Threose nucleic acid antisense oligonucleotides and methods thereof

Abstract

Antisense oligonucleotides containing one or more α-L-threofuranosyl (TNA) nucleosides linked to adjacent nucleosides via phosphodiester (PO) internucleoside linkages, as well as methods for modulating the properties of antisense oligonucleotides by the incorporation of such TNA nucleosides, are described, which are particularly applicable to antisense gapmer oligonucleotides.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antisense oligonucleotides comprising one or more α-L-threofuranosyl (TNA) nucleosides linked to adjacent nucleosides by phosphodiester internucleoside linkages, as well as methods for modulating the properties of antisense oligonucleotides by the incorporation of such TNA nucleosides. The present invention is particularly applicable to antisense gapmer oligonucleotides. [Background technology]

[0002] Background of the Invention Synthetic oligonucleotides as therapeutic agents have made significant progress in recent years, resulting in a broad portfolio of clinically validated molecules that act by diverse mechanisms, including ribonuclease H (RNase H)-activating gapmers, splice-switching oligonucleotides, microRNA inhibitors, small interfering RNAs (siRNAs), and antisense oligonucleotides such as aptamers (S.T. Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008).

[0003] Arguably, one of the most successful modifications is the introduction of phosphorothioate (PS) linkages, in which one of the non-bridging phosphate oxygen atoms is replaced with a sulfur atom (Eckstein, Antisense and Nucleic Acid Drug Development 2009; 10: 117-121 (Non-Patent Document 2)). Phosphorothioate oligodeoxynucleotides exhibit increased protein binding and significantly higher stability against nucleic acid degradation, and therefore longer half-lives in plasma, tissues, and cells than their unmodified phosphodiester analogs. For example, a recent review (Crooke et al., Nucleic Acids Research 2020; 48(10): 5235-5253 (Non-Patent Document 3)) notes that the PS moiety is the primary determinant of the distribution of single-stranded antisense oligonucleotides after all administration routes. Other modifications include locked nucleic acids (LNAs) and various other modified nucleosides. For example, TNAs have been used in the form of double-stranded siRNA molecules and oligomers (Matsuda et al., XXIII International Round Table on Nucleosides, Nucleotides and Nucleic acids; 2018 (Non-Patent Document 4), Liu et al., ACS Appl. Mater. Interfaces 2018; 10:9736-9743 (Non-Patent Document 5), WO2012 / 078536 (Patent Document 1), WO2012 / 118911 (Patent Document 2), and WO2013 / 179292 (Patent Document 3)).

[0004] However, there remains a need for stable, safe, and effective antisense oligonucleotide-based therapeutics. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2012 / 078536 [Patent Document 2] WO2012 / 118911 [Patent Document 3] WO2013 / 179292 [Non-patent literature]

[0006] [Non-Patent Document 1] STC Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008 [Non-patent document 2] Eckstein,Antisense and Nucleic Acid Drug Development2009;10:117-121 [Non-patent document 3] Crooke et al., Nucleic Acids Research2020;48(10):5235-5253 [Non-patent document 4] Matsuda et al.,XXIII International Round Table on Nucleosides,Nucleotides and Nucleic acids;2018 [Non-patent document 5] Liu et al.,ACS Appl.Mater.Interfaces2018;10:9736-9743 Summary of the Invention

[0007] The present inventors have discovered that one or more α-L-threofuranosyl (TNA) nucleosides can be introduced into antisense oligonucleotides, particularly antisense gapmer oligonucleotides, via linkages other than PS linkages to modulate the properties of the antisense oligonucleotide. Surprisingly, TNA nucleosides, particularly when introduced into gapmer designs via phosphodiester (PO) internucleoside linkages as described herein, can result in potent molecules with favorable properties for therapeutic use.

[0008] Therefore, the present invention relates to antisense oligonucleotides containing at least one TNA nucleoside linked to at least one adjacent nucleoside via an internucleoside bond other than a PS bond, particularly antisense gapmer oligonucleotides containing at least one such TNA nucleoside. PO internucleoside bond is particularly preferred. TNA nucleosides linked to at least one adjacent nucleoside via a PO bond may be referred to hereinafter as TNA(PO) nucleosides. Possible TNA(PO) nucleosides include those linked via a 2'-PO bond, a 3'-PO bond, or both, and are referred to hereinafter as 2'-PO linked TNA nucleosides, 3'-PO linked TNA nucleosides, and 2',3'-PO linked TNA nucleosides, respectively.

[0009] The present invention also relates to antisense gapmer oligonucleotides capable of recruiting RNase H, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I), wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside linked to adjacent nucleosides by a linkage other than a PS internucleoside linkage, e.g., a PO internucleoside linkage.

[0010] The present invention also relates to an antisense gapmer oligonucleotide capable of recruiting ribonuclease (RNase) H, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I), wherein: G is a gap region of up to 18 linked nucleosides containing at least three consecutive DNA nucleosides; each of F and F' is a flanking region of up to 15 linked nucleosides, independently comprising or consisting of 1 to 15 sugar-modified nucleosides; At least one of F, F', and G comprises a sugar-modified nucleoside, which is an α-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside bond that is different from a PS internucleoside bond, for example, a PO internucleoside bond.

[0011] The present invention also relates to a conjugate comprising an antisense gapmer oligonucleotide according to the present invention and at least one conjugate moiety covalently attached to the antisense gapmer oligonucleotide, optionally via a linker.

[0012] The present invention also relates to pharmaceutically acceptable salts of the antisense gapmer oligonucleotides or conjugates according to the invention.

[0013] The present invention also relates to a pharmaceutical composition comprising an antisense gapmer oligonucleotide, conjugate, or pharmaceutically acceptable salt thereof according to the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0014] The present invention also relates to an antisense oligonucleotide, a conjugate, a pharmaceutically acceptable salt or a pharmaceutical composition according to the invention for use as a medicament.

[0015] The present invention also provides a method for preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of the formula 5'FG-F'3'(I) capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides, and each of F and F' is a flanking region of up to 8 linked nucleosides independently comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, wherein in the modified version at least one nucleoside in F, F' and / or G of the parent antisense gapmer oligonucleotide is replaced with a TNA nucleoside linked to the adjacent nucleoside by an internucleoside linkage different from a PS internucleoside linkage, e.g., a PO internucleoside linkage; producing a modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked consecutive nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside; Optionally, the method includes purifying or isolating the modified antisense gapmer oligonucleotide.

[0016] The present invention also relates to antisense gapmer oligonucleotides obtained or obtainable by the methods of the present invention.

[0017] The present invention also relates to the use of TNA nucleotides in the preparation of antisense gapmer oligonucleotides according to the invention.

[0018] Further details of these and other aspects and embodiments of the present invention are provided in the detailed disclosure and claims that follow. [Brief explanation of the drawings]

[0019] [Figure 1] Relative MALAT1 RNA expression in C57BL / 6J mice. Antisense oligonucleotides were administered by subcutaneous injection at a dose of 10 mg / kg, and RNA levels were measured on day 9. For details, see Example 6. [Figure 2-1] Tissue concentrations and pharmacokinetic / pharmacodynamic (PK / PD) relationships. Antisense oligonucleotide concentrations in the liver (A), kidney (B), lung (C), and muscle (D) are shown, as well as the PK / PD relationships for the liver (E), kidney (F), lung (G), and muscle (H). See Example 6 for details. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Disclosure definition In order that the present invention may be more readily understood, certain terms are defined and explained below.

[0021] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be interpreted to mean the inclusion of one integer (or component) or group of integers (or components) but not other integers (or components) or groups of integers (or components).

[0022] Oligonucleotides As used herein, the term "oligonucleotide" is defined as commonly understood by those skilled in the art as a molecule containing two or more covalently linked nucleosides, including modified nucleosides or nucleotides. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically produced in laboratories by solid-phase chemical synthesis followed by purification. When referring to an oligonucleotide sequence, reference is made to the sequence or order of the nucleobase moieties or modifications of the covalently linked nucleotides or nucleosides. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The nucleosides may be linked by phosphodiester (PO) linkages or modified internucleoside linkages.

[0023] antisense oligonucleotides As used herein, the term " antisense oligonucleotide " is defined as an oligonucleotide that can regulate the expression of target gene by hybridizing to target nucleic acid, particularly to the continuous sequence on target nucleic acid.The intended antisense oligonucleotide is essentially not double-stranded, and therefore is not siRNA or short hairpin RNA (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 (duplex between two molecules of the same oligonucleotide) when the degree of intra or inter-complementarity is more than 50% over the entire length of the oligonucleotide.

[0024] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to the region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence." For example, all nucleotides of an oligonucleotide may constitute a contiguous nucleotide sequence. Alternatively, an oligonucleotide may comprise a contiguous nucleotide sequence such as an FG-F' gapmer region, and may optionally comprise a nucleotide linker region that can be used to attach additional nucleotides, such as functional groups, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. Advantageously, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid.

[0025] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring 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 can also be referred to interchangeably as "units" or "monomers."

[0026] 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 context of the present invention, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but function during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring 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.

[0027] The nucleobase moiety may optionally be modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, such as 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.

[0028] Nucleobase moieties may be represented by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include functionally equivalent modified nucleobases. For example, in some oligonucleotides, nucleobase moieties include A, T, G, C, and 5-methylcytosine ( m C) is selected.

[0029] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that has been modified by introducing one or more modifications to the sugar moiety or (nucleic acid) base moiety, compared to an equivalent DNA or RNA nucleoside. Preferably, the modified nucleoside contains a modified sugar moiety. The term "modified nucleoside" may also be used herein interchangeably with the term "nucleoside analog" or modified "unit" or modified "monomer." Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if Watson-Crick base pairing is possible.

[0030] Sugar-modified nucleosides The antisense oligonucleotides of the invention may comprise one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety compared to the ribose sugar moiety found in DNA and RNA.

[0031] Numerous nucleosides with modifications in the ribose sugar moiety have been created primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0032] Such modifications include those in which the ribose ring structure is modified, for example, by replacing it with a bicyclic ring having a bridge between the C2 and C4 carbons of the ribose ring (HNA), or typically a bicyclic ring having a bridge between the C2 and C4 carbons of the ribose ring (LNA), 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.

[0033] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0034] Non-limiting examples of modified sugar moieties include: α-L-threofuranosyl (found in threose nucleic acid; TNA), 2'-methoxy-ribose (2'-OMe), 2'-O-Methoxyethyl-ribose (2'-O-MOE), 5'-methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE), 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE), 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE), 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA), 2'-deoxy-2'-fluoro-ribose (found in 2'-deoxy-2'-fluororibonucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (found in 2'-fluoro-2'-arabinose nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxyβ-D-locked dribose (found in β-D-LNA), amino β-D-locked dribose (found in amino-β-D-LNA), Thioβ-D-locked dribose (found in thio-β-D-LNA), Oxyβ-L-locked dribose (found in β-L-LNA), amino β-L-locked dribose (found in amino β-L-LNA), Thioβ-L-locked dribose (found in thio-β-L-LNA), Oxyα-L-locked dribose (found in α-L-LNA), amino α-L-locked dribose (found in amino α-L-LNA), Thio α-L-locked dribose (found in thio-α-L-LNA), 2',4'-constrained 2'-O-ethyl ribose (as found in constrained ethyl-locked nucleic acid; cEt), tricyclodeoxyribose (tricyclodeoxyribose DNA; found in TcDNA), 3'-deoxy-ribose (found in 3'-deoxy-ribose DNA; 3'-DNA), unlocked dribose (found in unlocked nucleic acid; UNA), glycol (found in glycol nucleic acid; GNA), hexitols (found in hexitol nucleic acids; HNA), 3'-fluorohexitol (found in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabinofluorohexitol (found in 3'-arabinofluorohexitol nucleic acid; Ara-FHNA), cyclohexene (found in cyclohexene nucleic acid; CeNA), Fluoro-cyclohexenyl (found in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA), Serinol (found in serinol nucleic acid; SNA), 2'-O,4'-C-ethylene-bridged ribose (2'-O,4'-C-ethylene-linked nucleic acid; found in ENA) Acyclic (L)-threoninol (found in acyclic (L)-threoninol nucleic acid; aTNA) 2',4'-constrained 2'-O-methoxyethyl ribose (as found in cMOE), and 7',5'-alpha-bicyclosugar unit (7',5'-alpha-bicycloDNA; found in bcDNA).

[0035] Unless otherwise specified or contradicted by context, as used herein, the term "MOE" may refer to any nucleoside containing an O-methoxyethyl group at the 2' position of the ribose ring, including, but not limited to, 2'-O-MOE and 5'-Me-2'-O-MOE.

[0036] Threose nucleic acid (TNA) As used herein, "α-L-threofuranosyl nucleoside," "α-L-threose nucleic acid nucleoside," "TNA nucleoside," "TNA modified nucleoside," "TNA unit," "TNA moiety," and the like refer to a sugar-modified nucleoside that includes an α-L-threofuranosyl moiety.

[0037] TNA nucleosides are linked to adjacent nucleosides by a (2'→3') internucleoside linkage, eg, a phosphodiester (PO) or modified internucleoside linkage, as shown below for two adjacent TNA nucleosides: TIFF2025528464000001.tif66128

[0038] Sugar-modified nucleosides containing an α-L-threofuranosyl moiety and linked to at least one adjacent nucleoside via a PO linkage may be referred to herein as "α-L-threofuranosyl (PO) nucleosides," "α-L-threose nucleic acid (PO) nucleosides," "TNA(PO) nucleosides," "TNA(PO)-modified nucleosides," "TNA(PO) units," "TNA(PO) moieties," etc. Contemplated TNA(PO) nucleosides include those linked by a 2'-PO linkage, a 3'-PO linkage, or both, and are hereinafter referred to as "2'-PO linked TNA nucleosides," "3'-PO linked TNA nucleosides," and "2',3'-PO linked TNA nucleosides," respectively.

[0039] When the nucleobase (B) is cytosine, the TNA or TNA(PO) nucleoside is advantageously 5-methyl-cytosine ( m C) It is a TNA or TNA(PO) nucleoside.

[0040] 2' sugar-modified nucleosides 2'-Sugar-modified nucleosides are nucleosides having a substituent other than H or -OH at the 2' position (2'-substituted nucleosides). This includes nucleosides containing 2'-linked biradicals that can form a bridge between the 2'-carbon and a second carbon in the ribose ring, such as LNA (2'-4' bridged) nucleosides.

[0041] For purposes of this disclosure, a TNA or TNA(PO) nucleoside is not a 2'-substituted nucleoside.

[0042] Many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides.For example, 2'-modified sugars can improve the binding affinity to oligonucleotides and / or improve nuclease resistance.Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'-O-MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-F-ANA, and 2'-bridged molecules such as LNA.For further examples, see, for example, Freier & Altmann; Nucl.Acid Res., 1997,25,4429-4443 and Uhlmann; Curr.Opinion in Drug Development, 2000,3(2),293-213, and Deleavy and Damha, Chemistry and Biology 2012,19,937. Scheme 1 below shows examples of some 2'-substituted modified nucleosides. Scheme 1: TIFF2025528464000002.tif76128

[0043] Locked Nucleic Acid (LNA) "LNA nucleosides" are 2'-modified nucleosides containing a biradical (also referred to as a "2'-4' bridge") linking the C2' and C4' ends of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). The fixation of the ribose conformation is associated with improved hybridization affinity (duplex stabilization) when LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0044] Non-limiting, exemplary LNA nucleosides are those 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. 2002, 12, 73-76, Seth et al., J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0045] Further non-limiting exemplary LNA nucleosides are set forth in Scheme 2. Scheme 2: TIFF2025528464000003.tif191128TIFF2025528464000004.tif204129TIFF2025528464000005.tif239127

[0046] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA. A particularly advantageous LNA is β-D-oxy-LNA.

[0047] internucleoside bond The term " internucleoside bond " is generally understood by those skilled in the art and is defined as the bond that covalently connects two nucleosides together.In the antisense oligonucleotides described herein, internucleoside bond covalently connects adjacent nucleosides together, and typically forms a bond between the sugar moieties of adjacent nucleosides.Non-limiting examples of internucleoside bond include phosphodiester (PO) bond and modified internucleoside bond.

[0048] Modified internucleoside linkages The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides together, as commonly understood by those skilled in the art. Modified internucleoside linkages can improve the nuclease resistance of oligonucleotides compared to phosphodiester (PO) linkages. Modified internucleoside linkages can stabilize oligonucleotides for in vivo use and can protect against nuclease cleavage in regions of DNA or RNA nucleosides in oligonucleotides, such as the gap region of gapmer oligonucleotides, and in regions of modified nucleosides such as regions F and F'.

[0049] 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. In some oligonucleotides, all internucleoside linkages of the oligonucleotide or its consecutive nucleotide sequence can be nuclease-resistant internucleoside linkages. It is also contemplated that the nucleoside linking the oligonucleotide to a non-nucleotide functional group, such as a conjugate, can be a phosphodiester.

[0050] phosphorothioate internucleoside linkages The preferred modified internucleoside bond is phosphorothioate (PS).Phosphorothioate internucleoside bond can be useful due to its nuclease resistance, favorable pharmacokinetics and ease of manufacture.In some oligonucleotides, all modified internucleoside bonds of the oligonucleotide or its consecutive nucleotide sequence are phosphorothioate bonds.

[0051] Nuclease-resistant linkages such as phosphorothioate linkages can be useful in regions of an oligonucleotide that can recruit nucleases when duplexed with a target nucleic acid, such as region G for gapmers. However, phosphorothioate linkages can also be useful in non-RNase H recruiting regions and / or affinity-enhancing regions, such as regions F and F' for gapmers.

[0052] Non-bridging phosphorodithioate internucleoside linkages Another preferred modified internucleoside linkage is a non-bridging phosphorodithioate internucleoside linkage (PS2). PS2 internucleoside linkages have two identical sulfur atoms bonded to the phosphorus atom, achieved by replacing the non-bridging oxygen atom in the phosphorothioate linkage with a sulfur atom. Phosphorodithioate internucleoside linkages can be useful due to their beneficial nuclease resistance, favorable pharmacokinetics, and ease of manufacture. In some oligonucleotides or consecutive nucleotide sequences thereof, the 3'-most or 5'-most internucleoside linkages are phosphorodithioate internucleoside linkages. In some oligonucleotides or consecutive nucleotide sequences thereof, the 3'-most and 5'-most internucleoside linkages are phosphorodithioate internucleoside linkages. In some oligonucleotides or consecutive nucleotide sequences thereof, all modified internucleoside linkages of the oligonucleotide or consecutive nucleotide sequences thereof are phosphorodithioate linkages.

[0053] Nuclease-resistant linkages such as phosphorodithioate linkages may be useful in regions of an oligonucleotide that can recruit nucleases when duplexed with a target nucleic acid, such as, for example, region G for gapmers. However, phosphorodithioate linkages may also be useful in non-RNase H recruiting regions and / or affinity-enhancing regions, such as regions F and F' for gapmers.

[0054] Complementarity The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. 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; therefore, the term "complementarity" is understood to encompass Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al. (2012) Accounts of Chemical Research, vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Suppl. 37, 1.4.1).

[0055] As used herein, the term "% complementarity" refers to the number of nucleotides in a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an oligonucleotide) that are complementary (i.e., form Watson-Crick base pairs) at a given position to the contiguous sequence of nucleotides at a given position in an individual nucleic acid molecule (e.g., a target nucleic acid or target sequence), expressed as a percentage. The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when the target sequence 5'-3' and the oligonucleotide sequence 3'-5' are aligned), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form base pairs) are referred to as mismatches. Preferably, insertions and deletions are not allowed in calculating the % complementarity of contiguous nucleotide sequences.

[0056] The term "fully complementary" refers to 100% complementarity.

[0057] identity The term "identity" as used herein refers to the percentage (expressed as a percentage) of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned bases between two sequences (e.g., the contiguous nucleotide sequence of the compound of the present invention and the reference sequence), dividing this number by the total number of nucleotides in the aligned region, and multiplying by 100. Thus, the percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It should be understood that in determining identity, chemical modifications of nucleobases are ignored as long as the nucleobases retain their functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating identity percentage).

[0058] Hybridization As used herein, the term "hybridize" or "hybridizing" should be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is determined 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 ) is often explained by the following: Under physiological conditions, T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The Gibbs free energy ΔG° at standard conditions more accurately represents binding affinity, ΔG° = -RTln(K d ) to calculate the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at an aqueous concentration of 1 M, pH 7, and temperature of 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 experimentally measured, for example, by isothermal titration calorimetry (ITC), as described in Hansen et al., 1965, Chem. Comm. 36-38, and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will know that commercially available devices are available for measuring ΔG°. ΔG° can also be numerically estimated using the nearest neighbor model described in Santa Lucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. To ensure the potential for hybridization modulation of the intended nucleic acid target, 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. For example, the degree or strength of hybridization can be measured by the standard-state Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with an estimated ΔG° value in the range of less than -10 kcal, for example, less than -15 kcal, for example, less than -20 kcal, and for example, less than -25 kcal for oligonucleotides 8 to 30 nucleotides in length. The oligonucleotides may hybridize to the target nucleic acid with an estimated ΔG° value of, for example, -10 to -60 kcal, for example, -12 to -40 kcal, for example, -15 to -30 kcal, or 16 to -27 kcal, for example, -18 to -25 kcal.

[0059] target nucleic acid Target nucleic acid is the nucleic acid that antisense oligonucleotide can hybridize with, thereby regulating the expression of target gene.Target nucleic acid can be, for example, gene, RNA, mRNA, pre-mRNA, long non-coding RNA (lncRNA), mature mRNA or cDNA sequence, or synthetic nucleic acid derived from DNA or RNA.Target nucleic acid that is RNA can be referred to as "RNA target sequence", "target RNA sequence" etc.

[0060] Target sequence As used herein, the term "target sequence" refers to a sequence of nucleotides present in a target nucleic acid that contains a nucleobase sequence complementary to an antisense oligonucleotide described herein. The target sequence can, for example, consist of a region on the target nucleic acid that is complementary to the contiguous nucleotide sequence of an oligonucleotide of the present invention.

[0061] target cell As used herein, the term "target cell" refers to a cell that expresses a target nucleic acid. Suitably, the target cell contains at least one copy of the target gene in its genome. The target cell can be in vivo or in vitro. The target cell can be, for example, a mammalian cell, such as a rodent cell, for example, a mouse cell or a rat cell, or a primate cell, such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0062] Regulation of expression As used herein, the term "modulation of expression" should be understood as a general term for the ability of an oligonucleotide to change the amount of protein expressed or RNA transcribed from a target gene. Modulation of expression can be determined by reference to a control experiment. The control can be an individual cell or target cell treated with a saline composition, or an individual cell or target cell treated with a non-targeting oligonucleotide (mock).

[0063] High-affinity modified nucleosides High affinity modified nucleosides, when incorporated into oligonucleotides, can, for example, increase the melting temperature (T m High affinity modified nucleosides are modified nucleotides that increase the affinity of the oligonucleotide for its complementary target as measured by the affinity index (A.I.). High affinity modified nucleosides preferably have a A.I. of +0.5 to +12 per modified nucleoside. ℃ , more preferably +1.5 to +10 ℃ , most preferably +3 to +8 ℃ Many high-affinity modified nucleosides are known in the art, including, for example, many 2'-sugar-substituted nucleosides, such as 2'-O-MOE, 2'-F-RNA and LNA, and their analogs (see, for example, Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0064] RNase H activity and recruitment The ribonuclease (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 determine the ability to recruit RNase H. Recombinant human RNase H1 is available from Lubio Science GmbH (Lucerne, Switzerland). Typically, an oligonucleotide is considered to be capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, the initial cleavage rate of the target RNA molecule, measured in pmol / l / min, is at least 5%, e.g., at least 10% or more than 20% of the initial cleavage rate determined using an oligonucleotide having the same base sequence as the antisense oligonucleotide being tested but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, using the method provided in Examples 91-95 of WO 01 / 23613 (incorporated herein by reference).

[0065] Gapmar The antisense oligonucleotide or its contiguous nucleotide sequence may be or include a gapmer. Gapmers are commonly used to inhibit target nucleic acids via RNase H-mediated degradation. Gapmers comprise at least three distinct structural regions in a "5→3" orientation: a 5'-flank, a gap, and a 3'-flank, represented herein as 5'-FG-F'-3' (Formula I). ​​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 and a 3'-flanking region (F') containing one or more sugar-modified nucleosides. One or more sugar-modified nucleosides in regions F and F' may increase the affinity of the oligonucleotide for the target nucleic acid (i.e., they are affinity-enhancing sugar-modified nucleosides, such as high-affinity modified nucleosides) or may modulate other properties as desired.

[0066] In a gapmer design, the 5'-most and 3'-most nucleosides of the gap region are typically DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. Flanks can be further defined by having at least one sugar-modified nucleoside at the terminus furthest from the gap region, i.e., at the 5' end of the 5' flanking section and the 3' end of the 3' flanking section.

[0067] The regions FG-F' form a contiguous nucleotide sequence. The antisense oligonucleotide or its contiguous nucleotide sequence may comprise or consist of a gapmer of formula I, ie, FG-F'.

[0068] The total length of the gapmer design FG-F' is typically 12 to 32 nucleosides, for example 12 to 28, for example 12 to 26, for example 14 to 26, for example 14 to 24, for example 14 to 22, for example 16 to 22, for example 16 to 20 nucleosides.

[0069] Conventional gapmer designs that do not contain TNA nucleosides include, for example, F 1-8 -G 5-18 -F' 1-8 (II), e.g., F 1-8 -G 7-16 -F' 2-8 (III), provided that the total length of the gapmer region FG-F' is typically at least 12, for example at least 14 nucleotides in length.

[0070] Suitable designs for TNA(PO) gapmers according to the present invention include, for example, F 1-15 -G 3-18 -F' 1-15 (IV), typically provided that the total length of the gapmer region FG-F' is at least 12, e.g., at least 14, nucleotides in length. Additional designs of such gapmers (e.g., Formulas IV-VII) are described in further detail elsewhere herein.

[0071] Regions F, G and F' are further described below and may be incorporated into any of the FG-F' formulas.

[0072] Gapmer region G Region G (gap region) of a gapmer is a region of nucleosides, typically DNA nucleosides, that allows the oligonucleotide to recruit RNase H, such as human RNase H1. RNase H is a cellular enzyme that recognizes duplexes between DNA and RNA and enzymatically cleaves RNA molecules.

[0073] A conventional gapmer that does not contain TNA nucleosides, for example, comprises a gap region (G) of at least 5 or 6 consecutive DNA nucleosides, for example, 5 to 16 consecutive DNA nucleosides, for example, 6 to 15 consecutive DNA nucleosides, for example, 7 to 14 consecutive DNA nucleosides, for example, 8 to 12 consecutive DNA nucleotides, for example, 8 to 12 consecutive DNA nucleotides in length.

[0074] Suitable gapmers according to the present invention, particularly gapmers comprising one or more TNA(PO) nucleosides as described herein, can have a gap region (G) comprising at least three consecutive DNA nucleosides. Gap region G can comprise or consist of, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive DNA nucleosides. Preferably, gap region G comprises at least 4, at least 5, or at least 6 consecutive DNA nucleosides.

[0075] A gap region (G) containing one or more TNA(PO) nucleosides described herein 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'), typically retaining a region of at least three or four consecutive DNA nucleosides at either the 5' end, the 3' end, or both of the gap region.

[0076] The total length of the gap region G is typically up to 18 consecutive nucleosides. For example, the total length of the gap region G can be 3 to 18 consecutive nucleosides, e.g., 3 to 16 consecutive nucleosides, e.g., 4 to 18, 4 to 16, 4 to 14, 4 to 12, or 4 to 10 consecutive nucleosides, e.g., 5 to 18, 5 to 16, 5 to 14, 5 to 12, or 5 to 10 consecutive nucleosides, e.g., 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 consecutive nucleosides. Shorter gap regions, e.g., regions G comprising or consisting of 4, 5, 6, 7, 8, or 9 consecutive nucleosides, e.g., consecutive DNA nucleosides, are also contemplated.

[0077] One or more cytosine (C) DNA nucleosides in the gap region may optionally be methylated (e.g., a C DNA nucleoside followed by a guanine (G) DNA nucleoside, resulting in 5-methyl-cytosine ( me C or m C) when annotated as

[0078] Contemplated oligonucleotides include those in which all internucleoside linkages in the gap are phosphorothioate linkages, or those in which all modified internucleoside linkages in the gap are phosphorothioate linkages.

[0079] Conventional gapmers have a DNA gap region, and there are numerous examples of modified nucleosides that, when used within the gap region, allow for the recruitment of RNase H. Modified nucleosides that have been reported to be able to recruit RNase H when contained within the gap region include, for example, alpha-L-LNA, C4' alkylated DNA (as described in PCT / EP2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both of which are incorporated herein by reference), 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) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which is incorporated herein by reference). A UNA or "unlocked nucleic acid" typically occurs when the bond between C2 and C3 of the ribose is removed to form an unlocked "sugar" residue. The modified nucleosides used in gapmers can be nucleosides that adopt a 2'-terminal (DNA-like) structure when introduced into the gap region, allowing RNase H recruitment. The DNA Gap region (G) described herein can contain, for example, one or more (e.g., 1 to 3) sugar-modified nucleosides. Any two or more sugar-modified nucleosides in the gap can be contiguous or separated by one or more DNA nucleosides.

[0080] As described herein, modified nucleosides that can be used in the gap region include TNA(PO) nucleosides.

[0081] Area G - "Gap Breaker" Numerous reports have also been published on the insertion of modified nucleosides into the gap region of gapmers to confer a 3'-end conformation while retaining some RNase H activity. Gapmers with a gap region containing one or more 3'-end modified nucleosides are referred to as "gap breaker" or "gap-broken" gapmers; see, for example, International Publication No. WO 2013 / 022984. Gap breaker oligonucleotides retain a sufficient region of DNA nucleosides within the gap region to allow RNase H recruitment. The ability of gap breaker oligonucleotide designs to recruit RNase H is typically sequence- or even compound-specific—see Rukov et al., 2015, Nucl. Acids Res., Vol. 43, pp. 8476-8487, which describes "gap breaker" oligonucleotides that recruit RNase H, in some instances providing more specific cleavage of target RNA. Modified nucleosides used within the gap region of a gap breaker oligonucleotide may be, for example, modified nucleosides that impart a 3'-endo conformation, such as 2'-O-methyl (Ome) or 2'-O-MOE (MOE) nucleosides, or beta-D LNA nucleosides (in which the bridge between C2' and C4' of the ribose sugar ring of the nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

[0082] TNA(PO) nucleosides may also be contemplated as gap breakers.

[0083] Similar to the gapmers containing region G described above, the gap region of a gap breaker or gap disrupting 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' end, the 3' end, or both of the gap region.

[0084] Exemplary designs of gap breaker gapmers described herein include: F 1-15 -[D 3-4 -E1-D 3-4 ] - F' 1-15 F 1-15 -[D 1-4 -E1-D 3-4 ]-F' 1-15 F 1-15 -[D 3-4 -E1-D 1-4 ]-F' 1-15 The region G is the square bracket [D n -E r -D m ], where D is a consecutive 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.

[0085] Region G of the gap-breaking gapmers described herein can comprise at least four DNA nucleosides, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. As noted above, the DNA nucleosides can be contiguous or, optionally, intermixed with one or more modified nucleosides, provided that the gap region G can mediate RNase H recruitment.

[0086] Gapmer-flanking regions, F and F' Region F is positioned immediately adjacent to the 5' DNA nucleoside of region G. The 3'-most nucleoside of region F is a sugar-modified nucleoside. Advantageously, one or two of the 5'-most nucleosides of region F are also sugar-modified nucleosides. In gapmers described herein, particularly gapmers comprising one or more TNA(PO) nucleosides, region F is at least one, e.g., at least two, e.g., at least three contiguous nucleotides in length. Typically, region F is up to 15 contiguous nucleotides in length. For example, region F can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous nucleotides in length.

[0087] Region F' is positioned immediately adjacent to the 3' DNA nucleoside of region G. The 5'-most nucleoside of region F' is a sugar-modified nucleoside. Advantageously, one or two of the 3'-most nucleosides of region F' are also sugar-modified nucleosides. In gapmers described herein, particularly gapmers containing one or more TNA(PO) nucleosides, region F' is at least one, e.g., at least two, e.g., at least three contiguous nucleotides in length. Typically, region F' is up to 15 contiguous nucleotides in length. For example, region F' can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous nucleotides in length.

[0088] As described herein, any sugar-modified nucleotide may be used in regions F and / or F' of an antisense oligonucleotide, so long as the antisense oligonucleotide retains the ability to recruit RNase H and any other desired properties. Examples of sugar-modified nucleosides for use in F, F', or both F and F' are described herein and include, but are not limited to, those described in the section entitled "Sugar-Modified Nucleosides," and those described in more detail in the sections entitled "Threose Nucleic Acids (TNAs)," "2'-Sugar-Modified Nucleosides," and "Locked Nucleic Acids." The TNA(PO) gapmers described herein can include, for example, one or more TNA nucleosides, LNA nucleosides, MOE nucleosides, or mixtures thereof.

[0089] LNA gapmers An LNA gapmer is a gapmer in which one or both of regions F and F' comprise or consist of LNA nucleosides. A beta-D-oxy LNA gapmer is a gapmer in which one or both of regions F and F' comprise or consist of beta-D-oxy LNA nucleosides. An LNA gapmer can have, for example, the formula: [LNA] 1-5 -[Area G]-[LNA] 1-5 (wherein region G is as described in the section entitled "Gapmer-Region G.") An example of a specific LNA gapmer design is 3-10-3 (LNA-DNA-LNA).

[0090] cEt gapmer A cEt gapmer is a gapmer in which one or both of regions F and F' comprise or consist of cEt nucleosides. A beta-D-oxy cEt gapmer is a gapmer in which one or both of regions F and F' comprise or consist of beta-D-oxy cEt nucleosides. A cEt gapmer can have the formula, for example: [cEt] 1-5 -[Area G]-[cEt] 1-5(wherein region G is as described in the section entitled "Gapmer-Region G.") An example of a specific cEt gapmer design is 3-10-3 (cEt-DNA-cEt).

[0091] MOE Gapmar An MOE gapmer is a gapmer in which one or both of regions F and F' comprise or consist of MOE nucleosides, e.g., 2'-O-MOE nucleosides. MOE gapmers can be, for example, those of the formula [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] 3-6 where region G is as described in the section entitled "Gapmer-Region G." MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0092] TNA gapmer A "TNA gapmer" or "TNA-modified gapmer" is a gapmer in which one or more linked nucleosides of regions F, F', and G comprises at least one TNA nucleoside.

[0093] A TNA gapmer can have the formula, for example, in which F, F', or both F and F' nucleosides consist of TNA nucleosides.

[0094] TNA(PO) gapmer A "TNA(PO) gapmer" or "TNA(PO) modified gapmer" is a gapmer in which one or more linking nucleosides of regions F, F', and G contain at least one TNA nucleoside linked to at least one adjacent nucleoside by a phosphodiester (PO) internucleoside linkage.

[0095] A TNA(PO) gapmer can have the formula, for example, consisting of TNA nucleosides, where F, F', or both F and F' nucleosides are linked by PO internucleoside linkages. These and other examples of specific designs of TNA(PO) gapmers are described elsewhere herein.

[0096] Mixed Wing Gappa A mixed-wing gapmer is a gapmer in which one or both of regions F and F' contain two or more types of sugar-modified nucleosides. Many sugar-modified nucleosides are known in the art and are contemplated for this purpose. The two or more different sugar-modified nucleosides in the flanking regions can be selected, for example, from those described in the section entitled "Sugar-Modified Nucleosides," including, but not limited to, those described in the sections entitled "Threose Nucleic Acids (TNAs)," "2'-Sugar-Modified Nucleosides," and "Locked Nucleic Acids."

[0097] Contemplated mixed wing gapmers include, for example, those in which at least one of region F and region F' comprises a TNA or TNA(PO) nucleoside. The other sugar-modified nucleoside may then be selected from, for example, 2'-substituted nucleosides, such as 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, e.g., 2'-substituted nucleosides independently selected from the group consisting of MOE nucleosides.

[0098] Mixed wing gapmers are also contemplated in which, when at least one of regions F and F', or both regions F and F', comprise at least one TNA or TNA(PO) nucleoside, the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. When at least one of regions F and F', or both regions F and F', comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F' may, for example, be independently selected from the group consisting of MOE and LNA. In some mixed wing gapmers, one or both of regions F and F' may further comprise one or more DNA nucleosides.

[0099] Mixed wing gapmer designs are described in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0100] Alternating Flank Gap Mar An oligonucleotide having alternating flanks is a gapmer oligonucleotide, wherein at least one of the flanks (F or F') is composed of DNA in addition to sugar-modified nucleosides, e.g., selected from those described in the section entitled "Sugar-Modified Nucleosides," including, but not limited to, those described in the section entitled "Threose Nucleic Acids," "2'-Sugar-Modified Nucleosides," and "Locked Nucleic Acids." For example, apart from DNA, an alternating flank gapmer can include TNA, TNA(PO), LNA, and / or MOE nucleosides.

[0101] For example, at least one of regions F or F', or both regions F and F', can contain both sugar-modified nucleosides and DNA nucleosides. Flanking regions F or F', or both F and F', typically contain at least three nucleosides, with the 5'- and 3'-most nucleosides of the F and / or F' regions being sugar-modified nucleosides.

[0102] Regions D' and D'' The antisense oligonucleotides described herein may contain additional 5' and / or 3' nucleosides that are not perfectly complementary to the target nucleic acid. The additional 5' and / or 3' nucleosides are sometimes referred to herein as regions D' and D''.

[0103] The addition of region D' or D" can be used for the purpose of linking 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 function as a cleavable linker. It can also, or alternatively, be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0104] Regions D' and D'' may be attached to the 5' end of region F or the 3' end of region F', respectively, to generate 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 regions D' or D'' constitute separate portions of the oligonucleotide.

[0105] Regions D' or D" independently comprise or consist of 1, 2, 3, 4, or 5 additional nucleotides and may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides such as DNA or RNA, or base-modified versions thereof. The D' or D' region can function as a nuclease-sensitive biocleavable linker. For example, the additional 5' and / or 3' terminal nucleotides can be DNA or RNA nucleotides and can be linked by a phosphodiester bond.

[0106] Nucleotide-based biocleavable linkers suitable for use as regions D' or D" are described in WO 2014 / 076195 and include, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is described in WO 2015 / 113922 and is used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0107] Conjugates As used herein, the term "conjugate" refers to an oligonucleotide covalently attached to a non-nucleotide moiety (conjugate moiety or region C or third region).

[0108] The conjugation of the antisense oligonucleotide described herein to one or more non-nucleotide moieties can improve the pharmacology of oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake or stability of oligonucleotide.Conjugation can modify or enhance the pharmacokinetic properties of oligonucleotide, for example, by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of oligonucleotide.In particular, conjugates can target oligonucleotides to specific organs, tissues or cell types, thereby enhancing the effectiveness of oligonucleotides in those organs, tissues or cell types.At the same time, conjugates can help reduce the activity of oligonucleotides in non-target cell types, tissues or organs (for example, off-target activity or activity in non-target cell types, tissues or organs).

[0109] The non-nucleotide moiety (conjugate moiety) may be selected from the group consisting of, for example, a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0110] Linker A bond or linker is a connection between two atoms that connects a desired chemical group or segment to another desired chemical group or segment through one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker serves to covalently attach a third region, for example, the conjugate moiety (region C), to the first region, for example, the oligonucleotide or a continuous nucleotide sequence or gapmer region FG-F' (region A).

[0111] The conjugate or oligonucleotide conjugate may optionally include a linker region (second region or region B and / or region Y) located 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).

[0112] Region B refers to a biocleavable linker that contains or consists of a physiologically labile bond that is cleavable under conditions normally encountered in a mammalian body or similar conditions. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidizing or reducing conditions, or drugs, as well as salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. A biologically cleavable linker can be, for example, susceptible to S1 nuclease cleavage. DNA phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195 (incorporated herein by reference); see also region D' or D'' herein.

[0113] Region Y refers to a linker that is not necessarily biologically cleavable but primarily serves to covalently link the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). The linker of region Y may comprise a chain structure or oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. Oligonucleotide conjugates may be constructed from the following region elements: AC, ABC, ABYC, AYBC, or AYC. The linker (region Y) may be, for example, C6-C 12 C2-C containing aminoalkyl groups 36 It may be an aminoalkyl, such as an aminoalkyl group. Preferably, the linker (region Y) is a C6 aminoalkyl group.

[0114] treatment The term "treatment," as used herein, refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Accordingly, it will be recognized that the treatment referred to herein can be prophylactic.

[0115] TNA-modified antisense oligonucleotides Despite being unnatural, threose nucleic acids (TNAs) can form stable Watson-Crick duplexes and exhibit strong affinity and specificity for complementary RNA targets. As shown herein, TNA(PO)-modified gapmers offer a new design strategy for antisense oligonucleotide applications, particularly as an alternative to traditional gapmers that use only PS linkages. Using caspase 3 / 7 activation, in vitro target knockdown, and thermal melting assays, we demonstrated that TNA(PO) modifications can be used to mitigate toxicity while still maintaining target knockdown efficacy and affinity for the target nucleic acid. For example, in state-of-the-art gapmer designs, such as LNA or MOE gapmers, TNA(PO) units can replace one or more or all but three or four consecutive DNA units within the gap region of state-of-the-art gapmer designs, effectively extending the 5' or 3' flank and narrowing the gap. Furthermore, TNAs are poorly recognized by nucleases. Thus, when designed into gapmers, the TNA(PO) units may provide maintained or increased metabolic stability, maintained or extended duration of action, or both, while reducing sulfur content compared to gapmer designs that use only PS linkages. Thus, TNA(PO)-modified gapmers may provide long-acting therapeutic agents with increased therapeutic indices compared to classical gapmer designs.

[0116] Therefore, the present invention provides antisense oligonucleotides, such as antisense gapmer oligonucleotides, comprising TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.TNA nucleosides can be linked to adjacent nucleosides by 2'-PO internucleoside linkages or 3'-PO internucleoside linkages.TNA nucleosides can also be linked to any adjacent nucleoside by 2'-PO or 3'-PO internucleoside linkages.Non-limiting examples of adjacent nucleosides include sugar-modified nucleosides and DNA nucleosides.For example, TNA nucleosides can be linked to the first and second adjacent nucleosides by 2'-PO internucleoside linkages and 3'-modified internucleoside linkages, respectively; can be attached to 2'-modified internucleoside linkages and 3'-PO internucleoside linkages, respectively; or can be linked to 2'-PO and 3'-PO internucleoside linkages, respectively; Optionally, any 2'- or 3'-modified internucleoside linkage is a PS internucleoside linkage, or any 2'- or 3'-modified internucleoside linkage is a PS2 internucleoside linkage.

[0117] The present invention also provides one or more TNA (PO) nucleotides, typically selected from the group consisting of 2'-PO-linked, 3'-PO-linked, and 2',3'-PO-linked TNA nucleosides, e.g., antisense gapmer oligonucleotides. Antisense gapmer oligonucleotides may, in particular, comprise a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I) that is capable of recruiting ribonuclease (RNase) H. A contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I) that comprises at least one TNA(PO) nucleoside may be referred to herein as a "TNA(PO) gapmer." Contemplated designs for TNA(PO) gapmers include: G is a gap region of up to 18 linked nucleosides containing at least three consecutive DNA nucleosides; each of F and F' is a flanking region of up to 15 linked nucleosides, independently comprising or consisting of 1 to 15 sugar-modified nucleosides; At least one of F, F' and G comprises a sugar-modified nucleoside that is a TNA(PO) nucleoside.

[0118] The following sections provide further details regarding TNA(PO) gapmers, but unless otherwise indicated or contradicted by context, they should be understood to apply equally to antisense gapmer oligonucleotides or conjugates thereof that comprise or consist of TNA(PO) gapmers.

[0119] Advantageously, TNA(PO) gapmers can regulate the expression of target genes by reducing or inhibiting their expression into mRNA and / or protein, typically by hybridizing to the target nucleic acid. When the target nucleic acid is RNA, such as a pre-mRNA, mRNA, viral RNA, microRNA, or lncRNA target nucleic acid, the TNA(PO) gapmer can reduce or inhibit the expression of the target RNA. This is achieved by complementarity between the TNA(PO) gapmer and the target RNA and, appropriately, by recruiting cellular RNases such as RNase H. TNA(PO) gapmers can also reduce or inhibit the expression of target RNA through non-RNase H-mediated mechanisms, such as steric blocking mechanisms resulting in microRNA inhibition, reduced splice regulation of pre-mRNA, or blocking the interaction between lncRNA and chromatin.

[0120] Preferably, the TNA(PO) gapmer is capable of reducing the expression level of the target by at least about 20% compared to the normal expression level of the target, more preferably by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. The TNA(PO) gapmer is preferably, or alternatively, capable of inhibiting expression of the target by at least about 20% compared to the normal expression level of the target, more preferably by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the normal expression level of the target.

[0121] Assays for assessing the reduction of expression level or inhibition of the expression of a specific target are known to those skilled in the art. Suitable assays include in vitro assays using target cells that contain at least one copy of the target gene in their genome and express a target, for example, a target RNA. For example, in an in vitro assay in which target cells are incubated with about 25 μM of TNA(PO) gapmer, the TNA(PO) gapmer can reduce the expression level of the RNA target by at least about 50%, for example, at least about 60%, compared to the normal expression level of the RNA target. In such an assay, the TNA(PO) gapmer can alternatively inhibit the expression of the RNA target by at least about 50%, for example, at least about 60%, compared to the normal expression level of the RNA target. At a concentration of about 25 μM, the TNA(PO) gapmer can also reduce or inhibit the expression level of the RNA target by at least about 70%, for example, at least about 80%, for example, at least about 90%, compared to the normal expression level of the target.

[0122] The normal expression level of an RNA target can be determined using a control in which target cells are incubated without the TNA(PO) gapmer (e.g., in the presence of vehicle alone) or with an unrelated control oligonucleotide. Target cells for in vitro assays can be obtained from commercial sources (e.g., in the form of cell lines) or isolated from human or experimental animal blood or other tissues. The target cells can be incubated, for example, with the TNA(PO) gapmer or control for about 1 to 5 days, e.g., about 2, 3, or 4 days, e.g., about 3 days. RNA can then be extracted, and the level of remaining target RNA in the test and control samples can be measured by gene expression analysis. Alternatively, instead of determining the remaining target RNA, the level of RNA species (e.g., mRNA) or protein derived from the target RNA can be measured in the test and control samples. An example of a typical assay for assessing the reduction of expression levels or inhibition of target RNA expression by TNA(PO) gapmers, which can be adapted for other targets and target cells, is provided in Example 2.

[0123] The ability of a TNA(PO) gapmer to reduce the expression level of a target or inhibit the expression of a target can also be evaluated by measuring the IC50 value, i.e., the concentration of the TNA(PO) gapmer at which the expression level of the target nucleic acid is reduced by half. In an in vitro assay using a target cell containing at least one copy of the target gene in its genome and expressing a target, e.g., a target RNA, the IC50 is preferably about 20 μM or less, for example, about 10 μM or less, for example, about 5 μM or less. Typically, the IC50 value is measured in a cellular assay similar to that described above, except that the target cells are incubated with a dilution series of the TNA(PO) gapmer spanning the IC50 value. An example of a typical assay for evaluating the IC50 of the reduction or inhibition of the expression level of a target RNA by a TNA(PO) gapmer, which can be adapted to other targets and target cells, is provided in Example 3.

[0124] The potency of a TNA(PO) gapmer may also be assessed relative to a control or "parent" gapmer from which the TNA(PO) gapmer is derived and which does not contain any TNA nucleosides. The IC50 value of the TNA(PO) gapmer is preferably no more than about 10-fold, no more than about 8-fold, no more than about 6-fold, no more than about 4-fold, or no more than about 2-fold that of the control or "parent" gapmer.

[0125] TNA(PO) gapmers can also be characterized by low toxicity. For example, TNA(PO) gapmers can have lower toxicity than corresponding control gapmers, such as state-of-the-art reference gapmers or "parent" gapmers that differ from TNA(PO) gapmers in that their nucleosides do not contain any TNA nucleosides. Suitable assays for assessing the toxicity of gapmers or antisense nucleotides are known in the art, including in vitro assays such as the caspase 3 / 7 assay. The caspase 3 / 7 assay reflects the level of apoptosis induced by a compound and is suitable for assessing the risk of hepatotoxicity of a compound, for example, based on testing on hepatocytes or cell lines. Briefly, HepG2 cells from a commercial source can be transfected with 100 nM TNA(PO) or a control gapmer in an appropriate vehicle, and caspase 3 / 7 activation can be measured approximately 24 hours after transfection. Preferably, caspase 3 / 7 activation from transfection with a TNA(PO) gapmer is about 70% or less, such as about 60% or less, such as about 50% or less, such as about 40% or less, such as about 30% or less, for example about 20% or less, of the corresponding control gapmer. Alternatively, the percentage (% assay window reflecting apoptotic cells relative to total cells) determined for a TNA(PO) gapmer using the caspase 3 / 7 assay described in Example 4 is preferably about 250% or less, more preferably about 200% or less, more preferably about 150% or less, about 100% or less, about 80% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 10% or less. Preferably, the percentage (% assay window) determined for a TNA(PO) gapmer using the caspase 3 / 7 assay described in Example 4 is at most about 80%, for example at most about 60%.

[0126] TNA(PO) gapmers can hybridize to target nucleic acids, such as target RNA, particularly to target sequences whose nucleobase sequences are complementary. The ability of a TNA(PO) gapmer to hybridize to its target nucleic acid can be evaluated according to any assay known in the art. Advantageously, thermal melting (Tm) analysis can be used to determine the temperature at which the duplex between the TNA(PO) gapmer and its RNA target sequence is expressed, which can be referred to as the melting temperature or simply Tm. A typical assay for measuring the Tm of a TNA(PO) gapmer (i.e., in the form of a duplex with a complementary RNA target sequence) is described in Example 5. Briefly, the TNA(PO) gapmer and the RNA target sequence are added to 20 mM disodium phosphate buffer, 200 mM NaCl, and 0.2 mM EDTA (pH 7) to obtain a final concentration of 1.5 μM. The sample may be heated to 95°C for 5 minutes, then slowly cooled to room temperature over 1 hour, and a thermal melting curve may be recorded at 260 nm using a temperature gradient, for example, increasing from 25°C to 95°C at 5°C / min and then decreasing to 25°C. The melting temperature (Tm) may be measured from the derivative of both curves. Preferably, the TNA(PO) gapmer has a Tm of at least about 50°C, such as at least about 52°C, for example at least about 54°C, such as at least about 56°C, for example at least about 58°C, such as at least about 60°C, for example at least about 65°C, for example at least about 70°C.

[0127] In some cases, there may be one or two mismatches between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to exhibit the desired ability to modulate the target.

[0128] Preferably, the TNA(PO) gapmer is (a) the expression level of the target nucleic acid can be reduced by at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, compared to the normal expression level of the target; (b) has an IC50 of about 20 μM or less, e.g., about 10 μM or less, e.g., about 5 μM or less, for reducing the expression level of the target nucleic acid; (c) when using the caspase 3 / 7 assay described in Example 4, the percent assay window (%AW) is preferably about 60% or less, such as about 40% or less, such as about 20% or less, for example about 10% or less; (d) has a melting temperature (Tm) in the duplex form between the antisense gapmer oligonucleotide and the RNA target sequence of at least about 50°C, such as at least about 52°C, for example at least about 54°C, such as at least about 56°C, for example at least about 58°C, for example at least about 60°C; or (e) A combination of two or more of (a) to (d).

[0129] For example, a preferred TNA(PO) gapmer may be characterized by both features (a) and (b). Another preferred TNA(PO) gapmer may be characterized by both features (a) and (c). Another preferred TNA(PO) gapmer may be characterized by both features (a) and (d). Another preferred TNA(PO) gapmer may be characterized by both features (b) and (c). Another preferred TNA(PO) gapmer may be characterized by both features (b) and (d). Another preferred TNA(PO) gapmer may be characterized by both features (c) and (d). Another preferred TNA(PO) gapmer may be characterized by features (a), (b), and (c). Another preferred TNA(PO) gapmer may be characterized by features (a), (b), and (d). Another preferred TNA(PO) gapmer may be characterized by features (a), (c), and (c). Another preferred TNA(PO) gapmer may be characterized by features (b), (c) and (d). Another preferred TNA(PO) gapmer may be characterized by all of features (a)-(d).

[0130] Preferably, the target nucleic acid of (a) and (b) is RNA, and the RNA target sequence of (c) has a nucleobase sequence complementary to the contiguous nucleotide sequence of the TNA(PO) gapmer. The reduction in the expression level of (a) and (b) can be measured, for example, in target cells expressing the target nucleic acid and incubated with an antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days, as already described above.

[0131] In some TNA(PO) gapmers, region F comprises at least one TNA nucleoside. Region F of a TNA gapmer can, for example, comprise up to 15 TNA nucleosides.

[0132] In some TNA(PO) gapmers, region F comprises at least one TNA(PO) nucleoside. Region F can also comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 TNA(PO) nucleosides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA(PO) nucleosides. TNA(PO) gapmers in which region F comprises or consists of one TNA(PO) nucleoside are also contemplated.

[0133] In some TNA(PO) gapmers, region F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous TNA(PO) nucleosides, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA(PO) nucleosides. Region F may also consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA(PO) nucleosides.

[0134] In some TNA(PO) gapmers, at least the 3'-most nucleosides in F are TNA nucleosides. Suitably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the 3'-most nucleosides in F can be TNA nucleosides. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 3'-most nucleosides in F can be TNA nucleosides, e.g., TNA(PO) nucleosides.

[0135] In some TNA(PO) gapmers, at least the 5'-most nucleosides in F are TNA nucleosides. Suitably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the 5'-most nucleosides in F can be TNA nucleosides. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 5'-most nucleosides in F can be TNA nucleosides, e.g., TNA(PO) nucleosides.

[0136] In some TNA(PO) gapmers, both the 3'-most nucleoside and the 5'-most nucleoside in F are TNA nucleosides. For example, the 3'-most and / or 5'-most nucleosides in F can be independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides, such as TNA(PO) nucleosides.

[0137] Suitably, in a TNA(PO) gapmer, each TNA nucleoside in F can be a TNA(PO) nucleoside. However, TNA(PO) gapmers in which each TNA nucleoside in F is a TNA(PO) nucleoside except for the 5'-most TNA nucleoside in F are also contemplated. The 5'-most TNA nucleoside can be linked to the adjacent nucleoside via, for example, a PS or PS2 internucleoside linkage.

[0138] Any remaining nucleosides in F can be one or more other sugar-modified nucleosides other than TNA(PO) nucleosides (e.g., in the form of a mixed-wing gapmer) or one or more DNA nucleosides (e.g., in the form of alternating flank gaps). For example, F can further comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 2, 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled "Sugar-Modified Nucleosides," e.g., LNA, 2'-O-MOE, and TNA nucleosides, optionally linked by PS or PS2 internucleoside linkages.

[0139] TNA(PO) gapmers are also contemplated in which all sugar-modified nucleosides of F are TNA(PO) nucleosides. A TNA(PO) gapmer can be, for example, an alternating flank gapmer in which the nucleosides of F consist of DNA and TNA(PO), e.g., having one, two, or three DNA nucleosides. Suitably, at least the 5'-most and 3'-most nucleosides of F are TNA(PO) nucleosides.

[0140] The nucleosides of F can also consist of TNA(PO) nucleosides. Alternatively, region F can consist of multiple TNA(PO) nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA(PO) nucleosides. TNA(PO) nucleosides in which the nucleosides of F consist of 13, 14, or 15 TNA(PO) nucleosides are also contemplated. Typically, when the nucleosides of F consist of two or more TNA(PO) nucleosides, F is a contiguous sequence of linked TNA(PO) nucleosides.

[0141] In some TNA gapmers, F does not contain any TNA(PO) nucleosides.

[0142] In some TNA(PO) gapmers, region F' comprises at least one TNA nucleoside. Region F' of a TNA gapmer can, for example, comprise up to 15 TNA nucleosides.

[0143] In some TNA(PO) gapmers, region F' comprises at least one TNA(PO) nucleoside. Region F' may also comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 TNA(PO) nucleosides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA(PO) nucleosides. TNA(PO) gapmers in which region F' comprises or consists of one TNA(PO) nucleoside are also contemplated.

[0144] In some TNA(PO) gapmers, region F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous TNA(PO) nucleosides, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA(PO) nucleosides. Region F' may also consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA(PO) nucleosides.

[0145] In some TNA(PO) gapmers, at least the 5'-most nucleosides in F' are TNA nucleosides. Suitably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the 5'-most nucleosides in F' can be TNA nucleosides. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 5'-most nucleosides in F' can be TNA nucleosides, e.g., TNA(PO) nucleosides.

[0146] In some TNA(PO) gapmers, at least the 3'-most nucleosides in F' are TNA nucleosides. Suitably, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the 3'-most nucleosides in F' can be TNA nucleosides. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 3'-most nucleosides in F' can be TNA nucleosides, e.g., TNA(PO) nucleosides.

[0147] In some TNA(PO) gapmers, both the 3'-most nucleoside and the 5'-most nucleoside in F' are TNA nucleosides. For example, the 3'-most and / or 5'-most nucleosides in F' can be independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides, such as TNA(PO) nucleosides.

[0148] Suitably, in a TNA(PO) gapmer, each TNA nucleoside in F' can be a TNA(PO) nucleoside. However, TNA(PO) gapmers in which each TNA nucleoside in F, except for the 3'-most TNA nucleoside in F', is a TNA(PO) nucleoside are also contemplated. The 3'-most TNA nucleoside can be linked to the adjacent nucleoside via, for example, a PS or PS2 internucleoside linkage.

[0149] Any remaining nucleosides in F' can be one or more other sugar-modified nucleosides other than TNA(PO) nucleosides (e.g., in the form of a mixed-wing gapmer) or one or more DNA nucleosides (e.g., in the form of alternating flank gaps). For example, F' can further comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 2, 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled "Sugar-Modified Nucleosides," e.g., LNA, 2'-O-MOE, and TNA nucleosides, optionally linked by PS or PS2 internucleoside linkages.

[0150] TNA(PO) gapmers are also contemplated in which all sugar-modified nucleosides of F' are TNA(PO) nucleosides. A TNA(PO) gapmer can be, for example, an alternating flank gapmer in which the nucleosides of F' consist of DNA and TNA(PO), e.g., having one, two, or three DNA nucleosides. Suitably, at least the 5'-most and 3'-most nucleosides of F' are TNA(PO) nucleosides.

[0151] The nucleosides of F' can also consist of TNA(PO) nucleosides. Alternatively, region F' can consist of multiple TNA(PO) nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA(PO) nucleosides. TNA(PO) nucleosides in which the nucleosides of F' consist of 13, 14, or 15 TNA(PO) nucleosides are also contemplated. Typically, when the nucleosides of F' consist of two or more TNA(PO) nucleosides, F' is a contiguous sequence of linked TNA(PO) nucleosides.

[0152] In some TNA gapmers, F' does not contain any TNA nucleosides.

[0153] G comprises consecutive DNA nucleosides that enable the antisense oligonucleotide to recruit RNase H. Suitably, G can comprise up to 18 nucleosides, such as DNA nucleosides. Typically, at least the 5'-most nucleoside in G and the 3'-most nucleoside in G are DNA nucleosides.

[0154] In some TNA(PO) gapmers, the G does not include any TNA nucleosides. For example, the G can include at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive DNA nucleosides.

[0155] In some TNA(PO) gapmers according to the invention, G comprises at least one TNA nucleoside. For example, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth 5'-most nucleoside in G can be a TNA nucleoside. Alternatively, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth 3'-most nucleoside in G can be a TNA nucleoside.

[0156] TNA(PO) gapmers are also contemplated in which G comprises at least two or at least three TNA nucleosides. The at least two or at least three TNA nucleosides may be contiguous or non-contiguous. Region G may comprise, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides, optionally contiguous.

[0157] In some TNA(PO) gapmers according to the invention, G comprises at least one TNA(PO) nucleoside. For example, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth 5'-most nucleoside in G can be a TNA(PO) nucleoside. Alternatively, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth 3'-most nucleoside in G can be a TNA nucleoside.

[0158] TNA(PO) gapmers are also contemplated in which G comprises at least two or at least three TNA(PO) nucleosides. The at least two or at least three TNA(PO) nucleosides may be contiguous or non-contiguous. Region G may comprise, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA(PO) nucleosides, optionally contiguous.

[0159] Additionally, as described herein, other modified nucleosides have been reported to be capable of recruiting RNase H when contained within the gap region, and may be present or included in the gap region of a TNA(PO) gapmer. Preferably, in a TNA(PO) gapmer containing at least one, e.g., 1, 2, or 3, TNA(PO) nucleoside or other modified nucleoside within gap region G, gap region G still contains at least 3 consecutive DNA nucleosides, e.g., at least 4 consecutive DNA nucleosides, e.g., at least 5 consecutive DNA nucleosides, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive DNA nucleosides. In some TNA(PO) gapmers, except for the TNA(PO) nucleosides within gap region G, all nucleosides of G are DNA nucleosides.

[0160] In some TNA(PO) gapmers, any TNA(PO) nucleoside, particularly any contiguous stretch of two or more TNA or TNA(PO) nucleosides, is located closer to the 5' end of region G than to the 3' end of region G. Any contiguous stretch of two or more TNA or TNA(PO) nucleosides within region G may, for example, be located adjacent to the 5'-most nucleoside within region G, typically a DNA nucleoside.

[0161] As demonstrated herein, TNA(PO) gapmers can contain stretches of only three or four consecutive DNA nucleosides while still allowing for the recruitment of RNase H. Without being limited by theory, it is contemplated that TNA gapmers having a shorter gap region than that of conventional gapmer designs and / or having one or more TNA residues in the gap region may provide increased resistance to endonuclease-mediated degradation and / or reduced off-target binding compared to conventional gapmer designs. Consequently, in some TNA gapmers according to the present invention, G contains up to 10 DNA nucleosides, e.g., 9, 8, 7, 6, 5, or 4 DNA nucleosides. Optionally, G contains up to 10 consecutive DNA nucleosides, e.g., 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides. Furthermore, as described in more detail below, when G in a TNA(PO) gapmer comprises 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides, F' and F can optionally be of different lengths, e.g., F comprises more linked nucleosides than F'.

[0162] An antisense gapmer oligonucleotide can be designed, for example, such that, except for adjacent DNA nucleosides, all other nucleosides are DNA nucleosides, at least one of which is linked to at least one adjacent nucleoside by a 2'-PO or 3'-PO internucleoside linkage.

[0163] Also contemplated are antisense gapmer oligonucleotides wherein up to about 80%, such as up to about 70%, for example up to about 60%, for example up to about 50%, for example up to about 40%, for example up to about 30%, for example up to about 20%, for example up to about 15%, for example up to about 10%, for example up to about 5% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, and at least one TNA nucleoside is a 2'-PO linked, 3'-PO linked, or 2',3'-PO linked TNA nucleoside.

[0164] Antisense gapmer oligonucleotides in which up to about 50% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, and in which at least one TNA nucleoside is a 2'-PO linked, 3'-PO linked, or 2',3'-PO linked TNA nucleoside, are also contemplated. Details regarding the number and arrangement of at least one TNA(PO) nucleoside in regions F, G and / or F' are provided above and may be incorporated into the general gapmer formula 5'-FG-F'-3'(I). Specifically contemplated TNA(PO) gapmers include: (i) Region F contains at least one TNA(PO) nucleoside, but regions F' and G contain no TNA(PO) nucleosides; (ii) region F' contains at least one TNA(PO) nucleoside, but regions F and G contain no TNA(PO) nucleosides; (iii) region G contains at least one TNA(PO) nucleoside, but regions F and F′ do not; (iv) regions F and F' each contain at least one TNA(PO) nucleoside, but region G does not contain any TNA(PO) nucleosides; (v) regions F and G each contain at least one TNA(PO) nucleoside, but region F′ does not contain any TNA(PO) nucleosides; (vi) regions G and F' each contain at least one TNA(PO) nucleoside, but region F does not contain any TNA(PO) nucleosides; and (vii) Regions F, G, and F' each contain at least one TNA(PO) nucleoside. Examples include:

[0165] As an example, in a TNA(PO) gapmer according to item (iv) or (vii), F and F' can each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA nucleosides, at least one of which is a TNA(PO) nucleoside. Also contemplated are TNA(PO) gapmers according to item (iv) or (vii), in which F and F' each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive TNA nucleosides, at least one of which is a TNA(PO) nucleoside.

[0166] Alternatively, in a TNA(PO) gapmer according to any item other than (iii), F and F' together comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 TNA nucleosides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 TNA nucleotides, at least one of which is a TNA(PO) nucleoside.

[0167] For example, F and F' together can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TNA nucleosides, at least one of which is linked to an adjacent TNA nucleoside by a PO internucleoside linkage.

[0168] As another example, in a TNA(PO) gapmer described in paragraph (iv) or (vii), F and F' can each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA(PO) nucleosides. Also contemplated are TNA(PO) gapmers described in paragraph (iv) or (vii) in which F and F' each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive TNA(PO) nucleosides.

[0169] Alternatively, in a TNA(PO) gapmer according to any item other than (iii), F and F' together may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 TNA(PO) nucleosides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 TNA(PO) nucleosides.

[0170] For example, F and F' together can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 TNA nucleosides linked to adjacent TNA nucleosides by PO internucleoside linkages.

[0171] For example, in a TNA(PO) gapmer according to item (iv), F and F' together can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12 TNA(PO) nucleosides.

[0172] In some TNA(PO) gapmers, each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a PO internucleoside linkage. However, TNA(PO) gapmers in which each TNA nucleoside in F and F', except for the 5'-most TNA nucleoside in F and the 3'-most TNA nucleoside in F', is a TNA(PO) nucleoside are also contemplated.

[0173] In some TNA(PO) gapmers, each of regions F and F' can independently comprise or consist of a contiguous sequence of linked sugar-modified nucleosides.

[0174] In some TNA(PO) gapmers, at least one of regions F and F' can consist of only one type of sugar-modified nucleoside. For example, the sugar-modified nucleoside can be a high-affinity nucleoside or a TNA(PO) nucleoside.

[0175] In some TNA(PO) gapmers, both regions F and F' can consist of only one type of sugar-modified nucleoside (uniform flank or uniform gapmer design). For example, the sugar-modified nucleoside can be a high-affinity nucleoside or a TNA(PO) nucleoside. In some TNA gapmers, the nucleosides in regions F and F' are all TNA(PO) nucleosides. In some TNA(PO) gapmers, the nucleosides in regions F and F' are all sugar-modified nucleosides other than TNA nucleosides.

[0176] In some TNA(PO) gapmers, one or both of regions F and F' can independently comprise two different sugar-modified nucleosides (mixed wing designs). One of the two different sugar-modified nucleosides can be a TNA(PO) nucleoside, and the other sugar-modified nucleoside can be, for example, a high-affinity nucleoside or a TNA nucleoside linked by a modified internucleoside linkage.

[0177] In some TNA(PO) gapmers, all nucleosides in region F can be TNA(PO) nucleosides. The nucleosides in region F' can then comprise or consist of, for example, sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 2'-sugar-modified nucleosides such as high-affinity nucleosides. Optionally, region F' can comprise two different sugar-modified nucleosides, one of which can be TNA(PO). For example, F' can comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides. Alternatively, F' can consist of 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides.

[0178] In some TNA(PO) gapmers, all nucleosides in region F' can be TNA(PO) nucleosides. The nucleosides in region F can then comprise or consist of, for example, sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 2'-sugar-modified nucleosides such as high-affinity nucleosides. Optionally, region F can comprise two different sugar-modified nucleosides, one of which can be TNA(PO). For example, F can comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides. Alternatively, F can consist of 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, e.g., 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides.

[0179] In TNA(PO) gapmers where F, F', or both F and F' comprise or consist of one or more sugar-modified nucleosides other than TNA nucleosides, non-limiting examples of sugar-modified nucleosides include: 2'-methoxy-ribose (2'-OMe), 2'-O-Methoxyethyl-ribose (2'-O-MOE), 5'-methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE), 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE), 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE), 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA), 2'-deoxy-2'-fluoro-ribose (found in 2'-deoxy-2'-fluororibonucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (found in 2'-fluoro-2'-arabinose nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxyβ-D-locked dribose (found in β-D-LNA), amino β-D-locked dribose (found in amino-β-D-LNA), Thioβ-D-locked dribose (found in thio-β-D-LNA), Oxyβ-L-locked dribose (found in β-L-LNA), amino β-L-locked dribose (found in amino β-L-LNA), Thioβ-L-locked dribose (found in thio-β-L-LNA), Oxyα-L-locked dribose (found in α-L-LNA), amino α-L-locked dribose (found in amino α-L-LNA), Thio α-L-locked dribose (found in thio-α-L-LNA), 2',4'-constrained 2'-O-ethyl ribose (as found in constrained ethyl-locked nucleic acid; cEt), tricyclodeoxyribose (tricyclodeoxyribose DNA; found in TcDNA), 3'-deoxy-ribose (found in 3'-deoxy-ribose DNA; 3'-DNA), unlocked dribose (found in unlocked nucleic acid; UNA), glycol (found in glycol nucleic acid; GNA), hexitols (found in hexitol nucleic acids; HNA), 3'-fluorohexitol (found in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabinofluorohexitol (found in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA), cyclohexene (found in cyclohexene nucleic acid; CeNA), Fluoro-cyclohexenyl (found in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA), Serinol (found in serinol nucleic acid; SNA), 2'-O,4'-C-ethylene-bridged ribose (2'-O,4'-C-ethylene-linked nucleic acid; found in ENA) Acyclic (L)-threoninol (found in acyclic (L)-threoninol nucleic acid; aTNA) 2',4'-constrained 2'-O-methoxyethyl ribose (as found in cMOE), and 7',5'-alpha-bicyclosugar unit (7',5'-alpha-bicycloDNA; found in bcDNA) and those having a modified sugar moiety selected from the group consisting of:

[0180] Specifically contemplated are TNA(PO) gapmers in which the F, F', or both F and F' sugar-modified nucleosides comprise or consist of one or more 2'-sugar-modified nucleosides, e.g., high affinity 2'-sugar-modified nucleosides.

[0181] In some TNA(PO) gapmers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more LNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be LNA nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 LNA nucleosides, e.g., 3, 4, or 5 LNA nucleosides, respectively. Suitable LNA nucleosides include those having a modified sugar moiety selected from oxy, amino, or thio β-D-locked dribose (β-D-LNA) or oxy, amino, or thio β-L-locked dribose (β-L-LNA) or oxy, amino, or thio α-L-locked dribose (α-L-LNA), e.g., β-D-oxy-LNA, e.g., 6′-methyl-β-D-oxy LNA, such as (S)-6′-methyl-β-D-oxy-LNA (ScET), and ENA, as well as the LNA nucleosides described in Scheme 2. A particularly contemplated LNA nucleoside is β-D-oxy-LNA.

[0182] In some TNA(PO) gapmers, sugar-modified nucleosides in F, F', or both F and F' comprise one or more MOE nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be MOE nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 MOE nucleosides, e.g., 3, 4, or 5 MOE nucleosides, respectively. Suitable MOE nucleosides include 2'-O-MOE, 5'-Me-2'-O-MOE, and 2',4'-constrained 2'-O-methoxyethyl (cMOE). A particularly contemplated MOE nucleoside is 2'-O-MOE.

[0183] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-OMe nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-OMe nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, or 3, 4, or 5 2'-OMe nucleosides, respectively.

[0184] In some TNA(PO) gapmers, sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-O-MTE nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-O-MTE nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, or 3, 4, or 5 2'-O-MTE nucleosides, respectively.

[0185] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-O-MCE nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-O-MCE nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can each comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3, 4, or 5 2'-O-MCE nucleosides.

[0186] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-O-NMA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-O-NMA nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, e.g., 3, 4, or 5 2'-O-NMA nucleosides, respectively.

[0187] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-deoxy-2'-fluoro-ribose nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-deoxy-2'-fluoro-ribose nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, or 3, 4, or 5 2'-deoxy-2'-fluoro-ribose nucleosides, respectively.

[0188] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-fluoro-2'-arabinose nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-fluoro-2'-arabinose nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 3, 4, or 5 2'-fluoro-2'-arabinose nucleosides, respectively.

[0189] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 2'-O-benzyl-ribose nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 2'-O-benzyl-ribose nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, or 3, 4, or 5 2'-O-benzyl-ribose nucleosides, respectively.

[0190] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more cEt nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be cEt nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 cEt nucleosides, e.g., 3, 4, or 5 cEt nucleosides, respectively.

[0191] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more TcDNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be TcDNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can each comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 TcDNA nucleosides, e.g., 3, 4, or 5 TcDNA nucleosides.

[0192] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more 3'-DNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be 3'-DNA nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, or 3'-DNA nucleosides, respectively, e.g., 3, 4, or 5 3'-DNA nucleosides.

[0193] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more UNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be UNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 UNA nucleosides, e.g., 3, 4, or 5 UNA nucleosides, respectively.

[0194] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more GNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be GNA nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 GNA nucleosides, e.g., 3, 4, or 5 GNA nucleosides, respectively.

[0195] In some TNA(PO) gapmers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more HNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be HNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can each comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 HNA nucleosides, for example, 3, 4, or 5 HNA nucleosides. Suitable HNA nucleosides include HNA, FHNA, and Ara-FHNA.

[0196] In some TNA(PO) gapmers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more CeNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be CeNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can each comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 CeNA nucleosides, e.g., 3, 4, or 5 CeNA nucleosides. Suitable CeNA nucleosides include CeNA and F-CeNA.

[0197] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more SNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be SNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides, e.g., 3, 4, or 5 SNA nucleosides, respectively.

[0198] In some TNA(PO) gapmers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more ENA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be ENA nucleosides. In TNA(PO) gapmers in which only region G comprises one or more TNA(PO) nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 ENA nucleosides, e.g., 3, 4, or 5 ENA nucleosides, respectively.

[0199] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more SNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be SNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides, e.g., 3, 4, or 5 SNA nucleosides, respectively.

[0200] In some TNA(PO) gapmers, the sugar-modified nucleosides in F, F', or both F and F' comprise one or more aTNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be aTNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 aTNA nucleosides, e.g., 3, 4, or 5 aTNA nucleosides, respectively.

[0201] In some TNA(PO) gapmers, sugar-modified nucleosides in F, F', or both F and F' comprise one or more bcDNA nucleosides. For example, 1, 2, 3, 4, 5, 6, 7, 8, or all nucleosides in F', F, or both F and F', except for any at least one TNA(PO) nucleoside, can be bcDNA nucleosides. In TNA(PO) gapmers in which only region G contains one or more TNA(PO) nucleosides, regions F and F' can, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 bcDNA nucleosides, e.g., 3, 4, or 5 bcDNA nucleosides, respectively.

[0202] For any TNA(PO) gapmer described herein, particularly a TNA(PO) gapmer in which the sugar-modified nucleosides of F, F', or both F and F' comprise or consist of one or more 2' sugar-modified nucleosides, such as high-affinity 2' sugar-modified nucleosides, it is contemplated that at least one internucleoside linkage can be a PS2 internucleoside linkage. For example, in some TNA(PO) gapmers, (i) the 5'-most nucleoside of F is linked to the adjacent nucleoside in F by a PS2 internucleoside linkage, (ii) the 3'-most nucleoside of F' is linked to the adjacent nucleoside in F' by a PS2 internucleoside linkage, or (iii) both (i) and (ii) apply. Optionally, the nucleosides other than the at least one TNA(PO) nucleoside in a TNA(PO) gapmer may be linked to each other by PS internucleoside linkages.

[0203] TNA(PO) gapmers in which the contiguous nucleotide sequence of formula 5'-FG-F'-3'(I) has a length of 12 to 32 nucleosides, for example, 12 to 28 nucleosides, for example, 12 to 26 nucleosides, for example, 14 to 26 nucleosides, for example, 14 to 24 nucleosides, for example, 14 to 22 nucleosides, for example, 16 to 22 nucleosides, for example, 16 to 20 nucleosides are particularly contemplated. However, any suitable length can be used in the FG-F' design, including, but not limited to, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 linked nucleosides.

[0204] By way of example, a TNA(PO) gapmer according to the invention may be represented by one or more of the following formulas for region FG-F' (Formula I), provided that the total length of region FG-F' is at least 12, for example at least 14 nucleotides in length: F 1-15 -G 3-18 -F' 1-15 (IV), e.g., 1-15 -G 3-18-F' 1-12 (IVa), or 1-12 -G 3-18 -F' 1-15 (IVb); F 1-12 -G 3-18 -F' 1-12 (V), e.g., F 1-12 -G 3-18 -F' 1-9 (Va), or F 1-9 -G 3-18 -F' 1-12 (Vb); F 1-12 -G 4-16 -F' 1-12 (VI), e.g., F 1-12 -G 4-16 -F' 1-9 (VIa), or F 1-9 -G 4-16 -F' 1-12 (VIb); and F 3-12 -G 4-10 -F' 3-12 (VII), e.g., F 3-12 -G 4-10 -F' 3-9 (VIIa), or F 3-9 -G 4-10 -F' 3-12 (VIIb).

[0205] Each of the regions F, G and F' described herein can be incorporated into any of the FG-F' formulas.

[0206] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IVa has a length of at least 16 nucleosides, (a) the 5'-most nucleoside in F and the nucleoside in F' are independently 3, 4, or 5 high affinity sugar-modified nucleosides; (b) the remaining nucleosides in F are TNA(PO) nucleosides; (c) All nucleosides in G are DNA nucleosides.

[0207] For example, G can contain up to 10 consecutive DNA nucleosides, e.g., 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides, e.g., 4, 5, or 6 consecutive DNA nucleosides. F can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive TNA(PO) nucleosides.

[0208] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, (a) F and F′ each independently consist of 3, 4, or 5 nucleosides, wherein at least one of the nucleosides in F and / or F′ is a TNA(PO) nucleoside, and any remaining nucleosides are high-affinity sugar-modified nucleosides; (b) All nucleosides in G are DNA nucleosides.

[0209] For example, all nucleosides in either F or F' can be TNA(PO) nucleosides. Alternatively, all nucleosides in F and F' can be TNA(PO) nucleosides. Alternatively, each TNA nucleoside in F and F', except for the 5'-most TNA nucleoside in F and the 3'-most TNA nucleoside in F', can be linked to adjacent nucleosides, particularly any adjacent TNA nucleosides, by a PO internucleoside linkage.

[0210] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, (a) F and F' each independently comprise or consist of 3, 4, or 5 linked high-affinity sugar-modified nucleosides, and do not comprise any TNA nucleosides; (b) The second, third, fourth, or fifth 5'-most nucleoside in G is a TNA(PO) nucleoside, and the remaining nucleosides in G are DNA nucleosides.

[0211] The TNA(PO) nucleoside can be located, for example, closer to the 5' end of the region G than to the 3' end of the G.

[0212] Particularly contemplated designs of TNA(PO) gapmers include those also referred to as Design A and Design B in Example 1.

[0213] TTTTTddddddddddTTTTTT (where T is TNA and d is DNA) with a backbone (internucleoside linkage) pattern SOOOSSSSSSSSSSSOOOS (where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO)) (Design A).

[0214] MMMMMTTTTTTddddMMMMM (where M is MOE (e.g., 2'-O-MOE), T is TNA, and d is DNA) with a backbone (internucleoside linkage) pattern SSSSOOOOOOSSSSSSSSS (where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO)) (Design B).

[0215] The following designs are also specifically contemplated:

[0216] TTTTTTTTTTdddddMMMMM (where M is 2'-O-MOE, T is TNA, and d is DNA) with a backbone (internucleoside linkage) pattern of OOOOOOOOOSSSSSSSSSS (where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO)) (Design C).

[0217] MMMMMddddddddddMMMMM (where M is 2'-O-MOE, T is TNA, and d is DNA) with 1, 2, 3, 4, 5, 6, or 7 d's substituted with T's, having a backbone (internucleoside linkage) pattern of SSSSSSSSSSSSSSSSSSSS (where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO)), except that O is substituted with S as the 2' and 3'-internucleoside linkage of each T (Design D).

[0218] More specifically contemplated are the following designs:

[0219] TTTTTddddddddddTTTTTT (where T is TNA and d is DNA) with the backbone (internucleoside linkage) pattern POOOSSSSSSSSSSSOOOP (where P corresponds to PS2, S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO)) (Design A').

[0220] MMMMMTTTTTTddddMMMMM (where M is MOE (e.g., 2'-O-MOE), T is TNA, and d is DNA) with a backbone (internucleoside linkage) pattern PSSSOOOOOOSSSSSSSSP (where P corresponds to PS2, S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO)) (Design B').

[0221] It has a backbone (internucleoside linkage) pattern of OOOOOOOOOSSSSSSSSSP (where P corresponds to PS2, S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO)), TTTTTTTTTTdddddMMMMM (where M is 2'-O-MOE, T is TNA, and d is DNA) (Design C').

[0222] It has a backbone (internucleoside linkage) pattern of PSSSSSSSSSSSSSSSSSP (where O substitutes for S as any 2' and 3'-internucleoside linkage of each T, S corresponds to phosphorothioate (PS), O corresponds to phosphodiester (PO), and P corresponds to phosphorodithioate (PS2)), MMMMMddddddddddMMMMM (where 1, 2, 3, 4, 5, 6, or 7 d's are replaced by T's, M is 2'-O-MOE, T is TNA, and d is DNA) (Design D').

[0223] Antisense gapmer oligonucleotides containing TNA(PO) gapmers, i.e., contiguous nucleotide sequences of the formula 5'-FG-F'-3'(I), can include additional linked nucleosides, e.g., 1 to 100, 1 to 40, 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 linked nucleosides, at the 3' and / or 5' ends of the TNA(PO) gapmer. The additional linked nucleosides can, for example, facilitate delivery of the antisense gapmer oligonucleotide to the intended site or target a second molecule. Antisense gapmer oligonucleotides can be, or be part of, a longer nucleic acid construct. However, it is also contemplated that antisense gapmer oligonucleotides can consist of TNA(PO) gapmers.

[0224] Particularly contemplated for the present invention are TNA(PO) gapmer and antisense gapmer oligonucleotides that are single-stranded antisense oligonucleotides. In preparing TNA(PO) gapmer or antisense gapmer oligonucleotides according to the present invention, the TNA(PO) gapmer or antisense gapmer oligonucleotide is essentially single-stranded, such that the majority of the TNA(PO) gapmer molecule or antisense gapmer oligonucleotide molecule is in single-stranded form.

[0225] Manufacturing method Also provided is a method for producing the oligonucleotides of the present invention, comprising reacting nucleotide units to form covalently linked consecutive nucleotide units contained in the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al., 1987, Methods in Enzymology, vol. 154, pages 287-313).

[0226] The synthesis of TNA monomers and their incorporation into oligonucleotides is described, for example, in Zhang and Chaput, "Synthesis of Three Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012," International Publication Nos. WO 2012 / 078536, WO 2012 / 118911, and WO 2013 / 179292.

[0227] In particular, a method for preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of the formula 5'FG-F'3'(I) capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides, and each of F and F' is a flanking region of up to 8 linked nucleosides independently comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, wherein in the modified version at least one nucleoside in F, F' and / or G of the parent antisense gapmer oligonucleotide is replaced with a TNA nucleoside linked to the adjacent nucleoside by an internucleoside linkage, e.g., a PO internucleoside linkage; producing a modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked consecutive nucleotide units comprised in the oligonucleotide, at least one of the nucleotide units comprising a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage; Optionally, methods are provided that include purifying or isolating the modified antisense gapmer oligonucleotide.

[0228] In one embodiment, the modified antisense gapmer oligonucleotide has lower toxicity, and optionally lower hepatotoxicity, compared to the parent antisense gapmer oligonucleotide. In one embodiment, the modified antisense gapmer oligonucleotide has lower toxicity to HepG2 cells than the parent antisense gapmer oligonucleotide, optionally as determined by caspase 3 / 7 assay. In one embodiment, the modified antisense gapmer oligonucleotide has enhanced exonuclease resistance compared to the parent antisense gapmer oligonucleotide. In one embodiment, the modified antisense gapmer oligonucleotide has enhanced endonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0229] Optionally, the parent antisense gapmer oligonucleotide may be an LNA gapmer or MOE gapmer, for example an LNA gapmer or MOE gapmer in which all internucleoside linkages are phosphorothioate linkages. Furthermore, the nucleotide units used in the manufacturing process are advantageously nucleoside phosphoramidites.

[0230] The modified antisense gapmer oligonucleotide may include any of the TNA(PO) gapmer features described herein, for example, with respect to regions F, G and F' and the FG-F' design.

[0231] The present invention also provides an antisense gapmer oligonucleotide obtained or obtainable by the above method.

[0232] The method may further comprise reacting the contiguous nucleotide sequence with a conjugate moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide.

[0233] In a further aspect, there is provided a method of making a composition comprising mixing an oligonucleotide or a conjugated oligonucleotide with a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0234] Pharmaceutical Composition In a further aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof, and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0235] In a further aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates, or salts thereof, and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.

[0236] Pharmaceutically acceptable diluents include phosphate buffered saline (PBS), and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50-300 μM solution.

[0237] The oligonucleotide or oligonucleotide conjugate according to the present invention can exist in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a conventional acid or base addition salt that retains the biological effectiveness and properties of the compound of the present invention and is formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base. Acid addition salts include, for example, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a technique well known to medicinal chemists to improve the physical and chemical stability, hygroscopicity, flowability, and solubility of compounds. This is described, for example, in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, a pharmaceutically acceptable salt of the compounds provided herein can be a sodium salt.

[0238] Formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides additional suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Appropriate dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0239] The oligonucleotide or oligonucleotide conjugate of the present invention can be mixed with pharmaceutically acceptable active or inactive substances to prepare pharmaceutical compositions or formulations. The composition and method for the preparation of pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0240] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, e.g., 7 to 7.5. The resulting solid form compositions may be packaged in multiple single-dose units, each containing a fixed amount of the agent or agents, such as a sealed package of tablets or capsules. The solid form compositions may also be packaged in flexible volume containers, such as squeezable tubes designed for topically applied creams or ointments.

[0241] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. Particularly with respect to oligonucleotide conjugates, once the prodrug is delivered to the site of action, e.g., a target cell, the conjugate moiety is cleaved from the oligonucleotide.

[0242] Purpose The oligonucleotides or oligonucleotide conjugates described herein may be utilized as research reagents or as diagnostic, therapeutic and prophylactic agents.

[0243] In research, oligonucleotide or oligonucleotide conjugate can be used to specifically regulate the expression of target nucleic acid in cells (for example, in vitro cell culture) and experimental animals, thereby facilitating the functional analysis of target or the evaluation of its usefulness as a target for therapeutic intervention.Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces protein, thereby preventing protein formation, or by degrading or inhibiting the modulator of the gene or mRNA that produces protein.

[0244] When using oligonucleotides or oligonucleotide conjugates in research or diagnostics, the target nucleic acid can be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0245] Also provided is an in vivo or in vitro method for modulating expression of a target gene in a target cell, comprising administering to said cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the invention, comprising administering to said cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the invention.

[0246] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell culture or in vivo cells that form part of the tissue of a mammal, such as a human.

[0247] Diagnostic applications are also provided in which oligonucleotides or oligonucleotide conjugates may be used to detect and quantitate the expression of target genes in cells and tissues by Northern blotting, in situ hybridization or similar techniques.

[0248] Also provided is an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition described herein for use as a medicament.

[0249] The disease or disorder for which the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition is used is typically associated with expression of the target gene. Preferably, the disease or disorder is one that can be treated by modulating expression of the target gene.

[0250] The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition can be used to treat or prevent diseases or disorders caused by, for example, abnormal levels and / or activity of target genes or expression products derived from target genes, such as RNA or protein.Diseases or disorders can also or alternatively be associated with mutations in target genes.Thus, in some embodiments, the target nucleic acid is a mutant form of target genes.Non-limiting examples of target genes include genes associated with one or more cancers, infectious diseases, neurological diseases or disorders, eye diseases or disorders, or cardiovascular diseases or disorders.

[0251] The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition according to the invention is typically administered in an effective amount.

[0252] Therapeutic applications are also provided for the use of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition in the treatment or prevention of a disease or disorder in an animal or human suffering from or suspected of having the disease or disorder. Typically, the disease or disorder is one that can be treated by modulating the expression of the target gene.

[0253] Also provided is the use of an oligonucleotide or oligonucleotide conjugate in the manufacture of a medicament for treating or preventing a disease or disorder in an animal or human suffering from or suspected of having the disease or disorder. Typically, the disease or disorder is one that can be treated by modulating expression of the target gene.

[0254] Also provided are methods for treating or preventing a disease or disorder, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition to a subject suffering from or susceptible to the disease or disorder.

[0255] Administration In some embodiments, the oligonucleotide or pharmaceutical composition of the present invention can be administered orally. In further embodiments, the oligonucleotide or pharmaceutical composition of the present invention can be administered topically, enterally, or parenterally (e.g., intravenously, subcutaneously, intramuscularly, intracerebrally, intracerebroventricularly, or intrathecally).

[0256] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the present invention is administered by parenteral route, for example, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intrathecal or intracranial administration, for example, intracerebral or intraventricular administration, or intravitreal administration.In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously.In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

[0257] 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, once a week, once every two weeks, once every three weeks, or once a month.

[0258] Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention is used in a combination treatment with another therapeutic agent, which may be, for example, the standard of care for the disease or disorder being treated with the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention.

[0259] Embodiment The following numbered embodiments are specifically contemplated:

[0260] 1. An antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I) that is capable of recruiting ribonuclease (RNase) H, G is a gap region of up to 18 linked nucleosides containing at least three consecutive DNA nucleosides; each of F and F' is a flanking region of up to 15 linked nucleosides, independently comprising or consisting of 1 to 15 sugar-modified nucleosides; At least one of F, F' and G comprises a sugar-modified nucleoside, which is an α-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside linkage that is different from a phosphorothioate (PS) internucleoside linkage; Antisense gapmer oligonucleotides.

[0261] 2. The antisense gapmer oligonucleotide of embodiment 1, wherein the TNA nucleosides are linked to adjacent nucleosides by phosphodiester (PO) internucleoside linkages.

[0262] 3. The antisense gapmer oligonucleotide of embodiment 1 or 2, wherein the TNA nucleosides are linked to adjacent nucleosides by 2'-PO or 3'-PO internucleoside linkages.

[0263] 4. The antisense gapmer oligonucleotide of any one of embodiments 1 to 3, wherein the TNA nucleosides are linked to any adjacent nucleosides by a 2'-PO or 3'-PO internucleoside linkage.

[0264] 5. The antisense gapmer oligonucleotide of any one of embodiments 1 to 4, wherein the adjacent nucleosides are sugar-modified nucleosides or DNA nucleosides.

[0265] 6. TNA nucleosides are (a) 2'-PO internucleoside linkage and 3'-modified internucleoside linkage, respectively; (b) a 2'-modified internucleoside linkage and a 3'-PO internucleoside linkage, respectively; or (c) the first and second adjacent nucleosides are linked by a 2'-PO internucleoside linkage and a 3'-PO internucleoside linkage, respectively; Optionally, the 2'-modified internucleoside linkage or the 3'-modified internucleoside linkage is a phosphorothioate (PS) internucleoside linkage or a phosphorodithioate (PS2) internucleoside linkage; 6. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 5.

[0266] 7. The antisense gapmer oligonucleotide of any one of embodiments 1 to 6, wherein F comprises at least one TNA nucleoside.

[0267] 8. The antisense gapmer oligonucleotide of any one of embodiments 1 to 7, wherein F comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

[0268] 9. The antisense gapmer oligonucleotide of any one of embodiments 1 to 8, wherein F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

[0269] 10. The antisense gapmer oligonucleotide of any one of embodiments 1 to 9, wherein F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 adjacent TNA nucleosides linked by PO internucleoside linkages.

[0270] 11. The antisense gapmer oligonucleotide of any one of embodiments 1 to 10, wherein F comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 adjacent TNA nucleosides linked by PO internucleoside linkages.

[0271] 12. The antisense oligonucleotide of any one of embodiments 1 to 11, wherein at least the 3'-most nucleoside in F is a TNA nucleoside.

[0272] 13. The antisense oligonucleotide of any one of embodiments 1 to 12, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the 3'-most nucleosides in F are TNA nucleosides.

[0273] 14. The antisense oligonucleotide of any one of embodiments 1 to 13, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 3'-most nucleosides in F are TNA nucleosides.

[0274] 15. The antisense oligonucleotide of any one of embodiments 1 to 14, wherein at least the 5'-most nucleoside in F is a TNA nucleoside.

[0275] 16. The antisense oligonucleotide of any one of embodiments 1 to 15, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the 5'-most nucleosides in F are TNA nucleosides.

[0276] 17. The antisense oligonucleotide of any one of embodiments 1 to 16, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 5'-most nucleosides in F are TNA nucleosides.

[0277] 18. The antisense gapmer oligonucleotide of any one of embodiments 1 to 17, wherein all sugar-modified nucleosides of F are TNA nucleosides.

[0278] 19. The antisense gapmer oligonucleotide of any one of embodiments 1 to 18, wherein all nucleosides of F are TNA nucleosides.

[0279] 20. (a) each TNA nucleoside in F is linked to an adjacent nucleoside by a PO internucleoside linkage; or (b) except for the 5'-most TNA nucleoside in F, each TNA nucleoside in F is linked to an adjacent nucleoside by a PO internucleoside linkage; 20. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 19.

[0280] 21. The antisense gapmer oligonucleotide of any one of embodiments 1-20, wherein F' does not comprise any TNA nucleosides.

[0281] 22. The antisense gapmer oligonucleotide of any one of embodiments 1-20, wherein F' comprises at least one TNA nucleoside.

[0282] 23. The antisense gapmer oligonucleotide of any one of embodiments 1 to 22, wherein F' comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

[0283] 24. The antisense gapmer oligonucleotide of any one of embodiments 1-23, wherein F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

[0284] 25. The antisense gapmer oligonucleotide of any one of embodiments 1-24, wherein F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 adjacent TNA nucleosides linked by PO internucleoside linkages.

[0285] 26. The antisense gapmer oligonucleotide of any one of embodiments 1 to 25, wherein F' comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 adjacent TNA nucleosides linked by PO internucleoside linkages.

[0286] 27. The antisense oligonucleotide of any one of embodiments 1 to 26, wherein at least the 5'-most nucleoside in F' is a TNA nucleoside.

[0287] 28. The antisense oligonucleotide of any one of embodiments 1-27, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the 5'-most nucleosides in F' are TNA nucleosides.

[0288] 29. The antisense oligonucleotide of any one of embodiments 1-28, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 5'-most nucleosides in F' are TNA nucleosides.

[0289] 30. The antisense oligonucleotide of any one of embodiments 1 to 29, wherein at least the 3'-most nucleoside in F' is a TNA nucleoside.

[0290] 31. The antisense oligonucleotide of any one of embodiments 1-30, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the 5'-most nucleosides in F' are TNA nucleosides.

[0291] 32. The antisense oligonucleotide of any one of embodiments 1-31, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 5'-most nucleosides in F' are TNA nucleosides.

[0292] 33. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 32, wherein all sugar-modified nucleosides of F' are TNA nucleosides.

[0293] 34. The antisense gapmer oligonucleotide of any one of embodiments 1-33, wherein all nucleosides of F' are TNA nucleosides.

[0294] 35. (a) each TNA nucleoside in F' is linked to an adjacent nucleoside by a PO internucleoside linkage; or (b) each TNA nucleoside in F is linked to an adjacent nucleoside by a PO internucleoside linkage, except for the 3'-most TNA nucleoside in F'; 35. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 34.

[0295] 36. The antisense gapmer oligonucleotide of any one of embodiments 1-6 and 21-35, wherein F does not comprise any TNA nucleosides.

[0296] 37. The antisense gapmer oligonucleotide of any one of embodiments 1-36, wherein F and F' each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

[0297] 38. The antisense gapmer oligonucleotide of any one of embodiments 1-37, wherein F and F' together comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

[0298] 39. The antisense gapmer oligonucleotide of any one of embodiments 1-38, wherein F and F' each independently comprise or consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 adjacent TNA nucleosides linked by PO internucleoside linkages.

[0299] 40. The antisense gapmer oligonucleotide of any one of embodiments 1-39, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 TNA nucleosides, wherein F and F' together are linked to adjacent TNA nucleosides by PO internucleoside linkages.

[0300] 41. (a) each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a PO internucleoside linkage; or (b) each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a P-O internucleoside linkage, except for the 5'-most TNA nucleoside in F and the 3'-most TNA nucleoside in F'; 41. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 40.

[0301] 42. The antisense gapmer oligonucleotide of any one of embodiments 1-41, wherein F, F', or both F and F' further comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, such as 3, 4, or 5 sugar-modified nucleosides other than TNA(PO) nucleosides.

[0302] 43. F, F', or both F and F' sugar-modified nucleosides are α-L-threofuranosyl (found in threose nucleic acid; TNA), 2'-methoxy-ribose (2'-OMe), 2'-O-Methoxyethyl-ribose (2'-O-MOE), 5'-methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE), 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE), 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE), 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA), 2'-deoxy-2'-fluoro-ribose (found in 2'-deoxy-2'-fluororibonucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (found in 2'-fluoro-2'-arabinose nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxyβ-D-locked dribose (found in β-D-LNA), amino β-D-locked dribose (found in amino-β-D-LNA), Thioβ-D-locked dribose (found in thio-β-D-LNA), Oxyβ-L-locked dribose (found in β-L-LNA), amino β-L-locked dribose (found in amino β-L-LNA), Thioβ-L-locked dribose (found in thio-β-L-LNA), Oxyα-L-locked dribose (found in α-L-LNA), amino α-L-locked dribose (found in amino α-L-LNA), Thio α-L-locked dribose (found in thio-α-L-LNA), 2',4'-constrained 2'-O-ethyl ribose (as found in constrained ethyl-locked nucleic acid; cEt), tricyclodeoxyribose (tricyclodeoxyribose DNA; found in TcDNA), 3'-deoxy-ribose (found in 3'-deoxy-ribose DNA; 3'-DNA), unlocked dribose (found in unlocked nucleic acid; UNA), glycol (found in glycol nucleic acid; GNA), hexitols (found in hexitol nucleic acids; HNA), 3'-fluorohexitol (found in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabinofluorohexitol (found in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA), cyclohexene (found in cyclohexene nucleic acid; CeNA), Fluoro-cyclohexenyl (found in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA), Serinol (found in serinol nucleic acid; SNA), 2'-O,4'-C-ethylene-bridged ribose (2'-O,4'-C-ethylene-linked nucleic acid; found in ENA) Acyclic (L)-threoninol (found in acyclic (L)-threoninol nucleic acid; aTNA) 2',4'-constrained 2'-O-methoxyethyl ribose (as found in cMOE), 7',5'-alpha-bicyclosugar unit (7',5'-alpha-bicycloDNA; found in bcDNA) comprising or consisting of at least one sugar-modified nucleoside comprising a modified sugar moiety selected from the group consisting of: 43. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 42.

[0303] 44. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 43, wherein the sugar-modified nucleosides of F, F', or both F and F' comprise or consist of one or more 2'-sugar-modified nucleosides, such as high-affinity 2'-sugar-modified nucleosides.

[0304] 45. The antisense gapmer oligonucleotide of any one of embodiments 1-44, wherein the F, F', or both F and F' sugar-modified nucleosides comprise one or more LNA nucleosides.

[0305] 46. ​​The antisense gapmer oligonucleotide of embodiment 45, wherein all nucleosides in F' and F are LNA nucleosides or 2'-O-MOE nucleosides, except for any at least one TNA nucleoside.

[0306] 47. The antisense gapmer oligonucleotide of any one of embodiments 1-46, wherein the F, F', or both F and F' sugar-modified nucleosides comprise one or more 2'-O-methoxyethyl-RNA (2'-O-MOE) nucleosides.

[0307] 48. The antisense gapmer oligonucleotide of embodiment 47, wherein all nucleosides in F' and F, except for any at least one TNA nucleoside, are 2'-O-MOE nucleosides.

[0308] 49. The antisense gapmer oligonucleotide of any one of embodiments 1-48, wherein G does not comprise any TNA nucleosides.

[0309] 50. The antisense gapmer oligonucleotide of any one of embodiments 1-48, wherein G comprises at least one TNA nucleoside, such as at least 1, 2 or 3 TNA nucleosides.

[0310] 51. The antisense gapmer oligonucleotide of any one of embodiments 1-50, wherein G comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

[0311] 52. The antisense gapmer oligonucleotide of embodiment 51, wherein the second, third, fourth, fifth, sixth, seventh, or eighth nucleoside from the 5'-most side in G is a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

[0312] 53. The antisense gapmer oligonucleotide of embodiment 51 or 52, wherein the second, third, fourth, fifth, sixth, seventh, or eighth 3'-most nucleoside in G is a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

[0313] 54. The antisense gapmer oligonucleotide according to any one of embodiments 1-53, wherein G comprises at least two, such as two or three, adjacent TNA nucleosides linked by PO internucleoside linkages.

[0314] 55. The antisense gapmer oligonucleotide of any one of embodiments 1-54, wherein at least the 5'-most and 3'-most nucleosides in G are DNA nucleosides.

[0315] 56. The antisense gapmer oligonucleotide of any one of embodiments 1 to 55, wherein all nucleosides of G, except for any at least one TNA(PO) nucleoside, are DNA nucleosides.

[0316] 57. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 56, wherein the gap region G comprises at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides.

[0317] 58. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 57, wherein G comprises at most 10 DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 DNA nucleosides.

[0318] 59. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 58, wherein up to about 80% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, such as up to about 70%, for example up to about 60%, such as up to about 50%, for example up to about 40%, such as up to about 30%, for example up to about 20%, such as up to about 15%, for example up to about 10%, for example up to about 5%.

[0319] 60. The antisense gapmer oligonucleotide of any one of embodiments 1-59, wherein up to about 50% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides and at least one TNA nucleoside is a 2'-PO-linked, 3'-PO-linked, or 2',3'-PO-linked TNA nucleoside.

[0320] 61. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 60, comprising at least one modified internucleoside linkage.

[0321] 62. The antisense gapmer oligonucleotide according to any one of embodiments 1-61, comprising nuclease-resistant modified internucleoside linkages.

[0322] 63. The antisense gapmer oligonucleotide of any one of embodiments 1 to 62, wherein all internucleoside linkages, except for any PO internucleoside linkages between the TNA nucleoside and the adjacent nucleoside, are phosphorothioate (PS) internucleoside linkages, phosphorodithioate (PS2) internucleoside linkages, or combinations thereof.

[0323] 64. (a) the 5'-most nucleoside of F is linked to the adjacent nucleoside in F by a PS2 internucleoside linkage; or (b) the 3'-most nucleoside of F' is linked to the adjacent nucleoside in F' by a PS2 internucleoside bond; or (c) Both (a) and (b); 64. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 63.

[0324] 65. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 64, wherein the contiguous nucleotide sequence of formula 5'-FG-F'-3'(I) has a length of 12 to 32 nucleosides, such as 12 to 28 nucleosides, for example 12 to 26 nucleosides, such as 14 to 26 nucleosides, for example 14 to 24 nucleosides, such as 14 to 22 nucleosides, for example 16 to 22 nucleosides, such as 16 to 20 nucleosides.

[0325] 66. A contiguous nucleotide sequence of the formula F 1-15 -G 3-18 -F' 1-15 (IV), e.g., 1-15 -G 3-18 -F' 1-12 (IVa), or 1-12 -G 3-18 -F' 1-15 (IVb); F 1-12 -G 3-18 -F' 1-12 (V), e.g., F 1-12 -G3-18 -F' 1-9 (Va), or F 1-9 -G 3-18 -F' 1-12 (Vb); F 1-12 -G 4-16 -F' 1-12 (VI), e.g., F 1-12 -G 4-16 -F' 1-9 (VIa), or F 1-9 -G 4-16 -F' 1-12 (VIb); or F 3-12 -G 4-10 -F' 3-12 (VII), e.g., F 3-12 -G 4-10 -F' 3-9 (VIIa) or F 3-9 -G 4-10 -F' 3-12 The antisense gapmer oligonucleotide of embodiment 65, which is of formula (VIIb), wherein the ranges of numbers represent the number of linked nucleosides in F, G, and F', respectively.

[0326] 67. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 66, which, optionally as determined in a target cell expressing the target nucleic acid, is capable of reducing the expression level of the target nucleic acid by at least about 50%, such as at least about 60%, for example at least about 70%, for example at least about 80%, for example at least about 90%, compared to the normal expression level of the target when incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days.

[0327] 68. Optionally, the antisense gapmer oligonucleotide of any one of embodiments 1 to 67 has an IC50 for reducing the expression level of the target nucleic acid of about 20 μM or less, such as about 10 μM or less, for example about 5 μM or less, when determined in target cells expressing the target nucleic acid and incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days.

[0328] 69. The antisense gapmer oligonucleotide of any one of embodiments 1 to 68, wherein when using the caspase 3 / 7 assay described in Example 4, the percent assay window (%AW) is preferably about 60% or less, such as about 40% or less, for example about 20% or less, for example about 10% or less.

[0329] 70. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 69, which in the duplex form between the antisense gapmer oligonucleotide and the RNA target sequence has a melting temperature (Tm) of at least about 50°C, such as at least about 52°C, for example at least about 54°C, such as at least about 56°C, for example at least about 58°C, such as at least about 60°C.

[0330] 71. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 66, characterized by embodiments 67 and 68, embodiments 67 and 69, embodiments 67 and 70, embodiments 68 and 69, embodiments 68 and 70, embodiments 67, 68 and 69; embodiments 67, 68 and 70; embodiments 67, 69 and 70, embodiments 68, 69 and 70; or a combination of embodiments 67 to 70.

[0331] 72. The antisense gapmer oligonucleotide according to any one of embodiments 67 to 71, wherein the target nucleic acid is an RNA target sequence having a nucleobase sequence complementary to the contiguous nucleotide sequence of Formula I.

[0332] 73. An antisense gapmer oligonucleotide capable of recruiting RNase H, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I), wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside linked to adjacent nucleosides by a linkage other than a PS internucleoside linkage, e.g., a PO internucleoside linkage.

[0333] 74. The antisense gapmer oligonucleotide of embodiment 73, wherein the TNA nucleoside is linked to an adjacent nucleoside by a PO internucleoside linkage.

[0334] 75. The antisense gapmer oligonucleotide according to embodiment 73 or 74, wherein the contiguous nucleotide sequence of formula 5'-FG-F'-3'(I) has a length of 12 to 32 nucleosides, such as 12 to 28 nucleosides, for example 12 to 26 nucleosides, such as 14 to 26 nucleosides, for example 14 to 24 nucleosides, such as 14 to 22 nucleosides, for example 16 to 22 nucleosides, such as 16 to 20 nucleosides.

[0335] 76. The contiguous nucleotide sequence is represented by the formula F 1-15 -G 3-18 -F' 1-15 (II), e.g., F 1-15 -G 3-18 -F' 1-12 (IIa) or F 1-12 -G 3-18 -F' 1-15 (IIb); F 1-12 -G 3-18 -F' 1-12 (III), e.g., F 1-12 -G 3-18 -F' 1-9 (IIIa), or F 1-9 -G 3-18 -F' 1-12 (IIIb); F 1-12 -G 4-16 -F' 1-12 (IV), e.g., F 1-12 -G 4-16 -F' 1-9 (IVa), or F 1-9 -G 4-16 -F' 1-12 (IVb); or F 3-12 -G 4-10 -F' 3-12 (V), e.g., F 3-12 -G 4-10 -F'3-9 (Va) or F 3-9 -G 4-10 -F' 3-12 The antisense gapmer oligonucleotide of any one of embodiments 73 to 75, wherein the range of values ​​represents the number of linked nucleosides in F, G, and F', respectively.

[0336] 77. The antisense gapmer oligonucleotide of any one of embodiments 73 to 76, further comprising the features of any one of embodiments 1 to 72.

[0337] 78. The contiguous nucleotide sequence, which may be of formula IV, such as IVa, has a length of at least 16 nucleosides; (a) the 5'-most nucleoside in F and the nucleoside in F' are independently 3, 4, or 5 high affinity sugar-modified nucleosides; (b) the remaining nucleosides in F are TNA nucleosides; (c) all nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77.

[0338] 79. The antisense gapmer oligonucleotide of embodiment 78, wherein G comprises up to 10 consecutive DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 consecutive DNA nucleosides.

[0339] 80. The contiguous nucleotide sequence may be of formula IV and has a length of at least 16 nucleosides; (a) F and F' each independently consist of 3, 4, or 5 nucleosides; (b) at least one of the nucleosides in F and F' is a TNA nucleoside, and the remaining nucleosides are high-affinity sugar-modified nucleosides; (c) all nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77.

[0340] 81. The antisense gapmer oligonucleotide of embodiment 80, wherein all of the 2' sugar-modified nucleosides in region F and region F' are TNA nucleosides.

[0341] 82. (a) each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a PO internucleoside linkage; or (b) each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a P-O internucleoside linkage, except for the 5'-most TNA nucleoside in F and the 3'-most TNA nucleoside in F'; 68. The antisense gapmer oligonucleotide of embodiment 66 or 67.

[0342] 83. The contiguous nucleotide sequence of formula IV has a length of at least 16 nucleosides; (a) F and F' each independently comprise or consist of 3, 4, or 5 linked high affinity sugar-modified nucleosides, and do not comprise any TNA nucleosides; (b) the second, third, fourth, or fifth nucleoside from the 5'-most side in G is a TNA nucleoside, e.g., a TNA(PO) nucleoside, and the remaining nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77.

[0343] 84. The antisense gapmer oligonucleotide according to any one of embodiments 78 to 83, wherein the high affinity sugar-modified nucleoside is selected from the sugar-modified nucleosides of embodiment 43.

[0344] 85. The antisense oligonucleotide according to any one of embodiments 1 to 84, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0345] 86. A conjugate comprising an antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker.

[0346] 87. The conjugate of embodiment 86, wherein the conjugate moiety is selected from carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins, vitamins, viral proteins, and combinations thereof.

[0347] 88. The conjugate of embodiment 86 or 87, wherein the conjugate moiety facilitates delivery across the blood-brain barrier.

[0348] 89. A pharmaceutically acceptable salt of the antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 or the conjugate according to any one of embodiments 86 to 88.

[0349] 90. A pharmaceutical composition comprising an antisense gapmer oligonucleotide according to any one of embodiments 1 to 85, or a conjugate according to any one of embodiments 86 to 88, a pharmaceutically acceptable salt according to embodiment 89, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0350] 91. An antisense oligonucleotide according to any one of embodiments 1 to 85, a conjugate according to any one of embodiments 86 to 88, a pharmaceutically acceptable salt according to embodiment 89 or a pharmaceutical composition according to embodiment 90 for use as a medicament.

[0351] 92. A method for preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of the formula 5'FG-F'3'(I) capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides, and each of F and F' is independently a flanking region of up to 8 linked nucleosides comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, wherein in the modified version at least one nucleoside in F, F' and / or G of the parent antisense gapmer oligonucleotide is replaced with a TNA nucleoside linked to the adjacent nucleoside by a PO internucleoside linkage; producing a modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked consecutive nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside; Optionally, the method includes purifying or isolating the modified antisense gapmer oligonucleotide.

[0352] 93. The method of embodiment 92, wherein the modified antisense gapmer oligonucleotide has reduced toxicity, and optionally reduced hepatotoxicity, compared to the parent antisense gapmer oligonucleotide.

[0353] 94. The method of embodiment 92 or 93, wherein the modified antisense gapmer oligonucleotide is less toxic to HepG2 cells than the parent antisense gapmer oligonucleotide, optionally as determined by a caspase 3 / 7 assay.

[0354] 95. The method of any one of embodiments 92-94, wherein the modified antisense gapmer oligonucleotide has enhanced exonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0355] 96. The method of any one of embodiments 92-95, wherein the modified antisense gapmer oligonucleotide has enhanced endonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0356] 97. The method of any one of embodiments 92-96, wherein the parent antisense gapmer oligonucleotide is an LNA gapmer or an MOE gapmer, and optionally, all internucleoside linkages are phosphorothioate linkages.

[0357] 98. The method of any one of embodiments 92-97, wherein the nucleotide unit is a nucleoside phosphoramidite.

[0358] 99. The method of any one of embodiments 92 to 98, wherein the modified antisense gapmer oligonucleotide comprises the features of any one of embodiments 1 to 84.

[0359] 100. An antisense gapmer oligonucleotide obtained or obtainable by a method according to any one of embodiments 92 to 99.

[0360] 101. Use of a TNA nucleotide in the preparation of an antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 or a conjugate according to any one of embodiments 86 to 88. [Example]

[0361] The following examples illustrate, but do not limit, specific embodiments of the present invention.Furthermore, when specific embodiments are provided, the inventors intend that these specific embodiments are generally applicable.For example, the disclosure of the oligonucleotide with TNA(PO) for one target provides reasonable support for other targets.

[0362] Example 1: Oligonucleotide synthesis with TNA modification Oligonucleotides were synthesized by BioAutomation on either a Mermade 12 or Mermade 192 automated DNA synthesizer. Synthesis was performed on a 1 μmol scale using controlled pore glass supports (500 Å) equipped with universal linkers.

[0363] For Mermade 12 synthesis: Standard cycling procedure for coupling of DNA, LNA and MOE phosphoramidites: DMT deprotection was performed with 3% (w / v) trichloroacetic acid in CHCl in six applications of 230 μL for 70 seconds. Each phosphoramidite was then deprotected with acetonitrile (or LNA- mThe C component was coupled three times with 95 μL of a 0.1 M solution in acetonitrile / CHCl (1:1) and 110 μL of a 0.3 M solution of 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile as the activating agent for a coupling time of 180 seconds. Freshly prepared TNA phosphoramidite was coupled three times with 95 μL of a 0.1 M solution in acetonitrile and 110 μL of a 0.3 M solution of 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile as the activating agent for a coupling time of 360 seconds. Sulfurization was carried out using a 0.1 M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine (1 / 1) with two 200 μL applications over 80 seconds. Oxidation was carried out using two applications of 0.02 M I in THF / pyr / H2O (88 / 10 / 2) for 80 seconds. Capping was performed twice for 85 seconds using THF / lutidine / AcO:8 / 1 / 1 (CapA, 125 μL) and THF / N-methylimidazole:84 / 16 (CapB, 125 μL). After synthesis, the CPG was carefully transferred to a 4 mL vial, 1 mL of 25% NH4OH was added, and the mixture was left at 55 °C for 16 hours. The crude DMT-on oligonucleotides were purified using a solid-phase extraction cartridge (Waters Oasis HLB 6cc extraction cartridge) or by preparative reverse-phase HPLC (RP-HPLC) purification (C18 column, NHOAc / CH3CN buffer system) followed by removal of DMT with 80% aqueous acetic acid. After HPLC purification, the oligonucleotides were desalted using an AKTA pure 25 HiPrep 26 / 10 desalting column and lyophilized. The oligonucleotides were characterized by reverse-phase liquid chromatography coupled with high-resolution electrospray mass spectrometry.

[0364] For Mermade192 synthesis: Standard cycling procedure for coupling DNA, LNA, MOE, and TNA phosphoramidites: DMT deprotection was performed with 3% (w / v) trichloroacetic acid in CHCl in three 180 μL applications for 75 s. Each DNA and LNA phosphoramidite was coupled three times with 95 μL of a 0.1 M solution in acetonitrile (or acetonitrile / CHCl 1:1 for the LNA-MeC component) and 110 μL of a 0.285 M solution of 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile as the activator, with a coupling time of 205 s. Freshly prepared TNA phosphoramidite was coupled twice with 95 μL of a 0.1 M solution in acetonitrile / DMF 95:5 and 110 μL of a 0.285 M solution of 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile / DMF 95:5 as the activator, with a coupling time of 385 seconds. Sulfurization was carried out using a 0.1 M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine:1 / 1 with two 190 μL applications for 385 seconds. Oxidation was carried out using 0.02 M I2 in THF / pyr / HO:88 / 10 / 2 with two 160 μL applications for 78 seconds. Capping was performed twice for 70 seconds using THF / lutidine / AcO 8:1:1 (CapA, 125 μL) and THF / N-methylimidazole: 84 / 16 (CapB, 125 μL). After synthesis, the oligonucleotides were cleaved in three batches from 200 μl of CPG using 25% NH4OH. After each step, the CPG was allowed to stand at room temperature for 10 minutes. After sealing the DWP, the solution was left at 55°C for 15 hours. The crude DMT-on oligonucleotides were then purified, and the final DMT was cleaved using cartridges (Agilent TOP-DNA Tubes, 150 mg resin / tube) using aqueous NH4Cl buffer, acetonitrile, and 5% TFA in water.

[0365] TNA phosphoramidite was synthesized as follows: "Synthesis of Three Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012." All other reagents were purchased from Sigma-Aldrich.

[0366] Tables 1-5 show molecules prepared according to the above procedures, with the designations shown below (also applicable to the molecules shown in Tables 7-14). TIFF2025528464000006.tif4128 represents TNA modification. TIFF2025528464000007.tif4128 represents MOE modification. A, G, m C and T represent LNA nucleotides. a, g, c, m c and t represent DNA nucleotides. TIFF2025528464000008.tif2128 represents a phosphodiester bond. * represents a phosphorodithioate bond. All other linkages are phosphorothioate linkages.

[0367] Table 1. Synthetic molecules containing a TNA(PO) moiety (targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)). CMP ID NO = Compound ID number. TIFF2025528464000009.tif92151TIFF2025528464000010.tif232151TIFF2025528464000011.ti f232151TIFF2025528464000012.tif232151TIFF2025528464000013.tif232151TIFF20255284640 00014.tif232151TIFF2025528464000015.tif232151TIFF2025528464000016.tif232151TIFF202 5528464000017.tif232151TIFF2025528464000018.tif232151TIFF2025528464000019.tif33151

[0368] We designed two libraries of antisense oligonucleotides against human MALAT1 (genetic code: ENSG00000251562). The compounds were 20 nucleotides in length and had two different sugar patterns: Design A: TTTTTddddddddddTTTTT (where T is TNA and d is DNA), with the backbone pattern SOOOSSSSSSSSSSSOOOS (where S corresponds to phosphorothioate and O corresponds to phosphodiester); and Design B: MMMMMTTTTTTddddMMMMM (where M is MOE, T is TNA, and d is DNA), with the backbone pattern SSSSOOOOOOSSSSSSSSS (where S corresponds to phosphorothioate and O corresponds to phosphodiester). Compounds were tested at two different concentrations (5 and 25 μM), and the 18 most potent oligonucleotides of Design A (#267-284) and the 19 most potent oligonucleotides of Design B (#285-303) were selected for full dose-response determination.

[0369] Table 2. Compounds selected from two synthesized libraries containing TNA PO (targeting MALAT1) TIFF2025528464000020.tif108160TIFF2025528464000021.tif208160

[0370] Table 3: TNA (PO) vs. DNA (PO) (targeting MALAT1) TIFF2025528464000022.tif102151

[0371] Antisense oligonucleotides targeting ceramide synthase 2 (CERS2), ceramide synthase 5 (CERS5), and ceramide synthase 6 (CERS6) were synthesized and are shown in Table 4. Synthetic molecules containing TNA (PO) and phosphorodithioate (PS2) moieties are shown in Table 5 (targeting MALAT1).

[0372] Table 4: Synthetic molecules containing a TNA(PO) moiety (targeting CERS2, CERS5, CERS6). CMP ID NO = Compound ID number. TIFF2025528464000023.tif206159

[0373] Table 5: Synthetic molecules containing a TNA (PO) moiety and a phosphorodithioate (PS2) moiety (targeting MALAT1). CMP ID NO = Compound ID number TIFF2025528464000024.tif63158TIFF2025528464000025.tif214158

[0374] Further details regarding the molecules in Tables 1-5 are provided in Table 6, where the structure of each synthesized molecule is defined by the Hierarchical Editing Language for Macromolecules (HELM) (for details, see Zhang et al., Chem. Inf. Model. 2012;52(10):2796-2806). Additionally, the SEQ ID NO of the nucleobase sequence on which each synthesized molecule is based is provided. The following HELM annotation key is used: [LR](G) is a beta-D-oxy-LNA guanine nucleoside; [LR](T) is beta-D-oxy-LNA thymine nucleoside; [LR](A) is a beta-D-oxy-LNA adenine nucleoside; [LR] ([5meC] is beta-D-oxy-LNA 5-methylcytosine nucleoside, [dR](G) is a DNA guanine nucleoside; [dR](T) is a DNA thymine nucleoside; [dR](A) is a DNA adenine nucleoside; [dR](C) is a DNA cytosine nucleoside; [dR]([5meC]) is a DNA 5-methylcytosine nucleoside, [MOE] ([5meC]) is 2'O-MOE[2'O-(2-methoxyethyl)]5-methylcytidine nucleoside; [MOE](A) is 2'O-MOE[2'O-(2-methoxyethyl)]adenine nucleoside; [MOE](T) is 2'-O-MOE[2'O-(2-methoxyethyl)]thymine nucleoside; [MOE](G) is 2'-O-MOE[2'O-(2-methoxyethyl)]guanine nucleoside; [TNA] ([5meC]) is α-L-threose nucleic acid 5-methylcytidine nucleoside, [TNA](A) is α-L-threose nucleic acid adenine nucleoside, [TNA] (T) is α-L-threose nucleic acid thymine nucleoside; [TNA](G) is α-L-threose nucleic acid guanine nucleoside; [sP] is a phosphorothioate internucleoside linkage; [PS2] is a phosphorodithioate internucleoside linkage, [P] is a phosphodiester internucleoside linkage.

[0375] Further information and open source tools for HELM can be found at the internet address www.pistoiaalliance.org / helm-tools / (accessed August 26, 2022).

[0376] Table 6: Synthetic molecules in HELM annotation TIFF2025528464000026.tif54158TIFF2025528464000027.tif231158TIFF2025528464000028.tif231158TIFF2025528464000029.tif231158TIFF2025528464000030.tif231158TIFF2025528464000031.tif229158TIFF2025528464000032.tif235158TIFF2025528464000033.tif237158TIFF2025528464000034.tif229158TIFF2025528464000035.tif236158TIFF2025528464000036.tif236158TIFF2025528464000037.tif236158TIFF2025528464000038.tif236158TIFF2025528464000039.tif236158TIFF2025528464000040.tif236158TIFF2025528464000041.tif236158TIFF2025528464000042.tif236158TIFF2025528464000043.tif236158TIFF2025528464000044.tif236158TIFF2025528464000045.tif236158TIFF2025528464000046.tif236158TIFF2025528464000047.tif236158TIFF2025528464000048.tif236158TIFF2025528464000049.tif236158TIFF2025528464000050.tif236158TIFF2025528464000051.tif236158TIFF2025528464000052.tif230158TIFF2025528464000053.tif236158TIFF2025528464000054.tif236158TIFF2025528464000055.tif236158TIFF2025528464000056.tif238158TIFF2025528464000057.tif230158TIFF2025528464000058.tif230158TIFF2025528464000059.tif236158TIFF2025528464000060.tif236158TIFF202 5528464000061.tif236158TIFF2025528464000062.tif236158TIFF2025528464000063.ti f236158TIFF2025528464000064.tif236158TIFF2025528464000065.tif236158TIFF20255 28464000066.tif232158TIFF2025528464000067.tif230158TIFF2025528464000068.tif23 6158TIFF2025528464000069.tif230158TIFF2025528464000070.tif236158TIFF20255284 64000071.tif236158TIFF2025528464000072.tif238158TIFF2025528464000073.tif2291 58TIFF2025528464000074.tif227158TIFF2025528464000075.tif238158TIFF2025528464 000076.tif229158TIFF2025528464000077.tif236158TIFF2025528464000078.tif185158.

[0377] Example 2: In vitro efficacy of oligonucleotides targeting MALAT1 RNA in A549 cells at two different concentrations (5 and 25 μM) The A549 cell line was purchased from ATCC and maintained in a humidified incubator at 37°C and 5% CO2, according to the supplier's recommendations. For the assay, 3000 cells / well were seeded into 96-multiwell plates in complete culture medium. After 24 hours of incubation, oligonucleotides dissolved in PBS were added at the indicated final concentrations. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the RNeasy96 RNA Purification Kit (Qiagen) according to the manufacturer's instructions and eluted in 50 μL of water. The RNA was then diluted 10-fold with DNase / RNase-free water and heated at 90°C for 1 minute.

[0378] For gene expression analysis, one-step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex setup. The following TaqMan primer assays were used for qRT-PCR: MALAT1, Hs00273907_s1 [FAM-MGB]; CERS2, Hs01017465_m1 [FAM-MGB]; CERS5, Hs00332291_m1 [FAM-MGB]; CERS6, Hs00826756_m1 [FAM-MGB]; and endogenous control GAPDH, Hs99999905_m1 [VIC-MGB-PL]. All primer sets were purchased from Thermo Fisher Scientific. The relative RNA expression levels of MALAT1, CERS2, CERS5, or CERS6, also called knockdown (KD) values, were calculated as a percentage of the control (PBS-treated cells).

[0379] KD values ​​were determined using GraphPad Prism.

[0380] The results are shown in Tables 7 to 10. Values ​​separated by " / " indicate individual results when a compound or control was tested in more than one test vial.

[0381] Table 7. MALAT1 target knockdown (KD) values TIFF2025528464000079.tif96158TIFF2025528464000080.tif234158TIFF2025528464000081.tif234158 TIFF2025528464000082.tif238158TIFF2025528464000083.tif234158TIFF2025528464000084.tif234158 TIFF2025528464000085.tif238158TIFF2025528464000086.tif238158TIFF2025528464000087.tif238158 TIFF2025528464000088.tif238158TIFF2025528464000089.tif234158TIFF2025528464000090.tif117158

[0382] Data demonstrated that TNA(PO) was well tolerated for KD in A549 cells when TNA was placed in the flank / interstitial space.

[0383] Table 8: Knockdown (KD) values ​​for targeting MALAT1; DNA PO vs. TNA PO TIFF2025528464000091.tif125153

[0384] Because TNA(PO) has better metabolic stability than DNA(PO), the data demonstrated that placing TNA in the DNA interstitial space is superior to DNA(PO).

[0385] Table 9. Knockdown (KD) values ​​for targeting CERS2 / 5 / 6 TIFF2025528464000092.tif139158TIFF2025528464000093.tif125158

[0386] The data demonstrated that the strategy of using TNAs to reduce DNA gaps close to the 5′ wing can be generalized to other targets beyond MALAT1.

[0387] Table 10: Knockdown (KD) values ​​of MALAT1 compounds containing TNA(PO) and phosphorodithioate TIFF2025528464000094.tif63141TIFF2025528464000095.tif214141

[0388] A novel structure containing TNA (PO) in the DNA gap and phosphorodithioate (PS2) at both ends was successfully demonstrated. The phosphorodithioate linkage significantly reduced MALAT1 expression at 25 μM in A549 cells.

[0389] Example 3: In vitro potency and efficacy of oligonucleotides targeting MALAT1 RNA in A549 cells at various concentrations for dose-response curves

[0390] The A549 cell line was purchased from ATCC and maintained in a humidified incubator at 37°C and 5% CO2, according to the supplier's recommendations. For the assay, 3500 cells / well (A549) were seeded into a 96-multiwell plate in culture medium. After 24 hours of incubation, oligonucleotides dissolved in PBS were added. Oligonucleotide concentrations ranged from a maximum of 25 μM to eight 1:1 dilutions. Three days after addition of the oligonucleotides, cells were harvested. RNA was extracted using the PureLink Pro96 RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions and eluted in 50 μL of water. The RNA was then diluted 10-fold with DNase / RNase-free water (Gibco) and heated at 90°C for 1 minute.

[0391] For gene expression analysis, one-step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplicate setup. The following TaqMan primer assays were used for qPCR: MALAT1, Hs00273907_s1 (FAM-MGB), and the endogenous control GAPDH. All primer sets were purchased from Thermo Fisher Scientific. Relative expression levels of MALAT1 RNA are shown as a percentage of the control (PBS-treated cells), and IC50 values ​​were determined using GraphPad Prism 7 on data from two biological replicates.

[0392] The results are shown below in Table 11. Values ​​separated by " / " indicate individual results when a compound or control was tested in more than one test vial.

[0393] Table 11. In vitro potency results (IC50) TIFF2025528464000096.tif76141TIFF2025528464000097.tif236141TIFF2025528464000098.tif236141 TIFF2025528464000099.tif236141TIFF2025528464000100.tif236141TIFF2025528464000101.tif236141 TIFF2025528464000102.tif236141TIFF2025528464000103.tif236141TIFF2025528464000104.tif236141 TIFF2025528464000105.tif236141TIFF2025528464000106.tif236141TIFF2025528464000107.tif225141

[0394] Example 4: Caspase 3 / 7 activation in HepG2 cells Caspase 3 / 7-activated HepG2 cells were cultured at approximately 70% confluence in MEM medium containing GlutaMax (Gibco #41090) supplemented with 10% heat-inactivated fetal bovine serum. Cells were detached with 0.25% trypsin-EDTA solution (Gibco #25200056) and cultured at 1 × 10 4 HepG2 cells were seeded into black, clear 96-well plates P37102-EP78 (Corning #3904, NY, USA) at a density of 100 cells / well. 24 hours after seeding, cells were transiently transfected with Lipofectamine 2000 (Life Technologies #11668019) using 100 nM oligonucleotides dissolved in Opti-MEM (Gibco #31985). Caspase-3 / 7 activity was determined using the Caspase-Glo® 3 / 7 assay (Promega Corporation, Madison, WI, USA). Reconstituted Caspase-Glo® 3 / 7 reagent was added to the cells 24 hours after transfection and incubated for 60 minutes. Cell lysates were transferred to opaque 96-well plates (Corning #3600, NY, USA), and luminescence was measured using an Enspire multimode plate reader (Perkin Elmer) according to the manufacturer's instructions. The results are shown in Table 12, where the percentage (% assay window) indicates the degree of cell apoptosis based on the vehicle (cells treated with PBS only). Higher values ​​indicate higher apoptotic activity and therefore higher in vitro cytotoxicity. The negative control is a non-toxic ASO. Some values ​​of the tested ASOs are negative due to the low background of these ASOs. Negative values ​​are not cytotoxic, while ASOs with values ​​around 300 are slightly cytotoxic. ASO = antisense oligonucleotide.

[0395] Table 12. Caspase 3 / 7 assay results TIFF2025528464000108.tif94158TIFF2025528464000109.tif237158TIFF2025528464000110.tif114158

[0396] Data demonstrated that even a single TNA(PO) in the 5′ flank or DNA gap significantly reduced caspase induction across targets (MALAT1, CERS2 / 5 / 6).

[0397] Example 5: Thermal Melting (Tm) of Oligonucleotides Containing TNA(PO) Modifications Hybridized to RNA The denaturation point (thermal melting point = Tm) was measured according to the following procedure. Gapmer ASOs and complementary RNAs were added to 20 mM disodium phosphate buffer, 200 mM NaCl, and 0.2 mM EDTA (pH 7) to obtain a final concentration of 1.5 μM. Samples were heated to 95°C for 5 minutes and then slowly cooled to room temperature over 1 hour. Thermal melting curves were recorded at 260 nm on an Agilent Cary 3500 equipped with a Peltier Temperature Programmer, using a temperature gradient that increased from 25°C to 95°C at 5°C / min and then decreased to 25°C. The melting temperature (Tm) was determined using the first derivative of both curves. Values ​​were averaged over three heating and cooling curves.

[0398] (Table 13) Thermal melting temperature (Tm) values TIFF2025528464000111.tif224138TIFF2025528464000112.tif232138TIFF2025528464000113.tif160138

[0399] The data demonstrated that TNA(PO) increased Tm when incorporated into the DNA gap, but Tm decreased when TNA was replaced with sugars with high binding affinity (MOE, LNA).

[0400] Example 6: Inhibition of MALAT1 expression in mice administered subcutaneously To compare TNA with other saccharides, the well-described MOE gapmer targeting MALAT1 was chosen as the parent antisense oligonucleotide for in vivo testing. Substitution of TNA for MOE in the flank resulted in the following series of antisense oligonucleotides, administered subcutaneously at 10 mg / kg to mice:

[0401] Table 14. Selected antisense oligonucleotides containing TNAs for in vivo testing in mice TIFF2025528464000114.tif70158ND=Undecided

[0402] In vivo experiments were performed in strict compliance with Swiss federal regulations regarding animal welfare and the rules of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and with the approval of the local veterinary authorities. Male C57BL / 6J mice (8-12 weeks old) were purchased from Charles River Laboratories, France. Mice were group-housed in open cages and maintained at constant temperature (21-24°C) and humidity (40-80%) on a 12:12-h light:dark cycle. Each cage was provided with tap water and sterilized food (Provimi Kliba 3436) ad libitum. All cages (except metabolic cages) were supplied with autoclaved sawdust bedding and environmental enrichment, which were rotated weekly in accordance with best practice animal welfare standards. Mice were allowed to acclimate for at least 1 week before the start of the study.

[0403] Animals were housed in groups of three and provided with water and standard chow ad libitum. The vivarium was maintained at a constant temperature (23±1°C) and humidity (40±5%) under a 12-hour light:12-hour dark cycle (lights on at 08:00) throughout the study. Mice were intravenously dosed on day 0 and anesthetized (70% CO2 / 30% O2) before termination by cervical dislocation on day 9. Treatment groups (n=4) received a subcutaneous injection of 0.9% saline or a gapmer diluted in saline at a dose of 10 mg / kg. At the end of the study, organs and plasma were collected at the termination time points. Plasma samples were stored at -20°C for further processing and analysis. Organ samples were collected in homogenization tubes (Precellys® CK28, Bertin Instruments, France).

[0404] Total RNA was isolated from liver and kidney using the RNeasy kit (Qiagen), and RNA quantification was performed using the TaqMan assay (Applied Biosystems). Reverse transcription reactions were performed using random decamers, 0.5 mg of total RNA, and M-MLV reverse transcriptase (Ambion) according to the protocol for single-stranded complementary DNA (cDNA) synthesis. Depending on the expression level, the cDNA was subsequently diluted 5-fold in nuclease-free water before being added to the RT-PCR reaction mixture. An Applied Biosystems 7500 / 7900 / ViiA real-time PCR instrument was used for amplification. In each experiment, mRNA levels were normalized to actin, beta (Actb) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and expressed as fold changes relative to the mean levels in saline controls.

[0405] As shown in Figure 1, all antisense oligonucleotides resulted in better reduction of MALAT1 RNA in the liver compared to other tissues. In the liver, at a dose level of 10 mg / kg, the parent gapmer (control #2) demonstrated approximately 70% RNA target reduction. The performance of the corresponding PO version (#346) was significantly reduced. Two TNA analogs with six POs (#100 and #76) clearly outperformed control #2. Even two ASOs with eight phosphates (#99 and #78) demonstrated comparable in vivo efficacy compared to the all-PS parent. The all-PS TNA gapmer (#347) performed best, achieving approximately 80% target knockdown. TNAs did not improve the reduction of MALAT1 expression in the lung and muscle compared to the MOE ASOs (controls #2 and #346).

[0406] Tissue samples were collected, weighed during necropsy, and stored at -80°C in homogenization tubes CK28 (Precellys®) for subsequent bioanalytical testing by LC coupled to tandem mass spectrometry (LC-MS / MS). Prior to extraction, tissue homogenates were diluted 5-fold with blank mouse plasma. Quantitation was performed against a mouse plasma calibration curve. After addition of an internal standard (2000 ng / mL), 50 microliters of calibration standards, quality control samples (freshly prepared in mouse plasma), and tissue homogenate samples diluted with blank mouse plasma were subjected to protein denaturation treatment with 150 μL of 4 M guanidine thiocyanate. A characterized 16-mer oligonucleotide (MW: 5460 Da) consisting of DNA nucleotides, LNA nucleotides, and a fully phosphorothioate backbone was used as an internal standard to eliminate potential variations during pipetting, solid-phase post-treatment, or LC-MS / MS sample injection. After vigorous mixing (1,600 rpm for 20 min), 200 μL of HO / HFIP / DIPEA solution (100:4:0.2, v / v / v) was added and mixed (1500 rpm for 15 min). After elution and evaporation to dryness (40 °C for 30–45 min), a cleanup step was performed using a solid-phase extraction cartridge (Waters, OASIS HLB, 30 μm). The sample was reconstituted in 200 μL of mobile phase (HO / MeOH / HFIP / DIPEA [95 / 5 / 1 / 0.2, v / v / v / v]). After vortex mixing (1500 rpm for 10 min), an aliquot (20 μL) was injected onto an analytical column (Waters, Acquity BEH C18, 1.7 μm, 50 × 2.1 mm, held at 60 °C). Analytes and internal standards were separated from matrix interferences using a gradient elution from HO / MeOH / HFIP / DIPEA (95 / 5 / 1 / 0.2, v / v / v / v) to HO / MeOH / HFIP / DIPEA (10 / 90 / 1 / 0.2, v / v / v / v) within 4 min at a flow rate of 0.4 mL / min. Mass spectrometric detection was performed on an AB-Sciex 6500+ mass spectrometer using selected reaction monitoring (SRM) in negative ion mode.The selected ion responses (m / z) were 680.6 / 94.8 and 658.9 / 94.8 for ASO1 and ASO5, respectively, and 596.1 / 94.8 for the internal standard. Detection was achieved using ion spray MS / MS in negative ion SRM mode.

[0407] As shown in Figure 2, the antisense oligonucleotide content in the liver and kidney, as well as the lung and muscle, on day 9 was quantified using LC-MS / MS, demonstrating the PK / PD relationship. The TNA gapmer (#347) had the highest liver concentration, which correlated strongly with its strong reduction in MALAT1 RNA in the liver. Antisense oligonucleotide #99 showed comparable efficacy despite its low liver content. Clearly, antisense oligonucleotides containing PO linkages were primarily excreted in the kidney, likely due to their low affinity for plasma proteins. As expected, all antisense oligonucleotides showed much lower concentrations in the lung and muscle, resulting in no significant mRNA reduction in these two tissues.

[0408] List of References Crooke et al., Nucleic Acids Research2020;48(10):5235-5253.DOI:10.1093 / nar / gkaa299 Eckstein, Antisense and Nucleic Acid Drug Development2009;10:117-121.DOI:10.1089 / oli.1.2000.10.117. Liu et al.,ACS Appl.Mater.Interfaces2018;10:9736-9743.DOI:10.1021 / acsami.8b01180 Matsuda et al.,Poster;XXIII International Round Table on Nucleosides,Nucleotides and Nucleic acids;August2018.DOI:10.13140 / RG.2.2.10627.45605 Zhang and Chaput, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012. DOI:10.1002 / 0471142700.nc0451s50 International Publication No. 2012 / 078536 (Quark Pharmaceuticals, Inc.) International Publication No. 2012 / 118911 (Quark Pharmaceuticals, Inc.) WO 2013 / 179292 (QBI Enterprises Ltd. and Bio-Lab Ltd.)

Claims

1. 1. An antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of the formula 5′-F-G-F′-3′(I) capable of recruiting ribonuclease (RNase) H, G is a gap region of up to 18 linked nucleosides comprising at least three consecutive DNA nucleosides; each of F and F' is a flanking region of up to 15 linked nucleosides, independently comprising or consisting of 1 to 15 sugar-modified nucleosides; At least one of F, F' and G comprises a sugar-modified nucleoside, which is an α-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside linkage that is different from a phosphorothioate (PS) internucleoside linkage; The antisense gapmer oligonucleotide.

2. 2. The antisense gapmer oligonucleotide of claim 1, wherein the TNA nucleosides are linked to adjacent nucleosides by phosphodiester (PO) internucleoside linkages.

3. 2. The antisense gapmer oligonucleotide of claim 1, wherein the TNA nucleosides are linked to any adjacent nucleoside by a 2'-PO or a 3'-PO internucleoside linkage.

4. 2. The antisense gapmer oligonucleotide of claim 1, wherein F comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

5. 10. The antisense gapmer oligonucleotide of claim 1, wherein F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

6. 10. The antisense gapmer oligonucleotide of claim 1, wherein all nucleosides of F are TNA nucleosides.

7. 2. The antisense gapmer oligonucleotide of claim 1, wherein F' comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

8. 2. The antisense gapmer oligonucleotide of claim 1, wherein F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides linked to adjacent nucleosides by PO internucleoside linkages.

9. 10. The antisense gapmer oligonucleotide of claim 1, wherein all nucleosides of F' are TNA nucleosides.

10. the sugar-modified nucleoside is F, F', or both F and F', α-L-threofuranosyl (found in threose nucleic acid; TNA), 2'-methoxy-ribose (2'-OMe), 2'-O-methoxyethyl-ribose (2'-O-MOE), 5'-methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE), 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE), 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE), 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA), 2'-deoxy-2'-fluoro-ribose (found in 2'-deoxy-2'-fluororibonucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (found in 2'-fluoro-2'-arabinose nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxy-β-D-locked dribose (found in β-D-LNA), Amino β-D-locked dribose (found in amino-β-D-LNA), Thio-β-D-locked dribose (found in thio-β-D-LNA), Oxy-β-L-locked dribose (found in β-L-LNA), amino β-L-locked dribose (found in amino β-L-LNA); Thio-β-L-locked dribose (found in thio-β-L-LNA), Oxy-α-L-locked dribose (found in α-L-LNA), amino α-L-locked dribose (found in amino α-L-LNA); Thio α-L-locked dribose (found in thio-α-L-LNA), 2',4'-constrained 2'-O-ethyl ribose (as found in constrained ethyl-locked nucleic acids; cEt), tricyclodeoxyribose (tricyclodeoxyribose DNA; found in TcDNA), 3'-deoxy-ribose (found in 3'-deoxy-ribose DNA; 3'-DNA), unlocked dribose (found in unlocked nucleic acid; UNA), glycol (found in glycol nucleic acid; GNA), hexitols (found in hexitol nucleic acids; HNAs), 3'-fluorohexitol (found in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabinofluorohexitol (found in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA), cyclohexene (found in cyclohexene nucleic acid; CeNA), fluoro-cyclohexenyl (found in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA); serinol (found in serinol nucleic acid; SNA), 2'-O,4'-C-ethylene-bridged ribose (found in 2'-O,4'-C-ethylene-linked nucleic acids; ENA) Acyclic (L)-threoninol (found in acyclic (L)-threoninol nucleic acid; aTNA) 2',4'-constrained 2'-O-methoxyethyl ribose (as found in cMOE), 7',5'-alpha-bicyclosugar unit (found in 7',5'-alpha-bicycloDNA; bcDNA) at least one sugar-modified nucleoside comprising a modified sugar moiety selected from the group consisting of: An antisense gapmer oligonucleotide according to any one of the preceding claims.

11. 2. The antisense gapmer oligonucleotide of claim 1, wherein all nucleosides in F' and F, except for any at least one TNA nucleoside, are LNA nucleosides or 2'-O-MOE nucleosides.

12. 2. The antisense gapmer oligonucleotide of claim 1, wherein G comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

13. All internucleoside linkages are phosphorothioate (PS) internucleoside linkages, phosphorodithioate (PS) internucleoside linkages, except for any PO internucleoside linkages between TNA nucleosides and adjacent nucleosides. ２ ) internucleoside linkages, or combinations thereof.

14. (a) The 5'-most nucleoside of F is PS ２ is linked to adjacent nucleosides in F by internucleoside linkages; or (b) The 3'-most nucleoside of F' is PS ２ linked to adjacent nucleosides in F' by internucleoside bonds, or (c) both (a) and (b); An antisense gapmer oligonucleotide according to any one of the preceding claims.

15. 10. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein the contiguous nucleotide sequence of formula 5'-F-G-F'-3'(I) has a length of 12 to 32 nucleosides, such as 12 to 28 nucleosides, for example 12 to 26 nucleosides, such as 14 to 26 nucleosides, for example 14 to 24 nucleosides, such as 14 to 22 nucleosides, for example 16 to 22 nucleosides, such as 16 to 20 nucleosides.

16. the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides; (a) the 5'-most nucleoside in F and the nucleoside in F' are independently 3, 4, or 5 high-affinity sugar-modified nucleosides, the remaining nucleosides in F are DNA nucleosides, and all nucleosides in G are DNA nucleosides; or (b) F and F' each independently consist of 3, 4, or 5 nucleosides, and all nucleosides in F and F' are DNA nucleosides, and all nucleosides in G are DNA nucleosides; or (c) F and F' each independently comprise or consist of 3, 4, or 5 linked high-affinity sugar-modified nucleosides and do not comprise any TNA nucleosides, the second, third, fourth, or fifth 5'-most nucleoside in G is a TNA(PO) nucleoside, and the remaining nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide of claim 15.

17. A conjugate comprising the antisense gapmer oligonucleotide of any one of the preceding claims and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker.

18. A pharmaceutically acceptable salt of the antisense gapmer oligonucleotide of any one of claims 1 to 16 or the conjugate of claim 17.

19. 19. A pharmaceutical composition comprising the antisense gapmer oligonucleotide of any one of claims 1 to 16, the conjugate of claim 17, or the pharmaceutically acceptable salt of claim 18, together with a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

20. 20. The antisense oligonucleotide according to any one of claims 1 to 16, the conjugate according to claim 17, the pharmaceutically acceptable salt according to claim 18 or the pharmaceutical composition according to claim 19 for use as a drug.

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