EA043736B1 - ANTISENSE OLIGOMERS AND THEIR CONJUGATES DIRECTED AT PROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 (PCSK9) - Google Patents

Description

Field of invention

The present invention relates to oligomeric compounds and their conjugates that target proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA in a cell, resulting in reduced expression of PCSK9. Reducing PCSK9 expression is beneficial for a number of diseases such as hypercholesterolemia and related disorders.

Prior Art

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for lowering low-density lipoprotein cholesterol (LDL cholesterol). PCSK9 increases LDL receptor degradation, resulting in high LDL cholesterol in individuals with high PCSK9 activity.

Lindholm et al., Molecular Therapy (2012), 20, 2, 376-381 describe two locked nucleic acid (LNA) antisense nucleotides targeting PCSK9 that provide prolonged LDL cholesterol reduction in non-human primates following a loading dose (20 mg /kg) and four weekly maintenance doses (5 mg/kg). The compounds used were SPC5001 14-mer (SEQ ID NO: 1) and SPC4061 13-mer. SPC5001 is also disclosed in WO 2011/009697. The effectiveness of these PCSK9 inhibitors is due to their short length (Krieg et al., Molecular Therapy Nucleic Acids (2012), 1, e6).

WO 2007/146511 describes short bicyclic (BNC) gapmer antisense oligonucleotides which are apparently more effective and less toxic than longer compounds. The exemplary compounds were found to be 14 nucleotides (nt) in length.

According to the article by van Poelgeest et al. (American Journal of Kidney Disease, 2013 Oct, 62(4): 796-800) administration of the antisense LNA oligonucleotide SPC5001 in human clinical trials may result in acute kidney injury.

According to publications EP 1984381B1, Seth et al., Nucleic Acids Symposium Series, 2008, No. 52, 553-554; and Swayze et al., Nucleic Acid Research, 2007, vol 35, p. 687-700 LNA oligonucleotides cause significant hepatotoxicity in animals. According to WO 2007/146511, the toxicity of LNA oligonucleotides can be avoided by using LNA gapmers as short as 12-14 nucleotides in length. EP 1984381 B1 recommends the use of 6'-substituted bicyclic nucleotides to reduce the hepatotoxic potential of LNA oligonucleotides. According to Hagedorn et al., Nucleic Acid Therapeutics, 2013, the hepatotoxic potential of an antisense oligonucleotide can be predicted based on its sequence and modification pattern.

Oligonucleotide conjugates have been extensively evaluated for use in small interfering RNA (siRNA) applications, where they are considered essential to obtain sufficient in vivo efficacy. For example, WO 2004/044141 relates to modified oligomeric compounds that modulate gene expression through the biochemical RNA interference pathway. Oligomeric compounds include one or more conjugate moieties that can modify or enhance the pharmacokinetic and pharmacodynamic properties of the attached oligomeric compound.

WO 2012/083046 describes a galactose cluster pharmacokinetic modulator targeting moiety for siRNA.

In contrast, single-stranded antisense oligonucleotides are typically administered therapeutically without conjugation or formulation. The main target tissues for antisense oligonucleotides are the liver and kidney, although a wide range of other tissues are also available for the antisense method, including lymph node, spleen and bone marrow.

WO 2005/086775 relates to targeted delivery of therapeutic agents to specific organs using a therapeutic chemical moiety, a cleavable linker and a tagged domain. The cleavable linker may be, for example, a disulfide group, a peptide, or a restriction enzyme cleavable oligonucleotide domain.

WO 2011/126937 relates to targeted intracellular delivery of oligonucleotides through conjugation with small molecule ligands.

WO 2009/025669 relates to polymeric (polyethylene glycol) linkers containing pyridyl disulfide moieties. See also Zhao et al., Bioconjugate Chem., 2005, 16, 758-766.

Chaltin et al., Bioconjugate Chem., 2005, 16, 827-836 describe cholesterol-modified mono-, di-, and tetrameric oligonucleotides used to incorporate antisense oligonucleotides into cationic liposomes to form a dendrimer delivery system. Cholesterol is conjugated to oligonucleotides via a lysine linker.

Other noncleavable cholesterol conjugates have been used to target siRNAs and antagomirs to the liver; see, for example, Soutscheck et al., Nature, 2004, vol. 432, 173-178; and Krhtzfeldt et al., Nature, 2005, vol. 438, 685-689. For partially phosphorothiolated siRNAs and antagomirs, the use of cholesterol as a liver targeting molecule was found to be essential for in vivo activity.

- 1 043736

Object of the invention

Thus, there is a need for antisense compounds that target

PCSK9, which are as effective as SPC5001 but have a reduced risk of toxicity, particularly reduced renal toxicity.

According to the present invention, this goal is achieved by identifying new human PCSK9 sequences that are partially effective for targeting using the antisense method (SEQ ID NO: 33 and SEQ ID NO: 34), as well as longer sequence variants of SPC5001 that retain significant efficiency or have improved efficiency compared to SPC5001 without toxicity issues. The antisense nucleotides of the invention can be further improved by the use of conjugates that have been found to significantly enhance the therapeutic index of antisense LNA oligonucleotides.

The compounds of the present invention are potent and non-toxic PCSK9 inhibitors useful in the treatment of hypercholesterolemia and related disorders.

The essence of the invention

The oligomer of the invention may contain from 10 to 22, for example from 12 to 18 nucleotides in length, which are either

a) a sequence of 10-16 contiguous nucleotides that is complementary to the corresponding length of SEQ ID NO: 33 or 34 or 45; or

b) a sequence of 16 contiguous nucleotides that is complementary to the corresponding length of SEQ ID NO: 31.

The invention provides an antisense oligonucleotide conjugate comprising an oligomer according to the invention and at least one non-nucleotide or non-polynucleotide moiety covalently attached to the oligomer.

The invention also provides an antisense oligonucleotide conjugate comprising an oligomer (A) according to the invention and at least one non-nucleotide or non-polynucleotide moiety covalently attached to the oligomer (C), optionally via a linker region (B and/or Y) located between the continuous nucleotide sequence of the oligomer and the conjugate group.

In some embodiments, the invention also provides an antisense oligonucleotide conjugate comprising an oligomer of 10-22, for example 12-18 nucleotides in length, wherein the oligomer contains

a) a sequence of 10-16 contiguous nucleotides that is complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45; or

b) a sequence of 16 contiguous nucleotides that is complementary to the corresponding length of SEQ ID NO: 31.

The invention also provides a compound selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 16, 18, 19, 20, 21, 22, 23 and 24.

The invention provides a pharmaceutical composition containing an oligomer or conjugate according to the invention and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides an oligomer or conjugate or pharmaceutical composition according to the invention, used as a drug, for example, for the treatment of hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, for example acquisition functional mutations in PCSK9, high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol imbalance, dyslipidemias such as familial hyperlipidemia (familial combined hyperlipidemia (FCH) or familial hypercholesterolemia (FH), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease arterial disease (CAD) and coronary heart disease (CHD).

The invention provides the use of an oligomer or conjugate or pharmaceutical composition of the invention for the preparation of a medicament for the treatment of hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, for example acquisition of functional mutations in PCSK9, imbalance high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol, dyslipidemias such as familial hyperlipidemia (familial combined hyperlipidemia (FCH)) or familial hypercholesterolemia (FH), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).

The invention provides a method for treating hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, for example, acquisition of functional mutations in PCSK9, high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol imbalance ), dyslipidemias, such as familial hyperlipidemia (familial combined hyperlipidemia (FCH)) or familial hypercholesterolemia (FHC), acquired hyperlipidemia,

- 2043736 statin-resistant hypercholesterolemia, coronary arterial disease (CAD) and coronary artery disease (CHD), where the method includes administering an effective amount of an oligomer or conjugate or pharmaceutical composition of the invention to a patient suffering or likely to suffer from hypercholesterolemia or a related disorder.

The invention provides a method for in vivo or in vitro inhibition of PCSK9 in a cell expressing PCSK9, wherein the method comprises introducing an oligomer or conjugate or pharmaceutical composition of the invention into the cell so as to inhibit PCSK9 in the cell.

The invention also provides an oligomer according to the invention, such as a LNA oligomer containing a region of 10-22, for example 12-18, for example 13, 14, 15, 16 or 17, contiguous nucleosides linked by a phosphorothioate bond (i.e. a region A, which is typically complementary to the corresponding region of the target sequence, such as SEQ ID NO: 46), and additionally containing from 1 to 6 DNA nucleosides adjacent to the LNA oligomer, where internucleoside bonds between the DNA and/or a DNA-adjacent nucleoside ( nucleosides) are physiologically labile, like phosphodiester bonds. Such an LNA oligomer may be in the form of a conjugate, as disclosed herein, or may be, for example, an intermediate for use in a subsequent conjugation step. When conjugated, the conjugate may, for example, be or contain a sterol, such as cholesterol or tocopherol, or may be or contain a (non-nucleotide) carbohydrate, such as a GalNAc conjugate or other conjugate as disclosed herein.

The invention also provides a gapmer oligomer comprising at least one cET nucleotide, such as (S)-cET, of 10-22, e.g. 12-18, e.g. 13, 14, 15, 16 or 17 nucleotides in length, which is directed (ie has a sequence complementary to the corresponding region) to human PCSK9.

Brief description of graphic materials

In fig. 1 provides examples of tri-GalNAc conjugates that can be used. Conjugates 1-4 illustrate 4 suitable groups of GalNAc conjugates, and conjugates 1a-4a are the same conjugates with an additional linker moiety (Y) that is used to cross-link the conjugate to the oligomer (site A or a biocleavable linker such as site B). The wavy line represents a covalent bond to the oligomer.

In fig. 2 - examples of cholesterol and tocopherol conjugate groups. Conjugates 5a and 6a are the same conjugates with an additional linker moiety (Y) that is used to cross-link the conjugate to the oligomer (site A or a biocleavable linker such as site B). The wavy line represents a covalent bond to the oligomer.

In fig. 3 - specific compounds of ZNK. Beta-O-hydroxy LNAs are identified by a superscript ^L (locked) after the letter, a subscript 'V represents a phosphorothioate bond, a superscript ^Me preceding a capital C represents a 5-methylcytosine-LNA, non-LNA nucleotides represent nucleotides DNA (no superscript ' ^L ').

In fig. 4 - examples of cholesterol conjugates of LNA compounds. Beta-O-hydroxy LNAs are identified by a superscript ^L (locked) after the letter, a subscript _s represents a phosphorothioate linkage, a superscript ' ^Me ' preceding a capital C represents a 5-methylcytosine-LNA, non-LNA nucleotides represent DNA nucleotides (no superscript ^L ).

In fig. 5 - examples of GalNAc conjugates of LNA compounds. The conjugates essentially correspond to Conj2a in the figure, where the wavy line is replaced by a LNA oligomer. Beta-O-hydroxy LNAs are identified by a superscript ^L (locked) after the letter, a subscript _s represents a phosphorothioate linkage, a superscript ' ^Me ' preceding a capital C represents a 5-methylcytosine-LNA, non-LNA nucleotides represent DNA nucleotides (no superscript ^L ).

In fig. 5A shows the detailed structure of SEQ ID NO: 18.

In fig. 5B - detailed structure of SEQ ID NO: 19.

In fig. Figure 6 shows an example of a FAM conjugate group.

In fig. Figure 7 shows LNA-FAM conjugates with and without cleavable phosphodiester bonds. Beta-O-hydroxy LNAs are identified by a superscript ^L (locked) after the letter, a subscript _s '' represents a phosphorothioate bond, a superscript ^Me preceding a capital C represents a 5-methylcytosine-LNA, non-LNA nucleotides represent DNA nucleotides (without the superscript ' ^L ').

In fig. 8 - anti-PCSK9 gapmers ranked by in vitro activity.

In fig. 9 - selected anti-PCSK9 gapmers ranked by in vitro activity.

In fig. 10 shows the in vitro activity of selected compounds against PCSK9 and IC ₅₀ calculations.

In fig. 11 - In vivo ALT data for selected anti-PCSK9 conjugates.

Fig. 12 is a non-limiting illustration of compounds of the invention. The internucleoside linkage L may be, for example, a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphate, for example phosphodiester. PO is a phosphodiester bond. Compound a) has a single DNA (or RNA) B region where the bond between the first and second region is a PO. Compound b) has two DNA/RNA (such as DNA) nucleosides linked by a phosphodiester bond. Compound c) has three DNA/RNA (such as DNA) nucleosides linked by phosphodiester bonds. In some embodiments of the invention, the B region may be further extended by additional phosphodiester DNA/RNA (such as DNA nucleosides). A conjugate group (Marked X, otherwise referred to herein as region C) is illustrated on the left side of each compound (e.g., cholesterol, GalNAc, Conj1-4, 1a-4a, and 5 or 6) and may optionally be covalently attached to the terminal nucleoside of region B by a phosphorus nucleoside linking group such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphate, or may be linked via an alternative linkage such as a thiazole linkage (see L in compounds d), e) and f).

Fig. 13 is a non-limiting illustration of compounds of the invention, where the compounds contain an optional linker (Y) between a third (conjugate) region (X) and a second region (B region). The nomenclature is the same as in Fig. 12. Suitable linkers are disclosed herein and include, for example, alkyl linkers, eg C6 linkers. In compounds a), b) and c), the linker between X and the B site is attached to the B site via a phosphorus nucleoside linking group such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methyl phosphate linkage, or may be linked via an alternative linkage such as a triazole linkage ( Li). In these compounds, Li represents the internucleoside linkage between the first (A) and second sites (B). Compounds d), e) and f) further contain a linker (Y) between the B site and the conjugate group, and the Y site may be linked to the B site via, for example, a phosphorous nucleoside linking group such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methyl phosphate, or in some embodiments a triazole linkage. In addition or alternatively, X may be an activating group or a reactive group. X may be covalently attached to the B site via a phosphorus nucleoside linking group such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methyl phosphate, or may be linked via an alternative linkage such as a triazole linkage.

In fig. 14 shows PCSK9 mRNA silencing by cholesterol conjugates in vivo. Mice were injected with a single dose of 10 mg/kg unconjugated LNA antisense oligonucleotide (#40) or equimolar amounts of LNA antisense oligonucleotides conjugated to cholesterol with various linkers and sacrificed on days 1, 3, 7, and 10 after dosing. RNA was isolated from liver and kidney and subjected to PCSK9-specific quantitative reverse transcription-polymerase chain reaction (RT-qPCR) A. Quantification of PCSK9 mRNA from liver samples normalized to BACT and shown as a percentage of the mean of equivalent saline controls B. Quantification determination of PCSK9 mRNA from kidney samples was normalized to BACT and shown as a percentage of the mean of equivalent saline controls.

In fig. 15 shows Kim-1 expression from a safety study in rats (see Example 5).

In fig. 16 - Serum PCSK9 and LDL cholesterol in samples from cynomolgus monkeys injected four times (one injection/week) with 0.5 or 1.5 mg/kg/week SEQ IDs 2 and 18.

In fig. 17 - Serum PCSK9 and LDL cholesterol in samples from cynomolgus monkeys injected four times (one injection/week) with 0.5 or 1.5 mg/kg/week SEQ ID 3 and 19.

Information confirming the possibility of implementing the invention

Various elements of the invention are described below under separate headings. It will be understood that an embodiment of the invention from one element can be combined with embodiments of the invention from other elements (eg, as illustrated in FIGS. 12 and 13) to produce a compound of the invention.

Oligomer (section A).

The term oligomer or oligonucleotide in the context of the present invention refers to a molecule formed through the covalent linkage of two or more nucleotides (ie, an oligonucleotide). As used herein, a single nucleotide (unit) may also be referred to as a monomer or unit. In some embodiments of the invention, the terms nucleoside, nucleotide, unit, and monomer are used interchangeably. It will be understood that when referred to, the sequence of nucleotides or monomers being referenced is a sequence of bases such as A, T, G, C or U.

The oligomer according to the invention may contain from 10 to 22, for example from 12 to 22 nucleotides, for example from 12 to 18 nucleotides in length. The oligomer contains either

a) a sequence of 10-16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 33 or 34 or 45; or

b) a sequence of 16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 31.

In some embodiments of the invention, the oligomer of the invention contains a contiguous nucleotide sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29 and 44.

A compound (eg, oligomer or conjugate) of the invention targets PCSK9 and is itself capable of down-regulating the expression of or inhibiting PCSK9, such as PCSK9 in a human or in a cell expressing PCSK9.

In some embodiments of the invention, the internucleoside linkages of a sequence of 10-16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45 may be phosphorothioate linkages.

In some embodiments of the invention, the oligomer of the invention contains the sequence or consists of a contiguous nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 and 40. In one embodiment of the invention, the oligomer contains the sequence or consists of a sequence chosen from

a) SEQ ID NO: 2 or 3; or

b) SEQ ID NO: 4, 5 or 6; or

c) SEQ ID NO: 7 or 8; or

d) SEQ ID NO: 40.

In some embodiments of the invention, the oligomer contains 10-16 nucleosides linked by a phosphorothioate bond.

In some embodiments of the invention, the oligomer of the invention contains a sequence of at least 10-16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45, or a sequence of 16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 31, wherein the contiguous nucleotide sequence contains nucleotide analogues. Preferably, the nucleotide analogs are affinity-enhancing nucleotide analogs.

In some embodiments of the invention, the nucleotide analogs are sugar-modified nucleotides, such as sugar-modified nucleotides, independently or dependently selected from the group consisting of locked nucleic acid (LNA) units, 2'-O-alkyl-RNA units, 2' units -OMe-RNA, 2'-amino-DNA units and 2'-fluoro-DNA units.

In some embodiments of the invention, the nucleotide analogs contain or are locked nucleic acid (LNA) units.

In some embodiments of the invention, the oligomer of the invention contains or is a gapmer, such as a LNA gapmer oligonucleotide.

In some embodiments of the invention, the gapmer contains on each side (5' and 3') a wing segment of 2 to 4 nucleotide analogs, preferably LNA analogs.

In some embodiments of the invention, the oligomer of the invention contains a sequence of 13, 14, 15 or 16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45, or a sequence of 16 contiguous nucleotides complementary to the corresponding length of SEQ ID NO: : 31, and may optionally contain an additional 1-6 nucleotides, which may form or contain a biocleavable nucleotide region, such as a nucleotide phosphate linker. Advantageously, this biocleavable nucleotide region is formed by a short fragment (eg 1, 2, 3, 4, 5 or 6) of nucleotides that is physiologically labile. This can be achieved by using phosphodiester linkages with DNA/RNA nucleosides, or if its physiological lability may persist, a different nucleoside can be used. Physiological lability can be measured using liver extract, as illustrated in Example 6.

The oligomer of the invention may thus consist of a sequence of 10-16 contiguous nucleotides (nt) in length complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45, or a sequence of 16 contiguous nucleotides complementary to the corresponding length length SEQ ID NO: 31 (first section or section A). The oligomer of the invention may contain an additional region of nucleotides. In some embodiments of the invention, the additional nucleotide region comprises a biocleavable nucleotide region, such as a nucleotide phosphate sequence (second region, region B), which can covalently link region A to a non-nucleotide moiety, such as a conjugate group (third region, or region C). In some embodiments of the invention, the contiguous nucleotide sequence of the oligomer of the invention (region A) is covalently linked directly to region C. In some embodiments of the invention, region C is biocleavable.

An oligomer consists of a sequence or contains a sequence of contiguous nucleotides of 12-22, for example 13, 14, 15, 16, 17, 18, 19, 20, 21 nucleotides in length, for example 14-16 nucleotides in length, for example 15 or 16 nucleotides in length. An oligomer may thus refer to the combined length of region A and region B, for example region A (10-16 nt) and region B (1-6 nt).

In various embodiments of the invention, the compound of the invention does not contain RNA units. In some embodiments of the invention, a compound of the invention, the first region or the first and second regions together (for example, as a single contiguous nucleotide sequence) is

- 5 043736 are a linear molecule or are synthesized as a linear molecule. The oligomer may thus be a single-chain molecule. In some embodiments of the invention, the oligomer does not contain short stretches of, for example, at least 3, 4 or 5 contiguous nucleotides that are complementary to equivalent stretches within the same oligomer (ie, duplexes). The oligomer in some embodiments of the invention may not be (substantially) double-stranded. In some embodiments of the invention, the oligomer is substantially non-double-stranded, such as non-siRNA.

Oligomer sequences.

The table below shows oligomers and conjugates of the oligomers of the invention and the PCSK9 target sequences of the invention.

Table 1

SEQ ID Subsequence P.O. Chol-C6 GalNAc Position on gene PCSK9SEQ ID NO 44 1 TGCtacaaaacCCA 3643-3656 2 AATgctacaaaaCCCA 3643-3658 3 AATgctacaaaacCCA 3643-3658 4 GCtgtgtgagcttGG 3251-3265 5 TGctgtgtgagctTGG 3251-3266 6 TGCtgtgtgagctTGG 3251-3266 7 TCCtggtctgtgtTCC 3373-3388 8 TCCtggtctgtgttCC 3373-3388 9 TGCtacaaaacCCA Yes Yes 3643-3656 10 AATgctacaaaaCCCA Yes Yes 3643-3658 eleven AATgctacaaaacCCA Yes Yes 3643-3658 12 GCtgtgtgagcttGG Yes Yes 3251-3265 13 TGctgtgtgagctTGG Yes Yes 3251-3266 14 TGctgtgtgagctTGG Yes Yes 3251-3266 15 TCCtggtctgtgtTCC Yes Yes 3373-3388 16 TCCtggtctgtgttCC Yes Yes 3373-3388 17 TGCtacaaaacCCA Yes 3643-3656 18 AATgctacaaaacCCA Yes 3643-3658 19 AATgctacaaaacCCA Yes 3643-3658 20 GCtgtgtgagcttGG Yes 3251-3265 21 TGctgtgtgagctTGG Yes 3251-3266 22 TGctgtgtgagctTGG Yes 3251-3266 23 TCCtggtctgtgtTCC Yes 3373-3388 24 TCCtggtctgtgttCC Yes 3373-3388 40 GTctgtggaaGCG 1005-1017 41 GTctgtggaaGCG Yes 1005-1017 42 GTctgtggaaGCG Yes Yes 1005-1017

-6043736

43 GTctgtggaaGCG Yes Yes 1005-1017 25 tgctacaaaaccca 3643-3656 26 aatgctacaaaaccca 3643-3658 27 gctgtgtgagcttgg 3251-3265 28 tgctgtgtgagcttgg 3251-3266 29 tcctggtctgtgttcc 3373-3388 44 gtctgtggaagcg 1005-1017 thirty UGGGUUUUGUAGCA 3643-3656 31 UGGGUUUUGUAGCAUU 3643-3658 32 CCAAGCUCACACAGC 3251-3265 33 CCAAGCUCACACAGCA 3251-3266 34 GGAACACAGACCAGGA 3373-3388 45 CGCUUCCACAGAC 1005-1017

SEQ ID NOs: 25-29 and 44 are nucleotide base sequence motifs.

SEQ ID NOs: 30-34 and 45 are target RNA sequences present in human PCSK9 mRNA.

SEQ ID NO: 1 is SPC5001.

SEQ ID NOs: 1-24 and 40-43 are oligomers containing nucleotide analogues, such as LNA gapmer oligomers, wherein the lowercase letters represent DNA units (nucleoside/nucleotide), wherein the uppercase letters represent LNA units.

In some embodiments of the invention, all LNA Cs are 5-methylcytosine. In some embodiments of the invention, all LNA units are beta-D-hydroxy-LNA. In some embodiments of the invention, all internucleoside linkages between nucleosides SEQ ID NO: 1-24 and 40-43 are phosphorothioate linkages.

SEQ ID NOs: 9-16 and 41-43 contain an oligomer (as indicated by the SEQ ID), as well as a cholesterol conjugate that may be covalently linked to the oligomer at the 5' or 3' end of the oligomer, optionally via a biocleavable linker such as nucleoside phosphate linker. In some embodiments of the invention, the cholesterol conjugate is linked to the 5' end of the oligomer.

SEQ ID NO: 17-24 contain an oligomer (as indicated by SEQ ID), as well as a GalNAc conjugate, which can be covalently linked to the oligomer at the 5' or 3' end of the oligomer, optionally via a biocleavable linker such as a nucleoside phosphate linker or a cleavable peptide linker. In some embodiments of the invention, the GalNAc conjugate is linked at the 5' end of the oligomer.

Specific oligomers and conjugates used herein are illustrated in FIG. 3 (oligomers), FIG. 4 (cholesterol conjugates), FIG. 5 (GalNAc conjugates). Other examples of conjugates that can be used with the oligomer of the invention are illustrated in FIG. 1 and 2 and are described in the section GalNAc conjugate groups.

In table Table 2 shows specific combinations of oligomer and conjugates.

table 2

Oligomer/conjugate combinations

SEQ ID Conjugate number (see Fig. 1) Conjl Conj2 Conj3 Conj4 Conjl a Conj2a Conj3a Conj4a 2 C1 C2 C3 C4 C5 C6 C7 C8 3 C11 C12 C13 C14 C15 C16 C17 C18 4 C12 C13 C14 C15 C16 C17 C18 C19 5 C30 C31 C32 C33 C34 C35 C36 C37 6 C40 C41 C42 C43 C44 C45 C46 C47 7 C50 C51 C52 C53 C54 C55 C56 C57 8 C60 C61 C62 C63 C64 C65 C66 C67

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SEQ ID Conjugate number (see Fig. 2) Conj5 Conj6 Conj5a Conj6a 2 C9 SJ S70 S71 3 C19 S20 S72 S73 4 S20 S21 S74 S75 5 C38 C39 S76 S77 6 C48 S49 S78 S79 7 C58 S59 S80 S81 8 C68 S69 S82 S83

All of these combinations can be visualized by replacing the wavy line in FIG. 1 or 2 oligomer sequence. In fig. 5 shows the combination of Conj2a with the above SEQ ID numbers. Fig. 5A and 5B are two detailed examples of the connections in FIG. 5. It should be noted that a biocleavable linker (B) may or may not be present between the conjugate moiety (C) and the oligomer (A). For Conj1-4 and 1a-4a, the GalNAc conjugate itself is biocleavable, the use of a peptide linker in the GalNAc cluster and as such a biocleavable linker (B) may or may not be used. However, preliminary data indicate that inclusion of a biocleavable linker (B), such as the nucleotide phosphate linkers disclosed herein, may enhance the activity of such oligomeric GalNAc cluster conjugates. In fig. 4 shows the combination of Conj5a of the above SEQ ID numbers with a biocleavable linker (B) consisting of two DNA monomers C and A linked by a phosphodiester bond. Since, when used with Conj 5 and Conj 6, the use of a biocleavable linker significantly enhances the activity of the compound, the inclusion of a biocleavable linker (B), such as the nucleotide phosphate linkers disclosed herein, is recommended.

The terms corresponding and corresponds refer to a comparison between an oligomer's nucleotide sequence (i.e., sequence of nucleotide bases or bases) or contiguous nucleotide sequence (first region/A region) and the reverse complement of a target nucleic acid or subregion thereof (e.g., SEQ ID NO: 31, 32, 33, 34 or 45). Nucleotide analogs are compared directly to their equivalent or corresponding nucleotides. In a preferred embodiment of the invention, the oligomers (or the first region thereof) are complementary, for example, fully complementary to the target region or a subregion thereof (eg, SEQ ID NO: 31, 32, 33, 34 or 45).

The terms reverse complement, reverse complementary, and reverse complementarity, as used herein, are interchangeable with the terms complement, complementary, and complementarity.

The term complementary means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel direction, with the 3' end of each sequence binding to the 5' end of the other sequence, and then each A, T(U), G and C of one sequence are aligned with T(U), A, C and G of another sequence, respectively. Typically, the complementary sequence of an oligonucleotide has at least 90%, preferably 95%, more preferably 100% complementarity to the defined sequence.

By the term "corresponding nucleotide analogue" and "corresponding nucleotide" is meant an indication that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical. For example, when the 2-deoxyribose unit of a nucleotide is linked to an adenine, the corresponding nucleotide analog contains a pentose unit (different from the 2-deoxyribose) linked to the adenine.

The term nucleotide base refers to the moiety of a nucleotide that is a base and covers both naturally occurring and non-naturally occurring variants. Thus, the nucleotide base covers not only the known purine and pyrimidine heterocycles, but also their heterocyclic analogues and tautomers. It is understood that DNA or RNA nucleosides of the B region may have naturally occurring and/or non-naturally occurring nucleotide base(s).

Examples of nucleotide bases include, but are not limited to, alenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2- chloro-6-aminopurine. In some embodiments of the invention, the nucleotide bases may be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, 5-methylcytosine. In some embodiments of the invention, the nucleotide bases may be independently selected from the group consisting of

- 8 043736 adenine, guanine, cytosine, thymidine and 5-methylcytosine.

In some embodiments of the invention, at least one of the nucleotide bases present in the oligomer is a modified nucleotide base selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,

5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine.

Target.

Advantageously, the oligomer according to the invention is capable of modulating the expression of the PCSK9 gene. Preferably, the oligomer is capable of down-regulating PCSK9 gene expression. In this regard, the oligomer of the invention can influence the expression of PCSK9, typically in a mammalian cell, such as a human cell, for example a liver cell. In some embodiments of the invention, the oligomers of the invention bind to a target nucleic acid and their effect on expression is a reduction of at least 10 or 20% compared to the normal expression level (for example, the expression level of an animal or human cell treated with saline ), more preferably at least 30, 40, 50, 60, 70, 80, 90 or 95% inhibition compared to normal expression levels. In some embodiments of the invention, such modulation is observed when using approximately 0.04 to 25 nM, such as 0.8 to 20 nM, concentration of a compound of the invention. In some embodiments of the invention, such modulation is observed when using from 0.01 to 15 mg/kg, for example from 0.05 to 10 mg/kg, for example from 0.1 to 7.5 mg/kg, for example from 0.25 to 5 mg/kg, for example, concentrations of 0.5 and 2.5 mg/kg of the compound of the invention. In the same or another embodiment of the invention, the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. Modulation of expression levels can be determined by measuring protein levels, for example, by methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using appropriate antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring mRNA levels, for example using Northern blotting or quantitative reverse transcription-polymerase chain reaction (RT-PCR). When measured using mRNA levels, the level of down-regulation when using the appropriate dosage, for example, a concentration of 0.04 to 25 nM, for example 0.8 to 20 nM, in some embodiments of the invention is typically up to 10-20% of normal levels in the absence of a compound of the invention.

Thus, the invention provides a method for down-regulating or inhibiting the expression of PCSK9 protein and/or mRNA in a cell expressing PCSK9 protein and/or mRNA, wherein said method comprises introducing into the cell an oligomer or conjugate according to the invention to down-regulate or inhibit the expression of the protein and/or or PCSK9 mRNA in the cell. Suitably the cell is a mammalian cell, such as a human cell. Administration in some embodiments of the invention can be carried out in vitro. Administration in some embodiments of the invention can be carried out in vivo.

The term target nucleic acid as used herein refers to DNA or RNA encoding a mammalian PCSK9 polypeptide, such as human PCSK9, such as National Center for Biotechnology Information accession number NM_174936 SEQ ID NO: 46. Nucleic acids encoding PCSK9 or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA. In some embodiments of the invention, for example, when used in research or diagnostics, the target nucleic acid may be a cDNA or a synthetic oligonucleotide formed from the above-mentioned DNA or RNA target nucleic acids. The oligomer according to the invention is preferably capable of hybridizing with a target nucleic acid. It will be understood that SEQ ID NO: 46 is a cDNA sequence and as such corresponds to the mature target mRNA sequence, although uracil is replaced by thymidine in the cDNA sequences.

The term naturally occurring variant refers to variants of the polypeptide or nucleic acid sequence of PCSK9 existing naturally within a particular taxonomic group, such as a mammal, eg mouse, monkey and preferably human. Typically, when referring to naturally occurring polynucleotide variants, the term may also include any allelic variant of the genomic DNA encoding PCSK9, which is located on chromosome 4 at the 4 C7 locus, as a result of chromosomal translocation or duplication, and the RNA derived therefrom, such as mRNA . Naturally occurring variants may also include variants generated by alternative splicing of PCSK9 mRNA. When referring to a specific polypeptide sequence, the term, for example, also includes naturally occurring forms of the protein that may thus undergo processing, for example, through co- or post-translational modifications such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc. .

- 9 043736

In some embodiments of the invention, the oligomer (or a portion of contiguous nucleotides thereof) is selected from one of the sequences or contains one of the sequences selected from the group consisting of SEQ ID NO: 28, or 29, or 44 or a subsequence thereof of at least contiguous nucleotides , where the oligomer (or a portion of contiguous nucleotides thereof) may optionally contain one, two, or three mismatches relative to the sequence.

In some embodiments of the invention, the target sequence is selected from sequences, or contains sequences or consists of sequences selected from the group consisting of SEQ ID NO: 31, 32, 33, 34 or 45, or a subsequence of at least 10 contiguous nucleotides of SEQ ID NO: 33, 34 or 45.

In some embodiments of the invention, the subsequence may consist of 11, 12, 13, 14, 15 or 16 contiguous nucleotides, such as 12 to 16 nucleotides. Suitably, in some embodiments of the invention, the subsequence is the same length as the contiguous nucleotide sequence of the oligomer of the invention (optionally excluding the B region when the B region is not complementary to the target).

However, it is recognized that in some embodiments of the invention the nucleotide sequence of the oligomer may contain additional 5' or 3' nucleotides, for example, independently 1, 2, 3, 4, 5 or 6 additional nucleotides at the 5' and/or 3' end, which are not complementary to the target sequence, and such non-complementary oligonucleotides may form the B region. In this regard, the oligomer of the invention in some embodiments of the invention may contain a contiguous nucleotide sequence flanked at the 5' and/or 3' end by additional nucleotides. In some embodiments of the invention, the additional 5' or 3' nucleotides are naturally occurring nucleotides such as DNA or RNA. In some embodiments of the invention, the additional 5' or 3' nucleotides may be region D, which is referred to in the context of gapmer oligomers herein.

In some embodiments of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 27 or a subsequence thereof of at least 10 or 12 nucleotide bases.

In some embodiments of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 28 or a subsequence thereof of at least 10 or 12 nucleotide bases. In a preferred embodiment of the invention, the oligomer according to the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 5 or 6. In another preferred embodiment of the invention, the oligomer conjugate according to the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 13, or 14, or 21, or 22.

In some embodiments of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 29 or a subsequence thereof of at least 10 or 12 nucleotide bases. In a preferred embodiment, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 7 or 8. In another preferred embodiment of the invention, the oligomer conjugate of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 15 , or 16, or 23, or 24.

In some embodiments of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 44 or a subsequence thereof of at least 10 or 12 nucleotide bases. In a preferred embodiment of the invention, the oligomer according to the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 40. In another preferred embodiment of the invention, the oligomer conjugate according to the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 41. 42 or 43.

In some embodiments of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 26. In a preferred embodiment of the invention, the oligomer of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 2 or 3. In another preferred embodiment of the invention, the oligomer conjugate of the invention consists of or contains a nucleotide sequence in accordance with SEQ ID NO: 10, or 11, or 18, or 19.

Length.

Oligomers may contain or consist of a contiguous nucleotide sequence of a total length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides. The lengths may include, for example, section A or sections A and B.

- 10 043736

In some embodiments of the invention, the oligomers contain or consist of a contiguous nucleotide sequence with a total length of 10-22, for example 12-18, for example 13-17 or 12-16, for example 13, 14, 15, 16, contiguous nucleotides. Preferably, the Region A oligomer contains or consists of a contiguous nucleotide sequence of 14 contiguous nucleotides in length, more preferably 15 contiguous nucleotides in length, and most preferably 16 contiguous nucleotides in length.

In some embodiments of the invention, the oligomer of the invention consists of no more than 22 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments of the invention, the oligomer of the invention contains less than 20 nucleotides.

Nucleotide analogues.

The term nucleotide as used herein refers to a glycoside containing a sugar moiety, a base moiety, and a covalently linked group, such as a phosphate or phosphorothioate internucleotide linking group, and covers both naturally occurring nucleotides, such as DNA or RNA, and those not found in naturally occurring nucleotides containing modified sugar moieties and/or base moieties, which are also referred to herein as nucleotide analogues. As used herein, a single nucleotide unit may also be referred to as a monomer or nucleic acid unit.

In the field of biochemistry, the term nucleoside is widely used to refer to a glycoside containing a sugar moiety and a base moiety. A covalent bond between two nucleosides may be called an internucleoside bond. Alternatively, the term internucleoside linkage may be used to characterize the linkage between nucleotides of an oligomer.

As will be appreciated by one of ordinary skill in the art, the 5' nucleotide of the oligonucleotide does not contain a 5' internucleotide linking group, although it may or may not contain a 5' terminal group, such as a phosphodiester or phosphorothioate, for linker conjugation (B or Y or conjugate moiety ).

Non-naturally occurring nucleotides include nucleotides having modified sugar moieties, such as bicyclic nucleotides or 2'-modified nucleotides, such as 2'-substituted nucleotides.

Nucleotide analogues are variations of naturally occurring nucleotides, such as DNA or RNA nucleotides, resulting from modifications in sugar moieties and/or base moieties. Analogues can in principle be exclusively silent or equivalent to natural nucleotides in the context of an oligonucleotide, i.e. do not have a functional effect on the pathway by which the oligomer inhibits target gene expression. Such equivalent analogues may still be useful if, for example, they can be produced more easily and less expensively, or are more stable under storage or production conditions, or are end-labeled or labeled. Preferably, however, the analogs have a functional effect on the pathway by which the oligomer inhibits target gene expression; for example, by obtaining increased binding affinity (affinity enhancement) for the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell. Specific examples of nucleoside analogues are described, for example, in Freier & Altmann, Nucl. Acid Res., 1997, 25, 4429-4443; and Uhlmann, Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in diagram 1.

- 11 043736

Boranophosphates

Scheme 1

The oligomer may thus comprise a simple sequence or be composed of a simple sequence of naturally occurring nucleotides, preferably 2'-deoxyribonucleotides (typically referred to herein as DNA), but also optionally ribonucleotides (typically referred to herein as RNA ) or combinations of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues. Such nucleotide analogues can suitably enhance the affinity of the oligomer for the target sequence. Examples of suitable and preferred nucleotide analogues are given in WO 2007/031091 or incorporated herein by reference.

Incorporation of affinity-enhancing nucleotide analogues, such as LNA or 2'-substituted sugars, into the oligomer may allow the size of the specifically binding oligomer to be reduced and may also reduce the upper limit of oligomer size up to which nonspecific or aberrant binding occurs.

In some embodiments of the invention, the oligomer contains at least 2 nucleotide analogues. In some embodiments of the invention, the oligomer contains 3-8 nucleotide analogs, for example 6 or 7 nucleotide analogs.

Examples of nucleotide analogues include modification of the sugar moiety to produce a 2'-substituted group or to produce a bicyclic structure, which enhances binding affinity and may also provide increased resistance to nucleases.

In some embodiments of the invention, the nucleotide analogues present within the antisense oligomer of the present invention (for example, in the X' and Y' regions mentioned in the Gapmer Construction section) are independently selected from, for example, 2'-O-alkyl-RNA units, 2 units '-OMeRNA, 2'-O-alkyl-DNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units , INA (intercalating nucleic acid; Christensen, 2002, Nucl. Acids. Res., 2002, 30: 4918-4925, incorporated herein by reference) units and 2'MOE units.

In some embodiments of the invention, the nucleotide analogs are 2'-Omethoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomers, or LNA nucleotide analogs, and the antisense oligonucleotide of the present invention may itself contain nucleotide analogs independently selected from these three types of analogue, or may contain only one type of analogue, selected from three types. In some embodiments of the invention, at least one of the nucleotide analogues is a 2'-MOE RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-MOE RNA nucleotide units. In some embodiments of the invention, at least one of the nucleotide analogues is 2'-fluoro-DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nucleotide units.

A preferred nucleotide analogue is an LNA, such as oxy-LNA (eg, beta-Doxy-LNA and alpha-L-hydroxy-LNA) and/or amino-LNA (eg, beta-D-amino-LNA and alpha-L-amino-LNA). ZNK)

- 12 043736 and/or thio-LNA (for example, beta-O-thio-LNA and alpha-L-thio-LNA) and/or ENA (for example, beta-D-ENA and alpha-L-ENA). The most preferred is beta-D-hydroxy-LNA.

In some embodiments of the invention, only one of the types of nucleotide analogues described above is present in the antisense oligonucleotide of the present invention or in the contiguous nucleotide sequence thereof.

In some embodiments, the antisense oligonucleotide of the present invention contains at least one locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as 3 to 7 or 4 to 8 ZNK links. In by far the most preferred embodiments of the invention, at least one of the nucleotide analogues is a locked nucleic acid (LNA); for example, at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or 8 of the nucleotide analogs may be LNAs. In some embodiments of the invention, all nucleotide analogues may be LNAs.

In some embodiments of the invention, the antisense oligonucleotide of the present invention may contain both nucleotide analogues (preferably LNA) and DNA units. Preferably, the sum of the combined nucleotide analogues (preferably LNA) and DNA units is 10-25, for example 10-24, preferably 10-20, for example 10-18, even more preferably 12-16. In some embodiments of the invention, the nucleotide sequence of an antisense oligonucleotide of the present invention, such as a contiguous nucleotide sequence, consists of at least one nucleotide analogue (preferably LNA), and the remaining nucleotide units are DNA units. In some embodiments of the invention, the antisense oligonucleotide of the present invention contains only LNA nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages, such as phosphorothioate.

It is understood that when referring to a preferred nucleotide sequence motif or a preferred nucleotide sequence consisting of nucleotides only, oligomers of the invention that are defined by that sequence may include a corresponding nucleotide analogue in place of one or more nucleotides present in the sequence, such as LNA units or other nucleotide analogs that increase duplex stability/melting temperature (Tm) of the oligomer/target duplex (ie, affinity-enhancing nucleotide analogs).

_Tm analysis. Oligonucleotide:oligonucleotide and target RNA (PO) duplexes are diluted to 3 mM in 500 ml of RNase-free water and mixed with 500 ml of 2x Tm buffer (200 mM NaCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 20 mM phosphate Na, pH 7.0). The solution is heated to 95°C for 3 minutes and then allowed to anneal at room temperature for 30 minutes. Melting points (Tm) of the duplexes were measured on a Lambda 40 UV/VIS spectrophotometer equipped with a PTP6 Peltier temperature programmer using PE Templab software (Perkin Elmer). The temperature is sharply raised from 20 to 95°C and then sharply reduced to 25°C, recording absorbance at 260 nm. To estimate the Tm of a duplex, the first derivative and local maxima of both melting and annealing are used.

In some embodiments of the invention, any mismatches between the oligomer nucleotide sequence and the target sequence are preferentially found at regions external to the affinity-enhancing nucleotide analogues, such as the Y' region referred to in the Gapmer Design section, and/or at the position of unmodified nucleotides for example, DNA, in an oligonucleotide and/or in regions located 5' or 3' to a contiguous nucleotide sequence.

ZNK.

The term LNA refers to a bicyclic nucleoside analogue that contains a bridge between the 2' and 4' positions in the ribose ring (2'-4' bicyclic nucleotide analogue) and is known as a locked nucleic acid). In the literature, LNA is sometimes referred to as bridged nucleic acid or bicyclic nucleic acid (BNA; bridged nucleic acid or bicyclic nucleic acid), and the two terms can be used interchangeably. The term LNA may refer to a LNA monomer or, when used in the context of a LNA oligonucleotide, LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. In some aspects, bicyclic nucleoside analogs are LNA nucleotides and the terms can thus be used interchangeably and represent those embodiments of the invention that are also characterized by the presence of a linker group (such as a bridging group) between C2' and C4' of the ribose sugar ring.

In some embodiments of the invention, the antisense oligonucleotide of the present invention may contain both of beta-D-hydroxy-LNA and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, 5'-methyl-LNA and/or ENA in either beta D or alpha configurations or combinations thereof. In some embodiments of the invention, all cytosine units of the LNA are

- 13 043736

5'-methyl-cytosine. In some embodiments of the invention, at least one nucleoside analog present in the first region (X') is a bicyclic nucleoside analog, for example, at least 2, at least 3, at least 4, at least 5, at least at least 6, at least 7, at least 8 (excluding DNA and/or RNA nucleosides of the Y' region) sugar-modified nucleoside analogues, for example bicyclic nucleoside analogues such as LNA, for example beta-D-X-LNA or alpha-L-X -LNA (where X is hydroxy, amino or thio) or other LNAs disclosed herein, including but not limited to (R/S) cET, cMOE or 5'-Me-LNA.

In some embodiments of the invention, the LNA used in the oligonucleotide compounds of the invention preferably has the structure of general formula II

where Y is selected from the group consisting of -O-, -CH ₂ O-, -S-, -NH-, N(R ^e ) and/or -CH ₂ -; Z and Z* are independently selected from an internucleotide linkage, ^RH , a terminal group or a protecting group;

B constitutes a natural or synthetic nucleotide base moiety (nucleotide base) and ^RH is selected from a hydrogen atom and C1. ₄ -alkyl;

R ^a , R ^b , R ^c , R ^d and R ^e are optionally independently selected from the group consisting of hydrogen, optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, hydroxy , C1-12-alkoxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryl-oxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy -carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-aminocarbonyl, amino-C1-6-alkylaminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, urea, C1-6-alkanoyloxy, sulfono, C1-6-alkylsulfonyloxy, nitro, azido, sulfanyl, C1 -6-alkylthio, halogen atom, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands, where aryl and heteroaryl may be optionally substituted and where two substituents R ^a and attached to the same atom R ^b together may represent optionally substituted methylene (=CH2); And

^RH is selected from hydrogen atom and C _1-4 -alkyl.

In some embodiments of the invention, R ^a , R ^b , R ^c , R ^d and R ^e are optionally independently selected from the group consisting of a hydrogen atom and a C _1-6 alkyl such as methyl. For all chiral centers, asymmetric groups can be in either the R or S orientation, for example, two exemplary stereochemical isomers include the beta-D and alpha-L isoforms, which can be illustrated as follows.

Specific illustrative links of the ZNK are shown below.

- 14 043736

The term thio-LNA includes a locked nucleotide in which Y in the above general formula is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-b configurations.

The term amino-LNA includes a locked nucleotide in which Y in the above general formula is selected from -N(H)-, N(R)-, CH2-N(H)- and -CH2-N(R)-, where R selected from hydrogen atom and C _1-4 -alkyl.

Amino-LNA can be in both beta-D and alpha-b configurations.

The term oxy-LNA includes a locked nucleotide in which Y in the above general formula is -O-. Oxy-LNA can be in both beta-D and alpha-b configurations.

The term ENA includes a locked nucleotide in which Y in the above general formula is -CH2-O- (where the oxygen atom -CH ₂ -O- is attached at the 2' position to base B). R ^e represents a hydrogen or methyl atom.

In some illustrative embodiments of the invention, the LNA is selected from beta-O-hydroxy-LNA, alpha-L-hydroxy-LNA, beta-D-amino-LNA, and beta-O-thio-LNA, in particular beta-D-hydroxy-LNA.

As used herein, bicyclic nucleosides refer to modified nucleosides containing a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides containing a bridge between the 4' and 2' atoms of the ribosyl ring. In some embodiments of the invention, the compounds provided herein include one or more bicyclic nucleosides, where the bridge contains 4'-2' bicyclic nucleosides. Examples of such 4'-2' bicyclic nucleosides include, but are not limited to, one of the formulas 4'-(CH2)-O-2'(LNA);4'-(CH2)-S-2';4-(CH2)2-O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'CH(CH2OCH ₃ )-O-2* and analogs thereof (see US patent 7399845, published July 15, 2008); 4'-C(CH ₃ )(CH ₃ )-O-2' and analogs thereof (see published international patent application PCT WO 2009/006478, published January 8, 2009); 4'-CH2-N(OCH ₃ )-2' and its analogs (see published international patent application PCT WO 2008/150729, published December 11, 2008); 4'-CH2-ON(CH ₃ )-2' (see published patent application US 2004/0171570, published September 2, 2004); 4'-CH2-N(R)-O-2', where R represents H, C ₁ -C ₁₀ -alkyl or a protecting group (see US patent 7427672, published September 23, 2008); 4'-CH2-C(H)(CH ₃ )-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH ₂ -C(=CH ₂ )-2' and analogs thereof (see published international patent application PCT WO 2008/154401, published December 8, 2008). Also see, for example, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci., USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett, 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc, 129(26)8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Then, 2001, 3, 239-243; US patents No. 6670461, 7053207, 6268490, 6770748, 6794499, 7034133, 6525191, 7399845; published international PCT applications WO 2004/106356, WO 94/14226, WO 2005/021570 and WO 2007/134181; US Patent Publications No. US 2004/0171570, US 2007/0287831 and US 2008/0039618; and US Patent Serial Nos. 12/129154, 60/989574, 61/026995, 61/026998, 61/056564, 61/086231, 61/097787 and 61/099844; and international PCT applications No. PCT/US2008/064591, PCT/US2008/066154 and PCT/US2008/068922. Each of the above bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations including, for example, aL-ribofuranose and eD-ribofuranose (see PCT International Application DK98/00393, published March 25, 1999 as WO 99 /14226).

In some embodiments of the invention, bicyclic sugar moieties of LNA nucleosides include, but are not limited to, compounds having at least one bridge linkage between the 4' and 2' positions of the pentose furanosyl sugar moiety, wherein such bridge linkages independently contain 1 or 2 to 4 linkage groups independently selected from -[CiRaXRb)]-, -C(R _a )=C(R _b )-, -C(R _a )=N-, -C(=NR _a )-, -C(=O)-, -C (=S)-, -O-, -Si(R _a ) ₂ -, -S(=O) _x - and -N(Ra)-, where x is 0, 1 or 2; n is 1, 2, 3 or 4; each R _a and R _b independently represents H, a protecting group, hydroxyl, C ₁ -C ₁₂ -alkyl, substituted C ₁ -C ₁₂ -alkyl, C ₂ -C ₁₂ -alkenyl, substituted C ₂ -C ₁₂ -alkenyl, C ₂ -C ₁₂ -alkynyl, substituted C ₂ -C ₁₂ -alkynyl, C5-C ₂₀ -aryl, substituted C5-C ₂₀ -aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C ₇ -alicyclic radical, substituted C5-C ₇ -alicyclic radical, halogen atom, OJ1, NJ1J2, SJ1, N ₃ , COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ - J1) or sulfoxyl (S(=O)-J1); and each J1 and _J2 independently represents H, C ₁ -C ₆ -alkyl, substituted C ₁ -C ₁₂ -alkyl, C ₂ -C ₁₂ -alkenyl, substituted C ₁ -C ₁₂ -alkenyl, C ₂ -C ₁₂ -alkynyl, substituted C ₁ -C ₁₂ -alkynyl, C5-C ₂₀ -aryl, substituted C5-C ₂₀ -aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C ₁ -C ₁₂ -aminoalkyl substituted with C ₁ -C ₁₂ -aminoalkyl or protecting group.

In some embodiments of the invention, the bridging linkage of the bicyclic sugar moiety is -[C(R _a )(R _b )] _n -, -[C(Ra)(R _b )] _n -O-, -C(R _a R _b ) -N(R)-O- or -C(R _a R _b )-ON(R)-. In some embodiments of the invention, the bridging linkage is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4* -(CH ₂ ) ₂ -O-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2'-, where each R independently represents H, protective group or C ₁ -C ₁₂ -alkyl.

In some embodiments of the invention, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside containing a 4'-2'-methylene-oxy bridged bond,

- 15 043736 can be in a-L configuration or in e-D configuration. Previously a-L-methyleneoxy (4'-CH2-O-2')

BNA was included in antisense oligonucleotides that exhibited antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In some embodiments of the invention, bicyclic nucleosides include, without limitation, (A) aL-methyleneoxy (4'-CH2-O-2')BNA, (B) eD-methyleneoxy (4'-CH2-O-2')BNA, (C) ethyleneoxy (4'-(CH ₂ ) ₂ O-2') BNA, (D) aminooxy (4'-CH2-ON(R)-2') BNA, (E) oxyamino (4'-CH2-N(R )-O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH ₃ )-O-2') BNA, (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methylcarbocyclic (4'-CH2-CH(CH ₃ )-2') BNA and (J) propylenecarbocyclic ( 4'-(CH2) ₃ -2') BNA as shown below.

where Bx represents the base grouping; And

R independently represents H, a protecting group or C ₁ -C ₂ -alkyl.

In some embodiments of the invention, the bicyclic nucleoside is defined by formula I

where Bx represents a heterocyclic base moiety;

-Q _a -Q _b -Q _c - represents -CH ₂ -N(R _c )-CH ₂ -, -C(=O)-N(R _c )-CH ₂ -, -CH ₂ -ON(R _c )-, -CH ₂ -N(R _c )-Oor -N(R _c )-O-CH ₂ ;

R _c represents a C ₁ -C ₁₂ alkyl or amino protecting group; And

T _a and T _b each independently represent H, a hydroxy protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support.

In some embodiments of the invention, the bicyclic nucleoside is defined by Formula II

where Bx represents a heterocyclic base moiety;

T _a and T _b each independently represent H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support;

Z _a represents C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, substituted C ₁ -C ₆ -alkyl, substituted C ₂ -C ₆ -alkenyl, substituted C ₂ - C ₆ -alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

In some embodiments of the invention, each of the substituted groups is independently mono or polysubstituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ _c , NJ _d , SJ _C , N ₃ , OC(=X)J _c and NJ _e C(= X)NJ _c J _d wherein J _c , J _d and J _e are each independently H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl and X is O or NJ _C .

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In some embodiments of the invention, the bicyclic nucleoside is defined by formula III

where Bx represents a heterocyclic base moiety;

Ta and T _b each independently represent H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support;

Rd represents C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, substituted C ₁ -C ₆ -alkyl, substituted C ₂ -C ₆ -alkenyl, substituted C ₂ -C ₆ -alkynyl or substituted acyl (C(=O)-).

In some embodiments of the invention, the bicyclic nucleoside is defined by formula IV

where Bx represents a heterocyclic base moiety;

Ta and Tb each independently represent H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support;

Rd is C ₁ -C ₆ -alkyl substituted with C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl substituted with C 2 -C ₆ -alkenyl, C _{2 -C 6} -alkynyl substituted with C ₂ -C ₆ -alkynyl;

each q _b , q _c and qd independently represents H, a halogen atom, C ₁ -C ₆ -alkyl, substituted C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, substituted C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl or substituted C ₂ -C ₆ -alkynyl, C ₁ -C ₆ -alkoxy, substituted C ₂ -C ₆ -alkoxy, acyl, substituted acyl, C ₁ -C ₆ -aminoalkyl or substituted C ₁ -C ₆ -aminoαalkyl.

In some embodiments of the invention, the bicyclic nucleoside is defined by formula V

where Bx represents a heterocyclic base moiety;

T _a and T _b each independently represent H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support;

qa, q _b , qc and qf each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₁₂ -alkyl substituted by a C ₁ -C ₁₂ -alkyl, a C ₂ -C ₁₂ -alkenyl substituted by a C ₂ -C ₁₂ - alkenyl, C ₂ -C ₁₂ -alkynyl, substituted C ₂ -C ₁₂ -alkynyl, C ₁ -C _{12 -alkoxy, substituted C 1 -C 12 -alkoxy ,} OJj, SJj, SOJj, SO ₂ Jj, NJjJk, N ₃ , CN, C(=O)OJj, C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH)NJ _j J _k , N(H)C(=O)NJ _j J _k or N(H)C(=S)NJ _j J _k ; or q _e and qf together represent =C(qg)(q _h );

qg and _qh each independently represent H, a halogen atom, a C ₁ -C ₁₂ alkyl, or a substituted C ₁ -C ₁₂ alkyl.

The synthesis and preparation of methyleneoxy (4'-CH2-O-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil in combination with their oligomerization and nucleic acid recognition properties are described (see, for example, the article by Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4'-CH2-O-2') BNA, methyleneoxy (4'-CH2-O-2') BNA and 2'-tuo-BNA have also been prepared (see, for example, Kumar et al., Bioorg Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of closed nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (see, for example, Wengel et al., WO 99/14226). In addition, the prior art describes the synthesis of 2'-amino-BNA, a new conformation-constrained high-affinity oligonucleotide analogue (see, for example, Singh et al., J. Org. Chem., 1998, 63, 10035-10039) . In addition, 2'-amino- and 2'-methylamino-BNA and the thermostability of their duplexes with complementary RNA and DNA have been previously described.

- 17 043736

In some embodiments of the invention, bicyclic nucleosides are defined by formula VI

where Bx represents a heterocyclic base moiety;

T _a and T _b each independently represent H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to an immobilizing support;

each qi, qj, q _k and ql independently represents H, a halogen atom, C ₁ -C ₁₂ -alkyl, substituted C ₁ -C ₁₂ -alkyl, C ₂ -C 12 -alkenyl, substituted C ₂ -C 12 -alkenyl, C ₂ -C12-alkynyl, substituted C ₂ -C12-alkynyl, C1-C ₁₂ -alkoxyl, substituted C ₂ -C ₁₂ -alkoxyl, OJj, SJj, SOJj, SO ₂ Jj, NJjJk, N ₃ , GN, C(= O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH)NJ _j J _k , N(H )C(=O)NJ _j J _k or (H)C(=S)NJ _j J _k ; and qi and qj or ql and qk together represent =C(q _g )(q _h ), where q _g and q _h each independently represent H, a halogen atom, a C1- _C12 alkyl or a substituted _C1 - _C6 -alkyl.

Other carbocyclic bicyclic nucleosides having a 4'-(CH2) ₃ -2' bridge and an alkenyl analogue, a 4'-CH=CH-CH2-2' bridge have been described (see, for example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; and Albaek et al., J. Org. Chem., 2006, 71, 7731-77 '40). The synthesis and preparation of carbocyclic bicyclic nucleosides in combination with their oligomerization and biochemical studies have also been described (see, for example, Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, 4'-2' bicyclic nucleoside or 4'-2' bicyclic nucleoside refers to bicyclic nucleosides containing a furanose ring containing a bridge connecting the 2' carbon atom and the 4' carbon atom.

As used herein, monocyclic nucleosides refer to nucleosides containing modified sugar moieties that are non-bicyclic sugar moieties. In some embodiments of the invention, the sugar moiety or sugar moiety analogue of the nucleoside may be modified or substituted at any position.

As used herein, '^'-modified sugar means a furanosyl sugar modified at the 2' position. In some embodiments of the invention, such modifications include substituents selected from a halide including, but not limited to, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In some embodiments of the invention, the 2' modifications are selected from substituents including, but not limited to, O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ )NH ₂ , O(CH ₂ )CH ₃ , O(CH ₂ )ONH ₂ , OCH ₂ C(=O)N(H)CH ₃ and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ ] ₂ , where n and m are from 1 to about 10. Other 2'-substituent groups may also be selected from C1- _C12 -alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH ₃ ; OCN; d; Br; CN; CF ₃ ; OCF ₃ ; SOCH ₃ ; SO ₂ CH ₃ ; ONO2; NO2; N3; _NH2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; R; leaving group; group reporter; intercalator; a group for improving the pharmacokinetic properties and a group for improving the pharmacodynamic properties of the antisense compound, as well as other substituents having similar properties. In some embodiments of the invention, the modified nucleosides contain a 2'-MOE side chain (see, for example, Baker et al., J. Biol. Chem., 1997, 272, 1, 1944-12000). This 2'-MOE substitution is described to have improved binding affinity compared to unmodified nucleosides and to other modified nucleosides such as 2'-O-methyl-, O-propyl- and O-aminopropyl-. Oligonucleotides having a 2'-MOE substituent have also been shown to be antisense inhibitors of gene expression with promising properties for in vivo use (see, for example, Martin, P., He/v. Chirm. Acta, 1995, 78, 486- 504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, tetrahydropyran modified nucleoside or THP modified nucleoside means a nucleoside having a tetrahydropyran sugar replacing the pentafuranosyl moiety in conventional nucleosides (sugar surrogate). TGP-modified nucleosides include nucleosides referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see article Leumann, C.J., Bioorg. and Med. Chem. (2002), 10: 841-854), fluoro-HNA (F-HNA) or compounds defined by formula X

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Formula X wherein, independently for each of the at least one tetrahydropyran nucleoside analogue of formula X, Bx represents a heterocyclic base moiety;

T ₃ and T4 are each independently an internucleoside linking group linking a tetrahydropyran nucleoside analog to an antisense compound, or one of T ₃ and T4 is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an antisense compound and the other of T ₃ and T4 is H, hydroxyl protecting group, conjugate-linked group, or 5' or 3' terminal group;

q1 q ₂ q ₃ q4 q ₅ , q ₆ and q ₇ are each independently H, C ₁ -C ₆ -alkyl substituted with C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl substituted with C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl or substituted C ₂ -C ₆ -alkynyl; and one of R1 and R2 is a hydrogen atom and the other is selected from a halogen atom, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N ₃ , OC(=X)J1, OC(=X)NJ1J2, NJ ₃ C(=X )NJ1J ₂ and CN, where X represents O, S or NJ1, and each J1, J2 and J ₃ independently represents H or C ₁ -C ₆ -alkyl.

Some embodiments of the invention provide THP-modified nucleosides of formula X, wherein qm, qn, qp, _qr , qs, _qt , and _qu are each _H. In some embodiments, at least one of qm, qn, qp, qr , qs, q _t and q _u is different from H. In some embodiments of the invention at least one of qm, qn, qp, qr, qs, qt and qu is methyl. Some embodiments of the invention provide THP nucleosides of formula X, where one of R1 and R2 is F. In some embodiments of the invention, R1 is a fluorine atom and R2 is H, R1 is methoxy, and R2 is H and R1 is methoxyethoxy and R2 is H.

As used herein, '^'-modified or 2'-substituted refers to a sugar-containing nucleoside that contains a substituent at the 2' position other than H or OH. 2'-Modified nucleosides include, but are not limited to, nucleosides with non-bridging 2' substituents such as allyl, amino, azido, thio, O-allyl, OC ₁ -C ₁ o-alkyl, -OCF ₃ , O-(CH ₂ ) ₂ -O-CH ₃ , 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn) or O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn independently represents H, either substituted or unsubstituted C ₁ -C ₁ o-alkyl. 2'-Modified nucleosides may further contain other modifications, for example, at other sugar positions and/or at the nucleotide base.

As used herein, 2'-F refers to a sugar containing a fluorine group at the 2' position.

As used herein, 2'-OMe or 2'-OCH ₃ or 2'-O-methyl each refers to a sugar-containing nucleoside that contains an -OCH ₃ group at the 2' position of the sugar ring.

As used herein, oligonucleotide refers to a compound containing a plurality of linked nucleosides.

In some embodiments of the invention, one or more of the plurality of nucleosides is modified. In some embodiments of the invention, the oligonucleotide contains one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclic and tricyclic ring sugar surrogate systems are also known in the art that can be used to modify nucleosides for inclusion in antisense compounds (see, for example, review article Leumann, J.C., Bioorganic and Medicinal Chemistry, 2002, 10, 841- 854). Such ring systems can undergo various additional substitutions to enhance activity. Methods for producing modified sugars are also known to those skilled in the art. In nucleotides having modified sugar groups, groups of nucleotide bases (natural, modified or combinations thereof) are retained for hybridization with the corresponding target nucleic acid.

In some embodiments of the invention, antisense compounds contain one or more nucleotides having modified sugar moieties. In some embodiments of the invention, the modified sugar moiety is 2'-MOE. In some embodiments of the invention, the modified 2'-MOE nucleotides are designed as a gapmer motif. In some embodiments of the invention, the modified sugar moiety is cEt. In some embodiments of the invention, the cEt-modified nucleotides are designed through gapmer motif wing segments.

In some embodiments of the invention, in the NNA, R ⁴ * and R ² * together represent the diradical -O-CH(CH ₂ OCH ₃ )-pIO-methoxyethyl-bicyclic nucleic acids; Seth at al., 2010, J. Org. Chem)

- 19 043736 in either R or S configuration.

In some embodiments of the invention in the ZNA, R ⁴ * and R ² * together represent a diradical

-O-CH(CH ₂ CH ₃ )- (2'O-ethyl-bicyclic nucleic acids; Seth at al., 2010, J. Org. Chem) or

R- or S-configuration.

In some embodiments of the invention, in the ZNA, R ^4* and R ^2* together represent the diradical -O-CH(CH3) in either the R- or S-configuration. In some embodiments of the invention, R ⁴ * and R ² * together represent the diradical -O-CH2-O-CH ₂ - (Seth at al., 2010, J. Org. Chem).

In some embodiments of the invention in the ZNA, R ⁴ * and R ² * together represent the diradical -O-NR-CH ₃ (Seth at al., 2010, J. Org. Chem).

In some embodiments of the invention, the ZNK units have a structure selected from the following group:

Incorporation of affinity-enhancing nucleotide analogues into an oligomer, such as LNA or 2'-substituted sugars, may allow the size of a specifically binding oligomer to be reduced and may also reduce the upper limit of oligomer size up to which nonspecific or aberrant binding occurs.

The inventors evaluated the nephrotoxicity of the cET compound (using (S)-cET with the sequence (compound ID 6/411847 WO 2009/12495 and the comparative compound beta-D-hydroxy-LNA 6/392063 WO 2009/12495) and found that the cET compounds caused unexpectedly high neurotoxicity compared to beta-D-hydroxy-LNA control.The study was a single dose kill study after 3 days (for methodology, see Example 41 of EP 1984381, although the inventors used NMRI mice).Nephrotoxicity was confirmed by histological analysis. In particular, we observed evidence of neurotoxicity at lower doses of the cET compound compared to doses at which serum alanine aminotransferase (ALT) was observed, indicating that nephrotoxicity of the cET compounds may be a particular concern. The present invention, such as trivalent GalNAc conjugates, is highly useful in reducing the nephrotoxicity of ZNA compounds, such as cET compounds.

In some embodiments of the invention, the oligomer contains at least 1 nucleoside analog. In some embodiments of the invention, the oligomer contains at least 2 nucleotide analogues. In some embodiments of the invention, the oligomer contains 3-8 nucleotide analogs, for example 6 or 7 nucleotide analogs. In by far the most preferred embodiments of the invention, at least one of the nucleotide analogs is a locked nucleic acid (LNA); for example, at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or 8 of the nucleotide analogues may be LNA. In some embodiments of the invention, all nucleotide analogues may be LNAs.

It should be understood that when referring to a preferred nucleotide sequence motif or a nucleotide sequence that consists only of nucleotides, oligomers of the invention that are defined by this sequence may contain a corresponding nucleotide analogue in place of one or more nucleotides present in said sequence, such as units LNAs or other nucleotide analogs that increase the stability of the duplex/Tm oligomer/target duplex (i.e., affinity-enhancing nucleotide analogs).

A preferred nucleotide analogue is an LNA such as hydroxy LNA (such as beta-D-hydroxy-LNA and alpha-b-hydroxy-LNA) and/or amino-LNA (such as beta-D-amino-LNA and alpha-LNA). L-aminoLNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-E-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA).

In some embodiments of the invention, the oligomer of the invention, such as region A, may contain LNA units and other nucleotide analogues. Additional nucleotide analogues present within the oligomer of the invention are independently selected from, for example, 2'-O-alkylRNA units, 2'-amino DNA units, 2'-fluoro-DNA units, LNA units, arabinonucleic acid (ANA) units , 2'-fluoro-ANA units, HNA units, INA units (intercalating nucleic acid; Christensen, 2002. Nucl. Acids. Res., 2002, 30: 4918-4925, incorporated herein by reference) and 2' units MOE. In some embodiments of the invention, only one type of nucleotide analogue is present in the oligomer of the invention, for example, in the first region, or in a contiguous nucleotide sequence.

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In some embodiments of the invention, the oligomer of the invention (region A) may thus contain at least one locked nucleic acid (LNA) unit, for example 1, 2, 3, 4, 5, 6, 7 or 8 LNA units, for example 3 -7 or 4-8 ZNK links or 3, 4, 5, 6 or 7 ZNK links. In some embodiments of the invention, all nucleotide analogues are LNAs. In some embodiments of the invention, the oligomers may contain both beta-O-oxy-LNA and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA and/or ENA in either beta-D or alpha Lconfigurations or combinations thereof. In some embodiments of the invention, all cytosine units of the LNA are 5'-methyl-cytosine. In some embodiments of the invention, the oligomer (such as the first and optionally second regions) may contain both LNA and DNA units. In some embodiments of the invention, the sum of the combined LNA and DNA units is 10-25, for example 10-24, preferably 10-20, for example 10-18, for example 12-16. In some embodiments of the invention, the nucleotide sequence of the oligomer, its first region, such as a contiguous nucleotide sequence, consists of at least one LNA unit and the remaining nucleotide units are DNA units. In some embodiments of the invention, the oligomer or a first region thereof contains only LNAs, nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA nucleotides, most preferably DNA), optionally with modified internucleotide linkages such as phosphorothioate.

RNase recruitment.

It is known that the oligomeric compound can function through non-RNase-mediated cleavage of target mRNA, for example, by steric hindrance of translation or other means. In some embodiments of the invention, the oligomers of the invention are capable of recruiting an endoribonuclease (RNase), such as RNase H.

Preferably, such oligomers, such as an A region or a contiguous nucleotide sequence, comprising a region of at least 4, for example at least 5, for example at least 6, for example at least 7, consecutive nucleotide units, for example at least 8 or at least 9 consecutive nucleotide units (residues) comprising 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with a complementary target RNA, are capable of recruiting RNase ( such as DNA links). The contiguous sequence capable of recruiting RNase may be a Y' region referred to in the context of a gapmer as disclosed herein. In some embodiments of the invention, the size of the contiguous sequence capable of recruiting RNase, such as the Y' region, may be larger, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1222309 proposes methods for determining RNase H activity in vitro that can be used to determine the ability to recruit RNase H. An oligomer is considered capable of recruiting RNase H if, when providing a complementary RNA target, it has an initial reaction rate measured in picomoles (pmol). /l/min, constituting at least 1%, for example at least 5%, for example at least 10% or more than 20%, of the initial reaction rate determined using only a DNA oligonucleotide having the same base sequence, but containing only DNA monomers without 2' substitutions with phosphorothioate linking groups between all monomers in the oligonucleotide, using the methodology proposed in examples 91-95 of EP 1222309.

In some embodiments of the invention, an oligomer is considered substantially incapable of recruiting RNase H if, when providing a complementary RNA target, it has an initial reaction rate, measured in pmol/L/min, of less than 1%, such as less than 5%, such as less than 10% or less 20%, the initial reaction rate determined using only a DNA oligonucleotide without 2' substitutions with phosphorothioate linking groups between all monomers in the oligonucleotide, using the methodology proposed in examples 91-95 of EP 1222309.

In other embodiments of the invention, an oligomer is considered capable of recruiting RNase H if, when provided with complementary RNA target and RNase H, the initial rate of the RNase H reaction, measured in pmol/L/min, is at least 20%, for example, at least 40% , for example, at least 60%, for example, at least 80% of the initial reaction rate using only a DNA oligonucleotide without 2' substitutions with phosphorothioate linking groups between all monomers in the oligonucleotide, using the methodology proposed in Examples 91-95 EP 1222309.

Typically, the region of the oligomer that forms successive nucleotide units that, when formed into a duplex with a complementary RNA target, is capable of recruiting RNase, consists of nucleotide units that form a DNA/RNA-like duplex with the RNA target. An oligomer of the invention, such as a first region, may contain a nucleotide sequence containing both nucleotides and nucleotide analogues, and may take the form of, for example, a gapmer.

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Gapmer design.

In some embodiments of the invention, the oligomer of the invention, such as the first region, contains or is a gapmer. A gapmer oligomer is an oligomer containing a contiguous stretch of nucleotides capable of recruiting an RNase, such as RNase H, for example, a stretch of at least 6 or 7 DNA nucleotides, referred to herein as the Y' region. ), where the Y' region is flanked at the 5' and 3' ends by sections of nucleotide analogues that enhance affinity, for example 1-6 nucleotide analogues in the 5' and 3' direction to a continuous stretch of nucleotides capable of recruiting RNase, where the data the plots are referred to as plots X' (X') and Z' (Z'), respectively. Regions X' and Z' may also be referred to as gapmer wing segments. Gapmer regions are disclosed in WO 2004/046160, WO 2008/113832 and WO 2007/146511.

In some embodiments of the invention, monomers capable of recruiting RNase are selected from the group consisting of DNA monomers, alpha-L-LNA monomers, C4'-alkylated DNA monomers (see documents PCT/EP2009/050349; and Vester et al., Bioorg Med. Chem. Lett., 18 (2008), 2296-2300, incorporated herein by reference) and unlinked nucleic acid (UNA) nucleotides (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference). UNA is an open-ended nucleic acid where, typically, the C-C bond of the C2-C3 ribose has been removed to form an open-ended sugar residue. Preferably the gapmer contains a (poly)nucleotide sequence of the formula (5'-3') X'-Y'-Z', wherein the X' (X') region (5' region) consists of at least one or contains at least one a nucleotide analogue, for example at least one LNA unit, for example from 1 to 6 nucleotide analogues, such as LNA units, and the Y' region (Y') consists of at least four or contains at least four or at least five consecutive nucleotides capable of RNase recruitment (when formed into a duplex with a complementary RNA molecule such as a target mRNA), such as DNA nucleotides, and a Z' (Z') region (3' region) consists of at least one or contains at least one nucleotide analogue, for example at least one LNA unit, for example from 1 to 6 nucleotide analogues, such as LNA units.

In some embodiments of the invention, the X' region consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, for example, 2-5 nucleotide analogues, such as 2-5 LNA units, for example 3 or 4 analogues nucleotides, such as 3 or 4 units of LNA; and/or the Z region consists of 1, 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, for example, 2-5 nucleotide analogues, such as 2-5 LNA units, for example 3 or 4 nucleotide analogues, such as 3 or 4 links ZNK.

In some embodiments of the invention, Y' consists of or contains 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive RNase-recruiting nucleotides, either 4-12, 6-10, or 7 -9, for example 8 consecutive nucleotides capable of RNase recruitment. In some embodiments of the invention, the Y' region consists of at least one or contains at least one DNA nucleotide unit, for example 1-12 DNA units, preferably 4-12 DNA units, more preferably 6-10 DNA units, for example 7 -10 DNA units, more preferably 8, 9 or 10 DNA units.

In some embodiments of the invention, the X' region consists of 3 or 4 or contains 3 or 4 nucleotide analogs, such as LNA, the X' region consists of 7, 8, 9 or 10 DNA units, and the Z' region consists of 3 or 4 nucleotide analogs , such as ZNK. Such designs include (X'-Y'-Z') 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3 -8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3. In a preferred embodiment of the invention, the gapmer is a 3-9-4 gapmer, even more preferably the gapmer is a 3-10-3 gapmer.

Additional gapmer constructs are disclosed in WO 2004/046160, incorporated herein by reference. WO 2008/113832, which claims priority to provisional application US 60/977,409, incorporated herein by reference, relates to short-length gapmer oligomers. In some embodiments of the invention, the oligomers provided herein may be such short-length gapmers.

In some embodiments of the invention, the oligomer, such as the X' region, consists of a contiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14 nucleotide units, where the contiguous nucleotide sequence includes or has the formula (5'-3')X'-Y' -Z', where X' consists of 1, 2 or 3 units of nucleotide analogues, such as LNA units;

Y' consists of 7, 8, or 9 contiguous nucleotide units capable of recruiting RNase when formed into a duplex with a complementary RNA molecule (such as target mRNA); And

Z' consists of 1, 2 or 3 nucleotide analogues, such as LNA units.

In some embodiments of the invention, X' consists of 1 ZNA unit. In some embodiments of the invention, X' consists of 2 ZNA units. In some embodiments of the invention, X' consists of 3 ZNA units. In some embodiments of the invention, Z' consists of 1 ZNA unit. In some embodiments, it is

- 22 043736 harness Z' consists of 2 ZNK links. In some embodiments of the invention, Z' consists of 3 ZNA units. In some embodiments of the invention, Y' consists of 7 nucleotide units. In some embodiments of the invention, Y' consists of 8 nucleotide units. In some embodiments of the invention, Y' consists of 9 nucleotide units. In some embodiments of the invention, the Y' region consists of 10 nucleoside monomers. In some embodiments of the invention, Y' consists of 1-10 or contains 1-10 DNA monomers. In some embodiments of the invention, Y' contains from 1-9 DNA units, such as 2, 3, 4, 5, 6, 7, 8 or 9 DNA units. In some embodiments of the invention, Y' consists of DNA units. In some embodiments of the invention, Y' contains at least one LNA unit in the alpha L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the alpha L configuration. In some embodiments of the invention, Y' contains at least one alpha-b-oxy-LNA unit, or wherein all LNA units in the alpha-b configuration are alpha-b-oxy-LNA units. In some embodiments of the invention, the number of nucleotides present in X'-Y'-Z' is selected from the group consisting of (nucleotide analog units - Y' region - nucleotide analog units) 1-8-1, 1-8-2, 2 -8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8 -4; or 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4 -9-1, 1-9-4; or 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-3, 3-10-2. In some embodiments of the invention, the number of nucleotides in X'-Y'-Z' is selected from the group consisting of 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3 , 3-7-2, 3-7-4 and 4-7-3. In some embodiments of the invention, the X' and Y' regions each consist of three LNA monomers, and the Y' region consists of 8, or 9, or 10 nucleoside monomers, preferably DNA monomers. In some embodiments of the invention, both X' and Z' consist of two DNA units each and Y' consists of 8 or 9 nucleotide units, preferably DNA units. In various embodiments of the invention, other gapmer constructs include constructs wherein the X' and/or Z' regions consist of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing the sugar 2'-O-methoxyethyl ribose (2'-MOE ), or monomers containing the sugar 2'-fluoro-deoxyribose, and the Y' region consists of 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where the X'-Y'-Z' regions have 3-9 -3, 3-10-3, 5-10-5 or 4-12-4 monomers. Additional gapmer constructs are disclosed in WO 2007/146511A2, incorporated herein by reference.

Gapmers ZNK. A LNA gapmer is a gapmer oligomer (region A) containing at least one LNA nucleotide. SEQ ID NOs: 1,2, 3, 4, 5, 6, 7, 8 and 40 are LNA gapmer oligomers. Oligomers with a contiguous sequence of 10-16 nucleotides complementary to the corresponding length of SEQ ID NO: 33 or 34 or 45 may also be gapmer oligomers, such as LNA gapmers.

Internucleotide connections.

The nucleoside monomers of the oligomers (eg, the first and second regions) described herein are linked together via internucleoside linking groups. Advantageously, each monomer is linked to 3'-adjacent monomers via a linking group.

One of ordinary skill in the art will appreciate that in the context of the present invention, the 5' monomer at the end of the oligomer does not contain a 5' linking group, although it may or may not contain a 5' end group or a linking group for conjugation.

The terms linking group or internucleoside linkage are intended to denote a group capable of covalently linking two nucleotides together. Specific and preferred examples include phosphate groups and phosphorothioate groups. An internucleoside linkage can be used interchangeably with an internucleotide linkage.

The nucleotides of the oligomer of the invention, or a contiguous nucleotide sequence thereof, are joined together by means of linking groups. Suitably, each nucleotide is linked to 3' adjacent nucleotides via a linking group.

Suitable internucleotide linkages include those listed within the scope of WO 2007/031091, for example the internucleotide linkages listed in the first paragraph c. 34 of WO 2007/031091 (incorporated herein by reference). In some embodiments of the invention, where they are linkages different from the phosphodiester linkage(s) of site B (where present), it is preferable to modify the internucleotide linkage from its normal phosphodiester to produce a linkage that is more resistant to nucleases, such as phosphorothioate or boranophosphate, where these two bonds are capable of being cleaved by RNase H, which also allows for antisense inhibition when expression of the target gene is reduced.

In some embodiments of the invention, the oligomer of the present invention contains one or more nucleoside linkages selected from the group consisting of phosphorothioate, phosphorodithioate and boranophosphate linkages.

Internucleotide linkages as provided herein containing sulfur (S), such as phosphorothioate or phosphorodithioate, may be preferred. Phosphorothioate internucleotide linkages are also preferred, particularly in the first region, for example in gapmers, mixers, antimer splice switch oligomers and totalmers.

The term mixer refers to oligomers containing both naturally occurring and non-naturally occurring nucleotides, where, in contrast to gapmers, tailmers and headmers, there is no continuous sequence of more than 5, and in some embodiments of the invention no more than 4, for example, no more than 3, consecutive naturally occurring nucleotides, such as DNA units.

The term totalmer refers to a single-stranded oligomer that contains only non-naturally occurring nucleosides, such as modified sugar nucleoside analogues.

For gapmers, the internucleotide linkages in the oligomer may be, for example, phosphorothioate or boranophosphate to allow cleavage of the target RNA by RNase H. Phosphorothioate is preferred for improved nuclease resistance and for other reasons such as ease of preparation.

In one aspect, with the exception of the phosphodiester linkage between the first and second regions and optionally within region B, the remaining internucleotide bonds of the oligomer of the invention, the nucleotides and/or nucleotide analogs are linked to each other via phosphorothioate groups. In some embodiments of the invention, at least 50%, e.g. at least 70%, e.g. at least 80%, e.g. at least 90%, e.g. all, of the internucleoside linkages between the nucleosides in the first region are different from phosphodiester (phosphate), e.g. selected from the group consisting of phosphorothioate, phosphorodithioate or boranophosphate. In some embodiments of the invention, at least 50%, such as at least 70%, such as at least 80%, such as at least 90%, such as all, of the internucleoside linkages between the nucleosides in the first region are phosphorothioate.

WO 09124238 relates to oligomeric compounds having at least one bicyclic nucleoside attached to the 3' or 5' end via a neutral internucleoside bond. The oligomers of the invention may thus have at least one bicyclic nucleoside attached to the 3' or 5' end by a neutral internucleoside bond, such as one or more phosphotriester, methylphosphonate, MMI, amide-3, formoacetal or thioformacetal. The remaining bonds may be phosphorothioate.

Oligomer conjugates (section C).

Another aspect of the invention is an antisense oligonucleotide conjugate comprising an oligomer of the invention and at least one non-nucleotide or non-polynucleotide moiety (C) covalently attached to the oligomer (A), optionally via a linker region located between the contiguous sequence of the oligomer and the conjugate moiety (B and /or Y).

Representative conjugate moieties that are used with oligonucleotides may include lipophilic molecules (aromatic and non-aromatic), including steroid molecules; proteins (eg antibodies, enzymes, whey proteins); peptides; vitamins (water-soluble or fat-soluble); polymers (water-soluble or fat-soluble); small molecules including drugs, toxins, reporter molecules and receptor ligands; carbohydrate complexes; complexes that cleave nucleic acids; metal chelators (eg porphyrins, texaphyrins, crown ethers, etc.); intercalators, including hybrid photonucleases/intercalators; cross-linking agents (eg, photoactive, redox-active) and combinations and derivatives thereof. Various suitable conjugate moieties, their preparation and association with oligomeric compounds are proposed, for example, in WO 93/07883 and US Pat. No. 6,395,492, each of which is incorporated herein by reference in its entirety. Oligonucleotide conjugates and their syntheses are also comprehensively reviewed by Manoharan, Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., ch. 16, Marcel Dekker, Inc., 2001; and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

In some embodiments of the invention, the oligomer of the invention is directed to the liver, i.e. after systemic administration, the compound accumulates in liver cells (such as hepatocytes). Targeting to the liver can be significantly enhanced by attaching a conjugate moiety (C). However, in order to obtain maximum efficiency of the oligomer, it is often desirable that the conjugate (or targeting moiety) be linked to the oligomer via a biocleavable linker (B), such as a nucleotide phosphate linker. Thus, it is desirable to use a conjugate moiety that enhances uptake and activity in hepatocytes. The increased activity may be a consequence of enhanced uptake, or may be a consequence of enhanced potency of the compound in hepatocytes.

In some embodiments of the invention, the oligomeric compound is a LNA oligomer, such as a gapmer, or, for example, an antisense LNA oligomer (which may be referred to herein as region A) containing an antisense oligomer, optionally a biocleavable linker, such as region B, and carbohydrate conjugate (which may be referred to as the C site). An antisense LNA oligomer may be 7-30, eg 8-26, nucleosides in length and contains at least one LNA unit (nucleoside).

In some embodiments of the invention, the conjugate is or may contain a carbohydrate, or contains a carbohydrate group. In some embodiments of the invention, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose and mannose-6-phosphate. In some embodiments of the invention, the conjugate group is or may contain mannose or mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in a number of tissues, such as liver and/or muscle. See, for example, EP 1495769; WO 99/65925; Yang et al., Bioconjug Chem. (2009), 20(2): 213-21; Zatsepin & Oretskaya Chem. Biodivers. (2004), 1(10): 1401-17.

In some embodiments of the invention, the carbohydrate moiety is a non-linear carbohydrate polymer. In some embodiments of the invention, the oligomeric compound is a LNA oligomer, such as an antisense LNA oligomer (which may be referred to herein as an A region) comprising an antisense oligomer, a B region as defined herein, and a conjugate moiety directed to an asialoglycoprotein receptor , such as the GalNAc moiety (which may be referred to as the C region). The carbohydrate moiety may be multivalent, such that 2, 3, 4 or 4 identical or non-identical carbohydrate moieties may be covalently attached to the oligomer, optionally via a linker or linkers (such as the Y region).

GalNAc conjugate groups.

In some embodiments of the invention, the carbohydrate moiety is a non-linear carbohydrate polymer. The carbohydrate moiety may, however, be multivalent, such that 2, 3, 4 or 4 identical or non-identical carbohydrate moieties may be covalently linked to the oligomer, optionally via a linker or linkers. In some embodiments, the invention provides a conjugate comprising an oligomer of the invention and a carbohydrate conjugate moiety. In some embodiments, the invention provides a conjugate comprising an oligomer of the invention and a conjugate moiety directed to an asialoglycoprotein receptor moiety, such as a GalNAc moiety, which may form part of an additional region (referred to as Region C).

The invention also provides antisense LNA oligonucleotides conjugated to a moiety directed to the asialoglycoprotein receptor. In some embodiments of the invention, the conjugate moiety (such as the third region or region C) contains a moiety directed to the asialoglycoprotein receptor, such as galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl -galactosamine and N-isobutanoylgalactosamine. In some embodiments of the invention, the conjugate contains a galactose cluster, such as an N-acetylgalactosamine trimer. In some embodiments of the invention, the conjugate moiety comprises GalNAc (N-acetylgalactosamine), such as monovalent, divalent, trivalent or tetravalent GalNAc. Trivalent GalNAc conjugates can be used to target the compound to the liver. GalNAc conjugates have been used with methylphosphonate and antisense peptide nucleic acid (PNA) oligonucleotides (eg, US 5994517; and Hangeland et al., Bioconjug. Chem., 1995 Nov-Dec, 6(6): 695-701) and siRNA (eg, WO 2009/126933, WO 2012/089352 and WO 2012/083046). References to GalNAc and to specific conjugates used therein are incorporated herein by reference. WO 2012/083046 discloses siRNAs with GalNAc conjugate moieties containing cleavable pharmacokinetic modulators that are suitable for use in the present invention, where the preferred pharmacokinetic modulators are C ₁₆ hydrophobic groups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E ,12E)-octadeca-9,12-dienoyl, dioctanoyl and C ₁₆ -C2 ₀ -acyl. '046 cleavable pharmacokinetic modulators may also be cholesterol.

The directing moieties (conjugate moieties) can be selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoyl-galactosamine, N-isobutanoylgalactosamine, galactose cluster and N- trimer acetylgalactosamine and may have a pharmacokinetic modulator selected from the group consisting of a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16-20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadec -9,12-dienoyl, dioctanoyl, C ₁₆ -C2 ₀ -acyl and cholesterol. Some GalNAc clusters disclosed in '046 include (E)-hexadec-8-enoyl ( _C16 ), oleyl ( _C18 ), (9E,12E)-octadeca-9,12-dienoyl ( _C18 ), octanoyl ( C ₈ ), dodecanoyl (C ₁₂ ), C ₂₀ -acyl, C ₂ 4-acyl, dioctanoyl (2x C ₈ ). Targeting moiety - The targeting moiety of a pharmacokinetic modulator can be linked to a polynucleotide via a physiologically labile bond, either, for example, a disulfide bond or a polyethylene glycol (PEG) linker. The invention also relates to the use of phosphodiester linkers between the oligomer and the conjugate group (herein referred to as the B region and suitably located between the LNA oligomer and the carbohydrate conjugate group).

For targeting hepatocytes in the liver, the preferred targeting ligand is a galactose cluster.

The galactose cluster contains a molecule having, for example, two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialog receptor

- 25 043736 lycoprotein, equal to or greater than the affinity of galactose. The terminal galactose derivative is attached to the molecule via its C-1 carbon atom. The asialoglycoprotein receptor (ASGPr) is expressed primarily on hepatocytes and binds branched glycoproteins to terminal galactose. A preferred galactose cluster has three terminal galactosamines or galactosamine derivatives, each of which has an affinity for the asialoglycoprotein receptor. A more preferred galactose cluster has three terminal N-acetyl-galactosamines. Other terms commonly used in the art include tri-antennary galactose, trivalent galactose, and galactose trimer. It is known that clusters of three-antennary galactose derivatives bind to ASGPr with greater affinity than the two-antennary and single-antennary galactose derivatives (Baenziger & Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, 1, Biol. Chern ., 257, 939-945). To achieve nM relatedness, multivalency is required. According to WO 2012/083046, the attachment of a single galactose derivative with affinity for the asialoglycoprotein receptor does not provide functional delivery of the RNAi polynucleotide to hepatocytes in vivo when co-administered with the delivery polymer.

The galactose cluster may contain two or preferably three galactose derivatives, each of which is associated with a central branch point. Galactose derivatives are attached to the central branch point via the C-1 carbon atoms of the saccharides. The galactose derivative is preferably linked to the branch point via a linker or spacer. A preferred spacer is a flexible hydrophilic spacer (US Pat. No. 5,885,968; Biessen et al., J. Med. Chern., 1995, vol. 39, p. 1538-1546). A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEGZ spacer. The branch point can be any small molecule that allows three galactose derivatives to be attached and further allows the branch point to be attached to the oligomer. An exemplary branch point group is di-lysine. The di-lysine molecule contains three amine groups, through which three galactose derivatives can be attached, and a carboxyl reactive group, through which di-lysine can be attached to the oligomer. The attachment of the branch point to the oligomer can occur through a linker or spacer. A preferred spacer is a flexible hydrophilic spacer. A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer (three ethylene units). The galactose cluster can be attached to the 3' or 5' end of the oligomer using methods known in the art.

A preferred galactose derivative is N-acetyl-galactosamine (GalNAc). Other saccharides having affinity for the asialoglycoprotein receptor may be selected from the list consisting of galactosamine, N-n-butanoylgalactosamine and N-iso-butanoylgalactosamine. The affinities of various galactose derivatives for the asialoglycoprotein receptor have been studied (see, for example, Jobst, S.T. & Drickamer, K., JB.C., 1996, 271, 6686) or are easily determined using methods known in the art.

One embodiment of the galactose cluster.

nono' he ko

- 26 043736

A galactose cluster with a PEG spacer between the branch point and the nucleic acid.

Additional examples of the conjugate of the invention are illustrated below.

- 27 043736

When used in the GalNAc cluster conjugates described above, a hydrophobic or lipophilic (or additional conjugate) moiety (ie, pharmacokinetic modulators) is optional when using LNA oligomers, such as LNA antisense oligonucleotides.

The figures show the specific GalNAc clusters used in the present study, Conj 1, 2, 3, 4 and Conj1a, 2a, 3a and 4a (which are shown with an optional C6 linker connecting the GalNAc cluster in the oligomer).

In a preferred embodiment of the invention, the conjugate moiety of the antisense oligonucleotide conjugate contains or consists of Conj 1,2,3,4 and Conjia, 2a, 3a and 4a. Most preferably, the conjugate moiety contains or consists of Conj 2a.

In another preferred embodiment of the invention, the antisense oligonucleotide conjugate is selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23 and 24.

Each carbohydrate moiety of the GalNAc cluster (eg, GalNAc) can thus be linked to the oligomer via a spacer such as a (poly)ethylene glycol (PEG) linker, such as a di-, tri-, tetra-, penta-, hexaethylene glycol linker. As shown above, the PEG moiety forms a spacer between the galactose sugar moiety and the peptide (trilysine shown) linker.

In some embodiments of the invention, the GalNAc cluster contains a peptide linker, for example, a Tyr-Asp(Asp) tripeptide or an Asp(Asp) dipeptide, which is attached to the oligomer (or to the Y site or B region) via a biradical linker, for example, a GalNAc cluster, which may contain the following biradical linkers:

R ¹ is a biradical, preferably selected from -C ₂ H ₄ -, -C ₃ H ₆ -, -C4H8-, -C ₅ H ₁₀ -, -C6H12-, 1,4-cyclohexyl (-CbHu-), 1 ,4-phenyl (-C6H4-), -C2H4OC2H4-, -C2H4(OC2H4)2- or -C2H4(OC2H4)3-, C(O)CH2-, -C(O)C2H4-, -C(O) C3H6-, -C(O)C4H8-, -C(O)C5H _ω -, -C(O)C6Hi2-, 1,4-cyclohexyl (-C(O)CbH _ω -), 1,4-phenyl ( -(O)SbH4-), -C(O)C2H4OC2H4-, -C(O)C2H4(OC2H4)2- or - C(O)C2H4(OC2H4) ₃ -.

In some embodiments of the invention, R ¹ is a diradical, preferably selected from -C ₂ H ₄ -, -C3H6-, -C4H8-, -C ₅ H ₁₀ -, -C6H12-, 1,4-cyclohexyl (-C6H10-), 1,4-phenyl (-C6H4-), -C2H4OC2H4-, -C ₂ H ₄ (OC ₂ H ₄ ) ₂ - or -C ₂ H ₄ (OC ₂ H ₄ ) ₃ -.

A carbohydrate conjugate (e.g., GalNAc) or carbohydrate linker moiety (e.g., a carbohydrate-PEG moiety) can be covalently linked to the oligomer via a branch point group, such as an amino acid or peptide, which suitably contains two or more amine groups (e.g., 3, 4 or 5), such as lysine, di-lysine or tri-lysine or tetralysine. The tri-lysine molecule contains four amine groups, through which three carbohydrate conjugate groups, such as galactose derivatives (eg, GalNAc), and an additional conjugate, such as a hydrophobic or lipophilic moiety/group, and a carboxyl reactive group, can be attached, through which three -lysine can be attached to the oligomer. An additional conjugate, such as a lipophilic/hydrophobic moiety, can be attached to a lysine residue attached to the oligomer.

The inventors of the present invention have unexpectedly discovered that GalNAc conjugates for use

- 28 043736 with HNA oligomers do not require a pharmacokinetic modulator (as described below) and in some embodiments of the invention the GalNAc conjugate itself is not covalently attached to a lipophilic or hydrophobic moiety, such as the moieties described herein, for example, does not contain C8-C36 fatty acid or sterol. Thus, the invention also provides conjugates of LNA oligomer with GalNAc that do not contain a lipophilic or hydrophobic pharmacokinetic modulator or conjugate moiety/group.

Pharmacokinetic modulators.

The compound of the invention may further contain one or more additional conjugate moieties, of which lipophilic or hydrophobic moieties are of particular interest, for example when the conjugate moiety is a carbohydrate moiety. Such lipophilic or hydrophobic moieties can act as pharmacokinetic modulators and can be covalently linked to either a carbohydrate conjugate, a linker linking a carbohydrate conjugate to an oligomer, or a linker linking multiple carbohydrate conjugates (multivalent conjugates), or to an oligomer, optionally via a linker , such as a biocleavable linker.

Thus, the oligomer or conjugate moiety may contain a pharmacokinetic modulator, such as lipophilic or hydrophobic moieties. Such moieties are disclosed in the context of siRNA conjugates in WO 2012/082046. The hydrophobic moiety may contain a C ₈ -C ₃₆ fatty acid, which may be saturated or unsaturated. In some embodiments of the invention, C ₁₀ , C ₁₂ , C ₁₄ , C ₁₆ , C ₁₈ , C ₂₀ , C 22, _{C 24, C 26, C 28, C 30 , C 32 and C 34} can be used. A hydrophobic group may have 16 or more carbon atoms. Exemplary suitable hydrophobic groups may be selected from the group consisting of: sterol, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl, dioctanoyl and C ₁₆ -C ₂₀ -acyl. According to WO'346, hydrophobic groups having less than 16 carbon atoms are less effective in enhancing the targeting of a polynucleotide, but they can be used in multiple copies (eg 2x, eg 2x C8 or _C10 , _C12 or _C14 ) to enhance effectiveness. Pharmacokinetic modulators useful as polynucleotide targeting moieties may be selected from the group consisting of cholesterol, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group and an aralkynyl group, each of which may be straight, branched or cyclical. Pharmacokinetic modulators are preferably hydrocarbons containing only carbon and hydrogen atoms. However, substitutions or heteroatoms that retain hydrophobicity, such as a fluorine atom, may be acceptable.

Lipophilic conjugates.

In some embodiments of the invention, the conjugate group is or may contain a lipophilic moiety such as a sterol (eg, cholesterol, cholesterol, cholestanol, stigmasterol, cholanic acid and ergosterol). In some embodiments of the invention, the conjugate is or contains tocopherol (examples are Conj 6 and Conj 6a in Fig. 2). In some embodiments of the invention, the conjugate is or may contain cholesterol (examples are Conj 5 and Conj 5a in Fig. 2).

In some embodiments of the invention, the conjugate is or may contain a lipid, phospholipid or lipophilic alcohol, such as a cationic lipid, neutral lipid, sphingolipid, and a fatty acid, such as stearic, oleic, elaidic, linoleic, linolelenalaidic, linolenic and myristic acid. In some embodiments of the invention, the fatty acid contains a C ₄ -C ₃₀ saturated or unsaturated alkyl chain. The alkyl chain can be straight or branched.

Lipophilic conjugate moieties can be used, for example, to measure the hydrophilic nature of an oligomeric compound and enhance cell penetration.

Lipophilic moieties include, for example, sterols, stanols and steroids and related compounds such as cholesterol (US Pat. No. 4,958,013; and Letsinger et al., Proc. Natl. Acad. Sci., USA, 1989, 86, 6553), thiocholesterol ( Oberhauser et al., Nucl. Acids. Res., 1992, 20, 533), lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, androsterone, deoxycorticosterone , cortisone, 17-hydroxycorticosterone, their derivatives, etc. In some embodiments of the invention, the conjugate may be selected from the group consisting of cholesterol, thiocholesterol, lanosterol, coprostanol, sigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone and 17-hydroxycorticosterone. Other lipophilic conjugate moieties include aliphatic groups, such as, for example, straight-chain, branched and cyclic alkyls, alkenyls and alkynyls. Aliphatic groups may have, for example, 5 to about 50, 6 to about 50, 8 to about 50, or 10 to about 50 carbon atoms. Examples of aliphatic groups include undecyl, dodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, terpenes, bornyl, adamantyl, derivatives thereof and the like. In some embodiments of the invention, one or

- 29043736 more carbon atoms in the aliphatic group may be replaced by a heteroatom such as O, S or N (eg geranyloxyhexyl). Additional suitable lipophilic conjugate moieties include aliphatic glycerol derivatives such as alkylglycerols, bis(alkyl)glycerols, tris(alkyl)glycerols, monoglycerides, diglycerides and triglycerides. In some embodiments of the invention, the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-Ohexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777) or their phosphonates. Saturated and unsaturated fatty functional groups, such as, for example, fatty acids, fatty alcohols, fatty esters and fatty amines, can also serve as lipophilic conjugate moieties. In some embodiments of the invention, the fatty functional groups may contain from about 6 carbon atoms to about 30, or from about 8 to about 22 carbon atoms. Examples of fatty acids include capric, caprylic, lauric, palmitic, myristic, stearic, oleic, linoleic, linolenic, arachidonic, eicosenoic acids and the like.

In additional embodiments of the invention, the lipophilic conjugate groups may be polycyclic aromatic groups having from 6 to about 50, from 10 to about 50, or from 14 to about 40 carbon atoms. Exemplary polycyclic aromatic groups include pyrene, purine, acridine, xanthine, fluorene, phenanthrene, anthracene, quinoline, isoquinoline, naphthalene, derivatives thereof, and the like. Other suitable lipophilic conjugate moieties include menthols, trityls (eg, dimethoxytrityl (DMT)), phenoxazines, lipoic acid, phospholipids, ethers, thioesters (eg, hexyl-S-tritylthiol), derivatives thereof, and the like. The preparation of lipophilic conjugates of oligomeric compounds is widely described in the art, for example, in the articles of Saison-Behmoaras et al., EMBO J., 1991, 10, 1111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al, Biochimie, 1993, 75, 49; Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229; and Manoharan et al., Tetrahedron Lett, 1995, 36, 3651.

Oligomeric compounds containing conjugate moieties with affinity for low-density lipoprotein (LDL) may help provide an effective targeted delivery system. The high levels of expression of LDL receptors on tumor cells makes LDL an attractive carrier for selective drug delivery to these cells (Rump et al., Bioconjugate Chem., 1998, 9, 341; Firestone, Bioconjugate Chem., 1994, 5, 105; Mishra et al., Bioconjugate Chem., 1998, 9, 341 al., Biochim. Biophys. Acta, 1995, 1264, 229). Groups with affinity for LDL include many lipophilic groups such as steroids (eg cholesterol), fatty acids, derivatives and combinations thereof. In some embodiments of the invention, conjugate moieties having affinity for LDL may be dioleyl esters of cholic acids, such as chenodeoxycholic acid and lithocholic acid.

In some embodiments of the invention, the lipophilic conjugates may be or may include biotin. In some embodiments of the invention, the lipophilic conjugates may be or may contain a glyceride or a glyceride ester.

Lipophilic conjugates such as sterols, stanols and stanines such as cholesterol or those disclosed herein can be used to enhance delivery of the oligonucleotide, for example, to the liver (typically to hepatocytes).

In a preferred embodiment of the invention, the conjugate moiety of the antisense oligonucleotide conjugate contains or consists of Conj 5, 5a, 6 or 6a. More preferably, the conjugate moiety contains or consists of Conj 5a.

In another preferred embodiment of the invention, the antisense oligonucleotide conjugate is selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 41, 42 and 43.

The following references also refer to the use of lipophilic conjugates: Kobylanska et al., Acta Biochim Pol. (1999), 46(3): 679-91; Felber et al., Biomaterials (2012), 33(25): 599-65; Grijalvo et al., J. Org. Chem. (2010), 75(20): 6806-13; Koufaki et al., Curr. Med. Chem. (2009), 16(35): 4728-42; Godeau et al., J. Med. Chem. (2008), 51(15): 4374-6.

Linkers (for example, region B or Y).

A bond or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. Conjugate moieties (or directing or blocking moieties) can be attached to the oligomeric compound directly or through a linker moiety (linker or tethering moiety), a linker. Linkers are bifunctional moieties that serve to covalently connect a third region, such as a conjugate moiety, to an oligomeric compound (eg, the A region). In some embodiments of the invention, the linker contains a chain oligomer structure of repeating units, such as ethylene glycol or amino acid units. The linker may have at least two functional groups, one for attachment to the oligomeric compound and the other for attachment to the conjugate moiety. Examples of linker functional groups may be electrophilic to interact with nucleophilic groups on the oligomer or conjugate group

- 30 043736 ke or nucleophilic for interaction with electrophilic groups. In some embodiments of the invention, linker functional groups include amino, hydroxyl, carboxylic acid, thiol, phosphoramidate, phosphorothioate, phosphate, phosphite, unsaturated bonds (eg, double or triple bonds), and the like. Some examples of linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxy-(6-amido-caproate) (LCSMCC), succinimidyl meta-maleimido-benzoylate (MBS), succinimidyl-N-e -maleimido-caproylate (EMCS), succinimidyl-6-(beta-maleimido-propionamido)hexanoate (SMPH), succinimidyl-N-(a-maleimidoacetate) (AMAS), succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) , beta-alanine (beta-ALA), phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC), beta-(cyclopropyl)alanine (beta-CYPR), aminododecanoic acid (ADC), allylenediols, polyethylene glycols, amino acids, etc. .

A wide variety of additional linker groups are known in the art that can be useful in attaching conjugate moieties to oligomeric compounds. A review of many of the useful linker groups can be found, for example, in the book. Antisense Research & Applications, S.T. Crooke & B. Lebleu, eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. A disulfide bond is used to link the 3' end of the oligonucleotide to the peptide (Corey et al., Science 1987, 238, 1401; Zuckermann et al., J Am. Chem. Soc., 1988, 110, 1614; and Corey et al., J. Am. Chem. Soc., 1989, 111, 8524). The article by Nelson et al., Nucl. Acids Res., 1989, 17, 7187 describes a coupling reagent for attaching biotin to the 3' end of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol, is commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3'-Amine. It is also commercially available as 3'-Amino-Modifier Reagent from Glen Research Corporation (Sterling, VA). This reagent was also used to couple a peptide to an oligonucleotide, as described in Judy et al., Tetrahedron. Letters, 1991, 32, 879. A similar commercial reagent for binding to the 5' end of an oligonucleotide is 5'-Amino-Modifier C6. These reagents are commercially available from Glen Research Corporation (Sterling, VA). These or similar compounds were used by Krieg et al., Antisense Research and Development 1991, 1, 161, to bind fluorescein to the 5' end of the oligonucleotide. Other compounds, such as acridine, are attached to the 3'-terminal phosphate group of the oligonucleotide via a polymethylene bond (Asseline et al., Proc. Natl. Acad. Sci., USA, 1984, 81, 3297). Any of the groups described above can be used as a single linker or in combination with one or more additional linkers.

Linkers and their use in the preparation of conjugates of oligomeric compounds are proposed in the prior art, for example in documents WO 96/11205; and WO 98/52614; and US patents No. 4948882; 5525465; 5541313; 5545730; 5552538; 5580731; 5486603; 5608046; 4587044; 4667025; 5254469; 5245022; 5112963; 5391723; 5510475; 5512667; 5574142; 5684142; 5770716; 6096875; 6335432 and 6335437, WO 2012/083046, each of which is incorporated by reference in its entirety.

As used herein, a physiologically labile linkage is a labile linkage that is cleavable under conditions normally encountered or similar to those found in a mammal (also referred to as a cleavable linker, illustrated as region B in FIGS. 12 and 13). Physiological labile linking groups are selected from groups that undergo chemical transformation (eg, cleavage) when exposed to certain physiological conditions. Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentration found in or similar to those found in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in a mammalian cell, such as the activity of proteolytic or hydrolytic enzymes. In some embodiments of the invention, the cleavable linker is sensitive to nuclease(s), which may, for example, be expressed in the target cell, and as such, as detailed herein, the linker may be a short stretch (for example, 1-10) of nucleosides, linked by phosphodiester bonds, such as DNA nucleosides.

The chemical transformation (cleavage of the labile bond) can be initiated by the addition of a pharmaceutically acceptable agent to the cell or can occur spontaneously when the molecule containing the labile bond reaches the appropriate intra- and/or extracellular environment. For example, a pH-labile bond can be cleaved when the molecule enters an acidified endosome. Thus, a pH-labile bond can be considered as an endosomally cleaved bond. Enzymatically cleavable bonds can be cleaved by enzymes, such as those present in the endosome, either in the lysosome or in the cytoplasm. The disulfide bond can be cleaved when the molecule penetrates into the more reducing environment of the cell cytoplasm. Thus, the disulfide can be considered as a cytoplasmically cleavable bond. As used herein, a pH-labile bond is a labile bond that is selectively cleaved under acidic conditions (pH less than 7). Such connections may also be called

- 31 043736 endosomally labile bonds, since cellular endosomes and lysosomes have a pH less than 7.

Biocleavable conjugates bound to an oligomer.

The oligomeric compound may optionally comprise a second region (Region B) located between the oligomer (referred to as Region A) and the conjugate (referred to as Region C) (see FIGS. 12 and 13 for illustrations). The B region may be a linker, such as a cleavable linker (also referred to as a physiologically labile linkage). The physiologically labile bonds that are sensitive to nucleases are as follows. In some embodiments of the invention, the oligomer (also referred to as an oligomeric compound) of the invention (or conjugate) contains three regions:

i) the first region (region A), containing 10-18 contiguous nucleotides;

ii) a second region (region B) containing a biocleavable linker;

iii) a third region (C) containing a conjugate moiety, a targeting moiety, an activation moiety, where the third region is covalently linked to the second region.

In some embodiments of the invention, region B may be a nucleotide phosphate linker. For example, such linkers can be used when the conjugate is a lipophilic conjugate such as a lipid, a fatty acid, a sterol such as cholesterol or tocopherol. Nucleotide phosphate linkers can also be used for other conjugates, for example carbohydrate conjugates such as GalNAc.

Peptide linkers.

In some embodiments of the invention, the biocleavable linker (site B) is a peptide, such as a trilysine peptide linker, which can be used in a polyGalNAc conjugate, such as a triGalNAc conjugate. See also peptide biradicals mentioned herein.

Other linkers known in the art that can be used include disulfide linkers.

Nucleotide phosphate linkers.

In some embodiments of the invention, region B contains 1-6 nucleotides covalently linked to a 5' or 3' nucleotide of the first region, for example, through an internucleoside linking group, such as a phosphodiester linkage, where either

a) the internucleoside bond between the first and second region is a phosphodiester bond, and the nucleoside of the second region (eg, immediately) adjacent to the first region is either DNA or RNA; and/or

b) at least 1 nucleoside of the second region is a DNA or RNA nucleoside linked by a phosphodiester bond;

In some embodiments of the invention, region A and region B form one contiguous nucleotide sequence of 12-22 nucleotides in length.

In some aspects, the internucleoside linkage between the first and second regions may be considered part of the second region.

In some embodiments of the invention, a phosphorus-containing linking group is located between the second and third regions. The phosphorus linking group may, for example, be a phosphate (phosphodiester), phosphorothioate, phosphorodithioate or boranophosphate group. In some embodiments of the invention, the phosphorus-containing linking group is located between the second region and a linker region that is attached to the third region. In some embodiments of the invention, the phosphate group is a phosphodiester.

Thus, in some aspects, the oligomeric compound contains at least two phosphodiester groups, where at least one is a group in accordance with the above summary of the invention, and the other is located between the second and third regions, optionally between the linker group and the second region.

In some embodiments of the invention, the third region is an activation group, such as an activation group for use in conjugation. In this aspect, the invention also provides activated oligomers comprising an A and B region and an activation group, eg an intermediate, which is suitable for subsequent coupling to a third site, eg suitable for conjugation.

In some embodiments of the invention, the third region is a reactive group, such as a reactive group for use in conjugation. In this aspect, the invention also provides oligomers comprising an A and B region and a reactive group, eg an intermediate, which is suitable for subsequent coupling to a third site, eg suitable for conjugation. The reactive group may, in some embodiments of the invention, contain an amine alcohol group, such as an amine group.

In some embodiments of the invention, region A contains at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 internucleoside linkages other than phosphodiester, such as internucleoside linkages that are (optionally independently) selected from

- 32 043736 group consisting of phosphorothioate, phosphorodithioate and boranophosphate, and methyl phosphate such as phosphorothioate. In some embodiments of the invention, region A contains at least one phosphorothioate bond. In some embodiments of the invention, at least 50%, for example at least 75%, for example at least 90%, for example, of all internucleoside linkages within region A are other than phosphodiester, for example phosphorothioate linkages. In some embodiments of the invention, all internucleoside bonds in region A are other than phosphodiester bonds.

In some embodiments of the invention, the oligomeric compound is an antisense oligonucleotide, such as an antisense oligonucleotide conjugate. The antisense oligonucleotide may be or may comprise a first region and optionally a second region. In this aspect, in some embodiments of the invention, the B region may form part of a contiguous sequence of nucleotide bases complementary to the target. In other embodiments of the invention, region B may lack target complementarity.

Alternatively, in some embodiments, the invention provides an oligonucleotide (e.g., an antisense oligonucleotide) linked by a non-phosphodiester bond, such as a phosphorothioate bond, that has at least one terminal (5' and/or 3') DNA or RNA nucleoside linked to an adjacent nucleoside oligonucleotide via a phosphodiester bond, wherein the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting moiety or a blocking moiety, optionally via a linker moiety.

In some embodiments of the invention, the oligomeric compound comprises an antisense oligonucleotide, such as an antisense oligonucleotide conjugate. The antisense oligonucleotide may be or may comprise a first region and optionally a second region. In this aspect, in some embodiments of the invention, region B may form part of a contiguous sequence of nucleotide bases complementary to the target. In other embodiments of the invention, region B may lack target complementarity.

In some embodiments of the invention, at least two consecutive nucleosides of the second region are DNA nucleosides (eg, at least 3, or 4, or 5 consecutive DNA nucleotides).

In such an embodiment of the invention, the oligonucleotide of the invention may be described according to the following formula:

5’-A-RO-B (Y)X-3’ or Z’-A-RO-B (Υ)Χ-δ’ where A represents section A,

PO is a phosphodiester bond,

B represents section B,

Y represents an optional linking group, and

X represents a conjugate, directing, blocking group, or a reactive or activation group.

In some embodiments of the invention, region B contains 3'-5' or 5'-3'

i) a phosphodiester linkage to the 5' or 3' nucleoside of site A;

ii) a DNA or RNA nucleoside, such as a DNA nucleoside; and iii) an additional phosphodiester linkage

5’-A-RO-V-RO-3’ or 3’-A-RO-V-RO-5’

The additional phosphodiester linkage links the B site nucleoside to one or more additional nucleosides, such as one or more DNA or RNA nucleosides, or may link to X (representing a conjugate, directing or blocking group, or a reactive or activation group), optionally via a linking group (Y ).

In some embodiments of the invention, region B contains 3'-5' or 5'-3'

i) a phosphodiester linkage to the 5' or 3' nucleoside of site A;

ii) 2-10 phosphodiester-linked DNA or RNA nucleosides, such as a DNA nucleoside; and optionally iii) an additional phosphodiester linkage

5’-A-[PO-B]n-[Y]-X 3’ or 3’-A-[PO-B]p -[Y]-X 5’

5’-Α-[ΡΟ-Β]η-ΡΟ-[Υ]-Χ 3’ or 3’-Α-[ΡΟ-Β]η-ΡΟ -[Υ]-Χ 5’ where A represents section A,

[PO-B]n represents region B, where n is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

PO is an optional phosphodiester linking group between site B and X (or Y, if present).

In some embodiments of the invention, the invention provides compounds according to one of (or containing one of) the following formulas:

- 33 043736

5’ [Section A]-PO-[Section B] 3’ -Y-X

5’ [Section A]-PO-[Section B]-PO 3’ -Y-X

5’ [Section A]-PO-[Section B] 3’ - X

5’ [Section A] - PO - [Section B] - PO 3’ - X

3’ [Section A]-PO-[Section B] 5’ -Y-X

3’ [Section A]-PO-[Section B]-PO 5’ -Y-X

3’ [Section A]-PO-[Section B] 5’ -X

3’ [Section A]-RO-[Section B]-RO 5’ - X

Section B may, for example, contain or consist of

5' DNZ'

3' DNA 5'

5’ DNA-RO-DNKZ’

3’ DNA-RO-DNA5’

5’ DNA-RO-DNA-RO-DNA 3’

3’ DNA-RO-DNA-RO-DNA 5’

5’ DNA-RO-DNA-RO-DNA-RO-DNA 3’

3’ DNA-RO-DNA-RO-DNA-RO-DNA 5’

5’ DNA-RO-DNA-RO-DNA-RO-DNA-RO-DNA 3’

3’ DNA-RO-DNA-RO-DNA-RO-DNA-RO-DNA 5’

It will be appreciated that nucleosides other than DNA and RNA can be used in phosphate bonded biocleavable linkers. Biocleavable nucleotide linkers can be identified using the assays in Example 6.

In some embodiments of the invention, a compound of the invention contains a biocleavable linker (also referred to as a physiologically labile linker, nuclease-sensitive physiologically labile linkages, or nuclease-sensitive linker), such as a nucleotide phosphate linker (such as region B) or a peptide linker that connects the oligomer (or a contiguous nucleotide sequence or region A) with a conjugate moiety (or region C).

The cleavage sensitivity in the assays shown in Example 6 can be used to determine whether a linker is biodegradable or physiologically labile.

Biocleavable linkers according to the present invention refer to linkers that are sensitive to cleavage in the target tissue (ie, physiologically labile), for example, in the liver and/or kidney. Preferably, the rate of degradation observed in the target tissue is higher than that found in serum. Suitable methods for determining the level (%) of degradation in tissue (eg, liver or kidney) and serum can be found in Example 6. In some embodiments of the invention, the biocleavable linker (also referred to as a physiologically labile linker or nuclease-sensitive linker), such as region B, in a compound of the invention is at least about 20% cleaved, e.g. at least about 30% cleaved, e.g. at least about 40% cleaved, e.g. at least about 50% cleaved, e.g. at least about 60%, e.g., at least about 70% digestible, e.g., at least about 75% digestible in the liver or kidney homogenate assay of Example 6. In some embodiments, the digestion (%) in serum used in the assay Example 6 is less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%.

In some embodiments of the invention, which may be the same or different, a biocleavable linker (also referred to as a physiologically labile linker or a nuclease-sensitive linker), such as region B, in a compound of the invention is susceptible to cleavage by S1 nuclease. S1 cleavage sensitivity can be assessed using the S1 nuclease assay shown in Example 6. In some embodiments of the invention, a biocleavable linker (also referred to as a physiologically labile linker or a nuclease-sensitive linker), such as region B, is cleaved in a compound of the invention at least about 30% cleaved, e.g. at least about 40% cleaved, e.g. at least about 50% cleaved, e.g. at least about 60% cleaved, e.g. at least about 70% cleaved, e.g. at least about 80%, e.g., at least about 90% cleaved, e.g., at least about 95% cleaved, after 120 min of incubation with S1 nuclease according to

- 34 043736 analysis used in example 6.

Sequence selection in the second section.

In some embodiments of the invention, the B region does not form a complementary sequence when the A and B regions of the oligonucleotide are aligned with the complementary sequence of the target.

In some embodiments of the invention, the B region does not form a complementary sequence when the A and B regions of the oligonucleotide are aligned with the complementary sequence of the target. In this aspect, region A and B together may form one contiguous sequence complementary to the target sequence.

In some embodiments of the invention, the base sequence in region B is selected to provide an optimal endonuclease cleavage site based on the predominant endonuclease cleavage enzymes present in the target tissue or cell or subcellular compartment. In this aspect, by isolating cellular extracts from target and non-target tissues, endonuclease digestion sequences for use in Site B can be selected based on preferential digestion activity in the desired target cell (eg, liver/hepatocytes) versus with a non-target cell (for example, in a kidney). In this aspect, the effectiveness of the compound for targeted down-regulation can be optimized for the desired tissue/cell.

In some embodiments of the invention, region B contains dinucleotide sequences AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC or GG, where C may be 5-methylcytosine and /or T may be replaced by U. In some embodiments of the invention, region B contains the trinucleotide sequences AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA , TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT , CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC and GGG, where C may represent 5-methylcytosine and/or T may be replaced by U. In some embodiments of the invention, region B contains the tetranucleotide sequences AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX , AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX , CCAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX and GGGX, where X may be selected from the group consisting of A, T, U, G, C and analogs thereof, wherein C may be 5-methylcytosine and/or T may be replaced by U. It will be understood that when referring to (occurring in nature) nucleotide bases A, T, U, G, C, they can be replaced by nucleotide base analogues that function as equivalents to natural nucleotide bases (for example, a base pair with a complementary nucleoside). In some embodiments of the invention, region B does not contain T or U.

Aminoalkyl intermediates.

The invention further provides intermediate LNA oligomers comprising an antisense LNA oligomer containing (e.g., a terminal, 5' or 3') aminoalkyl linker, such as a C2-C36 aminoalkyl group, e.g., a C6-C12 aminoalkyl group, including, for example, C6 and C12 aminoalkyl groups. An aminoalkyl group can be added to a LNA oligomer as part of an oligonucleotide synthesis, for example, using (eg, protected) aminoalkyl phosphoramidite. The linking group between the aminoalkyl and the LNA oligomer may be, for example, a phosphorothioate or phosphodiester, or one of other nucleoside linkage groups, such as those referred to herein. The aminoalkyl group can be covalently linked, for example, to the 5' or 3' end of the LNA oligomer, for example, via a nucleoside linkage group such as a phosphorothioate or phosphodiester linkage.

The invention also provides a method for synthesizing a LNA oligomer, comprising the sequential synthesis of a LNA oligomer, such as solid phase oligonucleotide synthesis, including the step of adding an aminoalkyl group to the oligomer, such as during the first or last round of oligonucleotide synthesis. The synthesis method may additionally include the stage of interaction of the conjugate with the aminoalkyl oligomer of LNA (conjugation stage). The conjugate may contain suitable linkers and/or branch point groups and optionally additional conjugate groups, such as hydrophobic or lipophilic groups, as described herein. The conjugation step can be performed while the oligomer is bound to the solid support (eg, after oligonucleotide synthesis but before elution of the oligomer from the solid support), or sequentially (ie, after elution). The invention proposes the use of an aminoalkyl linker in the synthesis of the oligomer according to the invention.

Method of preparation/synthesis.

The invention provides a method for synthesizing (or producing) an oligomeric compound, such as an oligomeric compound of the invention, wherein the method comprises either

- 35 043736

a) the step of providing a [solid phase] oligonucleotide synthesis carrier [third section], to which one of the following is attached:

i) a linker group (-Y-), ii) a group selected from the group consisting of a conjugate, a directing group, a blocking group, a reactive group [for example, amine or alcohol] or an activation group (X-), iii) a -Y-X group ; And

b) a step of [sequential] oligonucleotide synthesis of region B followed by synthesis of region (A); and/or

c) a step of [sequential] oligonucleotide synthesis of the first region (A) and the second region (B); where the synthesis step is followed by

d) stage of addition of the third section [phosphoramidite inclusions]

i) a linker group (-Y-), ii) a group selected from the group consisting of a conjugate, a directing group, a blocking group, a reactive group [for example, amine or alcohol] or an activation group (X-), iii) a -Y-X group ; followed by

e) cleaving the oligomeric compound from the [solid phase] support, where optionally said method further comprises a further step selected from the steps wherein:

f) the third group is an activation group, the steps of activating the activation group to produce a reactive group followed by attaching a conjugate, blocking or directing group to the reactive group, optionally via a linker group (Y);

g) the third region represents a reactive group, the steps of attaching a conjugate, blocking or directing group to the reactive group, optionally via a linker group (Y);

h) the third region is a linker group (Y), the steps of attaching a conjugate, blocking or directing group to the linker group (Y), where steps f), g) or h) are performed either before or after cleavage of the oligomeric compound from the oligonucleotide synthesis support .

In some embodiments of the invention, the method can be performed using standard phosphoramidite chemistry, and the X region itself and/or the X region, or the X and Y region, can be prepared prior to incorporation into the oligomer as phosphoramidite. Please see fig. 5-10, which illustrate non-limiting aspects of the method of the invention.

The invention provides a method for synthesizing (or producing) an oligomeric compound, such as an oligomeric compound of the invention, wherein the method comprises a) a step of sequential oligonucleotide synthesis of a first region (A) and a second region (B), wherein the synthesis step is followed by the step of joining a third region, a phosphoramidite-containing region X (also referred to as region C) or Y, such as a region containing a group selected from the group consisting of a conjugate group, a directing group, a blocking group, a functional group, a reactive group (for example, amine or alcohol ) or activation group (X), or group -Y-X, followed by cleavage of the oligomeric compound from the [solid phase] carrier.

However, it is recognized that the X or X-Y region may be attached after being cleaved from the solid support. Alternatively, the method may include the steps of synthesizing a first (A) and optionally a second region (B), followed by cleavage of the oligomer from the support, and followed by the step of attaching a third region, such as an X or X-Y group to the oligomer. Attachment of the third site can be achieved, for example, by attaching an aminophosphoramidite unit at the final stage of the synthesis of the oligomer (on the support), which, after cleavage from the support, is used to couple with the X or X-Y group, optionally via an activation group on the X or Y group (when present). In embodiments of the invention where the cleavable linker is a non-nucleotide region, region B may be a non-nucleotide cleavable linker, for example, a peptide linker, which may form part of the X region (also referred to as the C region) or be a Y region (or its Part).

In some embodiments of the method, the X region (such as C) or (X-Y) such as a conjugate (for example, a GalNAc conjugate) contains an activation group (activated functional group), and in the synthesis method, the activated conjugate (either the X or X-Y region) is attached to the first and second regions, such as an amino-linked oligomer. An amino group can be added to the oligomer using standard phosphoramidite chemistry, for example, as a final step in oligomer synthesis (typically resulting in an amino group at the 5' end of the oligomer). For example, during the last step of oligonucleotide synthesis, an amino-alkyl phosphoramidite, for example, TFA-amino-C6-phosphoramidite (6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,Ndiisopropyl)-phosphoramidite), is used.

The X site (or the C region referred to herein) such as a conjugate (eg, a GalNAc conjugate) can be activated by an N-hydroxysuccinimide (NHS) ester method and then attached to an amine-linked oligomer. For example, N-hydroxysuccinimide (NHS)

- 36 043736 can be used as an activating group for the X region (or C region, such as a conjugate, for example a GalNAc conjugate moiety).

The invention provides an oligomer obtained by the method according to the invention.

In some embodiments of the invention, the X region and/or the X region or the X and Y region may be covalently linked to the B region through a nucleoside phosphate bond, such as described herein, including a phosphodiester or phosphorothioate, or through an alternative group, such as a triazole group.

In some embodiments of the invention, the internucleoside linkage between the first and second regions is a phosphodiester linked to the first (or only) DNA or RNA nucleoside of the second region, or region B comprises at least one DNA or RNA nucleoside linked by a phosphodiester bond.

The second region in some embodiments of the invention may contain additional DNA or RNA nucleosides, which may be linked by a phosphodiester bond. The second region is further covalently linked to a third region, which may, for example, be a conjugate, a directing group, a reactive group and/or a blocking group.

In some aspects, the present invention is based on providing a labile region, a second region linking the first region, such as an antisense oligonucleotide, and a conjugate or functional group, such as a targeting or blocking group. The labile region contains at least one phosphodiester-linked nucleoside, such as a DNA or RNA nucleoside, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphodiester-linked nucleosides, such as DNA or RNA. In some embodiments of the invention, the oligomeric compound contains a cleavable (labile) linker. In this aspect, the cleavable linker is preferably present in region B (or in some embodiments of the invention between region A and B).

Alternatively, in some embodiments of the invention, the invention provides an oligonucleotide (e.g., an antisense oligonucleotide) linked by a non-phosphodiester bond, e.g., a phosphorothioate bond, having at least one terminal (5' and/or 3' nucleoside) DNA or RNA linked to an adjacent nucleoside oligonucleotide via a phosphodiester bond, wherein the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting moiety or a blocking moiety, optionally via a linker moiety.

Compositions.

The oligomer or oligomer conjugates of the invention can be used in pharmaceutical preparations and compositions. Suitably, such compositions contain a pharmaceutically acceptable diluent, carrier, salt or adjuvant. WO 2007/031091, incorporated by reference, proposes suitable and preferred pharmaceutically acceptable diluent, carrier and adjuvants. Suitable dosages, preparations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrugs are also suggested in WO 2007/031091, also incorporated herein by reference.

Applications.

The oligomers or conjugates of oligomers according to the invention can be used as experimental reagents, for example, diagnostic, therapeutic and prophylactic.

In research, such oligomers can be used to specifically inhibit PCSK9 protein synthesis (typically by degrading or inhibiting the mRNA and thereby preventing protein formation) in cells and experimental animals, thereby facilitating functional analysis of the target or assessment of its utility as a target for therapeutic intervention.

In diagnostics, such oligomers can be used to detect and quantify PCSK9 expression in cells and tissues using Northern blotting, in-situ hybridization, or similar methods.

For therapeutic applications, an animal or human suspected of having a disease or disorder that can be treated by modulating the expression of PCSK9 is treated by administering oligomeric compounds in accordance with this invention. Additionally provided are methods of treating a mammal, such as treating a human, suspected of having or being predisposed to a disease or condition associated with PCSK9 expression, by administering a therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention. The oligomer, conjugate or pharmaceutical composition of the invention is typically administered in an effective amount.

The invention also provides the use of a compound or conjugate of the invention, as described herein, for the preparation of a medicament for treating a disorder referred to herein, or for a method of treating a disorder referred to herein.

The invention also provides a method for the disorder referred to herein, comprising administering to a patient in need thereof a compound of the invention as described herein and/or a conjugate of the invention and/or a pharmaceutical composition of the invention.

Medical indications.

Oligomers, oligomer conjugates, and other compositions of the invention can be used to treat conditions associated with overexpression or expression of a mutated variant of PCSK9.

The invention further provides the use of a compound of the invention in the preparation of a medicament for the treatment of a disease, disorder or condition referred to herein.

In general terms, one aspect of the invention relates to a method of treating a mammal suffering from or suspected of having conditions associated with abnormal levels and/or activity of PCSK9, where the method includes administering to the mammal a therapeutically effective amount of an oligomer or oligomer conjugate targeting PCSK9 containing one or more ZNK units. The oligomer, conjugate or pharmaceutical composition of the invention is typically administered in an effective amount.

The disease or disorder referred to herein, in some embodiments of the invention, may be associated with a mutation in the PCSK9 gene or in a gene whose protein product associates with or interacts with PCSK9. Thus, in some embodiments of the invention, the target mRNA is a mutated form of the PCSK9 sequence.

An aspect of interest of the invention relates to the use of an oligomer (compound), as defined herein, or a conjugate, as defined herein, for the preparation of a medicament for the treatment of a disease, disorder or condition referred to herein.

The methods of the invention are preferably used for the treatment or prevention of diseases caused by abnormal levels and/or activity of PCSK9.

Alternatively, in some embodiments of the invention, the invention further provides a method of treating abnormal levels and/or activity of PCSK9, wherein the method comprises administering an oligomer of the invention, or a conjugate of the invention, or a pharmaceutical composition of the invention to a patient in need thereof.

The invention also relates to an oligomer, composition or conjugate, as defined herein, for use as a drug.

The invention further relates to the use of a compound, composition or conjugate, as defined herein, for the preparation of a medicament for the treatment of abnormal levels and/or activity of PCSK9 or the expression of mutant forms of PCSK9 (such as allelic variants, for example, associated with one of the diseases, on which are referenced in this document).

In addition, the invention relates to a method of treating a subject suffering from a disease or condition, such as those referred to herein.

A patient in need of treatment is a patient suffering or likely to suffer from a disease or disorder.

In some embodiments of the invention, the term treatment as used herein refers to both treating an existing disease (eg, a disease or disorder referred to herein) and preventing the disease, i.e. prevention. Thus, it is well known that the treatment referred to herein may, in some embodiments of the invention, be prophylactic.

In one embodiment, the invention relates to compounds or compositions containing compounds for the treatment of hypercholesterolemia and related disorders, or methods of treatment using such compounds or compositions for the treatment of hypercholesterolemia and related disorders, wherein the term related disorders when referring to hypercholesterolemia refers to one or more of the conditions , selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, e.g. accumulation of functional mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias, e.g. familial hyperlipidemia (FH) or familial hypercholesterolemia (FHC), acquired hyperlipidemia, statin -resistant hypercholesterolemia, coronary artery disease (CAD) and coronary heart disease (CHD).

Combination therapies.

In some embodiments of the invention, a compound of the invention is intended for use in combination treatment with another therapeutic agent. For example, HMGCoA reductase inhibitors, such as statins, are widely used in the treatment of metabolic disease (see WO 2009/043354, incorporated herein by reference as examples of combination treatments). Combination treatments may be other hypocholesterolemic compounds, such as a compound selected from the group consisting of bile acid mol-sequestrants (for example, cholestyramine, colestipol and colesevelam hydrochloride),

- 38 043736 HMGCoA reductase inhibitors (for example, lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin and fluvastatin), nicotinic acid, fibric acid derivatives (for example, clofibrate, gemfibrozil, fenofibrate, bezafibrate and ciprofibrate), probucol, neomycin, dextrothyroxine , plant stanol esters, cholesterol absorption inhibitors (eg, ezetimibe), implitapide, bile acid transporter inhibitors (apical sodium-dependent bile acid transporters), regulators of hepatic CYP7a, estrogen replacement therapies (eg, tamoxifen), and anti-inflammatory drugs (eg, glucocorticoids) . Combinations with statins may be particularly advantageous.

Specific embodiments of the invention

1. Antisense oligonucleotide conjugate containing

a) an antisense oligomer (A) of 12-22 nucleotides in length, containing a contiguous sequence of 10-16 nucleotides complementary to the corresponding length of SEQ ID NO: 30, or 31, or 32, or 33, or 34, or 45; And

b) at least one non-nucleotide or non-polynucleotide conjugate moiety (C) covalently attached to the oligomer (A).

2. An oligonucleotide conjugate according to embodiment 1, wherein the antisense oligomer contains a contiguous sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29 and 44.

3. An oligonucleotide conjugate according to any one of embodiments 1 or 0, wherein the antisense oligomer is directed to PCSK9.

4. An oligonucleotide conjugate according to any one of 1-3, wherein the antisense oligomer contains affinity-enhancing nucleotide analogues.

5. An oligonucleotide conjugate according to embodiment 1, wherein the nucleotide analogues are sugar-modified nucleotides, such as sugar-modified nucleotides independently or dependently selected from the group consisting of locked nucleic acid (LNA) units; 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino DNA units and 2'-fluoro-DNA units.

6. An oligonucleotide conjugate according to embodiment 4 or 5, wherein the nucleotide analogs contain or are locked nucleic acid (LNA) units.

7. An oligonucleotide conjugate according to any one of embodiments 1-6, wherein the antisense oligomer is a gapmer.

8. An oligonucleotide conjugate according to embodiment 7, wherein the gapmer contains a wing segment on each side (5' and 3') of nucleotide analogs, preferably LNA analogs, in an amount of 2 to 4.

9. An oligonucleotide conjugate according to embodiment 7 or 8, wherein the gapmer construct is selected from the group consisting of 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-2, 2 -8-4, 2-9-2, 2-9-3, 3-9-2, 3-9-3, 3-9-4, 4-9-3, 2-10-2, 2-10 -3, 3-10-2, 3-10-3, 3-10-4, 4-10-3, 2-11-2, 2-11-3, 3-11-2, 3-11-3 , 3-11-4, 4-11-3 and 4-11-4.

10. An oligonucleotide conjugate according to any one of embodiments 0-0, wherein the gapmer construct is selected from the group consisting of 2-8-3, 3-8-3, 3-9-4, 3-10-3, 2-11- 2, 2-11-3 and 3-11-2.

11. An oligonucleotide conjugate according to any one of embodiments 1-0, wherein the oligomer contains a contiguous sequence of 13, 14, 15 or 16 nucleotides.

12. An oligonucleotide conjugate according to any one of embodiments 1 to 11, wherein the oligomer contains one or more nucleoside linkages selected from the group consisting of phosphorothioate, phosphorodithioate and boranophosphate.

13. An oligonucleotide conjugate according to any one of embodiments 1-12, wherein the oligomer contains or consists of phosphorothioate nucleoside linkages.

14. An oligonucleotide conjugate according to any one of embodiments 1 to 13, wherein the oligomer contains a contiguous sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8.

15. An oligonucleotide conjugate according to any one of embodiments 1 to 14, wherein the conjugate moiety (C) is selected from the group consisting of a carbohydrate such as GalNAc or a GalNAc cluster, a lipophilic group such as a lipid, a fatty acid; a sterol such as cholesterol or tocopherol; or a statin.

16. An antisense oligonucleotide conjugate according to any one of embodiments 1-15, wherein the conjugate moiety (C) enhances delivery and/or uptake into liver cells.

17. An antisense oligonucleotide conjugate according to any one of embodiments 1-16, wherein the conjugate moiety (C) contains a sterol such as tocopherol, cholesterol, for example the conjugates shown as Conj 5, Conj 5a, Conj 6 or Conj 6a.

18. An antisense oligonucleotide conjugate according to embodiment 1-17, wherein the conjugate moiety (C) contains a carbohydrate, for example GalNAc or trivalent GalNAc, for example the conjugates shown as Conj 1, 2, 3 or 4 or 1a, 2a, 3a or 4a.

19. An antisense oligonucleotide conjugate according to embodiment 18, wherein the conjugate moiety (C) contains Conj 2a.

20. An antisense oligonucleotide conjugate according to any one of embodiments 1-19, selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

21. An antisense oligonucleotide conjugate according to any one of embodiments 1-20, where

- 39 043736 antisense oligomer (A) is conjugated to a conjugate moiety (C) via a linker region located between the continuous sequence of the oligomer and the conjugate moiety (B and/or Y).

22. An antisense oligonucleotide conjugate according to embodiment 21, wherein the linker is selected from the group consisting of aminoalkyl linkers, nucleotide phosphate linkers and peptide linkers.

23. An antisense oligonucleotide conjugate according to embodiment 21 or 22, wherein the linker is selected from C ₆ -C ₁₂ aminoalkyl groups.

24. An antisense oligonucleotide conjugate according to embodiment 21 or 22, wherein the linker is a biocleavable nucleotide phosphate linker containing from 1 to 6 nucleotides.

25. An antisense oligonucleotide conjugate according to any one of embodiments 21-24, wherein the linker (B) is a phosphodiester nucleotide linkage containing one or more contiguous DNA nucleotides linked by a phosphodiester linkage, for example, 1, 2, 3, 4, 5 or 6 contiguous DNA nucleotides linked by a phosphodiester bond that are adjacent to the 5' or 3' end of a contiguous oligomer sequence and that may or may not form complementary base pairings with the PCSK9 target sequence.

26. An antisense oligonucleotide conjugate according to embodiment 24 or 25, wherein the phosphodiester nucleotide linkage (or biocleavable linker) contains 1, 2 or 3 DNA nucleotides linked by a phosphodiester linkage, e.g. two DNA nucleotides linked by a phosphodiester linkage, e.g. a 5' CA 3' dinucleotide .

27. Oligomer of 12-22 nucleotides in length, which contains either

a) a contiguous sequence of 16 nucleotides complementary to the corresponding length of SEQ ID NO: 31, or

b) a continuous sequence of 10-16 nucleotides, complementary to the corresponding length of SEQ ID NO: 33, or 34, or 45.

28. An oligomer according to embodiment 27, which contains a contiguous sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29 and 44.

29. The oligomer according to any one of embodiments 27 or 28, wherein the oligomer is directed to PCSK9.

30. An oligomer according to any one of embodiments 27-29, wherein the contiguous sequence comprises affinity-enhancing nucleotide analogues.

31. The oligomer according to embodiment 30, wherein the nucleotide analogues are sugar-modified nucleotides, such as sugar-modified nucleotides, independently or dependently selected from the group consisting of locked nucleic acid (LNA) units, 2'-O-alkyl-RNA units , 2'-OMe-RNA units, 2'-amino-DNA units and 2'-fluoro-DNA units.

32. An oligomer according to embodiment 30 or 31, wherein the nucleotide analogs include or are locked nucleic acid (LNA) units.

33. The oligomer according to any one of embodiments 30-32, which is a gapmer, such as a gated nucleic acid gapmer oligonucleotide.

34. An oligonucleotide conjugate according to embodiment 33, wherein the gapmer comprises a wing segment on each side (5' and 3') of 2-4 nucleotide analogs, preferably LNA analogs.

35. An oligonucleotide conjugate according to embodiment 32 or 33, wherein the gapmer construct is selected from the group consisting of 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-2, 2 -8-4, 2-9-2, 2-9-3, 3-9-2, 3-9-3, 3-9-4, 4-9-3, 2-10-2, 2-10 -3, 3-10-2, 3-10-3, 3-10-4, 4-10-3, 2-11-2, 2-11-3, 3-11-2, 3-11-3 , 3-11-4, 4-11-3 and 4-11-4.

36. An oligonucleotide conjugate according to any one of embodiments 33-35, wherein the gapmer construct is selected from the group consisting of 2-8-3, 3-8-3, 3-9-4, 3-10-3, 2-11- 2, 2-11-3 and 3-11-2.

37. An oligomer according to any one of embodiments 27-36, wherein the oligomer comprises a contiguous sequence of 13, 14, 15 or 16 nucleotides.

38. The oligomer according to any one of embodiments 27-37, wherein the oligomer contains one or more nucleoside linkages selected from the group consisting of phosphorothioate, phosphorodithioate and boranophosphate.

39. The oligomer according to any one of embodiments 27-38, wherein the oligomer contains or consists of phosphorothioate nucleoside linkages.

40. An oligomer according to any one of embodiments 27-39, comprising a contiguous sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7 and 8.

41. A pharmaceutical composition comprising an oligomer or antisense oligonucleotide conjugate according to any one of embodiments 1-40 and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

42. An oligomer or antisense oligonucleotide conjugate or pharmaceutical composition according to any one of embodiments 1-40 for use as a medicament for the treatment of, for example, hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, eg accumulation of functional mutations in PCSK9, HDL/LDL imbalance

- 40 043736 cholesterol, dyslipidemias, for example familial hyperlipidemia (FH) or familial hypercholesterolemia (FHC), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD) and coronary heart disease (CHD).

43. Use of an oligomer or antisense oligonucleotide conjugate or pharmaceutical composition according to any one of embodiments 1-40 for the preparation of a medicament for the treatment of hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, for example accumulation of functional mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias such as familial hyperlipidemia (FH) or familial hypercholesterolemia (FHC), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD) and coronary artery disease (CHD).

44. A method of treating hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, such as the accumulation of functional mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias, such as familial hyperlipidemia (FH), or familial hypercholesterolemia (FCH), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary artery disease (CHD), wherein the method includes administering an effective amount of an oligomer conjugate or antisense oligonucleotide or a pharmaceutical composition according to any one of embodiments 1-40 a patient suffering or likely to suffer from hypercholesterolemia or a related disorder.

45. A method of inhibiting PCSK9 in vivo or in vitro in a cell expressing PCSK9, wherein the method comprises introducing into the cell an oligomer or antisense oligonucleotide conjugate or a pharmaceutical composition according to any one of embodiments 1-40 so as to inhibit PCSK9 in that cell.

Examples

Oligonucleotides were synthesized on uridine universal supports using the phosphoramidite method on an Expedite 8900/MOSS (Multiple Oligonucleotide Synthesis System) or Oligomaker 48 synthesizer on a synthesis scale of 4 or 1 μmol, respectively. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using aqueous ammonia for 5-16 hours at 60°C. Oligonucleotides were purified by reverse phase HPLC (RP-HPLC) or solid phase extractions and characterized by ultra-performance liquid chromatography (UHPLC) and molecular weight was further confirmed by electrospray ionization mass spectrometry (ESI-MS). See below for more details.

Oligonucleotide elongation.

Combination of β-cyanoethyl phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, ZNK-5methyl-C(Br), 3HK-A(Bz), ZNA- G(dmf), ZNA-T or C6-SS-C6 linker) is performed using a solution of 0.1 M 5'-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as an activator. For the final cycle, commercially available C ₆ -linked cholesterol with phosphoramidite was used at 0.1 M in DCM. Incorporation of thiol groups to introduce phosphorothioate bonds is accomplished by using xanthan hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphodiester bonds are introduced using 0.02 M iodine in a 7:2:1 THF/pyridine/water mixture. The remaining reagents are reagents typically used for oligonucleotide synthesis. For conjugation after solid-phase synthesis, a commercially available C ₆ -amino linker phosphoramidite is used in the last cycle of solid-phase synthesis, and after removal of the protection and cleavage from the solid support, the amino-linked unprotected oligonucleotide is isolated. Conjugates are introduced by activation of the functional group using standard synthetic methods.

Purification by RP-HPLC.

The crude compounds were purified using preparative RP-HPLC on a Phenomenex Jupiter C18 10 μm, 150 x 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile were used as buffers at a flow rate of 5 ml/min. The collected fractions were lyophilized to typically obtain the purified compound as a white solid.

Abbreviations.

DCI: 4,5-dicyanoimidazole;

DCM: dichloromethane;

DMF: dimethylformamide;

DMT: 4,4'-dimethoxytrityl;

THF: tetrahydrofuran;

Bz: benzoyl;

Ibu: isobutyryl;

RP-HPLC: Reverse phase high performance liquid chromatography.

- 41 043736

The compounds were synthesized as shown in table. 1 and also illustrated in the figures.

Example 1: Discovery of a new PCSK9 target motif.

521 antisense oligonucleotides against PCSK9 were designed and synthesized, all of which contained three closed nucleic acids flanking ten DNAs, i.e. a 16-mer LNA gapmer construct, where the oligonucleotide is specific to human and primate PCSK9. The human 15PC3 cell line was incubated for three days with either mock-modified oligonucleotides or locked nucleic acid-modified oligonucleotides targeting human PCSK9 at a concentration of 0.3 μM. Each anti-PCSK9 oligonucleotide was tested in three independent experiments. PCSK9 mRNA levels were quantified from extracted RNA using real-time PCR as described and are presented normalized to β-actin mRNA and relative to the mean levels in the twelve mock-treated samples in Fig. 8, where a subset of the most effective molecules is discussed in detail in FIG. 9.

Example 2: In vitro mRNA knockdown.

The human 15PC3 cell line was incubated for 3 days with either mock-modified oligonucleotides or locked nucleic acid-modified oligonucleotides of SEQ ID 1-8 targeting human PCSK9 at concentrations of 0.0012, 0.06, 0.3 and 1.5 µM. PCSK9 mRNA levels were quantified from extracted RNA using real-time PCR as described and are presented relative to the average levels in the four mock-treated samples in Fig. 10. For each oligonucleotide, the efficiency, defined as the half maximum effective concentration (EC50), was determined by least squares fitting the Hill equation in two-parameter logistic form with a fixed lower limit of 0% and a fixed upper limit of 100% as EC50=estimate±standard deviation.

Example 3 In Vivo ALT Levels.

Four female NMRI mice, four weeks old (Taconic, Denmark), weighing approximately 20 g on arrival, were injected intravenously with either saline or cholesterol-conjugated locked nucleic acid modified oligonucleotides of the sequences SEQ ID 9-16 targeting on human PCSK9 at doses of 7.5 and 15 mg/kg. Mice were sacrificed 7 days after administration and serum alanine aminotransferase (ALT) levels were determined using an enzymatic assay (Horiba ABX Diagnostics). For each experimental group of five mice, means and standard deviations were calculated and presented in Fig. 11 relative to average levels in saline-treated mice. Elevations of ALT were noted at both concentrations for some, but not all, cholesterol-conjugated molecules. Several of the compounds, such as SEQ ID NOs: 9 and 10, did not clinically significantly increase ALT in mice even when cholesterol was used as a conjugate to enhance hepatic uptake of the compounds.

Example 4: Non-human primate study.

The primary objective of this study was to examine selected lipid markers 7 weeks after a single bolus injection of anti-PCSK9 ZNA compounds in cynomolgus monkeys and to evaluate the potential toxicity of the compounds in monkeys. The compounds used in this study had the sequences SEQ ID NO: 10 13, 18, 19, 20 and 21, prepared in sterile saline (0.9%) at an initial concentration of 0.625 and 2.5 mg/ml.

Male monkeys were used and were at least 24 months old, provided with free access to running water and distributed 180 g of MVVM(E) SQC SHORT increased diet (Dietex France, SDS, Saint-Gratien, France) per animal per day. The total amount of food distributed into each cage is calculated in accordance with the number of animals in the cage on a given day. In addition, each animal was given fruit or vegetables daily. Animals were acclimatized to study conditions for a period of at least 14 days prior to the start of the experimental treatment period. During this period, preliminary studies were carried out. Animals were dosed intravenously (i.v.) at a single dose of 0.25, 1.0 or 2.5 mg/kg (SEQ ID NOs: 10, 13, 18 and 21) or at a single dose of 1.0 or 2.5 mg/kg (SEQ ID NO: 19 and 20). The dose volume was 0.4 ml/kg. 2 animals per group were used.

Drug doses were administered once on day 1. Animals were observed for a period of 7 weeks after treatment and were removed from the study on day 51. Day 1 corresponds to the first day of the experimental period. Clinical observations, body weight and food intake (per group) are recorded before and during the study.

Blood samples and tests were collected at the following time points.

- 42 043736

24 hours of research Options -8 RCP, L, Apo-B, PCSK9*, OA -1 L, Apo-B, PCSK9*, RK, OA 1 Dosing 4 LSB, L, Apo-B, PCSK9*, OA 8 LSB, L, Apo-B, PCSK9*, RK, OA 15 RCP, L, Apo-B, PCSK9* RK, OA 22 LSB, L, Apo-B, PCSK9* RK, OA 29 L, Apo-B, PCSK9* RK, OA 36 LSB, L, Apo-B, PCSK9* RK, OA 43 L, RK, Apo-B, PCSK9* RK, OA 50 RCP, L, Apo-B, PCSK9* RK, OA

RCP (routine clinical pathology): standard clinical pathology.

LSB (liver safety biochemistry): biochemical safety indicators for the liver.

PK: pharmacokinetics.

OA (other analysis): other analyses.

L (Lipids): lipids.

In the cases indicated below, the following parameters were determined for all surviving animals.

Full biochemical panel (full list below) - on days -8, 15 and 50.

Liver safety (aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin and gamma-glutamyltransferase (GGT) only) - on days 4, 8, 22 and 36.

Lipidogram (total cholesterol, HDL-C, LDL-C and triglycerides) and Apo-B only - on days -1, 4, 8, 22, 29, 36 and 43.

Blood (approximately 1.0 ml) was collected in lithium heparin tubes (using an ADVIA 1650 blood chemistry analyzer): Apo-B, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea , creatinine, total bilirubin, total cholesterol, triglycerides, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine kinase, gamma-glutamyltransferase (GGT), lactate dehydrogenase, total protein, albumin, albumin/globulin ratio.

PCSK9 Blood Assay: Blood samples for PCSK9 assay were collected on days -8, -1, 4, 8, 15, 22, 29, 36, 43 and 50. Venous blood (approximately 2 ml) was collected from the corresponding vein of each animal into a tube. Serum Separating Tube (SST) and allowed to clot for at least 60 ± 30 min at room temperature. Blood was centrifuged at 1000 g for 10 min under refrigerated conditions (set to maintain +4°C). Serum was transferred into 3 individual tubes and stored at -80°C until analyzed at CitoxLAB, France, using an enzyme-linked immunosorbent assay (ELISA) method (Circulex Human PCKS9 ELISA kit, CY-8079, validated for cynomolgus monkey samples).

Other tests. WO 2011009697 proposes methods for the following analysis: analysis of PCSK9 mRNA using quantitative PCR (qPCR). Other analysis included PCSK9 protein ELISA, serum Lp(a) ELISA (Mercodia No. 10-1106-01), tissue and plasma oligonucleotide analysis (drug content), sample extraction, standard and quality control samples, determination oligonucleotide content using ELISA.

Data for connections targeting PCSK9 is shown in the following table.

- 43 043736

Values for dose 2.5 mg/kg Max, PCSK9 exposure (data are percentage of pre-dose value) Max XSTD exposure (data are percentage of pre-dose value) Compound SEQ ID PCSK9 protein day 4 (percentage of pre-dosing value) Protein PCSK9 day 29 (percentage of value before dosing) 10 86 71.5 69% (d15) 87% (d29) 13 81 71 71% (d29) 84% (d22) 18 57 42 42% (d29) 71% (d15) 21 80.5 56 55% (d29) 84% (d15) 20 51 53 48% (d4) 94% (D8) 19 55 60 55% (d4) 89% (D4)

There was no evidence of hepatotoxicity or nephrotoxicity with compounds targeting PCSK9. Of note, PCSK9-GalNAc compounds resulted in a rapid and highly efficient down-regulation of PCSK9 that was maintained over an extended period of time (the entire duration of the study), illustrating that GalNAc-conjugated compounds are more effective, both in terms of rapid initial knockdown, and over an extended period, indicating that they can be dosed relatively infrequently and at a lower dose, both compared to unconjugated parent compounds and compared to compounds prepared using alternative conjugation technology, such as conjugation with cholesterol. SEQ ID NO: 18 resulted in rapid and consistent down-regulation of PCSK9 and HDL-X throughout the study (observed on day 34 at the 2.5 mg/kg dose with significant down-regulation of PCSK9 observed at 48 days after single dose 2. 5 mg/kg, where plasma PCSK9 protein levels were 71% of pre-dose levels).

Example 5: Assessment of liver and kidney toxicity in rats.

The compounds of the invention can be assessed for their toxicity profile in rodents, for example mice or rats. The protocol below can be used. Male Wistar Han Crl:WI(Han) rats approximately 8 weeks old were used. At this age, males weighed approximately 250 g. All animals had free access to pelleted balanced food SSNIFF R/M-H (SSNIFF Spezialdiaten GmbH, Soest, Germany) and to running water (filtered through a 0.22 μm filter) contained in bottles. Dose levels of 10 and 40 mg/kg/dose (subcutaneous administration) were used and dosed on days 1 and 8. Animals were euthanized on day 15. Urine and blood samples were collected on days 7 and 14. Clinical pathology examination was performed on day 14. Body weight is determined before the study, on the first day of administration and 1 week before the autopsy. Food intake per group was determined daily. Blood samples were taken from the tail vein after 6 h of fasting. The following analysis of blood samples was carried out: red blood cell count, mean red blood cell volume, sedimented red blood cell volume, hemoglobin, mean hemoglobin concentration in the cell, platelet count, white blood cell count, differential white blood cell count with cell morphology, reticulocyte count, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, albumin/globulin ratio. Urinalysis was performed: α-glutathione-S-transferase (GST), β-2 microglobulin, calbindin, clusterin, cystatin C, KIM-1, osteopontin, tissue inhibitor of metalloproteinases (TIMP; Tissue Inhibitor of Metalloproteinase-1), endothelial growth factor blood vessels (VEGF; vascular endothelial growth factor) and neutrophil gelatinase-associated lipocalin (NGAL; neutrophil gelatinase-associated lipocalin). Seven analytes (calbindin, clusterin, GST-α, KIM-1, osteopontin, TIMP-1, VEGF) were quantified in panel 1 (Rat Renal Toxicity Panel 1 on MILLIPLEX® MAP Magnetic Microcarriers, RKTX1MAG-37K). Three analytes (P-2 microglobulin, cystatin C, lipocalin-2/NGAL) were quantified in Panel 2 (Rat Renal Toxicity Panel 2 on Magnetic Microcarriers

- 44 043736

MILLIPLEX® MAP, RKTX2MAG-37K). The assay for determining the concentrations of these biomarkers in rat urine was based on Luminex xMAP® technology. Microcarriers coated with antibodies against a-GST/e-2 microglobulin/calbindin/clusterin/cystacin C/KIM-1/osteopontin/TIMP-1/VEGF/NGAL were color coded with two different fluorescent dyes. The following parameters were determined (urine using ADVIA 1650): urine protein, urine creatinine. Quantitative parameters: volume, pH (using 10-Multistix SG test strips/Clinitek 500 urine analyzer), specific gravity (using a refractometer). Semi-quantitative parameters (using 10-Multistix SG test strips/Clinitek 500 urine analyzer): proteins, glucose, ketones, bilirubin, nitriles, blood, urobilinogen, sediment cytology (via microscopic examination). Qualitative parameters: appearance, color. After killing, body weight and the weight of the kidneys, liver and spleen are determined and the ratio of body weight to organ weight is calculated. Kidney and liver samples were collected and either frozen or stored in formaldehyde. Microscopic analysis was performed. Data for Kim-1 expression are shown in FIG. 15, where all molecules except SEQ ID NO: 4 are demonstrated to have lower urinary kim-1 signal than SEQ ID NO: 1, demonstrating lower renal impairment compared to the original and previously characterized unconjugated molecule.

Example 6: Analysis of cleavable linkers.

FAM-labeled antisense oligomers (ASOs) with various DNA/PO linkers were digested in vitro, either in S1 nuclease extract (table below), liver or kidney homogenates, or serum. _______________________________________________

No. Seq (5’-3’) Cleavable linker (B) Conjugate (C) 35 GCattggtatTCA ZPO-DNA (5’tca3’) FAM 36 GCattggtatTCA 2PO-DNA (5’caZ’) FAM 37 GCattggtatTCA 1PO-DNA (5’aZ’) FAM 38 GCattggtatTCA ZRO-DNA (5’dasZ’) FAM 39 GCattggtatTCA No FAM

Capital letters represent LNA nucleosides (such as LNA beta-D-hydroxy), lowercase letters represent DNA nucleosides. The subscript s represents phosphorothioate internucleoside bonds. LNA cytosines are not necessarily 5-methyl cytosine. The FAM conjugate moiety is shown in FIG. 6 and the molecules are shown in FIG. 7.

FAM-labeled 100 μM ASOs with various DNA/PO linkers were digested in vitro with S1 nuclease in nuclease buffer (60 U per 100 μl) for 20 and 120 min (A). Enzymatic activity was stopped by adding ethylenediaminetetraacetic acid (EDTA) in a buffer solution.

The solutions were then analyzed by AIE HPLC on a Dionex Ultimate 3000 using a Dionex DNApac p-100 column and a gradient ranging from 10 mM to 1 M sodium perchlorate at pH 7.5. The content of cleaved and uncleaved oligonucleotides was determined against a standard using both detectors, a fluorescence detector at 615 nm and a UV detector at 260 nm.

SEQ ID NO Linker sequence % splitting after 20 min with S1 % splitting after 120 min with S1 39 - 2 5 37 A 29.1 100 36 sa 40.8 100 35 tea 74.2 100 38 gac 22.9 BUT

Conclusion. The result of inclusion of the PO linkers (or the B region referred to herein) is the cleavage of the conjugate (or C group), and both parameters from the length and/or sequence of the linker composition can be used to modulate the sensitivity to nucleolytic cleavage of the B region. DNA linker sequence /PO may modulate the rate of cleavage, as observed after 20 min in the S1 nuclease extract. The choice of sequence for the B region (eg, for the DNA/PO linker) can thus also be used to modulate the level of cleavage in serum and in target tissue cells.

The exact amount of compound SEQ ID NO: 35 was added to liver and kidney homogenates to concentrations of 200 μg/g tissue. Liver and kidney samples collected from NMRI mice were homogenized in homogenization buffer (0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH). Homogenates were incubated for 24 h at 37° C., and then the homogenates were extracted with phenol - chloroform.The content of cleaved and uncleaved oligonucleotides in the extract from the liver and kidneys and from the serum was determined against the standard using the HPLC method described above.

- 45 043736

Seq ID Linker sequence % digestion after 24 h with liver homogenate % digestion after 24 h with kidney homogenate % splitting after 24 hours serum 35 tea 83 95 0

Conclusion. Incorporation of PO linkers (or the B region referred to herein) results in cleavage of the conjugate (or group C) in the liver or kidney homogenate, but not in the serum. The cleavage sensitivity in the assays shown in Example 6 can be used to determine whether a linker is biodegradable or physiologically labile. It should be noted that cleavage in the above assays refers to cleavage of the cleavable linker, and the oligomer or A region must remain functionally intact.

Example 7 Knockdown of PCSK9 mRNA with cholesterol conjugates in vivo PCSK9 - mouse-specific compounds. _____________________________________________________

No. Seq (5’-3’) (A) Cleavable linker (B) Conjugate (C) 40 GTctgtggaaGCG No No 41 GTctgtggaaGCG No Cholesterol 42 GTctgtggaaGCG 2PO-DNA (5’caZ’) Cholesterol 43 GTctgtggaaGCG 2PO-DNA (5’ct3’) Cholesterol

NMRI mice were injected with a single dose of saline, either 10 mg/kg unconjugated LNA antisense oligonucleotide (SEQ ID 40), or equivalent amounts of LNA antisense oligonucleotides conjugated to cholesterol with various linkers and sacrificed on days 1-10 according to Table 1. 3.

RNA was isolated from liver and kidney and subjected to qPCR with PCSK9-specific primers and a probe to analyze PCSK9 mRNA knockdown. The results are shown in Fig. 14.

Conclusions. Cholesterol conjugated to an antisense LNA oligonucleotide to PCSK9 with a 2-DNA phosphodiester backbone linker (SEQ ID NO: 42 and SEQ ID NO: 43) showed increased knockdown of PCSK9 in the liver (Fig. 14) compared to the unconjugated compound ( SEQ ID NO: 40), and also compared to cholesterol conjugates with a stable linker (SEQ ID NO: 41).

Materials and methods.

Table 3

Experimental design

Part Group No. Animal No. Number of animals Line/sex/feeding of the animal Dose level of compound per day Conc, at a dose volume of 10 ml/kg Route of administration Daily dosing Body weight, day Day of killing A 1 1-3 3 NMRI/DSukhoi feed Physiological solution - IV 0 0, 1 1 2 4-6 3 NMRI/2/Dry food SEQ ID NO 40 10 mg/kg 1 mg/ml IV 0 0, 1 1 3 7-9 3 NMRI/y/Dry food SEQ ID NO 41 equimolar 11.3 mg/kg 1.13 mg/ml IV 0 0, 1 1 5 13-15 3 NMRI/5/Dry feed SEQ ID NO 42 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0, 1 1 6 16-18 3 NMRI/DSukhoi feed SEQ ID NO 43 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0, 1 1

- 46 043736

V 7 19-21 3 NMRI/^/Sukhoi feed Physiological solution - IV 0 0, 3 3 8 22-24 3 NMRI/^/Sukhoi feed SEQ ID NO 40 10mg/kg 1 mg/ml IV 0 0, 3 3 9 25-27 3 NMRI/^/Sukhoi feed SEQ ID NO 41 equimolar 11.3 mg/kg 1.13 mg/ml IV 0 0, 3 3 eleven 31-33 3 NMRI/^/Sukhoi feed SEQ ID NO 42 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0, 3 3 12 34-36 3 NMRI/^/Sukhoi feed SEQ ID NO 43 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0, 3 3 With 13 37-39 3 NMRI/^/Sukhoi feed Physiological solution - IV 0 0.7 7 14 40-42 3 NMRI/^/Sukhoi feed SEQ ID NO 40 10 mg/kg 1 mg/ml IV 0 0.7 7 15 43-45 3 NMRI/^/Sukhoi feed SEQ ID NO 41 equimolar 11.3 mg/kg 1.13 mg/ml IV 0 0.7 7 17 49-51 3 NMRI/^/Sukhoi feed SEQ ID NO 42 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0.7 7 18 52-54 3 NMRI/^/Sukhoi feed SEQ ID NO 43 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0.7 7 D 19 55-57 3 NMRI/^/Sukhoi feed Physiological solution - IV 0 0, 7, 10 10 20 58-60 3 NMRI/^/Sukhoi feed SEQ ID NO 40 10 mg/kg 1 mg/ml IV 0 0, 7, 10 10 21 61-63 3 NMRI/^/Dry food SEQ ID NO 41 equimolar 11.3 mg/kg 1.13 mg/ml IV 0 0, 7, 10 10 24 70-72 3 NMRI/^/Dry food SEQ ID NO 42 equimolar 12.7 mg/kg 1.27 mg/ml IV 0 0, 7, 10 10 A 25 73-75 3 NMRI/^/Sukhoi feed Physiological solution - IV 0 0, 1 1

Dose administration. Female NMRI animals, approximately 20 g on arrival, were dosed with 10 ml per kg body weight (BW) (based on bodyweight on day 0) intravenously (i.v.) of compound included in saline or saline alone as appropriate. with the table above.

Taking liver and kidney tissue samples. Animals were anesthetized with 70% CO ₂ -30% O ₂ and sacrificed by cervical dislocation according to the table. Half of the liver lobe and one kidney were minced and immersed in RNAIater RNA stabilization solution.

Total RNA was extracted from a maximum of 10 mg of tissue homogenized by ball milling in the presence of MagNA Pure LC Tissue RNA Isolation Buffer (Roche, cat. no. 03604721001), using the MagNa Pure 96 Large Volume Cellular RNA Isolation Kit (Roche, no. according to catalog 5467535001) according to the manufacturer's instructions. First-strand synthesis was performed using reverse transcriptase reagents from Ambion according to the manufacturer's instructions.

For each sample, 0.5 μM total RNA was brought to (10.8 μL) RNase-free H2O and mixed with 2 μL random decamers (50 μM) and 4 μL dNTP mixture (2.5 mM each dNTP) and heated to 70°C for 3 min, after which the samples were quickly cooled on ice. To each sample, 2 μl of 10x reverse transcription (RT) buffer, 1 μl of MMLV reverse transcriptase (100 units/μl) and 0.25 μl of RNase inhibitor (10 units/μl) were added, followed by incubation at 42°C for 60 min, thermal inactivation of the enzyme at 95°C for 10 min, and then the sample was cooled to 4°C. cDNA samples were diluted 1:5 and subjected to RT-qPCR using the basic universal rapid mixture.

- 47 043736

Taqman 2x PCR (Applied Biosystems Cat# 4364103) and Taqman Gene Expression Assay (mPCSK9, Mn00463738_m1, and mActin #4352341E) following the manufacturers' protocol and reactions were performed on an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7) in fast mode.

Example 8: Non-Human Primate Study; multiple subcutaneous (s.c.) injections.

The purpose of this non-human primate study was to evaluate the efficacy and safety of compounds targeting PCSK9 under repeated dosing conditions when the compounds were administered by subcutaneous injection (s.c.). The compounds used in this study had the sequences SEQ ID NO: 2, 3, 18 and 19, prepared in sterile saline (0.9%) at an initial concentration of 0.625 and 2.5 mg/ml.

Female cynomolgus monkeys of at least 24 months of age were used and provided with free access to running water and distributed 180 g of the increased MVVM(E) SQC SHORT diet (Dietex France, SDS, Saint-Gratien, France) per animal per day. In addition, each animal was given fruit or vegetables daily. Animals were acclimatized to study conditions for a period of at least 14 days prior to the start of the experimental treatment period. During this period, preliminary studies were carried out. Animals were dosed with s.c. once a week for four weeks at a dose of 0.5 mg/kg (SEQ ID NOs: 2, 3, 18 and 19) or 1.5 mg/kg/injection (SEQ ID NOs: 18 and 19), for a total of four injections over a period of four weeks. The dose volume was 0.4 ml/kg/injection. Six animals per group were used. After the fourth and final dose, animals were observed for a week, after which half of the animals were sacrificed to study hepatic apoB transcript regulation, lipid parameters, liver and kidney histology, and tissue distribution in the liver and kidney. Day 1 corresponds to the first day of the experimental period. Clinical observations, body weight and food intake (per group) were recorded before and during the study.

Blood and tissue samples were collected and analyzed at the following time points.

Day of study Parameters “76 L, Aro-B, OA

-5 EevUTSARo-'V, OA ' -Ϊ RC R' L, Aro-V' RK, O A

Dosing before dosing LSB, L, Apo-B, PK, OA

Dosing before dosing LSB, L, Aro-B, RK, OA

Dosing before dosing LSB, L, Aro-B, RK, OA

Dosing

RCP, RK, 0A + main autopsy (recovered animals) LSB, L, Apo-B, RK, OA (recovered animals) RCP, RK, Aro-B, RK, OA (recovered animals) LSB, L, Aro-B, RK, OA (recovered animals) LSB, L, Aro-B, RK, OA (recovered animals) LSB, L, Aro-B, RK, OA (recovered animals) LSB, L, Aro-B, RK, OA (recovered animals) animals) RCP, L, Aro-B, RK, OA + necropsy recovery

RCP: standard clinical pathology.

LSB: biochemical liver safety indicators.

PK: pharmacokinetics.

OA: other tests.

L: lipids.

Blood (approximately 1.0 ml) was collected in lithium heparin tubes (using an ADVIA 1650 blood chemistry analyzer): Apo-B, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, HDL-X, LDL-X, urea , creatinine, total bilirubin, total cholesterol, triglycerides, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine kinase, gamma-glutamyltransferase (GGT), lactate dehydrogenase, total protein, albumin, albumin/globulin ratio.

Blood analysis. Blood samples for ApoB analysis were collected only from animals in groups 1-16 (i.e. animals treated with compounds directed against ApoB) on days -8, -1, 4, 8, 15, 22, 29, 36, 43 and 50 Venous blood (approximately 2 ml) was collected from the corresponding vein in each animal at

- 48 043736 serum separator tube (SST) and allowed to clot for at least

60±30 min at room temperature. Blood was centrifuged at 1000 g for 10 min under refrigerated conditions (set to maintain +4°C). Serum was transferred into 3 individual tubes and stored at -80°C until ApoB protein was analyzed by ELISA.

Another analysis. WO 2010142805 proposes methods for the following analysis: qPCR, ApoB mRNA analysis. Other tests include serum Lp(a) ELISA (Mercodia No. 10-1106-01), tissue and serum oligonucleotide (drug content), sample extraction, standard and quality control samples, oligonucleotide ELISA .

The intended pharmacological action of the anti-PCSK9 oligonucleotide is to lower LDL cholesterol by reducing circulating PCSK9 (serum PCSK9). Conjugated GalNAc molecules demonstrated enhanced potency compared to unconjugated molecules when both serum PCSK9 and LDL cholesterol were examined (Figures 16 and 17). In fig. 16 illustrates that four weekly injections of 0.5 mg/kg/injection of unconjugated SEQ ID NO: 2 had only minor effects on serum PCSK9 and LDL cholesterol, whereas GalNAc conjugates of the same LNA gapmer (SEQ ID 18) had a strong reduction effects on both serum PCSK9 and LDL cholesterol. The same relationship was observed when comparing data for multiple injections of SEQ ID NO: 3 and SEQ ID NO: 19 (Fig. 17): only minor effects of the unconjugated molecule and strong down-regulation of PCSK9 and serum LDL cholesterol by the corresponding GalNAc conjugate (SEQ ID NO : 19). It should be noted that the effects of SEQ IDs 18 and 19 on serum PCSK9 and LDL cholesterol were dose-dependent over long durations of action, with serum PCSK9 and LDL cholesterol levels below average baseline levels for at least seven weeks after the last injection (last injection on day 22, data are illustrated for the recovery period up to day 71).

The content of oligonucleotides in the liver and kidneys was analyzed one week after the last injection, i.e. on day 29 of the study. Oligonucleotide content was analyzed using hybridization ELISA (essentially as described in Lindholm et al., Mol Ther., 2012 Feb, 20(2): 376-81) using SEQ ID NO: 2 to generate a standard curve for samples from animals treated with SEQ ID NO: 2 and SEQ ID NO: 18, after ensuring that no change in results occurs if SEQ ID NO: 18 (conjugated) was used to prepare the standard curve. SEQ ID NO: 3 was used in the same way to obtain a standard curve for SEQ ID NO: 3 and SEQ ID NO: 19 after checking that there was no difference in the results using SEQ ID NO: 19 to obtain a standard curve for the ELISA analysis of these samples.___________________________________________

Oligonucleotide content in tissues one week after the last injection Liver (µg oligonucleotide/g fresh tissue) Kidney (µg oligonucleotide/g fresh tissue) Liver/kidney ratio Average SD Average SD SEQIDNO2, 4x0.5 mg/kg 0.260 0.14 30.3 4.8 0.008 SEQ ID NO 18, 4x0.5 mg/kg 3.57 0.61 11.5 2.5 0.310 SEQ ID NO 18, 4x1.5 mg/kg 18.8 1.7 26.8 6.6 0.701 SEQIDNO3, 4x0.5 mg/kg 0.149 0.059 38.2 0.72 0.004 SEQ ID NO 19, 4x0.5 mg/kg 2.72 0.69 16.3 1.5 0.167 SEQ ID NO 19, 4x1.5 mg/kg 12.2 3.44 41.2 6.5 0.296

As illustrated in the table above, conjugation of SEQ ID NO: 2 and SEQ ID NO: 3 resulted in higher liver/kidney ratios for the conjugated molecules (SEQ ID NO: 18 and SEQ ID 19) than for the corresponding unconjugated molecules through one week after the last injection, when animals were injected with s.c. once a week for four weeks. Given that evidence of tubulotoxicity has been demonstrated with other unconjugated anti-PCSK9 molecules (such as SEQ ID NO: 1, as illustrated in FIG. 15), and given that the liver is a target organ for anti-PCSK9 therapies , expect that the shift to a higher liver/kidney ratio will result in increased safety for the conjugated SEQ ID NOs: 18 and 19 compared to the unconjugated SEQ ID NOs: 2 and 3.

As illustrated in FIG. 16 and 18, SEQ ID NO: 18 and 19 were dosed at pharmacologically relevant levels. Clinical chemistry profiles of the same animals during the experimental and convalescent phases demonstrated no relevant increase in safety parameters in the liver or kidney.

Sequence listing < 110> Santaris Pharma A/S < 120> ANTISENSE OLIGOMERS AND THEIR CONJUGATES DIRECTED AT PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 (PCSK9) < 130> 1141WO < 160>46 < 170> PatentIn version 3.5 < 210>1 < 211>14 <212> DNA <213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

<221> misc_feature <222> (1) .. (14) <223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (12)..(14) < 223> LNA nucleosides, LNA C are 5-methyl C < 400>1 tgctacaaaa ccca14 < 210>2 < 211>16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides

- 50 043736 <220>

< 221> misc_feature < 222> (13)..(16) < 223> LNA nucleosides, LNA C are 5-methyl C < 400>2 aatgctacaa aaccca < 210>3 < 211>16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(3) < 223> LNA nucleosides <220>

<221> misc_feature <222> (14)..(16) <223> LNA nucleosides, LNA C are 5-methyl C <400>3 aatgctacaa aaccca <210>4 <211>15 <212> DNA <213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(15) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(2) <223> LNA nucleosides, LNA C is 5-methyl C <220>

<221> misc_feature <222> (14)..(15) <223> LNA nucleosides

- 51 043736 <400>4 gctgtgtgag cttgg <210>5 <211>16 <212> DNA <213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(2) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (14)..(16) <223> LNA nucleosides <400>5 tgctgtgtga gcttgg16 <210>6 <211>16 <212> DNA <213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

<221> misc_feature <222> (14)..(16) <223> LNA nucleosides <400>6 tgctgtgtga gcttgg16 <210>7 <211>16 <212> DNA <213> ARTIFICIAL

- 52 043736 <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides, LNA C are 5-methyl C < 400>7 tcctggtctg tgttcc16 < 210>8 < 211>16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (15)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

tcctggtctg tgttcc < 210> 9 < 211> 16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6

- 53 043736 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>9 catgctacaa aaccca16 < 210>10 < 211>18 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(5) < 223> LNA nucleosides <220>

<221> misc_feature <222> (15)..(18) <223> LNA nucleosides, LNA C is 5-methyl C <400>

caaatgctac aaaaccca

- 54 043736 <210> 11 <211> 18 <212> DNA <213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(5) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (16)..(18) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>11 caaatgctac aaaaccca <210>12 <211>17 <212> DNA <213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

<221> misc_feature < 222> (3)..(17)

- 55 043736 <223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(4) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (16)..(17) < 223> LNA nucleosides < 400>12 cagctgtgtg agcttgg17 < 210>13 < 211>18 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(4) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (16)..(18) < 223> LNA nucleosides <400>13 catgctgtgt gagcttgg18 <210>14 <211>18 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

<221> misc_feature

- 56 043736 <222> (1) .. (1) <223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (16)..(18) < 223> LNA nucleosides < 400>14 catgctgtgt gagcttgg18 < 210>15 < 211>18 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (16)..(18) < 223> LNA nucleosides, LNA C is 5-methyl C <400> 15

- 57 043736 catcctggtc tgtgttcc <210> 16 <211> 18 <212> DNA <213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol^ <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(18) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

<221> misc_feature <222> (17) .. (18) <223> LNA nucleosides, LNA C is 5-methyl C <400>16 catcctggtc tgtgttcc18 <210>17 <211>14 <212>DNA <213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(14) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(3) < 223> LNA nucleosides, LNA C is 5-methyl C

- 58 043736 <220>

<221> misc_feature <222> (12) .. (14) <223> LNA nucleosides, LNA C is 5-methyl C <400>17 tgctacaaaa ccca14 <210>18 <211>16 <212>DNA <213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(3) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (13)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

aatgctacaa aaccca <210> 19 <211> 16 <212> DNA <213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature

- 59 043736 <222> (1) .. (3) <223> LNA nucleosides <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>19 aatgctacaa aaccca16 < 210>20 < 211>15 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(15) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(2) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(15) < 223> LNA nucleosides < 400>20 gctgtgtgag cttgg15 < 210>21 < 211>16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(2) < 223> LNA nucleosides

- 60 043736 <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides < 400>21 tgctgtgtga gcttgg16 < 210>22 < 211>16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides <400>

tgctgtgtga gcttgg < 210> 23 < 211> 16 < 212> DNA < 213> ARTIFICIAL <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a <220>

<221> misc_feature

- 61 043736 <222> (1) .. (16) <223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

<221> misc_feature <222> (14)..(16) <223> LNA nucleosides, LNA C is 5-methyl C <400>23 tcctggtctg tgttcc16 <210>24 <211>16 <212>DNA <213> artificial <220>

<223>LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> GalNAc Conj2a < 220>

< 221> misc_feature < 222> (1)..(16) < 223> phosphorothioate internucleoside bonds < 220>

< 221> misc_feature < 222> (1)..(3) < 223> LNA nucleosides, LNA C is 5-methyl C < 220>

< 221> misc_feature < 222> (15)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

tcctggtctg tgttcc <210> 25 <211> 14 <212> DNA <213> artificial sequence <220>

<223> nucleobase motif <400> 25 tgctacaaaa ccca 14 <210> 26

- 62 043736 <211> 16 < 212> DNA < 213> ARTIFICIAL <220>

<223> nucleic base motif <400> 26 aatgctacaa aaccca 16 <210> 27 <211> 15 <212> DNA <213> ARTIFICIAL <220>

<223> nucleobase motif <400> 27 gctgtgtgag cttgg <210> 28 <211> 16 <212> DNA <213> ARTIFICIAL <220>

<223> nucleic base motif <400> 28 tgctgtgtga gcttgg <210> 29 <211> 16 <212> DNA <213> ARTIFICIAL <220>

<223> nucleobase motif <400> 29 tcctggtctg tgttcc <210>30 <211>14 <212> RNA <213> Homo sapiens <400>30 uggguuuugu agca <210>31 <211>16 <212> RNA <213> Homo sapiens <400>31 uggguuuugu agcauu

- 63 043736 <210>32 <211>15 <212> RNA <213> Homo sapiens <400>32 ccaagcucac aagc15 <210>33 <211>16 <212> RNA <213> Homo sapiens <400>

ccaagcucac acagca < 210>34 < 211>16 < 212> RNA < 213> Homo sapiens < 400>34 ggaacacaga ccadga16 < 210>35 < 211>16 < 212> DNA < 213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(3) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(1) < 223> FAM conjugate <220>

< 221> misc_feature < 222> (4)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (4)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

- 64 043736 tcagcattgg tattca <210> 36 <211> 15 <212> DNA <213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(1) < 223> FAM conjugate <220>

< 221> misc_feature < 222> (3)..(15) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (3)..(4) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (13)..(15) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>36 cagcattggt attca15 < 210>37 < 211>14 < 212> DNA < 213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(1) < 223> FAM conjugate <220>

< 221> misc_feature < 222> (2)..(14)

- 65 043736 <223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (2)..(3) <223> LNA nucleosides, LNA C is 5-methyl C <220>

<221> misc_feature <222> (12) .. (14) <223> LNA nucleosides, LNA C is 5-methyl C <400>37 agcattggta ttca14 <210>38 <211>16 <212>DNA <213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(3) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(1) < 223> FAM conjugate <220>

< 221> misc_feature < 222> (4)..(16) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (4)..(5) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (14)..(16) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>38 gacgcattgg tattca16 < 210>39 < 211>13 < 212> DNA < 213> artificial <220>

<223>LNA conjugate antisense gapmer oligonucleotide

- 66 043736 <220>

< 221> misc_feature < 222> (1)..(13) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (1)..(2) <223> LNA nucleosides, LNA C is 5-methyl C <220>

< 221> misc_feature < 222> (1)..(1) < 223> FAM conjugate <220>

< 221> misc_feature < 222> (11)..(13) < 223> LNA nucleosides, LNA C is 5-methyl C < 400>39 gcattggtat tca13 < 210>40 < 211>13 < 212> DNA < 213> artificial <220>

<223> LNA antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(13) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(2) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (11)..(13) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

gtctgtggaa gcg <210> 41 <211> 13 <212> DNA <213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

- 67 043736 <221> misc_feature <222> (1)..(1) <223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(13) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (1)..(2) < 223> LNA nucleosides <220>

<221> misc_feature <222> (11)..(13) <223> LNA nucleosides, LNA C is 5-methyl C <400>41 gtctgtggaa gcg13 <210>42 <211>15 <212>DNA <213> artificial <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(4) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (3)..(15) < 223> phosphorothioate internucleoside bonds <220>

< 221> misc_feature < 222> (13)..(15) < 223> LNA nucleosides, LNA C is 5-methyl C <400>

cagtctgtgg aagcg <210>

<211>

<212>

<213>

Artificial DNA

- 68 043736 <220>

<223> LNA conjugate antisense gapmer oligonucleotide <220>

< 221> misc_feature < 222> (1)..(1) < 223> cholesterol-C6 <220>

< 221> misc_feature < 222> (1)..(2) < 223> phosphodiester internucleoside bonds <220>

< 221> misc_feature < 222> (3)..(4) < 223> LNA nucleosides <220>

< 221> misc_feature < 222> (3)..(15) < 223> phosphorothioate internucleoside bonds <220>

<221> misc_feature <222> (13)..(15) <223> LNA nucleosides, LNA C is 5-methyl C <400>43 ctgtctgtgg aagcg15 <210>44 <211>13 <212> DNA <213> artificial <220>

<223> nucleobase motif <400>

gtctgtggaa gcg <210>45 <211>13 <212> RNA <213> Homo sapiens <400>45 cgcuuccaca gac13 <210>46 <211>3731 <212> DNA <213> homo sapiens <400>

gtccgatggg gctctggtgg cgtgatctgc gcgccccagg cgtcaagcac ccacacccta gaaggtttcc gcagcgacgt cgaggcgctc atggttgcag gcgggcgccg ccgttcagtt

120

- 69 043736

cagggtctga gcctggagga gtgagccagg cagtgagact ggctcgggcg ggccgggacg 180 cgtcgttgca gcagcggctc ccagctccca gccaggattc cgcgcgcccc ttcacgcgcc 240 ctgctcctga acttcagctc ctgcacagtc ctccccaccg caaggctcaa ggcgccgccg 300 gcgtggaccg cgcacggcct ctaggtctcc tcgccaggac agcaacctct cccctggccc 360 tcatggggcac cgtcagctcc aggcggtcct ggtggccgct gccactgctg ctgctgctgc 420 tgctgctcct gggtcccgcg ggcgcccgtg cgcaggagga cgaggacggc gactacgagg 480 agctggtgct agccttgcgt tccgaggagg acggcctggc cgaagcaccc gagcacggaa 540 ccacagccac cttccaccgc tgcgccaagg atccgtggag gttgcctggc acctacgtgg 600 tggtgctgaa ggagaggacc cacctctcgc agtcagagcg cactgcccgc cgcctgcagg 660 cccaggctgc ccgccgggga tacctcacca agatcctgca tgtcttccat ggccttcttc 720 ctggcttcct ggtgaagatg agtggcgacc tgctggagct ggccttgaag ttgccccatg 780 tcgactacat cgaggagac tcctctgtct ttgcccagag catcccgtgg aacctggagc 840 ggattacccc tccacggtac cggggcggatg aataccagcc ccccgacgga ggcagcctgg 900 tggaggtgta tctcctagac accagcatac agagtgacca ccgggaaatc gagggcaggg 960 tcatggtcac cgacttcgag aatgtgcccg aggagaggacgg gacccgcttc cacagacagg 1020 ccagcaagtg tgacagtcat ggcacccacc tggcaggggt ggtcagcggc cgggatgccg 1080 gcgtggccaa gggtgccagc atgcgcagcc tgcgcgtgct caactgccaa gggaagggca 1140 cggttagcgg caccctcata ggcctggagt ttattcggaa aagccagctg gtccagcctg 1200 tggggccact ggtggtgctg ctgcccctgg cggggtgggta cagccgcgtc ctcaacgccg 1260 cctgccagcg cctggcgagg gctggggtcg tgctggtcac cgctgccggc aacttccggg 1320 acgatgcctg cctctactcc ccagcctcag ctcccgaggt catcacagtt ggggccacca 1380 atgcccaaga ccagccggtg accctgggga ctttggggac caactttggc cgctgtgtgg 1440 acctctttgc cccaggggag gacatcattg gtgcctccag cgactgcagc acctgctttg 1500 tgtcacagag tgggacatca caggctgctg cccacgtggc tggcattgca gccatgatgc 1560 tgtctgccga gccggagctc accctggccg agttgaggca gagactgatch cacttctctg 1620 ccaaagatgt catcaatgag gcctggttcc ctgaggacca gcgggtactg acccccaacc 1680 tggtggccgc cctgcccccc agcacccatg gggcaggttg gcagctgttt tgcaggactg 1740 tatggtcagc acactcgggg cctacacgga tggccacagc cgtcgcccgc tgcgccccag 1800 atgaggagct gctgagctgc tccagtttct ccaggagtgg gaagcggcgg ggcgagcgca 1860 tggaggccca aggggggcaag ctggtctgcc gggcccacaa cgcttttgggg ggtgaggtg 1920 tctacgccat tgccaggtgc tgcctgctac cccaggccaa ctgcagcgtc cacacagctc 1980

- 70 043736

caccagctga ggccagcatg gggacccgtg tccactgcca ccaacagggc cagtcctca 2040 caggctgcag ctccactgg gaggtggagg accttggcac ccacaagccg cctgtgctga 2100 ggccacgagg tcagcccaac cagtgcgtgg gccacaggga ggccagcatc cacgcttcct 2160 gctgccatgc cccaggtctg gaatgcaaag tcaagggagca tggaatcccg gcccctcagg 2220 agcaggtgac cgtggcctgc gagggagct ggaccctgac tggctgcagt gccctccctg 2280 ggacctccca cgtcctgggg gcctacgccg tagacaacac gtgtgtagtc aggagccggg 2340 acgtcagcac tacaggcagc accagcgaag gggccgtgac agccgttgcc atctgctgcc 2400 ggagccggca cctggcgcag gcctcccagg agctccagtg acagccccat cccaggatgg 2460 gtgtctgggg agggtcaagg gctggggctg agctttaaaa tggttccgac ttgtccctct 2520 ctcagccctc catggcctgg cacgagggga tggggatgct tccgcctttc cggggctgct 2580 ggcctggccc ttgagtgggg cagcctcctt gcctggaact cactcactct gggtgcctcc 2640 tccccaggtg gaggtgccag gaagctccct ccctcactgt ggggcatttc accattcaaa 2700 caggtcgagc tgtgctcggg tgctgccagc tgctcccaat gtgccgatgt ccgtggggcag 2760 aatgactttt attgagctct tgttccgtgc caggcattca atcctcaggt ctccaccaag 2820 gaggcaggat tcttcccatg gataggggag ggggcggtag gggctgcagg gacaaacatc 2880 gttggggggt gagtgtgaaa ggtgctgatg gccctcatct ccagctaact gtggagaagc 2940 ccctggggggc tccctgatta atggaggctt agctttctgg atggcatcta gccagaggct 3000 ggagacaggt gcgcccctgg tggtcacagg ctgtgccttg gtttcctgag ccacctttac 3060 tctgctctat gccaggctgt gctagcaaca cccaaaggtg gcctgcgggg agccatcacc 3120 taggactgac tcggcagtgt gcagtggtgc atgcactgtc tcagccaacc cgctccacta 3180 cccggcaggg tacacattcg cacccctact tcacagga agaaacctgg aaccagagg 3240 ggcgtgcctg ccaagctcac acagcaggaa ctgagccaga aacgcagatt gggctggctc 3300 tgaagccaag cctcttctta cttcacccgg ctggggctcct catttttacg ggtaacagtg 3360 aggctgggaa ggggaacaca gaccaggaag ctcggtgagt gatggcagaa cgatgcctgc 3420 aggcatggaa ctttttccgt tatcaccag gcctgattca ctggcctggc ggagatgctt 3480 ctaaggcatg gtcggggggag aggggccaaca actngtccctc cttgagcacc agccccacc 3540 aagcaagcag acattattct tttgggtctg tcctctctgt tgccttttta cagccaactt 3600 ttctagacct gttttgcttt tgtaacttga agatatttat tctggggtttt gtagcatttt 3660 tattaatatg gtgacttttt aaaataaaaa caaacaaacg ttgtcctaac aaaaaaaaaa 3720 aaaaaaaaaa a 3731

-

Claims (50)

CLAIM 1. A compound consisting of an antisense oligomer, the length of which is 16-22 contiguous nucleotides, where the sequence of the antisense oligomer contains a continuous sequence of 16 nucleotides in length, 100% complementary to the sequence of SEQ ID NO: 31, and the antisense oligomer is a gapmer containing at least one locked nucleic acid (LNA) unit, wherein the antisense oligomer is directed to an mRNA encoding proprotein convertase subtilisin/kexin type 9 (PCSK9), and wherein said 16-22 contiguous nucleotides are 100% complementary to the mRNA encoding PCSK9. 2. The compound of claim 1, wherein the locked nucleic acid (LNA) is hydroxy-LNA, thio-LNA, amino-5-LNA, 5'-methyl-LNA, ENA, cET, cMOE, or a combination thereof. 3. A compound according to claim 1, wherein the LNA is a stereoisomer in the beta-O configuration or alpha-L configuration. 4. A compound according to claim 1, wherein the antisense oligomer contains at least one cET unit. 5. A compound according to claim 1, where the antisense oligomer contains 2, 3, 4, 5, 6 or 7 LNA units. 6. The compound of claim 5, wherein each LNA unit in the antisense oligomer is a stereoisomer in the same configuration. 7. The compound of claim 5, wherein each LNA unit in the antisense oligomer is a beta-O-oxy-LNA unit or each LNA unit in the antisense oligomer is an alpha-Loxy LNA unit. 8. A compound according to claim 1, wherein the antisense oligomer sequence contains at least one phosphorothioate, phosphorodithioate or boranophosphate internucleoside linkage. 9. The compound according to claim 8, where all internucleoside bonds are phosphorothioate. 10. The compound according to claim 8, where one or more internucleoside bonds contain a chiral center in the R-conformation and/or in the S-conformation. 11. The compound of claim 1, wherein the compound and the oligomer of SEQ ID NO: 31 can form a duplex with increased thermal stability compared to a corresponding duplex containing a corresponding antisense oligomer without the LNA. 12. An antisense oligonucleotide conjugate containing (i) an antisense oligomer comprising 16-22 contiguous nucleotides, wherein the sequence of the antisense oligomer contains a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31, wherein the antisense oligomer is a gapmer, containing at least one locked nucleic acid (LNA) unit, and wherein said 16-22 contiguous nucleotides are 100% complementary to the mRNA encoding PCSK9; and (ii) at least one conjugate moiety directed to an asialoglycoprotein receptor covalently attached to said antisense oligomer, directly or through a linker located between the contiguous sequence of the oligomer and said moiety, wherein the antisense nucleotide conjugate is directed to an mRNA encoding PCSK9. 13. The antisense oligonucleotide conjugate of claim 12, wherein the asialoglycoprotein receptor-targeted conjugate moiety comprises a monovalent, divalent, trivalent or tetravalent GalNAc cluster. 14. The antisense oligonucleotide conjugate of claim 13, wherein each GalNAc in the GalNAc cluster is attached to a branch point group via a spacer. 15. The antisense oligonucleotide conjugate of claim 14, wherein the branch point group comprises di-lysine. 16. The antisense oligonucleotide conjugate of claim 14, wherein the spacer includes a polyethylene glycol (PEG) spacer. 17. The antisense oligonucleotide conjugate of claim 12, wherein the linker includes a C ₆ -C ₁₂ aminoalkyl group or a biocleavable nucleotide phosphate linker containing from 1 to 6 nucleotides. 18. The antisense oligonucleotide conjugate of claim 13, wherein the trivalent GalNAc cluster contains Conj 1, Conj 2, Conj 1a or Conj 2a - 72 043736 Conj 2 Conjla Conj 2a. 19. The antisense oligonucleotide conjugate of claim 12, wherein said moiety is covalently attached to the antisense oligomer via a covalent bond. 20. Antisense oligonucleotide conjugate of the sequence SEQ ID NO: 18 or SEQ ID NO: 19 - 73 043736 Η ^Ν· G3INAC2 J o a Ί ^: с Λ.Γ \ ΛΛ l /О 1 _?Ч . _{( L} 1 j SEQ W 1" n GtilNAc2 _HS 3s l _L \Λ. ΟΝ -o - _;/ \O SEO II) 14 where the superscript ^L denotes the beta-D-hydroxy-ZNA unit; the superscript ^Me preceding the capital C indicates the 5-methylcytosine unit; the subscript _s denotes a phosphorothioate internucleoside linkage; And GalNAc2 is a conjugate group Conj 2a directed to the asialoglycoprotein receptor, and wherein the antisense nucleotide conjugate is directed to the mRNA encoding PCSK9. 21. A pharmaceutical composition having inhibitory activity against proprotein convertase subtilisin/kexin type 9 (PCSK9), containing a compound according to claim 1 or an antisense oligonucleotide conjugate according to claim 12 and a pharmaceutically acceptable diluent, carrier, salt or adjuvant. 22. A method of treating a disease or condition caused by abnormal levels of expression and/or activity of PCSK9 in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1 to 11, an antisense oligonucleotide conjugate of any of claims 12 to 20 or a pharmaceutical composition according to claim 21, wherein the abnormal levels and/or activity of PCSK9 are caused by a gene whose protein product associates with or interacts with PCSK9, wherein administration of the compound, antisense oligonucleotide conjugate or pharmaceutical composition reduces the level of PCSK9 in serum and/or reduces the level serum low-density lipoprotein (LDL) cholesterol. 23. The method of claim 22, wherein PCSK9 is (i) an allelic variant of PCSK9; (ii) PCSK9 gain-of-function mutant; or (iii) the expression product of a mutant PCSK9 gene. 24. The method of claim 22, wherein the disease or condition is selected from the group consisting of atherosclerosis, hypercholesterolemia, high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol imbalance, dyslipidemia, coronary artery disease (CAD), and coronary artery disease (IHD). 25. The method of claim 24, wherein the dyslipidemia is familial hyperlipidemia (familial combined hyperlipidemia (FCHL)) or acquired hyperlipidemia. 26. The method of claim 24, wherein the hypercholesterolemia is familial hypercholesterolemia or statin-resistant hypercholesterolemia. 27. The method of claim 22, further comprising administering a second therapeutic agent selected from the group consisting of a statin, a bile acid sequestrant resin, nicotinic acid, a fibric acid derivative, probucol, neomycin, dextrothyroxine, a plant stanol ester, a cholesterol absorption inhibitor , implitapide, a bile acid transporter inhibitor, a regulator of hepatic CYP7a (cholesterol 7-alpha hydroxylase), an estrogen replacement therapy and an anti-inflammatory agent. 28. The method of claim 27, wherein the statin is selected from the group consisting of lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin, rosuvastatin and fluvastatin. - 74 043736 29. The method of claim 22, wherein the compound, antisense oligonucleotide conjugate or pharmaceutical composition is administered intravenously or subcutaneously. 30. The method of claim 22, wherein the compound, antisense oligonucleotide conjugate or pharmaceutical composition is administered in single dose form or in multiple dose form. 31. A method of treating a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol imbalance, coronary artery disease (CAD), or coronary artery disease (CAD) in a subject, in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1 to 11, an antisense oligonucleotide conjugate of any of claims 12 to 20, or a pharmaceutical composition of claim 21. 32. An in vitro method for reducing the levels of expression and/or activity of PCSK9 in a cell, comprising introducing into the cell an effective amount of a compound according to any one of claims 1 to 11, an antisense oligonucleotide conjugate according to any one of claims 12 to 20, or a pharmaceutical composition according to claim 21 . 33. A method of reducing the levels of expression and/or activity of PCSK9 in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 11, an antisense oligonucleotide conjugate according to any one of claims 12 to 20, or a pharmaceutical composition according to claim. 21. 34. A method of lowering cholesterol in a subject in need thereof, comprising administering to said subject an effective amount of a compound of any one of claims 1 to 11, an antisense oligonucleotide conjugate of any of claims 12 to 20, or a pharmaceutical composition of claim 21. 35. A method for producing a compound consisting of an antisense oligomer with a length of 16-26 contiguous nucleotides, including chemical synthesis of the compound using sequential solid-phase synthesis of oligonucleotides, where the sequence of the antisense oligomer contains a continuous sequence of 16 nucleotides in length, which is 100% complementary to the sequence SEQ ID NO: 31 wherein the antisense oligomer is a gapmer containing at least one LNA unit, wherein the antisense oligomer is directed to the mRNA encoding PCSK9, and wherein said 16-22 contiguous nucleotides are 100% complementary to the mRNA encoding PCSK9. 36. A method for producing an antisense oligonucleotide conjugate, including covalent attachment of at least one non-nucleotide or non-polynucleotide group to an antisense oligomer with a length of 16-26 adjacent nucleotides directly or through a linker located between the sequence of the antisense oligomer and the non-nucleotide or non-polynucleotide group, wherein the sequence of the antisense oligomer contains a contiguous sequence of 16 nucleotides in length that is 100% complementary to the sequence of SEQ ID NO: 31, wherein the antisense oligomer is a gapmer containing at least one LNA unit, wherein the antisense nucleotide conjugate is directed to the mRNA encoding PCSK9, and wherein these 16-22 contiguous nucleotides are 100% complementary to the mRNA encoding PCSK9. 37. The method according to claim 36, where the covalent attachment of at least one non-nucleotide or non-polynucleotide group to the antisense oligomer includes (i) chemical synthesis of the antisense oligomer; and (ii) adding, by chemical synthesis or conjugation, at least one non-nucleotide or non-polynucleotide moiety to the antisense oligonucleotide conjugate. 38. The method of claim 37, wherein adding by chemical synthesis or conjugation at least one non-nucleotide or non-polynucleotide moiety to the antisense oligonucleotide conjugate comprises (i) incorporating by chemical synthesis or conjugation at least one non-nucleotide or non-polynucleotide moiety into the antisense oligonucleotide conjugate; (ii) incorporating, by chemical synthesis or conjugation, at least one linker into the antisense oligonucleotide conjugate; (iii) incorporating, by chemical synthesis or conjugation, at least one branch point into an antisense oligonucleotide conjugate; (iv) incorporating, by chemical synthesis or conjugation, at least one spacer into an antisense oligonucleotide conjugate; or (v) some combination of the above. 39. The method according to claim 38, where (i) at least one linker is located between the antisense oligomer and the branch point; (ii) at least one branch point is located between the linker and at least one non-nucleotide or non-polynucleotide moiety; (iii) at least one, two or three non-nucleotide or non-polynucleotide moieties are attached to the branch point; - 75 043736 (iv) at least one PEG spacer is located between the non-nucleotide or non-polynucleotide groups and the branch point; or (v) any combination of the above. 40. A compound according to any one of claims 1 to 11, wherein the antisense oligomer is an intermediate in the synthesis of an antisense oligonucleotide conjugate containing an antisense oligomer. 41. The method of claim 35, wherein the antisense oligomer is an intermediate in the synthesis of an antisense oligonucleotide conjugate containing the antisense oligomer. 42. A compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 21, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 26. 43. An antisense oligonucleotide conjugate according to any one of claims 12 to 20, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 26. 44. The method according to any one of claims 22 to 39, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 26. 45. A compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 21, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 3. 46. The antisense oligonucleotide conjugate of any one of claims 12 to 20, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 3. 47. The method according to any one of claims 22 to 39, wherein the antisense oligomer sequence comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 3. 48. The antisense oligonucleotide conjugate according to any one of claims 12 to 20, wherein the antisense oligonucleotide conjugate consists of SEQ ID NO: 18 or SEQ ID NO: 19. 49. The pharmaceutical composition according to claim 21, wherein the antisense oligonucleotide conjugate consists of SEQ ID NO: 18 or SEQ ID NO: 19. 50. The method according to any one of claims 22 to 39, wherein the antisense oligonucleotide conjugate consists of SEQ ID NO: 18 or SEQ ID NO: 19. -