Novel thiophosphoramidites

Abstract

To provide oligonucleotides with improved physiochemical and pharmacological properties, including improved potency.SOLUTION: The present invention relates to a compound of formula (II) or (IIb). The compound of formula (II) can be used in the manufacture of oligonucleotides.SELECTED DRAWING: None

Description

[Background Technology]

[0001] background The use of synthetic oligonucleotides as therapeutic agents has witnessed significant progress in recent decades, and molecules acting through diverse mechanisms have been developed, including RNase H-activating gapmers, splice-switching oligonucleotides, microRNA inhibitors, siRNAs, and aptamers (ST Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. ed., Boca Raton, FL: CRC Press, 2008 (Non-Patent Document 1)). However, oligonucleotides are inherently unstable to nucleolytic degradation in biological systems. Furthermore, oligonucleotides exhibit significantly unfavorable pharmacokinetic behavior. To ameliorate these shortcomings, a wide variety of chemical modifications have been investigated in recent decades. Arguably, one of the most successful modifications is the introduction of a phosphorothioate bond, in which one of the non-bridging phosphate oxygen atoms is replaced with a sulfur atom (F. Eckstein, Antisense and Nucleic Acid Drug Development 2009, 10, 117-121 (Non-Patent Document 2)). Such phosphorothioate oligodeoxynucleotides exhibit significantly greater stability against increased protein binding and nucleolytic degradation than their unmodified phosphodiester analogs, and therefore substantially longer half-lives in plasma, tissues, and cells. These crucial features enabled the development of first-generation oligonucleotide therapeutics and opened the door for further improvement through later-generation modifications such as locked nucleic acids (LNAs). However, the replacement of the phosphodiester bond with phosphorothioate creates a chiral center at the phosphorus atom. As a result, every approved phosphorothioate oligonucleotide therapeutic is used as a mixture of vast quantities of diastereoisomeric compounds, all of which potentially possess different (and possibly opposing) physicochemical and pharmacological properties.

[0002] Although the stereospecific synthesis of single stereochemically defined phosphorothioate oligonucleotides is now possible (N. Oka, M. Yamamoto, T. Sato, T. Wada, J. Am. Chem. Soc. 2008, 130, 16031-16037), identifying the stereoisomer with optimal properties from the vast number of possible diastereoisomers remains a challenge. In this context, there has been considerable interest in reducing the diastereomeric complexity by using achiral phosphorothioate linkages. For example, symmetric non-bridging dithioate modifications, in which both non-bridging oxygen atoms in the phosphate linkage are replaced with sulfur (see, e.g., W.T. Wiesler, M.H. Caruthers, J. Org. Chem. 1996, 61, 4272-4281 (Non-Patent Document 4)), have been used to enhance the functionality of immunostimulatory oligonucleotides (A.M. Krieg, S. Matson, E. Fisher, Antisense Nucleic Acid Drug Dev. 1996, 6, 133-139 (Non-Patent Document 5)), siRNAs (e.g., X. Yang, M. Sierant, M. Janicka, L. Peczek, C. Martinez, T. Hassell, N. Li, X. Li, T. Wang, B. Nawrot, ACS Chem. Biol. 2012, 7, 1214-1220 (Non-Patent Document 6)), and aptamers (e.g., X. Yang, S. Fennewald, B.A. Luxon, This method is applied in J. Aronson, NK Herzog, DG Gorenstein, Bioorg. Med. Chem. Lett. 1999, 9, 3357-3362 (Non-Patent Document 7).Interestingly, attempts to utilize this non-chiral modification in the context of antisense oligonucleotides have met with limited success to date (see, e.g., M.K. Ghosh, K. Ghosh, O. Dahl, J.S. Cohen, Nucleic Acids Res. 1993, 21, 5761-5766 (Non-Patent Document 8) and J.P. Vaughn, J. Stekler, S. Demirdji, J.K. Mills, M.H. Caruthers, J.D. Iglehart, J.R. Marks, Nucleic Acids Res. 1996, 24, 4558-4564 (Non-Patent Document 9)).

[0003] Surprisingly, the present inventors have now discovered that non-bridging phosphorodithioates can be introduced into oligonucleotides, particularly into general oligonucleotide gapmers or mixers, particularly LNA-DNA-LNA gapmers or LNA / DNA mixers. This modification is well tolerated, and the resulting molecules show great potential for therapeutic applications, with each non-bridging phosphorodithioate modification reducing the overall library size of potential diastereoisomers by 50%. When this modification is placed in the LNA flank of a gapmer, the resulting oligonucleotide is typically more potent than any of the corresponding parent phosphorothioates. This modification is typically more well tolerated within the gap region, and even more surprisingly, when placed appropriately, can also result in improved potency.

[0004] Therefore, the present inventors have surprisingly discovered that the present invention provides oligonucleotides with improved physicochemical and pharmacological properties, including, for example, improved efficacy.In some aspects, the oligonucleotides of the present invention retain activity or efficacy and can be as potent as or more potent than the same compounds (phosphorothioate reference compounds) in which the phosphodithioate linkages of formula (IA or IBIB) are replaced with conventional stereo-random phosphorothioate linkages.The introduction of any non-bridging phosphorodithioate modification removes one of the chiral centers at the phosphorus position, thereby reducing the diastereomeric complexity of the compound by 50%.In addition, whenever a dithioate modification is introduced, the oligonucleotide appears to be significantly better taken up by cells, particularly hepatocytes, muscle cells, cardiac cells, etc.

[0005] The introduction of non-bridging dithioate modifications into the LNA flanks of gapmers appears to be particularly beneficial, resulting in molecules that exhibit greater target reduction as well as substantially better uptake behavior, greater stability, and a better stability profile.

[0006] The chemical synthesis of non-bridging phosphorodithioate linkages in oligonucleotides is best achieved using solid-phase oligonucleotide synthesis techniques with appropriate thiophosphoramidite building blocks. The successful application of such thiophosphoramidites has been described for conventional DNA (X. Yang, Curr Protoc Nucleic Acid Chem 2016, 66, 4.71.71-74.71.14. (Non-Patent Document 10)) and RNA (X. Yang, Curr Protoc Nucleic Acid Chem 2017, 70, 4.77.71-74.77.13. (Non-Patent Document 11)), and the required building blocks are available from commercial suppliers. Interestingly, the synthesis of the corresponding LNA thiophosphoramidites, which are more challenging, has not been reported. In this application, we report the successful synthesis of all four LNA thiophosphoramidites and their incorporation into oligonucleotides. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] ST Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. ed., Boca Raton, FL: CRC Press, 2008 [Non-patent document 2] F. Eckstein, Antisense and Nucleic Acid Drug Development 2009, 10, 117-121 [Non-patent document 3] N. Oka, M. Yamamoto, T. Sato, T. Wada, J. Am. Chem. Soc. 2008, 130, 16031-16037 [Non-patent document 4] WT Wiesler, MH Caruthers, J. Org. Chem. 1996, 61, 4272-4281

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[0008] Description of the invention The present invention relates to a compound of formula (I) TIFF0007724259000001.tif28128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 ) is an LNA nucleoside and R is a hydrogen or phosphate protecting group. The present invention further relates to gapmer oligonucleotides, in particular those comprising a phosphorodithioate internucleoside linkage of formula (I). The present invention also relates to methods for producing the oligonucleotides according to the invention, and to LNA nucleoside monomers that are particularly useful in producing the oligonucleotides according to the invention.

[0009] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000002.tif49128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 and wherein in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation. The present invention relates to oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0010] In other words, M is a metal, such as an alkali metal, such as Na or K, or M is NH4.

[0011] The present invention provides antisense oligonucleotides comprising phosphorodithioate internucleoside linkages of formula IA or IB as described herein. The oligonucleotides of the present invention are preferably single-stranded antisense oligonucleotides comprising one or more 2' sugar-modified nucleosides, such as one or more LNA nucleosides or one or more 2' MOE nucleosides. The antisense oligonucleotides of the present invention can regulate the expression of target nucleic acids, such as target pre-mRNAs or target microRNAs, in cells expressing the target RNA in vivo or in vitro. In some embodiments, the single-stranded antisense oligonucleotides further comprise phosphorothioate internucleoside linkages. The single-stranded antisense oligonucleotides can be in the form of, for example, gapmer oligonucleotides, mixmer oligonucleotides, or totalmer oligonucleotides. The single-stranded antisense oligonucleotide mixmers can be used to regulate splicing events in target pre-mRNAs. The single-stranded antisense oligonucleotide mixmers can be used to inhibit the expression of target microRNAs.

[0012] The present invention further relates to the use of the oligonucleotides of the invention, such as single-stranded antisense oligonucleotides, as therapeutic agents.

[0013] The present invention further relates to mixmer oligonucleotides, particularly those containing phosphorodithioate internucleoside linkages of formula (IA or IB).The present invention further relates to totalmer oligonucleotides, particularly those containing phosphorodithioate internucleoside linkages of formula (IA or IB).

[0014] The present invention also relates to methods for producing oligonucleotides according to the invention, and to LNA nucleoside monomers that are particularly useful in the production of oligonucleotides according to the invention.

[0015] The present invention also relates to methods for producing oligonucleotides according to the invention, and to MOE nucleoside monomers that are particularly useful in producing oligonucleotides according to the invention.

[0016] The present invention further provides novel MOE and LNA monomers that can be used in the manufacture of oligonucleotides according to the invention.

[0017] During oligonucleotide synthesis, the use of protective R group is often used.After oligonucleotide synthesis, protective group is usually exchanged with hydrogen atom, or for example, when oligonucleotide exists in the form of salt, it is exchanged with cation such as alkali metal cation or ammonium cation.Typically, salt comprises cation such as metal cation, for example, sodium cation or potassium cation, or ammonium cation.For antisense oligonucleotide, preferably, R is hydrogen, or antisense oligonucleotide exists in the form of salt (as shown in IB).

[0018] The phosphorodithioate internucleoside linkage of formula (IB) is, for example, TIFF0007724259000003.tif28153 (wherein M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation; or M+ is an ammonium cation). Thus, the oligonucleotide of the present invention may exist in the form of a salt, an alkali metal salt, for example, a sodium salt, a potassium salt, or an ammonium salt of the oligonucleotide.

[0019] Alternatively expressed, the oligonucleotides of the invention can be represented by the formula IA' or IB' TIFF0007724259000004.tif59128 may contain phosphorodithioate internucleoside linkages.

[0020] The present invention further relates to gapmer oligonucleotides comprising phosphorodithioate internucleoside linkages of formula (I), particularly to formula IA or IB or formula IA' or formula IB'.

[0021] The present invention further relates to mixer oligonucleotides comprising phosphorodithioate internucleoside linkages of formula (I), particularly to formula IA or IB or formula IA' or formula IB'.

[0022] The present invention further relates to totalmer oligonucleotides comprising phosphorodithioate internucleoside linkages of formula (I), in particular to formula IA or IB or to formula IA' or IB'.

[0023] In a preferred embodiment of the oligonucleotide of the present invention, two nucleosides (A 1 ) and (A 2 ) is an LNA nucleoside.

[0024] In a preferred embodiment of the oligonucleotide of the present invention, two nucleosides (A 1 ) and (A 2 ) is a 2'-O-MOE nucleoside.

[0025] In a preferred embodiment of the oligonucleotide of the present invention, the oligonucleotide is a single-stranded antisense oligonucleotide, which contains two nucleosides (A 1 ) and (A 2 ) is an LNA nucleoside.

[0026] In a preferred embodiment of the oligonucleotide of the present invention, the oligonucleotide is a single-stranded antisense oligonucleotide and contains two nucleosides (A 1 ) and (A 2 ) is a 2'-O-MOE nucleoside.

[0027] The present invention provides antisense oligonucleotides for inhibiting a target RNA in a cell, the antisense gapmer oligonucleotide having formula (IA) or (IB): TIFF0007724259000005.tif46128 (wherein in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na cation or a K cation, or M is an ammonium cation) and the antisense oligonucleotide is or comprises an antisense gapmer oligonucleotide (referred to herein as a gapmer or gapmer oligonucleotide).

[0028] Thus, the antisense oligonucleotides of the present invention may comprise or consist of gapmers.

[0029] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000006.tif45128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 ) is an LNA nucleoside, and in formula (IA), R is a hydrogen or phosphate protecting group, and in formula (IB), M is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na cation or a K cation, or M is an ammonium cation, and A 2 is the 3'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0030] The present invention relates to compounds of formula (IA) or (IB) TIFF0007724259000007.tif46128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 ) is an LNA nucleoside, and in formula (IA), R is a hydrogen or phosphate protecting group, and in formula (IB), M is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na cation or a K cation, or M is an ammonium cation, and A 1 is the 5'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0031] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000008.tif46128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 at least one of (IA) is a 2-O-MOE nucleoside, and in (IA), R is a hydrogen or phosphate protecting group, and in (IB), M is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na cation or a K cation, or M is an ammonium cation; 2 is the 3'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0032] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000009.tif46128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 at least one of (IA) is a 2-O-MOE nucleoside, and in (IA), R is a hydrogen or phosphate protecting group, and in (IB), M is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na cation or a K cation, or M is an ammonium cation; 1 is the 5'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0033] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000010.tif44128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 ) is a 2' sugar-modified nucleoside, and in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation, and A 2 is the 3'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0034] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000011.tif47128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and is attached to the 5' carbon atom of two nucleosides (A 1 ) and (A 2 ) is a 2' sugar-modified nucleoside, and in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation, and A 1 is the 5'-terminal nucleoside of the oligonucleotide) The present invention provides antisense oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0035] The 2' sugar-modified nucleosides may be independently selected from the group consisting of 2' sugar-modified nucleosides selected from the group consisting of 2'-alkoxy-RNA nucleosides, 2'-alkoxyalkoxy-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 2'-fluoro-ANA nucleosides, and LNA nucleosides.

[0036] The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000012.tif45128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 and wherein in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation. a single-stranded antisense oligonucleotide comprising at least one phosphorodithioate internucleoside linkage of The single-stranded oligonucleotide contains at least one stereodefined phosphorothioate internucleoside linkage (Sp, S) or (Rp, R) TIFF0007724259000013.tif45128 (N1 and N2 are nucleosides) Further includes:

[0037] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000014.tif45128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2), and R is a hydrogen or phosphate protecting group). is a phosphorodithioate bond.

[0038] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000015.tif45128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2); and in formula (IA), R is a hydrogen or phosphate protecting group; and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation). is a phosphorodithioate bond of The single-stranded antisense oligonucleotides are for use in modulating splicing of a target RNA that is a pre-mRNA.

[0039] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000016.tif45128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2); and in formula (IA), R is a hydrogen or phosphate protecting group; and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation). is a phosphorodithioate bond of The single-stranded antisense oligonucleotide is used to inhibit the expression of long non-coding RNA.For examples of lncRNA that can be targeted by the compounds of the present invention, see WO 2012 / 065143.

[0040] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000017.tif45128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2), and in the formula (IA), R is hydrogen or a phosphate protecting group, and in the formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation), The single-stranded antisense oligonucleotides are for use in inhibiting expression of a target that is a human mRNA or pre-mRNA.

[0041] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000018.tif45128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2); and in formula (IA), R is a hydrogen or phosphate protecting group; and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation). is a phosphorodithioate bond of The single-stranded antisense oligonucleotide is for use in inhibiting expression of a viral RNA target. Suitable viral RNA targets may be, for example, HCV or HBV.

[0042] The present invention also provides a single-stranded antisense oligonucleotide for modulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 7 to 30 nucleotides in length, the contiguous nucleotide sequence comprising one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000019.tif45128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 and the other is attached to the 3' carbon atom of another adjacent nucleoside (A2), and in the formula (IA), R is a hydrogen or phosphate protecting group, and in the formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation. is a phosphorodithioate bond of The single-stranded antisense oligonucleotide is for use in inhibiting the expression of a microRNA.

[0043] When targeting RNA targets, such as pre-mRNA targets, mRNA targets, viral RNA targets, microRNA targets, or long non-coding RNA targets, the oligonucleotides of the present invention can suitably inhibit the expression of target RNA.This is achieved by the complementarity of antisense oligonucleotides and target RNA.Inhibition of RNA targets can be achieved by reducing the level of RNA targets or by interfering with the function of RNA targets.The RNA inhibition of RNA targets can be suitably achieved by recruiting cellular RNases such as RNase H, for example, through the use of gapmers, or by non-nuclease-mediated mechanisms, such as steric interference mechanisms (for example, in the case of microRNA inhibition, in the case of pre-mRNA splicing regulation, or in the case of interfering with the interaction of long non-coding RNA with chromatin).

[0044] The present invention also relates to methods for producing oligonucleotides according to the invention, and to LNA or MOE nucleoside monomers that are particularly useful in the production of oligonucleotides according to the invention.

[0045] The present invention provides pharmaceutically acceptable salts of the oligonucleotides according to the invention or conjugates thereof, in particular the sodium, potassium or ammonium salts.

[0046] The present invention provides a conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt thereof and at least one conjugate moiety covalently attached to the oligonucleotide or the pharmaceutically acceptable salt thereof, optionally via a linker moiety.

[0047] The present invention provides a pharmaceutical composition comprising an oligonucleotide according to the invention, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier.

[0048] The present invention provides an oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to the invention for use as a therapeutically active substance.

[0049] The invention provides a method for modulating a target RNA in a cell expressing said RNA, the method comprising administering to the cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate, or composition according to the invention, wherein said oligonucleotide is complementary to said target RNA.

[0050] The present invention provides a method for modulating splicing of a target pre-mRNA in a cell expressing said pre-mRNA, the method comprising the step of administering to the cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate or composition according to the invention, wherein said oligonucleotide is complementary to said target RNA and is capable of modulating a splicing event in said pre-mRNA.

[0051] The invention provides the use of an oligonucleotide, pharmaceutical salt, conjugate, or composition of the invention to inhibit pre-mRNA, mRNA, or long non-coding RNA in a cell, such as a human cell.

[0052] The above method or use may be an in vitro method or an in vivo method.

[0053] The invention provides the use of an oligonucleotide, pharmaceutical salt, conjugate, or composition of the invention in the manufacture of a medicament.

[0054] The present invention provides the use of phosphorodithioate internucleoside linkages of formula IA or IB for use in enhancing the in vitro or in vivo stability of single-stranded phosphorothioate antisense oligonucleotides.

[0055] The present invention provides the use of phosphorodithioate internucleoside linkages of formula IA or IB for use in extending the in vitro or in vivo duration of action of single-stranded phosphorothioate antisense oligonucleotides.

[0056] The present invention provides the use of phosphorodithioate internucleoside linkages of formula IA or IB for use in increasing the cellular uptake or tissue distribution of single-stranded phosphorothioate antisense oligonucleotides.

[0057] The present invention provides the use of a phosphorodithioate internucleoside linkage of Formula IA or IB for use in increasing the uptake of single-stranded phosphorothioate antisense oligonucleotides into tissues selected from the group consisting of skeletal muscle, heart, epithelial cells, including retinal epithelial cells (e.g., in the case of Htra1 targeting compounds), liver, kidney, or spleen.

[0058] For in vivo use, single-stranded phosphorothioate antisense oligonucleotides may be therapeutic oligonucleotides. [The present invention 1001] Formula (II) Compound TIFF0007724259000020.tif35128: During the ceremony, 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 -CR a R b -, -N(R a )-O-, or -O-CRa R b - and; 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-, -Si(R a )2-, -SO2-, -NR a -, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O-, or -O-CR a R b - and; However, -XY- is -OO-, Si(R a )2-Si(R a )2-, -SO2-SO2-, -C(R a )=C(R b )-C(R a )=C(R b ), -C(R a )=NC(R a )=N-, -C(R a )=NC(R a )=C(R b ), -C(R a )=C(R b )-C(R a )=N- and -Se-Se-; J is oxygen, sulfur, =CH2, or =N(R a ) and; R a and R bis hydrogen, halogen, hydroxyl, cyano, thiohydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, carbamido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, thiohydroxyl sulfido alkylsulfanyl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC(=X a )R c , -OC(=X a )NR c R d , and -NR e C(=X a )NR c R d more independently selected, or Two geminal R's a and R b together form an optionally substituted methylene, or Two geminal R's a and R b together with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl having only one carbon atom, -XY-; 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, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl, and heteroaryl; X a is oxygen, sulfur, or -NRc and; R c , R d , and R e are independently selected from hydrogen and alkyl; and n is 1, 2, or 3; R 5 is a hydroxyl protecting group; R x is phenyl, nitrophenyl, phenylalkyl, halophenylalkyl, cyanoalkyl, phenylcarbonylsulfanylalkyl, halophenylcarbonylsulfanylalkylalkylcarbonylsulfanylalkyl, or alkylcarbonylcarbonylsulfanylalkyl; R y is dialkylamino or pyrrolidinyl; and Nu is a nucleobase or a protected nucleobase. [The present invention 1002] 1001. The compound of claim 1001, wherein XY- is -CH2-O-, -CH(CH3)-O-, or -CH2CH2-O-. [The present invention 1003] Formula (III) or (IV) TIFF0007724259000021.tif35128, where R 5 , R x , R y and Nu is as defined in invention 1001. [The present invention 1004] Formula (IIb) Compound TIFF0007724259000022.tif37128: During the ceremony, R 5 is a hydroxyl protecting group; R xis phenyl, nitrophenyl, phenylalkyl, halophenylalkyl, cyanoalkyl, phenylcarbonylsulfanylalkyl, halophenylcarbonylsulfanylalkylalkylcarbonylsulfanylalkyl, or alkylcarbonylcarbonylsulfanylalkyl; R y is dialkylamino or pyrrolidinyl; and Nu is a nucleobase or a protected nucleobase. [The present invention 1005] R x is phenyl, nitrophenyl, phenylmethyl, dichlorophenylmethyl, cyanoethyl, methylcarbonylsulfanylethyl, ethylcarbonylsulfanylethyl, isopropylcarbonylsulfanylethyl, tert-butylcarbonylsulfanylethyl, methylcarbonylcarbonylsulfanylethyl, or difluorophenylcarbonylsulfanylethyl. [The present invention 1006] R x The compound of any one of 1001 to 1005 of the present invention, wherein is phenylcarbonylsulfanylalkyl. [The present invention 1007] R x The compound of any one of 1001 to 1006 of the present invention, wherein is phenylcarbonylsulfanylethyl. [The present invention 1008] R y The compound of any one of claims 1001 to 1007, wherein is diisopropylamino or pyrrolidinyl. [The present invention 1009] R y The compound of any one of 1001 to 1008, wherein is pyrrolidinyl. [The present invention 1010] Formula (V) TIFF0007724259000023.tif47128, where R 5 and Nu is as defined in invention 1001. [The present invention 1011] Formula (Vb) TIFF0007724259000024.tif50128, where R 5 and Nu is as defined in invention 1004. [The present invention 1012] 10. The compound of any one of claims 1001 to 1011, wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil, or protected uracil. [The present invention 1013] Any of compounds 1001 to 1012 of the present invention selected from TIFF0007724259000025.tif110134 and TIFF0007724259000026.tif164139. [The present invention 1014] Any of compounds 1001 to 1012 of the present invention selected from TIFF0007724259000027.tif172139 and TIFF0007724259000028.tif113138. [The present invention 1015] A method for producing a compound of formula (II) or (IIb) of any of inventions 1001 to 1014, comprising reacting a 5'-protected LNA nucleoside or a 5'-protected MOE nucleoside with a phosphine and a monoprotected dithiol in the presence of an acidic coupling agent and a silylating agent. [The present invention 1016] in the presence of an acidic coupling agent and a silylating agent, TIFF0007724259000029.tif30128 compound with formula P(R y )3 and the compound of formula HSR x wherein X, Y, R 5 ,Nu,R. x , and R y The method of invention 1015, as defined in any one of inventions 1001 to 1012. [The present invention 1017] in the presence of an acidic coupling agent and a silylating agent, Compound TIFF0007724259000030.tif25128 and formula P(R y )3 and the compound of formula HSR x wherein R 5 ,Nu,R. x , and R y The method of invention 1015 or 1016, as defined in any of inventions 1001 to 1012. [The present invention 1018] The process of any of claims 1015 to 1017, wherein the crude compound of formula (II) or (IIb) is purified by preparative HPLC. [The present invention 1019] The process of claim 10, wherein the crude compound of formula (II) or (IIb) is eluted using a gradient of acetonitrile versus aqueous ammonium hydroxide. [The present invention 1020] Use of any of the compounds of the present invention 1001 to 1014 in the production of an oligonucleotide. [Brief explanation of the drawings]

[0059] [Figure 1] Figure 1 shows the levels of target mRNA in primary rat hepatocytes 24 and 74 hours after administration of an oligonucleotide gapmer according to the present invention, which has a phosphorodithioate internucleoside linkage in the gap. [Figure 2] Figure 2 shows the levels of target mRNA in primary rat hepatocytes 24 and 74 hours after administration of an oligonucleotide gapmer according to the present invention, which has a plurality of phosphorodithioate internucleoside linkages in the gap. [Figure 3]Figure 3 shows the levels of target mRNA in primary rat hepatocytes 24 and 74 hours after administration of an oligonucleotide gapmer according to the present invention, which has a plurality of phosphorodithioate internucleoside linkages in the gap. [Figure 4] Figure 4 shows the levels of target mRNA in primary rat hepatocytes 24 and 74 hours after administration of an oligonucleotide gapmer according to the invention, flanked by phosphorodithioate internucleoside linkages. [Figure 5] 1 shows the thermal melting (Tm) of oligonucleotides containing phosphorodithioate internucleoside linkages according to the present invention hybridized to RNA and DNA. [Figure 6] 1 shows the stability in rat serum of oligonucleotides containing phosphorodithioate internucleoside linkages according to the present invention. [Figure 7] Investigation of achiral phosphodithioates in the gap and flanking regions of gapmers - residual mRNA levels after treatment of primary rat hepatocytes. [Figure 8] Investigation into the positional dependence and optimization strategy of achiral phosphodithioates in the gap region of gapmers - residual mRNA levels after treatment of primary rat hepatocytes. [Figure 9A] Investigation of achiral phosphodithioates in the gap region of gapmers - effects on cellular uptake. [Figure 9B] Investigation of achiral phosphodithioates in the gap region of gapmers - effects on cellular uptake. [Figure 10A]Introducing achiral phosphorodithioates into the flank regions of gapmers increases potency, with a correlation between the amount of phosphorothioates and increased potency (number of phosphorodithioate linkages in the flank: 4 > 3 > 2 > 1 > no linkages). [Figure 10B] Introducing achiral phosphorodithioates into the flank regions of gapmers increases potency, with a correlation between the amount of phosphorothioates and increased potency (number of phosphorodithioate linkages in the flank: 4 > 3 > 2 > 1 > no linkages). [Figure 11] IC50 values in different cell types. [Figure 12] In vitro rat serum stability of 3'-end-protected LNA oligonucleotides. [Figure 13] In vivo evaluation of gapmers containing achiral phosphorodithioate linkages in the flank and gap regions—target inhibition. [Figure 14A] In vivo evaluation of gapmers containing achiral phosphorodithioate linkages in the flank and gap regions—tissue uptake. [Figure 14B] In vivo evaluation of gapmers containing achiral phosphorodithioate linkages in the flank and gap regions—liver / kidney ratio. [Figure 15A] In vivo evaluation of gapmers containing achiral phosphorodithioate linkages in the flank and gap regions—metabolite analysis. [Figure 15B] In vivo evaluation of gapmers containing achiral phosphorodithioate linkages in the flank and gap regions—metabolite analysis. [Figure 16] The long duration of action associated with antisense oligonucleotides containing achiral phosphorodithioate internucleoside linkages can be further extended by combining them with stereodefined phosphorothioate internucleoside linkages. [Figure 17A] In vitro EC50 determination of achiral phosphorodithioate gapmers targeting MALAT-1. [Figure 17B] In vivo efficacy of achiral phosphorodithioate gapmers targeting MALAT-1. [Figure 17C] In vivo study of achiral phosphorodithioate gapmers targeting MALAT-1—tissue content. [Figure 18A] In vitro studies of achiral monophosphorothioate-modified gapmer oligonucleotides targeting ApoB. Activity data. [Figure 18B] In vitro study of achiral monophosphorothioate-modified gapmer oligonucleotides targeting ApoB. Cellular content data. [Figure 19A] In vitro studies of chiral phosphorodithioate-modified gapmer oligonucleotides targeting ApoB. Activity data. [Figure 19B] In vitro studies of chiral phosphorodithioate-modified gapmer oligonucleotides targeting ApoB. Cellular content data. [Figure 20] Effect of achiral phosphorodithioate (P2S) internucleoside linkages present in splice-switching oligonucleotides targeting the 3' splice site of TNFRSF1B. Human Colo 205 cells were seeded in 96-well plates and exposed to 5 μM (A) and 25 μM (B) oligos, respectively. The percentage of exon 7 skipping was analyzed by droplet digital PCR using a probe targeting the exon 6-8 junction and compared with the total amount of TNFRSF1B measured by an assay targeting exons 2-3. SSO#26 is the parent oligo, and SSO#27 is a negative control that does not target TNFRSF1B. [Figure 21] Stability assay using S1 nuclease. Dithioate-containing oligos were incubated with S1 nuclease for 30 and 120 minutes, respectively. These oligos were visualized on a 15% TBE-urea gel. As a marker for migration of intact oligos, (SSO#14) was included without exposure to S1 nuclease. DETAILED DESCRIPTION OF THE INVENTION

[0060] definition As used herein, the term "alkyl," alone or in combination, refers to a straight-chain or branched-chain alkyl group having 1 to 8 carbon atoms, specifically a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, and more specifically a straight-chain or branched-chain alkyl group having 1 to 4 carbon atoms. Examples of straight-chain and branched-chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, isomeric hexyls, isomeric heptyls, and isomeric octyls, particularly methyl, ethyl, propyl, butyl, and pentyl. Specific examples of alkyl are methyl, ethyl, and propyl.

[0061] The term "cycloalkyl," alone or in combination, refers to a cycloalkyl ring having 3 to 8 carbon atoms, and specifically a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, more specifically cyclopropyl and cyclobutyl. A specific example of "cycloalkyl" is cyclopropyl.

[0062] The term "alkoxy", alone or in combination, means a group having the formula alkyl-O-, where the term "alkyl" has the meaning given above, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Particular "alkoxy" are methoxy and ethoxy. Methoxyethoxy is a particular example of "alkoxyalkoxy".

[0063] The term "oxy", alone or in combination, signifies the -O- group.

[0064] The term "alkenyl", alone or in combination, denotes a straight-chain or branched hydrocarbon residue containing an olefinic bond and up to 8, preferably up to 6, particularly preferably up to 4 carbon atoms. Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, and isobutenyl.

[0065] The term "alkynyl", alone or in combination, means a straight-chain or branched hydrocarbon residue containing a triple bond and up to 8, preferably 2, carbon atoms.

[0066] The terms "halogen" or "halo," alone or in combination, mean fluorine, chlorine, bromine, or iodine, and specifically fluorine, chlorine, or bromine, more specifically fluorine. The term "halo," in combination with another group, means substitution of the group with at least one halogen, specifically 1 to 5 halogens, particularly 1 to 4 halogens, i.e., substitution with 1, 2, 3, or 4 halogens.

[0067] The term "haloalkyl," alone or in combination, refers to an alkyl group substituted with at least one halogen, specifically substituted with 1 to 5 halogens, especially 1 to 3 halogens. Examples of haloalkyl include monofluoro-, difluoro-, or trifluoro-methyl, -ethyl, or -propyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. Fluoromethyl, difluoromethyl, and trifluoromethyl are specific "haloalkyl."

[0068] The term "halocycloalkyl," alone or in combination, means a cycloalkyl group as defined above substituted with at least one halogen, particularly 1 to 5 halogens, and especially 1 to 3 halogens. Specific examples of "halocycloalkyl" include halocyclopropyl, especially fluorocyclopropyl, difluorocyclopropyl, and trifluorocyclopropyl.

[0069] The terms "hydroxyl" and "hydroxy", alone or in combination, refer to an --OH group.

[0070] The terms "thiohydroxyl" and "thiohydroxy", alone or in combination, refer to an --SH group.

[0071] The term "carbonyl", alone or in combination, signifies the -C(O)- group.

[0072] The terms "carboxy" or "carboxyl," alone or in combination, refer to a --COOH group.

[0073] The term "amino," alone or in combination, means a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group (-N-).

[0074] The term "alkylamino", alone or in combination, means an amino group as defined above substituted with one or two alkyl groups as defined above.

[0075] The term "sulfonyl", alone or in combination, means the -SO2 group.

[0076] The term "sulfinyl", alone or in combination, signifies the -SO- group.

[0077] The term "sulfanyl", alone or in combination, signifies the -S- group.

[0078] The term "cyano", alone or in combination, signifies the -CN group.

[0079] The term "azido", alone or in combination, means the -N3 group.

[0080] The term "nitro," alone or in combination, means an NO2 group.

[0081] The term "formyl", alone or in combination, means the group -C(O)H.

[0082] The term "carbamoyl", alone or in combination, means the group -C(O)NH2.

[0083] The term "carbamido", alone or in combination, means the group -NH-C(O)-NH2.

[0084] The term "aryl," alone or in combination, means a monovalent aromatic carbocyclic monocyclic or bicyclic ring system containing 6 to 10 carbon ring atoms optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl include phenyl and naphthyl, especially phenyl.

[0085] The term "heteroaryl," alone or in combination, means a monovalent aromatic heterocyclic monocyclic or bicyclic ring system having 5 to 12 ring atoms containing 1, 2, 3, or 4 heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon, which may be optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of heteroaryl 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, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, carbazolyl, or acridinyl.

[0086] The term "heterocyclyl", alone or in combination, means a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system having 4 to 12, particularly 4 to 9, ring atoms containing 1, 2, 3, or 4 ring heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon, which may be optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of monocyclic saturated heterocyclyl are azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, 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 dihydrofuryl, imidazolinyl, dihydrooxazolyl, tetrahydropyridinyl, or dihydropyranyl.

[0087] The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the free base or free acid, but are biologically or otherwise undesirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, especially hydrochloric acid, as well as 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, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. Furthermore, these salts may be prepared by the addition of inorganic or organic bases to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary amines, secondary amines, and tertiary amines, substituted amines including natural substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resin salts.The oligonucleotides of the present invention can also exist in zwitterionic form.The particularly preferred pharmaceutically acceptable salts of the present invention are sodium salt, lithium salt, potassium salt, and trialkylammonium salt.

[0088] The term "protecting group" refers to groups that, alone or in combination, selectively block a reactive site in a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site. The protecting group can be removed. Exemplary protecting groups are amino-protecting groups, carboxy-protecting groups, or hydroxy-protecting groups.

[0089] 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.

[0090] A "hydroxyl protecting group" is a protecting group for a hydroxyl group, and is also used to protect a thiol group. Examples of hydroxyl 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 ethers (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), methyl ether, and ethoxyethyl ether (EE). Specific examples of hydroxyl protecting groups are DMT and TMT, especially DMT.

[0091] A "thiohydroxyl protecting group" is a protecting group for a thiohydroxyl group. Examples of thiohydroxyl protecting groups are those of a "hydroxyl protecting group."

[0092] If one of the starting materials or compounds of the present invention contains one or more functional groups that are not stable or reactive under the reaction conditions of one or more reaction steps, methods well known in the art may be applied to provide suitable protecting groups (e.g., those described in "Protective Groups in Organic Chemistry" by TW Greene and PGM Wuts, 3 rd(Ed., 1999, Wiley, New York) can be introduced before that critical step. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature. Examples of protecting groups are tert-butoxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), carbobenzyloxy (Cbz), and p-methoxybenzyloxycarbonyl (Moz).

[0093] The compounds described herein may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g., racemates, mixtures of diastereoisomers, diastereoisomeric racemates, or mixtures of diastereoisomeric racemates.

[0094] Oligonucleotides The term "oligonucleotide" used herein is defined as a molecule that contains two or more covalently linked nucleosides, as is commonly understood by those skilled in the art.Such covalently linked nucleosides can also be called nucleic acid molecules or nucleic acid oligomers.Generally, oligonucleotides are produced in laboratories by solid-phase chemical synthesis and subsequent purification.When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moiety of covalently linked nucleotides or nucleosides or their modification.The oligonucleotides of the present invention are artificial, are chemically synthesized, and are typically purified or isolated.The oligonucleotides of the present invention can contain one or more modified nucleosides or nucleotides.

[0095] antisense oligonucleotides The term " antisense oligonucleotide " used herein is defined as the oligonucleotide that can regulate the expression of target gene by hybridizing with target nucleic acid, particularly the continuous sequence of target nucleic acid.Antisense oligonucleotide is not essentially double-stranded, and therefore is not siRNA or shRNA.Preferably, the antisense oligonucleotide of the present invention is single-stranded.It is understood that the single-stranded oligonucleotide of the present invention can form hairpin or intermolecular duplex structure (the duplex between two molecules of the same oligonucleotide), as long as the degree of internal or mutual self-complementarity over the entire length of the oligonucleotide is less than 50%.

[0096] Regulation of expression The term "modulation of expression" used herein should be understood as a general term for the ability of an oligonucleotide to change the expression or level of a target nucleic acid. The modulation of expression can be determined by comparing the expression or level of the target nucleic acid before the administration of the oligonucleotide, or the modulation of expression can be determined by referring to a control experiment in which the oligonucleotide of the present invention is not administered. It is generally understood that the control is an individual or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting oligonucleotide (mock).

[0097] One type of regulation is the ability of oligonucleotide to inhibit, down-regulate, reduce, suppress, eliminate, stop, interfere, hinder, reduce, decrease, disable or terminate the expression of target nucleic acid, for example, by degrading target nucleic acid (for example, by RNase H1-mediated degradation) or interfering with transcription.Another type of regulation is the ability of oligonucleotide to restore, increase or enhance the expression of target RNA, for example, by regulating the splicing event of target pre-mRNA or by interfering with inhibitory mechanisms such as microRNA suppression of mRNA.

[0098] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to, e.g., completely complementary to, a target nucleic acid. This term is used herein synonymously with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence, such as an FG-F' gapmer region, and may optionally comprise a nucleotide linker region, e.g., region D or D', that can be used to attach another nucleotide, e.g., a functional group, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. The antisense oligonucleotide mixer referred to herein may comprise or consist of a contiguous nucleotide sequence.

[0099] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both natural and non-natural nucleotides.Naturally, nucleotides, such as DNA nucleotides and RNA nucleotides, contain a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0100] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that has been modified relative to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications to the sugar or (nucleic acid) base moiety. In a preferred embodiment, the modified nucleoside contains a modified sugar moiety. As used herein, the term "modified nucleoside" may also be used synonymously with the term "nucleoside analog" or modified "unit" or "monomer." Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Typically, nucleosides with modifications in the base region of a DNA or RNA nucleoside continue to be referred to as DNA or RNA if Watson-Crick base pairing is possible.

[0101] Modified internucleoside linkages The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently couples two nucleosides together, as commonly understood by those skilled in the art. Thus, the oligonucleotide of the present invention may contain a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. In the case of natural oligonucleotides, the internucleoside linkage comprises a phosphate group that creates a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and can serve to protect regions of DNA or RNA nucleosides in the oligonucleotide of the present invention, such as in the gap region of a gapmer oligonucleotide, and regions of modified nucleosides, such as regions F and F', from nuclease cleavage.

[0102] In some embodiments, the oligonucleotide comprises one or more internucleoside linkages modified from natural phosphodiester, such as one or more modified internucleoside linkages that are more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. An internucleoside linkage that can enhance the nuclease resistance of an oligonucleotide is called a nuclease-resistant internucleoside linkage. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or its consecutive nucleotide sequence are modified, for example, at least 60%, for example, at least 70%, for example, at least 80%, or for example, at least 90% of the internucleoside linkages in the oligonucleotide or its consecutive nucleotide sequence are nuclease-resistant internucleoside linkages. In some embodiments, all of the internucleoside linkages in the oligonucleotide or its consecutive nucleotide sequence are nuclease-resistant internucleoside linkages. It is recognized that in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be a phosphodiester.

[0103] A preferred modified internucleoside linkage for use in the oligonucleotides of the invention is phosphorothioate.

[0104] Phosphorothioate internucleoside linkages are particularly useful due to their nuclease resistance, favorable pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages in an oligonucleotide or its consecutive nucleotide sequence are phosphorothioate, for example, at least 60%, for example, at least 70%, for example, at least 80%, or for example, at least 90% of the internucleoside linkages in an oligonucleotide or its consecutive nucleotide sequence are phosphorothioate. In some embodiments, all of the internucleoside linkages in an oligonucleotide or its consecutive nucleotide sequence, except for the phosphorodithioate internucleoside linkages, are phosphorothioate. In some embodiments, the oligonucleotide of the present invention contains both phosphorothioate internucleoside linkages and at least one phosphodiester linkage, for example, two, three, or four phosphodiester linkages, in addition to phosphorodithioate linkages. In gapmer oligonucleotides, it is suitable that phosphodiester linkages, if present, are not located between consecutive DNA nucleosides in the gap region G.

[0105] Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of an oligonucleotide that can recruit nucleases upon duplexing with a target nucleic acid, e.g., region G in the case of a gapmer. However, phosphorothioate linkages may also be useful in regions that do not recruit nucleases and / or affinity-enhancing regions, e.g., regions F and F' in the case of a gapmer. In some embodiments, a gapmer oligonucleotide may contain one or more phosphodiester linkages in region F or F' or both regions F and F', and the internucleoside linkages in region G may be entirely phosphorothioate.

[0106] Advantageously, all internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide or all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0107] As disclosed in EP 2742135, it is recognized that antisense oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), such as alkylphosphonate / methylphosphonate internucleosides, which, according to EP 2742135, may be tolerated, for example, in gap regions that would otherwise be DNA phosphorothioates.

[0108] Sterically random phosphorothioate linkages A phosphorothioate linkage is an internucleoside phosphate linkage in which one of the non-bridging oxygens is replaced with sulfur. The replacement of one of the non-bridging oxygens with sulfur introduces a chiral center, and therefore, within a single phosphorothioate oligonucleotide, each phosphorothioate internucleoside linkage can exist in either the S (Sp) or R (Rp) stereoisoform. Such internucleoside linkages are called "chiral internucleoside linkages." In comparison, phosphodiester internucleoside linkages are not chiral because they have two non-terminal oxygen atoms.

[0109] The designation for the chirality of stereocenters is determined based on the standard Cahn-Ingold-Prelog (CIP) rules, first published in Cahn, RS; Ingold, CK; Prelog, V. (1966) "Specification of Molecular Chirality" Angewandte Chemie International Edition 5 (4): 385-415. doi:10.1002 / anie.196603851.

[0110] During standard oligonucleotide synthesis, the stereoselectivity of coupling and subsequent sulfurization is not controlled. Therefore, the stereochemistry of each phosphorothioate internucleoside linkage is randomly Sp or Rp; therefore, phosphorothioate oligonucleotides produced by conventional oligonucleotide synthesis are, in fact, 2X A stereoisomeric phosphorothioate oligonucleotide can exist as up to 20 different phosphorothioate diastereoisomers (where X is the number of phosphorothioate internucleoside linkages). Herein, such oligonucleotides are referred to as stereo-random phosphorothioate oligonucleotides and do not contain any stereo-defined internucleoside linkages. Therefore, a stereo-random phosphorothioate oligonucleotide is a mixture of individual diastereoisomers resulting from a non-stereo-defined synthesis. In this context, this mixture can contain up to 2 X They are defined as different phosphorothioate diastereoisomers.

[0111] Stereodefined internucleoside linkages A stereodefined internucleoside bond is a chiral internucleoside bond that exhibits a diastereomeric excess of one of the two diastereoisomeric forms, namely Rp or Sp.

[0112] It should be recognized that stereoselective oligonucleotide synthesis methods used in the art typically result in at least about 90% or at least about 95% stereoselectivity at each chiral internucleoside linkage, and thus, up to about 10%, e.g., about 5%, of the oligonucleotide molecules may have alternative diastereoisomeric forms.

[0113] In some embodiments, the ratio of diastereoisomers of each stereodefined chiral internucleoside linkage is at least about 90: 10. In some embodiments, the ratio of diastereoisomers of each chiral internucleoside linkage is at least about 95:5.

[0114] A stereodefined phosphorothioate linkage is a specific example of a stereodefined internucleoside linkage.

[0115] Stereodefined phosphorothioate linkages A stereodefined phosphorothioate bond is a phosphorothioate bond that exhibits a diastereomeric excess of one of the two diastereoisomeric forms, Rp or Sp.

[0116] The Rp and Sp configurations of the phosphorothioate internucleoside linkage are shown below: TIFF0007724259000031.tif32128In the above formula, 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).

[0117] As used herein, an Rp internucleoside linkage may be designated as srP, and an Sp internucleoside linkage may be designated as ssP.

[0118] In certain embodiments, the ratio of diastereoisomers of each stereodefined phosphorothioate linkage is at least about 90:10 or at least 95:5.

[0119] In some embodiments, the ratio of diastereoisomers of each stereodefined phosphorothioate linkage is at least about 97:3. In some embodiments, the ratio of diastereoisomers of each stereodefined phosphorothioate linkage is at least about 98:2. In some embodiments, the ratio of diastereoisomers of each stereodefined phosphorothioate linkage is at least about 99:1.

[0120] In some embodiments, the stereodefined internucleoside linkage is present in the same diastereomeric form (Rp or Sp) in at least 97%, such as at least 98%, such as at least 99%, or (almost) all, of the oligonucleotide molecules present in a population of oligonucleotide molecules.

[0121] Diastereomeric purity can be determined in a model system containing only an achiral backbone (i.e., a phosphodiester). For example, the diastereomeric purity of each monomer can be determined by coupling a monomer having a stereodefined internucleoside linkage to the following model system: 5't-po-t-po-t-po-t-po. The result would then be 5'DMTr-t-srp-t-po-t-po-t-po or 5'DMTr-t-ssp-t-po-t-po-t-po, which can be separated using HPLC. Diastereomeric purity is determined by integrating the UV signals from the two possible diastereoisomers and determining their ratio, e.g., 98:2, 99:1, or greater than 99:1.

[0122] It is understood that the diastereomeric purity of a particular single diastereoisomer (a single stereodefined oligonucleotide molecule) is considered to be a function of the coupling selectivity at the defined stereocenters at each internucleoside position and the number of stereodefined internucleoside linkages introduced. As an example, if the coupling selectivity at each position is 97%, the purity obtained for a stereodefined oligonucleotide having 15 stereodefined internucleoside linkages is 0.97%. 15 The purity of the defined diastereoisomers can be increased after synthesis by purification, for example, by HPLC, such as ion exchange chromatography or reverse phase chromatography.

[0123] In some embodiments, stereodefined oligonucleotides refers to a population of oligonucleotides, wherein at least about 40%, eg, at least about 50%, of the population is the desired diastereoisomer.

[0124] In other words, in some embodiments, a stereodefined oligonucleotide refers to a population of oligonucleotides, wherein at least about 40%, e.g., at least about 50%, of the population consists of a desired (specific) stereodefined internucleoside linkage motif (also called a stereodefined motif).

[0125] For stereodefined oligonucleotides containing both stereorandom and stereodefined internucleoside chiral centers, the purity of the stereodefined oligonucleotide is determined based on the percentage of the oligonucleotide population that retains the desired stereodefined internucleoside linkage motif; the stereorandom linkages are ignored in this calculation.

[0126] Nucleic acid bases The term "nucleobase" 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 during nucleic acid hybridization.In the present invention, the term "nucleobase" also includes modified nucleobases that may differ from natural nucleobases but are functional during nucleic acid hybridization.In this context, "nucleobase" refers to both natural nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants.Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, vol. 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Suppl. 37, 1.4.1.

[0127] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0128] Nucleobase moiety can be represented by the letter symbol for each corresponding nucleobase, for example, A, T, G, C or U, where each letter can optionally comprise the modified nucleobase with equivalent function.For example, in the exemplified oligonucleotide, nucleobase moiety is selected from A, T, G, C and 5-methylcytosine.Optionally, in the case of LNA gapmer, 5-methylcytosine LNA nucleoside can be used.

[0129] Modified Oligonucleotides The term "modified oligonucleotide" refers to an oligonucleotide containing one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term that has been used in the literature to refer to oligonucleotides having modified nucleosides.

[0130] Stereodefined oligonucleotides A stereodefined oligonucleotide is an oligonucleotide in which at least one of the internucleoside linkages is a stereodefined internucleoside linkage.

[0131] A stereodefined phosphorothioate oligonucleotide is an oligonucleotide in which at least one of the internucleoside linkages is a stereodefined phosphorothioate internucleoside linkage.

[0132] Complementarity The term "complementarity" refers to the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may contain nucleosides with modified nucleobases, for example, 5-methylcytosine is often used instead of cytosine, and therefore, the term "complementarity" is understood to encompass Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research vol. 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0133] As used herein, the term "complementarity (%)" refers to the percentage of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are complementary (i.e., form Watson-Crick base pairs) at a given position with the contiguous nucleotide sequence at a given position in another nucleic acid molecule (e.g., a target nucleic acid). This percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5'-3' and the oligonucleotide sequence 3'-5'), dividing by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form base pairs) are called mismatches. Preferably, insertions and deletions are not allowed in the calculation of the complementarity (%) of a contiguous nucleotide sequence.

[0134] The term "fully complementary" means 100% complementarity.

[0135] identity As used herein, the term "identity" refers to the number of nucleotides that are identical (i.e., capable of forming Watson-Crick base pairs with complementary nucleosides) at a given position to the contiguous nucleotide sequence of another nucleic acid molecule (e.g., a target nucleic acid), expressed as a percentage (%) of the contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide). This percentage is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Percent identity = (match x 100) / length of aligned region. Preferably, insertions and deletions are not allowed in calculating the complementarity (%) of a contiguous nucleotide sequence.

[0136] Hybridization As used herein, the term "hybridizing" or "hybridizing" is understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposing strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is often measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) in terms of physiological conditions. m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard Gibbs free energy ΔG° more accurately represents the binding affinity and is related to the dissociation constant (K d ) and ΔG°=-RTln(K d) (R is the gas constant, and T is the absolute temperature). Therefore, if the ΔG° of the reaction between an oligonucleotide and a target nucleic acid is very small, this reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction in which the aqueous solution concentration is 1M, pH is 7, and temperature is 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by using the isothermal titration calorimetry (ITC) method described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will be aware that commercially available instruments are available for measuring ΔG°. ΔG° can also be numerically estimated by using the nearest neighbor model described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405, with appropriately derived thermodynamic parameters, as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465. To have the potential to modulate the intended nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for oligonucleotides 10 to 30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured based on the standard Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with estimated ΔG° values below the range of −10 kcal, such as less than −15 kcal, such as less than −20 kcal, and such as less than −25 kcal for oligonucleotides 8 to 30 nucleotides in length.In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of −10 to −60 kcal, such as −12 to −40, such as −15 to −30 kcal, or −16 to −27 kcal, such as −18 to −25 kcal.

[0137] sugar modification Oligomers of the invention may include one or more nucleosides having modified sugar moieties, ie, modifications of the sugar moiety as compared to the ribose sugar moiety present in DNA and RNA.

[0138] A number of nucleosides have been made with modifications of the ribose sugar moiety, primarily aimed at improving some property of oligonucleotides, such as affinity and / or nuclease resistance.

[0139] Such modifications include, for example, those in which the ribose ring structure is modified by substitution with a hexose ring (HNA), or a bicyclic ring (LNA), which typically has a biradical bridge between the C2 and C4 carbons on the ribose ring, or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. 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 with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0140] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' position.

[0141] 2' sugar-modified nucleosides A 2' sugar modified nucleoside is a nucleoside that either has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or contains a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon in the ribose ring, such as an LNA (2'-4' biradical bridged) nucleoside.

[0142] Indeed, much attention has been focused on developing 2'-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer enhanced binding affinity and / or increased nuclease resistance to oligonucleotides.

[0143] Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides and 2'-F-ANA nucleosides.More examples can be found in, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavy and Damha, Chemistry and Biology 2012, 19, 937.Below are some examples of 2'-substituted modified nucleosides. TIFF0007724259000032.tif75128

[0144] In the context of the present invention, 2' substitutions do not include 2' bridging molecules such as LNA.

[0145] Locked nucleic acid nucleosides (LNA nucleosides) "LNA nucleosides" are 2'-modified nucleosides containing a biradical (also called a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, restricting or locking the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Locking the conformation of the ribose has been associated with enhanced hybridization affinity to complementary RNA or DNA molecules (duplex stabilization) when LNAs are incorporated into oligonucleotides. This can be routinely demonstrated by measuring the melting temperature of the oligonucleotide / complement duplex.

[0146] Non-limiting exemplary LNA nucleosides are described 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.

[0147] The 2'-4' bridge includes atoms bridging positions 2 and 4, and specifically has the formula -XY- (X is attached at C4' and Y is attached at C2'): During the ceremony X is oxygen, sulfur, -CR a R b -, -C(R a )=C(R b )-, -C(=CR a R b )-, -C(Ra )=N-, -Si(R a )2-, -SO2-, -NR a -, -O-NR a -, -NR a -O-, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O-, or -O-CR a R b - and; 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-, -Si(R a )2-, -SO2-, -NR a -, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O-, or -O-CR a R b - and; However, -XY- is -OO-, Si(R a )2-Si(R a )2-, -SO2-SO2-, -C(R a )=C(R b )-C(R a )=C(R b ), -C(R a )=NC(R a )=N-, -C(R a )=NC(R a )=C(R b ), -C(R a )=C(R b )-C(R a )=N- and -Se-Se-; J is oxygen, sulfur, =CH2, or =N(Ra ) and; R a and R b is hydrogen, halogen, hydroxyl, cyano, thiohydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, carbamido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, thiohydroxyl sulfido alkylsulfanyl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC(=X a )R c , -OC(=X a )NR c R d , and -NR e C(=X a )NR c R d more independently selected, or Two geminal R's a and R b together form an optionally substituted methylene, or Two geminal R's a and R b together with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl having only one carbon atom, -XY-; 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, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, heterosilyl, aryl, and heteroaryl; X a is oxygen, sulfur, or -NR c and; R c , R d , and R e are independently selected from hydrogen and alkyl; and n is 1, 2, or 3.

[0148] In yet another particular embodiment of the invention, X is oxygen, sulfur, -NR a -, -CR a R b - or -C(=CR a R b )—, specifically oxygen, sulfur, —NH—, —CH2—, or —C(═CH2)—, more specifically oxygen.

[0149] In another particular embodiment of the 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-.

[0150] In certain embodiments of the present invention, -X-Y- is -O-(CR a R b ) n -, -S-CRa 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 -It is.

[0151] In certain embodiments of the present invention, R a and R b are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl, and alkoxyalkyl, particularly hydrogen, halogen, alkyl, and alkoxyalkyl.

[0152] In another embodiment of the present invention, R a and R b are independently selected from the group consisting of hydrogen, fluoro, hydroxyl, methyl, and -CH2-O-CH3, in particular hydrogen, fluoro, methyl, and -CH2-O-CH3.

[0153] Advantageously, R of -XY- a and R b is as defined above and all the others are simultaneously hydrogen.

[0154] In yet another particular embodiment of the present invention, Ra is hydrogen or alkyl, in particular hydrogen or methyl.

[0155] In another particular embodiment of the present invention, R b is hydrogen or alkyl, in particular hydrogen or methyl.

[0156] In certain embodiments of the present invention, R a and R b One or both of is hydrogen.

[0157] In certain embodiments of the present invention, R a and R b Only one of the groups is hydrogen.

[0158] In one particular embodiment of the present invention, R a and R b One of the groups is methyl and the other is hydrogen.

[0159] In certain embodiments of the present invention, R a and R b are both methyl at the same time.

[0160] In certain embodiments of the 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- It is O-CH2-, -O-CH2-O-CH2-, -CH2-O-CH2-, -C(=CH2)CH2-, -C(=CH2)CH(CH3)-, -N(OCH3)CH2-, or -N(CH3)CH2-.

[0161] In certain embodiments of the present invention, -X-Y- is -O-CR a R b -(In the formula, R a and R b are independently selected from the group consisting of hydrogen, alkyl, and alkoxyalkyl, particularly hydrogen, methyl, and —CH 2 —O—CH 3 ).

[0162] In certain embodiments, -XY- is -O-CH2- or -O-CH(CH3)-, particularly -O-CH2-.

[0163] The 2'-4' bridge may be positioned either 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.

[0164] LNA nucleosides according to the present invention specifically have the formula (B1) or (B2): TIFF0007724259000033.tif38128In formula, W is oxygen, sulfur, -N(R a )-, or -CR a R b -, especially oxygen; B is a nucleobase or modified nucleobase; Z is an internucleoside linkage to an adjacent nucleoside or a 5'-terminal group; Z* is an internucleoside linkage to an adjacent nucleoside or a 3' terminal group; 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 X, Y, R a , and R b is as defined above.

[0165] In certain embodiments, in the definition of -XY-, R a is hydrogen or alkyl, particularly hydrogen or methyl. In another particular embodiment, in the definition of -XY-, R bis hydrogen or alkyl, in particular hydrogen or methyl. In yet another particular embodiment, in the definition of -XY-, R a and R b In certain embodiments, in the definition of -XY-, one or both of R a and R b In one particular embodiment, in the definition of -XY-, only one of R a and R b In certain embodiments, in the definition of -XY-, one of R a and R b are both methyl at the same time.

[0166] In yet another particular embodiment, in the definition of X, R a is hydrogen or alkyl, in particular hydrogen or methyl. In another particular embodiment, in the definition of X, R b is hydrogen or alkyl, in particular hydrogen or methyl. In certain embodiments, in the definition of X, R a and R b In certain embodiments, in the definition of X, one or both of R a and R b In one particular embodiment, in the definition of X, only one of R a and R b In certain embodiments, in the definition of X, one of R a and R b are both methyl at the same time.

[0167] In yet another particular embodiment, in the definition of Y, R a is hydrogen or alkyl, in particular hydrogen or methyl. In another particular embodiment, in the definition of Y, R b is hydrogen or alkyl, in particular hydrogen or methyl. In certain embodiments, in the definition of Y, R a and R b In certain embodiments, in the definition of Y, one or both of R a and Rb In one particular embodiment, in the definition of Y, only one of R a and R b In certain embodiments, in the definition of Y, one of R a and R b are both methyl at the same time.

[0168] In certain embodiments of the present invention, R 1 , R 2 , R 3 , R 5 , and R 5* are independently selected from hydrogen and alkyl, in particular hydrogen and methyl.

[0169] In yet another particularly advantageous embodiment of the present invention, R 1 , R 2 , R 3 , R 5 , and R 5* are all hydrogen at the same time.

[0170] In another particular embodiment of the present invention, R 1 , R 2 , R 3 are simultaneously all hydrogen and R 5 and R 5* One of the groups is hydrogen and the other is as defined above, specifically alkyl, more specifically methyl.

[0171] In certain embodiments of the present invention, R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, alkoxyalkyl, and azido, in particular from hydrogen, fluoro, methyl, methoxyethyl, and azido. In a particular advantageous embodiment of the invention, R 5 and R 5* one of R is hydrogen and the other is alkyl, particularly methyl, halogen, particularly fluoro, alkoxyalkyl, particularly methoxyethyl, or azido; or R 5 and R 5*are both simultaneously hydrogen or halogen, particularly both simultaneously hydrogen or fluoro. In certain such embodiments, W can advantageously be oxygen and -XY- can advantageously be -O-CH2-.

[0172] In certain embodiments of the invention, -XY- is -O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, and WO 2004 / 046160, all of which are incorporated herein by reference, and include those commonly known in the art as β-D-oxy LNA nucleosides and α-L-oxy LNA nucleosides.

[0173] In another particular embodiment of the invention, -X-Y- is -S-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such thioLNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are incorporated herein by reference.

[0174] In another particular embodiment of the invention, -XY- is -NH-CH, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such amino LNA nucleosides are disclosed in WO 99 / 014226 and WO 2004 / 046160, which are incorporated herein by reference.

[0175] In another specific embodiment of the invention, -XY- is -O-CH2CH2- or -OCH2CH2CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such LNA nucleosides are disclosed in WO 00 / 047599 and Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, which are incorporated herein by reference, and include those commonly known in the art as 2'-O-4'C-ethylene bridged nucleic acids (ENA).

[0176] In another particular embodiment of the invention, -XY- is -O-CH2-, W is oxygen, and R 1 , R 2 , R 3 are simultaneously all hydrogen and R 5 and R 5* is hydrogen and the other is not hydrogen but is, for example, alkyl, such as methyl. Such 5' substituted LNA nucleosides are disclosed in WO 2007 / 134181, which is incorporated herein by reference.

[0177] In another particular embodiment of the present invention, -X-Y- is -O-CR a R b -(In the formula, R a and R b is not hydrogen, specifically alkyl, e.g., methyl), W is oxygen, and R 1 , R 2 , R 3 are simultaneously all hydrogen and R 5 and R 5* is hydrogen and the other is not hydrogen, specifically alkyl, e.g., methyl. Such doubly modified LNA nucleosides are disclosed in WO 2010 / 077578, which is incorporated herein by reference.

[0178] In another particular embodiment of the present invention, -X-Y- is -O-CHR a -, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such 6'-substituted LNA nucleosides are disclosed in WO 2010 / 036698 and WO 2007 / 090071, both of which are incorporated herein by reference. In such 6'-substituted LNA nucleosides, R a is specifically C1-C6 alkyl, for example, methyl.

[0179] In another specific embodiment of the invention, -XY- is -O-CH(CH2-O-CH3)- ("2'O-methoxyethyl bicyclic nucleic acid", Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81).

[0180] In another specific embodiment of the invention, -XY- is -O-CH(CH2CH3)-.

[0181] In another particular embodiment of the invention, -XY- is -O-CH(CH2-O-CH3)-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such LNA nucleosides are also known in the art as cyclic MOEs (cMOEs) and are disclosed in WO 2007 / 090071.

[0182] 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(5) pp. 1569-81).

[0183] In another particular embodiment of the present invention, -X-Y- is -O-CH2- O-CH2- (Seth et al., J. Org. Chem 2010, op. cit.).

[0184] In another particular embodiment of the invention, -XY- is -O-CH(CH3)-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Such 6'-methyl LNA nucleosides are also known in the art as cET nucleosides and can be either the (S)-cET diastereoisomer or the (R)-cET diastereoisomer, as disclosed in WO 2007 / 090071 (β-D) and WO 2010 / 036698 (α-L), both of which are incorporated herein by reference.

[0185] In another particular embodiment of the present invention, -X-Y- is -O-CR a R b -(In the formula, R a MoR b (Neither is hydrogen), W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. a and R b are simultaneously both alkyl, in particular both methyl. Such 6' disubstituted LNA nucleosides are disclosed in WO 2009 / 006478, which is incorporated herein by reference.

[0186] In another specific embodiment of the present invention, -X-Y- is -S-CHR a -, W is oxygen, and R 1 , R 2 , R 3 , R 5 and R 5*are simultaneously all hydrogen. Such 6'-substituted thio LNA nucleosides are disclosed in WO 2011 / 156202, which is incorporated herein by reference. In certain embodiments of such 6'-substituted thio LNA nucleosides, R a is alkyl, in particular methyl.

[0187] In certain embodiments of the invention, -XY- is -C(=CH2)C(R a R b )-, -C(=CHF)C(R a R b )-, or -C(=CF2)C(R a R b )-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Advantageously, R a and R b are independently selected from hydrogen, halogen, alkyl, and alkoxyalkyl, particularly hydrogen, methyl, fluoro, and methoxymethyl. In particular, R a and R b are both hydrogen or methyl at the same time, or R a and R b One of the is hydrogen and the other is methyl. Such vinyl carbo LNA nucleosides are disclosed in WO 2008 / 154401 and WO 2009 / 067647, both of which are incorporated herein by reference.

[0188] In certain embodiments of the present invention, -X-Y- is -N(OR a )-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. ais alkyl, e.g., methyl. Such LNA nucleosides, also known as N-substituted LNAs, are disclosed in WO 2008 / 150729, which is incorporated herein by reference.

[0189] In certain embodiments 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* are simultaneously all hydrogen. Advantageously, R a and R b are independently selected from hydrogen, halogen, alkyl, and alkoxyalkyl, particularly hydrogen, methyl, fluoro, and methoxymethyl. a is alkyl, e.g., methyl, and R b is hydrogen or methyl, especially hydrogen (Seth et al., J. Org. Chem 2010 op. cit.).

[0190] In another particular embodiment of the invention, -XY- is -ON(CH3)- (Seth et al., J. Org. Chem 2010 op. cit.).

[0191] In certain embodiments of the present invention, R 5 and R 5* are simultaneously both hydrogen. In another particular embodiment of the present invention, R 5 and R 5* One of R is hydrogen and the other is alkyl, e.g., methyl. In such embodiments, R 1 , R 2 , and R 3can in particular be hydrogen, and -XY- is in particular -O-CH2- or -O-CHC(R a )3-, for example, -O-CH(CH3)-.

[0192] In certain embodiments of the invention, -X-Y- is -CR a R b -O-CR a R b -, for example -CH2-O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. In certain such embodiments, R a may in particular be alkyl, for example methyl, and R b can be hydrogen or methyl, in particular hydrogen. Such LNA nucleosides, also known as conformationally restricted nucleotides (CRNs), are disclosed in WO 2013 / 036868, which is incorporated herein by reference.

[0193] In certain embodiments of the present invention, -X-Y- is -O-CR a R b -O-CR a R b -, e.g., -O-CH 2- O-CH2-, W is oxygen, and R 1 , R 2 , R 3 , R 5 , and R 5* are simultaneously all hydrogen. Advantageously, R a and R b are independently selected from hydrogen, halogen, alkyl, and alkoxyalkyl, particularly hydrogen, methyl, fluoro, and methoxymethyl. In certain such embodiments, R a may in particular be alkyl, for example methyl, and R bcan be hydrogen or methyl, particularly hydrogen. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, which is incorporated herein by reference.

[0194] Unless specified, it is recognized that LNA nucleosides may exist in either β-D or α-L stereoisoforms.

[0195] Specific examples of LNA nucleosides of the present invention are presented in Scheme 1, where B is as defined above. Scheme 1 TIFF0007724259000034.tif251129TIFF0007724259000035.tif224127TIFF0007724259000036.tif109128

[0196] Particular LNA nucleosides are β-D-oxy-LNA, 6′-methyl-β-D-oxy LNA, for example (S)-6′-methyl-β-D-oxy-LNA ((S)-cET) and ENA.

[0197] MOE nucleosides The term "MOE" stands for "methoxyethyl" and refers by abbreviation to a nucleoside substituted at the 2' position with a methoxy-ethoxy group, as shown below. TIFF0007724259000037.tif37128

[0198] Thus, the above nucleosides can be designated either as "MOE" or as "2'-O-MOE nucleosides."

[0199] RNase H activity and recruitment The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when duplexed with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for measuring RNase H activity, which can be used to measure the ability to recruit RNase H. Typically, an oligonucleotide is considered to be able to recruit RNase H if it satisfies the following condition: the initial rate (measured in pmol / l / min) when provided with a complementary target nucleic acid sequence is at least 5%, for example, at least 10%, or more than 20% of the initial rate measured using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided in Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). Recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland, for use in measuring RNase H activity.

[0200] Gapmar The antisense oligonucleotide of the present invention, or its contiguous nucleotide sequence, may be a gapmer. Antisense gapmers are commonly used to inhibit target nucleic acids through RNase H-mediated degradation. Gapmer oligonucleotides contain at least three unique structural regions: a 5' flank, a gap, and a 3' flank, i.e., FG-F', in a 5'→3' orientation. The "gap" region (G) contains a stretch of contiguous DNA nucleotides that enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5'-flanking region (F) containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides, and a 3'-flanking region (F') containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides. The one or more sugar-modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity-enhancing sugar-modified nucleosides). In some embodiments, one or more sugar-modified nucleosides in region F and region F' are 2' sugar-modified nucleosides, eg, high affinity 2' sugar modifications, eg, independently selected from LNA and 2'-MOE.

[0201] In a gapmer design, the 5'-most and 3'-most nucleosides of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) region or 3' (F') region, respectively. These flanks may be further characterized by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5' end of the 5' flank and the 3' end of the 3' flank.

[0202] The region FG-F' forms a contiguous nucleotide sequence. The antisense oligonucleotide or contiguous nucleotide sequence thereof of the present invention may comprise a gapmer region of the formula FG-F'.

[0203] The total length of the gapmer design FG-F' may be, for example, 12 to 32 nucleosides, for example, 13 to 24, for example, 14 to 22 nucleosides, for example, 14 to 17, for example, 16 to 18 nucleosides.

[0204] By way of example, a gapmer oligonucleotide of the invention can be represented by the following formula: F 1~8 -G 5~16 -F' 1~8 , e.g., F 1~8 -G 7~16 -F' 2~8 However, the total length of the gapmer region FG-F' is at least 12, for example at least 14, nucleotides in length.

[0205] Regions F, G, and F' are further defined below and can be combined into the FG-F' formula.

[0206] Gapmer region G The region G (gap region) of a gapmer is a region of nucleosides, typically DNA nucleosides, that allows the oligonucleotide to recruit RNase H, e.g., human RNase H1. RNase H is a cellular enzyme that recognizes DNA and RNA duplexes and enzymatically cleaves RNA molecules. Suitable gapmers may have a gap region (G) of at least 5 or 6 consecutive DNA nucleosides in length, e.g., 5 to 16 consecutive DNA nucleosides, e.g., 6 to 15 consecutive DNA nucleosides, e.g., 7 to 14 consecutive DNA nucleosides, e.g., 8 to 12 consecutive DNA nucleotides, e.g., 8 to 12 consecutive DNA nucleotides. In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive DNA nucleosides. Cytosine (C) DNA in the gap region may be optionally methylated, and such residues are designated as 5-methyl-cytosine ( meC) or with e instead of c. Methylation of cytosine DNA in the gap is advantageous when a cg dinucleotide is present in the gap to reduce potential toxicity, and this modification does not significantly affect the efficacy of the oligonucleotide.

[0207] 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 of the internucleoside linkages in the gap are phosphorothioate linkages.

[0208] While traditional gapmers have a DNA gap region, there are many examples of modified nucleosides that allow for RNase H recruitment when used within the gap region. Modified nucleosides that have been reported to be capable of recruiting RNase H when contained within the gap region include, for example, α-L-LNA, C4'-alkylated DNA (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), arabinose-derived nucleosides such as ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), and UNA (unlocked nucleic acid) (described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which is incorporated herein by reference). UNAs are unlocked nucleic acids, typically in which the bond between C2 and C3 of the ribose has been removed to form an unlocked "sugar" residue. The modified nucleosides used in such gapmers can be nucleosides that adopt a 2'-endo (DNA-like) structure when introduced into the gap region, i.e., modifications that allow for RNase H recruitment. In some embodiments, the DNA gap region (G) described herein can optionally contain one to three sugar-modified nucleosides that adopt a 2'-endo (DNA-like) structure when introduced into the gap region.

[0209] Area G - "Gap Breaker" Alternatively, there have been numerous reports of inserting modified nucleosides into the gap region of a gapmer to impart a 3'-end conformation to the gap while retaining some RNase H activity. Such gapmers, having a gap region containing one or more 3'-end modified nucleosides, are also referred to as "gap breakers" or "gap-broken" gapmers. See, for example, WO 2013 / 022984. Gap breaker oligonucleotides retain a sufficient DNA nucleoside region within the gap region to allow RNase H recruitment. The ability of gap breaker oligonucleotide designs to recruit RNase H is typically 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, potentially providing more specific cleavage of target RNA. Modified nucleosides used within the gap region of a gap breaker oligonucleotide can be, for example, modified nucleosides that provide 3'-end confirmation, such as 2'-O-methyl (OMe) nucleosides or 2'-O-MOE (MOE) nucleosides, or β-D LNA nucleosides (in which the bridge between C2' and C4' of the ribose sugar ring of the nucleoside is in a β conformation), such as β-D-oxy LNA nucleosides or ScET nucleosides.

[0210] Similar to the gapmers containing region G described above, the gap region of a gap breaker or gap-disrupted 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'). Gapmers containing a disrupted gap typically retain a region of at least three or four contiguous DNA nucleosides at either the 5' or 3' end of the gap region.

[0211] Exemplary designs of gap breaker oligonucleotides include: F 1~8 -[D 3~4 -E1-D 3~4 ] - F' 1~8 F 1~8 -[D 1~4 -E1-D 3~4 ]-F' 1~8 F 1~8 -[D 3~4 -E1-D 1~4 ]-F' 1~8 Contains, where the region G is the region between the square brackets [D n -E r -D m ], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (gap breaker or gap disrupting nucleoside), and F and F' are flanking regions as defined herein, provided that the total length of the gapmer region FG-F' is at least 12, e.g., at least 14, nucleotides in length.

[0212] In some embodiments, region G of the gap-disrupted gapmer comprises at least six DNA nucleosides, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 DNA nucleosides. As noted above, the DNA nucleosides may be contiguous or, optionally, interrupted by one or more modified nucleosides, provided that gap region G is capable of mediating RNase H recruitment.

[0213] Gapmer-flanking regions F and F' Region F is located 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, e.g., a 2'-substituted nucleoside such as an MOE nucleoside, or an LNA nucleoside.

[0214] Region F' is located immediately adjacent to the 3' DNA nucleoside of region G. The 5'-most nucleoside of region F' is a sugar-modified nucleoside, such as a high-affinity sugar-modified nucleoside, e.g., a 2'-substituted nucleoside such as an MOE nucleoside, or an LNA nucleoside.

[0215] Region F is 1 to 8 contiguous nucleotides in length, e.g., 2 to 6, e.g., 3 to 4 contiguous nucleotides in length. Advantageously, the 5'-most nucleoside of region F is a sugar-modified nucleoside. In some embodiments, the two 5'-most nucleosides of region F are sugar-modified nucleosides. In some embodiments, the 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are 2'-substituted nucleosides, e.g., two 3' MOE nucleosides. In some embodiments, the 5'-most nucleoside of region F is a 2'-substituted nucleoside, such as an MOE nucleoside.

[0216] Region F' is 2 to 8 contiguous nucleotides in length, e.g., 3 to 6, e.g., 4 to 5 contiguous nucleotides in length. Advantageously, in some embodiments, 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, e.g., two 3' MOE nucleosides. In some embodiments, the 3'-most nucleoside of region F' is a 2'-substituted nucleoside, such as an MOE nucleoside.

[0217] It should be noted that when region F or region F' has a length of 1, it is advantageously an LNA nucleoside.

[0218] In some embodiments, region F and region F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F can 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, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0219] In some embodiments, region F and region F' independently comprise both LNA nucleosides and 2'-substituted modified nucleosides (mixed wing design).

[0220] In some embodiments, regions F and F' consist of only one type of sugar-modified nucleoside, for example, only MOE, or only β-D-oxy LNA, or only ScET. Such a design is also referred to as a uniform flank design or a uniform gapmer design.

[0221] In some embodiments, all nucleosides in regions F or F', or F and F', are LNA nucleosides, e.g., independently selected from β-D-oxyLNA nucleosides, ENA nucleosides, or ScET nucleosides. In some embodiments, region F consists of 1 to 5, e.g., 2 to 4, e.g., 3 to 4, e.g., 1, 2, 3, 4, or 5 consecutive LNA nucleosides. In some embodiments, all nucleosides in regions F and F' are β-D-oxyLNA nucleosides.

[0222] In some embodiments, all nucleosides in regions F or F', or F and F', are 2'-substituted nucleosides, e.g., OMe nucleosides or MOE nucleosides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7, or 8 consecutive OMe nucleosides or consecutive MOE nucleosides. In some embodiments, only one of the flanking regions can consist of 2'-substituted nucleosides, e.g., OMe nucleosides or MOE nucleosides. In some embodiments, it is the 5' (F) flanking region that consists of 2'-substituted nucleosides, e.g., OMe nucleosides or MOE nucleosides, while the 3' (F') flanking region contains at least one LNA nucleoside, e.g., a β-D-oxyLNA nucleoside or a cET nucleoside. In some embodiments, it is the 3' (F') flanking region that consists of 2' substituted nucleosides, e.g., OMe nucleosides or MOE nucleosides, while the 5' (F) flanking region comprises at least one LNA nucleoside, e.g., a β-D-oxyLNA nucleoside or a cET nucleoside.

[0223] In some embodiments, all modified nucleosides in regions F and F' are LNA nucleosides, e.g., independently selected from β-D-oxyLNA nucleosides, ENA nucleosides, or ScET nucleosides, wherein regions F or F', or F and F', may optionally comprise DNA nucleosides (alternating flanks, see these definitions for more details). In some embodiments, all modified nucleosides in regions F and F' are β-D-oxyLNA nucleosides, wherein regions F or F', or F and F', may optionally comprise DNA nucleosides (alternating flanks, see these definitions for more details).

[0224] In some embodiments, the 5'-most and 3'-most nucleosides of regions F and F' are LNA nucleosides, such as β-D-oxyLNA nucleosides or ScET nucleosides.

[0225] In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F' and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between the nucleosides of regions F or F', F and F' is a phosphorothioate internucleoside linkage.

[0226] Further gapmer designs are disclosed in WO 2004 / 046160, WO 2007 / 146511, and WO 2008 / 113832, which are incorporated herein by reference.

[0227] LNA gapmers An LNA gapmer is a gapmer in which either or both of regions F and F' comprise or consist of LNA nucleosides. A β-D-oxy gapmer is a gapmer in which either or both of regions F and F' comprise or consist of β-D-oxy LNA nucleosides.

[0228] In some embodiments, the LNA gapmer has the formula: [LNA] 1~5 -[Area G]-[LNA] 1~5 where region G is as defined in the definition of gapmer region G.

[0229] MOE Gapmar An MOE gapmer is a gapmer in which region F and region F' consist of MOE nucleosides. In some embodiments, an MOE gapmer has the design [MOE] 1~8 -[Area G]-[MOE] 1~8 , e.g. [MOE] 2~7 -[Area G] 5~16 -[MOE] 2~7 , e.g. [MOE] 3~6 -[Area G]-[MOE] 3~6where region G is as defined in the gapmer definition. MOE gapmers with 5 to 10 −5 designs (MOE-DNA-MOE) are widely used in the art.

[0230] Mixed Wing Gappa A mixed-wing gapmer is an LNA gapmer in which one or both of regions F and F' comprise 2'-substituted nucleosides, e.g., MOE 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, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments in which at least one of regions F and F' or both regions F and F' 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 in which 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.

[0231] Mixed wing gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0232] Alternating Flank Gap Mar The flanking regions may contain both LNA and DNA nucleosides, and are referred to as "alternating flanks" because they contain an alternating motif of LNA-DNA-LNA nucleosides. Gapmers containing such alternating flanks are referred to as "alternating flank gapmers." Thus, an "alternating flank gapmer" is an LNA gapmer oligonucleotide in which at least one of the flanks (F or F') contains DNA in addition to LNA nucleosides. In some embodiments, at least one of region F or region F', or both regions F and F', contain both LNA and DNA nucleosides. In such embodiments, flanking region F or F', or both F and F', contain at least three nucleosides, and the 5'-most and 3'-most nucleosides of the F region and / or F' region are LNA nucleosides.

[0233] Alternately flanked LNA gapmers are disclosed in WO 2016 / 127002.

[0234] The alternating flanking regions may comprise up to three consecutive DNA nucleosides, for example, 1 to 2 or 1 or 2 or 3 consecutive DNA nucleosides.

[0235] Alternating flanks include, for example, [L] 1~3 -[D] 1~4 -[L] 1~3 [L] 1~2 -[D] 1~2 -[L] 1~2 -[D] 1~2 -[L] 1~2 The comment can be added as a series of integers representing the number of LNA nucleosides (L) followed by the number of DNA nucleosides (D), such as:

[0236] In oligonucleotide design, these are often represented as numbers, with 2-2-1 representing 5'[L]2-[D]2-[L]3', 1-1-1-1-1 representing 5'[L]-[D]-[L]-[D]-[L]3', and so on. The length of the flanks (regions F and F') in oligonucleotides with alternating flanks can independently be 3 to 10 nucleosides, e.g., 4 to 8, e.g., 5 to 6 nucleosides, e.g., 4, 5, 6, or 7 modified nucleosides. In some embodiments, only one of the flanks in a gapmer oligonucleotide is alternating, while the other is composed of LNA nucleotides. To confer additional exonuclease resistance, it may be advantageous to have at least two LNA nucleosides at the 3' end of the 3' flank (F'). Some examples of oligonucleotides with alternating flanks are as follows: [L] 1~5 -[D] 1~4 -[L] 1~3 -[G] 5~16 -[L] 2~6 [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 [L] 1~5 -[G] 5~16 -[L]-[D]-[L]-[D]-[L]2 provided that the total length of the gapmer is at least 12, such as at least 14, nucleotides in length.

[0237] Region D' or region D'' in the oligonucleotide In some embodiments, the oligonucleotides of the invention may comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, e.g., a gapmer FG-F', and additional 5' and / or 3' nucleosides. The additional 5' and / or 3' nucleosides may or may not be fully complementary to the target nucleic acid. Such additional 5' and / or 3' nucleosides may be referred to herein as region D' and region D".

[0238] The addition of region D' or region D" can be used to link a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used to link a conjugate moiety to a conjugate moiety, it can serve as a biocleavable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0239] Region D' and region D" can be attached to the 5' end of region F or the 3' end of region F', respectively, to create designs of the following formula: D'-FG-F', FG-F'-D", or D'-FG-F'-D", where FG-F' is the gapmer portion of the oligonucleotide and region D' or region D" constitutes a separate portion of the oligonucleotide.

[0240] Region D' or region D" can independently comprise or consist of 1, 2, 3, 4, or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides but are, for example, DNA or RNA or base-modified versions thereof. The D' or D' region can serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the additional 5'- and / or 3'-terminal nucleotides are linked by a phosphodiester bond and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or region D" are disclosed in WO 2014 / 076195 and include, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, where they are used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0241] In one embodiment, the oligonucleotide of the present invention comprises a region D' and / or a region D" in addition to the contiguous nucleotide sequence constituting the gapmer.

[0242] In some embodiments, the oligonucleotides of the invention can be represented by the following formula: FG-F', especially F 1~8 -G 5~16 -F' 2~8 D'-FG-F', especially D' 1~3 -F 1~8 -G 5~16 -F' 2~8 FG-F'-D”, especially F 1~8 -G 5~16 -F' 2~8 -D” 1~3 D'-FG-F'-D", especially D' 1~3 -F 1~8-G 5~16 -F' 2~8 -D” 1~3 .

[0243] In some embodiments, the internucleoside linkage located between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside linkage located between region F' and region D" is a phosphodiester bond.

[0244] Total Mar In some embodiments, all of the nucleosides of the oligonucleotide or its contiguous nucleotide sequence are sugar-modified nucleosides. Such oligonucleotides are referred to herein as totalmers.

[0245] In some embodiments, all of the sugar-modified nucleosides of a totalmer comprise the same sugar modification, e.g., they may all be LNA nucleosides or all 2'O-MOE nucleosides. In some embodiments, the sugar-modified nucleosides of a totalmer may be independently selected from LNA nucleosides and 2'-substituted nucleosides, e.g., 2'-substituted nucleosides selected from the group consisting of 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises both an LNA nucleoside and a 2'-substituted nucleoside, for example, the 2'-substituted nucleoside is selected from the group consisting of 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 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 an (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.

[0246] In some embodiments, all of the nucleosides of the oligonucleotide or its consecutive nucleotide sequence are LNA nucleosides, such as β-D-oxy-LNA nucleosides and / or (S)cET nucleosides.In some embodiments, such LNA total mer oligonucleotides are 7-12 nucleosides long (see, for example, WO 2009 / 043353).Such short, completely LNA oligonucleotides are particularly effective in inhibiting microRNA.

[0247] Various totalmer compounds are remarkably effective as therapeutic oligomers, especially when targeting microRNAs (anti-miRs) or as splice-switching oligomers (SSOs).

[0248] In some embodiments, the totalmer comprises or consists of at least one XYX or YXY sequence motif, such as the repeated sequence XYX or YXY, where X is LNA and Y is another (i.e., non-LNA) nucleotide analog, such as a 2'-OMe RNA unit and a 2'-fluoro DNA unit. In some embodiments, the sequence motif may be, for example, XXY, XYX, YXY, or YYX.

[0249] In some embodiments, a totalmer may comprise or consist of a contiguous nucleotide sequence of 7 to 24 nucleotides, for example, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0250] In some embodiments, the contiguous nucleotide sequence of the totalmer comprises at least 30%, such as at least 40%, for example at least 50%, such as at least 60%, for example at least 70%, for example at least 80%, such as at least 90%, for example 95%, for example 100% LNA units. In the case of entirely LNA compounds, these are advantageously less than 12 nucleotides in length, such as 7-10 nucleotides in length.

[0251] The remaining units may be selected from the non-LNA nucleotide analogues referred to herein, such as 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 from the group of 2'-OMe RNA units and 2'-fluoro DNA units.

[0252] Mixmar The term "mixmer" refers to an oligomer containing both DNA nucleosides and sugar-modified nucleosides, where the length of consecutive DNA nucleosides is insufficient to recruit RNase H. Suitable mixmers may contain up to three or up to four consecutive DNA nucleosides. In some embodiments, a mixmer, or its consecutive nucleotide sequence, contains alternating regions of sugar-modified nucleosides and DNA nucleosides. By alternating regions of sugar-modified nucleosides that form an RNA-like (3'-endo) conformation when incorporated into an oligonucleotide with short regions of DNA nucleosides, oligonucleotides that do not recruit RNase H can be created. Advantageously, the sugar-modified nucleosides are affinity-enhancing sugar-modified nucleosides.

[0253] Oligonucleotide mixers are often used to provide target gene occupancy-based regulation, such as splice regulators or microRNA inhibitors.

[0254] In some embodiments, the sugar-modified nucleosides or contiguous nucleotide sequence in a mixmer include or are all LNA nucleosides, such as (S)cET LNA nucleosides or β-D-oxy LNA nucleosides.

[0255] In some embodiments, all of the sugar-modified nucleosides of a mixmer contain the same sugar modification, e.g., they may all be LNA nucleosides or all 2'O-MOE nucleosides. In some embodiments, the sugar-modified nucleosides of a mixmer can 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 nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises both LNA nucleosides and 2'-substituted nucleosides, for example, the 2'-substituted nucleosides are selected from the group consisting of 2'-O alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. In some embodiments, the oligonucleotide comprises LNA nucleosides and 2'-O-MOE nucleosides. In some embodiments, the oligonucleotide comprises (S)cET LNA nucleosides and 2'-O-MOE nucleosides.

[0256] In some embodiments, a mixmer or its contiguous nucleotide sequence comprises only LNA and DNA nucleosides, e.g., an LNA mixmer oligonucleotide can be, e.g., 8 to 24 nucleosides in length (see, e.g., WO 2007112754, which discloses LNA anti-miR inhibitors of microRNAs).

[0257] Various miXmer compounds have been remarkably effective as therapeutic oligomers, particularly when targeting microRNAs (anti-miRs) or as splice-switching oligomers (SSOs).

[0258] In some embodiments, the mixmer comprises the following motif: TIFF0007724259000038.tif85154 (wherein L represents a sugar-modified nucleoside such as an LNA or a 2'-substituted nucleoside (e.g., 2'-O-MOE), D represents a DNA nucleoside, each m is independently selected from 1 to 6, and each n is independently selected from 1, 2, 3, and 4, e.g., 1 to 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 an LNA nucleoside and a 2'-O-MOE nucleoside.

[0259] In some embodiments, a mixmer may comprise or consist of a contiguous nucleotide sequence of between 10 and 24 nucleotides, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0260] In some embodiments, the contiguous nucleotide sequence of the mixmer comprises at least 30%, such as at least 40%, such as at least 50% LNA units.

[0261] In some embodiments, a mixmer comprises or consists of a contiguous nucleotide sequence with a repeating pattern of nucleotide analogs and natural nucleotides, or one type of nucleotide analog and another type of nucleotide analog. The repeating pattern can be, for example, as follows: every other nucleotide or every third nucleotide is a nucleotide analog such as LNA, and the remaining nucleotides are natural nucleotides such as DNA, or 2'-substituted nucleotide analogs such as 2'MOE analogs or 2'fluoro analogs, as mentioned herein, or in some embodiments, selected from the group of nucleotide analogs mentioned herein. It is recognized that the repeating pattern of nucleotide analogs, such as LNA units, can be combined with nucleotide analogs at a defined position, for example, at the 5' or 3' end.

[0262] In some embodiments, the first nucleotide from the 3' end of the oligomer is a nucleotide analog, such as an LNA nucleotide or a 2'-O-MOE nucleoside.

[0263] In some embodiments, which may be the same or different, the second nucleotide from the 3' end of the oligomer is a nucleotide analog, such as an LNA nucleotide or a 2'-O-MOE nucleoside.

[0264] 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.

[0265] In some embodiments, the mixmer comprises at least one region comprising at least two consecutive nucleotide analogue units, for example at least two consecutive LNA units.

[0266] In some embodiments, the mixmer comprises at least one region comprising at least three consecutive nucleotide analogue units, for example at least three consecutive LNA units.

[0267] Exosomes Exosomes are natural biological nanovesicles, typically ranging from 30 to 500 nm, that are involved in intercellular communication via functionally active cargo (e.g., miRNA, mRNA, DNA, and proteins).

[0268] Exosomes are secreted by all types of cells and are abundant in bodily fluids such as saliva, blood, urine, and milk. Their primary role is to convey information between specific cells by delivering various effector or signaling molecules (Acta Pol Pharm. 2014 Jul-Aug;71(4):537-43). Such effector or signaling molecules can be, for example, proteins, miRNA, or mRNA. Currently, exosomes are being investigated as a delivery vehicle for various drug molecules, including therapeutic RNA molecules, to expand the therapeutic and diagnostic applications of such molecules. In the field of exosomes loaded with synthetic molecules such as siRNA, antisense oligonucleotides, and small molecules, there are disclosures suggesting or demonstrating that such molecules offer advantages in terms of delivery and efficacy compared to free drug molecules (e.g., Andaloussi et al. 2013 Advanced Drug Delivery Reviews 65: 391-397, WO 2014 / 168548, WO 2016 / 172598, WO 2017 / 173034, and See WO 2018 / 102397).

[0269] Exosomes can be isolated from biological sources such as milk (milk exosomes), and in particular, milk is a rich source for isolating milk exosomes. See, e.g., Manca et al., Scientific Reports (2018) 8:11321.

[0270] In some embodiments of the present invention, single-stranded oligonucleotides are encapsulated in exosomes (exosome formulations), and examples of loading single-stranded antisense oligonucleotides into exosomes are described in EP Application No. 18192614.8. In the methods of the present invention, the antisense oligonucleotides may be administered to cells or subjects in the form of exosome formulations, and oral administration of exosome formulations is particularly envisioned.

[0271] In some embodiments, antisense oligonucleotides may be linked to lipophilic conjugates such as cholesterol, which can be covalently linked to the antisense oligonucleotide via a biocleavable linker (e.g., a region of phosphodiester-linked DNA nucleotides). Such lipophilic conjugates can facilitate the formulation of antisense oligonucleotides into exosomes and can also facilitate delivery to target cells.

[0272] Conjugates As used herein, the term "conjugate" refers to an oligonucleotide that is covalently linked to a non-nucleotide moiety (the conjugate moiety or region C or the third region).

[0273] The conjugation of the oligonucleotide of the present invention to one or more non-nucleotide moieties can improve the pharmacological action of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide.In some embodiments, the conjugate moiety modifies 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 guide the oligonucleotide to a specific organ, tissue, or cell type, thereby increasing the effectiveness of the oligonucleotide in that 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, such as off-target activity, or activity in non-target cell types, tissues, or organs.

[0274] WO 93 / 07883 and WO 2013 / 033230, which are incorporated herein by reference, provide suitable conjugate moieties.Yet other suitable conjugate moieties are those that can bind to asialoglycoprotein receptor (ASGPR).In particular, trivalent N-acetylgalactosamine conjugate moieties are suitable for binding to ASGPR, for example, see WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620 (incorporated herein by reference).Such conjugates increase the uptake of oligonucleotide into the liver and simultaneously reduce its abundance in the kidney, thereby increasing the liver / kidney ratio of conjugated oligonucleotide compared to the same unconjugated oligonucleotide.

[0275] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

[0276] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophile, 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.

[0277] Linker Bond or linker is the bond between two atoms, which connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.Conjugate moiety can be directly or through linking moiety (for example, linker or tether) to oligonucleotide.Linker serves to covalently connect third region, for example, conjugate moiety (region C), to first region, for example, oligonucleotide or continuous nucleotide sequence (region A) that is complementary to target nucleic acid.

[0278] In some embodiments of the present invention, the conjugate or oligonucleotide conjugate of the present invention may optionally comprise a linker region (second region or region B and / or region Y) disposed between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

[0279] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions similar to those occurring normally or in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions, or agents, and salt concentrations found in mammalian cells or similar to those occurring in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker comprises 1 to 10 nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably 2 to 6 nucleosides, and most preferably 2 to 4 linked nucleosides, containing at least two consecutive phosphodiester bonds, e.g., at least 3, 4, or 5 consecutive phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Biocleavable linkers containing phosphodiesters are described in more detail in WO 2014 / 076195 (incorporated herein by reference).

[0280] Region Y refers to a linker that is not necessarily biocleavable but primarily functions to covalently link the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). The region Y linker may comprise a chain structure or an oligomer composed of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugates of the invention can be constructed from the following region elements: AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, such as a C2-C36 aminoalkyl group, including, for example, a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

[0281] Administration The oligonucleotides or pharmaceutical compositions of the invention may be administered topically (e.g., to the skin, by inhalation, to the eye, or to the ear), enterally (e.g., orally or via the digestive tract), or parenterally (e.g., intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, or intrathecally).

[0282] In some embodiments, the oligonucleotide or pharmaceutical composition of the present invention is administered by parenteral route, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, for example, intracerebral or intraventricular, or intravitreal administration.In one embodiment, active oligonucleotide or oligonucleotide conjugate is administered intravenously.In another embodiment, active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

[0283] In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention is administered at a dose of 0.1 to 15 mg / kg, e.g., 0.2 to 10 mg / kg, e.g., 0.25 to 5 mg / kg. Administration can be weekly, every two weeks, every three weeks, or monthly, or every other month.

[0284] The present invention also provides the use of the described oligonucleotides or oligonucleotide conjugates of the present invention for the manufacture of pharmaceuticals in ophthalmic dosage forms, such as intravitreal injections. In some embodiments, the oligonucleotide for ophthalmic targeting is Htra-1.

[0285] The present invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament in a dosage form (e.g., an injectable solution) for intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, or intrathecal administration.

[0286] Exemplary Benefits As demonstrated herein, the achiral phosphorodithioate internucleoside linkages used in the compounds of the present invention allow for reduced complexity of non-stereodefined phosphorothioate oligonucleotides while maintaining the activity, efficacy, or potency of the oligonucleotides.

[0287] Indeed, as demonstrated herein, those used in the compounds of the present invention offer unique advantages in combination with stereodefined phosphorothioates, providing the opportunity to further reduce the complexity of phosphorothioate oligonucleotides while retaining or improving their activity, efficacy, or potency.

[0288] As demonstrated herein, the achiral phosphorodithioate internucleoside linkages used in the compounds of the present invention allow for improved cellular uptake in vitro or in vivo.

[0289] As demonstrated herein, the achiral phosphorodithioate internucleoside linkages used in the compounds of the present invention allow for altered or improved in vitro biodistribution (measured either as tissue or cellular content or activity / potency in target tissues). Notably, the inventors have observed improved tissue uptake, content, and / or potency in skeletal muscle, heart, spleen, liver, kidney, fibroblasts, and epithelial cells.

[0290] In the context of mixmer oligonucleotides, the inventors have determined that the incorporation of phosphorodithioate linkages (as shown in IA or IB) between or adjacent to one or more DNA nucleosides provides improvements, such as enhanced stability and / or improved potency. In the context of gapmer oligonucleotides, the inventors have determined that the incorporation of phosphorodithioate linkages (as shown in IA or IB) between nucleosides in the flanking regions (e.g., between 2' sugar-modified nucleosides) also provides improvements, such as enhanced stability and / or improved potency.

[0291] As demonstrated herein, the achiral phosphorodithioate internucleoside linkages used in the compounds of the present invention can improve oligonucleotide stability. The incorporation of achiral phosphorodithioate internucleosides into the compounds of the present invention enhances resistance to exonucleases in serum and cells, particularly 3' exonucleases, but also 5' exonucleases, and the excellent stability of the compounds of the present invention further suggests that compounds incorporating achiral phosphorodithioate linkages are also resistant to endonucleases. Oligonucleotide stabilization is particularly important in reducing or preventing the accumulation of toxic degradation products and in extending the duration of action of antisense oligonucleotides. As demonstrated in the examples, improved stability can be analyzed using rat serum stability. To assess cellular stability, tissue (e.g., liver) homogenate extracts can be used—see, for example, WO 2014076195, which provides such a method. Other assays for measuring oligonucleotide stability include the snake venom phosphodiesterase stability assay and S1 nuclease stability.

[0292] The reduced toxicity risk of the claimed oligonucleotides is tested in an in vitro hepatotoxicity assay (e.g., as disclosed in WO 2017 / 067970), an in vitro nephrotoxicity assay (e.g., as disclosed in WO 2017 / 216340), or an in vitro neurotoxicity assay (e.g., as disclosed in WO 2016127000). Alternatively, toxicity may be analyzed in vivo, e.g., in mice or rats.

[0293] Enhanced stability can benefit the duration of action of the oligonucleotides of the invention, and is particularly beneficial when the route of administration is invasive, e.g., parenteral administration, such as intravenous, subcutaneous, intramuscular, intracerebral, intraocular, intracerebroventricular, or intrathecal administration.

[0294] General Oligonucleotide Aspects 1 Formula (IA) or (IB) TIFF0007724259000039.tif49128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2), at least one of the two nucleosides (A1) and (A2) is an LNA nucleoside, and in the formula (IA), R is a hydrogen or phosphate protecting group, and in the formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation) An oligonucleotide containing at least one phosphorodithioate internucleoside linkage of the formula: 2. The oligonucleotide of embodiment 1, wherein one of (A1) and (A2) is an LNA nucleoside, and the other is a DNA nucleoside, an RNA nucleoside, or a sugar-modified nucleoside. 3. The oligonucleotide according to embodiment 1 or 2, wherein one of (A1) and (A2) is an LNA nucleoside, and the other is a DNA nucleoside or a sugar-modified nucleoside. 4. The oligonucleotide according to any one of aspects 1 to 3, wherein one of (A1) and (A2) is an LNA nucleoside, and the other is a DNA nucleoside. 6. The oligonucleotide according to any one of aspects 1 to 3, wherein one of (A1) and (A2) is an LNA nucleoside, and the other is a sugar-modified nucleoside. 7. The oligonucleotide according to any one of aspects 2 to 6, wherein the sugar-modified nucleoside is a 2'-sugar-modified nucleoside. 8. The oligonucleotide of embodiment 7, wherein the 2' sugar-modified nucleoside is a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, or an LNA nucleoside. 9. The oligonucleotide of embodiment 7 or 8, wherein the 2' sugar modified nucleoside is an LNA nucleoside. The oligonucleotide of any one of embodiments 1 to 9, wherein the 10 LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 11. The oligonucleotide of embodiment 9 or 10, wherein both LNA nucleosides are β-D-oxy LNA. 12 The oligonucleotide of embodiment 7 or 8, wherein the 2' sugar modified nucleoside is 2'-alkoxyalkoxy-RNA. 13 The oligonucleotide of embodiment 10, wherein the 2'-alkoxy-RNA is 2'-methoxy-RNA. 14. The oligonucleotide of any one of aspects 1 to 12, wherein the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA. 15. The oligonucleotide according to any one of embodiments 1 to 14, comprising 1 to 15, particularly 1 to 5, more particularly 1, 2, 3, 4, or 5 phosphorodithioate internucleoside linkages of formula (IA) or (IB) as defined in embodiment 1. 16. The oligonucleotide of any one of embodiments 1 to 15, comprising a phosphodiester internucleoside linkage, a phosphorothioate internucleoside linkage, and a further internucleoside linkage independently selected from a phosphorodithioate internucleoside linkage of formula (IA) or (IB) as defined in embodiment 1. 17. The oligonucleotide according to embodiment 16, wherein the further internucleoside linkages are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (IA) or (IB) as defined in embodiment 1. 18. The oligonucleotide of embodiment 16 or 17, wherein all further internucleoside linkages are phosphorothioate internucleoside linkages. 19. The oligonucleotide according to embodiment 16 or 17, wherein all further internucleoside linkages are phosphorodithioate internucleoside linkages of formula (IA) or (IB) as defined in embodiment 1. 20. The oligonucleotide of any one of aspects 1 to 19, wherein the oligonucleotide is 7 to 30 nucleotides in length. 21. The oligonucleotide according to any one of aspects 1 to 20, wherein one or more nucleosides are nucleobase-modified nucleosides. 22. The oligonucleotide of any one of embodiments 1 to 21, which is an antisense oligonucleotide, an siRNA, a microRNA mimetic, or a ribozyme. 23. A pharmaceutically acceptable salt, in particular a sodium, potassium or ammonium salt, of an oligonucleotide according to any one of embodiments 1 to 22. 24. A conjugate comprising the oligonucleotide or pharmaceutically acceptable salt according to any one of embodiments 1 to 23, and at least one conjugate moiety covalently attached to the oligonucleotide or pharmaceutically acceptable salt, optionally via a linker moiety. 25. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 24, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 26. An oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 24 for use as a therapeutically active substance. 27. A method for producing an oligonucleotide according to any one of embodiments 1 to 24, comprising the steps of: (a) coupling a thiophosphoramidite nucleoside to the 5' terminal oxygen atom of a nucleotide or oligonucleotide to form a thiophosphite triester intermediate; (b) thio-oxidizing the thiophosphite triester intermediate obtained in step (a); and (c) optionally further extending the oligonucleotide. 28. An oligonucleotide produced by the method of embodiment 27.

[0295] Gapmer Aspects 1. An antisense gapmer oligonucleotide for inhibiting a target RNA in a cell, comprising: TIFF0007724259000040.tif49128 (wherein in formula (IA), R is hydrogen or a phosphate protecting group, and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation) An antisense gapmer oligonucleotide comprising at least one phosphorodithioate internucleoside linkage of the formula: 2. The antisense gapmer oligonucleotide of embodiment 1, wherein at least one phosphorodithioate internucleoside linkage has the formula (IA) and R is hydrogen; or at least one phosphorodithioate internucleoside linkage has the formula (IB) and M is Na, K, or ammonium. 3 One of the two oxygen atoms of at least one internucleoside bond in formula (I) is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and two nucleosides (A 1 ) and (A 2 3. The gapmer oligonucleotide of embodiment 1 or 2, wherein at least one of the nucleotides (a) to (c) is a 2' sugar-modified nucleoside. 4 (A 1 ) and (A 2 4. The gapmer oligonucleotide according to any one of Aspects 1 to 3, wherein one of said nucleosides is a 2' sugar-modified nucleoside, and the other is a DNA nucleoside. 5 (A 1 ) and (A 2 4. The gapmer oligonucleotide of any one of aspects 1 to 3, wherein: 6 (A 1 ) and (A 2 4. The gapmer oligonucleotide of any one of aspects 1 to 3, wherein: 7. The gapmer oligonucleotide of any one of aspects 1 to 6, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3', wherein G is a region of 5 to 18 nucleosides capable of recruiting RNase H, and region G is flanked at the 5' and 3' ends by flanking regions F and F', respectively, and regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, and wherein the nucleosides of region F adjacent to region G are 2'-sugar-modified nucleosides, and the nucleosides of region F' adjacent to region G are 2'-sugar-modified nucleosides. 8. The gapmer oligonucleotide of any one of aspects 1 to 7, wherein the 2' sugar-modified nucleosides are independently selected from 2'-alkoxy-RNA nucleosides, 2'-alkoxyalkoxy-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 2'-fluoro-ANA nucleosides, and LNA nucleosides. 9. The gapmer oligonucleotide of embodiment 8, wherein the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA (2'-O-MOE). 10. The gapmer oligonucleotide according to embodiment 7 or 8, wherein region F and region F' comprise or consist of 2'-methoxyethoxy-RNA nucleotides. 11. The gapmer oligonucleotide of any one of aspects 7 to 10, wherein at least one or all of the 2' sugar-modified nucleosides in region F or region F', or in both regions F and F', are LNA nucleosides. 12. The gapmer oligonucleotide according to any one of embodiments 7 to 11, wherein region F or region F' or both regions F and F' comprise at least one LNA nucleoside and at least one DNA nucleoside. 13. The gapmer oligonucleotide of any one of aspects 7 to 12, wherein region F or region F', or both region F and region F', comprises at least one LNA nucleoside and at least one non-LNA 2' sugar-modified nucleoside, such as at least one 2'-methoxyethoxy-RNA nucleoside. 14. The gapmer oligonucleotide of any one of embodiments 1 to 13, wherein the gap region comprises 5 to 16, particularly 8 to 16, more particularly 8, 9, 10, 11, 12, 13, or 14 consecutive DNA nucleosides. 15. The gapmer oligonucleotide of any one of embodiments 1 to 14, wherein region F and region F' are independently 1, 2, 3, 4, 5, 6, 7, or 8 nucleosides in length. 16. The gapmer oligonucleotide of any one of embodiments 1 to 15, wherein region F and region F' each independently comprise 1, 2, 3, or 4 LNA nucleosides. 17. The gapmer oligonucleotide of any one of embodiments 8 to 16, wherein the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 19. The gapmer oligonucleotide according to any one of embodiments 8 to 18, wherein the LNA nucleosides are β-D-oxy LNA. 19. The gapmer oligonucleotide according to any one of aspects 1 to 18, wherein the oligonucleotide or the consecutive nucleotide sequence (FG-F') thereof is 10 to 30 nucleotides in length, specifically 12 to 22, more specifically 14 to 20 oligonucleotides in length. 20. The gapmer oligonucleotide of any one of embodiments 1 to 19, wherein at least one of the flanking regions, such as region F and region F', comprises a phosphorodithioate linkage of formula (IA) or (IB) as defined in any one of embodiments 1 to 19. 21. A gapmer oligonucleotide according to any one of embodiments 1 to 19, wherein both flanking regions, such as region F and region F', comprise phosphorodithioate linkages of formula (IA) or (IB) as defined in any one of embodiments 1 to 19. 22. A gapmer oligonucleotide according to any one of embodiments 1 to 21, wherein at least one of the flanking regions, such as F or F', comprises at least two phosphorodithioate linkages of formula (IA) or (IB) as defined in any one of embodiments 1 to 19. 23. A gapmer oligonucleotide according to any one of embodiments 1 to 21, wherein both flanking regions, i.e., F and F', comprise at least two phosphorodithioate linkages of formula (IA) or (IB) as defined in any one of embodiments 1 to 19. 24. The gapmer oligonucleotide of any one of embodiments 1 to 23, wherein one or both of the flanking regions comprise LNA nucleosides having a phosphorodithioate linkage of formula (IA) or (IB) linking the LNA to the 3' nucleoside, respectively. 25. The gapmer oligonucleotide of any one of embodiments 1 to 24, wherein one or both flanking regions each comprise two or more adjacent LNA nucleosides linked by phosphorodithioate linkages of formula (IA) or (IB) linking the LNA to the 3' nucleoside. 26. The gapmer oligonucleotide of any one of embodiments 1 to 25, wherein one or both of the flanking regions comprise an MOE nucleoside having a phosphorodithioate linkage of formula (IA) or (IB) linking the MOE to the 3' nucleoside, respectively. 27. The gapmer oligonucleotide of any one of embodiments 1-26, wherein one or both flanking regions each comprise two or more adjacent MOE nucleosides linked by a phosphorodithioate linkage of formula (IA) or (IB) linking the MOE to the 3' nucleoside. 28. The gapmer oligonucleotide of any one of embodiments 1-27, wherein flanking regions F and F' together comprise 1, 2, 3, 4, or 5 phosphorodithioate internucleoside linkages of formula (IA) or (IB), and optionally the internucleoside linkage between the 3'-most nucleoside of region F and the 5'-most nucleoside of region G is also a phosphorodithioate internucleoside linkage of formula (IA) or (IB). 29. The gapmer oligonucleotide of any one of embodiments 1 to 28, comprising one phosphorodithioate internucleoside linkage of formula (IA) or (IB) located between adjacent nucleosides in region F or region F', between region F and region G, or between region G and region F'. 30. The gapmer region of any one of embodiments 1-29, wherein the gap region comprises one, two, three, or four phosphorodithioate internucleoside linkages of formula (IA) or (IB), and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. 31. The gapmer according to any one of embodiments 1 to 30, wherein the gap region comprises a region of at least 5 consecutive DNA nucleotides, such as a region of 6 to 18 consecutive DNA nucleotides or 8 to 14 consecutive DNA nucleotides. 32 One or more stereodefined phosphorothioate internucleoside linkages (Sp, S) or (Rp, R) TIFF0007724259000041.tif45128(in the formula, N 1 and N 2 32. The gapmer of any one of aspects 1 to 31, further comprising: 33. The gapmer of embodiment 32, comprising at least one stereodefined internucleoside bond (Sp, S) or (Rp, R) between two DNA nucleosides, e.g., between two DNA nucleosides in the gap region. 34. The gapmer oligonucleotide of embodiment 32 or 33, wherein the gap region comprises 2, 3, 4, 5, 6, 7, or 8 stereodefined phosphorothioate internucleoside linkages independently selected from Rp internucleoside linkages and Sp internucleoside linkages. 35. The gapmer oligonucleotide of embodiment 32 or 33, wherein region G further comprises at least two, three, or four internucleoside linkages of formula IB. 34. The gapmer oligonucleotide according to any one of embodiments 32 to 35, wherein (i) all of the remaining internucleoside linkages within region G (i.e., between nucleosides in region G) are any stereodefined phosphorothioate internucleoside linkages independently selected from Rp internucleoside linkages and Sp internucleoside linkages, or (ii) all of the internucleoside linkages within region G are any stereodefined phosphorothioate internucleoside linkages independently selected from Rp internucleoside linkages and Sp internucleoside linkages. 35. The gapmer oligonucleotide of any one of embodiments 1 to 34, wherein all of the internucleoside linkages in the flank regions are phosphorodithioate internucleoside linkages of formula (IA) or (IB), and optionally the internucleoside linkage between the 3'-most nucleoside of region F and the 5'-most nucleoside of region G is also a phosphorodithioate internucleoside linkage of formula (IA) or (IB), and the internucleoside linkage between the 3'-most nucleoside of region G and the 5'-most nucleoside of region F' is a stereodefined phosphorothioate internucleoside linkage. 36. The gapmer oligonucleotide of any one of embodiments 6 to 35, wherein the internucleoside linkages between the nucleosides of region G are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1. 37. The gapmer oligonucleotide according to any one of aspects 7 to 36, wherein the internucleoside linkages between the nucleosides of region G comprise zero, one, two, or three phosphorodithioate internucleoside linkages of formula (I) as defined in aspect 1, in particular zero phosphorodithioate internucleoside linkages of formula (I). 38. The gapmer oligonucleotide of any one of embodiments 1 to 37, wherein the remaining internucleoside linkages are independently selected from the group consisting of phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, and phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1. 39. The gapmer oligonucleotide of any one of embodiments 7 to 38, wherein the internucleoside linkages between the nucleosides of region F and the internucleoside linkages between the nucleosides of region F' are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1. 40. The gapmer oligonucleotide of any one of embodiments 7 to 39, wherein each flanking region F and F′ independently comprises 1, 2, 3, 4, 5, 6, or 7 phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1. 41. A gapmer oligonucleotide according to any one of embodiments 7 to 40, wherein all of the internucleoside linkages of flanking regions F and / or F' are phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1. 42. The gapmer oligonucleotide of any one of embodiments 1 to 41, comprising at least one stereodefined internucleoside linkage, e.g., at least one stereodefined phosphorothioate internucleoside linkage. 43. The gapmer oligonucleotide of any one of embodiments 1 to 42, wherein the gap region comprises 1, 2, 3, 4, or 5 stereodefined phosphorothioate internucleoside linkages. 44. The gapmer oligonucleotide of any one of embodiments 1 to 43, wherein all of the internucleoside linkages between the nucleosides in the gap region are stereodefined phosphorothioate internucleoside linkages. 45. The gapmer oligonucleotide of any one of embodiments 7 to 44, wherein at least one phosphorodithioate internucleoside linkage of formula (IA) or (IB) is located between nucleosides of region F, between nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages within regions F and F', between region F and region G, and between region G and region F' are independently selected from stereodefined phosphorothioate internucleoside linkages, stereorandom internucleoside linkages, phosphorodithioate internucleoside linkages of formula (IA) or (IB), and phosphodiester internucleoside linkages. 46. The gapmer oligonucleotide of embodiment 45, wherein the remaining internucleoside linkages within region F, region F', or both region F and region F' are all phosphorodithioate internucleoside linkages of formula (IA) or (IB). 47. The gapmer oligonucleotide of any one of embodiments 6 to 33, wherein the internucleoside linkages between the nucleosides of region G comprise zero, one, two, or three phosphorodithioate internucleoside linkages of formula (I) as defined in embodiment 1, and the remaining internucleoside linkages in region G are independently selected from stereodefined phosphorothioate internucleoside linkages, stereorandom internucleoside linkages, and phosphodiester internucleoside linkages. 48. The gapmer oligonucleotide of any one of embodiments 1 to 47, wherein the 3' terminal nucleoside of the antisense oligonucleotide is an LNA nucleoside or a 2'-O-MOE nucleoside. 49. The gapmer oligonucleotide of any one of embodiments 1 to 48, wherein the 5' terminal nucleoside of the antisense oligonucleotide is an LNA nucleoside or a 2'-O-MOE nucleoside. 50. The gapmer oligonucleotide of any one of embodiments 1 to 49, wherein the two 3'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 51. The gapmer oligonucleotide of any one of embodiments 1 to 50, wherein the two 5'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 52. The gapmer oligonucleotide of any one of embodiments 1 to 51, wherein the three 3'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 53. The gapmer oligonucleotide of any one of embodiments 1 to 52, wherein the three 5'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 54. The gapmer oligonucleotide of any one of embodiments 1 to 53, wherein the two 3'-most terminal nucleosides of the antisense oligonucleotide are LNA nucleosides. 55. The gapmer oligonucleotide of any one of embodiments 1 to 54, wherein the two 5'-most terminal nucleosides of the antisense oligonucleotide are LNA nucleosides. 56 Nucleosides of formula (IA) or (IB) (A 2 56. The gapmer oligonucleotide of any one of aspects 1 to 55, wherein: 57 Nucleosides of formula (IA) or (IB) (A 1 57. The gapmer oligonucleotide of any one of aspects 1 to 56, wherein: 58. A gapmer oligonucleotide according to any one of embodiments 7 to 57, comprising a contiguous nucleotide sequence of the formula 5'-D'-FG-F'-D''-3' (wherein F, G, and F' are as defined in any one of embodiments 7 to 45, and wherein regions D' and D'' each independently consist of 0 to 5 nucleotides, particularly 2, 3, or 4 nucleotides, particularly DNA nucleotides, such as phosphodiester-linked DNA nucleosides) [oligonucleotide, including gapmer oligonucleotide and flanking sequence]. 59. A gapmer oligonucleotide according to any one of aspects 1 to 58, which is capable of recruiting human RNase H1. 60. A gapmer oligonucleotide according to any one of embodiments 1 to 59, which is for the in vitro or in vivo inhibition of a mammalian, e.g. human, mRNA or pre-mRNA target, a viral target, or a long non-coding RNA. 61. A pharmaceutically acceptable salt, particularly a sodium or potassium salt, of a gapmer oligonucleotide according to any one of embodiments 1 to 60. 62. A conjugate comprising a gapmer oligonucleotide or a pharmaceutically acceptable salt according to any one of embodiments 1 to 61, and at least one conjugate moiety covalently attached to the oligonucleotide or the pharmaceutically acceptable salt, optionally via a linker moiety. 63. A pharmaceutical composition comprising a gapmer oligonucleotide according to any one of embodiments 1 to 62, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 64. A gapmer oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 63 for use as a therapeutically active substance.

[0296] Antisense Oligonucleotide Embodiments The present invention relates to a compound of formula (IA) or (IB) TIFF0007724259000042.tif49128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2); and in formula (IA), R is a hydrogen or phosphate protecting group; and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation). The present invention relates to oligonucleotides containing at least one phosphorodithioate internucleoside linkage of the formula:

[0297] In other words, M is a metal, such as an alkali metal, such as Na or K, or M is NH4.

[0298] For example, oligonucleotide can be a single-stranded antisense oligonucleotide, which can regulate the expression of target nucleic acid, such as target microRNA, or can regulate the splicing of target pre-mRNA, and comprises a continuous nucleotide sequence.The antisense oligonucleotide of the present invention is complementary to target nucleic acid, and comprises a continuous nucleotide sequence that can hybridize with target nucleic acid and regulate its expression.In a preferred embodiment, antisense oligonucleotide or its continuous nucleotide sequence is a mixer oligonucleotide, in which either (A1) or (A2) is DNA nucleoside, or both (A1) and (A2) are DNA nucleoside.

[0299] In the present invention, antisense oligonucleotides are single-stranded oligonucleotides that are complementary to a nucleic acid target, e.g., a target RNA, and can modulate (e.g., splice modulation of a pre-mRNA target) or inhibit expression of the nucleic acid target (e.g., an mRNA target, a pre-mRNA target, a viral RNA target, or a long non-coding RNA target). Depending on the target, the length of the oligonucleotide or region that is complementary (i.e., antisense—preferably, the complementary region is fully complementary to the target) can be 7 to 30 nucleotides (a region referred to as a contiguous nucleotide sequence). For example, LNA nucleotide inhibitors of microRNAs can be as short as 7 contiguous complementary nucleotides (or as long as 30 nucleotides), while oligonucleotides that recruit RNase H are typically at least 12 contiguous complementary nucleotides in length, e.g., 12 to 26 nucleotides in length. Splice-modulating antisense oligonucleotides typically have a contiguous nucleotide region consisting of 10 to 30 complementary nucleotides.

[0300] Splice-modulating oligonucleotides, also known as splice-switching oligonucleotides (SSOs), are short, synthetic, antisense-modified nucleic acids that base-pair with pre-mRNA and disrupt the normal splicing repertoire of a transcript by interfering with RNA-RNA base pairing or protein-RNA binding interactions between components of the splicing machinery and the pre-mRNA. Pre-mRNA splicing is required for the proper expression of the vast majority of protein-coding genes; therefore, targeting this process provides a means to manipulate protein production from genes. Splicing modulation is particularly beneficial in diseases caused by mutations that disrupt normal splicing or in cases where interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs provide an effective and specific method for therapeutically targeting and altering splicing. See Haven's and Hasting, NAR (2016) 44, 6549-6563. SSOs may be complementary to exon / intron boundaries of the target pre-mRNA, or may target splicing enhancer or silencer elements (collectively referred to as cis-acting splice elements) within the pre-mRNA that control pre-mRNA splicing. Splice modulation can result in exon skipping or exon inclusion, thereby regulating alternative splicing of the pre-mRNA.SSOs function by modulating target pre-mRNAs without nuclease mediation and thus cannot recruit RNase H. They are often either fully modified oligonucleotides, i.e., those in which each nucleoside contains a modified sugar moiety, such as a 2'-sugar-substituted sugar moiety (e.g., a fully 2'-O-MOE oligonucleotide based on a phosphorothioate backbone, e.g., 15-25 nucleotides in length, often 18-22 or 20 nucleotides in length), or LNA mixer oligonucleotides (oligonucleotides 10-30 nucleotides in length containing DNA and LNA nucleosides, and optionally other 2'-sugar-modified nucleosides, such as 2'-O-MOE). LNA oligonucleotides that do not contain DNA nucleosides but contain LNA nucleosides and other 2'-sugar-modified nucleosides, such as 2'-O-MOE nucleosides, are also contemplated. Table 1 of Haven's and Hasting NAR (2016) 44, 6549-6563, incorporated herein by reference, provides a range of SSO targets and the chemistries of the oligonucleotides used that have reported activity in vivo, and is reproduced below in Table A.

[0301] (Table A) TIFF0007724259000043.tif127157TIFF0007724259000044.tif234157TIFF00077242590 00045.tif233157TIFF0007724259000046.tif253158TIFF0007724259000047.tif133157

[0302] In some embodiments of the present invention, the antisense oligonucleotide is a splice-regulatory oligonucleotide complementary to a pre-mRNA selected from the group consisting of HBB, FKTN, LMNA, CEP290, CLCN1, USH1C, BTK, LRP8, CTLA4, BCL2L1, ERBB4, MDM4, STAT3, IL1RAP, TNFRSFIB, FLT1, KDR, SMN2, MYBPC3, TTN, DMD, NBN, IL10, HTT, APOB, MSTN, GYS2, and ATXN3. Exemplary diseases that can be treated using the SSO of the present invention are listed in Table A, by individual target.

[0303] The following embodiments relate generally to the single-stranded antisense oligonucleotides of the invention, and in particular to splice control antisense oligonucleotides (SSOs). 1. A single-stranded antisense oligonucleotide for regulating an RNA target in a cell, wherein the antisense oligonucleotide comprises or consists of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprises one or more 2' sugar-modified nucleosides, and at least one of the internucleoside linkages present between the nucleosides of the contiguous nucleotide sequence is represented by formula (IA) or (IB): TIFF0007724259000048.tif55128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) is attached to the 5' carbon atom of ) and R is a hydrogen or phosphate protecting group) by a phosphorodithioate linkage. 2 Two nucleosides (A 1 ) and (A 2 2. The antisense oligonucleotide of embodiment 1, wherein at least one of the nucleotides (a) to (c) is a 2' sugar-modified nucleoside. 3 Nucleosides (A 1 ) and (A 2 2. The antisense oligonucleotide of embodiment 1, wherein both of said nucleotides are 2' sugar-modified nucleosides. 4 Two nucleosides (A 1 ) and (A 2 ) or at least one of nucleosides (A 1 ) and (A 2 4. The antisense oligonucleotide of any one of Aspects 1 to 3, wherein both of the nucleotides (a) and (b) are DNA nucleosides. 5 (A 1 ) and (A 2 5. The antisense oligonucleotide of any one of Aspects 1 to 4, wherein at least one of the 2'-sugar-modified nucleosides is a 2'-sugar-modified nucleoside, or the nucleosides are independently selected from a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, or an LNA nucleoside. 6 (A 1 ) and (A 2 6. The antisense oligonucleotide according to any one of aspects 1 to 5, wherein at least one of the following is an LNA nucleoside: 7 (A 1 ) and (A 2 6. The antisense oligonucleotide of any one of aspects 1 to 5, wherein both of said amino acid residues are LNA nucleosides. 8 (A 1 ) and (A 2 7. The antisense oligonucleotide of any one of Aspects 1 to 6, wherein at least one of the following is a 2'-O-methoxyethyl nucleoside: 9 (A 1 ) and (A 2 6. The antisense oligonucleotide of any one of Aspects 1 to 5, wherein both of Aspects 1 and 2 are 2'-O-methoxyethyl nucleosides. The antisense oligonucleotide of any one of aspects 1 to 8, wherein the 10 LNA nucleosides are selected from the group consisting of β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 11. The antisense oligonucleotide according to any one of aspects 1 to 8, wherein the LNA nucleoside is β-D-oxy LNA. 12. The antisense oligonucleotide of any one of aspects 1 to 11, wherein the contiguous nucleotide sequence comprises one or more additional 2' sugar-modified nucleosides, e.g., one or more additional 2' sugar-modified nucleosides selected from the group consisting of 2'-alkoxy-RNA nucleosides, 2'-alkoxyalkoxy-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 2'-fluoro-ANA nucleosides, or LNA nucleosides. 13. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and DNA nucleosides. 14. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and 2'-O-methoxyethyl nucleosides. The antisense oligonucleotide of any one of embodiments 1 to 13, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and 2' fluoro RNA nucleosides. 16. The antisense oligonucleotide according to any one of aspects 1 to 13, wherein the contiguous nucleotide sequence comprises any of the following: (i) LNA nucleosides and DNA nucleosides only (ii) LNA nucleosides and 2'-O-methoxyethyl nucleosides only (iii) LNA nucleosides, DNA nucleosides, and 2'-O-methoxyethyl nucleosides only (iv) LNA nucleosides, 2'-fluoro RNA nucleosides, and 2'-O-methoxyethyl nucleosides only (v) only LNA nucleosides, DNA nucleosides, 2'-fluoro RNA nucleosides, and 2'-O-methoxyethyl nucleosides, or only LNA nucleosides, 2'-fluoro RNA nucleosides, and 2'-O-methoxyethyl nucleosides (vi) 2'-O-methoxyethyl nucleosides only. 17. The antisense oligonucleotide of any one of aspects 1 to 16, wherein the contiguous nucleotide sequence does not comprise a sequence of 4 or more contiguous DNA nucleosides, or does not comprise a sequence of 3 or more contiguous DNA nucleosides. 18. The antisense oligonucleotide according to any one of aspects 1 to 17, wherein the antisense oligonucleotide or the contiguous nucleotide sequence thereof is a mixmer oligonucleotide or a totalmer oligonucleotide. 19. The antisense oligonucleotide according to any one of aspects 1 to 18, which is unable to recruit human RNase H1. 20. The antisense oligonucleotide according to any one of aspects 1 to 19, wherein the nucleoside (A2) is the 3′-terminal nucleoside of the consecutive nucleotide sequence of the oligonucleotide. The antisense oligonucleotide according to any one of aspects 1 to 20, wherein the 21st nucleoside (A1) is the 5'-terminal nucleoside of the consecutive nucleotide sequence of the oligonucleotide. 22. The antisense oligonucleotide of any one of embodiments 1 to 21, comprising at least two phosphorodithioate internucleoside linkages of formula I, e.g., two, three, four, five, or six phosphorodithioate internucleoside linkages of formula I. 23. The antisense oligonucleotide of any one of aspects 1 to 22, wherein the internucleoside linkage between the two 3'-most nucleosides of the contiguous nucleotide sequence is a phosphorodithioate internucleoside linkage of Formula I, and the internucleoside linkage between the two 5'-most nucleosides of the contiguous nucleotide sequence is a phosphorodithioate internucleoside linkage of Formula I. 24. The antisense oligonucleotide of any one of embodiments 1 to 23, further comprising phosphorothioate internucleoside linkages. 25. The antisense oligonucleotide of any one of embodiments 1 to 24, further comprising stereodefined phosphorothioate internucleoside linkages. 26. The antisense oligonucleotide of any one of aspects 1 to 25, wherein the remaining internucleoside linkages are independently selected from the group consisting of phosphorodithioate internucleoside linkages, phosphorothioate internucleoside linkages, and phosphodiester internucleoside linkages. 27. The antisense oligonucleotide of any one of aspects 1 to 26, wherein the remaining internucleoside linkages are phosphorothioate internucleoside linkages. 28. The antisense oligonucleotide of any one of embodiments 1 to 27, wherein the contiguous nucleotide sequence is complementary, e.g., 100% complementary, to a mammalian pre-mRNA, a mammalian mature mRNA target, a viral RNA target, or a mammalian long non-coding RNA. 29. The antisense oligonucleotide of any one of embodiments 28, wherein the RNA target is a human RNA target. 30. The antisense oligonucleotide of any one of embodiments 1 to 29, which modulates splicing of a mammalian, e.g., human, pre-mRNA target, e.g., is a splice-skipping antisense oligonucleotide or a splicing-modulating antisense oligonucleotide. 31. The antisense oligonucleotide of any one of embodiments 1 to 30, which is complementary, eg, 100% complementary, to an intron / exon splice junction of a human pre-mRNA or a splice control region of a human pre-mRNA. 32. The antisense oligonucleotide of any one of aspects 1 to 30, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof is complementary, e.g., fully complementary, to a human pre-mRNA sequence selected from the group consisting of TNFR2, HBB, FKTN, LMNA, CEP290, CLCN1, USH1C, BTK, LRP8, CTLA4, BCL2L1, ERBB4, MDM4, STAT3, IL1RAP, TNFRSFlB, FLT1, KDR, SMN2, MYBPC3, TTN, DMD, NBN, IL10, HTT, APOB, MSTN, GYS2, and ATXN3. 33. The antisense oligonucleotide according to any one of aspects 1 to 32, consisting of or comprising a contiguous nucleotide sequence selected from the group consisting of SSO#1 to SSO#25. 34. The antisense oligonucleotide according to any one of embodiments 1 to 33, wherein the cell is a human cell. 35. The antisense oligonucleotide of any one of aspects 1 to 34, wherein the antisense oligonucleotide is 10 to 30 nucleotides in length. 36. The antisense oligonucleotide of any one of embodiments 1 to 34, wherein the antisense oligonucleotide is 12 to 24 nucleotides in length. 37. The antisense oligonucleotide according to any one of aspects 1 to 36, wherein the 3' terminal nucleoside of the antisense oligonucleotide or the contiguous nucleotide sequence thereof is either an LNA nucleoside or a 2-O-methoxyethyl nucleoside. 38. The antisense oligonucleotide according to any one of aspects 1 to 27, wherein the 5' terminal nucleoside of the antisense oligonucleotide or its consecutive nucleotide sequence is either an LNA nucleoside or a 2-O-methoxyethyl nucleoside. 39. The antisense oligonucleotide according to any one of embodiments 1 to 38, wherein the 5'-terminal nucleoside and the 3'-terminal nucleoside of the antisense oligonucleotide or its consecutive nucleotide sequence are both LNA nucleosides. 40. The antisense oligonucleotide of any one of aspects 1 to 39, wherein the consecutive nucleotide sequence comprises at least one region consisting of two or three consecutive LNA nucleotides and / or at least one region consisting of two or three consecutive 2'-O-methoxyethyl nucleotides. 41. A pharmaceutically acceptable salt, in particular a sodium, potassium or ammonium salt, of an oligonucleotide according to any one of embodiments 1 to 40. 42. A conjugate comprising the oligonucleotide or pharmaceutically acceptable salt according to any one of embodiments 1 to 41 and at least one conjugate moiety covalently attached to the oligonucleotide or pharmaceutically acceptable salt, optionally via a linker moiety. 43. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 42, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 44. An oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 43 for use as a therapeutically active substance. 45. A method for modulating a target RNA in a cell expressing said target RNA, comprising administering to said cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate, or composition of any one of aspects 1 to 44. 46. A method for modulating splicing of a target pre-mRNA in a cell expressing the target pre-mRNA, the method comprising administering to the cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate, or composition according to any one of aspects 1 to 44. 47. The method of embodiment 45 or 46, which is an in vitro method or an in vivo method. 48. Use of an oligonucleotide, pharmaceutical salt, conjugate, or composition according to any one of embodiments 1 to 44 for inhibiting RNA in a cell, such as a human cell, wherein the use is in vitro or in vivo.

[0304] Specific Aspects of Mixmar 1. A single-stranded antisense oligonucleotide for regulating an RNA target in a cell, the antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length, the contiguous nucleotide sequence comprising alternating regions of 2' sugar-modified nucleosides, the maximum length of contiguous DNA nucleosides comprising the contiguous nucleotide sequence being 3 or 4, and at least one of the internucleoside linkages present between nucleosides of the contiguous nucleotide sequence being represented by formula (IA) or (IB): TIFF0007724259000049.tif55128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) is attached to the 5' carbon atom of ) and R is a hydrogen or phosphate protecting group) by a phosphorodithioate linkage. 2 Two nucleosides (A 1 ) and (A 2 2. The antisense oligonucleotide of embodiment 1, wherein at least one of the nucleotides (a) to (c) is a 2' sugar-modified nucleoside. 3 Nucleosides (A 1 ) and (A 2 2. The antisense oligonucleotide of embodiment 1, wherein both of said nucleotides are 2' sugar-modified nucleosides. 4 Two nucleosides (A 1 ) and (A 2 ) or at least one of nucleosides (A 1 ) and (A 2 4. The antisense oligonucleotide of any one of Aspects 1 to 3, wherein both of the nucleotides (a) and (b) are DNA nucleosides. 5 (A 1 ) and (A 25. The antisense oligonucleotide of any one of Aspects 1 to 4, wherein at least one of the 2'-sugar-modified nucleosides is a 2'-sugar-modified nucleoside, or the nucleosides are independently selected from a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, or an LNA nucleoside. 6 (A 1 ) and (A 2 6. The antisense oligonucleotide according to any one of aspects 1 to 5, wherein at least one of the following is an LNA nucleoside: 7 (A 1 ) and (A 2 6. The antisense oligonucleotide of any one of aspects 1 to 5, wherein both of said amino acid residues are LNA nucleosides. 8 (A 1 ) and (A 2 7. The antisense oligonucleotide of any one of Aspects 1 to 6, wherein at least one of the following is a 2'-O-methoxyethyl nucleoside: 9 (A 1 ) and (A 2 6. The antisense oligonucleotide of any one of Aspects 1 to 5, wherein both of Aspects 1 and 2 are 2'-O-methoxyethyl nucleosides. The antisense oligonucleotide of any one of aspects 1 to 8, wherein the 10 LNA nucleosides are selected from the group consisting of β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 11. The antisense oligonucleotide according to any one of aspects 1 to 8, wherein the LNA nucleoside is β-D-oxy LNA. 12. The antisense oligonucleotide of any one of aspects 1 to 11, wherein the contiguous nucleotide sequence comprises one or more additional 2' sugar-modified nucleosides, e.g., one or more additional 2' sugar-modified nucleosides selected from the group consisting of 2'-alkoxy-RNA nucleosides, 2'-alkoxyalkoxy-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 2'-fluoro-ANA nucleosides, or LNA nucleosides. 13. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and DNA nucleosides. 14. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and 2'-O-methoxyethyl nucleosides. The antisense oligonucleotide of any one of embodiments 1 to 13, wherein the contiguous nucleotide sequence comprises both LNA nucleosides and 2' fluoro RNA nucleosides. 16. The antisense oligonucleotide according to any one of aspects 1 to 13, wherein the contiguous nucleotide sequence comprises any of the following: (i) LNA nucleosides and DNA nucleosides (ii) LNA nucleosides, DNA nucleosides, and 2'-O-methoxyethyl nucleosides (iii) LNA nucleosides, DNA nucleosides, 2'-fluoro RNA nucleosides, and 2'-O-methoxyethyl nucleosides. 17. The antisense oligonucleotide of any one of aspects 1 to 16, wherein the contiguous nucleotide sequence does not comprise a sequence of 3 or more contiguous DNA nucleosides, or does not comprise a sequence of 2 or more contiguous DNA nucleosides. 18. The antisense oligonucleotide according to any one of aspects 1 to 17, wherein the antisense oligonucleotide or the contiguous nucleotide sequence thereof is a mixmer oligonucleotide, such as a splice regulator oligonucleotide or a microRNA inhibitor oligonucleotide. 19 Mixmar, alternating area motif 19. The antisense oligonucleotide of embodiment 18, consisting of or comprising: TIFF0007724259000050.tif87143, wherein L represents a 2' sugar-modified nucleoside; D represents a DNA nucleoside; and each m is independently selected from 1 to 6; and each n is independently selected from 1, 2, 3, and 4, e.g., 1 to 3. 20. The antisense oligonucleotide of embodiment 19, wherein each L nucleoside is independently selected from the group consisting of an LNA nucleoside, a 2'-O-MOE nucleoside, or a 2'-fluoro nucleoside, or each L is independently LNA or 2'-O-MOE. 21. The antisense oligonucleotide of embodiment 20, wherein each L is an LNA. 22. The antisense oligonucleotide according to any one of aspects 1 to 21, which is unable to recruit human RNase H1. 23 Nucleosides (A 2 23. The antisense oligonucleotide according to any one of Aspects 1 to 22, wherein: 24 Nucleosides (A 1 24. The antisense oligonucleotide according to any one of aspects 1 to 23, wherein: 25. The antisense oligonucleotide of any one of embodiments 1 to 24, comprising at least two phosphorodithioate internucleoside linkages of Formula I, e.g., two, three, four, five, or six phosphorodithioate internucleoside linkages of Formula I. 26. The antisense oligonucleotide according to any one of aspects 1 to 25, wherein the consecutive nucleotide sequence comprises two consecutive DNA nucleotides, wherein the nucleoside linkage between the two consecutive DNA nucleotides is a phosphorodithioate internucleoside linkage of formula (IA) or (IB), i.e., a P2S-linked DNA nucleotide pair. 27. The antisense oligonucleotide of any one of embodiments 1 to 26, wherein the contiguous nucleotide sequence comprises a plurality of P2S-bound DNA nucleotide pairs. 28. The antisense oligonucleotide of any one of embodiments 1 to 26, wherein all nucleoside linkages between two consecutive DNA nucleotides present in the consecutive nucleotide sequence are phosphorodithioate internucleoside linkages of formula (IA) or (IB). 29. The antisense oligonucleotide of any one of embodiments 1 to 27, wherein at least one internucleoside linkage between a 2' sugar-modified nucleoside and a DNA nucleoside is a phosphorodithioate internucleoside linkage of formula (IA) or (IB). 30 The antisense oligonucleotide of any one of embodiments 1 to 27, wherein a plurality of internucleoside linkages between 2' sugar-modified nucleosides and DNA nucleosides are phosphorodithioate internucleoside linkages of formula (IA) or (IB). 31 The antisense oligonucleotide of any one of embodiments 1 to 27, wherein all internucleoside linkages between 2' sugar-modified nucleosides and DNA nucleosides are phosphorodithioate internucleoside linkages of formula (IA) or (IB). 32 The antisense oligonucleotide of any one of aspects 1 to 27, wherein at least one of the internucleoside linkages between two 2' sugar-modified nucleosides is not a phosphorodithioate internucleoside linkage of formula (IA) or (IB), e.g., a phosphorothioate internucleoside linkage. 33 The antisense oligonucleotide of any one of aspects 1 to 27, wherein all of the internucleoside linkages between the two 2' sugar-modified nucleosides are not phosphorodithioate internucleoside linkages of formula (IA) or (IB), e.g., phosphorothioate internucleoside linkages. 34. The antisense oligonucleotide of any one of aspects 1 to 33, wherein the internucleoside linkage between the two 3'-most nucleosides of the contiguous nucleotide sequence is a phosphorodithioate internucleoside linkage of Formula I, and the internucleoside linkage between the two 5'-most nucleosides of the contiguous nucleotide sequence is a phosphorodithioate internucleoside linkage of Formula I. 35. The antisense oligonucleotide of any one of embodiments 1 to 34, further comprising a phosphorothioate internucleoside linkage. 36. The antisense oligonucleotide of any one of embodiments 1 to 35, further comprising stereodefined phosphorothioate internucleoside linkages. 37. The antisense oligonucleotide of any one of embodiments 1 to 35, wherein the remaining internucleoside linkages are independently selected from the group consisting of phosphorodithioate internucleoside linkages, phosphorothioate internucleoside linkages, and phosphodiester internucleoside linkages. 38. The antisense oligonucleotide of any one of aspects 1 to 36, wherein the remaining internucleoside linkages are phosphorothioate internucleoside linkages. 39. The antisense oligonucleotide according to any one of embodiments 1 to 37, wherein the contiguous nucleotide sequence is complementary, eg 100% complementary, to a mammalian, eg human, pre-mRNA. 40. The antisense oligonucleotide of any one of embodiments 1 to 38, which modulates splicing of a mammalian, e.g., human, pre-mRNA target, e.g., is a splice-skipping antisense oligonucleotide or a splice-regulatory antisense oligonucleotide. 41. The antisense oligonucleotide of any one of embodiments 1 to 39, which is complementary, eg, 100% complementary, to an intron / exon splice junction of a human pre-mRNA or a splice control region of a human pre-mRNA. 42. The antisense oligonucleotide of any one of aspects 1 to 41, wherein the antisense oligonucleotide or contiguous nucleotide sequence thereof is complementary, e.g., fully complementary, to a human pre-mRNA sequence selected from the group consisting of TNFR2, HBB, FKTN, LMNA, CEP290, CLCN1, USH1C, BTK, LRP8, CTLA4, BCL2L1, ERBB4, MDM4, STAT3, IL1RAP, TNFRSFlB, FLT1, KDR, SMN2, MYBPC3, TTN, DMD, NBN, IL10, HTT, APOB, MSTN, GYS2, and ATXN3. 43. The antisense oligonucleotide according to any one of aspects 1 to 42, consisting of or comprising a contiguous nucleotide sequence selected from the group consisting of SSO#1 to SSO#25. 44. The antisense oligonucleotide of any one of embodiments 1 to 43, wherein the cell is a mammalian cell. 45. The antisense oligonucleotide of any one of aspects 1 to 44, wherein the antisense oligonucleotide is 10 to 30 nucleotides in length. 46. The antisense oligonucleotide of any one of embodiments 1 to 44, wherein the antisense oligonucleotide is 12 to 24 nucleotides in length. 47. The antisense oligonucleotide according to any one of aspects 1 to 46, wherein the 3' terminal nucleoside of the antisense oligonucleotide or the contiguous nucleotide sequence thereof is either an LNA nucleoside or a 2-O-methoxyethyl nucleoside. 48. The antisense oligonucleotide according to any one of aspects 1 to 47, wherein the 5' terminal nucleoside of the antisense oligonucleotide or its contiguous nucleotide sequence is either an LNA nucleoside or a 2-O-methoxyethyl nucleoside. 49. The antisense oligonucleotide according to any one of embodiments 1 to 48, wherein the 5'-terminal nucleoside and the 3'-terminal nucleoside of the antisense oligonucleotide or its consecutive nucleotide sequence are both LNA nucleosides. 50. The antisense oligonucleotide of any one of aspects 1 to 49, wherein the contiguous nucleotide sequence comprises at least one region consisting of two or three consecutive LNA nucleotides and / or at least one region consisting of two or three consecutive 2'-O-methoxyethyl nucleotides. 51. A pharmaceutically acceptable salt, in particular a sodium, potassium or ammonium salt, of an oligonucleotide according to any one of embodiments 1 to 50. 52. A conjugate comprising the oligonucleotide or pharmaceutically acceptable salt according to any one of embodiments 1 to 51, and at least one conjugate moiety covalently attached to the oligonucleotide or pharmaceutically acceptable salt, optionally via a linker moiety. 53. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 52, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 54. An oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 53 for use as a therapeutically active substance. 55. A method for modulating a target RNA in a cell expressing said RNA, comprising administering to said cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate, or composition of any one of aspects 1 to 54. 56. A method for modulating splicing of a target pre-mRNA in a cell expressing the target pre-mRNA, comprising administering to the cell an effective amount of an oligonucleotide, pharmaceutically acceptable salt, conjugate, or composition according to any one of aspects 1 to 54. 57. The method of embodiment 55 or 56, which is an in vitro method or an in vivo method. 58. Use of an oligonucleotide, pharmaceutical salt, conjugate, or composition according to any one of embodiments 1 to 54 for inhibiting RNA in a cell, such as a mammalian cell, wherein the use is in vitro or in vivo.

[0305] Specific Aspects Regarding 3'-End Protection 1 Formula (IA) or (IB) TIFF0007724259000051.tif49128 (wherein one of the two oxygen atoms is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ), and in the formula (IA), R is hydrogen or a phosphate protecting group, and in the formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation, and two nucleosides (A 1 ) and (A 2 ) is a 2' sugar-modified nucleoside, e.g., an LNA nucleoside or a 2'-O-MOE nucleoside, R is a hydrogen or phosphate protecting group, and A 2 is the 3'-terminal nucleoside of the oligonucleotide) A single-stranded antisense oligonucleotide comprising at least one phosphorodithioate internucleoside linkage of the formula: 2 (A 2 ) is an LNA nucleoside, or (A 1 ) and (A 2 2. The single-stranded antisense of embodiment 1, wherein both of said amino acid residues are LNA nucleosides. 3 (A 2 ) is an LNA nucleoside, and (A 1 The single-stranded antisense according to embodiment 1, wherein the nucleotide sequence of 4 (A 2 ) is an LNA nucleoside, and (A 1 2. The single-stranded antisense oligonucleotide of embodiment 1, wherein: 5 (A 1) is an LNA nucleoside, and (A 2 The single-stranded antisense according to embodiment 1, wherein the nucleotide sequence of 6 (A 1 ) is an LNA nucleoside, and (A 2 2. The single-stranded antisense of embodiment 1, wherein: 7. The single-stranded antisense according to embodiment 3 or 5, wherein the sugar-modified nucleoside is a 2' sugar-modified nucleoside. 8. The single-stranded antisense according to embodiment 7, wherein the 2' sugar-modified nucleoside is a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, or an LNA nucleoside. 9. The single-stranded antisense according to embodiment 7 or 8, wherein the 2' sugar-modified nucleoside is a 2'-O-methoxyethyl nucleoside. 10. The single stranded antisense of any one of aspects 1 to 9, wherein the LNA nucleoside or LNA nucleotide is in the β-D-configuration. 11. The single-stranded antisense of any one of aspects 1 to 10, wherein the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 12. The single-stranded antisense according to any one of aspects 1 to 11, wherein the LNA is β-D-oxy LNA. 13. The single-stranded antisense according to any one of aspects 1 to 12, consisting of or comprising 7 to 30 consecutive nucleotides (referred to as the consecutive nucleotide sequence of the antisense single-stranded antisense) that are complementary to a target nucleic acid, for example, a target nucleic acid selected from the group consisting of pre-mRNA and mRNA, microRNA, viral RNA, and long non-coding RNA. 14. The single-stranded antisense of any one of aspects 1 to 13, wherein the contiguous nucleotide sequence comprises a gapmer region of the formula 5'-FG-F'-3', wherein G is a region of 5 to 18 nucleosides capable of recruiting RNase H, and region G is flanked at the 5' and 3' ends by flanking regions F and F', respectively, and regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, and the nucleosides of region F flanking region G are 2'-sugar-modified nucleosides, and the nucleosides of region F' flanking region G are 2'-sugar-modified nucleosides. 14. The single-stranded antisense of any one of aspects 1 to 13, wherein the consecutive nucleotide sequence is a mixmer oligonucleotide, the mixmer oligonucleotide comprising both LNA nucleosides and DNA nucleosides, and optionally 2' sugar-modified nucleosides (e.g., those described in aspect 8 or 9), and the single-stranded antisense does not comprise a region consisting of four or more consecutive DNA nucleosides. 15. The single-stranded antisense according to any one of aspects 1 to 13, wherein the contiguous nucleotide sequence comprises only sugar-modified nucleosides. 16. The oligonucleotide according to embodiment 14 or 15, which is a splice regulator oligonucleotide (capable of regulating the splicing of a pre-mRNA splice event). 17. The oligonucleotide of embodiment 14 or 15, which is complementary to a microRNA, e.g., is a microRNA inhibitor. 18. The oligonucleotide of any one of embodiments 1 to 17, comprising an additional internucleoside linkage independently selected from a phosphodiester internucleoside linkage, a phosphorothioate internucleoside linkage, and a phosphorodithioate internucleoside linkage, or wherein the additional internucleoside linkages within the oligonucleotide or within the contiguous nucleotide sequence thereof are independently selected from a phosphorothioate internucleoside linkage and a phosphorodithioate internucleoside linkage. 18. The oligonucleotide according to any one of embodiments 1 to 18, wherein all further internucleoside linkages of the oligonucleotide or of the contiguous nucleotide sequence thereof are phosphorothioate internucleoside linkages. 19. The oligonucleotide of any one of aspects 1 to 18, wherein the oligonucleotide comprises a 5' region that is 5' to the contiguous nucleotide sequence, the 5' nucleoside region comprising at least one phosphodiester bond. 20. The oligonucleotide according to embodiment 19, wherein the 5' region comprises 1 to 5 phosphodiester-linked DNA nucleosides, and optionally the oligonucleotide or the consecutive nucleotide sequence thereof may be linked to a conjugate moiety. 21. The oligonucleotide according to any one of aspects 1 to 20, wherein one or more nucleosides are nucleobase-modified nucleosides. 22. The oligonucleotide according to any one of aspects 1 to 21, wherein one or more nucleosides is 5-methylcytosine, eg, LNA 5-methylcytosine or DNA 5-methylcytosine. 23. A pharmaceutically acceptable salt, in particular a sodium, potassium or ammonium salt, of an oligonucleotide according to any one of embodiments 1 to 22. 24. A conjugate comprising the oligonucleotide or pharmaceutically acceptable salt according to any one of embodiments 1 to 23, and at least one conjugate moiety covalently attached to the oligonucleotide or pharmaceutically acceptable salt, optionally via a linker moiety. 25. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 24, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 26. An oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 25 for use as a therapeutically active substance. 27. The oligonucleotide, pharmaceutically acceptable salt, or conjugate according to any one of aspects 1 to 24 for use in therapy, for administration to a subject by parenteral administration, such as intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, or intrathecal administration.

[0306] Embodiments relating to oligonucleotides with achiral phosphorodithioate linkages and stereodefined phosphorothioate linkages 1 Formula (IA) or (IB) TIFF0007724259000052.tif58128 (wherein one of the two oxygen atoms is bonded to the 3' carbon atom of an adjacent nucleoside (A1) and the other is bonded to the 5' carbon atom of another adjacent nucleoside (A2); and in formula (IA), R is a hydrogen or phosphate protecting group; and in formula (IB), M+ is a cation, for example, a metal cation, for example, an alkali metal cation, for example, a Na+ cation or a K+ cation, or M+ is an ammonium cation). and at least one stereodefined phosphorothioate internucleoside linkage (Sp, S) or (Rp, R) TIFF0007724259000053.tif45128(N 1 and N 2 is a nucleoside) (Note: In some non-limiting embodiments, N 1 and / or N 2 is a DNA nucleotide, Single-stranded antisense oligonucleotide. 2. The single-stranded antisense oligonucleotide of embodiment 1, wherein A2 is the 3' terminal nucleoside of the oligonucleotide. 3. The single-stranded antisense oligonucleotide of embodiment 1, wherein A1 is the 5'-terminal nucleoside of the oligonucleotide. 4 A single-stranded antisense oligonucleotide according to any one of embodiments 1 to 3, comprising 1, 2, 3, 4, 5, or 6 internucleoside linkages of formula IB. 5. The single-stranded antisense oligonucleotide of any one of aspects 1 to 4, wherein both the 5'-most internucleoside linkage of the antisense oligonucleotide and the 3'-most internucleoside linkage of the antisense oligonucleotide are internucleoside linkages of formula IB. 6. The single-stranded antisense oligonucleotide according to any one of Aspects 1 to 5, wherein in at least one of the internucleoside linkages of Formula IB, at least one of the two nucleosides (A1) and (A2) is a 2'-sugar-modified nucleoside, for example, a 2'-sugar-modified nucleoside selected from the group consisting of a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, and an LNA nucleoside. 7. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 6, wherein in at least one of the internucleoside linkages of formula IB, at least one of the two nucleosides (A1) and (A2) is an LNA nucleoside. 8. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 6, wherein in at least one of the internucleoside linkages of formula IB, at least one of the two nucleosides (A1) and (A2) is a 2'-O-MOE nucleoside. 9. The single-stranded antisense oligonucleotide according to any one of Aspects 1 to 8, wherein the 3'-terminal nucleoside of the antisense oligonucleotide is an LNA nucleoside or a 2'-O-MOE nucleoside. 10. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 9, wherein the 5'-terminal nucleoside of the antisense oligonucleotide is an LNA nucleoside or a 2'-O-MOE nucleoside. 11. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 10, wherein the two 3'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 12. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 11, wherein the two 5'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 13. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 12, wherein the three 3'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 14. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 13, wherein the three 5'-most terminal nucleosides of the antisense oligonucleotide are independently selected from LNA nucleosides and 2'-O-MOE nucleosides. 15. The single-stranded antisense oligonucleotide according to any one of embodiments 1 to 14, wherein the two 3'-most terminal nucleosides of the antisense oligonucleotide are LNA nucleosides. 16. The single-stranded oligonucleotide according to any one of embodiments 1 to 15, wherein the two 5'-most terminal nucleosides of the antisense oligonucleotide are LNA nucleosides. 17. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 16, wherein the antisense oligonucleotide further comprises a region consisting of 2 to 16 DNA nucleotides, and wherein the internucleoside linkages between the DNA nucleotides are stereodefined phosphorothioate internucleoside linkages. 18. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 17, wherein the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA. 19. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 17, wherein the LNA nucleoside is β-D-oxy LNA. 20. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 19, consisting of or comprising 7 to 30 contiguous nucleotides (antisense oligonucleotide) that are complementary, e.g., perfectly complementary, to a target nucleic acid, e.g., a target nucleic acid selected from the group consisting of pre-mRNA and mRNA, microRNA, viral RNA, and long non-coding RNA. 21. A single-stranded antisense oligonucleotide according to any one of embodiments 1 to 20, which is capable of modulating an RNA target. 22. A single stranded antisense oligonucleotide according to any one of embodiments 1 to 20, which is capable of inhibiting an RNA target, for example by RNase H1 recruitment. 23. The single-stranded antisense oligonucleotide of any one of aspects 1 to 22, wherein the contiguous nucleotide sequence of the oligonucleotide comprises a gapmer region of the formula 5'-FG-F'-3', wherein G is a region of 5 to 18 nucleosides capable of recruiting RNase H1, and region G is flanked 5' and 3' by flanking regions F and F', respectively, and regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, and the nucleosides of region F flanking region G are 2'-sugar-modified nucleosides, and the nucleosides of region F' flanking region G are 2'-sugar-modified nucleosides. 24. The single-stranded antisense oligonucleotide according to embodiment 23, wherein region F or region F' comprises an internucleoside linkage of formula IB according to any one of embodiments 1 to 19. 25. The single-stranded antisense oligonucleotide according to embodiment 24, wherein both regions F and F′ comprise an internucleoside linkage of formula IB according to any one of embodiments 1 to 19. 26. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 25, wherein all internucleoside linkages within region F and / or F' are internucleoside linkages of formula IB according to any one of embodiments 1 to 19. 27. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 26, wherein regions F and F' both comprise or consist of LNA nucleosides. 28. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 27, wherein regions F and F' both comprise or consist of MOE nucleosides. 29. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 28, wherein region F comprises LNA nucleosides and region F' comprises or consists of MOE nucleosides. 30. The single-stranded antisense oligonucleotide of any one of embodiments 23 to 29, wherein region G further comprises at least one internucleoside linkage of formula IB located between the 3'-most nucleoside of region F and the 5'-most nucleoside of region G. 31. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 30, wherein region G comprises at least one stereodefined phosphorothioate bond located between two DNA nucleosides. 32. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 31, wherein region G comprises at least one internucleoside linkage of formula IB located between two DNA nucleosides. 33. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 32, wherein region G further comprises at least two, three, or four internucleoside linkages of formula IB. 34. The single-stranded antisense oligonucleotide according to any one of embodiments 23 to 31, wherein all remaining internucleoside linkages within region G are stereodefined phosphorothioate internucleoside linkages independently selected from Rp internucleoside linkages and Sp internucleoside linkages. 35. The single-stranded antisense oligonucleotide of any one of embodiments 23 to 31, wherein, optionally, all internucleoside linkages within region G other than the internucleoside linkage between the 3'-most nucleoside of region F and the 5'-most nucleoside of region G are stereodefined phosphorothioate internucleoside linkages independently selected from an Rp internucleoside linkage and an Sp internucleoside linkage. 36 The single-stranded antisense oligonucleotide according to any one of embodiments 1 to 22, comprising fewer than four consecutive DNA nucleotides. 37. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 22 or 36, which is a mixmer oligonucleotide or a totalmer oligonucleotide. 38. The single-stranded oligonucleotide of embodiment 37, wherein the mixmer oligonucleotide comprises both LNA and DNA nucleosides, and optionally 2' sugar-modified nucleosides (see, e.g., the list of embodiment 6), such as 2'-O-MOE nucleosides. 39. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 38, wherein the antisense oligonucleotide comprises a region of three or more consecutive MOE nucleosides, and optionally, all nucleosides of the oligonucleotide are 2'MOE nucleosides. 40. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 39, wherein the target is an mRNA target or a pre-mRNA target. 41. A single stranded antisense oligonucleotide according to any one of embodiments 1 to 40, which targets a pre-mRNA splice site or a region of the pre-mRNA that controls splicing events at the pre-mRNA splice site. 42. A single-stranded antisense oligonucleotide according to any one of embodiments 1 to 41, which is a splice regulator oligonucleotide capable of regulating splicing of a pre-mRNA target. 43. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 42, wherein the target is a microRNA. 44. The single-stranded antisense oligonucleotide according to any one of aspects 1 to 42, wherein the antisense oligonucleotide is 10 to 20 nucleotides in length, for example 12 to 24 nucleotides in length. 45. The single-stranded antisense oligonucleotide of embodiment 43, wherein the antisense oligonucleotide is 7 to 30, such as 8 to 12 or 12 to 23 nucleotides in length. 46. A single-stranded antisense oligonucleotide comprising the antisense oligonucleotide of any one of embodiments 1 to 45, wherein the oligonucleotide further comprises a 5' region located 5' to the contiguous nucleotide sequence, the 5' nucleoside region comprising at least one phosphodiester bond. 47. The single-stranded antisense oligonucleotide according to embodiment 46, wherein the 5' region comprises 1 to 5 phosphodiester-linked DNA nucleosides, and optionally the oligonucleotide or its consecutive nucleotide sequence may be linked to a conjugate moiety. 48. The single-stranded antisense oligonucleotide of any one of embodiments 1 to 47, wherein one or more nucleosides are nucleobase-modified nucleosides. 49. The single stranded antisense oligonucleotide according to any one of embodiments 1 to 48, wherein one or more nucleosides is 5-methylcytosine, eg, LNA 5-methylcytosine or DNA 5-methylcytosine. 50. A pharmaceutically acceptable salt, in particular a sodium, potassium, or ammonium salt, of the single-stranded antisense oligonucleotide according to any one of embodiments 1 to 49. 51. A conjugate comprising the single-stranded antisense oligonucleotide or pharmaceutically acceptable salt according to any one of embodiments 1 to 49, and at least one conjugate moiety covalently attached to the oligonucleotide or pharmaceutically acceptable salt, optionally via a linker moiety. 52. A pharmaceutical composition comprising a single-stranded antisense oligonucleotide according to any one of embodiments 1 to 51, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier. 53. A single-stranded antisense oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to any one of embodiments 1 to 52 for use as a therapeutically active substance. 54. The single-stranded antisense oligonucleotide, pharmaceutically acceptable salt, or conjugate of any one of aspects 1 to 53 for use in therapy, for administration to a subject by parenteral administration, such as intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, or intrathecal administration. 55. In vitro use of a single-stranded antisense oligonucleotide, salt, or composition according to any one of the preceding aspects for use in inhibiting a target RNA in a cell, wherein the single-stranded antisense oligonucleotide is complementary, e.g., perfectly complementary, to the target RNA. 56. An in vivo or in vitro method for inhibiting a target RNA in a cell expressing said target RNA, comprising administering to said cell an effective amount of an antisense oligonucleotide, salt, conjugate, or composition of any one of the preceding embodiments to inhibit said target RNA. 57. In vitro or in vivo use of a single-stranded antisense oligonucleotide, salt, or composition according to any one of the preceding aspects for use in modulating splicing of a target pre-mRNA in a cell. 58. An in vivo or in vitro method for modulating splicing of a target RNA precursor in a cell expressing said target RNA precursor, comprising administering to said cell an effective amount of an antisense oligonucleotide, salt, conjugate, or composition of any one of the preceding embodiments to modulate splicing of the target RNA.

[0307] Htra-1-targeting antisense oligonucleotides of the present invention In some embodiments, the antisense oligonucleotides of the invention are complementary to the mRNA or pre-mRNA encoding human high-temperature-requiring serine protease A1 (Htra1)—see, e.g., WO 2018 / 002105. Inhibiting Htra1 expression using antisense oligonucleotides of the invention that target Htra1 mRNA or pre-mRNA is beneficial for treating a range of medical disorders, such as macular degeneration, e.g., age-related macular degeneration (geographic atrophy). The target sequences for human Htra1 pre-mRNA and mRNA are available at: TIFF0007724259000054.tif65158

[0308] Compounds of the present invention that target Htra-1 are listed in the Examples as Htra1#1 to 38. 1. An antisense oligonucleotide of the present invention having a length of 10 to 30 nucleotides, wherein the antisense oligonucleotide targets human HTRA1 mRNA or pre-mRNA, and the antisense oligonucleotide is at least 90%, for example 100%, complementary to SEQ ID NO 1 or 2 of WO 2018 / 002105 and comprises a contiguous nucleotide region of 10 to 22 nucleotides as disclosed in the Sequence Listing as SEQ ID NO 9 and 10, and wherein the antisense oligonucleotide comprises at least one phosphorodithioate internucleoside linkage of Formula IA or Formula IB. 2 consecutive nucleotide regions are shown in SEQ ID NOs 11, 12, 13, 14, 15, 16, 17, and 18: 3. The antisense oligonucleotide of embodiment 1 or 2, wherein the antisense oligonucleotide is identical to a sequence present in a sequence selected from the group consisting of: TIFF0007724259000055.tif78128. A region of 3 consecutive nucleotides has the sequence The antisense oligonucleotide of any one of embodiments 1 to 3, comprising TIFF0007724259000056.tif4128. 4. The antisense oligonucleotide according to any one of aspects 1 to 4, wherein the contiguous nucleotide region of the oligonucleotide consists of or comprises a sequence selected from any one of SEQ ID NOs 11, 12, 13, 14, 15, 16, 17, and 18. 5. The antisense oligonucleotide of any one of aspects 1 to 5, wherein the contiguous nucleotide region of the oligonucleotide comprises one or more 2'-sugar-modified nucleosides, e.g., one or more 2'-sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-DNA nucleosides, arabinonucleic acid (ANA) nucleosides, 2'-fluoro-ANA nucleosides, and LNA nucleosides. 6. The antisense oligonucleotide of any one of aspects 1 to 5, wherein the contiguous nucleotide region of the oligonucleotide comprises at least one modified internucleoside linkage, e.g., one or more phosphorothioate internucleoside linkages, or, e.g., all internucleoside linkages within the contiguous nucleotide region are phosphorothioate internucleoside linkages. 7. The antisense oligonucleotide of any one of aspects 1 to 6, wherein the oligonucleotide or contiguous nucleotide sequence thereof is or comprises a gapmer, such as a gapmer of the formula 5'-FG-F'-3', wherein regions F and F' independently comprise 1 to 7 sugar-modified nucleotides, and G is a region of 6 to 16 nucleosides capable of recruiting RNase H, and the nucleosides of regions F and F' adjacent to region G are sugar-modified nucleosides. 8. The antisense oligonucleotide of embodiment 7, wherein at least one or both of regions F and F' each comprises at least one LNA nucleoside. 9. The antisense oligonucleotide according to any one of aspects 1 to 8, selected from the group selected from Htra1#1 to 38, wherein uppercase letters represent β-D-oxy LNA nucleoside units, lowercase letters represent DNA nucleoside units, subscript s represents a phosphorothioate internucleoside linkage, all LNA cytosines are 5-methylcytosines, P represents a phosphorodithioate internucleoside linkage of formula IB, S represents a stereodefined phosphorothioate internucleoside linkage of the Sp type, R represents a stereodefined phosphorothioate internucleoside linkage of the Rp type, and X represents a stereorandom phosphorothioate linkage. 10. The antisense oligonucleotide of any one of the preceding embodiments, in the form of a salt, such as a sodium salt, a potassium salt, or an ammonium salt (e.g., a pharmaceutically acceptable salt). 11. A conjugate comprising an oligonucleotide according to any one of embodiments 1 to 10 and at least one conjugate moiety covalently attached to said oligonucleotide or a salt thereof. 12. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 10 or a conjugate according to embodiment 11, and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant. 13. An in vivo or in vitro method for modulating HTRA1 expression in a target cell expressing HTRA1, the method comprising administering to the cell an effective amount of an oligonucleotide according to any one of aspects 1 to 10, or a conjugate according to aspect 11, or a pharmaceutical composition according to aspect 12. 14. A method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide according to any one of aspects 1 to 10, or a conjugate according to aspect 11, or a pharmaceutical composition according to aspect 12 to a subject suffering from or susceptible to the disease. 15. An oligonucleotide according to any one of aspects 1 to 10, or a conjugate according to aspect 11, or a pharmaceutical composition according to aspect 12, for use in medicine. 16. An oligonucleotide according to any one of aspects 1 to 10, or a conjugate according to aspect 11, or a pharmaceutical composition according to aspect 12, for use in the treatment or prevention of a disease selected from the group consisting of macular degeneration (e.g. wet AMD, dry AMD, geographic atrophy, moderate dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Duchenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic cerebral small vessel disease. 17. Use of an oligonucleotide according to aspects 1 to 10, or a conjugate according to aspect 11, or a pharmaceutical composition according to aspect 12, for the preparation of a medicament for the treatment or prevention of a disease selected from the group consisting of macular degeneration (e.g. wet AMD, dry AMD, geographic atrophy, moderate dAMD, diabetic retinopathy), Parkinson's disease, Alzheimer's disease, Duchenne muscular dystrophy, arthritis, such as osteoarthritis, and familial ischemic cerebral small vessel disease. 18. The oligonucleotide, conjugate, salt, or composition or use according to any one of the preceding aspects for use in the treatment of geographic atrophy.

[0309] Further aspects of the invention Thus, the present invention relates in particular to: an oligonucleotide according to the present invention which is an antisense oligonucleotide capable of regulating expression of a target RNA in a cell expressing said target RNA; an oligonucleotide according to the present invention which is an antisense oligonucleotide capable of inhibiting expression of a target RNA in a cell expressing said target RNA; (A 1 ) and (A 2 ) an oligonucleotide according to the present invention, wherein one of said nucleosides is an LNA nucleoside and the other is a DNA nucleoside, an RNA nucleoside, or a sugar-modified nucleoside; (A 1 ) and (A 2 an oligonucleotide according to the present invention, wherein one of said nucleosides is an LNA nucleoside and the other is a DNA nucleoside or a sugar-modified nucleoside; (A 1 ) and (A 2 an oligonucleotide according to the present invention, wherein one of said nucleosides is an LNA nucleoside and the other is a DNA nucleoside; (A 1 ) and (A 2 ) an oligonucleotide according to the present invention, wherein one of said nucleosides is an LNA nucleoside and the other is a sugar-modified nucleoside; The oligonucleotide according to the present invention, wherein the sugar-modified nucleoside is a 2' sugar-modified nucleoside. The oligonucleotide according to the present invention, wherein the 2' sugar-modified nucleoside is a 2'-alkoxy-RNA nucleoside, a 2'-alkoxyalkoxy-RNA nucleoside, a 2'-amino-DNA nucleoside, a 2'-fluoro-RNA nucleoside, a 2'-fluoro-ANA nucleoside, or an LNA nucleoside; the oligonucleotide according to the present invention, wherein the 2' sugar-modified nucleoside is an LNA nucleoside; an oligonucleotide according to the invention, wherein the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA; an oligonucleotide according to the invention, wherein both LNA nucleosides are β-D-oxyLNA; the oligonucleotide according to the present invention, wherein the 2' sugar-modified nucleoside is 2'-alkoxyalkoxy-RNA; the oligonucleotide according to the invention, wherein the 2'-alkoxy-RNA is 2'-methoxy-RNA; the oligonucleotide according to the invention, wherein the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA; an oligonucleotide according to the invention comprising 1 to 15, in particular 1 to 5, more in particular 1, 2, 3, 4 or 5 phosphorodithioate internucleoside linkages of formula (I) as defined above; an oligonucleotide according to the present invention comprising further internucleoside linkages independently selected from phosphodiester internucleoside linkages, phosphorothioate internucleoside linkages, and phosphorodithioate internucleoside linkages of formula (I) as defined above; an oligonucleotide according to the present invention, wherein the further internucleoside linkages are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I) as defined above; an oligonucleotide according to the invention, wherein all further internucleoside linkages are phosphorothioate internucleoside linkages; an oligonucleotide according to the invention, wherein all further internucleoside linkages are phosphorodithioate internucleoside linkages of formula (I) as defined above; an oligonucleotide according to the invention which is a gapmer, in particular an LNA gapmer, a mixed wing gapmer, an alternating flank gapmer, a splice-switching oligomer, a mixmer, or a totalmer; an oligonucleotide according to the invention which is a gapmer and which comprises at least one phosphorodithioate internucleoside linkage of formula (I) in the gap region and / or in one or more flanking regions of said gapmer; an oligonucleotide according to the invention, in which a contiguous nucleotide sequence, e.g., a gapmer region FG-F', is flanked by flanking regions D' or D'' or D' and D'' comprising one or more DNA nucleosides linked to the remainder of the oligonucleotide by phosphodiester internucleoside bonds; an oligonucleotide according to the invention which is a gapmer, wherein one or both, in particular one, of the flanking regions F and F' is further flanked by phosphodiester-linked DNA nucleosides, in particular 1 to 5 phosphodiester-linked DNA nucleosides (regions D' and D''); The oligonucleotide according to the present invention, which is 7 to 30 nucleotides in length.

[0310] When the oligonucleotide of the invention is a gapmer, it is advantageously of 12 to 26 nucleotides in length, 16 nucleotides being a particularly advantageous length for gapmer oligonucleotides.

[0311] When the oligonucleotide is a completely LNA oligonucleotide, it is advantageously of 7 to 10 nucleotides in length.

[0312] When the oligonucleotide is a mixmer oligonucleotide, it is advantageously of length 8 to 30 nucleotides.

[0313] The present invention relates in particular to: an oligonucleotide according to the invention, wherein one or more nucleosides are nucleobase-modified nucleosides; an oligonucleotide according to the invention which is an antisense oligonucleotide, an siRNA, a microRNA mimetic, or a ribozyme; pharmaceutically acceptable salts of the oligonucleotides according to the invention, in particular sodium or potassium salts; a conjugate comprising an oligonucleotide or a pharmaceutically acceptable salt thereof according to the present invention and at least one conjugate moiety covalently attached to said oligonucleotide or said pharmaceutically acceptable salt thereof, optionally via a linker moiety; a pharmaceutical composition comprising an oligonucleotide according to the present invention, a pharmaceutically acceptable salt, or a conjugate, and a therapeutically inert carrier; An oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to the present invention for use as a therapeutically active substance; and Use of an oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to the present invention as a medicament.

[0314] In some embodiments, the oligonucleotides of the present invention have increased activity in modulating a target nucleic acid compared to the corresponding oligonucleotides that are fully phosphorothioate-linked. In some embodiments, the present invention provides oligonucleotides with enhanced activity, enhanced potency, enhanced specific activity, or increased cellular uptake. In some embodiments, the present invention provides oligonucleotides with an altered in vitro or in vivo duration of action, e.g., a prolonged in vitro or in vivo duration of action. In some embodiments, increased activity in modulating a target nucleic acid is measured in vitro or in vivo in cells expressing the target nucleic acid.

[0315] In some embodiments, the oligonucleotides of the present invention have altered pharmacological properties, such as reduced toxicity, for example, reduced nephrotoxicity, reduced hepatotoxicity, or reduced immune stimulation. Hepatotoxicity can be measured, for example, in vivo or by using the in vitro assay method disclosed in WO 2017 / 067970, which is incorporated herein by reference. Nephrotoxicity can be measured, for example, in vitro or by using the assay method disclosed in PCT / EP2017 / 064770, which is incorporated herein by reference. In some embodiments, the oligonucleotides of the present invention comprise a 5'CG3' dinucleotide, for example, a DNA 5'CG3' dinucleotide, wherein the internucleoside linkage between C and G is a phosphorodithioate internucleoside linkage of formula (I) as defined above.

[0316] In some embodiments, the oligonucleotides of the present invention have improved nuclease resistance, for example, improved in vivo stability in serum. In some embodiments, the 3'-terminal nucleoside of the oligonucleotide of the present invention has an A base or a G base, for example, a 3'-terminal LNA-A nucleoside or a 3'-terminal LNA-G nucleoside. Optionally, the internucleoside linkage between the two 3'-most nucleosides of the oligonucleotide may be a phosphorodithioate internucleoside linkage of formula (I) as defined above.

[0317] In some embodiments, the oligonucleotides of the invention have enhanced bioavailability, ie, greater exposure to blood, e.g., longer residence time in blood.

[0318] Non-bridging phosphorodithioate modifications are introduced into oligonucleotides by solid-phase synthesis using the phosphoramidite method. Synthesis is carried out using controlled pore glass (CPG) with a universal linker as the support. On such a solid support, oligonucleotides are typically constructed in the 3' to 5' direction using sequential cycles of coupling 5'O-DMT-protected nucleoside phosphoramidite building blocks, followed by (thio)oxidation, capping, and deprotection of the DMT group. Introduction of non-bridging phosphorodithioates is achieved by using the appropriate thiophosphoramidite building block, followed by thiooxidation of the primary intermediate.

[0319] Although the corresponding DNA thiophosphoramidites are commercially available, the individual LNA building blocks have not been previously described. They can be prepared, for example, from 5'-O-DMT-protected nucleoside 3'-alcohols by reaction with monobenzoyl-protected ethanedithiol and tripyrrolidin-1-ylphosphane.

[0320] Thus, the oligonucleotide according to the present invention can be prepared, for example, according to Scheme 2, where R1 , R 2a , R 2b , R 4a , R 4b , R 5 , R x , R y , and V are as defined below.

[0321] Scheme 2 TIFF0007724259000057.tif84155

[0322] Therefore, the present invention also relates to a method for producing an oligonucleotide according to the invention, comprising the following steps: (a) coupling a thiophosphoramidite nucleoside to the 5' terminal oxygen atom of a nucleotide or oligonucleotide to form a thiophosphite triester intermediate; (b) thio-oxidizing the thiophosphite triester intermediate obtained in step (a); and (c) optionally, further extending the oligonucleotide.

[0323] The present invention particularly relates to a method for producing an oligonucleotide according to the invention, comprising the following steps: (a1) Equation (A) The compound of formula (B) coupling to the 5' oxygen atom of the nucleotide or oligonucleotide of TIFF0007724259000059.tif48128; (b1) thio-oxidizing the thiophosphite triester intermediate obtained in step (a1); and (c) optionally, further extending the oligonucleotide; In the above equation, R 2a and R 4a taken together form -XY- as defined above; or R 4a is hydrogen and R 2ais selected from alkoxy, in particular methoxy, halogen, in particular fluoro, alkoxyalkoxy, in particular methoxyethoxy, alkenyloxy, in particular allyloxy, and aminoalkoxy, in particular aminoethyloxy; R 2b and R 4b taken together form -XY- as defined above; or R 2b and R 4b are both hydrogen at the same time; or R 4b is hydrogen and R 2b is selected from alkoxy, in particular methoxy, halogen, in particular fluoro, alkoxyalkoxy, in particular methoxyethoxy, alkenyloxy, in particular allyloxy, and aminoalkoxy, in particular aminoethyloxy; V is oxygen or sulfur; and R 5 , R x , R y , and Nu are as defined below.

[0324] The present invention particularly relates to a method for producing an oligonucleotide according to the invention, comprising the following steps: (a2) Formula (II) The compound of formula (IV) coupling to the 5' oxygen atom of the nucleotide or oligonucleotide of TIFF0007724259000061.tif47128; (b2) thio-oxidizing the thiophosphite triester intermediate obtained in step (a2); and (c) optionally, further extending the oligonucleotide; In the above equation, R 2b and R 4b taken together form -XY- as defined above; or R 2b and R 4b are both hydrogen at the same time; or R4b is hydrogen and R 2b is selected from alkoxy, in particular methoxy, halogen, in particular fluoro, alkoxyalkoxy, in particular methoxyethoxy, alkenyloxy, in particular allyloxy, and aminoalkoxy, in particular aminoethyloxy; and R 5 , R x , R y , and Nu are as defined below.

[0325] The present invention also relates to oligonucleotides produced according to the methods of the present invention.

[0326] The present invention further relates to: Formula (I) TIFF0007724259000062.tif28128 (wherein R is a hydrogen or phosphate protecting group) a gapmer oligonucleotide comprising at least one phosphorodithioate internucleoside linkage of a gapmer oligonucleotide as defined above, which is an antisense oligonucleotide capable of modulating expression of a target RNA in a cell expressing said target RNA; a gapmer oligonucleotide as defined above, which is an antisense oligonucleotide capable of inhibiting expression of a target RNA in a cell expressing said target RNA; a gapmer oligonucleotide as defined above, capable of recruiting RNase H, such as human RNase H1; At least one of the two oxygen atoms of the internucleoside bond of formula (I) is an adjacent nucleoside (A 1 ) and the other is attached to the 3' carbon atom of another adjacent nucleoside (A 2 ) and two nucleosides (A 1 ) and (A 2 a gapmer oligonucleotide according to the present invention, wherein at least one of the nucleotides (a) to (c) is a 2' sugar-modified nucleoside; (A 1 ) and (A2 a gapmer oligonucleotide according to the present invention, wherein one of said nucleotides is a 2' sugar-modified nucleoside and the other is a DNA nucleoside; (A 1 ) and (A 2 ) but simultaneously both 2'-modified nucleosides; (A 1 ) and (A 2 ) but at the same time both are DNA nucleosides; a gapmer oligonucleotide according to the invention, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3', wherein G is a region of 5 to 18 nucleosides capable of recruiting RNase H, and region G is flanked 5' and 3' by flanking regions F and F', respectively, and regions F and F' independently comprise or consist of 1 to 7 2'-sugar-modified nucleotides, and wherein the nucleosides of region F flanking region G are 2'-sugar-modified nucleosides, and the nucleosides of region F' flanking region G are 2'-sugar-modified nucleosides; a gapmer oligonucleotide according to the present invention, wherein the 2' sugar-modified nucleosides are independently selected from 2'-alkoxy-RNA nucleosides, 2'-alkoxyalkoxy-RNA nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, 2'-fluoro-ANA nucleosides, and LNA nucleosides; a gapmer oligonucleotide according to the present invention, wherein the 2'-alkoxyalkoxy-RNA is 2'-methoxyethoxy-RNA (2'-O-MOE); A gapmer oligonucleotide according to the invention, wherein region F and region F' comprise or consist of 2'-methoxyethoxy-RNA nucleotides; Formula [MOE] 3~8 [DNA] 8~16 [MOE] 3~8 , e.g., [MOE]5[DNA] 10a gapmer oligonucleotide according to the invention, wherein both regions F and F' consist of 2'-methoxyethoxy-RNA nucleotides, such as a gapmer comprising FG-F' having [MOE]5 (i.e., region F and region F' each consist of 5 2'-methoxyethoxy-RNA nucleotides, and region G consists of 10 DNA nucleotides); a gapmer oligonucleotide according to the present invention, wherein at least one or all of the 2' sugar-modified nucleosides in region F or region F' or in both region F and region F' are LNA nucleosides; a gapmer oligonucleotide according to the invention, wherein region F or region F' or both region F and region F' comprise at least one LNA nucleoside and at least one DNA nucleoside; a gapmer oligonucleotide according to the invention, wherein region F or region F' or both region F and region F' comprise at least one LNA nucleoside and at least one non-LNA 2' sugar modified nucleoside, such as at least one 2'-methoxyethoxy-RNA nucleoside; A gapmer oligonucleotide according to the present invention, wherein the gap region comprises 5 to 16, particularly 8 to 16, more particularly 8, 9, 10, 11, 12, 13, or 14 consecutive DNA nucleosides; a gapmer oligonucleotide according to the invention, wherein region F and region F' are independently 1, 2, 3, 4, 5, 6, 7, or 8 nucleosides in length; A gapmer oligonucleotide according to the invention, wherein region F and region F' each independently comprise 1, 2, 3, or 4 LNA nucleosides; a gapmer oligonucleotide according to the invention, wherein the LNA nucleosides are independently selected from β-D-oxy LNA, 6'-methyl-β-D-oxy LNA, and ENA; a gapmer oligonucleotide according to the present invention, wherein the LNA nucleoside is β-D-oxy LNA; A gapmer oligonucleotide according to the present invention, wherein the oligonucleotide or its consecutive nucleotide sequence (FG-F') is 10 to 30 nucleotides in length, particularly 12 to 22, more particularly 14 to 20 oligonucleotides in length; a gapmer oligonucleotide according to the invention, comprising a contiguous nucleotide sequence of the formula 5'-D'-FG-F'-D''-3', wherein F, G and F' are as defined in any one of claims 4 to 17, and wherein the regions D' and D'' each independently consist of 0 to 5 nucleotides, in particular 2, 3 or 4 nucleotides, in particular DNA nucleotides, such as phosphodiester-linked DNA nucleosides; a gapmer oligonucleotide according to the invention, which is capable of recruiting human RNase H1; a gapmer oligonucleotide according to the present invention, wherein at least one phosphorodithioate internucleoside linkage of formula (I) as defined above is located between adjacent nucleosides in region F or region F', between region F and region G, or between region G and region F'; a gapmer oligonucleotide according to the present invention, further comprising phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the present invention, wherein the internucleoside linkages between the nucleosides of region G are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein the internucleoside linkages between the nucleosides of region G comprise 0, 1, 2 or 3 phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the present invention, wherein the remaining internucleoside linkages are independently selected from the group consisting of phosphorothioate linkages, phosphodiester linkages, and phosphorodithioate internucleoside linkages of formula (I) defined above; a gapmer oligonucleotide according to the present invention, wherein the internucleoside linkages between the nucleosides of region F and the internucleoside linkages between the nucleosides of region F' are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein each flanking region F and F' independently comprises 1, 2, 3, 4, 5, 6, or 7 phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein the flanking regions F and F' together or independently comprise 1, 2, 3, 4, 5, or 6 phosphorodithioate internucleoside linkages of formula (I) as defined above, or all internucleoside linkages in region F and / or region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein the flanking regions F and F' together comprise 1, 2, 3, or 4 phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein the flanking regions F and F' each comprise two phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the present invention, wherein all of the internucleoside linkages of flanking region F and / or flanking region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention comprising at least one stereodefined internucleoside linkage, for example at least one stereodefined phosphorothioate internucleoside linkage; A gapmer oligonucleotide according to the present invention, wherein the gap region comprises 1, 2, 3, 4, or 5 stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the present invention, wherein all of the internucleoside linkages between the nucleosides in the gap region are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the present invention, wherein at least one phosphorodithioate internucleoside linkage of formula (I) as defined above is located between nucleosides of region F, between nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages within regions F and F', between region F and region G, and between region G and region F' are independently selected from stereodefined phosphorothioate internucleoside linkages, stereorandom internucleoside linkages, phosphorodithioate internucleoside linkages of formula (I), and phosphodiester internucleoside linkages; A gapmer oligonucleotide according to the present invention, wherein at least one phosphorodithioate internucleoside linkage of formula (I) defined above is located between at least two adjacent nucleosides of region F, between two adjacent nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages between nucleotides of regions F and F' are independently selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages of formula (I), and phosphodiester internucleoside linkages. The phosphorothioate internucleoside linkages of regions F and F' may be either sterically random or sterically defined, or may be independently selected from sterically random and sterically defined; A gapmer oligonucleotide according to the present invention, wherein at least one phosphorothioate internucleoside linkage of formula (I) as defined above is located between at least two adjacent nucleosides of region F, between at least two adjacent nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages between nucleotides of regions F and F' are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I). The phosphorothioate internucleoside linkages of regions F and F' may be either sterically random or sterically defined, or may be independently selected from sterically random and sterically defined; a gapmer oligonucleotide according to the present invention, wherein at least one phosphorothioate internucleoside linkage of formula (I) as defined above is located between at least two adjacent nucleosides of region F, between at least two adjacent nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages between the nucleotides of regions F and F', between region F and region G, and between region G and region F' are independently selected from phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I); the phosphorothioate internucleoside linkages of regions F and F' may be either sterically random or sterically defined, or may be independently selected from sterically random and sterically defined; a gapmer oligonucleotide according to the present invention, wherein at least one phosphorodithioate internucleoside linkage of formula (I) as defined above is located between at least two adjacent nucleosides of region F, between at least two adjacent nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages between the nucleotides of regions F and F', between region F and region G, and between region G and region F' are independently selected from stereodefined phosphorothioate internucleoside linkages and phosphorodithioate internucleoside linkages of formula (I); a gapmer oligonucleotide according to the present invention, wherein at least one phosphorodithioate internucleoside linkage of formula (I) as defined above is located between at least two adjacent nucleosides of region F, between at least two adjacent nucleosides of region F', between region F and region G, or between region G and region F', and the remaining internucleoside linkages within regions F and F', between region F and region G, and between region G and region F' are phosphorothioate internucleoside linkages, which may be all stereo-random phosphorothioate internucleoside linkages, all stereo-defined phosphorothioate internucleoside linkages, or independently selected from stereo-random phosphorothioate internucleoside linkages and stereo-defined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein all remaining internucleoside linkages within region F, within region F', or within both region F and region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above; a gapmer oligonucleotide according to the invention, wherein the internucleoside linkages between the nucleosides of region G comprise 0, 1, 2, or 3 phosphorodithioate internucleoside linkages of formula (I) as defined above, and the remaining internucleoside linkages in region G are independently selected from stereodefined phosphorothioate internucleoside linkages and stereorandom phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein the internucleoside linkages between the nucleosides of region G comprise 0, 1, 2 or 3 phosphorodithioate internucleoside linkages of formula (I) as defined above, and at least one of the remaining internucleoside linkages in region G or all of the remaining internucleoside linkages in region G are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein the internucleoside linkages between the nucleosides of region G comprise 0, 1, 2, or 3 phosphorodithioate internucleoside linkages of formula (I) as defined above, and the remaining internucleoside linkages within region G are phosphorothioate internucleoside linkages, e.g., stereorandom phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the present invention, wherein at least one of region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all internucleoside linkages within region G are phosphorothioate internucleoside linkages, e.g., stereorandom phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein at least one of region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all internucleoside linkages within region G are phosphorothioate internucleoside linkages, and at least one of the phosphorothioate internucleoside linkages within region G is a stereodefined phosphorothioate internucleoside linkage; a gapmer oligonucleotide according to the invention, wherein at least one of region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all internucleoside linkages within region G are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the present invention, wherein the internucleoside linkage between region F and region G, or the internucleoside linkage between region G and region F', or both the internucleoside linkages between region F and region G and between region G and region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above, and when only one of the internucleoside linkages between region F and region G and region G and region F' is a phosphorodithioate internucleoside linkage of formula (I) as defined above, the other internucleoside linkage between region F and region G or region G and region F' is a phosphorothioate internucleoside linkage; a gapmer oligonucleotide according to the present invention, wherein at least one of region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and wherein the internucleoside linkage between region F and region G, or the internucleoside linkage between region G and region F', or both the internucleoside linkages between region F and region G and region G and region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above, and when only one of the internucleoside linkages between region F and region G and region G and region F' is a phosphorodithioate internucleoside linkage of formula (I) as defined above, the other internucleoside linkage between region F and region G or region G and region F' is a phosphorothioate internucleoside linkage; the internucleoside linkages between the nucleosides of region G contain 0, 1, 2, or 3 phosphorodithioate internucleoside linkages of formula (I) as defined above, and the remaining internucleoside linkages in region G are phosphorothioate internucleoside linkages; the internucleoside linkages between region F and region G, or between region G and region F', or both the internucleoside linkages between region F and region G and region G and region F' is a phosphorodithioate internucleoside linkage of formula (I) as defined above, and when only one of the internucleoside linkages between region F and region G and between region G and region F' is a phosphorodithioate internucleoside linkage of formula (I) as defined above, then the other internucleoside linkage between region F and region G or between region G and region F' is a phosphorothioate internucleoside linkage; At least one of region F or region F' contains at least one phosphorodithioate internucleoside linkage of formula (I) defined above, the internucleoside linkages between the nucleosides of region G contain 0, 1, 2, or 3 phosphorodithioate internucleoside linkages of formula (I) defined above, and the remaining internucleoside linkages in region G are phosphorothioate internucleoside linkages, and the internucleoside linkage between region F and region G, or the internucleoside linkage between region G and region F', or region F a gapmer oligonucleotide according to the present invention, wherein both internucleoside linkages between region F and region G and between region G and region F' are phosphorodithioate internucleoside linkages of formula (I) as defined above, and when only one of the internucleoside linkages between region F and region G and between region G and region F' is a phosphorodithioate internucleoside linkage of formula (I) as defined above, the other internucleoside linkage between region F and region G or between region G and region F' is a phosphorothioate internucleoside linkage; a gapmer oligonucleotide according to the present invention, wherein region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, or the internucleoside linkage between region F and region G or between region G and region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and region G comprises one, two, or three phosphorodithioate internucleoside linkages of formula (I) as defined above, and the remaining internucleoside linkages in region G are phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, or the internucleoside linkage between region F and region G or between region G and region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all of the internucleoside linkages in region G are phosphorothioate internucleoside linkages, and at least one of the phosphorothioate internucleoside linkages in region G is a stereodefined phosphorothioate internucleoside linkage; a gapmer oligonucleotide according to the present invention, wherein region F or region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, or the internucleoside linkage between region F and region G or between region G and region F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all of the internucleoside linkages within region G are phosphorothioate internucleoside linkages, and all of the phosphorothioate internucleoside linkages within region G are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein, apart from at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, all remaining internucleoside linkages within the gapmer region FG-F' are phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein at least one of regions F or F' comprises at least one phosphorodithioate internucleoside linkage of formula (I) as defined above, and all internucleoside linkages within region G are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention, wherein, except for at least one phosphorodithioate internucleoside linkage of formula (I), all remaining internucleoside linkages within the gapmer region FG-F' are stereodefined phosphorothioate internucleoside linkages; a gapmer oligonucleotide according to the invention which is an LNA gapmer, a mixed wing gapmer, an alternating flank gapmer, or a gap breaker gapmer; a pharmaceutically acceptable salt of the gapmer oligonucleotide according to the invention, in particular a sodium or potassium salt; a conjugate comprising a gapmer oligonucleotide or a pharmaceutically acceptable salt thereof according to the present invention and at least one conjugate moiety covalently bonded to said oligonucleotide or said pharmaceutically acceptable salt, optionally via a linker moiety, in particular via a biocleavable linker, in particular via 2 to 4 phosphodiester-linked DNA nucleosides (e.g., region D' or region D''); a pharmaceutical composition comprising a gapmer oligonucleotide according to the invention, a pharmaceutically acceptable salt, or conjugate, and a therapeutically inert carrier; A gapmer oligonucleotide, a pharmaceutically acceptable salt, or a conjugate according to the invention for use as a therapeutically active substance; Use of a gapmer oligonucleotide, a pharmaceutically acceptable salt, or a conjugate as a medicament. a method of modulating expression of a target RNA in a cell, comprising administering to a cell expressing said target RNA an oligonucleotide or gapmer oligonucleotide according to the present invention, such that expression of the target RNA is modulated; A method of inhibiting expression of a target RNA in a cell, comprising administering to a cell expressing said target RNA an oligonucleotide or gapmer oligonucleotide according to the invention, such that expression of said target RNA is inhibited; and An in vitro method of modulating or inhibiting a target RNA in a cell, comprising the step of administering to a cell expressing said target RNA an oligonucleotide or gapmer oligonucleotide according to the invention, such that the target RNA is modulated or inhibited in the cell.

[0327] The target RNA can be, for example, a mammalian mRNA, such as a pre-mRNA or mature mRNA, a human mRNA, a viral RNA, or a non-coding RNA, such as a microRNA or a long non-coding RNA.

[0328] In some embodiments, the modulation is splice modulation of a target pre-mRNA, resulting in a change in the splicing pattern of the target pre-mRNA.

[0329] In some embodiments, modulation is inhibition, which can occur via target degradation (e.g., by recruitment of RNase H, such as RNase H1, or RISC), or inhibition can occur by an occupancy-mediated mechanism that inhibits the normal biological function of the target RNA (e.g., inhibition by mixmers or totalmers of microRNAs or long non-coding RNAs).

[0330] Human mRNA can be mature RNA or pre-mRNA.

[0331] The present invention further relates to compounds of formula (II): TIFF0007724259000063.tif35128In formula, 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 -CR a R b -, -N(R a )-O-, or -O-CR a R b - and; 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-, -Si(R a )2-, -SO2-, -NR a -, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O-, or -O-CR a R b - and; However, -XY- is -OO-, Si(R a )2-Si(R a )2-, -SO2-SO2-, -C(R a )=C(R b )-C(R a )=C(R b ), -C(R a )=NC(R a )=N-, -C(R a )=NC(R a )=C(R b ), -C(R a )=C(R b )-C(R a )=N- and -Se-Se-; J is oxygen, sulfur, =CH2, or =N(R a ) and; R a and R bis hydrogen, halogen, hydroxyl, cyano, thiohydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, carbamido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, thiohydroxyl sulfido alkylsulfanyl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC(=X a )R c , -OC(=X a )NR c R d , and -NR e C(=X a )NR c R d more independently selected, or Two geminal R's a and R b together form an optionally substituted methylene, or Two geminal R's a and R b together with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl having only one carbon atom, -XY-; 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, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, heterocyclyl, aryl, and heteroaryl; X a is oxygen, sulfur, or -NRc and; R c , R d , and R e are independently selected from hydrogen and alkyl; n is 1, 2, or 3. R 5 is a hydroxyl protecting group; R x is phenyl, nitrophenyl, phenylalkyl, halophenylalkyl, cyanoalkyl, phenylcarbonylsulfanylalkyl, halophenylcarbonylsulfanylalkylalkylcarbonylsulfanylalkyl, or alkylcarbonylcarbonylsulfanylalkyl; R y is dialkylamino or pyrrolidinyl; and Nu is a nucleobase or a protected nucleobase.

[0332] The present invention further relates to: Compounds of formula (II) wherein -XY- is -CH2-O-, -CH(CH3)-O-, or -CH2CH2-O-; The present invention further provides a compound of formula (IIb): TIFF0007724259000064.tif37128In formula, R 5 is a hydroxyl protecting group; R x is phenyl, nitrophenyl, phenylalkyl, halophenylalkyl, cyanoalkyl, phenylcarbonylsulfanylalkyl, halophenylcarbonylsulfanylalkylalkylcarbonylsulfanylalkyl, or alkylcarbonylcarbonylsulfanylalkyl; R y is dialkylamino or pyrrolidinyl; and Nu is a nucleobase or a protected nucleobase; R 5 , R x , R y and Nu is as defined above, a compound of formula (II) according to TIFF0007724259000065.tif35128; R x is phenyl, nitrophenyl, phenylmethyl, dichlorophenylmethyl, cyanoethyl, methylcarbonylsulfanylethyl, ethylcarbonylsulfanylethyl, isopropylcarbonylsulfanylethyl, tert-butylcarbonylsulfanylethyl, methylcarbonylcarbonylsulfanylethyl, or difluorophenylcarbonylsulfanylethyl; R x is phenyl, 4-nitrophenyl, 2,4-dichlorophenylmethyl, cyanoethyl, methylcarbonylsulfanylethyl, ethylcarbonylsulfanylethyl, isopropylcarbonylsulfanylethyl, tert-butylcarbonylsulfanylethyl, methylcarbonylcarbonylsulfanylethyl, or 2,4-difluorophenylcarbonylsulfanylethyl; R x is phenylcarbonylsulfanylalkyl; R x is phenylcarbonylsulfanylethyl; R y is diisopropylamino or pyrrolidinyl; R y is pyrrolidinyl; R 5 and Nu is as defined above, A compound of formula (II) according to TIFF0007724259000066.tif47128; R 5 and Nu is as defined above, A compound of formula (IIb) according to TIFF0007724259000067.tif50128; A compound of formula (II), (IIb), (III), (IV), or (V), or (Vb), wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil, or protected uracil; A compound of formula (IIb) wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil, or protected uracil; A compound of formula (Vb), wherein Nu is thymine, protected thymine, adenosine, protected adenosine, cytosine, protected cytosine, 5-methylcytosine, protected 5-methylcytosine, guanine, protected guanine, uracil, or protected uracil; TIFF0007724259000068.tif165134TIFF0007724259000069.tif109140. A compound of formula (IIb) selected from TIFF0007724259000070.tif113139TIFF0007724259000071.tif172139.

[0333] The presence of impurities in the compounds of formula (II) and (IIb) can lead to by-products during the preparation of oligonucleotides, hindering successful synthesis. Furthermore, when impurities are present, the compounds of formula (II) or (IIb) are unstable during storage.

[0334] In particular, compounds of formula (X1), (X2), X(11), and (X21) TIFF0007724259000072.tif78128 is an example of such an impurity.

[0335] Thus, there was a need for compounds of formula (II) or (IIb) in a form sufficiently pure for storage purposes and for oligonucleotide manufacturing purposes.

[0336] Therefore, the present invention also relates to compounds of formula (II)(IIb) having a purity of at least 98%, particularly 99%, more particularly 100%.

[0337] The present invention therefore particularly relates to compounds of formula (II) which contain less than 1%, in particular 0%, of compounds of formula (X1) and / or (X2) as impurities.

[0338] The present invention further relates to a process for preparing a compound of formula (II) as defined above, which comprises reacting a 5'-protected LNA nucleoside with a phosphine and a monoprotected dithiol in the presence of an acidic coupling agent and a silylating agent.

[0339] The present invention further relates to a process for preparing a compound of formula (IIb) as defined above, which comprises reacting a 5'-protected MOE nucleoside with a phosphine and a monoprotected dithiol in the presence of an acidic coupling agent and a silylating agent.

[0340] The present invention relates to a method for preparing a compound of formula (C) in the presence of an acidic coupling agent and a silylating agent. Compound TIFF0007724259000073.tif25128 and formula P(R y )3 and the compound of formula HSR x where X, Y, R 5 ,Nu,R. x , and R y is as defined above), relates to a process for preparing a compound of formula (II).

[0341] The present invention further relates to a method for preparing a compound of formula (C1) in the presence of an acidic coupling agent and a silylating agent: Compound TIFF0007724259000074.tif25128 and formula P(R y )3 and the compound of formula HSRx where R 5 ,Nu,R. x , and R y is as defined above), also relates to a process for preparing a compound of formula (II).

[0342] The present invention also relates to a method for preparing a compound of formula (Cb) in the presence of an acidic coupling agent and a silylating agent: Compound TIFF0007724259000075.tif30128 and formula P(R y )3 and the compound of formula HSR x where R 5 ,Nu,R. x , and R y is as defined above), also relates to a process for preparing a compound of formula (IIb).

[0343] Examples of acidic coupling agents, also known as acidic activators, include tetrazole, 5-nitrophenyl-1H-tetrazole (NPT), 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 5-methylthio-1H-tetrazole (MTT), 5-mercapto-tetrazole (MCT), 5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole, and azole-based activators such as 4,5-dicyanoimidazole (DCI), or acid salts such as pyridinium hydrochloride, imidazolium triflate, benzimidazolium triflate, 5-nitrobenzimidazolium triflate, or weak acids such as 2,4-dinitrobenzoic acid or 2,4-dinitrophenol. Tetrazole is a specific acidic coupling agent.

[0344] Examples of silylating agents, also known as hydroxyl group deactivators, include bis(dimethylamino)dimethylsilane, N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)carbamate (BSC), N,N-bis(trimethylsilyl)methylamine, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N,N′-bis(trimethylsilyl)urea (BSU), bromotrimethylsilane (TMBS), N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), chlorodimethyl(pentafluorophenyl)silane, chlorotriethylsilane (TESCI), Examples of suitable silanes include chlorotrimethylsilane (TMCS), 1,3-dimethyl-1,1,3,3-tetraphenyldisilazane (TPDMDS), N,N-dimethyltrimethylsilylamine (TMSDMA), hexamethyldisilazane (HMDS), hexamethyldisiloxane (HMDSO), N-methyl-N-trimethylsilylacetamide (MSA), N-methyl-N-trimethylsilylheptafluorobutyramide (MSHFA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), 1,1,3,3-tetramethyl-1,3-diphenyldisilazane (DPTMDS), 4-(trimethylsiloxy)-3-penten-2-one (TMS acac), 1-(trimethylsilyl)imidazole (TMSI), and trimethylsilyl methallyl sulfinate (SILMAS-TMS). 1-(trimethylsilyl)imidazole is an exemplary silylating agent.

[0345] The present invention further relates to a process for preparing a compound of formula (II), (IIb), or (III), wherein the crude compound of formula (II) or (IIb) is purified by preparative HPLC.

[0346] The present invention further relates to a process for preparing a compound of Formula (II), (IIb), or (III), wherein the crude compound of Formula (II), (IIb), or (III) is purified by preparative HPLC, eluting with a gradient of acetonitrile versus aqueous ammonium hydroxide.

[0347] The ammonium hydroxide content in the water is particularly at least about 0.05% v / v, in particular about 0.05% to 1% v / v, more particularly 0.05% to 0.5% v / v, and even more particularly about 0.05% v / v.

[0348] The acetonitrile gradient is specifically from 0%-25% to 75%-100% acetonitrile, particularly within 20 to 120 minutes, more specifically from 10%-20% to 75%-90% acetonitrile, particularly within 25 to 60 minutes, and more specifically about 25%-75% acetonitrile, particularly within 30 minutes.

[0349] The present invention also relates to the use of compounds of formula (II), (IIb) or (III) in the manufacture of oligonucleotides, in particular oligonucleotides or gapmer oligonucleotides according to the invention.

[0350] The invention will now be illustrated by the following examples, which have no limiting character. [Example]

[0351] Example 1: Monomer synthesis 1.1: S-(2-sulfanylethyl)benzenecarbothioate To a solution of 1,2-ethanedithiol (133.57 mL, 1592 mmol, 1 equiv.) and pyridine (64.4 mL, 796 mmol, 0.5 equiv.) in chloroform (200 mL), benzoyl chloride (92.4 mL, 796 mmol, 0.5 equiv.) dissolved in chloroform (200 mL) was added dropwise, and the reaction was stirred at 0 °C for 1 h. The mixture was washed with water (300 mL) and brine (300 mL). The organic phase was dried over NaSO and concentrated to a yellow oil. This oil was distilled (135–145 °C) to give S-(2-sulfanylethyl)benzenecarbothioate (40 g, 202 mmol, 13% yield) as a colorless oil. TIFF0007724259000077.tif18153

[0352] 1.2: S-[2-[[(1R,3R,4R,7S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-(5-methyl-2,4-dioxo-pyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy-pyrrolidin-1-yl-phosphanyl]sulfanylethyl]benzenecarbothioate TIFF0007724259000078.tif391541-[(1R,4R,6R,7S)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-6-yl]-5-methyl-pyrimidine-2,4-dione (2.29 g, 4.00 mmol, 1.0 equiv.) was dissolved in 60 mL of anhydrous dichloromethane and a spatula of 3 Å molecular sieves was added. Tripyrrolidin-1-ylphosphane (960 mg, 3.98 mmol, 0.99 equiv.) was added via syringe, followed by the addition of seven 0.1 mmol aliquots of tetrazole (seven 0.4 mL portions of a 0.5 M solution in anhydrous acetonitrile preserved with 3 Å molecular sieves) at 2 min intervals. To the reaction mixture was then added N-trimethylsilylimidazole (56.0 mg, 0.400 mmol, 0.1 equiv.). After 5 min, tetrazole (21.6 mL of a 0.5 M solution in anhydrous acetonitrile) was added, followed immediately by S-(2-sulfanylethyl)benzenecarbothioate (1.04 g, 5.24 mmol, 1.31 equiv.). The reaction was allowed to proceed for 120 s. Four identical batches of the reaction mixture were combined and quenched by pouring the solution into 600 mL of dichloromethane containing 40 mL of triethylamine. The mixture was immediately washed with saturated sodium bicarbonate (800 mL), followed by 10% sodium carbonate (2 × 800 mL) and brine (800 mL). The organic layer was dried using Na2SO4. After 10–15 min, the drying agent was removed by filtration. Triethylamine (40 mL) was added to the solution and concentrated to a syrup using a rotary evaporator. The syrup was dissolved in toluene (200 mL) and triethylamine (40 mL), and the solution was pipetted into 4500 mL of vigorously stirred heptane to precipitate a fluffy white product. After decanting most of the heptane, the white precipitate was collected by filtration through a medium sintered glass funnel and subsequently dried under reduced pressure to yield a white solid.The solid was purified by preparative HPLC (Phenomenex Gemini C18, 250 x 50 mm, 10 mm column, 0.05% ammonium hydroxide in water / CH3CN) and lyophilized to give 4.58 g of the target compound as a white solid. TIFF0007724259000079.tif30150

[0353] 1.3: S-[2-[[(1R,3R,4R,7S)-3-(6-benzamidopurin-9-yl)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy-pyrrolidin-1-yl-phosphanyl]sulfanylethyl]benzenecarbothioate N-[9-[(1R,4R,6R,7S)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-6-yl]purin-6-yl]benzamide (2.74 g, 4.00 mmol, 1.0 equiv.) was dissolved in 60 mL of anhydrous dichloromethane and a spatula of 3 Å molecular sieves was added. Tripyrrolidin-1-ylphosphane (960 mg, 3.98 mmol, 0.99 equiv.) was added via syringe, followed by seven 0.1 mmol aliquots of tetrazole (seven 0.4 mL portions of a 0.5 M solution in anhydrous acetonitrile preserved with 3 Å molecular sieves) at 2 min intervals. To the reaction was then added 1-(trimethylsilyl)-1H-imidazole (56.0 mg, 0.400 mmol, 0.1 equiv.). After 5 min, tetrazole (21.6 mL of a 0.5 M solution in anhydrous acetonitrile) was added, followed immediately by S-(2-sulfanylethyl)benzenecarbothioate (1.04 g, 5.24 mmol, 1.31 equiv.). The reaction was allowed to proceed for 120 s.

[0354] Four identical batches of reactants were combined and quenched by pouring the solution into 600 mL of dichloromethane containing 40 mL of triethylamine. The mixture was immediately washed with saturated sodium bicarbonate (800 mL), followed by 10% sodium carbonate (2 × 800 mL) and brine (800 mL). The organic layer was dried with Na2SO4. After 10–15 min, the drying agent was removed by filtration. Triethylamine (10 mL) was added to the solution, which was then concentrated to a syrup using a rotary evaporator. The syrup was dissolved in toluene (100 mL) and triethylamine (20 mL), and the solution was pipetted into 4500 mL of vigorously stirred heptane to precipitate a fluffy white product. After decanting most of the heptane, the white precipitate was collected by filtration through a medium sintered glass funnel and subsequently dried under reduced pressure to yield a white solid. The solid was purified by preparative HPLC (Phenomenex Gemini C18, 250 x 50 mm, 10 mm column, 0.05% ammonium hydroxide in water / CH3CN) and lyophilized to give 5.26 g of the target compound as a white solid. TIFF0007724259000081.tif30151

[0355] 1.4: S-[2-[[(1R,3R,4R,7S)-3-(4-benzamido-5-methyl-2-oxo-pyrimidin-1-yl)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy-pyrrolidin-1-yl-phosphanyl]sulfanylethyl]benzenecarbothioate TIFF0007724259000082.tif43153 N-[1-[(1R,4R,6R,7S)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-6-yl]-5-methyl-2-oxo-pyrimidin-4-yl]benzamide (2.70 g, 4.00 mmol, 1.0 equiv) was dissolved in 60 mL of anhydrous dichloromethane, to which was added a spatula of 3 Å molecular sieves. Tripyrrolidin-1-ylphosphane (965 mg, 4.00 mmol, 1.0 equiv.) was added via syringe, followed by seven 0.1 mmol aliquots of tetrazole (seven 0.4 mL portions of a 0.5 M solution in anhydrous acetonitrile preserved with 3 Å molecular sieves) at 2 min intervals. 1-(trimethylsilyl)-1H-imidazole (56.0 mg, 0.400 mmol, 0.1 equiv.) was then added to the reaction. After 5 min, tetrazole (21.6 mL of a 0.5 M solution in anhydrous acetonitrile) was added, followed immediately by S-(2-sulfanylethyl)benzenecarbothioate (1.04 g, 5.24 mmol, 1.31 equiv.). The reaction was allowed to proceed for 120 s. Four identical batches of reaction mixture were quenched and combined by pouring the solution into 600 mL of dichloromethane containing 40 mL of triethylamine. The mixture was immediately washed with saturated sodium bicarbonate (800 mL), followed by 10% sodium carbonate (2 × 800 mL) and brine (800 mL). The organic layer was dried with Na2SO4. After 10–15 min, the drying agent was removed by filtration. Triethylamine (40 mL) was added to the solution, which was then concentrated to a syrup using a rotary evaporator. The syrup was dissolved in toluene (100 mL) and triethylamine (30 mL), and the solution was pipetted into 4500 mL of vigorously stirred heptane to precipitate a fluffy white product. After decanting most of the heptane, the white precipitate was collected by filtration through a medium sintered glass funnel and subsequently dried under reduced pressure to yield a white solid.The solid was purified by preparative HPLC (Phenomenex Gemini C18, 250 x 50 mm, 10 mm column, 0.05% ammonium hydroxide in water / CH3CN) and lyophilized to give 2.05 g of the target compound as a white solid. TIFF0007724259000083.tif30154

[0356] 1.5: S-[2-[[(1R,3R,4R,7S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-[(E)-dimethylaminomethyleneamino]-6-oxo-1H-purin-9-yl]-2,5-dioxabicyclo[2.2.1]heptan-7-yl]oxy-pyrrolidin-1-yl-phosphanyl]sulfanylethyl]benzenecarbothioate TIFF0007724259000084.tif36153 N'-[9-[(1R,4R,6R,7S)-4-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-7-hydroxy-2,5-dioxabicyclo[2.2.1]heptan-6-yl]-6-oxo-1H-purin-2-yl]-N,N-dimethyl-formamidine (2.62 mg, 4.00 mmol, 1.0 equiv) was dissolved in 200 mL of anhydrous dichloromethane, to which was added a spatula of 3 Å molecular sieves. Tripyrrolidin-1-ylphosphane (965 mg, 4.00 mmol, 1.0 equiv.) was added via syringe, followed by seven 0.1 mmol aliquots of tetrazole (seven 0.4 mL aliquots of a 0.5 M solution in anhydrous acetonitrile preserved with 3 Å molecular sieves) at 2 min intervals. 1-(trimethylsilyl)-1H-imidazole (56.0 mg, 0.400 mmol, 0.1 equiv.) was then added to the reaction. After 5 min, tetrazole (21.6 mL of a 0.5 M solution in anhydrous acetonitrile) was added, followed immediately by S-(2-sulfanylethyl)benzenecarbothioate (1.04 g, 5.24 mmol, 1.31 equiv.). The reaction was allowed to proceed for 180 s.

[0357] Four identical batches were combined and quenched by pouring the solution into 600 mL of dichloromethane containing 40 mL of triethylamine. The mixture was immediately washed with saturated sodium bicarbonate (800 mL), followed by 10% sodium carbonate (2 × 800 mL) and brine (800 mL). The organic layer was dried with Na2SO4. After 10–15 min, the drying agent was removed by filtration. Triethylamine (40 mL) was added to the solution, which was then concentrated to a syrup using a rotary evaporator. The syrup was dissolved in toluene (100 mL) and triethylamine (30 mL), and the solution was pipetted into 4500 mL of vigorously stirred heptane to precipitate a fluffy white product. After decanting most of the heptane, the white precipitate was collected by filtration through a medium sintered glass funnel and subsequently dried under reduced pressure to yield a white solid. The solid was purified by preparative HPLC (Phenomenex Gemini C18, 250 x 50 mm, 10 mm column, 0.05% ammonium hydroxide in water / CH3CN) and lyophilized to give 3.82 g of the target compound as a yellow solid. TIFF0007724259000085.tif30153

[0358] Example 2: Oligonucleotide synthesis Oligonucleotides were synthesized using a MerMade 12 automated DNA synthesizer from Bioautomation. Synthesis was performed on a 1 μmol scale using controlled pore glass supports (500 Å) with universal linkers.

[0359] In the standard cycling procedure for coupling DNA and LNA phosphoramidites, DMT deprotection was performed by applying 200 μL of 3% (w / v) trichloroacetic acid in CHCl for 30 seconds three times. Each phosphoramidite was then washed with acetonitrile (or LNA- MeFor the C building block, coupling was performed three times using 100 μL of a 0.1 M solution in 1:1 acetonitrile / CHCl and 110 μL of a 0.1 M solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile as the activating agent, with a coupling time of 180 s. For thioxidation, a 0.1 M solution of 3-amino-1,2,4-dithiazole-5-thione in 1:1 acetonitrile / pyridine was used (3 × 190 μL, 55 s). Capping was performed for 55 s with 8:1:1 THF / lutidine / AcO (CapA, 75 μmol) and 8:2 THF / N-methylimidazole (CapB, 75 μmol).

[0360] The synthesis cycle for the introduction of thiophosphoramidites included DMT deprotection by applying 200 μL of 3% (w / v) trichloroacetic acid in CHCl three times for 30 seconds. Commercially available DNA thiophosphoramidites or freshly prepared LNA thiophosphoramidites were coupled three times with 100 μL of a 0.15 M solution in 10% (v / v) CHCl in acetonitrile and 110 μL of a 0.1 M solution of 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole in acetonitrile as the activating agent, each time with a coupling time of 600 seconds. Thioxidation was carried out by applying a 0.1 M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine three times for 55 seconds. Capping was performed with 8:1:1 THF / lutidine / Ac2O (CapA, 75 μmol) and 8:2 THF / N-methylimidazole (CapB, 75 μmol) for 55 seconds.

[0361] Upon completion of the automated synthesis, removal of the nucleobase protecting groups and cleavage from the solid support is carried out using ammonia (32%):ethanol mixture (3:1, v:v) containing 20 mM DTT at 55 °C for 15-16 h.

[0362] The crude DMT-tagged oligonucleotides were purified either by repurification using solid-phase extraction cartridges and ion-exchange chromatography or by RP-HPLC purification using a C18 column, followed by DMT removal using 80% aqueous acetic acid and ethanol precipitation.

[0363] In the examples below, we used the following thio-linked chemical structure: TIFF0007724259000086.tif79142

[0364] In the following examples, unless otherwise specified, achiral phosphorodithioate linkages (also referred to as P2S) are non-bridging dithioates (as shown in formula (IA) or (IB)) and are marked with an *. Compounds used in the examples include compounds having the following nucleobase sequences: TIFF0007724259000087.tif46128

[0365] Following the above procedure, the following molecules were prepared: TIFF0007724259000088.tif222159TIFF0007724259000089.tif141159*Dithioate modification between adjacent nucleotides A, G, m C and T represent LNA nucleotides. a, g, c, t represent DNA nucleotides. All other linkages were prepared as phosphorothioates.

[0366] Example 3: In vitro studies of efficacy and cellular uptake Primary rat hepatocytes were seeded in 96-well plates and treated in William's medium E containing 10% FCS without antibiotics. The cells were treated with the indicated concentrations of LNA solutions in complete cell culture medium. After 24 and 72 h of incubation, respectively, the Ca 2+ and Mg 2+Cells were washed three times with PBS containing 1000 kJ / ml and lysed with 165 μL of PureLink Pro Lysis Buffer. Total RNA was isolated using the Thermo Fisher PureLink PRO 96 RNA Kit according to the manufacturer's instructions, and RT-qPCR was performed using the LightCycler Multiplex RNA Virus Master (Roche) with a primer probe set for RnApoB (Invitrogen). Data were normalized to Ribogreen.

[0367] Intracellular concentrations of LNA oligonucleotides were measured using a hybridization-based ELISA assay for various compounds. All data points were performed in triplicate and data are presented as means.

[0368] These results are shown in Figures 1-4.

[0369] Example 4: Thermal Melting (Tm) of Oligonucleotides Containing Phosphorodithioate Internucleoside Linkages Hybridized to RNA and DNA The following oligonucleotides were prepared: Phosphorothioate linkages are designated with the subscript S, and phosphorodithioate linkages according to the invention are designated with the subscript PS2. TIFF0007724259000090.tif108128 Compounds 1-6 have the sequence motif SEQ ID NO 1.

[0370] The thermal melting (Tm) of compounds 1-6 hybridized to RNA and DNA was measured according to the following procedure.

[0371] Equimolar solutions of RNA or DNA and LNA oligonucleotides (1.5 μM) in buffer (100 mM NaCl, 0.1 mM EDTA, 10 mM NaHPO, pH 7) were heated to 90°C for 1 minute and then allowed to cool to room temperature. UV absorbance at 260 nm was recorded using a Cary series UV-Vis spectrophotometer (heating rate 1°C / min, reading rate 1 / min). Absorbance was plotted against temperature, and Tm values were calculated by taking the first derivative of each curve.

[0372] The results are summarized in the table below and in FIG. TIFF0007724259000091.tif101128Td: dissociation (denaturation) temperature; Ta: association (renaturation) temperature

[0373] The compounds according to the invention retain high affinity for control RNA and DNA.

[0374] Example 5: Serum stability of oligonucleotides containing phosphorodithioate internucleoside linkages The stability of oligonucleotides 1-6 in the serum of male Sprague-Dawling rats was determined according to the following procedure.

[0375] A 25 μM solution of oligonucleotides dissolved in a 3:1 mixture of rat serum and nuclease buffer (30 mM sodium acetate, 1 mM zinc sulfate, 300 mM NaCl, pH 4.6) was incubated at 37°C for 0, 5, 25, 52, or 74 hours. 2 μL of each sample was transferred to a Water Acquisition BEH C 18 The resulting oligonucleotides were injected for UPLC-MS analysis on a Water Acquity UPLC equipped with a 1.7 μm column. The analog peak areas measured at 260 nm, corrected for the elongation constants for different degradation lengths, were used to determine the percentage of uncleaved oligonucleotide.

[0376] UPLC eluent: A: 2.5%MeOH, 0.2M HEP, 16.3mM TEA B: 60%MeOH, 0.2M HEP, 16.3mM TEA TIFF0007724259000092.tif118132

[0377] These results are summarized in FIG.

[0378] Compounds according to the present invention having at least one phosphorodithioate internucleoside linkage have superior nuclease resistance than compounds having only phosphorothioate internucleoside linkages.

[0379] The initial oligonucleotide degradation observed after 5 hours for compounds 1-6 was found to be due to the presence of a monothioate impurity.

[0380] Example 7: Dithioate-modified gapmers: Investigation of dithioates in the gap region of LNA gapmers Compounds tested TIFF0007724259000093.tif153128 Compounds #1-16 and references have the sequence motif shown in SEQ ID NO 1. Uppercase letters: β-D-oxyLNA nucleosides; lowercase letters: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0381] Experimental: The above compounds targeting ApoB mRNA were tested in primary rat hepatocytes by gymnastic uptake after incubation at a compound concentration of 2 μM for 72 hours. Target mRNA levels were then measured using RT-PCR. The results are shown in Figure 7.

[0382] The results shown in Figure 7 indicate that both single and multiple achiral phosphorodithioates are acceptable in the gap and flanking regions. The use of more than three or four achiral phosphorodithioates in the gap may tend to reduce drag compared to the use of multiple achiral phosphorodithioates in the flanking regions.

[0383] Example 8: Position Dependence of Activity - Design Optimization Compounds tested TIFF0007724259000094.tif174128 Compounds #1-16 and references have the sequence motif shown in SEQ ID NO 1. Uppercase letters: β-D-oxyLNA nucleosides; lowercase letters: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0384] Experimental: The above compounds targeting ApoB mRNA were tested in primary rat hepatocytes by gymnastic uptake after incubation at a compound concentration of 2 μM for 72 hours. Target mRNA levels were then measured using RT-PCR. The results are shown in Figure 8.

[0385] Example 9: Cellular uptake of achiral phosphorodithioate gapmers Compounds tested TIFF0007724259000095.tif81128 Compounds #1-16 and references have the sequence motif shown in SEQ ID NO 1. Uppercase letters: β-D-oxyLNA nucleosides; lowercase letters: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0386] Experimental: The above compounds targeting ApoB mRNA were tested in primary rat hepatocytes using gymnostic uptake after incubation for 72 hours at a compound concentration of 2 μM. Oligonucleotide content was measured using a hybridization-based ELISA assay. The results are shown in Figures 9A and 9B.

[0387] Without exception, the inclusion of an achiral phosphorodithioate increased cellular uptake, although the improvement in uptake varied depending on the position of the achiral phosphorodithioate linkage.

[0388] Example 10: Increasing Achiral Phosphorodithioate Amounts in the Flanking Regions of a Gapmer Compounds tested (sequence motif = SEQ ID NO 1) TIFF0007724259000096.tif94143Uppercase: β-D-oxyLNA nucleosides; Lowercase: DNA nucleosides; * = achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0389] Experimental: The above compounds targeting ApoB mRNA were tested in primary rat hepatocytes using gymnastic uptake after incubation at a compound concentration of 2 μM for 72 hours. Target mRNA levels were then measured using RT-PCR. The results are shown in Figures 10A and 10B.

[0390] Introducing achiral phosphorodithioate modifications into the flanking regions of gapmers resulted in a significant increase in potency and a 3- to 7-fold decrease in IC50. Interestingly, increasing the number of chiral phosphorodithioate modifications in the flanks resulted in a decrease in IC50.

[0391] Example 11: Effect of achiral phosphorodithioate linkages in different cell types in vitro Compounds tested (sequence motif = SEQ ID NO 3) TIFF0007724259000097.tif92138Uppercase: β-D-oxyLNA nucleosides; Lowercase: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0392] The above compounds targeting Malat-1 were tested in three in vitro cell lines: human primary skeletal muscle, human primary bronchial epithelial cells, and mouse fibroblasts (LTK cells) using 72-hour gymnosic uptake at a range of concentrations to determine compound potency (IC50).

[0393] Concentration range for LTK cells: 50 μM; 1 / 2log dilution, 8 concentrations.

[0394] Malat1 RNA levels were quantified using qPCR (normalized to GAPDH levels) and IC50 values were determined.

[0395] The IC50 results are shown in Figure 11. Introduction of achiral phosphorodithioates resulted in a reliable increase in potency in skeletal muscle cells and generally improved potency in mouse fibroblasts. Effects in human bronchial epithelial cells tended to be compound-specific, although some compounds (#5) were significantly more potent than the reference compound.

[0396] Example 12: In vitro rat serum stability of 5'- and 3'-protected LNA oligonucleotides Compounds tested (sequence motif = SEQ ID NO 1) TIFF0007724259000098.tif113128Uppercase: β-D-oxyLNA nucleosides; Lowercase: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate.

[0397] Experimental—See Example 5.

[0398] These results are shown in Figure 12. We confirmed that the 3' ends of LNA phosphorothioate oligonucleotides are more susceptible to serum nucleases than previously thought, as indicated by the rapid cleavage of parent oligonucleotide #1 by 50%, and that this appears to be related to the chirality of the phosphorothioate bond at the 3' end of the oligonucleotide. Protecting the 5' end with an achiral phosphorodithioate improved protection. Protecting the 3' end with an achiral phosphorodithioate provided complete protection from rat serum exonucleases—the slight reduction observed for compounds #4-8 correlated with the monothioate impurity.

[0399] Therefore, protection of the 5' and / or 3' ends of antisense oligonucleotides with achiral phosphorothioate linkages is considered to provide a solution to the major problem of instability associated with stereo-random and stereo-defined phosphorothioates.

[0400] Example 13: In vivo evaluation of gapmers with achiral phosphorodithioate linkages in the flanks Compounds tested (sequence motif = SEQ ID NO 1) TIFF0007724259000099.tif144128Uppercase: β-D-oxyLNA nucleosides; Lowercase: DNA nucleosides; *=achiral phosphorodithioate modified bond; all other bonds are phosphorothioate. Note that the underlined bold nucleosides are linked at the 3' position by a stereodefined phosphorothioate internucleoside bond. Compound #7 has a stereodefined motif in the gap region of SSRSSRSR (S = Sp, R = Rp). The backbone motif of compound #9 = RRSPRSSPSPSS (where S = Sp, R = Rp, and P = achiral PS2 bond (*)).

[0401] Experiment: The above compounds targeting ApoB were administered to female C57BL / 6JBom mice using a single intravenous dose of 1 mg / kg and sacrificed on day 7 (n=5). mRNA reduction in the liver was measured using RT-PCR, and the results are shown in Figure 13.

[0402] These results indicate that the introduction of achiral phosphorodithioate internucleoside ...

Claims

1. In the production of an oligonucleotide, a compound represented by the following formula (III) or formula (IV): (In the formula, R 5 is a hydroxyl protecting group selected from 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 ethers (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), methyl ether, and ethoxyethyl ether (EE); R x is phenyl, nitrophenyl, phenylalkyl, halophenylalkyl, cyanoalkyl, phenylcarbonylsulfanylalkyl, halophenylcarbonylsulfanylalkylalkylcarbonylsulfanylalkyl, or alkylcarbonylcarbonylsulfanylalkyl; R y is dialkylamino or pyrrolidinyl; and Nu is a nucleobase) Use of a compound of the formula The oligonucleotide comprises a gapmer oligonucleotide having the following formula (IA) or (IB): wherein in formula (IA), R is hydrogen or a phosphate protecting group selected from 2-cyanoethyl and methyl, and in formula (IB), M is a cation, such as a metal cation, for example an alkali metal cation, for example a Na cation or a K cation, or M is an ammonium cation. an antisense gapmer oligonucleotide comprising a phosphorodithioate internucleoside linkage of the formula: the oligonucleotide further comprises phosphorothioate internucleoside linkages; Use of a compound of formula (III) or formula (IV).

2. R x 2. The use of claim 1, wherein is phenyl, nitrophenyl, phenylmethyl, dichlorophenylmethyl, cyanoethyl, methylcarbonylsulfanylethyl, ethylcarbonylsulfanylethyl, isopropylcarbonylsulfanylethyl, tert-butylcarbonylsulfanylethyl, methylcarbonylcarbonylsulfanylethyl, or difluorophenylcarbonylsulfanylethyl.

3. R x 3. The use according to claim 1 or 2, wherein is phenylcarbonylsulfanylalkyl.

4. R x The use according to any one of claims 1 to 3, wherein is phenylcarbonylsulfanylethyl.

5. R y The use according to any one of claims 1 to 4, wherein is diisopropylamino or pyrrolidinyl.

6. R y The use according to any one of claims 1 to 5, wherein is pyrrolidinyl.

7. The compound has the formula (V) wherein R 5 and Nu are as defined in claim 1.

8. 8. The use according to any one of claims 1 to 7, wherein Nu is thymine, adenosine, cytosine, 5-methylcytosine, guanine, or uracil.

9. The compound is The use according to any one of claims 1 to 8, wherein the compound is selected from the group consisting of:

10. The use according to any one of claims 1 to 9, wherein the phosphorothioate internucleoside linkage further comprised in the oligonucleotide is independently selected from a phosphorodithioate internucleoside linkage and a phosphorothioate internucleoside linkage of formula (IA) or (IB) as defined in claim 1.

11. 11. The use according to any one of claims 1 to 10, wherein all of the internucleoside linkages between nucleosides in the gap region of the oligonucleotide are phosphorothioate internucleoside linkages.

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