Threose nucleic acid antisense oligonucleotides and methods thereof

Abstract

Antisense oligonucleotides containing one or more alpha-L-threofuranosyl (TNA) nucleosides are described, as well as methods for modulating the properties of antisense oligonucleotides by the incorporation of TNA nucleosides, which are particularly applicable to antisense gapmer oligonucleotides.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antisense oligonucleotides comprising one or more alpha-L-threofuranosyl (TNA) nucleosides, as well as methods for modulating the properties of antisense oligonucleotides by the incorporation of TNA nucleosides. The present invention is particularly applicable to antisense gapmer oligonucleotides. [Background technology]

[0002] 2. Background of the Invention Synthetic oligonucleotides as therapeutic agents have made significant advances in recent years, resulting in a broad portfolio of clinically validated molecules that act by diverse mechanisms, including ribonuclease H (RNase H)-activatable gapmers, splice-switching oligonucleotides, microRNA inhibitors, small interfering RNA (siRNA) and antisense oligonucleotides such as aptamers (S. T. Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008 (Non-Patent Document 1)).

[0003] Arguably, one of the most successful modifications is the introduction of phosphorothioate linkages, in which one of the non-bridging phosphate oxygen atoms is replaced by 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 apparently higher stability against nucleic acid degradation, and therefore higher half-life in plasma, tissues and cells than their unmodified phosphodiester analogues. These key features enabled the development of the first generation of oligonucleotide therapeutics and opened the door for further improvement by later generation modifications such as locked nucleic acids (LNA). Other modifications include locked nucleic acids (LNA) as well as a variety of other modified nucleosides. For example, TNAs have been used, for example, in double-stranded siRNA molecules and in the form of oligomers (Matsuda et al., XXIII International Round Table on Nucleosides, Nucleotides and Nucleic acids; 2018 (Non-Patent Document 3); Liu et al., ACS Appl. Mater. Interfaces 2018; 10: 9736-9743 (Non-Patent Document 4); WO 2012 / 078536 (Patent Document 1); WO 2012 / 118911 (Patent Document 2); and WO 2013 / 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] International Publication No. 2012 / 078536 [Patent Document 2] International Publication No. 2012 / 118911 [Patent Document 3] International Publication No. 2013 / 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 Development 2009;10:117-121 [Non-Patent Document 3] 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 Summary of the Invention

[0007] The present inventors have found that one or more alpha-L-threofuranosyl (TNA) nucleosides can be introduced into antisense oligonucleotides, particularly antisense gapmer oligonucleotides, to modulate the properties of antisense oligonucleotides.Surprisingly, when TNA modifications are introduced into gapmer designs as described herein, they can result in potent molecules with favorable properties for therapeutic use.

[0008] Thus, the present invention relates to antisense oligonucleotides comprising at least one TNA nucleoside, in particular antisense gapmer oligonucleotides comprising at least one TNA nucleoside.

[0009] The present invention also relates to an antisense gapmer oligonucleotide capable of recruiting ribonuclease (RNase) H, the antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I), the contiguous nucleotide sequence comprising at least one TNA nucleoside.

[0010] The present invention also relates to an antisense gapmer oligonucleotide capable of recruiting 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 that contains at least three consecutive DNA nucleosides; each of F and F' is independently a flanking region of up to 15 linked nucleosides that comprises or consists of 1 to 15 sugar-modified nucleosides; and At least one of F, F', and G comprises a sugar-modified nucleoside that is an α-L-threofuranosyl (TNA) nucleoside.

[0011] The present invention also relates to a conjugate comprising an antisense gapmer oligonucleotide according to the 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 pharma- ceutically acceptable salts of the antisense gapmer oligonucleotides or antisense gapmer conjugates according to the invention.

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

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

[0015] The invention also provides a method of preparing a modified version of a parent antisense gapmer oligonucleotide, the parent antisense gapmer comprising 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; The method includes producing a modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, at least one of the nucleotide units comprising 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 invention are provided in the detailed disclosure and claims which follow. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Disclosure definition In order that the present invention may be more readily understood, certain terms are defined and explained below.

[0020] Throughout this specification, the word "comprise" or grammatical variations thereof includes a stated integer (or component) or group of integers (or components) and does not exclude any other integer (or component) or group of integers (or components).

[0021] Oligonucleotides As used herein, the term "oligonucleotide" is defined as commonly understood by those skilled in the art as a molecule that contains 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 usually made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to an oligonucleotide sequence, it refers 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. Nucleosides may be linked by phosphodiester (PO) bonds or modified internucleoside bonds.

[0022] Antisense oligonucleotides As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid.The contemplated antisense oligonucleotide is not double-stranded in nature, 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 a hairpin or intermolecular duplex structure (a duplex between two molecules of the same oligonucleotide) if the degree of complementarity between itself or between itself is more than 50% over the entire length of the oligonucleotide.

[0023] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a 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 may be used to attach additional nucleotide(s), such as a functional group, 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.

[0024] 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 may also be referred to interchangeably as "units" or "monomers".

[0025] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds 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 are functional 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.

[0026] The nucleobase moiety can 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.

[0027] The nucleobase moieties may be designated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include modified nucleobases of equivalent function. For example, in some oligonucleotides, the nucleobase moieties may include A, T, G, C, and 5-methylcytosine ( m C) is selected.

[0028] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified compared to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or (nucleic acid) base moieties. Preferably, the modified nucleoside comprises a modified sugar moiety. The term "modified nucleoside" may also be used herein indistinguishable from the term "nucleoside analogue" 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 they are capable of Watson-Crick base pairing.

[0029] Sugar-modified nucleosides The antisense oligonucleotides of the invention may contain 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.

[0030] A number of nucleosides with modifications in the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0031] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA) or bicyclic ring (typically 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 has been replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0032] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

[0033] Non-limiting examples of modified sugar moieties include: α-L-threofuranosyl (as 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 (as in 2'-deoxy-2'-fluororibo-nucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinose-nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxy-, amino- or thio-β-D-locked ribose (as in β-D-LNA); Oxy, amino or thio α-L-locked dribose (as in α-L-LNA), 2',4' constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), Tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA), 3'-deoxy-ribose (as in 3'-deoxy-ribose DNA; 3'-DNA), Unlocked ribose (as in Unlocked Nucleic Acid; UNA), glycol (as in glycol nucleic acid; GNA), hexitols (as in hexitol nucleic acids; HNA), 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabino-fluoro-hexitol (as in 3'-arabino-fluoro-hexitol nucleic acid; Ara-FHNA), cyclohexene (as in cyclohexene nucleic acid; CeNA), and Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA).

[0034] Unless otherwise specified or contradicted by context, the term "MOE," as used herein, 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.

[0035] 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 sugar-modified nucleosides that include an α-L-threofuranosyl moiety.

[0036] The TNA nucleosides are linked to adjacent nucleosides by a (2'→3') internucleoside linkage, such as a phosphodiester (PO) or modified internucleoside linkage, as shown below for two adjacent TNA nucleosides. TIFF2024547071000001.tif64128

[0037] When the nucleobase (B) is cytosine, the TNA nucleoside is advantageously 5-methyl-cytosine ( m C) TNA nucleoside.

[0038] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2' substituted nucleoside). This includes nucleosides that contain a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon in the ribose ring, such as LNA (2'-4' bridged) nucleosides.

[0039] For purposes of this disclosure, TNA nucleosides are not 2'-substituted nucleosides.

[0040] Many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer improved binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (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 Deleavey and Damha, Chemistry and Biology 2012,19,937. Scheme 1 below shows examples of some 2'-substituted modified nucleosides. Scheme 1: TIFF2024547071000002.tif85128

[0041] Locked Nucleic Acid (LNA) "LNA nucleosides" are 2' modified nucleosides that contain a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' 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). Fixation of the ribose conformation is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0042] Non-limiting exemplary LNA nucleosides are described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 07757 8, International Publication No. 2010 / 036698, International Publication No. 2007 / 090071, International Publication No. 2009 / 006478, International Publication No. 2011 / 156202, International Publication No. 2008 / 154401, International Publication No. 2009 / 067647, International Publication No. 2008 / 150729, Morita See, e.g., Seth et al., Bioorganic & Med. Chem. Lett. 2002, 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0043] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 2. Scheme 2: TIFF2024547071000003.tif126128

[0044] 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 beta-D-oxy-LNA.

[0045] Internucleoside bond The term "internucleoside bond" is defined as the bond that covalently connects two nucleosides to each other, as generally understood by those skilled in the art.In the antisense oligonucleotides described herein, internucleoside bond covalently connects adjacent nucleosides to each other, 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.

[0046] Modified Internucleoside Linkages The term "modified internucleoside bond" is defined as a bond other than a phosphodiester (PO) bond that covalently connects two nucleosides to each other, as generally understood by those skilled in the art. Modified internucleoside bond can increase the nuclease resistance of oligonucleotides compared to phosphodiester bond. Modified internucleoside bond is particularly useful for stabilizing oligonucleotides for in vivo use, and can protect against nuclease cleavage in regions of DNA or RNA nucleosides in oligonucleotides, such as in the gap region of gapmer oligonucleotides, and in regions of modified nucleosides, such as regions F and F'.

[0047] Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using nuclease resistance assays (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 contemplated that the nucleoside that links the oligonucleotide to a non-nucleotide functional group, e.g., a conjugate, can be a phosphodiester.

[0048] Phosphorothioate Internucleoside Linkages The preferred modified internucleoside bond is phosphorothioate (PS).Phosphorothioate internucleoside bond is particularly useful due to its nuclease resistance, favorable pharmacokinetics, and ease of manufacture.In some oligonucleotides, all internucleoside bonds of the oligonucleotide or its consecutive nucleotide sequence are phosphorothioate bonds.

[0049] Nuclease-resistant linkages, such as phosphorothioate linkages, are particularly useful in regions of oligonucleotides that can recruit nucleases when they form a duplex with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate linkages may also be useful in non-RNase H recruiting and / or affinity enhancing regions, such as regions F and F' of a gapmer.

[0050] Complementarity The term "complementarity" refers to the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It will be understood that oligonucleotides may contain nucleosides with modified nucleobases, e.g., 5-methylcytosine is often used in place of cytosine, and thus the term complementarity encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., 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).

[0051] As used herein, the term "complementarity %" refers to the number of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is complementary (i.e., forms Watson Crick base pairs) at a given position to the contiguous sequence of nucleotides at a given position of 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 this 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 the calculation of the complementarity % of a contiguous nucleotide sequence.

[0052] The term "fully complementary" refers to 100% complementarity.

[0053] identity As used herein, the term "identity" refers to the proportion (expressed as a percentage) of nucleotides of 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) over the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of aligned bases that are identical (matching) between two sequences (e.g., a contiguous nucleotide sequence of a compound of the invention and a reference sequence), dividing that 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 will be understood that in determining identity, chemical modifications of the nucleobases are disregarded so long as the functional ability of the nucleobases to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

[0054] Hybridization As used herein, the term "hybridize" or "hybridize" should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming 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), defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often explained by 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 the binding affinity, ΔG°=-RTln(K d ) where R is the gas constant and T is the absolute temperature, giving the dissociation constant (K d) is associated with the reaction temperature. Thus, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction with an aqueous solution concentration of 1M, pH of 7, and temperature of 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and the ΔG° in the case of a spontaneous reaction 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 Discov Today. Those skilled in the art will know that commercially available equipment is available for measuring ΔG°. ΔG° can also be numerically estimated by using the nearest neighbor model described by SantaLucia, 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 possibility of modulating its intended nucleic acid target by hybridization, the oligonucleotides of the present invention hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for oligonucleotides of 10-30 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 estimated ΔG° values ​​in the range of less than -10 kcal, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal for oligonucleotides of 8 to 30 nucleotides in length. The oligonucleotides may hybridize to the target nucleic acid with estimated ΔG° values ​​of, for example, -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal, or -16 to -27 kcal, such as -18 to -25 kcal.

[0055] target nucleic acid Target nucleic acid is the nucleic acid that antisense oligonucleotide can hybridize to and thereby regulate 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.

[0056] 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 that is complementary to the 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 the oligonucleotide of the present invention.

[0057] target cell As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. Suitably, a target cell contains at least one copy of a target gene in its genome. A target cell can be in vivo or in vitro. A 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, for example, a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0058] 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 or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting oligonucleotide (mock).

[0059] High-affinity modified nucleosides High affinity modified nucleosides are modified nucleotides which, when incorporated into an oligonucleotide, e.g., have a higher melting temperature (T m ) increases the affinity of the oligonucleotide for its complementary target. The high affinity modified nucleoside preferably provides an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. 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 & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0060] RNase H activity and recruitment Ribonuclease (RNase) H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining 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 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 bonds between all monomers in the oligonucleotide, using the methodology provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference).

[0061] Gapmar The antisense oligonucleotide or its contiguous nucleotide sequence may be or include a gapmer. Gapmers are typically used to inhibit target nucleic acids via RNase H-mediated degradation. Gapmers are represented herein as 5'-FG-F'-3' (Formula I) and include at least three distinct structural regions in a "5→3" orientation: a 5'-flank, a gap, and a 3'-flank. The "gap" region (G) includes a stretch of contiguous DNA nucleotides that allow the oligonucleotide to recruit RNase H. The gap region is adjacent to a 5'-flanking region (F) that includes one or more sugar-modified nucleosides and a 3'-flanking region (F') that includes one or more sugar-modified nucleosides. The 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., affinity-enhancing sugar-modified nucleosides, such as high-affinity modified nucleosides) or modulate other properties as desired.

[0062] 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 of the 5'(F) or 3'(F') regions, respectively. The flanks may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5'-end of the 5' flank and at the 3'-end of the 3' flank.

[0063] 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, namely FG-F'.

[0064] The total length of the gapmer designed FG-F' is typically 12 to 32 nucleosides, such as 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.

[0065] 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 typically the total length of the gapmer region FG-F' is at least 12, such as at least 14, nucleotides in length.

[0066] Suitable designs for TNA gapmers according to the invention include, for example, F 1~15 -G 3~18 -F' 1~15 (IV), 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.

[0067] Regions F, G, and F' are described further below and may be incorporated into any of the FG-F' formulas.

[0068] Gapmer region G Region G (gap region) of a gapmer is a region of nucleosides, typically DNA nucleosides, that enable 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 the RNA molecule.

[0069] 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, such as 5-16 consecutive DNA nucleosides, such as 6-15 consecutive DNA nucleosides, such as 7-14 consecutive DNA nucleosides, such as 8-12 consecutive DNA nucleotides, for example 8-12 consecutive DNA nucleotides in length.

[0070] Suitable gapmers according to the invention, particularly gapmers comprising one or more TNA nucleosides as described herein, may have a gap region (G) comprising at least three consecutive DNA nucleosides. Gap region G may 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.

[0071] A gap region (G) containing one or more TNA nucleosides as 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 contiguous DNA nucleosides at either the 5' end, the 3' end, or both of the gap region.

[0072] 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, such as 3 to 16 consecutive nucleosides, such as 4 to 18, 4 to 16, 4 to 14, 4 to 12, or 4 to 10 consecutive nucleosides, such as 5 to 18, 5 to 16, 5 to 14, 5 to 12, or 5 to 10 consecutive nucleosides, such as 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 consecutive nucleosides. Shorter gap regions, such as regions G that include or consist of 4, 5, 6, 7, 8, or 9 consecutive nucleosides, such as consecutive DNA nucleosides, are also contemplated.

[0073] One or more cytosine (C) DNA nucleosides in the gap region may be optionally methylated (e.g., a C DNA nucleoside followed by a guanine (G) DNA nucleoside, such as a 5-methyl-cytosine ( me C or m C) is annotated as

[0074] Contemplated oligonucleotides include those in which all modified internucleoside linkages in the gap are phosphorothioate linkages, or those in which all internucleoside linkages in the gap are phosphorothioate linkages.

[0075] Although traditional gapmers have a DNA gap region, there are many examples of modified nucleosides that allow for the recruitment of RNase H when used within the gap region. Modified nucleosides that have been reported to be able to recruit RNase H when included 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), 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" is typically 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'-endo (DNA-like) structure when introduced into the gap region, allowing for RNase H recruitment. The DNA Gap region (G) described herein, for example, can optionally contain one or more (e.g., 1-3) sugar-modified nucleosides. Any two or more sugar-modified nucleosides in the gap can be contiguous or separated by one or more DNA nucleosides.

[0076] As described herein, modified nucleosides that can be used in the gap region include TNA nucleosides.

[0077] Area G - "Gap Breaker" There are also numerous reports on the insertion of modified nucleosides that confer a 3'-end conformation to the gap region of a gapmer 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-disrupting" gapmers, see, for example, WO2013 / 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-specific or even compound-specific (see Rukov et al. 2015 Nucl.Acids Res.Vol.43 pp.8476-8487), and the literature discloses "gap breaker" oligonucleotides that recruit RNase H, in some cases providing more specific cleavage of target RNA. Modified nucleosides used within the gap region of a gap breaker oligonucleotide may be, for example, a modified nucleoside that confers a 3' endo conformation, such as a 2'-O-methyl (OMe) or 2'-O-MOE (MOE) nucleoside, or a beta-D LNA nucleoside (in which the bridge between the C2' and C4' of the ribose sugar ring of the nucleoside is in a beta conformation), such as a beta-D-oxy LNA or ScET nucleoside.

[0078] TNA nucleosides may also be contemplated as gap breakers.

[0079] 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.

[0080] 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 region of the square bracket [D n -E r -D m ], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (gap breaker or gap disrupting nucleoside), and F and F' are flanking regions as defined herein, with the proviso that the total length of the gapmer region FG-F' is at least 12, e.g., 14, nucleotides in length.

[0081] Region G of the gap breaking gapmers described herein can comprise at least four DNA nucleosides, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 DNA nucleosides. As noted above, the DNA nucleosides may be contiguous and, optionally, interspersed with one or more modified nucleosides, provided that the gap region G is capable of mediating RNase H recruitment.

[0082] Gapmer-flanking regions, F and F' Region F' is located immediately adjacent to the 5' DNA nucleoside of region G. The 3' most terminal nucleoside of region F is a sugar-modified nucleoside. Advantageously, the 5' most terminal one or two nucleosides of region F are also sugar-modified nucleosides. In gapmers described herein, particularly gapmers comprising one or more TNA nucleosides, region F is at least 1, such as at least 2, such as at least 3 contiguous nucleotides long. Typically, region F is up to 15 contiguous nucleotides long. For example, region F can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides long, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous nucleotides long.

[0083] Region F' is located immediately adjacent to the 3' DNA nucleoside of region G. The 5' most terminal nucleoside of region F' is a sugar-modified nucleoside. Advantageously, the 3' most terminal one or two nucleosides of region F' are also sugar-modified nucleosides. In gapmers described herein, particularly gapmers comprising one or more TNA nucleosides, region F' is at least 1, such as at least 2, such as at least 3 contiguous nucleotides long. Typically, region F' is up to 15 contiguous nucleotides long. For example, region F' can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides long, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous nucleotides long.

[0084] Any sugar-modified nucleotide can be used in regions F and / or F' of the antisense oligonucleotides described herein, provided that 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 disclosed in the section entitled "Sugar-Modified Nucleosides," including those described in more detail in the sections entitled "Threose Nucleic Acids (TNA)," "2' Sugar-Modified Nucleosides," and "Locked Nucleic Acids." The TNA gapmers described herein can include, for example, one or more TNA nucleosides, LNA nucleosides, MOE nucleosides, or mixtures thereof.

[0085] LNA gapmer 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 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).

[0086] MOE Gapmar An MOE gapmer is a gapmer in which one or both of regions F and F' comprise or consist of an MOE nucleoside, e.g., a 2'-O-MOE nucleoside. An MOE gapmer can be, for example, 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.

[0087] TNA gapmer A "TNA gapmer" or "TNA modified gapmer" is a gapmer in which one or more of the linked nucleosides of regions F, F', and G contains at least one TNA nucleoside.

[0088] A TNA gapmer can have a formula, for example, where F, F', or both F and F' nucleosides consist of TNA nucleosides. Examples of specific designs of TNA gapmers are provided elsewhere herein.

[0089] Mixed Wing Gappar 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 from those disclosed in the section entitled "Sugar Modified Nucleosides", including, but not limited to, those disclosed in the sections entitled "Threose Nucleic Acids (TNA)", "2' Sugar Modified Nucleosides" and "Locked Nucleic Acids".

[0090] Contemplated mixed wing gapmers include, for example, those in which at least one of region F and region F' comprises a TNA nucleoside. The other sugar modified nucleoside(s) may then be selected from 2'-substituted nucleosides, such as, for example, 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.

[0091] 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 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' can, 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.

[0092] Mixed wing gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0093] Alternating Flank Gappar An oligonucleotide having alternating flanks is a gapmer oligonucleotide, in which at least one of the flanks (F or F') comprises DNA in addition to a sugar-modified nucleoside selected from those described herein in the section entitled "Sugar-Modified Nucleosides," including but not limited to those described in the sections entitled "Threose Nucleic Acids," "2' Sugar-Modified Nucleosides," and "Locked Nucleic Acids." For example, apart from DNA, an alternating flank gapmer may comprise TNA, LNA and / or MOE nucleoside(s).

[0094] 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' majority of nucleosides of the F and / or F' regions being sugar-modified nucleosides.

[0095] Area D' or D'' The antisense oligonucleotides described herein may contain additional 5' and / or 3' nucleosides that are not completely complementary to the target nucleic acid. The additional 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0096] 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 serve as a cleavable linker. It can also, or alternatively, be used to provide exonuclease protection or to facilitate synthesis or manufacture.

[0097] Regions D' and D'' can 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.

[0098] The region D' or D" independently comprises or consists of 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides, such as DNA or RNA or base-modified versions thereof. The D' or D" region may serve as a nuclease-sensitive biocleavable linker. For example, the additional 5' and / or 3' terminal nucleotides may be DNA or RNA nucleotides and may be linked by a phosphodiester bond.

[0099] Nucleotide-based biocleavable linkers suitable for use as region D' or D'' are disclosed in WO 2014 / 076195 and include, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, where they have been used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0100] Conjugates As used herein, the term conjugate refers to an oligonucleotide covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or a third region).

[0101] 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 efficacy of oligonucleotides in those organs, tissues or cell types.At the same time, conjugates can help to 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).

[0102] The non-nucleotide moiety (conjugate moiety) can be selected from the group consisting of, for example, carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0103] Linker Bond or linker is the connection between two atoms, which connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.Conjugate moiety can be directly or through a linking moiety (e.g., linker or tether) to oligonucleotide.Linker serves to covalently connect third region, e.g., conjugate moiety (region C), to first region, e.g., oligonucleotide or continuous nucleotide sequence or gapmer region FG-F' (region A).

[0104] The conjugate or oligonucleotide conjugate may optionally comprise 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).

[0105] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally or similar to those encountered in a mammalian body. 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 oxidizing or reducing agents, and 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. Biocleavable linkers can be, for example, susceptible to S1 nuclease cleavage. Biocleavable linkers that include DNA phosphodiesters are described in more detail in WO 2014 / 076195, which is incorporated herein by reference. See also region D' or D'' herein.

[0106] Region Y refers to a linker, not necessarily biocleavable, that serves primarily to covalently connect the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers can include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units or aminoalkyl groups. Oligonucleotide conjugates can be constructed from the following region elements AC, ABC, ABYC, AYBC or AYC. Linkers (region Y) can 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.

[0107] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., referred to herein as a disease or disorder) or the prevention of disease, i.e., prophylaxis. Thus, it will be recognized that the treatment referred to herein can be prophylactic.

[0108] TNA-modified antisense oligonucleotides Despite being unnatural, threose nucleic acid (TNA) can form stable Watson-Crick duplexes and exhibit strong affinity and specificity for complementary RNA targets. As shown herein, TNA modified gapmers offer a new design strategy for antisense oligonucleotide applications. Using caspase 3 / 7 activation, in vitro target knockdown and thermal melting assays, it was demonstrated that TNA 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 designed gapmers, such as LNA or MOE gapmers, TNA units can replace one or more or all but three or four consecutive DNA units in the gap region of state of the art designed gapmers, effectively resulting in, for example, an extension of the 5' or 3' flank and a narrowing of the gap. Furthermore, TNA is poorly recognized by nucleases. Thus, when designed into an antisense oligonucleotide sequence, the TNA unit may provide increased metabolic stability, extended duration of action, or both. Thus, TNA-modified gapmers can provide long-acting therapeutic agents with increased therapeutic index compared to classical gapmer designs.

[0109] Thus, the present invention provides antisense oligonucleotides comprising one or more TNA nucleosides, particularly antisense gapmer oligonucleotides comprising one or more TNA nucleosides. Antisense gapmer oligonucleotides may in particular comprise a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I) that can recruit ribonuclease (RNase) H. Contiguous nucleotide sequences of the formula 5'-FG-F'-3'(I) that comprise at least one TNA residue may be referred to herein as "TNA gapmers". Contemplated designs for TNA gapmers include: G is a gap region of up to 18 linked nucleosides containing at least 3 consecutive DNA nucleosides; each of F and F' is independently a flanking region of up to 15 linked nucleosides that comprises or consists of 1 to 15 sugar-modified nucleosides; At least one of F, F', and G comprises a sugar-modified nucleoside that is a TNA nucleoside. This includes:

[0110] The following sections provide further details regarding TNA gapmers according to the invention, but unless otherwise indicated or contradicted by context, they should be understood to apply equally to antisense gapmer oligonucleotides or conjugates thereof which comprise or consist of a TNA gapmer.

[0111] Advantageously, TNA 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 an RNA, such as a pre-mRNA, mRNA, viral RNA, microRNA or lncRNA target nucleic acid, the TNA gapmer can reduce or inhibit the expression of the target RNA. This is achieved by the complementarity between the TNA gapmer and the target RNA, and suitably by the recruitment of cellular RNases, such as RNase H. TNA gapmers may further be able to reduce or inhibit the expression of the target RNA by non-RNase H mediated mechanisms, such as microRNA inhibition, reduced splice regulation of pre-mRNA, or steric blocking mechanisms that result in blocking the interaction between lncRNA and chromatin.

[0112] Preferably, the TNA 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% compared to the normal expression level of the target. The TNA gapmer is preferably also, 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.

[0113] Assays for assessing the reduction of expression level or inhibition of expression of a particular 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, such as a target RNA. For example, in an in vitro assay in which target cells are incubated with about 25 μM of TNA gapmer, the TNA gapmer can reduce the expression level of the RNA target by at least about 50%, such as at least about 60%, compared to the normal expression level of the RNA target. In such an assay, the TNA gapmer can also or alternatively inhibit the expression of the RNA target by at least about 50%, such as at least about 60%, compared to the normal expression level of the RNA target. At a concentration of about 25 μM, the TNA gapmer can also reduce or inhibit the expression level of the RNA target by 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.

[0114] Normal expression levels of the RNA target can be determined using controls in which target cells are incubated without the TNA gapmer (e.g., in the presence of vehicle only) 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 blood or other tissues of humans or experimental animals. Target cells can be incubated, for example, with the TNA gapmer or control for about 1-5 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 determined 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 determined in the test and control samples. An example of a typical assay for assessing the reduction in expression levels or inhibition of expression of a target RNA by TNA gapmers, which can be adapted for other targets and target cells, is provided in Example 2.

[0115] The ability of TNA gapmers to reduce the expression level of a target or inhibit the expression of a target can also be evaluated by determining the IC50 value, i.e., the concentration of TNA gapmer at which the expression level of the target nucleic acid is reduced by half. In an in vitro assay using a target cell that contains at least one copy of the target gene in its genome and expresses a target, e.g., a target RNA, the IC50 is preferably about 20 μM or less, e.g. about 10 μM or less, e.g. about 5 μM or less. Typically, the IC50 value is determined in a cell assay similar to that described above, except that the target cells are incubated with a dilution series of TNA gapmers that span the IC50 value. An example of a typical assay for evaluating the IC50 reduction of expression level or inhibition of expression of a target RNA by TNA gapmers that can be adapted to other targets and target cells is provided in Example 3.

[0116] The potency of a TNA gapmer can also be assessed relative to a control or "parent" gapmer from which the TNA gapmer is derived and which does not contain any TNA nucleosides. The IC50 value of the TNA gapmer is preferably about 10-fold or less, about 8-fold or less, about 6-fold or less, about 4-fold or less, or about 2-fold or less than the IC50 value of the control or "parent" gapmer.

[0117] The TNA gapmers described herein can also be characterized as having low toxicity. For example, the TNA gapmers can have lower toxicity than a corresponding control gapmer, such as a state-of-the-art reference gapmer or a "parent" gapmer that differs from the TNA gapmer in that its nucleosides do not contain any TNA nucleosides. Suitable assays for assessing the toxicity of gapmers or antisense nucleotides are known in the art, and include, for example, 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 or a control gapmer in an appropriate vehicle, and caspase 3 / 7 activation can be determined approximately 24 hours after transfection. Preferably, caspase 3 / 7 activation from transfection with TNA gapmers is at most about 70%, such as at most about 60%, such as at most about 50%, such as at most about 40%, such as at most about 30%, such as at most about 20% of the corresponding control gapmer. Alternatively, the percentage (assay window %) determined for the TNA gapmer using the caspase 3 / 7 assay described in Example 4 is preferably at most about 200%, more preferably at most about 150%, at most about 100%, at most about 80%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20% or at most about 10%. Preferably, the percentage (assay window %) determined for the TNA gapmer using the caspase 3 / 7 assay described in Example 4 is at most about 60%.

[0118] The TNA gapmers described herein can hybridize to a target nucleic acid, such as a target RNA, particularly a target sequence whose nucleobase sequence is complementary. The ability of a TNA gapmer to hybridize to its target nucleic acid can be assessed according to any assay known in the art. Advantageously, thermal melting (Tm) analysis can be used to determine at what temperature the duplex between the TNA gapmer and its RNA target sequence denatures, which can be expressed as melting temperature or simply Tm. A typical assay for determining the Tm of a TNA gapmer (i.e., in the form of a duplex with a complementary RNA target sequence) is described in Example 5. Briefly, the TNA gapmer and the RNA target sequence can be 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 can be heated to 95°C for 5 minutes, then slowly cooled to room temperature over 1 hour, and a thermal melting curve can be recorded at 260 nm using a temperature gradient, e.g., increasing from 25°C to 95°C at 5°C / min, then decreasing to 25°C. From the derivative of both curves, the melting temperature (Tm) can be determined. Preferably, the TNA gapmer has a Tm of at least about 50°C, such as at least about 52°C, e.g., at least about 54°C, e.g., at least about 56°C, e.g., at least about 58°C, e.g., at least about 60°C, e.g., at least about 65°C, e.g., at least about 70°C.

[0119] In some cases, there may be mismatches, such as one or two mismatches, between the oligonucleotide and the target nucleic acid. Despite the mismatches, hybridization to the target nucleic acid may still be sufficient to exhibit the desired ability to modulate the target.

[0120] Preferably, the TNA gapmer according to the invention comprises: (a) reducing the expression level of the target nucleic acid 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 assay window percentage (AW%) is preferably at most about 60%, such as at most about 40%, such as at most about 20%, such as at most about 10%, (d) has a melting temperature (Tm) in the form of a duplex between the antisense gapmer oligonucleotide and the RNA target sequence of at least about 50°C, such as at least about 52°C, such as at least about 54°C, such as at least about 56°C, such as at least about 58°C, such as at least about 60°C; or (e) A combination of two or more of (a) to (d).

[0121] For example, in some embodiments, preferred TNA gapmers may be characterized by both features (a) and (b); in some embodiments, preferred TNA gapmers may be characterized by both features (a) and (c); in some embodiments, preferred TNA gapmers may be characterized by both features (a) and (d). In some embodiments, a preferred TNA gapmer may be characterized by both features (b) and (c). In some embodiments, a preferred TNA gapmer may be characterized by both features (b) and (d). In some embodiments, a preferred TNA gapmer may be characterized by both features (c) and (d). In some embodiments, a preferred TNA gapmer may be characterized by features (a), (b), and (c). In some embodiments, a preferred TNA gapmer may be characterized by features (a), (b), and (d). In some embodiments, a preferred TNA gapmer may be characterized by features (a), (b), and (c). In some embodiments, a preferred TNA gapmer may be characterized by features (a), (c), and (c). In some embodiments, a preferred TNA gapmer may be characterized by features (b), (c), and (d). Further, in some embodiments, a preferred TNA gapmer may be characterized by all of features (a)-(d).

[0122] 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 gapmer. The reduction in the expression level of (a) and (b) can be determined, 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.

[0123] In some TNA gapmers according to the invention, region F comprises at least one TNA nucleoside. Region F of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

[0124] In some TNA gapmers, the F nucleoside may comprise at least one TNA nucleoside. The F nucleoside 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 nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TNA nucleosides. TNA gapmers in which the F nucleoside comprises or consists of one TNA nucleoside are also contemplated.

[0125] In some TNA gapmers, the F nucleoside comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous TNA nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA nucleosides. The F nucleoside may also consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA nucleosides.

[0126] In some TNA gapmers, at least the 3'-most nucleosides in F are TNA nucleosides. Suitably, at least the 2, at least the 3'-most nucleosides in F can be TNA nucleosides. For example, the 3'-most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleosides in F can be TNA nucleosides.

[0127] In some TNA gapmers, at least the 5'-most nucleosides in F' are TNA nucleosides. Suitably, at least the 2, at least the 3, at least the 4, at least the 5, at least the 6, at least the 7, at least the 8, at least the 9, at least the 10, at least the 11, or at least the 12 nucleosides at the 5'-most end in F' can be TNA nucleosides. For example, the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleosides at the 5'-most end in F' can be TNA nucleosides.

[0128] In some TNA gapmers, both the 3'-most and the 5'-most nucleosides 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.

[0129] Any remaining nucleoside(s) in F can be one or more other sugar-modified nucleosides other than TNA nucleosides (e.g., in the form of a mixed wing gapmer) or one or more DNA nucleosides (e.g., in the form of an alternating flank gapmer). For example, F can further comprise 1 to 8 sugar-modified nucleosides other than TNA nucleosides, such as 2, 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled "Sugar-Modified Nucleosides."

[0130] Also contemplated are TNA gapmers in which all sugar-modified nucleosides of F are TNA nucleosides. TNA gapmers can be, for example, alternating flank gapmers in which the nucleosides of F consist of DNA and TNA with, for example, 1, 2, or 3 DNA nucleosides. Suitably, at least the 5'-most and 3'-most nucleosides of F are TNA nucleosides.

[0131] The nucleoside of F can also consist of a TNA nucleoside. Alternatively, the nucleoside of F may consist of two or more TNA nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides. TNA nucleosides in which the nucleoside of F consists of 13, 14, or 15 TNA nucleosides are also contemplated. Typically, when the nucleoside of F consists of two or more TNA nucleosides, F is a contiguous sequence of linked TNA nucleosides.

[0132] In some TNA gapmers, F does not contain a TNA nucleoside.

[0133] In some TNA gapmers according to the invention, region F' comprises at least one TNA nucleoside. Region F' of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

[0134] In some TNA gapmers, the F' nucleosides may comprise at least one TNA nucleoside. The F' nucleosides 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, at least 12, 13, 14, or 15 TNA nucleosides. TNA gapmers in which the F' nucleosides comprise or consist of one TNA nucleoside are also contemplated.

[0135] In some TNA gapmers, the nucleoside of F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous TNA nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA nucleosides. The nucleoside of F' may also consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous TNA nucleosides.

[0136] In some TNA gapmers, the nucleoside of F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 adjacent TNA nucleosides. The nucleoside of F' may also consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 adjacent TNA nucleosides.

[0137] In some TNA 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, the 5'-most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleosides in F' can be TNA nucleosides.

[0138] In some TNA gapmers, at least the 3'-most nucleoside in F' is a TNA nucleoside. 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, the 3'-most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleosides in F' can be TNA nucleosides.

[0139] In some TNA gapmers, both the 3'-most and the 5'-most nucleosides 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.

[0140] Any remaining nucleoside(s) in F' can be one or more other sugar-modified nucleosides other than TNA nucleosides (e.g., in the form of a mixed wing gapmer) or one or more DNA nucleosides (e.g., in the form of an alternating flank gapmer). For example, F' can further comprise 1 to 8 sugar-modified nucleosides other than TNA nucleosides, such as 2, 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled "Sugar-Modified Nucleosides."

[0141] Also contemplated are TNA gapmers in which all sugar-modified nucleosides of F' are TNA nucleosides. TNA gapmers can be, for example, alternating flank gapmers in which the nucleosides of F' consist of DNA and TNA with, for example, one, two, or three DNA nucleosides. Suitably, at least the 5'-most and 3'-most nucleosides of F' are TNA nucleosides.

[0142] The nucleosides of F' can also consist of TNA nucleosides. Alternatively, the nucleosides of F' can consist of two or more TNA nucleosides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides. TNA nucleosides in which the nucleosides of F' consist of 13, 14, or 15 TNA nucleosides are also contemplated. Typically, when the nucleosides of F' consist of two or more TNA nucleosides, F' is a contiguous sequence of linked TNA nucleosides.

[0143] In some TNA gapmers, F' does not contain any TNA nucleosides.

[0144] G comprises a stretch of contiguous DNA nucleosides that enables the antisense oligonucleotide to recruit RNase H. Suitably, G may 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.

[0145] In some TNA gapmers according to the invention, the G does not include any TNA nucleosides. For example, the G may 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.

[0146] In some TNA gapmers according to the invention, G comprises at least one TNA nucleoside. For example, depending on the total length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth nucleoside from the 5'-most end in G can be a TNA nucleoside. Alternatively, depending on the total length of region G, the second, third, fourth, fifth, sixth, seventh, or eighth nucleoside from the 3'-most end in G can be a TNA nucleoside.

[0147] TNA gapmers in which G comprises two or three TNA nucleosides are also contemplated. The two or three TNA nucleosides may be contiguous or non-contiguous.

[0148] Also contemplated are TNA gapmers 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, for example, comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides, optionally contiguous.

[0149] Additionally, as described herein, other modified nucleosides have been reported to be capable of recruiting RNase H when included within the gap region, and may also or alternatively be included in the gap region of a TNA gapmer. Preferably, in a TNA gapmer that includes at least one, e.g., 1, 2 or 3 TNA nucleosides or other modified nucleosides, e.g., in gap region G, the gap region G still includes 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 gapmers, all nucleosides of G, except for any 1, 2, or 3 TNA nucleosides, are DNA nucleosides. In some TNA gapmers, all nucleosides are DNA nucleosides.

[0150] In some TNAs, any one, two, or three TNA nucleosides, and in particular any contiguous stretch of two or more TNA nucleosides, are 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 nucleosides within region G may, for example, be located adjacent to the 5'-most nucleoside in region G, which is typically a DNA nucleoside.

[0151] As shown herein, TNA gapmers may contain a stretch of only three or four consecutive DNA nucleosides while still allowing recruitment of RNase H. Without being limited by theory, it is contemplated that TNA gapmers with shorter gap regions than those of conventional gapmer designs and / or with one or more TNA residues within the gap region may provide increased resistance to endonuclease-mediated degradation and / or reduced off-target binding compared to conventional gapmer designs. As a result, in some TNA gapmers according to the 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 gapmer comprises 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides, F' and F can optionally be of different lengths, such that, for example, F comprises more linked nucleosides than F'.

[0152] Details regarding the number and arrangement of at least one TNA nucleoside in each respective region F, G, and / or F' are described above and can be incorporated into the general gapmer formula 5'-FG-F'-3'(I). Specifically contemplated TNA gapmers include: (i) region F contains at least one TNA nucleoside, but regions F' and G contain no TNA nucleosides; (ii) region F' contains at least one TNA nucleoside, but regions F and G contain no TNA nucleosides; (iii) region G contains at least one TNA nucleoside, but regions F and F′ contain no TNA nucleosides; (iv) regions F and F′ each contain at least one TNA nucleoside, but region G does not contain any TNA nucleosides; (v) regions F and G each contain at least one TNA nucleoside, but region F′ does not contain any TNA nucleosides; (vi) regions G and F' each contain at least one TNA nucleoside, but region F does not contain any TNA nucleosides; and (vii) regions F, G, and F' each contain at least one TNA nucleoside; Some examples include:

[0153] For example, in a TNA gapmer according to item (iv) or (vii), F and F' may each comprise or consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides.

[0154] In a TNA gapmer according to paragraph (iv) or (vii), for example, 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. Also contemplated are TNA gapmers according to 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 nucleosides.

[0155] Alternatively, in a TNA 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 nucleosides, for example 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. For example, in a TNA gapmer according to item (iv), F and F' together can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 TNA nucleosides.

[0156] In some TNA gapmers, each of regions F and F' can independently comprise or consist of a contiguous sequence of linked sugar-modified nucleosides.

[0157] In some TNA gapmers, at least one of regions F and F' may consist of only one type of sugar-modified nucleoside. For example, the sugar-modified nucleoside may be a high affinity nucleoside or a TNA nucleoside.

[0158] In some TNA 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 nucleoside. In some TNA gapmers, the nucleosides in regions F and F' are all TNA nucleosides. In some TNA gapmers, the nucleosides in regions F and F' are all sugar-modified nucleosides other than TNA nucleosides.

[0159] In some TNA gapmers, one or both of regions F and F' can independently comprise two different sugar-modified nucleosides (mixed wing design). One of the two different sugar-modified nucleosides can be a TNA nucleoside and the other sugar-modified nucleoside can be, for example, a high affinity nucleoside.

[0160] In some TNA gapmers, all nucleosides of region F can be TNA nucleosides. The nucleosides of region F' can then comprise or consist of, for example, sugar-modified nucleosides other than TNA nucleosides, such as 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. For example, F' can comprise 1-8 sugar-modified nucleosides other than TNA nucleosides, such as 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides. Alternatively, F' can consist of 1-8 sugar-modified nucleosides other than TNA nucleosides, such as 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides.

[0161] In some TNA gapmers, all nucleosides of region F' may be TNA nucleosides. The nucleosides of region F may then comprise or consist of, for example, sugar-modified nucleosides other than TNA nucleosides, such as 2' sugar-modified nucleosides, such as high affinity nucleosides. Optionally, region F may comprise two different sugar-modified nucleosides, one of which may be TNA. For example, F may comprise 1-8 sugar-modified nucleosides other than TNA nucleosides, such as 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides. Alternatively, F may consist of 1-8 sugar-modified nucleosides other than TNA nucleosides, such as 3, 4, or 5 sugar-modified nucleosides other than TNA nucleosides.

[0162] In TNA 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 those having a modified sugar moiety selected from the group consisting of: 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 (as in 2'-deoxy-2'-fluororibo-nucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinose-nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxy-, amino- or thio-β-D-locked ribose (as in β-D-LNA); Oxy, amino or thio α-L-locked dribose (as in α-L-LNA), 2',4' constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), Tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA), 3'-deoxy-ribose (as in 3'-deoxy-ribose DNA; 3'-DNA), Unlocked ribose (as in Unlocked Nucleic Acid; UNA), glycol (as in glycol nucleic acid; GNA), hexitols (as in hexitol nucleic acids; HNA), 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabino-fluoro-hexitol (as in 3'-arabino-fluoro-hexitol nucleic acid; Ara-FHNA), cyclohexene (as in cyclohexene nucleic acid; CeNA), and Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA).

[0163] Specifically contemplated are TNA gapmers in which F, F', or both F and F' sugar modified nucleosides comprise or consist of one or more 2' sugar modified nucleosides, such as high affinity 2' sugar modified nucleosides.

[0164] In some TNA 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 at least one of any TNA nucleoside, can be LNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 LNA nucleosides, such as 3, 4, or 5 LNA nucleosides, respectively. Suitable LNA nucleosides include those selected from oxy, amino or thio β-D-locked dribose (β-D-LNA) or those with modified sugar moieties selected from oxy, amino or thio α-L-locked dribose (α-L-LNA), such as β-D-oxy-LNA, 6′-methyl-β-D-oxy LNA, such as (S)-6′-methyl-β-D-oxy-LNA (ScET) and ENA, as well as the LNA nucleosides disclosed in Scheme 2. A particularly contemplated LNA nucleoside is β-D-oxy-LNA.

[0165] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be MOE nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may 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 and 5'-Me-2'-O-MOE. A particularly contemplated MOE nucleoside is 2'-O-MOE.

[0166] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be 2'-OMe nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-OMe nucleosides, e.g., 3, 4, or 5 2'-OMe nucleosides, respectively.

[0167] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be 2'-O-MTE nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-MTE nucleosides, e.g., 3, 4, or 5 2'-O-MTE nucleosides, respectively.

[0168] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of the TNA nucleosides, can be 2'-O-MCE nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-MCE nucleosides, e.g., 3, 4, or 5 2'-O-MCE nucleosides, respectively.

[0169] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be 2'-O-NMA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-NMA nucleosides, e.g., 3, 4, or 5 2'-O-NMA nucleosides, respectively.

[0170] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of the TNA nucleosides, can be 2'-deoxy-2'-fluoro-ribose nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 2'-deoxy-2'-fluoro-ribose nucleosides, such as 3, 4, or 5 2'-deoxy-2'-fluoro-ribose nucleosides, respectively.

[0171] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of the TNA nucleosides, can be 2'-fluoro-2'-arabinose nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 2'-fluoro-2'-arabinose nucleosides, such as 3, 4, or 5 2'-fluoro-2'-arabinose nucleosides, respectively.

[0172] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of the TNA nucleosides, can be 2'-O-benzyl-ribose nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 2'-O-benzyl-ribose nucleosides, such as 3, 4, or 5 2'-O-benzyl-ribose nucleosides, respectively.

[0173] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be cEt nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 cEt nucleosides, e.g., 3, 4, or 5 cEt nucleosides, respectively.

[0174] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be TcDNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 TcDNA nucleosides, e.g., 3, 4, or 5 TcDNA nucleosides, respectively.

[0175] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be 3'-DNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 3'-DNA nucleosides, e.g., 3, 4, or 5 3'-DNA nucleosides, respectively.

[0176] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be UNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' can comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 UNA nucleosides, e.g., 3, 4, or 5 UNA nucleosides, respectively.

[0177] In some TNA gapmers, the sugar-modified nucleosides of 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 at least one of any TNA nucleoside, can be GNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may, for example, comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 GNA nucleosides, e.g., 3, 4, or 5 GNA nucleosides, respectively.

[0178] In some TNA 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 at least one of any TNA nucleoside, can be HNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 HNA nucleosides, such as 3, 4, or 5 HNA nucleosides, respectively. Suitable HNA nucleosides include HNA, FHNA, and Ara-FHNA.

[0179] In some TNA 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 at least one of any TNA nucleoside, can be CeNA nucleosides. In TNA gapmers in which only region G comprises one or more TNA nucleosides, regions F and F' may comprise or consist of, for example, 1, 2, 3, 4, 5, 6, 7, 8 CeNA nucleosides, e.g., 3, 4, or 5 CeNA nucleosides, respectively. Suitable CeNA nucleosides include CeNA and F-CeNA.

[0180] TNA gapmers are specifically contemplated in which 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, such as 12 to 26 nucleosides, such as 14 to 26 nucleosides, such as 14 to 24 nucleosides, such as 14 to 22 nucleosides, such as 16 to 22 nucleosides, such as 16 to 20 nucleosides. 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.

[0181] By way of example, a TNA gapmer according to the invention can be represented by one or more of the following formulae (Formula I) for the region FG-F', provided that the total length of the region FG-F' is at least 12, such as at least 14 nucleotides in length: F 1~15 -G 3~18 -F' 1~15 (IV), e.g., F 1~15 -G 3~18 -F' 1~12 (IVa) or F 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), for example, F 3~12 -G 4-10 -F' 3~9 (VIIa) or F 3~9 -G 4~10 -F' 3~12 (VIIb).

[0182] Each of the regions F, G, and F' described herein can be incorporated into any of the FG-F' formulas.

[0183] In some TNA gapmers, the contiguous nucleotide sequence of formula IVa has a length of at least 16 nucleosides; and (a) the 5'-most nucleoside in F' and the nucleosides in F are independently 3, 4, or 5 high affinity sugar-modified nucleosides; (b) the remaining nucleosides in F are TNA nucleosides, and (c) All nucleosides in G are DNA nucleosides.

[0184] For example, G can contain up to 10 consecutive DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides, such as 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 nucleosides.

[0185] In some TNA gapmers, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides; and (a) F and F' each independently consist of 3, 4, or 5 nucleosides, at least one of the nucleosides in F and F' is a TNA nucleoside, and the remaining nucleosides are high affinity sugar-modified nucleosides; and (b) All nucleosides in G are DNA nucleosides.

[0186] For example, all nucleosides in F and F' can be TNA nucleosides.

[0187] In some TNA gapmers, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides; and (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; and (b) the second, third, fourth, or fifth nucleoside from the 5'-most end in G is a TNA nucleoside, and the remaining nucleosides in G are DNA nucleosides.

[0188] For example, a TNA nucleoside can be located closer to the 5' end of a region G than to the 3' end of the G.

[0189] Particularly contemplated designs of TNA gapmers are as follows: TTTTTddddddddddTTTTT (Design A), where T is TNA and d is DNA. MMMMMTTTTTTddddMMMMM (Design B), where M is MOE (e.g., 2'-O-MOE), T is TNA and d is DNA. TTTTTTTTdddddMMMMM (Design C), where M is 2'-O-MOE, T is TNA and d is DNA.

[0190] Antisense gapmer oligonucleotides comprising TNA gapmers, i.e., contiguous nucleotide sequences of formula 5'-FG-F'-3'(I), may contain additional linked nucleosides, e.g., 1-100, 1-40, 40, 1-30, 1-20, 1-10, or 1-5 linked nucleosides, at the 3' and / or 5' ends of the TNA gapmer. The additional linked nucleosides may, for example, facilitate delivery of the antisense gapmer oligonucleotide to an intended site or target a second molecule. The antisense gapmer oligonucleotide may be, or may be part of, a longer nucleic acid construct. However, it is also contemplated that the antisense gapmer oligonucleotide may consist of a TNA gapmer.

[0191] TNA gapmers and antisense gapmer oligonucleotides that are single-stranded antisense oligonucleotides are specifically contemplated for the present invention. In preparing TNA gapmer or antisense gapmer oligonucleotides according to the present invention, the TNA gapmer or antisense gapmer oligonucleotides are essentially single-stranded, such that the majority of the TNA gapmer or antisense gapmer oligonucleotide molecules are in single-stranded form.

[0192] Manufacturing method Also provided is a method for producing the oligonucleotide of the invention, comprising reacting nucleotide units to form covalently linked consecutive nucleotide units comprising the oligonucleotide.Preferably, the method uses phosphoramidite chemistry (see, for example, Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).

[0193] The synthesis of TNA monomers and their incorporation into oligonucleotides is disclosed, for example, in Zhang and Chaput, "Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012", WO 2012 / 078536, WO 2012 / 118911, and WO 2013 / 179292 A1.

[0194] In particular, a method of 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; The method includes producing a modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, at least one of the nucleotide units comprising a TNA nucleoside; Optionally, methods are provided which include purifying or isolating the modified antisense gapmer oligonucleotide.

[0195] In one embodiment, the modified antisense gapmer oligonucleotide has reduced toxicity, optionally hepatotoxicity, compared to the parent antisense gapmer oligonucleotide.In one embodiment, the modified antisense gapmer oligonucleotide has less 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 increased exonuclease resistance compared to the parent antisense gapmer oligonucleotide.In one embodiment, the modified antisense gapmer oligonucleotide has increased endonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0196] Optionally, the parent antisense gapmer oligonucleotide may be an LNA gapmer or an MOE gapmer, for example an LNA gapmer or an MOE gapmer in which all internucleoside linkages are phosphorothioate linkages.Furthermore, the nucleotide units used in the manufacturing process are advantageously nucleoside phosphoramidites.

[0197] The modified antisense gapmer oligonucleotide may contain any of the TNA gapmer features described herein, for example with respect to regions F, G, and F', and the FG-F' design.

[0198] The present invention also provides an antisense gapmer oligonucleotide obtained or obtainable by the above method.

[0199] The method may further comprise reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugated moiety to the oligonucleotide.

[0200] In a further aspect, a method is provided for producing a composition comprising mixing an oligonucleotide or a conjugated oligonucleotide with a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0201] Pharmaceutical Compositions 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 pharma- ceutically acceptable diluent, carrier, salt, and / or adjuvant.

[0202] 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 pharma- ceutically acceptable diluent, carrier, salt or adjuvant.

[0203] Pharmaceutically acceptable diluents include phosphate buffered saline (PBS) and pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma- ceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in the pharma- ceutically acceptable diluent at a concentration of 50-300 μM solution.

[0204] The oligonucleotide or oligonucleotide conjugate according to the present invention may be in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to conventional acid or base addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. 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, fumaric acid, and the like. 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 pharmaceutical scientists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of the 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, the pharma- ceutically acceptable salt of the compounds provided herein can be a sodium salt.

[0205] 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 of drug delivery, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides further suitable and preferred examples of pharma-ceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0206] The oligonucleotide or oligonucleotide conjugate of the present invention can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations.The composition and method for the preparation of pharmaceutical compositions depend on many criteria, including but not limited to, the route of administration, the extent of disease, or the dose to be administered.

[0207] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3-11, more preferably 5-9 or 6-8, most preferably 7-8, e.g., 7-7.5. The resulting solid form compositions may be packaged in a plurality of 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 applicable creams or ointments.

[0208] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. Particularly with respect to oligonucleotide conjugates, when the prodrug is delivered to the site of action, e.g., a target cell, the conjugate moiety is cleaved from the oligonucleotide.

[0209] Purpose The oligonucleotides or oligonucleotide conjugates described herein may be utilized as research reagents or as diagnostic, therapeutic and prophylactic agents.

[0210] In research, oligonucleotides or oligonucleotide conjugates can be used to specifically regulate the expression of target nucleic acids in cells (e.g., in in vitro cell cultures) and experimental animals, facilitating the functional analysis of targets or the evaluation of their usefulness as targets 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.

[0211] When using oligonucleotides or oligonucleotide conjugates in research or diagnostics, the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0212] Also provided is an in vivo or in vitro method for modulating expression of a target gene in a target cell, comprising a target nucleic acid, comprising administering to said cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the invention.

[0213] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells that form part of the tissue of a mammal, such as a human.

[0214] 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.

[0215] Also provided is an oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition described herein for use as a medicament.

[0216] 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 can be treated by modulating expression of the target gene.

[0217] The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition can be used to treat or prevent disease or disorder caused by, for example, abnormal levels and / or activity of target gene or expression product from target gene, such as RNA or protein.Disease or disorder can also or alternatively be associated with mutation in target gene.Thus, in some embodiments, the target nucleic acid is a mutant form of target gene.Non-limiting examples of target gene include genes associated with one or more cancers, infectious diseases, neurological diseases or disorders, eye diseases or disorders, or cardiovascular diseases or disorders.

[0218] The oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions according to the invention are typically administered in effective amounts.

[0219] Therapeutic applications are also provided for the use of the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition for the treatment or prevention of a disease or disorder in an animal or human suffering from or suspected of having a disease or disorder. Typically, the disease or disorder is one that can be treated by modulating expression of a target gene.

[0220] 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 a disease or disorder. Typically, the disease or disorder is one that can be treated by modulating expression of a target gene.

[0221] Also provided is a method of 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.

[0222] Administration In some embodiments, the oligonucleotide or pharmaceutical composition of the present invention may be administered orally. In further embodiments, the oligonucleotide or pharmaceutical composition of the present invention may be administered topically or enterally or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, or intrathecally).

[0223] 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.

[0224] In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention is administered at a dose of 0.1-15 mg / kg, such as 0.2-10 mg / kg, such as 0.25-5 mg / kg. Administration can be once a week, once every two weeks, once every three weeks, or once a month.

[0225] Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention is used in a combination treatment with another therapeutic agent, which may be, for example, a standard of care for the disease or disorder being treated with the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the present invention.

[0226] Embodiment The following numbered embodiments are specifically contemplated:

[0227] 1. An antisense gapmer oligonucleotide capable of recruiting ribonuclease (RNase) H, comprising a contiguous nucleotide sequence of the formula 5'-FG-F'-3'(I), G is a gap region of up to 18 linked nucleosides containing at least 3 consecutive DNA nucleosides; each of F and F' is independently a flanking region of up to 15 linked nucleosides that comprises or consists of 1 to 15 sugar-modified nucleosides; At least one of F, F', and G comprises a sugar-modified nucleoside that is an α-L-threofuranosyl (TNA) nucleoside; Antisense gapmer oligonucleotides.

[0228] 2. The antisense gapmer oligonucleotide of embodiment 1, wherein F comprises at least one TNA nucleoside.

[0229] 3. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

[0230] 4. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein F comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

[0231] 5. The antisense oligonucleotide of any one of the previous embodiments, wherein at least the 3'-most nucleoside in F is a TNA nucleoside.

[0232] 6. The antisense oligonucleotide of any one of the previous embodiments, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, or 14 of the 3'-most nucleosides in F are TNA nucleosides.

[0233] 7. The antisense oligonucleotide of any one of the previous embodiments, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, or 14 of the 3'-most nucleosides in F are TNA nucleosides.

[0234] 8. The antisense oligonucleotide of any one of the previous embodiments, wherein at least the 5'-most nucleoside in F is a TNA nucleoside.

[0235] 9. The antisense oligonucleotide of any one of the previous embodiments, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, or 14 of the 5'-most nucleosides in F are TNA nucleosides.

[0236] 10. The antisense oligonucleotide of any one of the previous embodiments, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 5'-most nucleosides in F are TNA nucleosides.

[0237] 11. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein all sugar modified nucleosides of F are TNA nucleosides.

[0238] 12. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein all nucleosides of F are TNA nucleosides.

[0239] 13. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 12, wherein F' does not comprise any TNA nucleosides.

[0240] 14. The antisense gapmer oligonucleotide of any one of embodiments 1 to 12, wherein F' comprises at least one TNA nucleoside.

[0241] 15. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

[0242] 16. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein F' comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

[0243] 17. The antisense oligonucleotide of any one of the previous embodiments, wherein at least the 5'-most nucleoside in F' is a TNA nucleoside.

[0244] 18. The antisense oligonucleotide of any one of the previous embodiments, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 5'-most nucleosides in F' are TNA nucleosides.

[0245] 19. The antisense oligonucleotide of any one of the previous embodiments, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 5'-most nucleosides in F' are TNA nucleosides.

[0246] 20. The antisense oligonucleotide of any one of the previous embodiments, wherein at least the 3'-most nucleoside in F' is a TNA nucleoside.

[0247] 21. The antisense oligonucleotide of any one of the previous embodiments, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 3'-most nucleosides in F' are TNA nucleosides.

[0248] 22. The antisense oligonucleotide of any one of the previous embodiments, wherein the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 3'-most nucleosides in F' are TNA nucleosides.

[0249] 23. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein all sugar modified nucleosides at F' are TNA nucleosides.

[0250] 24. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein all nucleosides of F' are TNA nucleosides.

[0251] 25. The antisense gapmer oligonucleotide of any one of embodiments 1 and 14-24, wherein F' does not comprise any TNA nucleosides.

[0252] 26. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein each of F and F' independently comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

[0253] 27. The antisense gapmer oligonucleotide of any one of the previous embodiments, 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.

[0254] 28. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein F, F', or both F and F' further comprise 1 to 8 sugar modified nucleosides other than TNA nucleosides, such as 3, 4, or 5 sugar modified nucleosides other than TNA nucleosides.

[0255] 29. The F, F', or both F and F' sugar modified nucleosides are: 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 (as in 2'-deoxy-2'-fluororibo-nucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinose-nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxy-, amino- or thio-β-D-locked ribose (as in β-D-LNA); Oxy, amino or thio α-L-locked dribose (as in α-L-LNA), 2',4' constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), Tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA), 3'-deoxy-ribose (as in 3'-deoxy-ribose DNA; 3'-DNA), Unlocked ribose (as in Unlocked Nucleic Acid; UNA), glycol (as in glycol nucleic acid; GNA), hexitols (as in hexitol nucleic acids; HNA), 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabino-fluoro-hexitol (as in 3'-arabino-fluoro-hexitol nucleic acid; Ara-FHNA), cyclohexene (as in cyclohexene nucleic acid; CeNA), and Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA) The antisense gapmer oligonucleotide of any one of the preceding embodiments, comprising or consisting of at least one sugar modified nucleoside comprising a modified sugar moiety selected from the group consisting of:

[0256] 30. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein F, F', or both F and F' sugar modified nucleosides comprise or consist of one or more 2' sugar modified nucleosides, such as high affinity 2' sugar modified nucleosides.

[0257] 31. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein F, F', or both F and F' sugar modified nucleosides comprise one or more LNA nucleosides.

[0258] 32. The antisense gapmer oligonucleotide of embodiment 31, wherein all nucleosides in F' and F, except for at least one any TNA nucleoside, are LNA nucleosides.

[0259] 33. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein the sugar modified nucleosides of F, F', or both F and F' comprise one or more 2'-O-methoxyethyl-RNA (2'-O-MOE) nucleosides.

[0260] 34. The antisense gapmer oligonucleotide of embodiment 33, wherein all nucleosides in F' and F, except for at least one of the TNA nucleosides, are 2'-O-MOE nucleosides.

[0261] 35. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein G does not comprise any TNA nucleosides.

[0262] 36. The antisense gapmer oligonucleotide of any one of embodiments 1-34, wherein G comprises at least one TNA nucleoside, such as at least 1, 2, or 3 TNA nucleosides.

[0263] 37. The antisense gapmer oligonucleotide of embodiment 36, wherein the 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th nucleoside from the 5'-most end in the G is a TNA nucleoside.

[0264] 38. The antisense gapmer oligonucleotide of embodiment 36 or 37, wherein the 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th nucleoside from the 3'-most end in the G is a TNA nucleoside.

[0265] 39. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein at least the 5'-most and 3'-most nucleosides in the G are DNA nucleosides.

[0266] 40. The antisense gapmer oligonucleotide of any one of the previous embodiments, wherein all nucleosides of G, except for at least one any TNA nucleoside, are DNA nucleosides.

[0267] 41. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein the gap region G comprises at least 4 DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive DNA nucleosides.

[0268] 42. The antisense gapmer according to any one of the previous embodiments, wherein G comprises up to 10 DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 DNA nucleosides.

[0269] 43. The antisense gapmer oligonucleotide according to any one of the previous embodiments, comprising at least one modified internucleoside linkage.

[0270] 44. The antisense gapmer oligonucleotide according to any one of the previous embodiments, comprising nuclease-resistant modified internucleoside linkages.

[0271] 45. The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein all internucleoside linkages are phosphorothioate internucleoside linkages.

[0272] 46. ​​The antisense gapmer oligonucleotide according to any one of the previous embodiments, wherein said 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.

[0273] 47. The contiguous nucleotide sequence has the following formula: F 1~15 -G 3~18 -F' 1~15 (IV), e.g., F 1~15 -G 3~18 -F' 1~12 (IVa) or F 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); or F 3~12 -G 4~10 -F' 3~12 (VII), for example, F 3~12 -G 4~10 -F' 3~9 (VIIa) or F 3~9 -G 4~10 -F' 3~12 (VIIb) 47. The antisense gapmer oligonucleotide of embodiment 46, wherein the numerical ranges represent the number of linked nucleosides in F, G and F', respectively.

[0274] 48. The antisense gapmer oligonucleotide, (a) capable of reducing said expression level of a target nucleic acid 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 a normal expression level of said 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 assay window percentage (AW%) is preferably at most about 60%, such as at most about 40%, for example at most about 20%, for example at most about 10%; (d) has a melting temperature (Tm) in the form of a duplex between the antisense gapmer oligonucleotide and the RNA target sequence of at least about 50°C, such as at least about 52°C, such as at least about 54°C, such as at least about 56°C, such as at least about 58°C, such as at least about 60°C; or (e) A combination of (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), (a) and (b) and (c), (a) and (b) and (d), (a) and (c) and (d), (b) and (c) and (d), or all of (a) through (d). The antisense gapmer oligonucleotide according to any one of the preceding embodiments.

[0275] 49. The antisense gapmer oligonucleotide of embodiment 48, wherein the target nucleic acid is an RNA target sequence, the RNA target sequence having a nucleobase sequence complementary to the contiguous nucleotide sequence of formula I, and optionally (a) and (b) are determined in a target cell expressing the target nucleic acid and incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days.

[0276] 50. An antisense gapmer oligonucleotide capable of recruiting ribonuclease (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.

[0277] 51. The antisense gapmer oligonucleotide according to embodiment 50, wherein said 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, for example 16 to 20 nucleosides.

[0278] 52. The contiguous nucleotide sequence has the following formula: F 1~15 -G 3~18 -F' 1~15 (IV), e.g., F 1~15 -G 3~18 -F' 1~12 (IVa) or F 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); or F 3~12 -G 4~10 -F' 3~12 (VII), for example, F 3~12 -G 4~10 -F' 3~9 (VIIa) or F 3~9 -G 4~10 -F' 3~12 (VIIb) 52. The antisense gapmer oligonucleotide of any one of embodiments 50 and 51, wherein the numerical ranges represent the number of linked nucleosides in F, G and F', respectively.

[0279] 53. An antisense gapmer according to any one of embodiments 50 to 52, further comprising the features of any one of embodiments 2 to 49.

[0280] 54. The contiguous nucleotide sequence of formula IVa has a length of at least 16 nucleosides; and (a) the 5'-most nucleoside in F and the nucleosides in F are, independently, 3, 4, or 5 high affinity sugar-modified nucleosides; (b) the remaining nucleosides in F are TNA nucleosides, and (c) all nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide according to any one of embodiments 47 to 53.

[0281] 55. The antisense gapmer oligonucleotide according to embodiment 54, wherein G comprises up to 10 consecutive DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides.

[0282] 56. The contiguous nucleotide sequence of formula IV has a length of at least 16 nucleosides; and (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 47 to 53.

[0283] 57. The antisense gapmer oligonucleotide of embodiment 56, wherein all nucleosides in F and F' are TNA nucleosides.

[0284] 58. The contiguous nucleotide sequence of formula IV has a length of at least 16 nucleosides; and (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; and (b) the second, third, fourth, or fifth nucleoside from the 5'-most end in G is a TNA nucleoside, and the remaining nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide according to any one of embodiments 47 to 53.

[0285] 59. The antisense gapmer oligonucleotide of any one of embodiments 54 to 58, wherein said high affinity sugar modified nucleoside is selected from the sugar modified nucleosides of embodiment 29.

[0286] 60. The antisense oligonucleotide of any one of the previous embodiments, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0287] 61. A conjugate comprising an antisense gapmer oligonucleotide according to any one of the preceding embodiments and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker.

[0288] 62. The conjugate of embodiment 61, 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.

[0289] 63. The conjugate of embodiment 61 or 62, wherein the conjugate moiety facilitates delivery across the blood-brain barrier.

[0290] 64. A pharma- ceutically acceptable salt of the antisense gapmer oligonucleotide according to any one of embodiments 1 to 60, or the conjugate according to any one of embodiments 61 to 63.

[0291] 65. A pharmaceutical composition comprising an antisense gapmer oligonucleotide according to any one of embodiments 1 to 60, a conjugate according to any one of embodiments 61 to 63, or a pharma- ceutically acceptable salt according to embodiment 64, and a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0292] 66. An antisense oligonucleotide according to any one of embodiments 1 to 60, a conjugate according to any one of embodiments 61 to 63, a pharma- ceutically acceptable salt according to embodiment 64, or a pharmaceutical composition according to embodiment 65, for use as a medicament.

[0293] 67. A method for preparing a modified version of a parent antisense gapmer oligonucleotide, said parent antisense gapmer comprising a contiguous nucleotide sequence of the formula 5'FG-F'3'(I) capable of recruiting RNase H, wherein G is a gap region of 5-18 linked DNA nucleosides and each of F and F' is independently a flanking region of up to 8 linked nucleosides comprising or consisting of 1-8 sugar-modified nucleosides other than TNA nucleosides, wherein in said modified version at least one nucleoside in F, F' and / or G of said parent antisense gapmer oligonucleotide is replaced with a TNA nucleoside; The method includes producing the modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, at least one of the nucleotide units comprising a TNA nucleoside; Optionally, the method comprises purifying or isolating said modified antisense gapmer oligonucleotide.

[0294] 68. The method of embodiment 67, wherein the modified antisense gapmer oligonucleotide has reduced toxicity, optionally hepatotoxicity, compared to the parent antisense gapmer oligonucleotide.

[0295] 69. The method of any one of embodiments 67 and 68, wherein the modified antisense gapmer oligonucleotide is, optionally, less toxic to HepG2 cells than the parent antisense gapmer oligonucleotide, as determined by a caspase 3 / 7 assay.

[0296] 70. The method of any one of embodiments 67-69, wherein the modified antisense gapmer oligonucleotide has increased exonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0297] 71. The method of any one of embodiments 67-70, wherein the modified antisense gapmer oligonucleotide has increased endonuclease resistance compared to the parent antisense gapmer oligonucleotide.

[0298] 72. The method of any one of embodiments 67 to 71, wherein said parent antisense gapmer oligonucleotide is an LNA gapmer or an MOE gapmer, and optionally, all internucleoside linkages are phosphorothioate linkages.

[0299] 73. The method of any one of embodiments 67 to 72, wherein the nucleotide unit is a nucleoside phosphoramidite.

[0300] 74. The method of any one of embodiments 67 to 73, wherein the modified antisense gapmer oligonucleotide comprises the features of any one of embodiments 1 to 60.

[0301] 75. An antisense gapmer oligonucleotide obtained or obtainable by a method according to any one of embodiments 67 to 74.

[0302] 76. Use of a TNA nucleotide in the preparation of an antisense gapmer oligonucleotide according to any one of embodiments 1 to 60 or a conjugate according to any one of embodiments 61 to 63. EXAMPLES

[0303] Example 1: Oligonucleotide synthesis with TNA modifications Oligonucleotides were synthesized using a MerMade12 automated DNA synthesizer by Bioautomation. Synthesis was performed on a 1 μmol scale using a controlled pore glass support (500 Å) equipped with a universal linker.

[0304] In a standard cycling procedure for coupling of DNA, MOE and LNA phosphoramidites, 4,4-dimethoxytrityl (DMT) deprotection was performed with 3% (w / v) trichloroacetic acid in CHCl for 70 seconds in eight applications of 230 μL. Each phosphoramidite was then cooled to room temperature in acetonitrile (or LNA- m The C component was coupled three times with 95 μL of a 0.1 M solution in acetonitrile / CH 2 Cl 2 1:1) and 110 μL of a 0.3 M solution of 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile as activator with a coupling time of 180 s.

[0305] Freshly prepared α-L-threofuranosyl (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 activator, with a coupling time of 360 s. Sulfurization was performed with two applications of 200 μL for 80 s using a 0.1 M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine:1 / 1. Oxidation was performed with two applications of 0.02 M I2 in THF / pyr / H2O:88 / 10 / 2 for 80 s. Capping was performed twice for 85 s using THF / lutidine / Ac2O 8:1:1 (CapA, 125 μL) and 0.625% DMAP in pyridine (CapB, 125 μL). After synthesis, the controlled pore glass (CPG) was then carefully transferred into a 4 mL vial, 1 mL of 25% NH4OH was added, and left at 55 °C for 24 h. The crude DMT-on oligonucleotides were purified by RP-HPLC purification using a C18 column followed by removal of DMT with 80% aqueous acetic acid or by cartridge purification. The oligonucleotides were characterized by reversed-phase ultra-performance liquid chromatography coupled with high-resolution electrospray mass spectrometry.

[0306] TNA phosphoramidites were synthesized as described in Zhang and Chaput, ''Synthesis of Threose 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.

[0307] Following the procedures described above, the molecules shown in Table 1 were prepared.

[0308] Table 1. Synthetic molecules containing a TNA moiety (targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat-1)) CMP ID number = Compound ID number TIFF2024547071000004.tif148151TIFF2024547071000005.tif237151TIFF2024547071000006.tif23415 1TIFF2024547071000007.tif237151TIFF2024547071000008.tif238151TIFF2024547071000009.tif31151

[0309] In the sequences of Tables 1 to 6, A, G, m C and T (bold) represent α-L-threofuranosyl (TNA) nucleosides; A, G, m C and T (underlined) represent 2'-O-MOE nucleosides; A, G, m C and T represent β-D-oxy-LNA nucleosides; The a, g, c and t represent DNA nucleosides.

[0310] All linkages were prepared as phosphorothioate linkages.

[0311] Further details regarding the molecules in Table 1 are provided in Table 2, 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). In addition, 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] (where [C] is a DNA cytosine 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 TNA 5-methylcytidine nucleoside. [TNA](A) is TNA adenine nucleoside, [TNA] (T) is TNA thymine nucleoside, [TNA](G) is TNA guanine nucleoside; [sP] is a phosphorothioate internucleoside linkage.

[0312] Table 2: Synthetic molecules in HELM annotation TIFF2024547071000010.tif222158TIFF2024547071000011.tif237158TIFF2024547 071000012.tif237158TIFF2024547071000013.tif231158TIFF2024547071000014.t if235158TIFF2024547071000015.tif235158TIFF2024547071000016.tif232158TIF F2024547071000017.tif232158TIFF2024547071000018.tif235158TIFF20245470710 00019.tif235158TIFF2024547071000020.tif232158TIFF2024547071000021.tif23 7158TIFF2024547071000022.tif239158TIFF2024547071000023.tif239158TIFF202 4547071000024.tif231158TIFF2024547071000025.tif237158TIFF20245470710000 26.tif235158TIFF2024547071000027.tif237158TIFF2024547071000028.tif130158

[0313] Further information and open source tools for HELM can be found at the internet addresses www.pistoiaalliance.org / helm-tools / and www.pistoiaalliance.org / membership / (both accessed on December 2, 2022).

[0314] Example 2: In vitro efficacy of oligonucleotides targeting Malat1 RNA in A549 cells at two different concentrations (5 and 25 μM) A549 cell line was purchased from ATCC and maintained in a humidified incubator at 37°C and 5% CO2 as recommended by the supplier. For the assay, 3000 cells / well were seeded in 96 multi-well plates in complete culture medium. Cells were incubated for 24 h before the addition of oligonucleotides dissolved in PBS at the indicated final concentrations. Three days after addition of oligonucleotides, cells were harvested. RNA was extracted using RNeasy 96 RNA purification kit (Qiagen) according to the manufacturer's instructions and eluted in 50 μl water. RNA was then diluted 10-fold in DNase / RNase-free water and heated at 90°C for 1 min.

[0315] 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] and endogenous control GAPDH, Hs99999905_m1 [VIC-MGB-PL]. All primer sets were purchased from Thermo Fisher Scientific. Relative MALAT1 RNA expression levels, also called knockdown (KD) values, were calculated as a percentage of control (PBS-treated cells).

[0316] The results are shown in Table 3. Values ​​separated by " / " indicate individual results when a compound or control was tested in more than one test vial.

[0317] Table 3. In vitro efficacy results (KD) TIFF2024547071000029.tif163153TIFF2024547071000030.tif234153TIFF2024547071000031.tif23415 3TIFF2024547071000032.tif234153TIFF2024547071000033.tif234153TIFF2024547071000034.tif47153

[0318] Example 3: In vitro potency of oligonucleotides targeting MALAT1 mRNA in A549 cells at different concentrations versus dose response curve The A549 cell line was purchased from ATCC and maintained in a humidified incubator at 37 °C and 5% CO2 as recommended by the supplier. For the assay, 3500 cells / well (A549) were seeded in 96 multiwell plates in culture medium. Cells were incubated for 24 h before the addition of oligonucleotides dissolved in PBS. Oligonucleotide concentration range: highest concentration 25 μM, 1:1 dilution in eight steps. Cells were harvested 3 days after addition of oligonucleotides. RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer's instructions and eluted in 50 μl water. RNA was then diluted 10-fold in DNase / RNase-free water (Gibco) and heated at 90 °C for 1 min.

[0319] 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 qPCR: MALAT1, Hs00273907_s1 (FAM-MGB), and GAPDH as an endogenous control. All primer sets were purchased from Thermo Fisher Scientific. IC 50 Values ​​were determined using GraphPad Prism 8 on data from n=2 biological replicates. Results are shown in Table 4.

[0320] Table 4: In vitro potency results (IC50) TIFF2024547071000035.tif39128TIFF2024547071000036.tif235121TIFF2024547071000037.tif169128

[0321] Example 4: Caspase 3 / 7 Activation HepG2 cells were cultured at approximately 70% confluence in MEM medium containing GlutaMax (Gibco #41090) supplemented with 10% heat-inactivated fetal bovine serum. The cells were detached with 0.25% trypsin-EDTA solution (Gibco #25200056) and cultured at 1 × 10 4 HepG2 cells were seeded in black clear 96-well plates (Corning #3904, NY, USA) at a density of 100 cells / well. 24 hours after HepG2 seeding, cells were transiently transfected with Lipofectamine 2000 (Life Technologies #11668019) using 100 nM oligonucleotide 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 cells 24 hours after transfection, incubated for 60 minutes, and cell lysates were transferred to opaque 96-well plates (Corning #3600, NY, USA) before luminescence was determined on an Enspire multimode plate reader (Perkin Elmer) according to the manufacturer's instructions. The results are shown in Table 5, 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.

[0322] (Table 5) In vitro cytotoxicity results ASO = antisense oligonucleotide TIFF2024547071000038.tif169160TIFF2024547071000039.tif231160TIFF2024547071000040.tif46160

[0323] Example 5: Thermal Melting Temperatures (Tm) of Oligonucleotides Containing TNA Modifications Hybridized to RNA The denaturation point (thermal melting temperature = 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 give a final concentration of 1.5 μM. Samples were heated to 95°C for 5 min and then slowly cooled to room temperature over 1 h. 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 first derivative of both curves was used to determine the melting temperature (Tm). Values ​​are averaged over three heating and cooling curves (reported as values ​​± standard deviation). Results are shown in Table 6.

[0324] (Table 6) Thermal melting temperature (Tm) results TIFF2024547071000041.tif76128TIFF2024547071000042.tif233118TIFF2024547071000043.tif233118TIFF2024547071000044.tif221118

[0325] References Crooke et al., Nucleic Acids Research 2020;48(10):5235-5253.DOI:10.1093 / nar / gkaa299 Eckstein, Antisense and Nucleic Acid Drug Development 2009;10:117-121.DOI:10.1089 / oli.1.2000.10.117. Liu et al.,ACS Appl.Mater.Interfaces 2018;10:9736-9743.DOI:10.1021 / acsami.8b01180 Matsuda et al.,Poster;XXIII International Round Table on Nucleosides,Nucleotides and Nucleic acids;August 2018.DOI:10.13140 / RG.2.2.10627.45605 International Publication No. 2012 / 078536 (Quark Pharmaceuticals, Inc.) International Publication No. 2012 / 118911 (Quark Pharmaceuticals, Inc.) International Publication No. 2013 / 179292 A1 (QBI Enterprises Ltd. and Bio-Lab Ltd.) Zhang and Chaput,''Synthesis of Threose Nucleic Acid(TNA)Phosphoramidite Monomers and Oligonucleotide Polymers,Current Protocols in Nucleic Acid Chemistry,4.51.1-4.51.26,2012''.

Claims

1. An antisense gapmer oligonucleotide capable of recruiting ribonuclease (RNase) H, comprising a contiguous nucleotide sequence of the formula 5'-F-G-F'-3'(I), G is a gap region of up to 18 linked nucleosides comprising at least 3 consecutive DNA nucleosides; each of F and F' is independently a flanking region of up to 15 linked nucleosides 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 an α-L-threofuranosyl (TNA) nucleoside; and The sugar-modified nucleosides F, F', or both F and F' are selected from the group consisting of: 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 (as in 2'-deoxy-2'-fluororibo-nucleic acid; 2'-F-RNA), 2'-fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinose-nucleic acid; 2'-F-ANA), 2'-O-benzyl-ribose, Oxy-, amino- or thio-β-D-locked dribose (as in β-D-LNA), Oxy, amino or thio α-L-locked dribose (as in α-L-LNA), 2',4' constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA), 3'-deoxy-ribose (as in 3'-deoxy-ribose DNA; 3'-DNA), unlocked ribose (as in unlocked nucleic acid; UNA), glycol (as in glycol nucleic acid; GNA), hexitols (as in hexitol nucleic acids; HNA), 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabino-fluorohexitol (as in 3'-arabino-fluoro-hexitol nucleic acid; Ara-FHNA), cyclohexene (as in cyclohexene nucleic acid; CeNA), and Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA) at least one sugar-modified nucleoside comprising a modified sugar moiety selected from the group consisting of: Antisense gapmer oligonucleotides.

2. The antisense gapmer oligonucleotide of claim 1 , wherein F comprises at least one TNA nucleoside.

3. 2. The antisense gapmer oligonucleotide of claim 1, wherein F comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

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

5. The antisense gapmer oligonucleotide of claim 1 , wherein F′ comprises at least one TNA nucleoside.

6. 2. The antisense gapmer oligonucleotide of claim 1, wherein F' comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 TNA nucleosides.

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

8. 2. The antisense gapmer oligonucleotide of claim 1, wherein the sugar-modified nucleosides of F, F', or both F and F' comprise one or more 2'-O-methoxyethyl-RNA (2'-O-MOE) nucleosides.

9. 9. The antisense gapmer oligonucleotide of claim 8, wherein all nucleosides in F' and F, except for at least one of any TNA nucleoside, are 2'-O-MOE nucleosides.

10. 2. The antisense gapmer oligonucleotide of claim 1, wherein G comprises at least one TNA nucleoside, such as at least 1, 2, or 3 TNA nucleosides.

11. 2. The antisense gapmer oligonucleotide of claim 1, wherein at least the 5'-most and 3'-most nucleosides in the G are DNA nucleosides.

12. 2. The antisense gapmer oligonucleotide of claim 1, wherein all nucleosides of G, except for at least one of any TNA nucleoside, are DNA nucleosides.

13. 2. The antisense gapmer oligonucleotide of claim 1, 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.

14. The contiguous nucleotide sequence has the following formula: F １～１５ -G ３～１８ -F' １～１５ (IV), for example, F １～１５ -G ３～１８ -F' １～１２ (IVa) or F １～１２ -G ３～１８ -F' １～１５ (IVb); F １～１２ -G ３～１８ -F' １～１２ (V), for example, 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), for example, 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), for example, F ３～１２ -G ４～１０ -F' ３～９ (VIIa) or F ３～９ -G ４～１０ -F' ３～１２ (VIIb) 14. The antisense gapmer oligonucleotide of claim 13, wherein the numerical ranges represent the number of linked nucleosides in F, G and F', respectively.

15. the contiguous nucleotide sequence of Formula IVa has a length of at least 16 nucleosides; and (a) the 5'-most nucleoside in F and the nucleosides in F are independently 3, 4, or 5 high-affinity sugar-modified nucleosides; (b) the remaining nucleosides in F are TNA nucleosides, and (c) all nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide of claim 14.

16. 16. The antisense gapmer oligonucleotide of claim 15, wherein G comprises up to 10 consecutive DNA nucleosides, e.g., 9, 8, 7, 6, 5, or 4 consecutive DNA nucleosides.

17. the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides; and (a) F and F' each independently consist of 3, 4, or 5 nucleosides; (b) all nucleosides in F and F' are TNA nucleosides; (c) all nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide of claim 14.

18. the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides; and (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; and (b) the second, third, fourth, or fifth nucleoside from the 5'-most end in G is a DNA nucleoside, and the remaining nucleosides in G are DNA nucleosides; The antisense gapmer oligonucleotide of claim 14.

19. The antisense gapmer oligonucleotide of claim 1, which is a single-stranded antisense oligonucleotide.

20. A conjugate comprising an antisense gapmer oligonucleotide according to any one of claims 1 to 19 and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker.

21. An antisense gapmer oligonucleotide according to any one of claims 1 to 19, or A conjugate comprising the antisense gapmer oligonucleotide of any one of claims 1 to 19 and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker. A pharmaceutically acceptable salt of

22. The antisense gapmer oligonucleotide according to any one of claims 1 to 19. A conjugate comprising the antisense gapmer oligonucleotide of any one of claims 1 to 19 and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker; or 20. The antisense gapmer oligonucleotide of any one of claims 1 to 19 or a pharmaceutically acceptable salt of a conjugate comprising the antisense gapmer oligonucleotide of any one of claims 1 to 19 and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker; and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant. A pharmaceutical composition comprising:

23. 23. A pharmaceutical composition according to claim 22 for use as a medicament.