterized in that the gapmer oligonucleotide is an LNA gapmer, a mixed flank gapmer or a 2'-substituted gapmer, in particular a 2'-O-methoxyethyl gapmer.

[0405] 22. A gapmer oligonucleotide according to any one of embodiments 19 to 21, characterized in that the gapmer oligonucleotide comprises a continuous nucleotide sequence of the formula 5'-FG-F'-3', wherein G is a region having 5 to 18 nucleosides capable of recruiting ribonuclease H, and the region G has flanking regions F and F' on the 5' and 3' sides, respectively, wherein regions F and F' independently contain or consist of 1 to 7 2'-sugar modified nucleotides, wherein the nucleosides of region F adjacent to region G are 2'-sugar modified nucleosides and wherein the nucleosides of region F' adjacent to region G are 2'-sugar modified nucleosides.

[0406] 23. A gapmer oligonucleotide according to embodiment 22, characterized in that the at least one trithioate phosphorothioate internucleoside bond of formula (I) as defined in embodiment 1 is positioned between adjacent nucleosides in region G or between region G and region F'.

[0407] 24. The oligonucleotide according to any one of embodiments 1 to 23, characterized in that the oligonucleotide is an antisense oligonucleotide mixed polymer or full polymer, in particular a splice-switching oligonucleotide or a microRNA inhibitor oligonucleotide.

[0408] 25. A pharmaceutically acceptable salt, in particular a sodium or potassium salt, of the oligonucleotide according to any one of embodiments 1 to 24.

[0409] 26. A conjugate comprising the oligonucleotide or the pharmaceutically acceptable salt according to any one of embodiments 1 to 25 and at least one conjugate moiety covalently linked to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety.

[0410] 27. A pharmaceutical composition comprising the oligonucleotide according to any one of embodiments 1 to 26, a pharmaceutically acceptable salt or conjugate and a therapeutically inert carrier.

[0411] 28. An oligonucleotide, a pharmaceutically acceptable salt or a conjugate according to any one of embodiments 1 to 26 for use as a therapeutically active substance.

[0412] 29. The oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of embodiments 1 to 26, for use in treating or preventing a heart disease or a blood disease.

[0413] 30. Use of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of embodiments 1 to 26 for the preparation of a medicament for treating or preventing heart disease or blood disease.

[0414] 31. Use of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of embodiments 1 to 26 in the treatment or prevention of heart disease or blood disease.

[0415] 32. A method for treating or preventing a heart disease or a blood disease, comprising administering an effective amount of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of embodiments 1 to 26 to a patient in need thereof.

[0416] 33. A method of making an oligonucleotide according to any one of embodiments 1 to 26, comprising the following steps:

[0417] (a) coupling a 3'S-modified nucleoside phosphoramidite to the terminal 5' sulfur atom of a 5'S-modified nucleoside or oligonucleotide to generate a dithiophosphite triester intermediate;

[0418] (b) sulfur-oxidizing the dithiophosphite triester intermediate obtained in step a); and

[0419] (c) optionally further extending the oligonucleotide.

[0420] 34. An oligonucleotide produced according to the method of embodiment 33.

[0421] 35. The invention as hereinbefore described.

[0422] The invention will now be illustrated by the following examples, which are not limiting.

[0423] Examples

[0424] Example 1

[0425] Oligonucleotide synthesis

[0426] Oligonucleotides were synthesized using the MerMade 12 automated DNA synthesizer from Bioautomation. Controlled pore glass supports with universal adapters were used. The synthesis was performed on a 1 μmol scale.

[0427] In the standard cycling procedure for coupling DNA and LNA phosphoramidites, DMT deprotection was performed by three applications of 200 μL of 3% (w / v) trichloroacetic acid in CH2Cl2 for 30 seconds. Me C building block, acetonitrile / CH2Cl2 1:1) and 110 μL of 0.1M 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole solution in acetonitrile as activator and coupling for 180 seconds, the corresponding phosphoramidite was coupled three times. For sulfur oxidation, 0.1M 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine 1:1 was used (3×190 μL, 55 seconds). Endcapping was performed using THF / lutidine / Ac2O 8:1:1 (CapA, 75 μmol) and THF / N-methylimidazole 8:2 (CapB, 75 μmol) for 55 seconds.

[0428] The synthetic cycle incorporating 2',3'-dideoxy-3'-mercaptophosphoramidite included three applications of 200 μL for 30 seconds with 3% (w / v) trichloroacetic acid in CH2Cl2 for DMT deprotection. Phosphoramidite coupling was performed ten times with 40 μL of 0.1 M acetonitrile and 44 μL of 0.1 M 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile for 900 seconds. Sulfur oxidation was performed immediately after coupling with three applications of 190 μL of 0.1 M 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine 1:1 for 55 seconds. Capping was performed using THF / lutidine / Ac2O 8:1:1 (CapA, 75 μmol) and THF / N-methylimidazole 8:2 (CapB, 75 μmol) for 55 seconds.

[0429] The synthetic cycle incorporating 2',5'-deoxy-5'-mercaptophosphoramidite included treatment with 100 μL of 0.1 M acetonitrile solution and 110 μL of 0.1 M 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole solution in acetonitrile for 180 seconds to couple the phosphoramidite building block. The coupling was performed three times. Sulfur oxidation was performed using 0.1 M 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine 1:1 (3×190 μL, 55 seconds). For end-capping, THF / lutidine / Ac2O 8:1:1 (CapA, 75 μmol) and THF / N-methylimidazole 8:2 (CapB, 75 μmol) solutions were applied for 55 seconds. 3% (w / v) trichloroacetic acid in CH2Cl2 solution was applied 15 times by 200 μL for 30 seconds to carry out DMT deprotection and release of thiol. It can also be advantageous to apply 3-6 times of 200 μL (for 45 seconds) of 1-10% (v / v) trifluoroacetic acid or 5-10% (w / v) trichloroacetic acid in the presence of 5-30% (v / v) triethylsilane in CH2Cl2 solution and / or 2-10% p-methoxythiophenol in CH2Cl2 solution to carry out DMT deprotection and release of thiol.

[0430] Under standard conditions (32% ammonia at 55°C for at least 8 h), the nucleobase protecting groups are removed and cleaved from the solid support. The crude DMT-on oligonucleotides are purified using solid phase extraction cartridges and then repurified by ion exchange chromatography or RP-HPLC purification using a C18 column, followed by removal of DMT using 80% aqueous acetic acid and ethanol precipitation.

[0431] The following molecules were prepared according to the general procedure described above.

[0432]

[0433] Trithioester modifications between bold and underlined nucleotides

[0434] A.G. m C and T represent LNA nucleotides

[0435] a, g, c, t represent DNA nucleotides

[0436] All other bonds were prepared as phosphorothioates

[0437] Compounds #1-8 are based on SEQ ID NO: 1. As shown in the table above, they differ in the position of the phosphorothioate internucleoside linkage of formula (I).

[0438] Example 2

[0439] In vitro mRNA reduction and intracellular concentration (uptake)

[0440] Primary rat hepatocytes were plated in 96-well plates and treated in Williams medium E without antibiotics containing 10% FCS. Cells were treated with LNA solutions at the indicated concentrations in complete cell culture medium. After 24 and 72 hours of incubation, cells were incubated with Ca 2+ and Mg 2+ The cells were washed 3 times with PBS and lysed with 165uL PureLink Pro lysis buffer. Total RNA was isolated using the PureLink PRO 96 RNA kit from Thermo Fisher according to the manufacturer's instructions, and RT-qPCR was performed using the LightCycler Multiple RNA Virus Master Mix (Roche) with a primer probe set for RnApoB (Invitrogen). The data obtained were normalized to Ribogreen.

[0441] The intracellular concentration of LNA oligonucleotides was determined using a hybridization-based ELISA assay. All data points were processed in triplicate and the data are presented as their mean.

[0442] The results are as follows Figure 1 shown.

[0443] From the data, it can be seen that the modifications according to the present invention are well tolerated and the oligonucleotides exhibit good efficacy and suitable intracellular concentrations.

[0444] Example 3

[0445] In vivo mRNA reduction in liver and kidney

[0446] C57BL / 6JBomTac female mice weighing about 20 g received a single dose of 1 mg / kg (concentration 0.1 mg / kg, volume 10 ml / kg) on ​​day 0, and the animals were sacrificed on day 7. Serum, liver, kidney and heart tissues were collected at the end.

[0447] The mRNA in the tissue was analyzed using the Qantigene mRNA quantification kit ("bDNA-assay", Panomics / Affimetrix) according to the manufacturer's protocol. For tissue lysates, 50-80 mg of tissue was lysed by ultrasonic treatment in 1 ml of lysis buffer containing proteinase K. The lysate was used directly for bDNA determination without RNA extraction. The probes for the target and GAPDH were custom designed by Panomics. During the analysis, the luminescence units of the target gene obtained were normalized to the housekeeper GAPDH.

[0448] The results are as follows Figure 2 shown.

[0449] Figure 2 It is shown that oligonucleotides carrying modifications according to the invention improve the reduction of target concentration in the liver compared to unmodified phosphorothioate controls. These findings are all the more surprising considering the fact that this particular molecule showed the lowest reduction of target concentration in vitro in this series.

[0450] Example 4

[0451] Serum cholesterol level

[0452] C57BL / 6JBomTac female mice weighing about 20 g received a single dose of 1 mg / kg (concentration 0.1 mg / kg, volume 10 ml / kg) on ​​day 0, and the animals were sacrificed on day 7. Serum, liver, kidney and heart tissues were collected at the end.

[0453] Cholesterol in serum was analyzed using 10 μL of serum on the "Cobas INTEGRA 400plus" clinical chemistry platform (Roche Diagnostics).

[0454] The results are as follows Figure 3 shown.

[0455] The data show that a higher reduction in target concentration was observed for oligonucleotides carrying modifications according to the invention ( Figure 2 , Example 3) improved the reduction of serum cholesterol levels, which is a functional indicator of activity of this particular sequence. These findings are even more surprising considering the fact that this particular molecule showed the lowest in vitro target concentration reduction in this series.

[0456] Example 5

[0457] In vivo tissue uptake in liver, kidney, and heart

[0458] C57BL / 6JBomTac female mice weighing about 20 g received a single dose of 1 mg / kg (concentration 0.1 mg / kg, volume 10 ml / kg) on ​​day 0, and the animals were sacrificed on day 7. Serum, liver, kidney and heart tissues were collected at the end.

[0459] For oligonucleotide quantification, fluorescently labeled PNA probes are hybridized to target oligonucleotides in tissue lysates. The same lysates as for the bDNA assay are used, except that the tissue content is accurately weighed. Heteroduplexes are quantified by AEX-HPLC and fluorescence detection.

[0460] The results are as follows Figure 4 shown.

[0461] Figure 4 The beneficial effects of the modifications according to the invention on tissue uptake are demonstrated. Not only do the data indicate high tissue content in the liver and kidney, but the modifications have the potential to surprisingly improve tissue distribution in the heart. These findings are even more surprising considering the fact that this particular molecule showed the lowest decrease in in vitro target concentration in this series.

Claims

1. An oligonucleotide comprising at least one phosphorothioate internucleoside bond of formula (I) One of the two bridging sulfur atoms is bound to a DNA or RNA nucleoside (A 1 ) and the other is connected to the 3' carbon atom of another nucleoside (A 2 ), and wherein R is hydrogen or a phosphate protecting group; The oligonucleotide is an antisense gapmer oligonucleotide; The gapmer oligonucleotide comprises a continuous nucleotide sequence of the formula 5'-FG-F'-3', wherein G is a region having 5 to 18 nucleosides capable of recruiting RNase H, and the region G has flanking regions F and F' on the 5' and 3' sides, respectively, wherein regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, wherein the nucleosides of region F adjacent to region G are 2'-sugar-modified nucleosides and wherein the nucleosides of region F' adjacent to region G are 2'-sugar-modified nucleosides; and The at least one phosphorothioate internucleoside bond is positioned between adjacent nucleosides in region G or between region G and region F'.

2. The oligonucleotide according to claim 1, characterized in that The nucleoside (A 1 ) are DNA nucleosides.

3. The oligonucleotide according to claim 1 or 2, characterized in that The nucleoside (A 2 ) is a DNA nucleoside, an RNA nucleoside or a sugar-modified nucleoside.

4. The oligonucleotide according to any one of claims 1 to 3, characterized in that The nucleoside (A 2 ) is a DNA nucleoside or a sugar-modified nucleoside.

5. The oligonucleotide according to claim 3 or 4, characterized in that The sugar-modified nucleoside is a 2' sugar-modified nucleoside.

6. The oligonucleotide according to any one of claims 1 to 5, characterized in that The nucleoside (A 2 ) is: 2'-alkoxy-RNA; 2'-alkoxyalkoxy-RNA; 2'-amino- DNA; 2'-Fluoro-RNA; or 2'-Fluoro-ANA.

7. The oligonucleotide according to claim 6, characterized in that the nucleoside (A 2 ) is: 2'-methoxy-RNA or 2'-methoxyethoxy-RNA.

8. The oligonucleotide according to any one of claims 1 to 5, characterized in that The nucleoside (A 2 ) is an LNA nucleoside.

9. The oligonucleotide according to claim 8, characterized in that The LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA and ENA.

10. The oligonucleotide according to claim 8, characterized in that The LNA nucleosides are independently selected from β-D-oxy LNA.

11. The oligonucleotide according to claim 1, characterized in that The nucleoside (A 1 ) and nucleosides (A 2 ) is 2'-alkoxyalkoxy-RNA.

12. The oligonucleotide according to claim 11, characterized in that The 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA.

13. The oligonucleotide according to any one of claims 1 to 4, characterized in that The nucleoside (A 1 ) and nucleosides (A 2 ) Both are DNA nucleosides.

14. An oligonucleotide according to any one of claims 1 to 13, comprising other internucleoside bonds selected from phosphodiester internucleoside bonds, phosphorothioate internucleoside bonds and trithiophosphate internucleoside bonds of formula (I) as defined in claim 1.

15. The oligonucleotide according to any one of claims 1 to 14, comprising further internucleoside linkages selected from phosphorothioate internucleoside linkages and trithiophosphate internucleoside linkages of formula (I) as defined in claim 1.

16. An oligonucleotide according to any one of claims 1 to 15 comprising between 1 and 15 phosphorothioate internucleoside linkages of formula (I) as defined in claim 1.

17. An oligonucleotide according to claim 16 comprising between 1 and 5 phosphorothioate internucleoside linkages of formula (I) as defined in claim 1.

18. The oligonucleotide according to claim 16, comprising 1, 2, 3, 4 or 5 phosphorothioate internucleoside linkages of formula (I) as defined in claim 1.

19. The oligonucleotide according to any one of claims 1 to 18, characterized in that The other internucleoside bonds are all phosphorothioate internucleoside bonds of the formula -P(=S)(OR)O2-, wherein R is as defined in claim 1.

20. The oligonucleotide according to any one of claims 1 to 19, comprising other nucleosides selected from DNA nucleosides, RNA nucleosides and sugar-modified nucleosides.

21. The oligonucleotide according to any one of claims 1 to 20, characterized in that One or more of the nucleosides are nucleobase modified nucleosides.

22. The oligonucleotide according to any one of claims 1 to 21, characterized in that The phosphorothioate internucleoside linkage of formula (I) is in the gap region of the gapmer oligonucleotide.

23. The oligonucleotide according to claim 22, characterized in that The gapmer oligonucleotide is an LNA gapmer, a mixed flank gapmer or a 2'-substituted gapmer.

24. The oligonucleotide according to claim 22, characterized in that The gapmer oligonucleotide is a 2'-O-methoxyethyl gapmer.

25. A pharmaceutically acceptable salt of the oligonucleotide according to any one of claims 1 to 24.

26. A pharmaceutically acceptable salt of the oligonucleotide according to claim 25, which is a sodium salt or a potassium salt.

27. A conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt according to any one of claims 1 to 26 and at least one conjugate moiety covalently linked to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety.

28. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 27, a pharmaceutically acceptable salt or conjugate and a therapeutically inert carrier.

29. An oligonucleotide, a pharmaceutically acceptable salt or a conjugate according to any one of claims 1 to 27 for use as a therapeutically active substance.

30. The oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 27 for use in treating or preventing a heart disease or a blood disease.

31. Use of the oligonucleotide, pharmaceutically acceptable salt or conjugate according to any one of claims 1 to 27 for preparing a medicament for treating or preventing heart disease or blood disease.

32. A method for producing an oligonucleotide according to any one of claims 1 to 27, comprising the steps of: (a) coupling a 3'S-modified nucleoside phosphoramidite to the terminal 5' sulfur atom of a 5'S-modified nucleoside or oligonucleotide to generate a dithiophosphite triester intermediate; (b) sulfur-oxidizing the dithiophosphite triester intermediate obtained in step a); and (c) optionally further extending the oligonucleotide.

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