Threose nucleic acid antisense oligonucleotides and methods thereof

Abstract

The present invention describes antisense oligonucleotides comprising one or more alpha-L-threofuranosyl (TNA) nucleosides linked to adjacent nucleosides via phosphodiesters (PO) inter-nucleoside linkages and methods of modulating the properties of antisense oligonucleotides by introducing such TNA nucleosides. These are particularly useful for antisense spacer oligonucleotides.

Description

Technical Field

[0001] The present invention relates to antisense oligonucleotides comprising one or more α-L-threofuranosyl (TNA) nucleosides linked to adjacent nucleosides by phosphodiester internucleoside bonds and methods for modulating the properties of antisense oligonucleotides by incorporating such TNA nucleosides. The present invention is particularly applicable to antisense gapper oligonucleotides. Background Art

[0002] In recent years, significant progress has been made in the use of synthetic oligonucleotides as therapeutic agents, resulting in a broad portfolio of clinically validated molecules, including antisense oligonucleotides (such as ribonuclease H (RNase H)-activating spacers), splice-switching oligonucleotides, microRNA inhibitors, small interfering RNA (siRNA), and aptamers, which act through different mechanisms (ST Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008).

[0003] Arguably, one of the most successful modifications is the introduction of a phosphorothioate (PS) bond, 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). Compared to unmodified phosphodiester analogs, phosphorothioate oligodeoxynucleotides exhibit increased protein binding and significantly higher nuclear lytic degradation stability, and therefore have a higher half-life in plasma, tissues, and cells. For example, a recent review (Crooke et al., Nucleic Acids Research 2020; 48 (10): 5235–5253) describes the PS moiety as the main determinant of the distribution of single-stranded antisense oligonucleotides after all routes of administration. Other modifications include locked nucleic acids (LNA) and various 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; Liu et al., ACS Appl. Mater. Interfaces 2018; 10:9736-9743; WO 2012 / 078536; WO 2012 / 118911; and WO 2013 / 179292).

[0004] However, there remains a need for stable, safe, and effective antisense oligonucleotide-based therapeutics. Summary of the invention

[0005] The present inventors have found that one or more α-L-threofuranosyl (TNA) nucleosides can be introduced into antisense oligonucleotides, particularly into antisense spacer oligonucleotides, via bonds other than PS bonds, to adjust the properties of antisense oligonucleotides. Surprisingly, when TNA nucleosides, particularly via phosphodiester (PO) internucleoside bonds, are introduced into the spacer designs as described herein, these nucleosides can produce effective molecules with properties that are advantageous for therapeutic use.

[0006] Thus, the present invention relates to an antisense oligonucleotide comprising at least one TNA nucleoside, which is linked to at least one adjacent nucleoside via an internucleoside bond different from a PS bond, and in particular to an antisense spacer oligonucleotide comprising at least one such TNA nucleoside. Particularly preferred is a PO internucleoside bond. TNA nucleosides linked to at least one adjacent nucleoside via a PO bond may be referred to hereinafter as TNA(PO) nucleosides. Contemplated TNA(PO) nucleosides include those linked by 2'-PO bonds, 3'-PO bonds, or both; hereinafter referred to as 2'-PO linked nucleosides, 3'-PO linked nucleosides, and 2',3'-PO linked TNA nucleosides, respectively.

[0007] The present invention also relates to an antisense spacer oligonucleotide comprising a contiguous nucleotide sequence of formula 5'-FG-F'-3' (I) capable of recruiting RNase H, wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside, which TNA nucleoside is linked to an adjacent nucleoside by a bond different from a PS internucleoside bond, such as a PO internucleoside bond.

[0008] The present invention also relates to an antisense spacer oligonucleotide comprising a continuous nucleotide sequence of formula 5'-FG-F'-3' (I) capable of recruiting ribonuclease (RNase) H, wherein

[0009] G is a spacer region of up to 18 linked nucleosides, said spacer region comprising at least 3 consecutive DNA nucleosides,

[0010] Each of F and F' is a flanking region of up to 15 linked nucleosides, the flanking region independently comprising or consisting of 1 to 15 sugar-modified nucleosides,

[0011] At least one of F, F' and G comprises a sugar modified nucleoside that is an alpha-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside bond other than a PS internucleoside bond, such as by a PO internucleoside bond.

[0012] The present invention also relates to a conjugate comprising an antisense gapper oligonucleotide according to the present invention and at least one conjugate moiety covalently linked to the antisense gapper oligonucleotide, optionally via a linker.

[0013] The present invention also relates to a pharmaceutically acceptable salt of the antisense gapmer oligonucleotide or conjugate according to the present invention.

[0014] The present invention also relates to a pharmaceutical composition comprising the antisense spacer oligonucleotide, conjugate or pharmaceutically acceptable salt according to the present invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0015] The present invention also relates to an antisense oligonucleotide, a conjugate, a pharmaceutically acceptable salt or a pharmaceutical composition according to the present invention for use as a medicament.

[0016] The present invention also relates to a method for preparing a modified form of a parent antisense spacer oligonucleotide, wherein the parent antisense spacer comprises a contiguous nucleotide sequence of formula 5'FG-F'3' (I) capable of recruiting RNase H, wherein G is a spacer region of 5 to 18 linked DNA nucleosides, and each of F and F' is a flanking region of up to 8 linked nucleosides, the flanking regions independently comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified form, at least one nucleoside among F, F' and / or G of the parent antisense spacer oligonucleotide has been replaced by a TNA nucleoside linked to an adjacent nucleoside by an internucleoside bond different from a PS internucleoside bond, such as by a PO internucleoside bond,

[0017] The method comprises the steps of making a modified antisense gapper oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside, and,

[0018] The modified antisense gapmer oligonucleotide is optionally purified or isolated.

[0019] The present invention also relates to an antisense gapmer oligonucleotide obtained or obtainable by the method of the present invention.

[0020] The present invention also relates to the use of TNA nucleotides in the preparation of an antisense gapmer oligonucleotide according to the present invention.

[0021] Further details of these and other aspects and embodiments of the invention are provided in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1. Relative MALAT1 RNA expression in C57BL / 6J mice. Antisense oligonucleotides were administered at a dose of 10 mg / kg by subcutaneous injection and RNA levels were determined on day 9. See Example 6 for details.

[0023] Figure 2. Tissue concentration and pharmacokinetic / pharmacodynamic (PK / PD) relationships. Concentrations of antisense oligonucleotides in liver (A), kidney (B), lung (C) and muscle (D), as well as PK / PD relationships in liver (E), kidney (F), lung (G) and muscle (H) are shown. See Example 6 for details. DETAILED DESCRIPTION

[0024] definition

[0025] In order that the present invention may be more readily understood, certain terms are defined and described below.

[0026] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated integers (or components) or groups of integers (or components) but not the exclusion of any other integers (or components) or groups of integers (or components).

[0027] Oligonucleotides

[0028] As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides (including modified nucleosides or nucleotides) as generally understood by those skilled in the art. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically prepared in a laboratory by solid phase chemical synthesis followed by purification. When referring to an oligonucleotide sequence, reference is made to the core base portion of a covalently linked nucleotide or nucleoside or its modified sequence or order. The oligonucleotides of the present invention are artificial, chemically synthesized, and are typically purified or separated. Nucleosides may be linked by phosphodiester (PO) bonding or by modified internucleoside bonding.

[0029] Antisense Oligonucleotides

[0030] As used herein, the term "antisense oligonucleotide" is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing with a target nucleic acid, particularly with a continuous sequence on the target nucleic acid. The expected antisense oligonucleotide is not substantially double-stranded, and therefore is not siRNA or short hairpin RNA (shRNA). Preferably, the antisense oligonucleotide of the present invention is single-stranded. It should be understood that if the degree of intramolecular or intermolecular self-complementarity is greater than 50% across the full length of the oligonucleotide, 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).

[0031] Continuous nucleotide sequence

[0032] The term "continuous nucleotide sequence" refers to an oligonucleotide region that is complementary to a target nucleic acid. The term is used interchangeably herein with the term "continuous core base sequence" and the term "oligonucleotide motif sequence". For example, all nucleotides of an oligonucleotide can constitute a continuous nucleotide sequence. Alternatively, an oligonucleotide can comprise a continuous nucleotide sequence, such as a FG-F' spacer region, and can optionally comprise other nucleotides, such as a nucleotide joint region that can be used to attach a functional group to a continuous nucleotide sequence. The nucleotide joint region may be complementary or non-complementary to the target nucleic acid. Advantageously, the continuous nucleotide sequence is 100% complementary to the target nucleic acid.

[0033] Nucleotides

[0034] Nucleotides are structural units of oligonucleotides and polynucleotides, and for purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a core base moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0035] Nucleobase

[0036] The term nucleobase includes purines (e.g., adenine and guanine) and pyrimidines (e.g., uracil, thymine and cytosine) present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases, which may be different from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Supplement 37, 1.4.1.

[0037] The nucleobase moiety may optionally be modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-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.

[0038] The nucleobase moiety can be indicated by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, wherein each letter can optionally include a modified nucleobase having an equivalent function. For example, in some oligonucleotides, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine ( m C).

[0039] Modified Nucleosides

[0040] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by introducing one or more modifications of a sugar moiety or (core) base moiety compared to an equivalent DNA or RNA nucleoside. Preferably, the nucleoside modified comprises a modified sugar moiety. The term "modified nucleoside" may also be used interchangeably with the term "nucleoside analog" or modified "unit" or modified "monomer" in this article. Nucleosides with unmodified DNA or RNA sugar moieties are referred to as DNA or RNA nucleosides in this article. Nucleosides with modifications in the base region of a DNA or RNA nucleoside are still generally referred to as DNA or RNA if Watson-Crick base pairing is allowed.

[0041] Sugar-modified nucleosides

[0042] The antisense oligonucleotides of the invention may comprise one or more nucleosides having modified sugar moieties (ie, modifications of the sugar moiety) when compared to the ribose moieties found in DNA and RNA.

[0043] A number of modified nucleosides having a ribose moiety have been produced, primarily for the purpose of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance.

[0044] Such modifications include those in which the ribose ring structure is modified, for example, by replacing with a hexose ring (HNA) or a bicyclic (it usually has a bridge between the C2 carbon atom and the C4 carbon atom of a ribose ring (LNA)) or an unconnected ribose ring (it usually lacks a key between the C2 carbon and the C3 carbon) (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is substituted for a non-sugar moiety, for example, with respect to peptide nucleic acids (PNA) or morpholino nucleic acids.

[0045] Sugar modifications also include modifications by changing the substituents on the ribose ring to groups other than hydrogen or the 2'-OH group naturally present in DNA and RNA nucleosides. For example, substituents can be introduced at the 2', 3', 4' or 5' positions.

[0046] Non-limiting examples of modified sugar moieties include the following:

[0047] α-L-threofuranosyl (as in threose nucleic acid; TNA),

[0048] 2'-Methoxy-ribose (2'-OMe),

[0049] 2'-O-methoxyethyl-ribose (2'-O-MOE),

[0050] 5'-Methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE),

[0051] 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE),

[0052] 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE),

[0053] 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA),

[0054] 2'-deoxy-2'-fluoro-ribose (as in 2'-deoxy-2'-fluoro-ribose-nucleic acid; 2'-F-RNA),

[0055] 2'-Fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinonucleotide; 2'-F-ANA),

[0056] 2'-O-Benzyl-ribose,

[0057] Oxylated β-D-locked ribose (such as in β-D-LNA),

[0058] Amino-β-D-locked ribose (as in amino-β-D-LNA),

[0059] Thio-β-D-locked ribose (such as in thio-β-D-LNA),

[0060] Oxylated β-L-locked ribose (such as in β-L-LNA),

[0061] Amino-β-L-locked ribose (as in amino-β-L-LNA),

[0062] Thio-β-L-locked ribose (such as thio-β-L-LNA),

[0063] Oxylated α-L-locked ribose (such as in α-L-LNA),

[0064] Amino α-L-locked ribose (such as amino-α-L-LNA),

[0065] Thio-α-L-locked ribose (such as thio-α-L-LNA),

[0066] 2',4'-constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt),

[0067] Tricyclic-deoxyribose (such as tricyclic-deoxyribose DNA; TcDNA),

[0068] 3'-deoxy-ribose (such as 3'-deoxy-ribose DNA; 3'-DNA),

[0069] Unlocked ribose (as in unlocked nucleic acid; UNA),

[0070] Diols (as in diol nucleic acids; GNA),

[0071] Hexitols (as in hexitolide nucleic acid; HNA),

[0072] 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA),

[0073] 3'-arabino-fluorohexitol (as in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA),

[0074] Cyclohexene (as in cyclohexene nucleic acid; CeNA),

[0075] Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA),

[0076] Serinol (as serinol nucleic acid; SNA),

[0077] 2'-O,4'-C-ethylene bridged ribose (as in 2'-O,4'-C-ethylene linked nucleic acid; ENA),

[0078] acyclic (L)-threonine (as in acyclic (L)-threonine nucleic acid; aTNA),

[0079] 2',4'-constrained 2'-O-methoxyethyl ribose (as in cMOE), and

[0080] 7',5'-α-bicyclic sugar unit (such as 7',5'-α-bicyclic DNA; in bcDNA).

[0081] Unless otherwise indicated or contradicted by context, the term "MOE" may refer herein to any nucleoside comprising 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.

[0082] Threonose nucleic acid (TNA)

[0083] As used herein, "α-L-threofuranosyl nucleoside", "α-L-threose nucleic acid nucleoside", "TNA nucleoside", "TNA modified nucleoside", "TNA unit", "TNA moiety" and the like refer to a sugar-modified nucleoside comprising an α-L-threofuranosyl moiety.

[0084] The TNA nucleosides are linked to adjacent nucleosides via a (2'->3') internucleoside bond, such as a phosphodiester (PO) or modified internucleoside bond, as shown below for two adjacent TNA nucleosides.

[0085]

[0086] Sugar-modified nucleosides comprising an α-L-threofuranosyl moiety and linked to at least one adjacent nucleoside via a PO bond may be referred to herein as "α-L-threofuranosyl (PO) nucleosides," "α-L-threose nucleic acid (PO) nucleosides," "TNA (PO) nucleosides," "TNA (PO)-modified nucleosides," "TNA (PO) units," "TNA (PO) moieties," etc. Contemplated TNA (PO) nucleosides include those linked by a 2'-PO bond, a 3'-PO bond, or both; hereinafter referred to as "2'-PO linked TNA nucleosides," "3'-PO linked TNA nucleosides," and "2', 3'-PO linked TNA nucleosides," respectively.

[0087] When the nucleobase (B) is cytosine, the TNA or TNA(PO) nucleoside is advantageously 5-methyl-cytosine ( m C) TNA or TNA(PO) nucleoside.

[0088] 2'-Sugar-modified nucleosides

[0089] 2'-sugar modified nucleosides are nucleosides with a substituent other than H or -OH at the 2'-position (2'-substituted nucleosides). This includes nucleosides containing a 2'-linked diradical capable of forming a bridge between the 2'-carbon and a second carbon in the ribose ring, such as LNA (2'-4' bridged) nucleosides.

[0090] For the purposes of this disclosure, a TNA or TNA(PO) nucleoside is not a 2'-substituted nucleoside.

[0091] It has been found that many 2' substituted nucleosides have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can provide enhanced binding affinity to oligonucleotides and / or increased nuclease resistance. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'-O-MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-F-ANA, and 2'-bridging molecules (such as LNA). For further examples, see, for example, Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Scheme 1 below shows schematic diagrams of some 2' substituted modified nucleosides.

[0092] Scenario 1:

[0093]

[0094] Locked Nucleic Acid (LNA)

[0095] "LNA nucleosides" are 2'-modified nucleosides that contain a double radical (also called a "2'-4' bridge") connecting the C2' and C4' ribose sugar rings of the nucleoside, which restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). When LNAs are incorporated into oligonucleotides for complementary RNA or DNA molecules, locking the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0096] Non-limiting exemplary LNA nucleosides are disclosed in WO 99 / 014226; WO 00 / 66604; WO 98 / 039352; WO 2004 / 046160; WO 00 / 047599; WO 2007 / 134181; WO 2010 / 077578; WO 2010 / 036698; WO 2007 / 090071; WO 2009 / 006478; WO 2011 / 156202; WO 2008 / 154401; WO 2009 / 067647; WO 2008 / 150729; Morita et al., Bioorganic & Med. Chem. Lett. 2002, 12, 73-76; Seth et al. J. Org. Chem. 2010, volume 75(5), pages 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.

[0097] By way of further non-limiting illustration, exemplary LNA nucleosides are disclosed in Scheme 2.

[0098] Scenario 2:

[0099]

[0100]

[0101]

[0102] Specific LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy-LNA, such as (S)-6'-methyl-β-D-oxy-LNA (ScET) and ENA. A particularly advantageous LNA is β-D-oxy-LNA.

[0103] Internucleoside bonding

[0104] As commonly understood by the skilled artisan, the term "internucleoside linkage" is defined as a linkage that covalently couples two nucleosides together. In antisense oligonucleotides as described herein, the internucleoside linkage covalently couples adjacent nucleosides together such that a bond is generally formed between the sugar moieties of adjacent nucleosides. Non-limiting examples of internucleoside linkages include phosphodiester (PO) linkages and modified internucleoside linkages.

[0105] Modified internucleoside bonds

[0106] As is generally understood by the skilled artisan, the term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently couples two nucleosides together. Compared to a phosphodiester (PO) linkage, a modified internucleoside linkage can increase the nuclease resistance of an oligonucleotide. The modified internucleoside linkage can stabilize an oligonucleotide for in vivo use and can protect against nuclease cleavage in regions of DNA or RNA nucleosides in an oligonucleotide (e.g., in the spacer region of a spacer oligonucleotide) and in regions of modified nucleosides (such as region F and region F').

[0107] Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. In some oligonucleotides, all internucleoside linkages in the oligonucleotide or its contiguous nucleotide sequence can be nuclease-resistant internucleoside linkages. It is contemplated that the nucleoside that connects the oligonucleotide to a non-nucleotide functional group (such as a conjugate) can be a phosphodiester.

[0108] Phosphorothioate internucleoside linkage

[0109] Preferred modified internucleoside linkages are phosphorothioate (PS). Phosphorothioate internucleoside linkages may be useful due to nuclease resistance, beneficial pharmacokinetics, and ease of manufacture. In some oligonucleotides, all modified internucleoside linkages in an oligonucleotide or its contiguous nucleotide sequence are phosphorothioate linkages.

[0110] Nuclease-resistant bonds, such as phosphorothioate bonds, may be useful, for example, in regions of the oligonucleotide that are capable of recruiting nucleases when forming a duplex with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate bonds may also be used in non-RNase H recruitment regions and / or affinity enhancing regions, such as region F and region F' of a gapmer.

[0111] Non-bridged phosphorodithioate internucleoside linkages

[0112] Another preferred modified internucleoside bond is a non-bridged dithiophosphate internucleoside bond (PS2). PS2 internucleoside bond has two identical sulfur atoms attached to the phosphorus atom, which is achieved by replacing the non-bridged oxygen atom in the phosphorothioate bonding with a sulfur atom. Dithiophosphate internucleoside bonds may be useful due to beneficial nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some oligonucleotides or their continuous nucleotide sequences, the most 3' end or the most 5' end internucleoside bond is a dithiophosphate internucleoside bond. In some oligonucleotides or their continuous nucleotide sequences, the most 3' end and the most 5' end internucleoside bond is a dithiophosphate internucleoside bond. In some oligonucleotides, all modified internucleoside bonds in an oligonucleotide or its continuous nucleotide sequence are dithiophosphate bonds.

[0113] Nuclease-resistant bonds, such as phosphorodithioate bonds, may be useful, for example, in regions of the oligonucleotide that are capable of recruiting nucleases when forming a duplex with a target nucleic acid, such as region G of a gapmer. However, phosphorodithioate bonds may also be used in non-RNase H recruitment regions and / or affinity enhancing regions, such as region F and region F' of a gapmer.

[0114] Complementarity

[0115] The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It should be understood that oligonucleotides may contain nucleosides with modified nucleobases, such as 5-methylcytosine is often used instead of cytosine, and therefore, the term complementarity encompasses Watson-Crick base pairing between unmodified nucleobases and modified nucleobases (see, e.g., Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Supplement 37 1.4.1).

[0116] As used herein, the term "complementarity % " refers to the number of nucleotides in a continuous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) as a percentage, which are complementary to the continuous nucleotide sequence at a given position with different nucleic acid molecules (e.g., target nucleic acids or target sequences) at a given position (i.e., forming Watson Crick base pairs with them). By counting the number of aligned bases formed between the two sequences (when aligned from 3'-5' with the target sequence 5'-3' and the oligonucleotide sequence), it is divided by the total number of nucleotides in the oligonucleotide and multiplied by 100 to calculate the percentage. In such comparisons, the core base / nucleotide that is not aligned (forming base pairs) is called a mismatch. Preferably, insertions and deletions are not allowed when calculating the complementarity % of continuous nucleotide sequences.

[0117] The term "fully complementary" refers to 100% complementarity.

[0118] Identity

[0119] As used herein, the term "identity" refers to the ratio (expressed as a percentage) of nucleotides identical to a reference sequence (e.g., sequence motif) of a continuous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide), and the nucleic acid molecule spans a continuous nucleotide sequence. Therefore, by counting the number of aligned bases that are identical (matched) between two sequences (e.g., in a continuous nucleotide sequence of a compound of the present invention and in a reference sequence), the number is divided by the total number of nucleotides in the alignment region and multiplied by 100 to calculate the identity percentage. Therefore, the length of the alignment region (e.g., continuous nucleotide sequence) is calculated as (number of matches × 100) / alignment region (e.g., continuous nucleotide sequence). When calculating the identity percentage of a continuous nucleotide sequence, insertions and deletions are not allowed. It should be understood that when determining the identity, as long as the functional ability of the core bases forming Watson Crick base pairing is retained, the chemical modification of the core base is not considered (e.g., 5-methylcytosine is considered to be the same as cytosine in order to calculate the identity%).

[0120] Hybridization

[0121] As used herein, the term "hybridizing" or "hybridizes" is understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. The binding affinity between the two nucleic acid strands is the strength of the hybridization. It is usually measured in terms of the melting temperature (T m ), the melting temperature is defined as the temperature at which half of the oligonucleotides and the target nucleic acid form a duplex. Under physiological conditions, T m It is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515–537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity and is proportional to the dissociation constant (K) of the reaction. d ) has ΔG°=-RTln(K d), where R is the gas constant and T is the absolute temperature. Therefore, the very low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with the reaction in which the aqueous concentration is 1M, the pH is 7 and the temperature is 37°C. The hybridization of the oligonucleotide with the target nucleic acid is a spontaneous reaction, and the ΔG° of a spontaneous reaction is less than zero. For example, ΔG° can be measured experimentally using an isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36–38 and Holdgate et al., 2005, Drug Discov Today. Technicians will know that commercial equipment can be used for ΔG° measurement. ΔG ° can also be estimated numerically by using the nearest neighbor model as described in SantaLucia 1998, Proc Natl Acad Sci USA.95:1460–1465, using Sugimoto et al., 1995, Biochemistry 34:11211–11216 and McTigue et al., 2004, Biochemistry 43:5388–5405, describing appropriately derived thermodynamic parameters. In order to have the possibility of regulating its expected nucleic acid target by hybridization, for oligonucleotides of 10-30 nucleotides in length, the oligonucleotides of the present invention hybridize with the target nucleic acid with an estimated ΔG ° of less than -10 kcal. For example, the degree or intensity of hybridization can be measured by the standard state Gibbs free energy ΔG °. For oligonucleotides of 8 to 30 nucleotides in length, the oligonucleotides can hybridize with the target nucleic acid at an estimated ΔG ° value of less than -10 kcal, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal. The oligonucleotide may, for example, hybridize to the target nucleic acid with a ΔG° estimate of -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal or -16 to -27 kcal, such as -18 to -25 kcal.

[0122] Target nucleic acid

[0123] Target nucleic acid is a nucleic acid with which antisense oligonucleotides can hybridize and thereby regulate the expression of a target gene. Target nucleic acid can be, for example, a gene, RNA, mRNA, pre-mRNA, long non-coding RNA (lncRNA), mature mRNA or cDNA sequence or a synthetic nucleic acid derived from DNA or RNA. Target nucleic acid as RNA can be referred to as "RNA target sequence", "target RNA sequence", etc.

[0124] Target sequence

[0125] As used herein, the term "target sequence" refers to a sequence of nucleotides present in a target nucleic acid, which comprises a nucleobase sequence complementary to an antisense oligonucleotide as described herein. The target sequence may, for example, consist of a region on the target nucleic acid that is complementary to a contiguous nucleotide sequence of an oligonucleotide of the invention.

[0126] Target cells

[0127] As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. Suitably, the target cell comprises at least one copy of the target gene in its genome. The target cell may be in vivo or in vitro. The target cell may, for example, be a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell; or a primate cell, such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0128] Regulation of expression

[0129] As used herein, the term "modulation of expression" is understood as a general term for the ability of an oligonucleotide to alter the amount of protein expressed by a target gene 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).

[0130] High affinity modified nucleosides

[0131] High affinity modified nucleosides are modified nucleotides that, when incorporated into an oligonucleotide, enhance the affinity of the oligonucleotide for its complementary target, for example as determined by the melting temperature (T m ). High affinity modified nucleosides preferably increase the melting temperature of each modified nucleoside by between +0.5°C and +12°C, more preferably between +1.5°C and +10°C and most preferably between +3°C and +8°C. Many high affinity modified nucleosides are known in the art, and include, for example, many 2'-sugar substituted nucleosides, such as 2'-O-MOE, 2'-F-RNA and LNA and their analogs (see, e.g., Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443, and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0132] RNase H activity and recruitment

[0133] The ribonuclease (RNase) H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when forming 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 can be obtained from Lubio Science GmbH in Lucerne, Switzerland. Generally, if an oligonucleotide has an initial cleavage rate of a target RNA molecule when provided with a complementary target nucleic acid sequence, then the oligonucleotide is considered to be able to recruit RNase H, the initial cleavage rate being measured in pmol / l / min as a unit, for at least 5% of the initial cleavage rate determined when using an oligonucleotide having the same base sequence as the tested antisense oligonucleotide but containing only DNA monomers having phosphorothioate bonds between all monomers in the oligonucleotide and using the methods provided in Examples 91 to 95 of WO 01 / 23613 (incorporated herein by reference).

[0134] Gap polymer

[0135] Antisense oligonucleotides or their continuous nucleotide sequences can be or include spacer polymers. Spacer polymers are generally used to inhibit target nucleic acids via RNAse H-mediated degradation. Spacer polymers include at least three different structural regions in the '5->3' direction, i.e., 5'-flank, spacer and 3'-flank, represented herein as 5'-FG-F'-3' (Formula I). ​​The "spacer" region (G) includes a section of continuous DNA nucleotides that enable oligonucleotides to recruit RNAse H. The spacer region is flanked by a 5' flank region (F) comprising one or more sugar-modified nucleosides, and a 3' flank region (F') comprising one or more sugar-modified nucleosides. One or more sugar-modified nucleosides in region F and region F' can enhance the affinity of the oligonucleotide to the target nucleic acid (i.e., a sugar-modified nucleoside with enhanced affinity, such as a high-affinity modified nucleoside) or can adjust other desired characteristics.

[0136] In the spacer polymer design, the closest 5' nucleoside and the closest 3' nucleoside of the spacer region are usually DNA nucleosides and are located near the sugar-modified nucleosides of the 5' (F) or 3' (F') regions, respectively. The flanks can be further defined as having at least one sugar-modified nucleoside at the ends farthest from the spacer region, i.e., at the 5' end of the 5' flank and at the 3' end of the 3' flank.

[0137] The region FG-F' forms a continuous nucleotide sequence. The antisense oligonucleotide or its continuous nucleotide sequence may comprise or consist of a gapmer of formula I (ie FG-F').

[0138] The total length of the gapmer design FG-F' is typically 12 to 32 nucleosides, such as 12 to 28, such as 12 to 26, such as 14 to 26, such as 14 to 24, such as 14 to 22, such as 16 to 22 nucleosides, such as 16 to 20 nucleosides.

[0139] Traditional gapmer designs that do not contain TNA nucleosides include, for example, F 1-8 -G 5-18 -F' 1-8 (II), such as F 1-8 -G 7-16 -F' 2-8 (III), usually with the proviso that the total length of the gapmer region FG-F' is at least 12, such as at least 14 nucleotides in length.

[0140] Suitable designs for TNA(PO) spacers according to the present invention include, for example, F 1-15 -G 3-18 -F' 1-15 (IV), generally with the proviso that the total length of the gapmer region FG-F' is at least 12, such as at least 14 nucleotides in length. Additional designs of such gapmers (eg, Formula IV to Formula VII) are described in more detail elsewhere herein.

[0141] Region F, region G, and region F' are further described below, and may be incorporated into any of the FG-F' formulas.

[0142] Spacer-region G

[0143] Region G (spacer region) of the gapmer is a region of nucleosides that enables the oligonucleotide to recruit RNase H, such as human RNase H1, typically to DNA nucleosides. RNase H is a cellular enzyme that recognizes duplexes between DNA and RNA and enzymatically cleaves RNA molecules.

[0144] Traditional gapmers that do not contain TNA nucleosides include, for example, a gap region (G) of at least 5 or 6 consecutive DNA nucleosides in length, such as 5 to 16 consecutive DNA nucleosides, such as 6 to 15 consecutive DNA nucleosides, such as 7 to 14 consecutive DNA nucleosides, such as 8 to 12 consecutive DNA nucleotides, such as 8 to 12 consecutive DNA nucleotides.

[0145] Suitable spacer polymers according to the invention, in particular spacer polymers comprising one or more TNA (PO) nucleosides as described herein, may have a spacer region (G) comprising at least 3 consecutive DNA nucleosides. The spacer region G may, for example, comprise or consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive DNA nucleosides.

[0146] Preferably, the spacer region G comprises at least 4, at least 5 or at least 6 consecutive DNA nucleosides.

[0147] The spacer region (G) comprising one or more TNA(PO) nucleosides as described herein has a DNA nucleoside at the 5' end of the spacer (the 3' nucleoside adjacent to region F), and a DNA nucleoside at the 3' end of the spacer (the 5' nucleoside adjacent to region F'), typically retaining a region of at least 3 or 4 consecutive DNA nucleosides at the 5' end, the 3' end, or both of the spacer region.

[0148] The total length of the spacer region G is generally up to 18 continuous nucleosides. For example, the total length of the spacer region G can be 3 to 18 continuous nucleosides, such as 3 to 16 continuous nucleosides, such as 4 to 18, 4 to 16, 4 to 14, 4 to 12 or 4 to 10 continuous nucleosides, such as 5 to 18, 5 to 16, 5 to 14, 5 to 12 or 5 to 10 continuous nucleosides, such as 6 to 18, 6 to 16, 6 to 14, 6 to 12 or 6 to 10 continuous nucleosides. Shorter spacers are also envisioned, such as comprising 4, 5, 6, 7, 8 or 9 continuous nucleosides (e.g., continuous DNA nucleosides) or the region G consisting thereof.

[0149] In some cases, one or more cytosine (C) DNA nucleosides in the intervening region may be methylated (e.g., when a C DNA nucleoside is followed by a guanine (G) DNA nucleoside) and annotated as 5-methyl-cytosine ( me C or m C).

[0150] Contemplated oligonucleotides include oligonucleotides in which all internucleoside linkages in the gap are phosphorothioate linkages, or oligonucleotides in which all modified internucleoside linkages in the gap are phosphorothioate linkages.

[0151] Although traditional gapmers have a DNA gap region, there are many examples of modified nucleosides that, when used within the gap region, allow for the recruitment of RNase H. Modified nucleosides that have been reported to be able to recruit RNase H when included within the gap region include, for example, α-L-LNA, C4' alkylated DNA (as described in PCT / EP2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both of which are incorporated herein by reference), arabinose-derivatized 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)). UNA or "unlocked nucleic acid" is generally one in which the bond between C2 and C3 of the ribose has been removed, thereby forming a non-locked "sugar" residue. The modified nucleosides used in the spacer polymer can be nucleosides that adopt a 2'-endo (DNA-like) structure when introduced into the spacer region, thereby allowing RNase H recruitment. The DNA spacer region (G) described herein can, for example, optionally contain 1 or more (e.g., 1 to 3) sugar-modified nucleosides. Any two or more sugar-modified nucleosides in the spacer can be continuous or separated by one or more DNA nucleosides.

[0152] As described herein, modified nucleosides useful in the spacer region include TNA(PO) nucleosides.

[0153] Zone G - "Interval Breaker"

[0154] There are also numerous reports on inserting modified nucleosides that confer a 3' endo conformation into the spacer region of the spacer polymer while retaining some RNase H activity. Spacers with the following spacer region are called "gap-breakers" or "gap-disrupted" spacers, which contain one or more 3' endo-modified nucleosides, see, for example, WO2013 / 022984. The spacer breaker oligonucleotide retains enough DNA nucleoside regions in the spacer region to allow the recruitment of RNase H. The ability of the spacer breaker oligonucleotide to recruit RNase H is usually sequence-specific or even compound-specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43, pp. 8476-8487, which discloses "gap-breaker" oligonucleotides that recruit RNase H that provide more specific target RNA cleavage in some cases. The modified nucleosides used in the spacer region of the spacer breaker oligonucleotide can be, for example, modified nucleosides that confer a 3' endo conformation, such as 2'-O-methyl (Ome) or 2'-O-MOE (MOE) nucleosides, or β-D LNA nucleosides (the bridge between C2' and C4' of the ribose sugar ring of the nucleoside is in the β conformation), such as β-D-oxy LNA or ScET nucleosides.

[0155] TNA(PO) nucleosides are also contemplated as spacer breakers.

[0156] As with the gapmer containing region G described above, the gap region of a gapbreaker gapmer or gapinterrupter 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'). A gapmer containing an interrupted gap typically retains a region of at least 3 or 4 consecutive DNA nucleosides at the 5' end, the 3' end, or both of the gap region.

[0157] Exemplary designs of spacer-destroying spacers as described herein include

[0158] F 1-15 -[D 3-4 -E1-D 3-4 ]-F' 1-15

[0159] F 1-15 -[D 1-4 -E1-D 3-4 ]-F' 1-15

[0160] F 1-15 -[D 3-4 -E1-D 1-4 ]-F' 1-15

[0161] The region G is in brackets [D n -E r -D m ], D is a continuous sequence of DNA nucleosides, E is a modified nucleoside (a spacer breaker or a spacer interrupting nucleoside), and F and F' are flanking regions as defined herein, with the proviso that the total length of the spacer region FG-F' is at least 12, such as at least 14 nucleotides in length.

[0162] Region G of the intermittent spacer as described herein may comprise at least 4 DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be continuous, or may be optionally interspersed with one or more modified nucleosides, provided that the spacer region G is capable of mediating RNase H recruitment.

[0163] Spacer-flanking regions, F and F′

[0164] Region F is located next to the 5'DNA nucleoside of region G. The nucleoside closest to 3' of region F is a sugar-modified nucleoside. Advantageously, one or two nucleosides closest to 5' of region F are also sugar-modified nucleosides. In the interval polymer as described herein, in particular, in the interval polymer comprising one or more TNA (PO) nucleosides, the length of region F is at least one, such as at least 2, such as at least 3 continuous nucleotides. Typically, the length of region F is up to 15 continuous nucleotides. For example, the length of region F can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 continuous nucleotides, such as a length of 3,4,5,6,7,8,9,10,11 or 12 continuous nucleotides.

[0165] Region F' is located next to the 3'DNA nucleoside of region G. The nucleoside closest to 5' of region F' is a sugar-modified nucleoside. Advantageously, one or two nucleosides closest to 3' of region F' are also sugar-modified nucleosides. In the interval polymer as described herein, in particular, in the interval polymer comprising one or more TNA (PO) nucleosides, the length of region F' is at least one, such as at least 2, such as at least 3 continuous nucleotides. Typically, the length of region F' is up to 15 continuous nucleotides. For example, the length of region F' can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous nucleotides, such as a length of 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 continuous nucleotides.

[0166] Any sugar-modified nucleotide can be used in region F and / or region F' of the antisense oligonucleotide as 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 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". TNA(PO) spacers as described herein may, for example, comprise one or more TNA nucleosides, LNA nucleosides, MOE nucleosides, or mixtures thereof.

[0167] LNA spacer

[0168] An LNA spacer is a spacer in which one or both of region F and region F' comprises or consists of an LNA nucleoside. A β-D-oxy LNA spacer is a spacer in which one or both of region F and F' comprises or consists of a β-D-oxy LNA nucleoside. An LNA spacer may, for example, have the formula: [LNA] 1–5 -[Region G]-[LNA] 1-5 , wherein region G is as described in the section entitled "Spacer - Region G". An example of a specific LNA spacer design is 3-10-3 (LNA-DNA-LNA).

[0169] cEt spacer

[0170] A cEt gapmer is a gapmer in which one or both of region F and region F' comprises or consists of a cEt nucleoside. A β-D-oxy cEt gapmer is a gapmer in which one or both of region F and F' comprises or consists of a β-D-oxy cEt nucleoside. A cEt gapmer may, for example, have the formula: [cEt] 1–5 -[Region G]-[cEt] 1-5 , wherein region G is as described in the section entitled "Spacer - Region G". An example of a specific cEt spacer design is 3-10-3 (cEt-DNA-cEt).

[0171] MOE spacer

[0172] A MOE gapmer is a gapmer in which one or both of region F and region F' comprises or consists of a MOE nucleoside (e.g., a 2'-O-MOE nucleoside). A MOE gapmer may, for example, have the formula [MOE] 1-8 -[Region G]-[MOE] 1-8 , such as [MOE] 2-7 -[Area G] 5-16-[MOE] 2-7 , such as [MOE] 3-6 -[Region G]-[MOE] 3-6 , wherein region G is as described in the section entitled "Gapsomer - Region G". MOE gapmers (MOE-DNA-MOE) with a 5-10-5 design have been widely used in the art.

[0173] TNA spacer

[0174] A "TNA gapmer" or "TNA-modified gapmer" is a gapmer wherein the linked nucleosides of one or more of region F, region F' and region G comprise at least one TNA nucleoside.

[0175] The TNA gapmer may, for example, have a formula wherein the nucleosides of F, F', or both F and F' consist of TNA nucleosides.

[0176] TNA(PO) spacer

[0177] A "TNA(PO) gapmer" or "TNA(PO)-modified gapmer" is a gapmer wherein the linked nucleosides of one or more of region F, region F' and region G comprise at least one TNA nucleoside linked to at least one adjacent nucleoside via a phosphodiester (PO) internucleoside bond.

[0178] The TNA(PO) spacer may, for example, have a formula wherein the nucleosides of F, F', or both F and F' consist of TNA nucleosides linked by PO internucleoside bonds. These and other examples of specific designs of TNA(PO) spacers are described elsewhere herein.

[0179] Hybrid wing spacer

[0180] Hybrid wing spacer polymers are such spacer polymers, wherein one or both of region F and region F' contain more than one type of sugar-modified nucleosides. Many sugar-modified nucleosides are known in the art and are considered for this purpose. Two or more different sugar-modified nucleosides in the wing region can be, for example, selected from those disclosed in the chapters and sections entitled "Sugar-modified Nucleosides", including but not limited to those disclosed in the chapters and sections entitled "Threose Nucleic Acids (TNA)", "2'-Sugar-Modified Nucleosides" and "Locked Nucleic Acids".

[0181] Contemplated hybrid wing spacers include, for example, those spacers in which at least one of region F and region F' comprises a TNA or TNA(PO) nucleoside. The other sugar-modified nucleosides may then be selected, for example, from 2'-substituted nucleosides, such as 2'-substituted nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units, and 2'-fluoro-ANA units, such as MOE nucleosides.

[0182] Such hybrid wing spacer polymers are also contemplated, wherein, when at least one of region F and region F' or both region F and region F' contain at least one TNA or TNA (PO) nucleoside, the remaining nucleosides of region F and region F' are independently selected from the group consisting of MOE and LNA. When at least one of region F and region F' or both region F and region F' contain at least two LNA nucleosides, the remaining nucleosides of region F and region F' can, for example, be independently selected from the group consisting of: MOE and LNA. In some hybrid wing spacer polymers, one or both of region F and region F' can further contain one or more DNA nucleosides.

[0183] Hybrid wing spacer designs have been disclosed in WO2008 / 049085 and WO2012 / 109395, both of which are incorporated herein by reference.

[0184] Alternating flanking spacer

[0185] Oligonucleotides with alternating flanks are such spacer oligonucleotides, wherein at least one of the flanks (F or F') comprises DNA in addition to sugar-modified nucleosides, e.g., sugar-modified nucleosides selected from those described in the section entitled "Sugar-modified Nucleosides" herein, including but not limited to those described in the section entitled "Threose Nucleic Acids," "2'-Sugar-Modified Nucleosides," and "Locked Nucleic Acids." For example, in addition to DNA, the alternating flank spacer may also comprise TNA, TNA(PO), LNA, and / or MOE nucleosides.

[0186] For example, at least one of region F or region F' or both region F and region F' may contain both sugar-modified nucleosides and DNA nucleosides. Then, flanking region F or flanking region F' or both F and F' generally contain at least three nucleosides, wherein the nucleoside closest to 5' and the nucleoside closest to 3' of region F and / or region F' are sugar-modified nucleosides.

[0187] Area D' or Area D"

[0188] The antisense oligonucleotides described herein may contain additional 5' nucleosides and / or 3' nucleosides that are not fully complementary to the target nucleic acid. The additional 5' nucleosides and / or 3' nucleosides may be referred to herein as region D' and region D".

[0189] For the purpose of joining a contiguous nucleotide sequence (such as a spacer) to a conjugate portion or another functional group, an added region D' or D" may be used. When this region is used to join a contiguous nucleotide sequence to a conjugate portion, this region may act as a cleavable linker. This region may also or alternatively be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0190] Regions D' and D" can be linked to the 5' end of region F or the 3' end of region F', respectively, to generate the following formulas: D'-FG-F', FG-F'-D", or D'-FG-F'-D". In this case, FG-F' is the spacer part of the oligonucleotide, while region D' or D" constitutes a separate part of the oligonucleotide.

[0191] Region D' or D" may independently contain or have 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 phosphodiester bonds.

[0192] WO2014 / 076195 discloses nucleotide-based biocleavable linkers suitable for use as region D' or region D", including, for example, phosphodiester-linked DNA dinucleotides. WO2015 / 113922 discloses the use of biocleavable linkers in oligonucleotide constructs, wherein these linkers are used to link multiple antisense constructs (e.g., spacer regions) within a single oligonucleotide.

[0193] Conjugate

[0194] As used herein, the term "conjugate" refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

[0195] The conjugation of antisense oligonucleotides as described herein to one or more non-nucleotide moieties can improve the pharmacology of oligonucleotides, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotides. Conjugation can, for example, modify or enhance the pharmacokinetic properties of oligonucleotides by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of the oligonucleotides. In particular, the conjugates can target the oligonucleotides to specific organs, tissues or cell types, thereby enhancing the effectiveness of the oligonucleotides in the organ, tissue or cell type. Meanwhile, the conjugates can be used to reduce the activity of the oligonucleotides in non-target cell types, tissues or organs, for example, off-target activity or activity in non-target cell types, tissues or organs.

[0196] The non-nucleotide moiety (conjugate moiety) can, for example, be selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0197] Connectors

[0198] A bond or linker is a connection between two atoms that connects one target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or via a linker (e.g., a linker or tether). The linker is used to covalently link a third region (e.g., a conjugate moiety (region C)) to a first region, such as an oligonucleotide or a continuous nucleotide sequence or a spacer region FG-F' (region A).

[0199] The conjugate or oligonucleotide conjugate may optionally comprise a linker region (second region or region B and / or region Y) between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate portion (region C or third region).

[0200] Region B refers to a biodegradable linker comprising or consisting of a physiologically unstable bond that is cleavable under conditions commonly encountered in mammals or under conditions similar to those encountered. Conditions under which a physiologically unstable linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentrations found in mammalian cells or salt concentrations similar thereto. Conditions within mammalian cells also include the presence of enzymatic activity (such as from proteases or hydrolases or nucleases) that is commonly present in mammalian cells. A biodegradable linker may, for example, be sensitive to cleavage by S1 nuclease. Biodegradable linkers containing DNA phosphodiesters are described in more detail in WO 2014 / 076195 (incorporated herein by reference), see also Region D' or Region D" herein.

[0201] Region Y refers to a linker that is not necessarily biocleavable but is primarily used to covalently link the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). The region Y linker may comprise a chain structure or oligomer of repeating units such as diols, amino acid units or aminoalkyl groups. The oligonucleotide conjugate may be composed of the following regional elements AC, ABC, ABYC, AYBC or AYC. The linker (region Y) may, for example, be an aminoalkyl group such as C2 to C 36 Aminoalkyl groups include, for example, C6 to C 12 Preferably, the linker (region Y) is a C6 aminoalkyl group.

[0202] treat

[0203] As used herein, the term 'treatment' refers to both the treatment of an existing condition (eg a disease or disorder as referred to herein) or the prevention (ie prophylaxis) of a disease. It will therefore be appreciated that treatment as referred to herein may be prophylactic.

[0204] TNA-modified antisense oligonucleotides

[0205] Although threose nucleic acid (TNA) is non-natural, threose nucleic acid can form a stable Watson Crick duplex and show strong affinity and specificity to complementary RNA targets. As shown herein, TNA (PO) modified spacer polymers provide new design strategies for antisense oligonucleotide applications, particularly as an alternative to traditional spacer polymers that only use PS bonds. Using caspase 3 / 7 activation, in vitro target knockdown and thermal melting assays, it is demonstrated that TNA (PO) modifications can be used to reduce toxicity while still maintaining the target knockdown efficacy and affinity for target nucleic acids. For example, in the spacer polymers (such as LNA or MOE spacer polymers) designed in the prior art, TNA (PO) units can replace one or more or all LNA or MOE units in the flanking region. TNA (PO) units can also replace one or more or up to all (except three or four) continuous DNA units in the spacer region of the spacer polymer designed in the prior art, for example, this can effectively produce extended 5' or 3' flanks and reduced intervals. In addition, TNA is difficult to be recognized by nucleases. Therefore, when designed as a spacer, the TNA (PO) unit can maintain or increase metabolic stability, maintain or extend the duration of action, or both, while reducing sulfur content compared to spacer designs using only PS bonds. Therefore, compared to the classical spacer design, TNA (PO) modified spacers can produce long-acting therapeutic agents with increased therapeutic index.

[0206] Thus, the present invention provides antisense oligonucleotides, such as antisense spacer oligonucleotides, comprising TNA nucleosides linked to adjacent nucleosides via PO internucleoside bonds. TNA nucleosides can be linked to adjacent nucleosides via 2'-PO internucleoside bonds or 3'-PO internucleoside bonds. TNA nucleosides can also be linked to any adjacent nucleoside via 2'-PO or 3'-PO internucleoside bonds. Non-limiting examples of adjacent nucleosides include sugar-modified nucleosides and DNA nucleosides. For example, TNA nucleosides can be linked to adjacent nucleosides via 2'-PO internucleoside bonds and 3'-modified internucleoside bonds, respectively;

[0207] through 2'-modified internucleoside bonds and 3'-PO internucleoside bonds, respectively;

[0208] or linked to the first and second adjacent nucleosides via a 2'-PO internucleoside bond and a 3'-PO internucleoside bond, respectively, optionally wherein any 2'-modified or 3'-modified internucleoside bond is a PS internucleoside bond, or wherein any 2'-modified or 3'-modified internucleoside bond is a PS2 internucleoside bond.

[0209] The present invention also provides antisense gapper oligonucleotides, such as antisense gapper oligonucleotides, comprising one or more TNA(PO) nucleotides, generally selected from the group consisting of 2'-PO linked, 3'-PO linked and 2',3'-PO linked TNA nucleosides.

[0210] The antisense gapmer oligonucleotide may specifically comprise a contiguous nucleotide sequence of formula 5′-FG-F′-3′(I) capable of recruiting ribonuclease (RNase) H. A contiguous nucleotide sequence of formula 5′-FG-F′-3′(I) comprising at least one TNA(PO) nucleoside may be referred to herein as a “TNA(PO) gapmer”. Contemplated designs of TNA(PO) gapmers include those wherein

[0211] G is a spacer region of up to 18 linked nucleosides, said spacer region comprising at least 3 consecutive DNA nucleosides,

[0212] Each of F and F' is a flanking region of up to 15 linked nucleosides, said flanking region independently comprising or consisting of 1 to 15 sugar-modified nucleosides, and

[0213] At least one of F, F' and G comprises a sugar-modified nucleoside which is a TNA(PO) nucleoside.

[0214] Although the following sections provide more details on TNA(PO) spacers, it should be understood that they also apply to antisense spacer oligonucleotides or conjugates thereof, which comprise or consist of a TNA(PO) spacer, unless otherwise indicated or contradicted by context.

[0215] Advantageously, TNA (PO) spacer polymers can regulate the expression of target genes by reducing or inhibiting the expression of target genes as mRNA and / or protein, usually by hybridizing with target nucleic acids. When the target nucleic acid is RNA (e.g., pre-mRNA, mRNA, viral RNA, microRNA or lncRNA target nucleic acid), TNA (PO) spacer polymers can reduce or inhibit the expression of target RNA. This is achieved by the complementarity between the TNA (PO) spacer polymer and the target RNA and by appropriately recruiting cellular RNA enzymes (such as RNAse H). TNA (PO) spacer polymers can also reduce or inhibit the expression of target RNA by non-RNAse H-mediated mechanisms, such as steric blocking mechanisms (causing microRNA inhibition, reduced splicing regulation of pre-mRNA or blocking of the interaction between lncRNA and chromatin).

[0216] Preferably, the TNA (PO) spacer can reduce 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 (PO) spacer can also preferably or alternatively inhibit the 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.

[0217] Determinations for evaluating the reduction of expression or inhibition of expression of a specific target are known to the skilled person. Suitable determinations include in vitro determinations using target cells that contain at least one target gene copy in the genome and express a target, such as a target RNA. For example, in an in vitro determination in which the target cells are incubated with about 25 μM TNA (PO) spacer polymers, the TNA (PO) spacer polymers may be able to 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 determinations, the TNA (PO) spacer polymers may also or alternatively be able to inhibit the expression of the RNA target by at least about 50%, such as at least about 60%, compared to the normal expression of the RNA target. At a concentration of about 25 μM, the TNA (PO) spacer polymers may also be able to 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.

[0218] Normal expression levels of RNA targets can be determined using controls, where target cells are incubated in the absence of TNA (PO) spacer polymers (e.g., in the presence of a vehicle alone) or with an unrelated control oligonucleotide. Target cells for in vitro assays can be obtained from commercial sources (e.g., in the form of cell lines) or isolated from blood or other tissues of humans or experimental animals. Target cells can be incubated with TNA (PO) spacer polymers or controls for about 1 to 5 days, such as about 2, 3 or 4 days, such as about 3 days, for example. RNA can then be extracted and the amount of the remaining target RNA in the test and control samples determined by gene expression analysis. Alternatively, the level of RNA species (e.g., mRNA) or protein derived from the target RNA can be determined in the test and control samples instead of determining the remaining target RNA. An example of a typical assay is provided in Example 2, which is used to evaluate the reduction of the expression level of the target RNA or the inhibition of expression by the TNA (PO) spacer polymer, and the typical assay can be applied to other targets and target cells.

[0219] The ability of a TNA(PO) spacer to reduce the expression level of a target or inhibit the expression of a target can also be evaluated by determining an IC50 value (i.e., the concentration of the TNA(PO) spacer when the expression level of the target nucleic acid is halved). In an in vitro assay using a target cell that contains at least one copy of a target gene in its genome and expresses a target (e.g., a target RNA), the IC50 is preferably no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM. Typically, the IC50 value is determined in a cell assay similar to the assay already described, except that the target cells are incubated with a dilution series of TNA(PO) spacers spanning the IC50 value. An example of a typical assay for evaluating the IC50 reduction in the expression level of a target RNA or the inhibition of expression by a TNA(PO) spacer is provided in Example 3, which can be applied to other targets and target cells.

[0220] The ability of the TNA(PO) gapmer can also be evaluated relative to a control or "parent" gapmer from which the TNA(PO) gapmer is derived and which does not contain any TNA nucleosides. The IC50 value of the TNA(PO) gapmer is preferably no more than about 10 times, no more than about 8 times, no more than about 6 times, no more than about 4 times, or no more than about 2 times that of the control or "parent" gapmer.

[0221] TNA (PO) spacer polymers may also be characterized by having low toxicity. For example, TNA (PO) spacer polymers may have lower toxicity than corresponding control spacer polymers, such as reference spacer polymers or "parent" spacer polymers of the prior art, which differ from TNA (PO) spacer polymers in that the nucleosides of the reference spacer polymers or "parent" spacer polymers do not include any TNA nucleosides. Suitable assays for assessing the toxicity of spacer polymers or antisense nucleotides are known in the art and include, for example, in vitro assays, such as caspase 3 / 7 assays. Caspase 3 / 7 assays reflect the level of apoptosis induced by the compound and are suitable for assessing the hepatotoxicity risk of the compound, for example, based on tests on hepatocytes or cell lines. Briefly, HepG2 cells from commercial sources can be transfected with 100nM TNA (PO) or control spacer polymers in a suitable vehicle, and caspase 3 / 7 activation is determined at about 24 hours after transfection. Preferably, the caspase 3 / 7 activation caused by transfection with the TNA(PO) spacer is at most about 70% of the corresponding control spacer, 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%. Alternatively, using the caspase 3 / 7 assay described in Example 4, the percentage determined for the TNA(PO) spacer (assay window%, reflecting the percentage of apoptotic cells to total cells) is preferably at most about 250%, more 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, using the Caspase 3 / 7 assay described in Example 4, the percentage determined for the TNA(PO) gapmer (Assay Window %) is at most about 80%, such as at most about 60%.

[0222] The TNA (PO) spacer polymer is capable of hybridizing with a target nucleic acid (e.g., a target RNA), in particular a target sequence complementary to its nucleobase sequence. The ability of the TNA (PO) spacer polymer to hybridize with its target nucleic acid can be evaluated according to any assay known in the art. Advantageously, a thermal melting (Tm) analysis can be used to determine at which temperature the duplex between the TNA (PO) spacer polymer and its RNA target sequence denatures, which can indicate the melting temperature or simply Tm. A typical assay for determining the Tm of a TNA (PO) spacer polymer (i.e., in the form of a duplex with a complementary RNA target sequence) is described in Example 5. Briefly, the TNA (PO) spacer polymer 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 produce a final concentration of 1.5 μM. The sample can be heated to 95°C for 5 min, and then slowly cooled to room temperature over a 1 hour period, and a thermal melting curve is recorded at 260 nm using a temperature gradient, which is, for example, increased from 25°C to 95°C at 5°C / min, and then dropped to 25°C. According to the derivative of the two curves, the melting temperature (Tm) can be determined. Preferably, the TNA(PO) spacer has a Tm 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, such as at least about 65°C, such as at least about 70°C.

[0223] In some cases, there may be mismatches between the oligonucleotide and the target nucleic acid, such as 1 or 2. Despite the mismatches, hybridization to the target nucleic acid may still be sufficient to exhibit the desired ability to modulate the target.

[0224] Preferably, the TNA(PO) spacer

[0225] (a) the expression level of the target nucleic acid can be reduced by at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as compared to the normal expression level of the target;

[0226] (b) having an IC50 of no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM, for reducing the expression of a target nucleic acid;

[0227] (c) Using the caspase 3 / 7 assay described in Example 4, the window percentage (AW%) was determined.

[0228] Preferably at most about 60%, such as at most about 40%, such as at most about 20%, such as at most about 10%;

[0229] (d) in the form of a duplex of an antisense spacer oligonucleotide and an RNA target sequence, having a melting temperature (Tm) 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

[0230] (e) A combination of any two or more of (a) to (d).

[0231] For example, a preferred TNA(PO) spacer polymer may be characterized by both features (a) and (b). Another preferred TNA(PO) spacer polymer may be characterized by both features (a) and (c). Another preferred TNA(PO) spacer polymer may be characterized by both features (a) and (d). Another preferred TNA(PO) spacer polymer may be characterized by both features (b) and (c). Another preferred TNA(PO) spacer polymer may be characterized by both features (b) and (d). Another preferred TNA(PO) spacer polymer may be characterized by both features (c) and (d). Another preferred TNA(PO) spacer polymer may be characterized by features (a), (b) and (c). Another preferred TNA(PO) spacer polymer may be characterized by features (a), (b) and (d). Another preferred TNA(PO) spacer polymer may be characterized by features (a), (c) and (c). Another preferred TNA(PO) spacer may be characterized by features (b), (c) and (d).Preferred TNA(PO) spacer may also be characterized by all of features (a) to (d).

[0232] Preferably, the target nucleic acid in (a) and (b) is RNA, and the RNA target sequence in (c) has a nucleobase sequence complementary to the contiguous nucleotide sequence of the TNA(PO) spacer. 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 the antisense spacer oligonucleotide at a concentration of about 25 μM for about 3 days, as already described above.

[0233] In some TNA(PO) gapmers, region F comprises at least one TNA nucleoside. Region F of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

[0234] In some TNA (PO) gapmers, region F comprises at least one TNA (PO) nucleoside. Region F may also comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA (PO) nucleosides. TNA (PO) gapmers are also envisioned, wherein region F comprises or consists of one TNA (PO) nucleoside.

[0235] In some TNA (PO) gapmers, region F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides. Region F may also consist of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides.

[0236] In some TNA(PO) spacers, at least the nucleoside closest to the 3' in F is a TNA nucleoside. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve nucleosides closest to the 3' in F may be TNA nucleosides. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen nucleosides closest to the 3' in F may be TNA nucleosides, such as TNA(PO) nucleosides.

[0237] In some TNA(PO) gapmers, at least the nucleoside closest to the 5' in F is a TNA nucleoside. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve nucleosides closest to the 5' in F may be TNA nucleosides. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen nucleosides closest to the 5' in F may be TNA nucleosides, such as TNA(PO) nucleosides.

[0238] In some TNA(PO) gapmers, both the 3'-most nucleoside and the 5'-most nucleoside in F are TNA nucleosides. For example, the 3'-most nucleoside and / or the 5'-most nucleoside in F can be independently selected from one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides, such as TNA(PO) nucleosides.

[0239] Suitably, in a TNA(PO) gapmer, each TNA nucleoside in F may be a TNA(PO) nucleoside. However, TNA(PO) gapmers are also envisioned in which, except for the TNA nucleoside closest to the 5' in F, each TNA nucleoside in F is a TNA(PO) nucleoside. For example, the TNA nucleoside closest to the 5' may be linked to the adjacent nucleoside via a PS or PS2 internucleoside bond.

[0240] Any remaining nucleosides in F can be one or more other sugar-modified nucleosides (e.g., in the form of mixed wing spacer polymers) or one or more DNA nucleosides (e.g., in the form of alternating flank spacer polymers) except TNA (PO) nucleosides. For example, F can further comprise 1 to 8 sugar-modified nucleosides except TNA (PO) nucleosides, such as two, three, four or five sugar-modified nucleosides except TNA (PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the chapters entitled "sugar-modified nucleosides", such as, for example, LNA, 2'-O-MOE and TNA nucleosides, optionally connected by PS or PS2 internucleoside bonds.

[0241] Also contemplated are TNA(PO) gapmers in which all sugar-modified nucleosides of F are TNA(PO) nucleosides. TNA(PO) gapmers may, for example, be alternating flanking gapmers in which the nucleosides of F consist of DNA and TNA(PO), with, for example, 1, 2 or 3 DNA nucleosides. Suitably, then at least the 5'-most proximal nucleoside and the 3'-most proximal nucleoside of F are TNA(PO) nucleosides.

[0242] The nucleoside of F can also be composed of TNA (PO) nucleosides. Alternatively, region F can be composed of more than one TNA (PO) nucleoside (such as two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA (PO) nucleosides). It is also envisioned that the nucleoside of F is composed of thirteen, fourteen or fifteen TNA (PO) nucleosides. Generally, when the nucleoside of F is composed of two or more TNA (PO) nucleosides, F is a continuous sequence of connected TNA (PO) nucleosides.

[0243] In some TNA gapmers, F does not contain any TNA(PO) nucleosides.

[0244] In some TNA(PO) gapmers, region F' comprises at least one TNA nucleoside.Region F' of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

[0245] In some TNA(PO) gapmers, region F' comprises at least one TNA(PO) nucleoside. Region F' may also comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve TNA(PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA(PO) nucleosides. TNA(PO) gapmers are also envisioned in which region F' comprises or consists of one TNA(PO) nucleoside.

[0246] In some TNA (PO) gapmers, region F 'contains at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides. Region F 'can also consist of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides.

[0247] In some TNA(PO) spacers, at least the nucleoside closest to 5' in F' is a TNA nucleoside. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve nucleosides closest to 5' in F' may be TNA nucleosides. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen nucleosides closest to 5' in F' may be TNA nucleosides, such as TNA(PO) nucleosides.

[0248] In some TNA(PO) spacers, at least the nucleoside closest to 3' in F' is a TNA nucleoside. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve nucleosides closest to 3' in F' may be TNA nucleosides. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen nucleosides closest to 3' in F' may be TNA nucleosides, such as TNA(PO) nucleosides.

[0249] In some TNA(PO) spacers, both the nucleoside closest to 3' and the nucleoside closest to 5' in F' are TNA nucleosides. For example, the nucleoside closest to 3' and / or the nucleoside closest to 5' in F' can be independently selected from one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides, such as TNA(PO) nucleosides.

[0250] Suitably, in a TNA(PO) spacer, each TNA nucleoside in F' may be a TNA(PO) nucleoside. However, TNA(PO) spacers are also envisioned in which, except for the TNA nucleoside closest to the 3' in F', each TNA nucleoside in F is a TNA(PO) nucleoside. For example, the TNA nucleoside closest to the 3' may be linked to the adjacent nucleoside via a PS or PS2 internucleoside bond.

[0251] Any remaining nucleosides in F' can be one or more other sugar-modified nucleosides (e.g., in the form of mixed wing spacer polymers) or one or more DNA nucleosides (e.g., in the form of alternating flank spacer polymers) other than TNA (PO) nucleosides. For example, F' can further comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as two, three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the chapters entitled "Sugar-modified Nucleosides", such as, for example, LNA, 2'-O-MOE and TNA nucleosides, optionally linked by PS or PS2 internucleoside bonds.

[0252] Also contemplated are TNA(PO) spacer polymers in which all sugar-modified nucleosides of F' are TNA(PO) nucleosides. TNA(PO) spacer polymers may, for example, be alternating flanking spacer polymers in which the nucleosides of F' consist of DNA and TNA(PO), with, for example, 1, 2 or 3 DNA nucleosides. Suitably, then at least the nucleoside closest to the 5' and the nucleoside closest to the 3' of F' are TNA(PO) nucleosides.

[0253] The nucleosides of F' may also consist of TNA(PO) nucleosides. Alternatively, region F' may consist of more than one TNA(PO) nucleosides (such as two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA(PO) nucleosides). It is also envisioned that the nucleosides of F' consist of thirteen, fourteen or fifteen TNA(PO) nucleosides. Typically, when the nucleosides of F' consist of two or more TNA(PO) nucleosides, F' is a continuous sequence of connected TNA(PO) nucleosides.

[0254] In some TNA gapmers, F' does not contain any TNA nucleosides.

[0255] G comprises a stretch of contiguous DNA nucleosides which enables the antisense oligonucleotide to recruit RNase H. Suitably, G may comprise up to eighteen nucleosides, such as DNA nucleosides. Typically, at least the 5'-most nucleoside in G and the 3'-most nucleoside in G are DNA nucleosides.

[0256] In some TNA(PO) gapmers, G does not contain any TNA nucleosides. For example, G can contain at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides.

[0257] In some TNA(PO) 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 5′-most nucleoside in G may be a TNA nucleoside. Alternatively, depending on the total length of region G, the second, third, fourth, fifth, sixth, seventh or eighth 3′-most nucleoside in G may be a TNA nucleoside.

[0258] Also contemplated are TNA(PO) 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 continuous or discontinuous. Region G may, for example, comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen TNA nucleosides, optionally continuous.

[0259] In some TNA(PO) gapmers according to the invention, G comprises at least one TNA(PO) nucleoside. For example, depending on the total length of region G, the second, third, fourth, fifth, sixth, seventh or eighth closest 5' nucleoside in G may be a TNA(PO) nucleoside. Alternatively, depending on the total length of region G, the second, third, fourth, fifth, sixth, seventh or eighth closest 3' nucleoside in G may be a TNA nucleoside.

[0260] Also contemplated are TNA (PO) spacers in which G comprises at least two or at least three TNA (PO) nucleosides. The at least two or at least three TNA (PO) nucleosides may be continuous or discontinuous. Region G may, for example, comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen TNA (PO) nucleosides, optionally in succession.

[0261] In addition, as described herein, other modified nucleosides have been reported to be able to recruit RNase H when included in the spacer region, and may also or alternatively be included in the spacer region of the TNA (PO) spacer polymer. Preferably, in a TNA (PO) spacer polymer comprising at least one (such as one, two or three) TNA (PO) nucleosides or other modified nucleosides, in the spacer region G, the spacer region G still comprises at least three consecutive DNA nucleosides, such as at least four consecutive DNA nucleosides, such as at least five consecutive DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides. In some TNA (PO) spacer polymers, except for the TNA (PO) nucleosides in the spacer region G, all nucleosides of G are DNA nucleosides.

[0262] In some TNA(PO) spacers, any TNA(PO) nucleoside (particularly any consecutive stretch of two or more TNA or TNA(PO) nucleosides) is located closer to the 5' end of region G than to the 3' end of region G. Any consecutive stretch of two or more TNA or TNA(PO) nucleosides in region G may, for example, be located adjacent to the 5'-most nucleoside (typically a DNA nucleoside) in region G.

[0263] As shown herein, a TNA(PO) spacer can comprise a stretch of only three or four consecutive DNA nucleosides while still enabling the recruitment of RNase H. Without being limited by theory, it is envisioned that TNA spacers having a shorter spacer region than a conventional spacer design and / or having one or more TNA residues in the spacer region can provide increased resistance to endonuclease-mediated degradation and / or reduced off-target binding compared to conventional spacer designs. Thus, in some TNA spacers according to the present invention, G comprises up to 10 DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 DNA nucleosides. Optionally, G comprises up to 10 consecutive DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 consecutive DNA nucleosides. In addition, as further described in more detail below, when G in a TNA(PO) spacer comprises 9, 8, 7, 6, 5 or 4 consecutive DNA nucleosides, F' and F may optionally have different lengths, for example such that F comprises more linked nucleosides than F'.

[0264] For example, an antisense gapmer oligonucleotide can be designed such that, except for consecutive DNA nucleosides, all other nucleosides are TNA nucleosides, at least one of which is linked to at least one adjacent nucleoside by a 2'-PO or 3'-PO internucleoside bond.

[0265] Also contemplated are antisense spacer oligonucleotides wherein up to about 80%, such as up to about 70%, such as up to about 60%, such as up to about 50%, such as up to about 40%, such as up to about 30%, such as up to about 20%, such as up to about 15%, such as up to about 10%, such as up to about 5%, of the nucleosides of the antisense spacer oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2′-PO linked, 3′-PO linked or 2′,3′-PO linked TNA nucleoside.

[0266] Also contemplated are antisense gapper oligonucleotides wherein up to about 50% of the nucleosides of the antisense gapper oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2'-PO linked, 3'-PO linked, or 2',3'-PO linked TNA nucleoside.

[0267] Details regarding the number and arrangement of at least one TNA(PO) nucleoside in regions F, G and / or F' have been set forth above and may be incorporated into the general gapmer formula 5'-FG-F'-3' (I). Specifically contemplated TNA(PO) gapmers include those wherein

[0268] (i) Region F contains at least one TNA(PO) nucleoside, but Region F' and Region G do not;

[0269] (ii) region F' contains at least one TNA(PO) nucleoside, but region F and region G do not;

[0270] (iii) region G contains at least one TNA(PO) nucleoside, but region F and region F' do not;

[0271] (iv) Region F and Region F' each contain at least one TNA(PO) nucleoside, but Region G does not;

[0272] (v) Region F and Region G each contain at least one TNA(PO) nucleoside, but Region F' does not;

[0273] (vi) Region G and Region F' each contain at least one TNA(PO) nucleoside, but Region F does not; and

[0274] (vii) Region F, region G and region F' each contain at least one TNA(PO) nucleoside.

[0275] As an example, in a TNA(PO) gapmer according to item (iv) or (vii), F and F' may each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA nucleosides, at least one of which is a TNA(PO) nucleoside. Also contemplated are TNA(PO) gapmers according to item (iv) or (vii), wherein F and F' each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen consecutive TNA nucleosides, at least one of which is a TNA(PO) nucleoside.

[0276] Alternatively, in the TNA(PO) gapmer according to any one of items except (iii), F and F' may together comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty TNA nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides, at least one of which is a TNA(PO) nucleoside.

[0277] For example, F and F' together can comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides, at least one of which is linked to an adjacent TNA nucleoside by a PO internucleoside bond.

[0278] As another example, in the TNA(PO) gapmer according to item (iv) or (vii), F and F' may each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA(PO) nucleosides. Also contemplated are TNA(PO) gapmers according to item (iv) or (vii), wherein F and F' each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen consecutive TNA(PO) nucleosides.

[0279] Alternatively, in the TNA(PO) gapmer according to any one of items except (iii), F and F' may together comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty TNA(PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA(PO) nucleosides.

[0280] For example, F and F' together can comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides linked to adjacent TNA nucleosides by PO internucleoside bonds.

[0281] For example, in the TNA(PO) gapmer according to item (iv), F and F' together may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12 TNA(PO) nucleosides.

[0282] In some TNA(PO) gapmers, each TNA nucleoside in F and F' is linked to an adjacent nucleoside by a PO internucleoside bond. However, TNA(PO) gapmers are also contemplated in which, except for the 5'-most TNA nucleoside in F and the 3'-most TNA nucleoside in F', each TNA nucleoside in F and F' is a TNA(PO) nucleoside.

[0283] In some TNA(PO) gapmers, each of region F and region F' can independently comprise or consist of a contiguous sequence of linked sugar-modified nucleosides.

[0284] In some TNA(PO) gapmers, at least one of region F and region 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(PO) nucleoside.

[0285] In some TNA(PO) spacer polymers, both region F and region F' may consist of only one type of sugar-modified nucleosides (uniform flanking or uniform spacer polymer design). For example, the sugar-modified nucleosides may be high-affinity nucleosides or TNA(PO) nucleosides. In some TNA spacer polymers, all nucleosides in region F and region F' are TNA(PO) nucleosides. In some TNA(PO) spacer polymers, all nucleosides in region F and region F' are sugar-modified nucleosides other than TNA nucleosides.

[0286] In some TNA(PO) spacers, one or both of region F and region F' can independently comprise two different sugar-modified nucleosides (hybrid wing design). One of the two different sugar-modified nucleosides can be a TNA(PO) nucleoside, while the other sugar-modified nucleoside can be, for example, a high-affinity nucleoside or a TNA nucleoside linked by a modified internucleoside bond.

[0287] In some TNA(PO) spacers, all nucleosides in region F may be TNA(PO) nucleosides. Then, the nucleosides of region F' may, for example, comprise or consist of sugar-modified nucleosides other than TNA(PO) 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(PO). For example, F' may comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA(PO) nucleosides. Alternatively, F' may consist of 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides (such as three, four or five sugar-modified nucleosides other than TNA(PO) nucleosides).

[0288] In some TNA (PO) spacers, all nucleosides in region F' may be TNA (PO) nucleosides. Then, the nucleosides of region F may, for example, comprise or consist of sugar-modified nucleosides (such as 2'-sugar-modified nucleosides, such as high-affinity nucleosides) other than TNA (PO) nucleosides. Optionally, region F may comprise two different sugar-modified nucleosides, one of which may be TNA (PO). For example, F may comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Alternatively, F may consist of 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides (such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides).

[0289] In TNA gapmers where F, F', or both F and F' comprise or consist of one or more sugar-modified nucleosides other than TNA(PO) nucleosides, non-limiting examples of sugar-modified nucleosides include those having a modified sugar moiety selected from the group consisting of:

[0290] 2'-Methoxy-ribose (2'-OMe),

[0291] 2'-O-methoxyethyl-ribose (2'-O-MOE),

[0292] 5'-Methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE),

[0293] 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE),

[0294] 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE),

[0295] 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA),

[0296] 2'-deoxy-2'-fluoro-ribose (as in 2'-deoxy-2'-fluoro-ribose-nucleic acid; 2'-F-RNA),

[0297] 2'-Fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinonucleotide; 2'-F-ANA),

[0298] 2'-O-Benzyl-ribose,

[0299] Oxylated β-D-locked ribose (such as in β-D-LNA),

[0300] Amino-β-D-locked ribose (as in amino-β-D-LNA),

[0301] Thio-β-D-locked ribose (such as in thio-β-D-LNA),

[0302] Oxylated β-L-locked ribose (such as in β-L-LNA),

[0303] Amino-β-L-locked ribose (as in amino-β-L-LNA),

[0304] Thio-β-L-locked ribose (such as thio-β-L-LNA),

[0305] Oxylated α-L-locked ribose (such as in α-L-LNA),

[0306] Amino α-L-locked ribose (such as amino-α-L-LNA),

[0307] Thio-α-L-locked ribose (such as thio-α-L-LNA),

[0308] 2',4'-constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt),

[0309] Tricyclic-deoxyribose (such as tricyclic-deoxyribose DNA; TcDNA),

[0310] 3'-deoxy-ribose (such as 3'-deoxy-ribose DNA; 3'-DNA),

[0311] Unlocked ribose (as in unlocked nucleic acid; UNA),

[0312] Diols (as in diol nucleic acids; GNA),

[0313] Hexitols (as in hexitolide nucleic acid; HNA),

[0314] 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA),

[0315] 3'-arabino-fluorohexitol (as in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA),

[0316] Cyclohexene (as in cyclohexene nucleic acid; CeNA),

[0317] Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA),

[0318] Serinol (as serinol nucleic acid; SNA),

[0319] 2'-O,4'-C-ethylene bridged ribose (as in 2'-O,4'-C-ethylene linked nucleic acid; ENA),

[0320] acyclic (L)-threonine (as in acyclic (L)-threonine nucleic acid; aTNA),

[0321] 2',4'-constrained 2'-O-methoxyethyl ribose (as in cMOE), and

[0322] 7',5'-α-bicyclic sugar unit (such as 7',5'-α-bicyclic DNA; in bcDNA).

[0323] Particularly contemplated are TNA(PO) gapmers wherein the sugar-modified nucleosides of F, F', or both F and F' comprise one or more 2'-sugar modified nucleosides (such as high affinity, 2'-sugar modified nucleosides), or consist thereof.

[0324] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more LNA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be LNA nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 LNA nucleosides (such as 3, 4 or 5 LNA nucleosides). Suitable LNA nucleosides include those with a modified sugar moiety selected from oxy, amino or thio β-D-locked ribose (β-D-LNA) or from oxy, amino or thio β-L-locked ribose (β-L-LNA) or from oxy, amino or thio α-L-locked ribose (α-L-LNA), such as β-D-oxy-LNA, for example, 6'-methyl-β-D-oxy-LNA, such as (S)-6'-methyl-β-D-oxy-LNA (ScET) and ENA and the LNA nucleosides disclosed in Scheme 2. Particularly contemplated LNA nucleosides are β-D-oxy-LNA.

[0325] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' include one or more MOE nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides in F', F' or both F and F' can be MOE nucleosides. In a TNA (PO) spacer polymer in which only region G includes one or more TNA (PO) nucleosides, region F and region F' can, for example, each include 1, 2, 3, 4, 5, 6, 7, 8 MOE nucleosides (such as 3, 4 or 5 MOE nucleosides) or consist of them. Suitable MOE nucleosides include 2'-O-MOE, 5'-Me-2'-O-MOE and 2', 4'-constrained 2'-O-methoxyethyl (cMOE). The MOE nucleoside specifically considered is 2'-O-MOE.

[0326] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 2'-OMe nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-OMe nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-OMe nucleosides (such as 3, 4 or 5 2'-OMe nucleosides).

[0327] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 2'-O-MTE nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-O-MTE nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-MTE nucleosides (such as 3, 4 or 5 2'-O-MTE nucleosides).

[0328] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 2'-O-MCE nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-O-MCE nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-MCE nucleosides (such as 3, 4 or 5 2'-O-MCE nucleosides).

[0329] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more 2'-O-NMA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F, or both F and F' may be 2'-O-NMA nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-NMA nucleosides (such as 3, 4 or 5 2'-O-NMA nucleosides).

[0330] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 2'-deoxy-2'-fluoro-ribonucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-deoxy-2'-fluoro-ribonucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-deoxy-2'-fluoro-ribonucleosides (such as 3, 4 or 5 2'-deoxy-2'-fluoro-ribonucleosides).

[0331] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 2'-fluoro-2'-arabinoside. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-fluoro-2'-arabinoside. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-fluoro-2'-arabinoside (such as 3, 4 or 5 2'-fluoro-2'-arabinoside).

[0332] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F', F or both F and F' comprise one or more 2'-O-benzyl-ribonucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 2'-O-benzyl-ribonucleosides. In TNA(PO) spacer polymers where only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2'-O-benzyl-ribonucleosides (such as 3, 4 or 5 2'-O-benzyl-ribonucleosides).

[0333] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F', F or both F and F' comprise one or more cEt nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be cEt nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 cEt nucleosides (such as 3, 4 or 5 cEt nucleosides).

[0334] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more TcDNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be TcDNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 TcDNA nucleosides (such as 3, 4 or 5 TcDNA nucleosides).

[0335] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more 3'-DNA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be 3'-DNA nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 3'-DNA nucleosides (such as 3, 4 or 5 3'-DNA nucleosides).

[0336] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more UNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be UNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 UNA nucleosides (such as 3, 4 or 5 UNA nucleosides).

[0337] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more GNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' can be GNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' can, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 GNA nucleosides (such as 3, 4 or 5 GNA nucleosides).

[0338] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more HNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' can be HNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' can, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 HNA nucleosides (such as 3, 4 or 5 HNA nucleosides). Suitable HNA nucleosides include HNA, FHNA and Ara-FHNA.

[0339] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more CeNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be CeNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 CeNA nucleosides (such as 3, 4 or 5 CeNA nucleosides). Suitable CeNA nucleosides include CeNA and F-CeNA.

[0340] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more SNA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be SNA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides (such as 3, 4 or 5 SNA nucleosides).

[0341] In some TNA (PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more ENA nucleosides. For example, in addition to any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be ENA nucleosides. In a TNA (PO) spacer polymer in which only region G comprises one or more TNA (PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 ENA nucleosides (such as 3, 4 or 5 ENA nucleosides).

[0342] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more SNA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 nucleosides or all nucleosides of F', F' or both F and F' may be SNA nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides (such as 3, 4 or 5 SNA nucleosides).

[0343] In some TNA(PO) gapmers, the sugar-modified nucleosides of F, F' or both F and F' comprise one or more aTNA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F or both F and F' may be aTNA nucleosides. In a TNA(PO) gapmer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 aTNA nucleosides (such as 3, 4 or 5 aTNA nucleosides).

[0344] In some TNA(PO) spacer polymers, the sugar-modified nucleosides of F, F', or both F and F' comprise one or more bcDNA nucleosides. For example, in addition to any at least one TNA(PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides of F', F, or both F and F' may be bcDNA nucleosides. In a TNA(PO) spacer polymer in which only region G comprises one or more TNA(PO) nucleosides, region F and region F' may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 bcDNA nucleosides (such as 3, 4 or 5 bcDNA nucleosides).

[0345] For any TNA(PO) spacer polymer described herein, in particular TNA(PO) spacer polymers wherein the sugar-modified nucleosides of F, F' or both F and F' comprise or consist of one or more 2'-sugar-modified nucleosides (such as high affinity 2'-sugar-modified nucleosides), it is contemplated that at least one internucleoside bond may be a PS2 internucleoside bond. For example, in some TNA(PO) spacer polymers, (i) the nucleoside closest to the 5' of F is linked to the adjacent nucleoside in F via a PS2 internucleoside bond, (ii) the nucleoside closest to the 3' of F' is linked to the adjacent nucleoside in F' via a PS2 internucleoside bond, or (iii) both (i) and (ii) are applicable. Optionally, nucleosides other than at least one TNA(PO) nucleoside in the TNA(PO) spacer polymer may be linked to each other via PS internucleoside bonds.

[0346] Particularly contemplated are TNA(PO) gapmers 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 may be used for 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.

[0347] For example, a TNA(PO) gapmer according to the present invention may be represented by one or more of the following formulae of region FG-F' (Formula I), provided that the total length of region FG-F' is at least 12, such as at least 14 nucleotides in length:

[0348] F 1-15 -G 3-18 -F' 1-15 (IV), such as F 1-15 -G 3-18 -F' 1-12 (IVa) or F 1-12 -G 3-18 -F' 1-15 (IVb);

[0349] F 1-12 -G 3-18 -F′ 1-12 (V), such as F 1-12 -G 3-18 -F' 1-9 (Va) or F 1-9 -G 3-18 -F' 1-12(Vb);

[0350] F 1-12 -G 4-16 -F' 1-12 (VI), such as 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), such as F 3-12 -G 4-10 -F' 3-9 (VIIa) or F 3-9 -G 4-10 -F' 3-12 (VIIb).

[0351] Each of Region F, Region G, and Region F' as described herein may be incorporated into any of the FGF' formulas.

[0352] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IVa has a length of at least 16 nucleosides, and

[0353] (a) the 5'-most nucleoside in F and the nucleosides in F' are independently 3, 4 or 5 high affinity, sugar-modified nucleosides,

[0354] (b) the remaining nucleosides in F are TNA(PO) nucleosides, and

[0355] (c) All nucleosides in G are DNA nucleosides.

[0356] For example, G may comprise 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 may, for example, comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 consecutive TNA(PO) nucleosides.

[0357] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, and

[0358] (a) F and F' each independently consist of 3, 4 or 5 nucleosides, wherein at least one nucleoside in F and / or F' is a TNA(PO) nucleoside, and any remaining nucleosides are high-affinity sugar-modified nucleosides, and

[0359] (b) All nucleosides in G are DNA nucleosides.

[0360] For example, all nucleosides in F or F' may be TNA(PO) nucleosides. Alternatively, all nucleosides in F and F' may be TNA(PO) nucleosides. Alternatively, except for the TNA nucleoside closest to the 5' in F and the TNA nucleoside closest to the 3' in F', each TNA nucleoside in F and F' may be linked to an adjacent nucleoside, in particular to any adjacent TNA nucleoside, via a PO internucleoside bond.

[0361] In some TNA(PO) gapmers, for example, the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, and

[0362] (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

[0363] (b) The second, third, fourth or fifth 5'-most nucleoside in G is a TNA(PO) nucleoside and the remaining nucleosides in G are DNA nucleosides.

[0364] For example, the TNA(PO) nucleoside may be positioned closer to the 5' end of region G than to the 3' end of G.

[0365] Particularly contemplated designs of TNA(PO) spacers also include those referred to as Design A and Design B in Example 1:

[0366] TTTTTddddddddddTTTTT (Design A), where T is TNA and d is DNA, and has a backbone (internucleoside bond) pattern SOOOSSSSSSSSSSSOOOS, where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO).

[0367] MMMMMTTTTTTddddMMMMM (Design B) where M is MOE (e.g., 2′-O-MOE), T is TNA, and d is DNA, and has a backbone (internucleoside bond) pattern SSSSOOOOOOSSSSSSSSS where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO).

[0368] Also specifically envisioned are the following designs:

[0369] TTTTTTTTTTdddddMMMMM (Design C) where M is 2′-O-MOE, T is TNA and d is DNA, and has a backbone (internucleoside linkage) pattern OOOOOOOOOSSSSSSSSSS where S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO).

[0370] MMMMMddddddddddMMMMM, one, two, three, four, five, six or seven d are replaced by T (Design D), wherein M is 2′-O-MOE, T is TNA and d is DNA, and has a backbone (internucleoside bond) pattern of SSSSSSSSSSSSSSSSSS, except that O replaces S as the 2′- and 3′-internucleoside bonds of each T, wherein S corresponds to phosphorothioate (PS) and O corresponds to phosphodiester (PO).

[0371] Further specifically envisaged are the following designs:

[0372] TTTTTddddddddddTTTTT (Design A), where T is TNA and d is DNA, and has a backbone (internucleoside bond) pattern POOOSSSSSSSSSSSOOOP, where P corresponds to PS2, where S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO).

[0373] MMMMMTTTTTTddddMMMMM (Design B), where M is MOE (e.g., 2′-O-MOE), T is TNA, and d is DNA, and has a backbone (internucleoside bond) pattern PSSSOOOOOOSSSSSSSSP, where P corresponds to PS2, S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO).

[0374] TTTTTTTTTTdddddMMMMM (Design C) where M is 2'-O-MOE, T is TNA and d is DNA, and has a backbone (internucleoside bond) pattern OOOOOOOOOSSSSSSSSSP where P corresponds to PS2, S corresponds to phosphorothioate (PS), and O corresponds to phosphodiester (PO).

[0375] MMMMMddddddddddMMMMM, wherein one, two, three, four, five, six or seven d are replaced by T (Design D), wherein M is 2′-O-MOE, T is TNA and d is DNA, and has a backbone (internucleoside bond) pattern of PSSSSSSSSSSSSSSSSSSP, except that O replaces S as any 2′- and 3′-internucleoside bonds of each T, wherein S corresponds to phosphorothioate (PS), O corresponds to phosphodiester (PO) and P corresponds to phosphorodithioate (PS2).

[0376] The antisense spacer polymer oligonucleotide comprising a TNA (PO) spacer polymer, i.e. a continuous nucleotide sequence of formula 5'-FG-F'-3' (I) can include additional nucleosides connected at the 3' end and / or 5' end of the TNA (PO) spacer polymer, for example 1 to 100, 1 to 40, 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 connected nucleosides. Additional connected nucleosides can, for example, promote the delivery of the antisense spacer polymer oligonucleotide to the expected site or the targeting second molecule. The antisense spacer polymer oligonucleotide can also be a part of a longer nucleic acid construct or can be a part of a longer nucleic acid construct. However, it is also envisioned that the antisense spacer polymer oligonucleotide can be composed of the TNA (PO) spacer polymer.

[0377] The present invention particularly contemplates TNA(PO) spacer and antisense spacer oligonucleotides that are single-stranded antisense oligonucleotides. In the preparation of TNA(PO) spacer or antisense spacer oligonucleotides according to the present invention, the TNA(PO) spacer or antisense spacer oligonucleotides are substantially single-stranded, such that the majority of TNA(PO) spacer molecules or antisense spacer oligonucleotide molecules are in single-stranded form.

[0378] Manufacturing method

[0379] Also provided is a method for making an oligonucleotide of the present invention, comprising reacting nucleotide units and forming the covalently linked continuous nucleotide units contained in the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al., 1987, Methods in Enzymology Vol. 154, pp. 287-313).

[0380] The synthesis of TNA monomers and the incorporation of TNA monomers into oligonucleotides are disclosed in, for example, 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 / 179292A1.

[0381] Particularly provided is a method for preparing a modified form of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of formula 5'FG-F'3' (I) capable of recruiting RNase H, wherein G is a spacer region of 5 to 18 linked DNA nucleosides, and each of F and F' is a flanking region of up to 8 linked nucleosides, the flanking regions independently comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified form, at least one nucleoside among F, F' and / or G of the parent antisense gapmer oligonucleotide has been replaced by a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond,

[0382] The method comprises the steps of making a modified antisense gapper oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond, and,

[0383] The modified antisense gapmer oligonucleotide is optionally purified or isolated.

[0384] In one embodiment, compared with the parent antisense interval polymer oligonucleotide, the antisense interval polymer oligonucleotide of modification has the toxicity of reduction, optionally hepatotoxicity.In one embodiment, the toxicity of the antisense interval polymer oligonucleotide of modification to HepG2 cells is lower than the parent antisense interval polymer oligonucleotide, optionally as determined by Caspase 3 / 7 assay.In one embodiment, compared with the parent antisense interval polymer oligonucleotide, the antisense interval polymer oligonucleotide of modification has the exonuclease resistance of increase.In one embodiment, compared with the parent antisense interval polymer oligonucleotide, the antisense interval polymer oligonucleotide of modification has the endonuclease resistance of increase.

[0385] Optionally, the parent antisense gapmer oligonucleotide may be an LNA gapmer or a MOE gapmer, such as an LNA gapmer or a MOE gapmer in which all internucleoside linkages are phosphorothioate linkages. Furthermore, the nucleotide units employed in the manufacturing steps are advantageously nucleoside phosphoramidites.

[0386] The modified antisense gapmer oligonucleotide may comprise features of any of the TNA(PO) gapmers described herein, such as features with respect to region F, region G and region F' and the FG-F' design.

[0387] The present invention also provides an antisense gapmer oligonucleotide obtained or obtainable by the method.

[0388] The method may further comprise reacting the contiguous nucleotide sequence with a conjugate moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide.

[0389] In another aspect, a method for making a composition is provided, comprising mixing an oligonucleotide or a conjugated oligonucleotide with a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0390] Pharmaceutical composition

[0391] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0392] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

[0393] Pharmaceutical diluents include phosphate buffered saline (PBS), and pharmaceutical salts include but are not limited to sodium and potassium salts. In some embodiments, the pharmaceutical diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in a pharmaceutical diluent at a concentration of 50-300 μM solution.

[0394] The oligonucleotide or oligonucleotide conjugate according to the present invention can exist in the form of its pharmaceutical salt. The term "pharmaceutical salt" refers to conventional acid addition salts or base addition salts formed by suitable non-toxic organic or inorganic acids or organic or inorganic bases that retain the biological effectiveness and characteristics of the compounds of the present invention. Acid addition salts include, for example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, aminosulfonic acid, phosphoric acid and nitric acid; and salts 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, etc. Base addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides (such as, for example, tetramethylammonium hydroxide). Chemical modification of drug compounds into salts in order to obtain improved physical and chemical stability, hygroscopicity, fluidity and solubility of the compound is a well-known technique for pharmaceutical chemists. This is described, for example, in Bastin, Organic Process Research & Development 2000, No. 4, pp. 427-435 or Ansel, Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th edition (1995) pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compound provided herein may be a sodium salt.

[0395] Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990). WO 2007 / 031091 (incorporated herein by reference) provides other suitable and preferred examples of pharmaceutical diluents, carriers and adjuvants. Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0396] The oligonucleotide or oligonucleotide conjugate of the present invention can be mixed with pharmaceutically active or inert substances to prepare pharmaceutical compositions or preparations. The composition and preparation method of the pharmaceutical composition depends on many criteria, including but not limited to the route of administration, the extent of the disease or the dosage of administration.

[0397] These compositions can be sterilized or can be aseptically filtered by conventional sterilization techniques. The aqueous solution gained can be directly used or lyophilized after packaging, and the lyophilized preparation is mixed with a sterile aqueous vehicle before application. The pH of the preparation is generally between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting composition of solid form can be packaged in multiple single-dose units, and each unit comprises one or more of the above-mentioned agents of fixed amount, such as in the sealed package of tablets or capsules. The composition of solid form can also be packaged in a container in a flexible amount, such as in a squeezable tube designed for a locally applicable cream or ointment.

[0398] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. In particular, with respect to oligonucleotide conjugates, once the prodrug is delivered to the site of action (eg, target cell), the conjugate portion is cleaved from the oligonucleotide.

[0399] application

[0400] The oligonucleotides or oligonucleotide conjugates described herein can be used as research reagents or as diagnostic, therapeutic and prophylactic agents.

[0401] In research, oligonucleotides or oligonucleotide conjugates can be used to specifically modulate the expression of a target nucleic acid in cells (e.g., in vitro cell culture) and experimental animals, thereby facilitating functional analysis of the target, or evaluating its usefulness as a target for therapeutic intervention. Typically, target modulation is achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting the gene or mRNA that produces the protein.

[0402] If an oligonucleotide or oligonucleotide conjugate is employed in research or diagnostics, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0403] Also provided is an in vivo or in vitro method for modulating the expression of a target gene in a target cell comprising a target nucleic acid, the method comprising administering to the cell an effective amount of an oligonucleotide or oligonucleotide conjugate of the invention.

[0404] In some embodiments, the target cell is a mammalian cell, particularly a human cell.The target cell may be an in vitro cell culture or an in vivo cell that forms part of a tissue in a mammal, such as a human.

[0405] Also provided are diagnostic applications in which the oligonucleotides or oligonucleotide conjugates can be used to detect and quantify the expression of target genes in cells and tissues by northern blotting, in situ hybridization or similar techniques.

[0406] Also provided is an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition as described herein for use as a medicament.

[0407] The disease or disorder for which the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition is used is generally associated with the expression of a target gene. Preferably, the disease or disorder is a disease or disorder that can be treated by regulating the expression of a target gene.

[0408] Oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions can be used, for example, to treat or prevent a disease or disorder caused by abnormal levels and / or activity of a target gene or an expression product (e.g., RNA or protein) from the target gene. The disease or disorder can also or alternatively be associated with a mutation in the target gene. Therefore, in some embodiments, the target nucleic acid is a mutant form of the target gene. Non-limiting examples of target genes include genes associated with one or more cancers, infectious diseases, neurological diseases or disorders, ophthalmic diseases or disorders, or cardiovascular diseases or disorders.

[0409] The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition according to the present invention is usually administered in an effective amount.

[0410] Also provided are therapeutic applications, wherein the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition is used to treat or prevent a disease or disorder in an animal or human suffering from or suspected of suffering from the disease or disorder. Typically, the disease or disorder is a disease or disorder that can be treated by regulating the expression of a target gene.

[0411] Also provided is the use of an oligonucleotide or oligonucleotide conjugate in the manufacture of a medicament for treating or preventing an animal or human suffering from or suspected of suffering from a disease or disorder. Typically, the disease or disorder is a disease or disorder that can be treated by regulating the expression of a target gene.

[0412] Also provided are methods for treating or preventing a disease or disorder, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition to a subject suffering from or suspected of suffering from the disease or disorder.

[0413] Application

[0414] In some embodiments, the oligonucleotides or pharmaceutical compositions of the invention can be administered orally. In other embodiments, the oligonucleotides or pharmaceutical compositions of the invention can be administered topically or enterally or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0415] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the present invention is administered by a parenteral route, such as, for example, by intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intrathecal or intracranial administration, such as 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.

[0416] In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the 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 may be once a week, once every two weeks, once every three weeks or even once a month.

[0417] Combination therapy

[0418] In some embodiments, the oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions of the invention are used in combination therapy with another therapeutic agent. The therapeutic agent may, for example, be the standard of care for the disease or disorder to be treated with the oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions of the invention.

[0419] Implementation

[0420] The following numbered embodiments are particularly contemplated.

[0421] 1. An antisense spacer oligonucleotide comprising a continuous nucleotide sequence of formula 5'-FG-F'-3' (I) capable of recruiting ribonuclease (RNase) H, wherein

[0422] G is a spacer region of up to 18 linked nucleosides, said spacer region comprising at least 3 consecutive DNA nucleosides,

[0423] Each of F and F' is a flanking region of up to 15 linked nucleosides, said flanking region independently comprising or consisting of 1 to 15 sugar-modified nucleosides,

[0424] At least one of F, F' and G comprises a sugar-modified nucleoside that is an α-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside bond other than a phosphorothioate (PS) internucleoside bond.

[0425] 2. The antisense gapmer oligonucleotide of embodiment 1, wherein the TNA nucleoside is linked to the adjacent nucleoside via a phosphodiester (PO) internucleoside bond.

[0426] 3. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleosides are linked to adjacent nucleosides via 2'-PO internucleoside bonds or 3'-PO internucleoside bonds.

[0427] 4. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleoside is linked to any adjacent nucleoside via a 2'-PO or 3'-PO internucleoside bond.

[0428] 5. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the adjacent nucleosides are sugar-modified nucleosides or DNA nucleosides.

[0429] 6. The antisense gap polymer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleoside

[0430] (a) through 2'-PO internucleoside linkage and 3'-modified internucleoside linkage, respectively;

[0431] (b) via a 2'-modified internucleoside linkage and a 3'-PO internucleoside linkage, respectively; or

[0432] (c) linked to the first and second adjacent nucleosides via a 2'-PO internucleoside bond and a 3'-PO internucleoside bond, respectively,

[0433] Optionally, wherein the 2'-modified internucleoside linkage or the 3'-modified internucleoside linkage is a phosphorothioate (PS) internucleoside linkage or a phosphorodithioate (PS2) internucleoside linkage.

[0434] 7. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least one TNA nucleoside.

[0435] 8. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

[0436] 9. The antisense spacer oligonucleotide according to any of the preceding embodiments, wherein F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

[0437] 10. The antisense gapmer oligonucleotide according to any of the preceding embodiments, wherein F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside bonds.

[0438] 11. The antisense gapper oligonucleotide according to any of the preceding embodiments, wherein F comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside bonds.

[0439] 12. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 3'-most nucleoside in F is a TNA nucleoside.

[0440] 13. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 3'-most nucleosides of F are TNA nucleosides.

[0441] 14. The antisense oligonucleotide according to any one of the preceding embodiments, wherein two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of the 3'-most nucleosides in F are TNA nucleosides.

[0442] 15. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 5'-most nucleoside in F is a TNA nucleoside.

[0443] 16. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the 5'-most nucleosides in F are TNA nucleosides.

[0444] 17. The antisense oligonucleotide according to any one of the preceding embodiments, wherein two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of the 5'-most nucleosides in F are TNA nucleosides.

[0445] 18. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all sugar-modified nucleosides of F are TNA nucleosides.

[0446] 19. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all nucleosides of F are TNA nucleosides.

[0447] 20. The antisense gapper oligonucleotide according to any one of the preceding embodiments, wherein

[0448] (a) each TNA nucleoside in F is linked to an adjacent nucleoside via a PO internucleoside bond; or

[0449] (b) Except for the TNA nucleoside closest to the 5' side in F, each TNA nucleoside in F is linked to the adjacent nucleoside via a PO internucleoside bond.

[0450] 21. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 20, wherein F′ does not comprise any TNA nucleosides.

[0451] 22. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 20, wherein F′ comprises at least one TNA nucleoside.

[0452] 23. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F' comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

[0453] 24. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F' comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

[0454] 25. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F' comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside bonds.

[0455] 26. The antisense gapmer oligonucleotide according to any of the preceding embodiments, wherein F' comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside bonds.

[0456] 27. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 5'-most nucleoside in F' is a TNA nucleoside.

[0457] 28. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the 5'-most nucleosides in F' are TNA nucleosides.

[0458] 29. The antisense oligonucleotide according to any one of the preceding embodiments, wherein two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of the nucleosides closest to 5' in F' are TNA nucleosides.

[0459] 30. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 3'-most nucleoside in F' is a TNA nucleoside.

[0460] 31. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 3'-most nucleosides in F' are TNA nucleosides.

[0461] 32. The antisense oligonucleotide according to any one of the preceding embodiments, wherein two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen of the 3'-most nucleosides in F' are TNA nucleosides.

[0462] 33. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all sugar-modified nucleosides of F' are TNA nucleosides.

[0463] 34. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all nucleosides of F' are TNA nucleosides.

[0464] 35. The antisense spacer oligonucleotide according to any one of the preceding embodiments, wherein

[0465] (a) each TNA nucleoside in F' is linked to an adjacent nucleoside via a PO internucleoside bond; or

[0466] (b) Except for the TNA nucleoside closest to the 3' side in F', each TNA nucleoside in F' is linked to the adjacent nucleoside via a PO internucleoside bond.

[0467] 36. The antisense gapmer oligonucleotide of any one of embodiments 1 to 6 and 21 to 35, wherein F does not comprise any TNA nucleosides.

[0468] 37. An antisense spacer oligonucleotide according to any of the preceding embodiments, wherein F and F′ each independently comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

[0469] 38. An antisense spacer oligonucleotide according to any of the preceding embodiments, wherein F and F' together comprise at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

[0470] 39. The antisense spacer oligonucleotide according to any of the preceding embodiments, wherein F and F' each independently comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA nucleosides linked by PO internucleoside bonds.

[0471] 40. The antisense spacer oligonucleotide according to any of the preceding embodiments, wherein F and F' together comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty TNA nucleosides linked to adjacent TNA nucleosides by PO internucleoside bonds.

[0472] 41. The antisense spacer oligonucleotide according to any one of the preceding embodiments, wherein

[0473] (a) each TNA nucleoside in F and F' is linked to the adjacent nucleoside via a PO internucleoside bond, or

[0474] (b) With the exception of the TNA nucleoside closest to the 5' end of F and the TNA nucleoside closest to the 3' end of F', each TNA nucleoside in F and F' is linked to an adjacent nucleoside via a PO internucleoside bond.

[0475] 42. The antisense spacer oligonucleotide according to any one of the preceding embodiments, wherein F, F′ or both F and F′ further comprise 1 to 8 sugar-modified nucleosides other than TNA(PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA(PO) nucleosides.

[0476] 43. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleoside of F, F′, or both F and F′ comprises or consists of at least one sugar-modified nucleoside comprising a modified sugar moiety selected from the group consisting of:

[0477] α-L-threofuranosyl (as in threose nucleic acid; TNA),

[0478] 2'-Methoxy-ribose (2'-OMe),

[0479] 2'-O-methoxyethyl-ribose (2'-O-MOE),

[0480] 5'-Methyl-2'-O-methoxyethyl ribose (5'-Me-2'-O-MOE),

[0481] 2'-O-[2-(methylthio)ethyl]-ribose (2'-O-MTE),

[0482] 2-(N-methylcarbamoyl)-ethyl]-ribose (2'-O-MCE),

[0483] 2'-O-[2-(methylamino)-2-oxoethyl]-ribose (2'-O-NMA),

[0484] 2'-deoxy-2'-fluoro-ribose (as in 2'-deoxy-2'-fluoro-ribose-nucleic acid; 2'-F-RNA),

[0485] 2'-Fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinonucleotide; 2'-F-ANA),

[0486] 2'-O-Benzyl-ribose,

[0487] Oxylated β-D-locked ribose (such as in β-D-LNA),

[0488] Amino-β-D-locked ribose (as in amino-β-D-LNA),

[0489] Thio-β-D-locked ribose (such as in thio-β-D-LNA),

[0490] Oxylated β-L-locked ribose (such as in β-L-LNA),

[0491] Amino-β-L-locked ribose (as in amino-β-L-LNA),

[0492] Thio-β-L-locked ribose (such as thio-β-L-LNA),

[0493] Oxylated α-L-locked ribose (such as in α-L-LNA),

[0494] Amino α-L-locked ribose (such as amino-α-L-LNA),

[0495] Thio-α-L-locked ribose (such as thio-α-L-LNA),

[0496] 2',4'-constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt),

[0497] Tricyclic-deoxyribose (such as tricyclic-deoxyribose DNA; TcDNA),

[0498] 3'-deoxy-ribose (such as 3'-deoxy-ribose DNA; 3'-DNA),

[0499] Unlocked ribose (as in unlocked nucleic acid; UNA),

[0500] Diols (as in diol nucleic acids; GNA),

[0501] Hexitols (as in hexitolide nucleic acid; HNA),

[0502] 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA),

[0503] 3'-arabino-fluorohexitol (as in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA),

[0504] Cyclohexene (as in cyclohexene nucleic acid; CeNA),

[0505] Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA),

[0506] Serinol (as serinol nucleic acid; SNA),

[0507] 2'-O,4'-C-ethylene-bridged ribose (as in 2'-O,4'-C-ethylene linked nucleic acid; ENA)

[0508] Acyclic (L)-threonine (as in acyclic (L)-threonine nucleic acid; aTNA)

[0509] 2',4'-Constrained 2'-O-methoxyethyl ribose (as in cMOE)

[0510] 7',5'-α-bicyclic sugar unit (such as 7',5'-α-bicyclic DNA; in bcDNA).

[0511] 44. The antisense spacer oligonucleotide according to any of the preceding embodiments, wherein the sugar-modified nucleosides of F, F', or both F and F' comprise or consist of one or more 2'-sugar-modified nucleosides (such as high affinity, 2'-sugar-modified nucleosides).

[0512] 45. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleosides of F, F' or both F and F' comprise one or more LNA nucleosides.

[0513] 46. ​​The antisense gapmer oligonucleotide of embodiment 45, wherein, except for any at least one TNA nucleoside, all nucleosides in F' and F are LNA nucleosides.

[0514] 47. The antisense gapmer oligonucleotide according to any one of the preceding 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.

[0515] 48. The antisense gapmer oligonucleotide of embodiment 47, wherein, except for any at least one TNA nucleoside, all nucleosides in F' and F are 2'-O-MOE nucleosides.

[0516] 49. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G does not contain any TNA nucleosides.

[0517] 50. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 48, wherein G comprises at least one TNA nucleoside, such as at least one, two or three TNA nucleosides.

[0518] 51. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

[0519] 52. The antisense gapmer oligonucleotide of embodiment 51, wherein the second, third, fourth, fifth, sixth, seventh or eighth 5'-most nucleoside in G is a TNA nucleoside linked to the adjacent nucleoside by a PO internucleoside bond.

[0520] 53. The antisense gapmer oligonucleotide of embodiment 51 or 52, wherein the second, third, fourth, fifth, sixth, seventh or eighth 3'-most nucleoside in G is a TNA nucleoside linked to the adjacent nucleoside by a PO internucleoside bond.

[0521] 54. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G comprises at least two, such as two or three, adjacent TNA nucleosides linked by PO internucleoside bonds.

[0522] 55. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein at least the 5'-most nucleoside and the 3'-most nucleoside in G are DNA nucleosides.

[0523] 56. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all nucleosides of G, except for any at least one TNA(PO) nucleoside, are DNA nucleosides.

[0524] 57. An antisense spacer oligonucleotide according to any one of the preceding embodiments, wherein the spacer region G comprises at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides.

[0525] 58. The antisense gapper oligonucleotide according to any one of the preceding embodiments, wherein G comprises up to 10 DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 DNA nucleosides.

[0526] 59. An antisense spacer oligonucleotide according to any of the preceding embodiments, wherein at most about 80%, such as 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%, such as at most about 15%, such as at most about 10%, such as at most about 5%, of the nucleosides of the antisense spacer oligonucleotide are TNA nucleosides.

[0527] 60. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein up to about 50% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2'-PO linked, 3'-PO linked or 2',3'-PO linked TNA nucleoside.

[0528] 61. The antisense gapmer oligonucleotide according to any of the preceding embodiments, comprising at least one modified internucleoside linkage.

[0529] 62. The antisense spacer oligonucleotide of any of the preceding embodiments, comprising nuclease-resistant, modified internucleoside linkages.

[0530] 63. An antisense spacer oligonucleotide according to any of the preceding embodiments, wherein, except for any PO internucleoside bonds between a TNA nucleoside and an adjacent nucleoside, all internucleoside bonds are phosphorothioate (PS) internucleoside bonds, phosphorodithioate (PS2) internucleoside bonds, or a combination thereof.

[0531] 64. The antisense spacer oligonucleotide according to any one of the preceding embodiments, wherein

[0532] (a) the nucleoside closest to the 5' side of F is linked to the adjacent nucleoside in F via a PS2 internucleoside bond;

[0533] (b) the nucleoside closest to the 3′ of F′ is linked to the adjacent nucleoside in F′ via a PS2 internucleoside bond; or

[0534] (c) Both (a) and (b).

[0535] 65. An antisense spacer oligonucleotide according to any of the preceding embodiments, wherein the continuous 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.

[0536] 66. The antisense gapper oligonucleotide of embodiment 65, wherein the contiguous nucleotide sequence has the formula

[0537] F 1-15 -G 3-18 -F' 1-15 (IV), such as F 1-15 -G 3-18 -F' 1-12 (IVa) or F 1-12 -G 3-18 -F′ 1-15 (IVb);

[0538] F 1-12 -G 3-18 -F' 1-12 (V), such as F 1-12 -G 3-18 -F' 1-9 (Va) or F 1-9 -G 3-18 -F' 1-12 (Vb);

[0539] F 1-12 -G 4-16 -F' 1-12 (VI), such as F 1-12 -G 4-16 -F′ 1-9 (VIa) or F 1-9 -G 4-16 -F′ 1-12 (VIb); or

[0540] F 3-12 -G 4-10 -F′ 3-12 (VII), such as F 3-12 -G 4-10 -F' 3-9 (VIIa) or F 3-9 -G 4-10 -F' 3-12 (VIIb), wherein the numerical ranges represent the number of linked nucleosides in F, G and F', respectively.

[0541] 67. According to any of the aforementioned embodiments, the antisense spacer oligonucleotide can reduce 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, optionally when determined in a target cell expressing the target nucleic acid and incubated with the antisense spacer oligonucleotide at a concentration of about 25 μM for about 3 days.

[0542] 68. An antisense spacer oligonucleotide according to any of the preceding embodiments, which has an IC50 of no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM, for reducing the expression level of a target nucleic acid, optionally when determined in a target cell expressing the target nucleic acid and incubated with the antisense spacer oligonucleotide at a concentration of about 25 μM for about 3 days.

[0543] 69. An antisense spacer oligonucleotide according to any of the preceding embodiments, which uses the caspase 3 / 7 assay described in Example 4, and 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%.

[0544] 70. According to any one of the preceding embodiments, the antisense spacer oligonucleotide is in the form of a duplex of the antisense spacer oligonucleotide and an RNA target sequence, having a melting temperature (Tm) 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.

[0545] 71. An antisense intercalator oligonucleotide according to any one of embodiments 1 to 66, characterized by embodiments 67 and 68; embodiments 67 and 69; embodiments 67 and 70; embodiments 68 and 69; embodiments 68 and 70; embodiments 67, 68 and 69; embodiments 67, 68 and 70; embodiments 67, 69 and 70; embodiments 68, 69 and 70; or a combination of embodiments 67 to 70.

[0546] 72. The antisense spacer oligonucleotide of any one of embodiments 67 to 71, wherein the target nucleic acid is an RNA target sequence having a nucleobase sequence complementary to the contiguous nucleotide sequence of Formula I.

[0547] 73. An antisense gapper oligonucleotide comprising a contiguous nucleotide sequence of formula 5'-FG-F'-3' (I) capable of recruiting RNase H, wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside linked to an adjacent nucleoside by a bond different from a PS internucleoside bond.

[0548] 74. The antisense gapmer oligonucleotide of embodiment 73, wherein the TNA nucleosides are linked to adjacent nucleosides by PO internucleoside bonds.

[0549] 75. The antisense spacer oligonucleotide according to any one of embodiments 73 and 74, wherein the contiguous nucleotide sequence of formula 5'-FG-F'-3' (I) has a length of 12 to 32 nucleosides, such as 12 to 28 nucleosides, 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.

[0550] 76. The antisense gapmer oligonucleotide of any one of embodiments 73 to 75, wherein the contiguous nucleotide sequence has the formula

[0551] F 1-15 -G 3-18 -F' 1-15 (II), such as F 1-15 -G 3-18 -F' 1-12 (IIa) or F 1-12 -G 3-18 -F' 1-15 (IIb);

[0552] F 1-12 -G 3-18 -F' 1-12 (III), such as F 1-12 -G3-18 -F' 1-9 (IIIa) or F 1-9 -G 3-18 -F' 1-12 (IIIb);

[0553] F 1-12 -G 4-16 -F′ 1-12 (IV), such as F 1-12 -G 4-16 -F′ 1-9 (IVa) or F 1-9 -G 4-16 -F′ 1-12 (IVb); or

[0554] F 3-12 -G 4-10 -F' 3-12 (V), such as F 3-12 -G 4-10 -F' 3-9 (Va) or F 3-9 -G 4-10 -F' 3-12 (Vb), wherein the numerical ranges represent the number of linked nucleosides in F, G, and F', respectively.

[0555] 77. The antisense gapmer of any one of embodiments 73 to 76, further comprising the features of any one of embodiments 1 to 72.

[0556] 78. The antisense gapper oligonucleotide of any one of embodiments 65 to 77, wherein optionally the contiguous nucleotide sequence of formula IV, such as IVa, has a length of at least 16 nucleosides, and

[0557] (a) the 5'-most nucleoside in F and the nucleosides in F' are independently 3, 4 or 5 high affinity, sugar-modified nucleosides,

[0558] (b) the remaining nucleosides in F are TNA nucleosides, and

[0559] (c) All nucleosides in G are DNA nucleosides.

[0560] 79. The antisense gapmer oligonucleotide of embodiment 78, wherein G comprises up to 10 consecutive DNA nucleosides, such as 9, 8, 7, 6, 5 or 4 consecutive DNA nucleosides.

[0561] 80. The antisense gapper oligonucleotide of any one of embodiments 65 to 77, wherein optionally the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, and

[0562] (a) F and F' each independently consist of 3, 4 or 5 nucleosides,

[0563] (b) wherein at least one of F and F' is a TNA nucleoside and the remaining nucleosides are high affinity, sugar-modified nucleosides,

[0564] (c) All nucleosides in G are DNA nucleosides.

[0565] 81. The antisense gapmer oligonucleotide of embodiment 80, wherein all nucleosides in F and F' are TNA nucleosides.

[0566] 82. The antisense gapmer oligonucleotide of any one of embodiments 66 to 77, wherein

[0567] (a) each TNA nucleoside in F and F' is linked to the adjacent nucleoside via a PO internucleoside bond, or

[0568] (b) With the exception of the TNA nucleoside closest to the 5' in F and the TNA nucleoside closest to the 3' in F', each TNA nucleoside in F and F' is linked to the adjacent nucleoside via a PO internucleoside bond.

[0569] 83. The antisense gapper oligonucleotide of any one of embodiments 65 to 77, wherein the contiguous nucleotide sequence of Formula IV has a length of at least 16 nucleosides, and

[0570] (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

[0571] (b) The second, third, fourth or fifth 5'-most nucleoside in G is a TNA nucleoside, such as a TNA(PO) nucleoside, and the remaining nucleosides in G are DNA nucleosides.

[0572] 84. The antisense gapmer oligonucleotide of any one of embodiments 78 to 83, wherein the high affinity, sugar-modified nucleosides are selected from the sugar-modified nucleosides of embodiment 43.

[0573] 85. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0574] 86. A conjugate comprising the antisense gapmer oligonucleotide according to any one of the preceding embodiments and at least one conjugate moiety covalently linked to the oligonucleotide, optionally via a linker.

[0575] 87. The conjugate of embodiment 86, wherein the conjugate moiety is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins, and combinations thereof.

[0576] 88. The conjugate of embodiment 86 or 87, wherein the conjugate moiety facilitates delivery across the blood-brain barrier.

[0577] 89. A pharmaceutically acceptable salt of the antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 or the conjugate according to any one of embodiments 86 to 88.

[0578] 90. A pharmaceutical composition comprising the antisense gapper oligonucleotide according to any one of embodiments 1 to 85, or the conjugate according to any one of embodiments 86 to 88, the pharmaceutically acceptable salt according to embodiment 89, and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0579] 91. The antisense oligonucleotide according to any one of embodiments 1 to 85, the conjugate according to any one of embodiments 86 to 88, the pharmaceutically acceptable salt according to embodiment 89 or the pharmaceutical composition according to embodiment 90 for use as a medicament.

[0580] 92. A method for preparing a modified form of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of formula 5'FG-F'3' (I) capable of recruiting RNase H, wherein G is a spacer region of 5 to 18 linked DNA nucleosides, and each of F and F' is a flanking region of up to 8 linked nucleosides, the flanking regions independently comprising or consisting of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified form, at least one nucleoside among F, F' and / or G of the parent antisense gapmer oligonucleotide has been replaced by a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond,

[0581] The method comprises the steps of making a modified antisense gapper oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside, and,

[0582] The modified antisense gapmer oligonucleotide is optionally purified or isolated.

[0583] 93. A method according to embodiment 92, wherein the modified antisense spacer oligonucleotide has reduced toxicity, optionally hepatotoxicity, compared to the parent antisense spacer oligonucleotide.

[0584] 94. The method of any one of embodiments 92 and 93, wherein the modified antisense spacer oligonucleotide is less toxic to HepG2 cells than the parent antisense spacer oligonucleotide, optionally as determined by a caspase 3 / 7 assay.

[0585] 95. A method according to any one of embodiments 92 to 94, wherein the modified antisense spacer oligonucleotide has increased exonuclease resistance compared to the parent antisense spacer oligonucleotide.

[0586] 96. A method according to any one of embodiments 92 to 95, wherein the modified antisense spacer oligonucleotide has increased endonuclease resistance compared to the parent antisense spacer oligonucleotide.

[0587] 97. A method according to any one of embodiments 92 to 96, wherein the parent antisense gapmer oligonucleotide is an LNA gapmer or a MOE gapmer, optionally wherein all internucleoside linkages are phosphorothioate linkages.

[0588] 98. The method of any one of embodiments 92 to 97, wherein the nucleotide unit is a nucleoside phosphoramidite.

[0589] 99. The method of any one of embodiments 92 to 98, wherein the modified antisense spacer oligonucleotide comprises the features of any one of embodiments 1 to 84.

[0590] 100. An antisense gapmer oligonucleotide obtained or obtainable by the method according to any one of embodiments 92 to 99.

[0591] 101. Use of a TNA nucleotide in the preparation of an antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 or a conjugate according to any one of embodiments 86 to 88.

[0592] Examples

[0593] The following examples illustrate certain embodiments of the invention and are not intended to be limiting. In addition, where specific embodiments are provided, the inventors have contemplated the general application of those specific embodiments. For example, disclosing an oligonucleotide with a TNA(PO) for one target provides reasonable support for other targets.

[0594] Example 1: Oligonucleotide synthesis using TNA modification

[0595] Oligonucleotides were synthesized by BioAutomation on a MerMade 12 or MerMade 192 automated DNA synthesizer. Controlled pore glass supports with universal adapters were used. The synthesis was performed on a 1 μmol scale.

[0596] For Mermade 12 : Standard cycling procedure for coupling of DNA, LNA and MOE phosphoramidites: DMT deprotection was performed using 3% (w / v) trichloroacetic acid in CH2Cl2, applied in six applications of 230 μL each for 70 seconds. The respective phosphoramidites were mixed with 95 μL of 0.1 M acetonitrile solution (or LNA- m Acetonitrile / CH2Cl2 1:1) of C structural unit and 110 μ L 0.3M solution of 5-benzylthio-1-H-tetrazole as activator in anhydrous acetonitrile were coupled three times, and the coupling time was 180 seconds. The freshly prepared TNA phosphoramidite was coupled three times with 95 μ L 0.1M acetonitrile solution and 110 μ L 0.3M 5-benzylthio-1-H-tetrazole as activator in anhydrous acetonitrile and the coupling time was 360 seconds. Use 0.1M 3-amino-1,2,4-dithiazole-5-thione solution in acetonitrile / pyridine: 1 / 1 for sulfidation, apply twice, 200 μ L each time, for 80 seconds. Use 0.02M I2 THF / pyr / H2O:88 / 10 / 2 solution for oxidation, apply twice, for 80 seconds. Capping was performed twice using THF / lutidine / Ac2O:8 / 1 / 1 (CapA, 125 μL) and THF / N-methylimidazole:84 / 16 (CapB, 125 μL), each for 85 seconds. After synthesis, the CPG was then carefully transferred to a 4 mL vial, to which 1 mL of 25% NH4OH was added and placed at 55°C for 16 hours. The crude DMT-on oligonucleotide was purified using a solid phase extraction column (Oasis HLB 6cc extraction column from Waters) or by preparative reverse phase HPLC (RP-HPLC) purification (C18 column, NH4OAc / CH3CN buffer system), followed by removal of DMT with 80% aqueous acetic acid. After HPLC purification, the oligonucleotide was purified using a HiPrep 26 / 10 desalting column. Oligonucleotides were desalted and lyophilized on a pure 25. Oligonucleotides were characterized by reversed-phase liquid chromatography coupled to high-resolution electrospray ionization mass spectrometry.

[0597] For Mermade 192: Standard cycling procedure for coupling of DNA, LNA, MOE and TNA phosphoramidites: DMT deprotection was performed using 3% (w / v) trichloroacetic acid in CH2Cl2, applied three times, 180 μL each time, for 75 seconds. The respective DNA and LNA phosphoramidites were coupled three times with 95 μL of 0.1 M acetonitrile (or acetonitrile / CH2Cl2 1:1 for LNA-MeC building blocks) and 110 μL of 0.285 M 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile as an activator, and the coupling time was 205 seconds. Freshly prepared TNA phosphoramidites were coupled twice with 95 μL of 0.1 M acetonitrile / DMF 95:5 and 110 μL of 0.285 M 5-benzylthio-1-H-tetrazole in anhydrous acetonitrile / DMF 95:5 as an activator, and the coupling time was 385 seconds. Sulfurization was performed using 0.1M 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine: 1 / 1, applied twice with 190 μL for 385 seconds. Oxidation was performed using 0.02M I2 in THF / pyr / H2O:88 / 10 / 2, applied twice with 160 μL for 78 seconds. Capping was performed twice using THF / dimethylpyridine / Ac2O 8:1:1 (CapA, 125 μL) and THF / N-methylimidazole: 84 / 16 (CapB, 125 μL) for 70 seconds each. After synthesis, the oligonucleotide was cleaved from the CPG using 25% NH4OH in 3 portions of 200 μl each. After each step, the CPG was left to stand at RT for 10 min. After the DWP was sealed, the solution was placed at 55°C for 15 hours. The crude DMT-on oligonucleotide was then purified and the final DMT was cleaved by passing through a column (Agilent TOP-DNA tubes, 150 mg resin / tube) using aqueous NH4Cl buffer, acetonitrile and 5% TFA in water.

[0598] The synthesis of TNA phosphoramidite was described as follows: “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.

[0599] Tables 1 to 5 show the molecules prepared according to the above procedures, using the nomenclature shown below (which also applies to the molecules shown in Tables 7 to 14).

[0600] A.G. mC, T (in bold): indicates TNA modification

[0601] A , G , m C , T (underlined): indicates MOE modification

[0602] A.G. m C, T represent LNA nucleotides

[0603] a, g, c, m c and t represent DNA nucleotides

[0604] o represents a phosphodiester bond

[0605] * indicates phosphorodithioate bond

[0606] All other bonds are phosphorothioate bonds.

[0607] Table 1: Synthetic molecules containing a TNA(PO) moiety (targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)). CMP ID NO = Compound ID number.

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616] We have designed two libraries of antisense oligonucleotides for human MALAT1 (Gencode: ENSG00000251562). The compound is 20 nucleotides long with two different sugar patterns: Design A: TTTTTddddddddddTTTTT, where T is TNA and d is DNA, and has a backbone pattern SOOOSSSSSSSSSSSOOOS, where S corresponds to phosphorothioate, and O corresponds to phosphodiester. Design B: MMMMTTTTTTddddMMMMM, where M is MOE, T is TNA and d is DNA, and has a backbone pattern SSSSOOOOOOSSSSSSSSSS, where S corresponds to phosphorothioate, and O corresponds to phosphodiester. These compounds were tested at two different concentrations (5 and 25 μM), and the 18 most effective oligonucleotides for Design A (#267-284) and the 19 most effective oligonucleotides for Design B (#285-303) were selected for full dose response determination.

[0617] Table 2: Compounds containing TNA PO (targeting MALAT1) selected from two synthetic libraries.

[0618]

[0619]

[0620] Table 3: TNA(PO) and DNA(PO) (targeting MALAT1)

[0621]

[0622] Antisense oligonucleotides targeting ceramide synthase 2 (CERS2), ceramide synthase 5 (CERS5), and ceramide synthase 6 (CERS6) were synthesized and are shown in Table 4. Synthetic molecules containing TNA (PO) and phosphorodithioate (PS2) moieties are shown in Table 5 (targeting MALAT1).

[0623] Table 4: Synthetic molecules containing TNA(PO) moieties (targeting CERS2, CERS5, CERS6). ID NO=compound Item ID number.

[0624]

[0625]

[0626] Table 5: Synthetic molecules containing TNA (PO) and phosphorodithioate (PS2) moieties (targeting MALAT1). NO = compound ID number.

[0627]

[0628]

[0629] Table 6 lists further details about the molecules in Tables 1 to 5, where the structure of each synthetic molecule is defined by the macromolecular hierarchical editing language (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 corresponding synthetic molecule is based is also indicated. The following HELM annotation legend is used:

[0630] [LR](G) is β-D-oxy-LNA guanosine,

[0631] [LR](T) is β-D-oxy-LNA thymidine,

[0632] [LR] (A) is β-D-oxy-LNA adenine nucleoside,

[0633] [LR]([5meC] is β-D-oxy-LNA 5-methylcytidine nucleoside,

[0634] [dR](G) is DNA guanosine nucleoside,

[0635] [dR](T) is DNA thymidine nucleoside,

[0636] [dR](A) is DNA adenine nucleoside,

[0637] [dR](C) is DNA cytosine nucleoside,

[0638] [dR]([5meC]) is DNA 5-methylcytosine nucleoside,

[0639] [MOE]([5meC]) is 2'O-MOE[2'O-(2-methoxyethyl)]5-methylcytidine nucleoside

[0640] [MOE] (A) is 2'O-MOE [2'O-(2-methoxyethyl)] adenine nucleoside

[0641] [MOE](T) is 2'-O-MOE[2'O-(2-methoxyethyl)]thymidine

[0642] [MOE](G) is 2'-O-MOE[2'O-(2-methoxyethyl)]guanosine

[0643] [TNA]([5meC]) is α-L-threonucleic acid 5-methylcytidine nucleoside

[0644] [TNA] (A) is α-L-threose nucleic acid adenine nucleoside

[0645] [TNA] (T) is α-L-threose nucleic acid thymidine

[0646] [TNA](G) is α-L-threonucleoside guanosine

[0647] [sP] is a phosphorothioate internucleoside linkage.

[0648] [PS2] is a phosphorodithioate internucleoside bond

[0649] [P] is a phosphodiester internucleoside bond

[0650] Further information about HELM as well as the open source tools can be found at the Internet address www.pistoiaalliance.org / helm-tools / (accessed on August 26, 2022).

[0651] Table 6: Synthetic molecules in HELM annotation

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704] Example 2: In vitro efficacy of oligonucleotides targeting MALAT1 RNA in A549 cells at two different concentrations (5 μM and 25 μM)

[0705] 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 determination, 3000 cells per well were seeded in complete medium in a 96-well plate. The cells were incubated for 24 hours and then oligonucleotides dissolved in PBS were added to the specified final concentration. Three days after the 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 subsequently diluted 10 times with DNase / RNase-free water and heated to 90°C for one minute.

[0706] For gene expression analysis, use qScript TM XLT One-step RT-qPCR Low ROX TMOne-step RT-qPCR was performed in a duplex setup (Quantabio). The following TaqMan primer assays were used for qRT-PCR: MALAT1, Hs00273907_s1[FAM-MGB], CERS2, Hs01017465_m1[FAM-MGB], CERS5, Hs00332291_m1[FAM-MGB], CERS6, Hs00826756_m1[FAM-MGB], and endogenous control GAPDH, Hs99999905_m1[VIC-MGB-PL]. All primer sets were purchased from Thermo Fisher Scientific. The relative RNA expression level of MALAT1, CERS2, CERS5 or CERS6, also known as the knockdown (KD) value, was calculated as a percentage of the control (PBS-treated cells).

[0707] KD values ​​were determined using GraphPad Prism.

[0708] The results are shown in Tables 7 to 10. Values ​​separated by " / " indicate individual results when a compound or control was tested in more than one test vial.

[0709] Table 7: Knockdown (KD) values ​​targeting MALAT1

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719] The data demonstrate that TNA(PO) is well tolerated for KD in A549 cells when placed in flanks / spacers.

[0720] Table 8: Knockdown (KD) values ​​targeting MALAT1; DNA PO vs. TNA PO

[0721]

[0722] The data demonstrate the superiority of TNA(PO) over DNA(PO) by virtue of its good metabolic stability when TNA is placed in the DNA compartment.

[0723] Table 9: Knockdown (KD) values ​​targeting CERS2 / 5 / 6

[0724]

[0725] The data demonstrate that the strategy of using TNAs to reduce DNA spacers close to the 5' flank can be generalized to other targets beyond MALAT1.

[0726] Table 10: Knockdown (KD) values ​​of MALAT1 compounds containing TNA(PO) and phosphorodithioate

[0727]

[0728] A novel structure containing TNA(PO) in the DNA spacer and phosphorodithioate(PS2) at both ends was successfully demonstrated. The phosphorodithioate bond significantly reduced MALAT1 expression at 25 μM in A549 cells.

[0729] Example 3: In vitro potency and efficacy of oligonucleotides targeting MALAT1 RNA in A549 cells at different concentrations in a dose-response curve

[0730] 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 (A549) per well were seeded in culture medium in a 96-well plate. The cells were cultured for 24 hours and then oligonucleotides dissolved in PBS were added. The concentration range of the oligonucleotides was: the highest concentration was 25 μM, and 1:1 dilution was performed in 8 steps. Three days after the addition of the oligonucleotides, the cells were harvested. 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 of water. The RNA was subsequently diluted 10-fold with DNase / RNase-free water (Gibco) and heated to 90°C for one minute.

[0731] For gene expression analysis, use qScript TM XLT One-step RT-qPCR Low ROX TMOne-step RT-qPCR was performed in a duplex setup (Quantabio). The following TaqMan primers were used for qPCR: MALAT1, Hs00273907_s1 (FAM-MGB) and endogenous control GAPDH. All primer sets were purchased from Thermo Fisher Scientific. The relative expression level of MALAT1 RNA is shown as a percentage of the control (PBS-treated cells), and IC50 values ​​have been determined using GraphPad Prism7 based on data from n=2 biological replicates.

[0732] The results are shown below in Table 11. Values ​​separated by " / " indicate individual results when a compound or control was tested in more than one test vial.

[0733] Table 11: In vitro potency results (IC50)

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746] Example 4: Caspase 3 / 7 activation of HepG2 cells

[0747] Caspase 3 / 7 activated HepG2 cells were cultured at approximately 70% confluence in MEM medium supplemented with 10% heat-inactivated fetal bovine serum and GlutaMax (Gibco #41090). Cells were detached using 0.25% trypsin-EDTA solution (Gibco #25200056) and plated at 1×10 4Cells were seeded at a density of 10 cells / well into black, transparent 96-well plates P37102-EP 78 (Corning #3904, NY, USA). 24 h after seeding, HepG2 cells were transiently transfected using Lipofectamine 2000 (Life Technologies #11668019) using 100 nM oligonucleotides dissolved in Opti-MEM (Gibco #31985). Caspase-3 / 7 activity was determined using the Promega 3 / 7 assay (Promega Corporation, Madison WI, USA). 3 / 7 reagent was added to cells 24 hours after transfection, incubated for 60 min, and the cell lysate was transferred to an opaque 96-well plate (Corning #3600, NY, USA), and then luminescence was measured on an Enspire multi-mode plate analyzer (Perkin Elmer) according to the manufacturer's instructions. The results are shown in Table 12, where the percentage (measurement window %) indicates the degree of apoptosis based on the vehicle (cells treated with PBS alone). The higher the value, the higher the apoptotic activity, and thus the higher the in vitro cytotoxicity. The negative control is a non-toxic ASO. Due to the low background of these ASOs, some values ​​of the tested ASOs are negative. Negative values ​​have no cytotoxicity, while ASOs with values ​​of about 300 have slight cytotoxicity. ASO = antisense oligonucleotide.

[0748] Table 12: Caspase 3 / 7 assay results

[0749]

[0750]

[0751] The data demonstrate that even a single TNA(PO) in the 5' flank or DNA spacer significantly reduces caspase induction across targets (MALAT1, CERS2 / 5 / 6).

[0752] Example 5: Thermal melting (Tm) of oligonucleotides containing TNA(PO) modifications hybridized to RNA

[0753] Denaturation point (thermal melting = Tm) is measured according to the following procedure: the spacer ASO and complementary RNA are added to 20mM disodium phosphate buffer, 200mM NaCl and 0.2mM EDTA (pH 7) to produce a final concentration of 1.5 μM. The sample is heated to 95°C for 5min and then slowly cooled to room temperature over a 1 hour period. Using a temperature gradient that increases from 25°C to 95°C at 5°C / min and then drops to 25°C, the thermal melting curve is recorded at 260nm on an Agilent Cary3500 equipped with a Peltier temperature programmer. The first derivative of the two curves is used to determine the melting temperature (Tm). These values ​​are the average values ​​on three heating and cooling curves.

[0754] Table 13: Thermal melting temperature (Tm) values

[0755]

[0756]

[0757]

[0758] The data demonstrate that when TNA(PO) is incorporated into the DNA spacer, the Tm increases. However, when TNA replaces sugars with high binding affinity (MOE, LNA), the Tm decreases.

[0759] Example 6: Inhibition of MALAT1 expression in mice administered subcutaneously

[0760] To compare TNA with other sugars, the well-described MOE spacer targeting MALAT1 was selected as the parent antisense oligonucleotide for in vivo studies. TNA substitution of MOE in the flank generated the following set of antisense oligonucleotides, which were administered subcutaneously to mice at 10 mg / kg.

[0761] Table 14: Selected TNA-containing antisense oligonucleotides used in in vivo studies in mice

[0762]

[0763] ND: Not Determined

[0764] In vivo experiments were performed. The study was performed with the approval of the local veterinary authorities and in strict compliance with the Swiss Federal Animal Protection Regulations and the rules of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Male C57BL / 6J mice (8 to 12 weeks old) were purchased from Charles River Laboratories, France. Mice were group housed in open cages and maintained with a 12:12-h light:dark cycle, constant temperature (21–24°C), and humidity (40–80%). Each cage had unrestricted access to municipal water and sterilized food (Provimi Kliba 3436). All cages (except metabolic cages) were equipped with autoclaved sawdust bedding and environmental enrichment, which were adapted to best practice animal welfare standards and rotated weekly. Mice were acclimated for at least 1 week before the start of the study.

[0765] The animals were housed in 3 groups and provided with water and a standard diet ad libitum. The enclosures were maintained at a constant temperature (23 ± 1 °C) and humidity (40 ± 5%) under a 12 h light: 12 h dark cycle (lights on at 08:00) throughout the study. Mice were dosed intravenously and anesthetized (70% CO2 / 30% O2) on day 0, then terminated by cervical dislocation on day 9. Treatment groups (n = 4) received 0.9% saline or saline-formulated spacers, administered by subcutaneous injection at a dose of 10 mg / kg. At the end of the study, organs and plasma were collected at the endpoint time points. Plasma samples were stored at -20 °C for further processing and analysis. Organ samples were collected into homogenizer tubes ( CK28, Bertin Instruments, France).

[0766] Total RNA was isolated from liver and kidney using the RNeasy kit (Qiagen), and RNA quantification was performed using the TaqMan assay (Applied Biosystems). Reverse transcription reactions were performed using random decamers, 0.5 mg of total RNA, and M-MLV reverse transcriptase (Ambion) according to the protocol for first-strand complementary DNA (cDNA) synthesis. Depending on expression levels, cDNA was then diluted 5-fold in nuclease-free water and added to the RT-PCR reaction mixture. Amplification was performed using an Applied Biosystems 7500 / 7900 / ViiA real-time PCR instrument. In each study, mRNA levels were normalized to actin, β (Actb), or 3-phosphoglyceraldehyde dehydrogenase (Gapdh) and presented as fold changes relative to the mean level of saline controls.

[0767] like Figure 1As shown, all antisense oligonucleotides can better reduce MALAT1RNA in the liver compared with other tissues. In the liver, at a dosage level of 10mg / kg, the parental interval polymer (control #2) demonstrated that the RNA target was reduced by about 70%. The corresponding PO version (#346) performed much lower. Two TNA analogs (#100 and #76) with six POs were introduced, which were significantly better than control #2. Compared with the full PS parent, even two ASOs (#99 and #78) with eight phosphates were inserted, which also demonstrated the same in vivo efficacy. The full PS TNA interval polymer (#347) performed best, and the target was knocked down by about 80%. Compared with MOEASO, TNA did not improve the reduction of MALAT1 expression in the lungs and muscles (control #2 and #346).

[0768] Tissue samples were collected and weighed during necropsy and stored in homogenizer tubes CK28 at -80 °C. for subsequent bioanalytical examination by LC coupled to tandem mass spectrometry (LCMS / MS). Prior to extraction, the tissue homogenate was diluted 5-fold in mouse blank plasma. Quantification was performed against a mouse plasma calibration curve. 50 microliters of calibration standards, quality control samples (freshly prepared in mouse plasma), and tissue homogenate samples diluted in mouse blank plasma were treated with 150 μL 4M guanidine thiocyanate for protein denaturation after the addition of internal standard (2000 ng / mL). The characteristic 16-mer oligonucleotide (MW: 5460 Da) consists of DNA nucleotides, LNA nucleotides, and a complete phosphorothioate backbone and is used as an internal standard to exclude possible changes during pipetting, solid phase post-processing, or LC-MS / MS sample injection. After vigorous mixing (at 1,600 rpm for 20 min), 200 μL of H2O / HFIP / DIPEA solution (100:4:0.2, v / v / v) was added, followed by mixing (at 1500 rpm for 15 min). Then, after elution and evaporation to dryness (30-45 min at 40°C), a cleanup step was performed using a solid phase extraction column (Waters, OASIS HLB, 30 μm), and the sample was reconstituted in 200 μL of mobile phase (HO / MeOH / HFIP / DIPEA [95 / 5 / 1 / 0.2, v / v / v / v]). After vortex mixing (10 min at 1500 rpm), an aliquot (20 μL) was injected into an analytical column (Waters, Acquity BEH C18, 1.7 μm, 50×2.1 mm, maintained at 60°C). Analytes and internal standards were separated from matrix interferences using a gradient from H2O / MeOH / HFIP / DIPEA (95 / 5 / 1 / 0.2, v / v / v / v) to H2O / MeOH / HFIP / DIPEA (10 / 90 / 1 / 0.2, v / v / v / v) at a flow rate of 0.4 mL / min over 4 min. Mass spectrometry detection was performed on an AB-Sciex 6500+ mass spectrometer using selected reaction monitoring (SRM) in negative ion mode. The selected ion reactions (m / z) for ASO 1 and ASO 5 were 680.6 / 94.8 and 658.9 / 94.8, respectively, and the selected ion reaction (m / z) for the internal standard was 596.1 / 94.8. Detection was accomplished using ion spray MS / MS in negative ion SRM mode.

[0769] As shown in Figure 2, quantification of antisense oligonucleotide levels in the liver and kidney, as well as in the lung and muscle at day 9 was demonstrated by using LCMS / MS and PK / PD relationships. The TNA spacer (#347) had the highest liver concentration, which strongly correlated with its strong reduction of MALAT1 RNA in the liver. Antisense oligonucleotide #99 showed comparable efficacy, albeit at lower liver levels. Apparently, antisense oligonucleotides containing PO bonds were primarily eliminated into the kidneys, likely due to lower affinity to plasma proteins. As expected, all antisense oligonucleotides were present at much lower concentrations in the lung and muscle and, therefore, did not result in meaningful reductions in mRNA in these two tissues.

[0770] References

[0771] Crooke et al., Nucleic Acids Research 2020;48(10):5235–5253. DOI:10.1093 / nar / gkaa299

[0772] Eckstein, Antisense and Nucleic Acid Drug Development 2009;10:117-121. DOI:10.1089 / oli.1.2000.10.117.

[0773] Liu et al., ACS Appl. Mater. Interfaces 2018;10:9736-9743. DOI:10.1021 / acsami.8b01180

[0774] Matsuda et al., Poster; XXIII International Round Table on Nucleosides, Nucleotides and Nucleic acids; August 2018. DOI: 10.13140 / RG.2.2.10627.45605

[0775] Zhang and Chaput, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012.DOI:10.1002 / 0471142700.nc0451s50

[0776] WO 2012 / 078536 (Quark Pharmaceuticals, Inc.)

[0777] WO 2012 / 118911 (Quark Pharmaceuticals, Inc.)

[0778] WO 2013 / 179292 A1 (QBI Enterprises Ltd. and Bio-Lab Ltd.)

Claims

1. An antisense spacer oligonucleotide comprising a continuous nucleotide sequence of formula 5'-FG-F'-3' (I) capable of recruiting ribonuclease (RNase) H, wherein G is a spacer region of up to 18 linked nucleosides, said spacer region comprising at least 3 consecutive DNA nucleosides, Each of F and F' is a flanking region of up to 15 linked nucleosides, said flanking region independently comprising or consisting of 1 to 15 sugar-modified nucleosides, At least one of F, F' and G comprises a sugar-modified nucleoside that is an α-L-threofuranosyl (TNA) nucleoside and is linked to an adjacent nucleoside by an internucleoside bond other than a phosphorothioate (PS) internucleoside bond.

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

3. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein the TNA nucleoside is linked to any adjacent nucleoside via a 2'-PO or 3'-PO internucleoside bond.

4. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein F comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

5. The antisense gapper oligonucleotide of any of the preceding claims, wherein F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

6. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein all nucleosides of F are TNA nucleosides.

7. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein F' comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

8. The antisense gapmer oligonucleotide of any of the preceding claims, wherein F' comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to adjacent nucleosides by PO internucleoside bonds.

9. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein all nucleosides of F' are TNA nucleosides.

10. The antisense gapmer oligonucleotide of any of the preceding claims, wherein the sugar-modified nucleosides of F, F', or both F and F' comprise at least one sugar-modified nucleoside comprising a modified sugar moiety selected from the group consisting of: 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'-fluoro-ribose-nucleic acid; 2'-F-RNA), 2'-Fluoro-2'-arabinose (as in 2'-fluoro-2'-arabinonucleotide; 2'-F-ANA), 2'-O-Benzyl-ribose, Oxylated β-D-locked ribose (such as in β-D-LNA), Amino-β-D-locked ribose (as in amino-β-D-LNA), Thio-β-D-locked ribose (such as in thio-β-D-LNA), Oxylated β-L-locked ribose (such as in β-L-LNA), Amino-β-L-locked ribose (as in amino-β-L-LNA), Thio-β-L-locked ribose (such as thio-β-L-LNA), Oxylated α-L-locked ribose (such as in α-L-LNA), Amino α-L-locked ribose (such as amino-α-L-LNA), Thio-α-L-locked ribose (such as thio-α-L-LNA), 2',4'-constrained 2'-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), Tricyclic-deoxyribose (such as tricyclic-deoxyribose DNA; TcDNA), 3'-deoxy-ribose (such as 3'-deoxy-ribose DNA; 3'-DNA), Unlocked ribose (as in unlocked nucleic acid; UNA), Diols (as in diol nucleic acids; GNA), Hexitols (as in hexitolide nucleic acid; HNA), 3'-fluorohexitol (as in 3'-fluorohexitol nucleic acid; FHNA), 3'-arabino-fluorohexitol (as in 3'-arabino-fluorohexitol nucleic acid; Ara-FHNA), Cyclohexene (as in cyclohexene nucleic acid; CeNA), Fluoro-cyclohexenyl (as in 2'-fluoro-cyclohexenyl nucleic acid; F-CeNA), Serinol (as serinol nucleic acid; SNA), 2'-O,4'-C-ethylene-bridged ribose (as in 2'-O,4'-C-ethylene linked nucleic acid; ENA) Acyclic (L)-threonine (as in acyclic (L)-threonine nucleic acid; aTNA) 2',4'-Constrained 2'-O-methoxyethyl ribose (as in cMOE) 7',5'-α-bicyclic sugar unit (such as 7',5'-α-bicyclic DNA; in bcDNA).

11. The antisense gapmer oligonucleotide according to any one of the preceding claims, wherein All nucleosides in F' and F, except any at least one TNA nucleoside, are LNA nucleosides or 2'-O-MOE nucleosides.

12. The antisense gapper oligonucleotide of any one of the preceding claims, wherein G comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside bond.

13. An antisense gapmer oligonucleotide according to any one of the preceding claims, wherein Except for any PO internucleoside linkages between a TNA nucleoside and an adjacent nucleoside, all internucleoside linkages are phosphorothioate (PS) internucleoside linkages, phosphorodithioate (PS2) internucleoside linkages, or a combination thereof.

14. An antisense gapmer oligonucleotide according to any one of the preceding claims, wherein (a) the nucleoside closest to the 5' side of F is linked to the adjacent nucleoside in F via a PS2 internucleoside bond; or (b) the nucleoside closest to the 3' side of F' is linked to the adjacent nucleoside in F' via a PS2 internucleoside bond; or (c) Both (a) and (b).

15. The antisense spacer oligonucleotide according to any one of the preceding claims, wherein the contiguous nucleotide sequence of formula 5'-FG-F'-3' (I) has a length of 12 to 32 nucleosides, such as 12 to 28 nucleosides, 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.

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

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

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

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

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

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