WO2015113922A1

WO2015113922A1 - Poly oligomer compound with biocleavable conjugates

Info

Publication number: WO2015113922A1
Application number: PCT/EP2015/051442
Authority: WO
Inventors: Nanna ALBÆK; Henrik Frydenlund Hansen; Susanne KAMMLER; Morten Lindow; Jacob Ravn; Mark Turner
Original assignee: Roche Innovation Center Copenhagen A/S
Priority date: 2014-01-30
Filing date: 2015-01-26
Publication date: 2015-08-06
Also published as: US20170349896A1; EP3099797A1; CA2935426A1; EP3099797B1; US10358643B2; JP2017505623A; KR20160117535A; KR102287532B1; CA2935426C

Abstract

The invention relates to the field of oligonucleotide therapeutics, and in particular to poly oligo oligonucleotides conjugates where two or more antisense oligonucleotides are covalently linked by physiologically labile linkers, and to a biocleavable functional group such as a conjugate group.

Description

POLY OLIGOMER COMPOUND WITH BIOCLEAVABLE CONJUGATES

FIELD OF INVENTION

The invention relates to the field of oligonucleotide therapeutics, and in particular to poly oligo oligonucleotides and conjugates where two or more antisense oligonucleotides are covalently linked by physiologically labile linkers, and a functional group such as a conjugate group.

BACKGROUND

Oligonucleotide conjugates have been extensively evaluated for use in siRNAs, where they are considered essential in order to obtain sufficient in vivo potency. For example, W 02004/044141 refers to modified oligomeric compounds that modulate gene expression via an RNA interference pathway. The oligomeric compounds include one or more conjugate moieties that can modify or enhance the pharmacokinetic and pharmacodynamic properties of the attached oligomeric compound.

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 the kidney, although a wide range of other tissues are also accessible by the antisense modality, including lymph node, spleen, bone marrow.

WO2008/1 13832 discloses LNA phosphorothioate gapmer oligonucleotides where the flanking regions comprise at least one phosphodiester between or adjacent to a LNA nucleoside. The oligomers were preferentially targeted to the kidney.

WO2004/087931 refers to oligonucleotides comprising an acid cleavable hydrophilic polymer (PEG) conjugate.

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

WO 2009/126933 refers to specific delivery of siRNA nucleic acids by combining targeting ligands with endosomolytic components.

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

WO2009/025669 refers to polymeric (polyethylene glycol) linkers containing pyridyl disulphide moieties. See also Zhao et al., Bioconjugate Chem. 2005 16 758 - 766.

WO2014/043544 and WO2014/076195 refer to multimeric oligonucleotide compounds which are linked via cleavable linkages, including DNA phosphodiester linkages. WO2014/076195 also refers to oligonucleotide conjugates which utilise biocleavable DNA phosphodiester linkages to link the conjuge to the oligonucleotide.

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

Other non-cleavable 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 Krutzfeldt et al., Nature 2005 vol 438, 685 - 689. For the partially phosphorothiolated siRNAs and antagomirs, the use of cholesterol as a liver targeting entity was found to be essential for in vivo activity.

Bhat et al., AASLD November 7 - 1 1^th 2013 (poster) disclosed data from the use of a GalNac conjugated anti-miR, RG-101 targeting miR-122 for reduction of HCV in preclinical studies. The identity of RG-101 was not disclosed.

The present invention refers to the use of such short regions, e.g. 1 - 5, of physiologically labile nucleotides, such as DNA phosphodiester, to link multiple single stranded antisense oligonucleotides, which enables a single drug entity to target mutiple targets, and the use of a single conjugate moiety to target multiple single stranded oligonucleotides to a target tissue or cell.

The present invention is also based upon the discovery that highly effective targeted delivery of multiple oligonucleotides is achieved by the use of a homing device linked to two or more oligonucleotides by means of a short region of nuclease labile nucleosides, such as phosphodiester linked DNA or RNA nucleosides, linking the oligonucleotides to the conjugate moiety.

RELATED APPLICATIONS

WO2014/076195, hereby incorporated by reference, discloses the use of short regions of physiologically labile nucleotides, such as DNA phosphodiesters, to link an antisense oligonucleotide to a conjugate, enabling efficient targeting of potent oligonucleotides to target cells.

SUMMARY OF INVENTION

Poly oligomeric compounds

The invention provides for an oligomeric compound (an oligomer) which comprises a first oligomer region (region A), a second oligomer region (Α') and a biocleavable linker region (region B), and a third region (region C), wherein the biocleavable linker region (B) is positioned between the first oligomer region (region A), a second oligomer region (Α').

The invention provides for an oligomeric compound (an oligomer) which comprises a first oligomer region (region A), a second oligomer region (Α') and a region of 1 - 10 physiologically labile nucleotides (region B), and a third region (region C), wherein the biocleavable linker region (B) is positioned between the first oligomer region (region A), a second oligomer region (Α').

The invention provides for an oligomeric compound (an oligomer) which comprises a first oligomer region (region A), a second oligomer region (Α') and a region of 1 - 10 phosphodiester linked DNA or RNA nucleotides (region B), and optionally a third region (region C), wherein the biocleavable linker region (B) is positioned between the first oligomer region (region A), a second oligomer region (Α'). Suitably, group C is covalently joined to the oligomeric complex via a further region B'.

The invention provides for an oligomeric compound (an oligomer) which comprises a first oligomer region (region A), a second oligomer region (Α') and a region of 1 - 10 phosphodiester linked DNA nucleotides (region B), and a third region (region C), wherein the biocleavable linker region (B) is positioned between the first oligomer region (region A), a second oligomer region (A^*). Suitably, group C is covalently joined to the oligomeric complex via a further region B^*.

The oligomer regions A and A', and if present A", may target the same nucleic acid target or diferrent nucleic acid targets. The oligomer regions A and A', and if present A", may comprise the same sequence of nucleobases or different sequence of nucleobases.

Region (C), when present, may comprise a conjugate moiety, a targeting moiety, a reactive group, an activation group, or a blocking moiety. For therapeutic use, conjugate groups are preferred, and as such the compound of the invention may comprise a conjugation group. The conjugation group may.for example, be a targeting moiety which enhances delivery and/or uptake of the oligomeric compound of the invention to the intended site of action. In some embodiments, the conjugate group is a liver-targeting group which enhances the delivery and/or uptake of the oligomeric compound of the invention to the liver, such as to hepatocytes. Sterols such as cholesterol and tocopherol, as well as GalNAc comjugates are know liver-targeting conjugates. Suitably, group C is covalently joined to the oligomeric complex via a further region B. The beneficial use of biocleavable or physiological labile linkers to join a functional group C to an oligomer is reported in WO2014/076195, which is hereby incorporated by reference. The use of a region B to link a region C or region C-Y to an oligomer allows for the predictable cleavage of the conjugation group at the intended target tissue/cell, allowing the delivery of active and potent oligomers. The linking of lipophilic conjugates, such as sterols, is particularly beneficial.

The invention provides for an oligomeric compound (an oligomer) which comprises a first oligomer region (region A), a second oligomer region (Α') and a region of 1 , 2, 3, 4 or 5 phosphodiester linked DNA nucleotides (region B), and a third region (region C), wherein the biocleavable linker region (B) is positioned between the first oligomer region (region A), a second oligomer region (A^*). Suitably, group C is covalently joined to the oligomeric complex via a further region B'.

Region C may, for example be covalently linked to region A or region A', or a linking group (Y) which is covalently linked to region A or region A'.

Region C, or C-Y, when present may, for example be covalently linked to region A or region A¹, or a linking group (Y) which via a further physiologically labilie group (Β'). Region B^* may be as according to region B, or may be a different linkage group.

Region B may, for example be a region of at least one phosphodiester linked DNA or RNA (such as DNA), such as two, three, four or five phosphodiester linked DNA or RNA nucleosides (such as DNA nucleosides). Regions B and B' may, in some embodiments have the same structure, e.g. the same number of DNA/RNA nucleosides and

phosphodiester linkages and/or the same nucleobase sequence. In other embodiments Regions B and B' may be different. By way of example such poly oligomeric compounds may have a structure such as: (5' - 3' or 3' - 5') Conjugate-PO-ON-PO'-ON', wherein conjugate is region C, PO is region B, PO' is region B', and ON 1 is region A, and ON^* is region A^*. In some embodiments, the functional group (C), such as a conjugate group may be covalently linked to a first oligomer via a non-nucleotide cleavable linker (Β') such as a peptide linker, such as a lysine linker such as mono or poly lysine, e.g. a tri-lysine or di- lysine linker. Such polylysine linkers may be used with e.g. carbohydrate conjugates such as GalNAc conjugates, such as trivalent GalNAc conjugates. The functional group, such as a conjugate group (C) and biocleavable linker (Β'), e.g. C-B'~ may further be joined to a further linker group (Y) which links region C with the first region.

By way of a non-limiting explaination, the poly oligomeric compounds of the invention are referred to as the oligomeric compound here - they are "poly oligomeric" as although they form a single covalently attached entity, upon delivery to a cell, which may be their intended target site in the body, for a non-limiting example, a hepatocyte, it is considered the linker groups (B) are cleaved, relaseing separate oligomers into the target cell.

It should be understood that region A' may, in some embodiments, comprise multiple further oligomeric compounds (such as a further 2 or 3 oligomeric compounds) linked in series via biocleavable linkers, for example: Conjugate-PO-ON-PO-ON'-PO"-ON", or Conjugate-PO- ON-[PO-ON']n, wherein n may, for example be 1 , 2 or 3, and each ON" may be the same or different, and if different may have the same or different targets. Alternatively two or more oligomer regions may be joined to a common linking group, each via a independent region B (i.e. the oligomer region's are linked in parallel).

When referring to oligomer regions, a first oligomer region may be designated A, and subsequent oligomer regions A', and if present A". In some non-limiting embodiments, one or more oligomer regions (such as A, A & A', or A& A'& A") comprise at least one sugar modified nucleoside analogue, for example at least one LNA unit. The oligomer region(s) may therefore be LNA oligomers.

Each oligomer region (A, A' or A") is 7 - 26 nucleosides in length, wherein the nucleosides within the oligomer region(s) are other than phosphodiester. In some emboidments, the nucleoside linkages, or at least 70% of the nucleoside linkages within each oligomer region (A, A^* and A") are phosphorothioate linkages.

The present invention provides for an oligonucleotide comprising i) a first region (A) of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; ii) a second region (A^*) of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; wherein the first and the second regions are covalently linked via iii) at least one region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [or RNA] nucleosides. The oligonucleotide (compound of the invention) may therefore be described as an oligonucleotide complex or poly-oligomer. In some embodiments, the compound of the invention comprises a single contiguous nucleotide sequence which comprises the first oligomer region (A) a region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides, and a second oligomer (Α') region (Α-Β-Α').

In some embodiments, the compound of the invention comprises a single contiguous nucleotide sequence which comprises the first oligomer region (A) a region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides, a second oligomer (Α') region followed by a further region (B) (which may be denoted B') of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides, followed by a third oligomer region (A"), i.e. A-B-A'-B'-A". Such linear compounds may further comprise a functional e.g. a conjugate group (C), which may, by example, be covalently attached to oligomer A or A", or a or A" (when present). The functional or conjugate group may be attached to the single contiguous nucleotide sequence (e.g. A-B-A' or A-B-A'-B'-A") via a linker (Y). The functional or conjugate group (C) or (C-Y) may further be attached to the single contiguous nucleotide sequence via a further region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides. In some embodiments, region A -region B and region A form a single contiguous nucleotide sequence of 15 - 50, such as 15 - 40, 15 - 35, 15 - 30, 15 - 25, 15 - 24 nucleotides in length.

In some embodiments, the compound of the invention comprises two or more oligomer regions (e.g.A, A and if present A") wherein each oligomer region is covalently attached to a linking group (F) (e.g. a branching group to which each of the oligomers are atatched) via a region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides. A functional or conjugate group may be attached to either one or more of oligomer regions or to the linking group. By way of a non-liming example a tri-lysine linker may be used to join two, three or four oligomers together, or optionally two or three oligomers and a functional /conjugate group. It will be recognized that such a peptide linking group may in itself be physiologically labile, and as such, a peptide linking group may, in some embodiments be the physiological labile linker (B) which joins the two or more oligomer regions. Alternatively, at least one or each oligomer region may be linked to such a peptide linker group via a region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides. The advantage of using a nucleotide based region B is that cleavage will result in a predictable oligomer product, and as such full efficacy of the oligomer can be retained and delivered to the desired site of therapeutic activity.

Peptide linkers, such as di and trilysine are used a scaffolds for conjugate delivery of siRNAs, and as such the linking group (F) may form part of or be attached to a conjugate group, for example a carbohydrate conjugate group, such as a galactose group, such as a GalNAc group, such as a GalNAc cluster.

In some embodiments the invention provides for a compound (an oligonucleotide) comprising i) a first region (A) of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; ii) a second region (A^*) of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; wherein the first and the second regions, and optionally further regions of 7 - 26 phosphorothioate linked nucleosides (e.g. A"), are covalently linked, via a non- nucleotide or conjugate (C) or linking moiety, wherein each of the first (A) and second (A^*) and optionally further (A") regions are independently or dependently linked to the conjugate or linking moiety via a region (B) of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [and/or RNA] nucleosides.

Region C~, or C-Y-, may, in some embodiments, be covalently attached to one of the regions A, A or A" (oligomer region), via a phosphorus containing linkage group (illustrated by the ~. The phosphorus linkage group, may, for example, be a phosphate (phosphodiester), a phosphorothioate, a phosphorodithioate or a bora nophosp hate group. In some embodiments, this phosphorus containing linkage group is positioned between the oligomer region and a linker region (Y) which is attached to region C, In some

embodiments, the phosphate group is a phosphodiester. In some embodiments, region C or C-Y~ may be covaientiy joined (linked) to region B' via a phosphate nucleoside linkage, such as those described herein, including phosphodiester or phosphorothioate, or via an alternative group, such as a triazol group.

In some embodiments, region C is an activation group, such as an activation group for use in conjugation. In this respect, the invention also provides activated oligomeric compound (the compound of the invention with an activation group), e.g. an intermediate which is suitable for subsequent linking to a conjugation or other functional group, such as suitable for conjugation.

In some embodiments, region C is a reactive group, such as a reactive group for use in conjugation. In this respect, the invention also provides an intermediate comprising the oligomer complex which is suitable for subsequent linking to a conjugation or other functional group, such as suitable for conjugation. The reactive group may, in some embodiments comprise an amine of alcohol group, such as an amine group.

In some embodiments the internucieoside linkages within regions A, A' and A" (i.e. the oligomer regions) each comprises at least 50%, such as at least 75%, such as at least 90% phosphorothioate linkages. In some embodiments, all the internucieoside linkages in the oligomer regions are other than phosphodiester, such as are phosphorothioate linkages.

In a preferred embodiment, region B (B' and B") each comprise 1 , 2, 3, 4 or 5 contiguous phosphodiester linked nucleotides, such as DNA nucleosides.

The oligomeric complex of theinvention may also be referred to as the oligomeric compound, or oligomeric compound conjugate (when C is present and is a conjugate group). The invention provides for a pharmaceutical composition comprising the oligomeric compound of the invention and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of a nucleic acid target in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo. The oligomer regions of the compound of the invention may, in some embodiments target the same nucleic acid target, for example a mRNA or viral RNA.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of two or more independent (i.e. different) nucleic acid targets in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo. When targeting two or more independent nucleic acid targets, the oligomer regions A, A' and if present A" may comprise non-identical nucleobase sequences. The contiguous nucleobase sequence of each oligomer region may therefore be different.

In some embodiments the compounds of the invention are capable of inhibiting the expression of one or two or three or more targets in a cell which is expressing said target(s). The cell, for example may be a mammalian cell, such as a human cell. In some

embodiments at least one of the targets is selected from the mRNA, viral and/or microRNA targets listed herein, including the targets listed in table 2 (microRNA targets).

The invention provides for the oligomeric compound according to the invention for use in the inhibition of a microRNA target in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo. In some emboidments the compounds of the invention are capable of inhibiting the expression of a (or more, such as 2 or 3) microRNA target(s) in a cell which is expressing said microRNA target(s). The cell, for example may be a mammalian cell, such as a human cell.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of two or more independent (i.e. different) microRNA targets in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of one or more mRNA targets in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo. In some emboidments the compounds of the invention are capable of inhibiting the expression of a (or more, such as 2 or 3) mRNA target(s) in a cell which is expressing said mRNA target(s). The cell, for example may be a mammalian cell, such as a human cell.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of a viral RNA target in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo.

The invention provides for the oligomeric compound according to the invention for use in the inhibition of two or more (such as three) independent (i.e. different) mRNA targets in a cell. In some embodiments the use is in vitro. In some embodiments the use is in vivo. In some embodiments at least one of the mRNA targets is selected from the mRNA targets listed herein.

The invention provides for the oligomeric compound of the invention for use in medicine, such as for use as a medicament.

The invention provides for the oligomeric compound of the invention for use in the treatment of a medical disease or disorder. The invention provides for the use of the oiigomeric compound of the invention for the preparation of a medicament for the treatment of a disease or disorder, such as a metabolic disease or disorder.

The invention provides for a method of treatment of a disease or disorder in a subject in need of treatment, said method comprising the steps of administering a pharmaceutical composition comprising the oiigomeric compound of the invention to said subject in a therapeutically effective amount.

The invention provides for a method of inhibiting the expression of one (or more, such as two or three) target gene(s) in a cell, said method comprising administering the oiigomeric compound according to the invention to a cell which is expressing said target gene(s), suitably in an amount effective to reduce the expression of the target gene in said cell. In some embodiments the method is in vitro (.e. not in an organism, but may be in a (e.g. ex- vivo) cell or tissue). In some embodiments the method is in vivo.

The oiigomeric compound of the invention may comprise an LNA oligomer (e.g. as region A, A^* and/or A"). In some embodiments, region A and region A^* are both LNA oligomers. In some embodiments, region A and region A' and A" are all LNA oligomers.

In some embodiments, such as in a non-limiting aspect when regions A and optionally A" (and if present optionally A") are LNA oligomers, region C may be a conjugate. Such as a targeting moiety, may, for example, be a conjugate which targets the compound of the invention to the liver (a liver-targeting conjugate moiety). The conjugate may, for example be or comprise a sterol, such as cholesterol or tocopherol, or may be or comprise a (non- nucleotide) carbohydrate, such as a GalNac conjugate, such as a GalNac cluster, e.g.

triGalNac, or another conjugate as described herein. Such compounds may comprise a linker group Y between the conjugate group and an oligomer region, optionally via a region B.

The compound of the invention may therefore, in some embodiments, comprise at least one LNA antisense oligomer region (which may be referred to as region A herein) covalently linked to an asialoglycoprotein receptor targeting moiety conjugate moiety, such as a GalNAc moiety, which may form part of a further region (referred to as region C). An LNA antisense oligomer comprises at least one LNA unit (nucleoside).

The compound of the invention may therefore comprise an LNA antisense oligomer region covalently joined to (e.g. linked to) a (non-nucleoside) carbohydrate or a sterol moiety, such as a carbohydrate conjugate moiety or a cholesterol moiety. In some embodiments the carbohydrate moiety is not a linear carbohydrate polymer. The

carbohydrate moiety may however be multi-valent, such as, for example 2, 3, 4 or 4 identical or non-identical carbohydrate moieties may be covalently joined to the oligomer, optionally via a linker or linkers.

The invention provides for a poly oligomeric complex of comprising a contiguous nucleotide sequence of formula [LNAsJ7.i8-p AJ₁.5-tL AsJ7.i8, and a non-nucleobase conjugate, such as a sterol (e.g cholesterol or tocopherol) or a GalNAc conjugate moeity, for example a trivalent GalNAc conjugate conjugate, such as a conjugate moeity selected from the group consisting of any one of Conjl , 2, 3, 4, 1a, 2a, 3a, 4a, or other trivalent GalNAc conjugates, such as those disclosed herein. Subscript s refers to a phosphorothioate linkage. At least one internucleoside linkage within or adjacent to the -[DNA]i.₅. region are phosphodiester linkages. In some embodiments, all internucleoside linkages within or adjacent to the -[DNA] 1.5. region are phosphodiester linkages. In some embodiments, the - [DNAJ-,.5. region has 2, 3, 4 or 5 contiguous DNA nucleoside which are joined by

phosphodiester linkages. In such an embodiment, the internucleoside linkages between the -[DNAJ₂_5_ are phosphodiester linkages, and optionally the internucleoside linkages between region -[DNA]i_₅ and the LNA regions [LNAJ₇.i₈ are independently phosphorothioate or phosphodiester linkages, such as both phosphodiester or both phosphorothioate, or one phosphodiester and one phosphorothioate. In the embodiment when the DNA region is a single DNA nucleoside, at least one or both the the internucleoside linkages adjacent to the DNA region is a phosphodiester, and if only a single phosphodiester, the other may be a phosphorothioate. The region -[DNAJ1.5 may be as defined as described by region B herein - i.e. may be a physiologically cleavable nucleoside linker region. Each [LNAJ₇.i₈ is a LNA phosphorothioate oligomer, and may for example be independently selected from the group consisting of an LNA gapmer, an LNA mixmer or an LNA totalmer. The GalNAc conjugate may for example be located 5' or 3' to the contiguous nucleotide sequence. In a preferred embodiment, at least one of the LNA oligomers, or both the poly oligomer conjugate is a

LNA totalmer of 7 - 12, such as 8, 9 or 10 nucleotides in length. In some embodiments, the LNA totalmer may comprise only LNA nucleotides, such as beta-D-oxy LNA nucleoside, which are linked by phosphorothioate linkages. For example the poly oligomer conjugate may comprise a

such as IL Asl7.10-tD AJ2-IL AJ7.10 or [LNAJ₇._ir[DNAJ₃-[LNA_sj7.io or [LNAsJ7.10-tDNAJ4-tLNAsJ7.10. in one embodiment the contiguous nucleositide sequence comprises

such as [LNA_S]₈-[DNA]₂-[LNA_S]₈, [LNA_S]₈-[DNA]₃-[LNAJ₈, or [LNA_sJ_r[DNAJ₄-[LNAJ₈. Such poly oligomeric complexes are particularly useful to target microRNAs, such as mature microRNAs. By utilising a first LNA oligomer region which targets a first target (e.g. a mRNA, a microRNA, or a viral sequence), and a second LNA oligomer region which targets a second target (e.g. a mRNA, a microRNA, or a viral sequence), single compounds can be made which target two distinct targets, for example, the first oligomer region may target ApoB, and the second oligomer region may target another mRNA, such as mtGPAT mRNA, for example:

By utilising a first LNA oligomer regions (e.g. [LNAJ7.10) which targets one microRNA, and a second LNA oligomer region which targets a second microRNA, single compounds can be made which target two different microRNA targets, for example miR-21 and miR-221, both of which are indicated in hepatocellular carcinoma. Alternatively the first and the second may target the same microRNA, such as e.g. miR-122, miR-21 , miR-155, miR-33, miR-221 , which allows two oligomers to be delivered to the target cell for a single conjugate moiety.

This of particular importance for receptor mediate conjugate targeting, such as with asialoglycoprotein receptor conjugates, where the receptor mediated uptake of e.g. GalNAc conjugated oligomers is limited by the availability of free receptors on the surface of the target cell, the use of poly-oligomer conjugates allows for enhanced delivery to the target cell. It is also important to avoid compelte saturation of cell -suface receptors which are performing an important biological function, the use of the poly-oligomer strategy therefore allows for effective delivery of sufficient compound to ensure relevant pharmacology, whilst reducing the risk of side effects due to receptor saturation/competition by the conjugate moiety. The use of the poly-oligomer conjugate therefore provides an effective solution for enhancing the therapeutic index - increased oligomer delivery and activity with a reduction of undesirable side-effects.

BRIEF DESCRIPTION OF FIGURES Figure 1: Examples of poly-oligomers using a bio-cleavable linker (B) between two oligomer regions (A and A), optionally covalently joined to a functional group (C), which may further be attached to the first (or the second) oligomer via a second bio-cleavable linker (B). A and A' may be LNA oligomers, such as LNA gapmers, mixmers or totalmers. Region C may be a conjugate, such as a targeting conjugate, e.g. (for liver targeting) a sterol or a GalNAc conjugate. Region B and B^* may be, for example a region of 1 , 2, 3, 4 or 5

phosphodiester linked DNA nucleosides.

Figure 2: Examples of branched poly-oligomers, where each oligomer (A, A' and A") is attached to a non-nucleotide linker (F) via a bio-cleavable region (B, B' and B"). The non- nucleotide linker (F) may be attached to a functional group (C), or may be a functional group (C). Region C may be a conjugate, such as a targeting conjugate, e.g. (for liver targeting) a sterol or a GalNAc conjugate. Region B, B" and B" may be, for example a region of 1 , 2, 3, 4 or 5 phosphodiester linked DNA nucleosides.

Figure 3: Examples of cholesterol, trivalent GalNac, FA , folic acid, monovalent GalNac and tocopherol conjugates.

Figure 4: Examples of tri-GalNac conjugates which may be used. Conjugates 1 - 4 illustrate 4 suitable GalNac conjugate moieties, and conjugates 1a - 4a refer to the same conjugates with an additional linker moiety (Y) which is used to link the conjugate to the oligomer (region A or to a biocleavable linker, such as region B). The wavy line represents the covalent link to the oligomer. Also shown are examples of cholesterol and tocopherol conjugate moieties (5a and 6a). The wavy line represents the covalent link to the oligomer.

Figure 5: Silencing of miR-122 in the mouse liver by seed-targeting tiny LNA. (a) RNA blot analysis of liver RNAs from mice after treatment with three intravenous doses of 20 mg/kg tiny antimiR-122, 15-mer antimiR-122 or LNA scramble control or with saline.

Figure 6: Total Cholesterol analysis at pre-dose, day 4 and day7. Cholesterol is upregulated due to decreased miR122.

Figure 7: Expression of Aldo A and Bckdk was measured by standard TaqMan Q-PCR assays. The mRNA levels of these genes are upregulated due to decreased miR122.

Figure 8: ALT was measured from final serum (day 7) to assess tolerability of the compounds.

Figure 9: Expression of Aldo A and Bckdk was measured by standard TaqMan Q-PCR assays. The mRNA levels of these genes are upregulated due to decreased miR122.

Figure 10: The ApoB/mtGPAT targeting compound SEQID NO 55. Other conjugate moieties may be used, and alternative cleavable linker may be used, e.g. between the conujugate moiety and 5' of region 1 , e.g. a PO linker which may comprise a region of 1 , 2, 3, 4 or 5 phosphodiester linked DNA nucleosides. Note GalNAc 1 comprises the

biocleavable dilysine linker.

Figure 11 : Results obtained using a polyoligo GalNAc conjugate targeting both ApoB and mtGPAT in the liver of mice in vivo. DESCRIPTION OF THE INVENTION

In some embodiments, the invention provides for a poly oligomeric compound which may comprise the first region (region A), the second region (region B) and the third region (region C), wherein the first region is covalently linked to at least one further oligomeric compound (region A'), wherein the first region (region A) and region A' are covalently linked via a biocleavable linker (region B'), which may be, by way of example, as according to the second region as disclosed here, for example a region of at least one phosphodiester linked DNA or RNA (such as DNA), such as two, three, four or five phosphodiester linked DNA or RNA nucleosides (such as DNA nucleosides). Regions B and B' may, in some

embodiments have the same structure, e.g. the same number of DNA/RNA nucleosides and phosphodiester linkages and/or the same nucleobase sequence. In other embodiments Regions B and B' may be different. By way of example such poly oligomeric compounds may have a structure such as: {5' - 3' or 3' - 5') Conjugate- PO-ON-PO'-ON', wherein conjugate is region C, PO is region B, PO' is region B\ and ON 1 is region A, and ON' is region A'

It should be understood that region A may, in some embodiments, comprise multiple further oligomeric compounds (such as a further 2 or 3 oligomeric compounds) linked in series {or in parallel) via biocleavable linkers, for example: Conjugate-ΡΟ-ΟΝ-ΡΟ-ΟΝ'-ΡΟ''-ΟΝ", or Conjugate-PO-ON-[PO-ON']n, wherein n may, for example be 1 , 2 or 3, and each ON' may be the same or different, and if different may have the same or different targets.

The Oligomer

The term "oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e. an oligonucleotide). Herein, a single nucleotide (unit) may also be referred to as a monomer or unit. In some

embodiments, the terms "nucleoside", "nucleotide", "unit" and "monomer" are used interchangeably. It will be recognized that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U.

In the contect of the present invention the term "oligomer", as used herein may refer to the contiguous oligonucleotide sequence of nucleotides or the compound of the invention, or a oligomer region which forms part of the compound of the invention, such as A, A and A", which may, in some embodiments form part of the contiguous oligonucleotide sequence of nucleotides or the compound of the invention.

The present invention employs poly-oligomeric compounds (also referred herein as oligomer compounds) for use in modulating, such as inhibiting a target nucleic acid in a cell. The oligomer compound comprises at least two oligomer regions, e.g. (A and A') and may comprise further oligomer regions (e.g. A") The oligomer regions may have a length of 7 - 26 contiguous nucleotides and each oligomer region may be flanked by a bio-cleavabe region (region B), which may, for example, be a further region of 1 - 10 contiguous nucleotides (region B), which comprise at least one phosphodiester linkage. Other physiological labile nucleoside regions may be used.

In some embodiments, the oligomer compounds of the invention are covalently linked to a conjugate group, a targeting group, a reactive group, an activation group, or a blocking group, optionally, via a short region comprising (e.g. 1 - 10) of phosphodiester linked DNA or RNA nucleoside(s).

In some embodiments, the compound of the invention does not comprise RNA (units). In some embodiments, the compound according to the invention forms a single contiguous sequence), optionally linked to a function group, such as a gonjugate group, and is such a linear molecule or is synthesized as a linear molecule. The oligomeric compound may therefore be single stranded molecule. In some embodiments, the oligomer does not comprise short regions of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to equivalent regions within the same oligomeric compound (i.e. duplexes). The oligomer, in some embodiments, may be not (essentially) double stranded. In some embodiments, the oligomer is essentially not double stranded, such as is not a siRNA.

Oligomer regions A, A^* and if present A" are phosphorothioate olgiomers, i.e. at least 70% of the internucleoside linkages within each oligomer region A, A^* and if present A", are phosphorothioate linkages, such as at least 80% or at least 90% or all of the internucleoside linkages present I oligomer regions A, A^* and A" (if present), are phosphorothioate.

In some embodiments, oligomer regions A, A^* and if present A" may form a single contiguous oligonucleotide sequence. Regions A, A' and A" are interspaced by regions B, for example regions of 1 , 2, 3, 4, or 5 phosphodiester linked DNA nucleosides.

When region B comprises only 1 nucleoside, at least one, or both of the

internucleoside linkages between the region B nucleoside (e.g. a DNA nucleoside) may be phosphodiester linkages. When region B comprises only 2 or more nucleosides, the internucleoside linkages between the region B nucleoside (e.g. the DNA nucleosides) may be phosphodiester linkages and/or may be another internucleoside linkage, such as phosphorothioate linkages.

The oligomers of the invention, such as A, A^* and if present A", do not form part of a siRNA complex.

The oligomers of the invention, such as A, A' and if present A", are non- complementary, e.g. they do not hybridize to one another to form a region of more than 8 or in some embodiments more than 6 contiguous base pairs. In some embodiments, regions A and A" do not hybridize to one another to form a region of more than 4 contiguous base pairs. Exemplary base pairs may be between A-T, G-C or A-U. In the case there are three oligomer regions, A, A' and A", the non-complementarity is between A and A', and A' and A", as well as A and A",

The oligomer regions A, A' and if present A"are not in the form of a duplex with a (substantially) complementary oligonucleotide - e.g. is not an siRNA. In some embodiments, oligomer regions A, A^* and A" share the same contiguous nucleotide sequence. In some embodiments, oligomer regions A and A' share the same contiguous nucleotide sequence. In this respect the invention provides for a single compound which can be used to deliver multiple copies of an oligomer (i.e. with the same contiguous nucleobase sequence and optionally the same chemical modifications) to the target tissue.

Length of Oligomer Regions

Each oligomer region (e.g. A, A' and A"), may be between 7 - 26 nucleotides, such as 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 26. It is recognized that in the embodiment where the oligomer regions, A and A" (and optionally A") form a single contiguous nucleotide sequence (see Figure 1 ), the use of shorter oligomer regions is highly preferred, such as between 7 and 18 nucleotides, such as 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 and 17 nucleotides, such as 7 - 16 nucleotides or 7 - 14 nucleotides, or 7 - 12, nucleotides, or in some embodiments, for example when using LNA totalmers, between 7 - 12 or 7, 8, 9 or 10 contiguous nucleotides. Suitably the combined length of the oligomer regions, and the cleavable region(s) B is less than 40 nucleotides, such as less than 38 nucleotides, such as less than 36 nucleotides, such as less than 34 nucleotides, such as less than 32

nucleotides, such as less than 30 nucleotides, such as less than 28 nucleotides, such as less than 26 nucleotides, such as less than 24 nucleotides, such as less than 22

nucleotides, such as less than 20 nucleotides. The minimum length of the the combined length of the oligomer regions, and the cleavable region(s) B is 15 nucleotides, and may be therefore 16 nucleotides, 17 nucleotides or 18 nucleotides.

In the embodiment where the oligomer regions do not form a single contiguous nucleotide sequence (e.g. see figure 2), such as are joined in parallel, the length of each oligomer region (A, A' or A"), may be between 7 and 26 nucleotides. In some embodiments the length of an (or all) oligomer region may be between 7 - 20 nucleotides, such as 7 - 18 nucleotides or 7 - 18 nucleotides or 7 - 16 nucleotides. In some embodiments the length of an (or all) oligomer region may be between 8 - 20 nucleotides, such as 8 - 18 nucleotides or 8 - 18 nucleotides or 8 - 16 nucleotides. In some embodiments the length of an (or all) oligomer region may be between 12 - 20 nucleotides, such as 12 - 18 nucleotides or 12 - 18 nucleotides or 12 - 16 nucleotides. Such lengths are particularly suited for use with gapmer oligomers, such as LNA gapmer oligomer (regions).

In some embodiments, when the oligomer regions are joined in series (Figure 1 ) or in parallel (Figure 2), the length of an (or all) oligomer regions may be 7 - 12 nucleotides, such as 7 - 10 nucleotide, such as 7, 8, 9 or 10 nucleotides. Such lengths are particularly useful when using LNA mixmer or toalmer oligomers, such as oligomers which target a microRNA, such as a microRNA seed region.

In some embodiments, the oligomer regions comprise or consist of a contiguous nucleotide sequence of a total of from 10 - 22, such as 12 - 18, such as 13 - 17 or 12 - 16, such as 13, 14, 15, 16 contiguous nucleotides in length.

In some embodiments, the oligomer regions comprise or consist of a contiguous nucleotide sequence of a total of 10, 11, 12, 13, or 14 contiguous nucleotides in length.

In some embodiments, the oligomer regions 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 the regions comprises less than 20 nucleotides. It should be understood that when a range is given for an oligomer, or contiguous nucleotide sequence length it includes the lower an upper lengths provided in the range, for example from (or between) 10 - 30, includes both 10 and 30. LNA Oligomer Regions

In some embodiments, at least one of the oligomer regions (A, A' and A" if present), is a LNA oligomer, for example an LNA antisense oligomer. In some embodiments, at least two of the oligomer regions (A and A) are LNA oligomers, such as an LNA antisense oligomer. In some embodiments, at least three of the oligomer regions (A, A" and A") are LNA oligomers, such as an LNA antisense oligomers.

In some embodiments the compound of the invention, such as the LNA oligomer, such as LNA antisense oligomer is conjugated to a carbohydrate moiety, such as a non-linear carbohydrate, such as a GalNac moietys, such as a tri-GalNac cluster. In some

embodiments the compound of the invention, such as the LNA oligomer, such as LNA antisense oligomer is conjugated to an asiaioglycoprotein receptor targeting moiety conjugate moiety, such as a Gal Ac moiety (which may be region C). The carbohydrate moiety may be multi-valent, such as, for example 2, 3, 4 or 4 identical or non-identical carbohydrate moieties may be covalently joined to the oligomer, optionally via a linker or linkers (such as region Y).

In some embodiments, the LNA oligomer region(s), for example an LNA antisense oligomer, (which may be referred to as region A, A' or A" herein) comprising an antisense oligomer, is covalently linked to an asiaioglycoprotein receptor targeting moiety conjugate moiety, such as a GalNAc moiety (which may be referred to as region C), optinally via a region B as defined herein. The carbohydrate moiety may be multi-valent, such as, for example 2, 3, 4 or 4 identical or non-identical carbohydrate moieties may be covalently joined to the oligomer ore region B, optionally via a (further) linker or linkers (such as region Y, e.g. a C6 aikyl linker).

Oligomer Regions (e.g. A, A^* and if present A")

In some embodiments, the each oligomer region may comprise a nucleic acid based oligomer, such as an antisense oligonucleotide. In some embodiments, each oligomer region comprises or consists of a phosphorothioate linked oligonucleotide, such as an antisense oligonucleotide, of 7 - 25 or 26 nucleotides in length. The oligomer regin may be referred to as a "first region" - it will be recognized that the invention refers to embodiments where there are multiple first regions which may be the same or different, each oligomer region may comprise at least one modified nucleoside (a nucleoside analogue), such as at least one bicyclic nucleoside (e.g. LNA) or 2' substituted nucleoside. In some embodiments, some or all of the nucleosides each oligomer region may be modified nucleosides, also referred to as nucleoside analogues herein. In some embodiments, the modified

nucleosides are sugar-modified (e.g. comprise a sugar or sugar surrogate moiety other than ribose or deoxyribose). LNA (also referred to as BNA is a preferred nucleoside modification. In some embodiments, at least one of the oligomer regions, such as all the oligomer regions are antisense oligomers (antisense oligonucleotide), such as a single stranded oligomer which comprises a sequence which is (independtently or dependently) complementary to a nucleic acid target.

In some embodiments at least one of the oligomer regions, such as all the oligomer regions is a gapmer. In some embodiments at least one of the oligomer regions, such as all the oligomer regions is a mixmer. In some embodiments at least one of the oligomer regions, such as all the oligomer regions a totalmer.

In some embodiments, each oligomer region (e.g. A, A^* and if present A") comprises at least one , such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or 25 nucleoside analogues. In some embodiments the nucleoside analogues are (optionally independently selected from the group consisting of bicyclic nucleoside analogues (such as LNA), and/or 2^* substituted nucleoside analogues, such as (optionally independently) selected from the group consisting of 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, 2'-AP. 2 -FANA, 2'-(3-hydroxy)propyl, and 2'-fluoro-DNA units, and/or other (optionally) sugar modified nucleoside analogues such as morpholino, peptide nucleic acid (PNA), CeNA, unlinked nucleic acid (UNA), hexitol nucleoic acid (HNA). bicyclo- HNA (see e.g. WO2009/100320), In some embodiments, the nucleoside analogues increase the affinity of the first region for its target nucleic acid (or a complementary DNA or RNA sequence). Various nucleoside analogues are disclosed in Freier & Altmann; Nucl. Acid Res,, 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, hereby incorporated by reference.

In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A"), such as the gapmer, mixmer or totalmer comprise at least one bicyclic nucleotide analogue, such as LNA. In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A") comprises of at least one bicyclic nucleoside analogues (e.g. LNA) and/or 2'substituted nucleoside analogues. In some embodiments, the nucleoside analogues present in at least one or each oligomer region (e.g. A, A* and if present A")all comprise the same sugar modification. In some embodiments, at least one nucleoside analogue present at least one or each oligomer region (e.g. A, A* and if present A") is a bicyclic nucleoside analogue, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, for example all nucleoside analogues (or in a totalmer all nucleosides) bicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA or alpha-L-X-LNA (wherein X is oxy, amino or thio), or other LNAs disclosed herein including, but not limited to,(R/S) cET, cMOE or 5'-Me- LNA. In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A"), comprises of DNA and sugar modified nucleoside analogues, such as bicyclic nucleoside analogues and/or 2'substituted nucleoside analogues. In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A") , comprises of DNA and LNA nucleoside analogues, . In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A") comprises LNA nucleoside analogues. In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A") .comprises only nucleoside analogues, and may include LNA nucleosides. In some embodiments, at least one or each oligomer region (e.g. A, A* and if present A") comprises only LNA nucleosides analogues.

WO05013901 , WO07/027775, WO07027894 refers to filly 2'substituted oligomers, such as fully 2'-0-MOE. In some embodiments, the first region of the oligomer may comprise of 2' substituted nucleosides. WO07/027775 also refers to MOE, LNA, DNA mixmers for use in targeting microRNAs.

In some embodiments, at least one or each oligomer region (e.g. A, A* and if present

A") do not comprise a region of more than 4 or 5 consecutive DNA units. Such oligomer regions may be (essentially) unable to recruit RNAseH.

The first region is covalently linked to a region B (may also be referred as the second region), such as via a 5' terminal or 3' terminal internucleoside linkage, such as a

phosphodiester linkage. A phosphodiester linkage may therefore be positioned between the

5' most nucleoside of region A and the 3' most nucleoside of region B, and/or between the 3^* most nucleoside of region A and the 5' most nucleoside of region B. In this respect, in some embodiments, there may be two region B covalently joined to (a) oligomer region A, one at the 5" terminus of a region A and one at the 3' terminus of a region A, The two region Bs may be the same or different. One region B mak be joined to a further oligomer region (e.g. region A") see Figure 1 , or a non-nucleotide linker group (see Figure 2), and the other may be joined to another further oligomer region (A"), or for example a functional group (C) optionally via a linker (Y), for example a sterol or GalNAc conjugate.

In some embodiments, some or all of the nucleosides of an or each oligomer region (e.g. A, A' oand/or A") may be modified nucleosides, also referred to as nucleoside analogues herein, such as sugar modified nucleoside analogues, for example bicyclic nucleoside analogues (e.g. LNA) and/or 2'substituted nucleoside analogues. In some embodiments, the nucleoside analogues present in an or each oligomer region (e.g. A, A^* oand/or A")all comprise the same sugar modification, for example are all bicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA or alpha-L-X-LNA (wherein X is oxy, amino or thio), or other LNAs disclosed herein including, but not limited to,(R/S) cET, cMOE or 5'-Me- LNA.

The internucleoside linkages of an or each oligomer region (e.g. A, A' oand/or A") comprise at t least 50%, such as at least 75%, such as at least 90%, such as 100% of the internucleoside linkages in the oligomer region are other than phosphodiester, such as phosphorothioate. In some embodiments, the internucleoside linkages other than phosphodiester are sulphur containing internucleoside linkages, such as phosphorothioate, phosphorodithioate and boranophosphate, such as phosphorothioate.

Region B (also referred to as the second region, region B" and region B", or Nuclease Susceptible Physiological Labile Linkages

The oligomer regions (A, A' and if present A") are linked via at least one biocleavable region, referred to as region B herein (and where there is more than one region B, region B' and region B"). In some embodiments, region B comprises 1 - 10 nucleosides which form a physiologically labile region between oligomer regions, or between an (or each) oligomer region and a linking group (see Figure 2). Regions of DNA phosphodiester nucleosides may be used, but other nucleotide regions may be used if they are suitably physiologically labile.

In some embodiments, the internucleoside linkage between the oligomer region (A, A^* or if present A") and (each) second region B, is a phosphodiester linked to the first (or only) DNA or RNA nucleoside of region B comprises at least one phosphodiester linked DNA or RNA nucleoside.. The region B may, in some embodiments, comprise further DNA or RNA nucleosides which may be phosphodiester linked.

As explained herein, region B may also be used to join a functional group to the oligomeric region(s), optionally via a further linkage group (Y). The use of region B as a cieavable linker to join functional groups to oligomer is described in detail in PCT/EP2013/073858, which is hereby incorporated by reference.

In some embodiments a region B is further covalently linked to a third region which may, for example, be a conjugate, a targeting group a reactive group, and/or a blocking group.

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

In some embodiments, one (or more or all) region B may comprise or consists of at least one DNA or RNA nucleosides linked to the first region via a phosphodiester linkage. In some aspects, the internucleoside linkage between an oligomer region and second region is considered as part of region B.

In some embodiments, a (or more or each) region B comprises or consists of at least between 1 and 10 linked nucleosides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 linked DNA or RNA nucleotides. Whilst a region of DNA RNA phosphodiester is considered important in the provision of a cieavable linker, it is possible that region B also comprises sugar-modified nucleoside analogues, such as those referred to under the first region above. However in some embodiments, the nucleosides of region B are (optionally independently) selected from the group consisting of DNA and RNA. In some embodiments, the nucleosides of region B are (optionally independently) DNA. It will be recognized that the nucleosides of region B may comprise naturally occurring or non-naturally occurring nucleobases.

Typically, region B comprises at least one phosphodiester linked DNA or RNA nucleoside (which may, in some embodiments, be the first nucleoside adjacent to an oligomer). If region B comprises other nucleosides, region B may also comprise of other nucleoside linkages other than phosphodiester, such as (optionally independently) phosphorothioate, phosphodithioate, boranophosphate or methyl phosphonate. However, in other exemplified embodiments, all the internucleoside linkages in region B are phosphorothioate. In some embodiments, all the nucleosides of region B comprise (optionally independently) either a 2'- OH ribose sugar (RNA) or a 2'-H sugar - i.e. RNA or DNA. Between 1 - 5, or 1 -4, such as 2, 3, 4 phosphate (phosphodiester) linked DNA nucleosides have been shown to be particularly useful in the compounds of the invention,

In some embodiments, the second region comprises or consists of at least between 1 and 10 (e.g. phosphodiester) linked DNA or RNA nucleosides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g. phosphodiester) linked DNA or RNA nucleotides.

In some embodiments, region B comprises no more than 3 or no more than 4 consecutive DNA or RNA nucleosides (such as DNA nucleosides). As such region B may be so short as it does not recruit RNAseH, an aspect which may be important in

embodiments when region B does not form a part of a single contiguous nucleobase sequence which is complementary to the target Shorter region Bs, e.g. of 1 - 4nts in length may also be preferable in some embodiments, as they are unlikely to be the target of sequence specific restriction enzymes. As such it is possible to vary the susceptibility of the region B to endonuclease cleavage, and thereby fine-tune the rate of activation of the active oligomer in vivo, or even intra-cellular. Suitably, if very rapid activation is required, longer region Bs may be employed and/or region Bs which comprise the recognition sites of (e.g. cell or tissue specific or differentially expressed) restriction enzymes.

In some embodiments, a region B may be conjugated to a functional group (C), such as a conjugate, targeting reactive group, an activation group, or blocking group, optinally via a linker group (Y)m such as those provided herein. Functional groups may also be joined to an oligomer region, or the compound of the invention via other means, e.g. via phosphate nucleoside linkage (e.g. phosphodiester, phosphorothioate, phosphodithioate,

boranophosphate or methylphosphonate) or a triazol group. In some aspects, the linkage group is the same as the region B between at least two of the oligomer regions, and as such may be a phosphodiester linkage.

In some embodiments the DNA or RNA nucleotides of an (or more or each) region B are independently selected from DNA and RNA nucleotides. In some embodiments the DNA or RNA nucleotides of an (or more or each) region B are DNA nucleotides. In some embodiments the DNA or RNA nucleotides of an (or more or each) region B are RNA nucleotides.

In the context of the second region, the term DNA and RNA nucleoside may comprise a naturally occurring or non-naturally occurring base (also referred to as a base analogue or modified base).

It will be recognized that, in some embodiments, an (or more or each) region B may further comprise other nucleotides or nucleotide analogues. In some embodiments, (or more or each) region B comprises only DNA or RNA nucleosides. In some embodiments, an (or more or each) region B comprises more than one nucleoside, the internucleoside linkages in an or each region B comprise phosphodiester linkages. In some embodiments, when an (or more or each) region B comprises more than one nucleoside, all the internucleoside linkages in the second region comprise phosphodiester linkages.

In some embodiments, at least two consecutive nucleosides of an (or more or each) region B are DNA nucleosides (such as at least 3 or 4 or 5 consecutive DNA nucleotides). In some embodiments the at least two consecutive nucleosides an (or more or each) region B are RNA nucleosides {such as at least 3 or 4 or 5 consecutive RNA nucleotides). In some embodiments the at least two consecutive nucleosides of the an (or more or each) region B are at least one DNA and at least one RNA nucleoside. The internucleoside linkage between a region A and region B may be a phosphodiester linkage. In some embodiments, when region B comprises more than one nucleoside, at least one further internucleoside linkage is phosphodiester - such as the linkage group(s) between the 2 (or 3 or 4 or 5) nucleosides adjacent to a region A.

A region B may be flanked on at least one side (either 5' or 3') by the first region, e.g. an antisense oligonucleotide, and on the other side (either 3' or 5' respectfully, via a further oligomer region (A^*), or a conjugate moiety or similar group (e.g. a blocking moiety/group, a targeting moiety/group or therapeutic small molecule moiety), optionally via a linker group (i.e. between the second region and the conjugate/blocking group etc. moiety).

Sequence selection in Region B:

In some embodiments, region B does not form a complementary sequence when the oligomer region (e.g. A, A' and/or A") and B is aligned to the complementary target sequence.

In some embodiments, region B does form a complementary sequence when the oligomer region {e.g. A, A' and/or A") and B is aligned to the complementary target sequence. In this respect region A and B together may form a single contiguous sequence which is complementary to the target sequence.

In some embodiments, the sequence of bases in region B is selected to provide an optimal endonuclease cleavage site, based upon the predominant endonuclease cleavage enzymes present in the target tissue or cell or sub-cellular compartment. In this respect, by isolating cell extracts from target tissues and non-target tissues, endonuclease cleavage sequences for use in region B may be selected based upon a preferential cleavage activity in the desired target cell (e.g. liver/hepatocytes) as compared to a non-target cell (e.g. kidney). In this respect, the potency of the compound for target down-regulation may be optimized for the desired tissue/ceil. In some embodiments region B comprises a dinucleotide of sequence AA, AT, AC, AG, TA, TT, TC, TG, GA, GT, CG, CG, GA, GT, GC, or GG, wherein C may be 5- mthylcytosine, and/or T may be replaced with U, In some embodiments region B comprises a trinucleotide of sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, AGA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAG, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGG, 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 wherein C may be 5-mthylcytosine and/or T may be replaced with U. In some embodiments region B comprises a trinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AG AX, 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, wherein X may be selected from the group consisting of A, T, U, G, C and analogues thereof, wherein C may be 5-mthylcytosine and/or T may be replaced with U. It will be recognized that when referring to (naturally occurring) nucleobases A, T, U, G, C, these may be substituted with nucleobase analogues which function as the equivalent natural nucleobase (e.g. base pair with the complementary nucleoside).

In some embodiments, the compound of the invention may comprise more than one conjugate group (or more than one functional group X - such as a conjugate, targeting, blocking or activated group or a reactive or activation group), such as 2 or 3 such groups. In some embodiments, region B is covalently linked, optionally via a [e.g. non-nucleotide] linker group), to at least one functional group, such as two or three functional groups. In some embodiments, the first region (A) may be covalently linked (e.g. via internucleoside linkages, such as phosphodiester linkages), to two region Bs, for example, one 5' and one 3^* to the first region A, wherein each region B may be (optionally independently) selected from the region B described herein.

Multi conjugate oligomeric compounds

In some embodiments, the compound of the invention comprise more than one conjugate region (region C), which may be the same or different. For example, in some embodiments, one of Conjugate 1 and Conjugate2 are a carbohydrate or sterol conjugates and the other is a lipophilic conjugate.

The carbohydrate conjugate moiety (represented by GalNac in the preceding formulas (e.g. when used as conjl or conj2) may for example be selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, Nacetylgalactosamine, N-propionyl- galactosamine, N-n-butanoyl-galactosamine, and N-isobutanoylgalactose-amine. The lipophilic conjugate (e.g. when used as conjl or conj2, and represented as palmotoyl in the preceding formulas) may be a hydrophobic group, such as a C16-20 hydrophobic group, a sterol, cholesterol. Other carbohydrate and lipophilic groups which may be used are, for example, disclosed herein.

The Target(s)

The poly oligomeric compounds of the invention may target one or more nucleic acid targets. In some embodiments each oligomer region targets the same nucleic acid target, and each oligomer region may therefore comprise the same nucleobase sequence (i.e. target the exact same nucleobase sequence of the target), or may have a different nucleobase sequence, i.e. when the nucleabase sequence of at least two, such as all, of the oligomer regions targets (i.e. is complementary to) the same nucleic acid target.

In some embodiments each oligomer region targets a different nucleic acid target, and each oligomer region may therefore comprise a different nucleobase sequence, wherein the nucleabase sequence of at least two, such as ail, of the oligomer regions targets different nucleic acid targets. It will be recognized that when there are more than 2 oligomeric regions, such as three oligomer regions, two of the oligomer regions may target the same nucleic acid target, and the third oligomer region may target a different nucleic acid target. Oligomer regions may, for a non-limiting example, target a nucleic acid selected from the group consisting of a mRNA, a microRNA, a IncRNA (long non-coding RNA), a snRNA, snoRNA, and a viral RNA.

Exemplary, but not limiting mRNA and microRNA targets include for example:

The genes indicated in cancer, such as H if 1 -alpha, survivin, BcI2, Mcl1 , Her2, androgen receptor, beta-catenin, human transforming growth factor TGF-beta2, ras, TNF- alpha, c-RAF, HSPs e.g. Hsp27, elF-4E (e.g. ISIS-EIF4ER_X) STAT3 (e.g. !SiS-STAT3Rx), clusterin (e.g. OGX-011 ), AurkB, AurkA, PBK, miR-155, miR-21 , miR-10b, mir-34 (see WO2011088309), miR-199a, miR-182. Other microRNA targets include miR-221.

The mRNAs of genes involved in inflammation, e.g. !CAM-1 (e.g. Alicoforsen), CD49d, VLA-4 osteopontin, miR-21 (psoriasis),

Other medically relevant mRNA targets include CTGF (local fibrosis) and c-Raf-kinase (ocular disease). miR-29 (cardiac fibrosis), Factor XI (clotting), factor VII (clotting) miR15 miR-159 (post-MI modeling (post-M! modeling), miR-138 (bone-loss), mir-21 (see

WOI 2148952) and mir214 (fibrosis) - see WO2012012716. Metabolic disease or disorders targets, such as Apo-B (high LDL cholesterol, ACS), ApoCIII (high serum TG, diabetes), Apo(a) (cardiovascular disease), FGFR4 (obesity), GCCR (T2 diabetes), GCGR (T2 diabetes), PTP1 B (T2 diabetes), DGAT2 (NASH), PCSK9 (hyperlipidaemia and related disorders), MtGPAT (obesity and NAFLD), miR-122 (high cholesterol), miR-33 (metabolic syndrome, atherosclerosis), miR-208 (chronic heart failure), miR-499 (chronic heart failure), miR-378 (cardio metabolic disease), mir-143 (vascular disease), miR-145 (vascular disease), miR-92 (peripheral arterial disease), miR-375 (diabetes), miR-27b (diabetes), miR-34a (diabetes), miR-199a, miR-27a (heart disease, ischemia), miR-338 (diabetes).

Metabolic diseases include, for examples, metabolic syndrome, obesity,

hyperlipidemia, HDULDL cholesterol imbalance, dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD)., atherosclerosis, heart disease, diabetes (I and/or II), NASH, acute coronary syndrome (ACS),

Viral diseases: miR-451 (polycythemia), miR-122 (HCV), HBV, HCV, BKV, etc. Severe and rare diseases include SMN2 (spinal muscular atrophy), TTR (TTR amyloidosis), GHr (acromegaly), AAT (AATD associated liver disease), Dystophin (Duchennes muscular dystrophy).

In some embodiments, the oligomer of the invention targets a liver expressed nucleic acid, such as a liver expressed mRNA, such as PCSK9, ApoB, or MtGPAT. In some

embodiments, the oligomer of the invention targets PCSK9 mRNA. In some embodiments, the oligomer of the invention targets ApoB mRNA. In some embodiments, the oligomer of the invention targets a liver expressed microRNA, such as miR-122.

Suitable Oligomer regions: In some embodiments, an (or more or all) oligomer region of the invention targets a liver expressed microRNA, such as miR-122 Oligomers targeting miR-122 are disclosed in WO2007/112754, WO2007/112753, WO2009/043353, and may be mixmers, such as SPC3649, also referred to as miravirsen (which has the sequence 5'- CcAttGTcaCaCtCC-3' (SEQ ID NO 1 ) , where capital letters are beta-D-oxy LNA, small letters are DNA, fully phosphorothioate and LNA C are 5-methyl cyctosine), or a tiny LNA, such as those disclosed in WO2009/043353 (e.g. 5'-ACACTCC-3', 5'-CACACTCC-3', 5'- TCACACTCC-3') where capital letters are (optionally beta-D_oxy) LNA, fully

phosphorothioate and LNA Cs are, optionally5-methyl cyctosine). In some embodiments, the miR-122 targeting oligomers have a length of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. In some embodiments, the miR-122 targeting oligomer region comprise a sequence which is fully complementary to miR-122 as measured accross the length of the oligomer, and preferably include the sequence 5'-CACACTCC-3'. According to miRBase, the mature microRNA-122 sequence is 5' uggagugugacaaugguguuugu 3" (SEQ ID NO 2). In some embodiments, the oligomer region targeting a microRNA such as miR-122, is complementary to a corresponding region of the microRNA accorss the length of the oligomer and in some embodiments the 3' nucleoside of the oligomer is compelmentary to ( .e. aligns to) the first, second, third or fourth 5' nucleotides of the microRNA, such as miR- 122, such as the second 5' nucleotide of the microRNA, such as miR-122.

In some embodiments, an (or more or all) oligomer of the invention targets a liver expressed microRNA, such as miR-33 (miR-33a and/or miR-33b), which may be used in treating metabolic disorders such as atherosclerosis (see for example WO2010/120508). Oligomer regions targeting miR-33a/b may comprise a nucleobase sequence selected from the group consisting of 5'-TACAATGCA-3', 5'-ACAATGCAC-3', 5'-ACAATGCA-3' & 5'- CAATGCA-3' , specific oligomer regions targeting miR-33a/b may be 5'-T ACAATGCA-3', 5'- ACAATGCA-3' & 5'-CAATGCA-3\ where capital letters are (optionally beta-D-oxy) LNA, fully phosphorothioate and LNA Cs are, optionally, 5-methyl cyctosine). According to miRBase, the mature microRNA-33a sequence is 5 -GUGCAUUGUAGUUGCAUUGCA-3' (SEQ ID NO 3) , and miR-33b is 5' GUGCAUUGCUGUUGCAUUGC-3' (SEQ ID NO 4).

In some embodiments, the oligomer of the invention targets a liver expressed microRNA, such as miR-21 , which may be used in treating diseases such as liver fibrosis or hepatocellular carcinoma. A compound of the invention may comprise (or more or all) oligomer regions targeting miR-21 may comprise a nucleobase sequence selected from the group consisting of 5'- TGATAAGCT-3', 5'- GATAAGCT-3', 5'- ATAAGCT-3', specific oligomer regions targeting miR-21 may be 5'- TGATAAGCT-3', 5'- GATAAGCT-3', 5'- ATAAGCT-3', or 5' TcAGtCTGaTaAgCT 3' (SEQ ID NO 5) where capital letters are

(optionally beta-D_oxy) LNA, lower case letters are DNA, fully phosphorothioate and LNA Cs are, optionally, 5-methyl cyctosine). A fully LNA oligomer phosphorothioate (e.g. beta-D- oxy-LNA) with sequence 5'- GATAAGCT-3' (LNA C are 5-methylcytosine) has been extensively used in vivo for inhibiting miR-21 (SEQ ID NO 399). According to miRBase, the mature microRNA-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA -3'. In some embodiments the oligomer of the invention comprises two oligomer regions, one which targets a microRNA-21 sequence and a further oligomer region which targets a micro RNA- 155 sequence.

In some embodiments, the oligomer of the invention targets a microRNA, such as miR- 155, which may be used in treating cancer. A compound of the invention may comprise (or more or all) oligomer regions targeting miR-155 which may comprise a nucleobase sequence selected from the group consisting of 5'- TTAGCATTA -3', 5'- TAG C ATT A -3', 5'- AG C ATT A -3', specific oligomer regions targeting miR-21 may be 5'- TTAGCATTA -3', 5'- TAGCATTA -3', 5 - AG C ATT A -3", or 5^* 5'-TcAcgATtaGcAtTA-3' (SEQ ID NO 7) where capital letters are (optionally beta-D-oxy) LNA, lower case letters are DNA, fully

phosphorothioate and LNA Cs are, optionally, 5-methyl cyctosine). SEQ ID NO 304 is a miR-155 sequence.

In some embodiments, a compound of the invention may comprise (or more or all) oligomer region which targets a liver expressed microRNA, such as miR-221 , which may be used in treating, for example, hepatocellular carcinoma. Oligomer regions targeting miR-221 may comprise a nucleobase sequence selected from the group consisting of 5^*- CAATGTAGC-3', 5'- AATGTAGC-3', and 5'- ATGTAGC-3' specific oligomer regions targeting miR-221 include 5^*- C AATGTAGC-3', 5'- AATGTAGC-3', and 5 - ATGTAGC-3', where capital letters are (optionally beta-D-oxy) LNA, fully phosphorothioate and LNA Cs are, optionally, 5-methyl cyctosine). According to miRBase, the mature microRNA-221 sequence is 5' AGCUACAUUGUCUGCUGGGUUUC 3' (SEQ ID NO 8).

Other suitable oligomer regions for targeting microRNAs are disclosed in table 2.

In some embodiments, the oligomer of the invention is capable of down-regulating

(e.g. reducing or removing) expression of the target (e.g. target nucleic acid). In this regards, the oligomer of the invention can affect the inhibition of the target. In some embodiments, the oligomers of the invention bind to the target nucleic acid and affect inhibition of expression of at least 10% or 20% compared to the normal expression level, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to the normal expression level (such as the expression level in the absence of the oligomer(s) or conjugate(s)). In some embodiments, such modulation is seen when using from 0.04 and 25nM, such as from 0.8 and 20nM concentration of the compound of the invention. In the same or a different embodiment, 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 level may be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR. When measuring via mRNA levels, the level of down-regulation when using an appropriate dosage, such as from 0.04 and 25nM, such as from 0.8 and 20nM concentration, is, in some embodiments, typically to a level of from 10- 20% the normal levels in the absence of the compound, conjugate or composition of the invention.

The invention therefore provides a method of down-regulating or inhibiting the expression of oneor more such as two or three target(s) in a cell which is expressing the target(s), said method comprising administering the oligomer or conjugate according to the invention to said cell to down-regulating or inhibiting the expression of the target(s) in said cell. Suitably the cell is a mammalian cell such as a human cell. The administration may occur, in some embodiments, in vitro. The administration may occur, in some embodiments, in vivo.

Oligomer regions in the compounds of the invention, such as the oligomers and conjugates thereof, may be targeted to different targets, such as mRNA or microRNA or other nucleic acid targets which are expressed in the liver (references to NCBI Genbank/Gene IDs are given as examples of sequences which may be targeted by the compounds of the invention - the Genbank / NCBI sequences are hereby incorporated by reference).

ApoB

In some embodiments, the first region (or first and second region) forms a single contiguous nucleobase sequence which is complementary, to a corresponding region of an ApoB mRNA target (i.e. targets) ApoB-100 (NCBI Genbank ID NM_000384.2 Gl:105990531 , hereby incorporated by reference).

Compounds of the invention which target ApoB may be used in the treatment of acute coronary syndrome (see WO20100076248). The invention therefore provides for the oligomer according to the invention which targets ApoB 100 for use in the treatment of acute coronary syndrome. The invention further provides for a method of treatment of acute coronary syndrome, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

Compounds of the invention which target ApoB may be used in the treatment

atherosclerosis. The invention therefore provides for the oligomer according to the invention which targets ApoB 100 for use in the treatment of atherosclerosis. The invention further provides for a method of treatment of atherosclerosis, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

Compounds of the invention which target ApoB may be used in the treatment

hypercholesterolemia or hyperlipidaemia. The invention therefore provides for the oligomer according to the invention which targets ApoB 100 for use in the treatment of

hypercholesterolemia or hyperlipidaemia. The invention further provides for a method of treatment of hypercholesterolemia or hyperlipidaemia, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

The invention provides for an in vivo or in vitro method for the inhibition of ApoB in a cell which is expressing ApoB, said method comprising administering an oligomer or conjugate or pharmaceutical composition according to the invention to said cell so as to inhibit ApoB in said cell. Examples of LNA oligomer regions which may be used as the first region in the oligomers/conjugates of the invention include, for example those disclosed in

WO2007/031081 , WO2008/1 13830, WO2007131238, and WO2010142805, which are hereby incorporated by reference. Specific preferred oligomer regions include the following: 5'- G_s ^mC_sa₈t_et_eg_sg_st_sa_st_sT₈ ^mC_iA -3' (SEQ ID NO 9)

5'- G,T,t .a.c,a_ec.Ug.T.^mC -3' (SEQ ID NO 10)

Wherein capital letters are beta-D-oxy LNA units (nucleosides), lower case letters are DNA units, subscript s is a phosphorothioate linkage, and a superscript m before the capital C illustrates that all LNA cytosines are 5-methyl cytosine. Compounds of theinvention may therefore comprise a first oligomer region which comprises of SEQ ID NO 9, and a second oligomer region which comprises SEQ ID NO 9 or SEQ ID NO 10. Compounds of theinvention may therefore comprise a first oligomer region which comprises of SEQ ID NO 10, and a second oligomer region which comprises SEQ ID NO 9 or SEQ ID NO 10.

Compounds of the invention targeting ApoB may be conjugated to a conjugate which targets the oligomer to the liver, as disclosed herein, such as a carbohydrate or lipophilic conjugate, such as a GalNac conjugate or a sterol conjugate (e.g. cholesterol or tocopherol). The conjugate may be, for example, at the 5' end or the 3^* end of the oligomer compound (suitably via region B). Other oligomers which target ApoB are disclosed in WO03/01 1887, WO04/044181. WO2006/020676, WO2007/131238, WO2007/031081 , and WO2010142805. PCSK9

In some embodiments, the first region (or first and second region) forms a single contiguous nucleobase sequence which is complementary, to a corresponding region of a PCSK9 mRNA target (i.e. targets), such as the human PCSK9 mR A: NCBI Genbank ID

NM_174936.3 Gl:299523249, hereby incorporated by reference.

The invention provides for an oligomer according to the invention which targets

PCSK9, for use as a medicament, such as for the treatment of hypercholesterolemia or related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidaemia, hypercholesterolemia, familiar hypercholesterolemia e.g. gain of function mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidaemia (FCHL). acquired hyperlipidaemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).

The invention provides for the use of an oligomer of the invention which targets PCSK9, for the manufacture of a medicament for the treatment of hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidaemia, hypercholesterolemia, familiar hypercholesterolemia e.g. gain of function mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidaemia (FCHL), acquired hypertipidaemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD),

The invention provides for a method of treating hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting atherosclerosis,

hyperlipidaemia, hypercholesterolemia, familiar hypercholesterolemia e.g. gain of function mutations in PCSK9, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidaemia (FCHL), acquired hyperlipidaemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD), said method comprising administering an effective amount of an oligomer according to the invention which targets PCSK9, to a patient suffering from, or likely to suffer from hypercholesterolemia or a related disorder.

The invention provides for an in vivo or in vitro method for the inhibition of PCSK9 in a cell which is expressing PCSK9, said method comprising administering an oligomer according to the invention which targets PCSK9 to said cell so as to inhibit PCSK9 in said cell.

The following is an oligomer which targets the human PCSK9 mRNA, and may be used as region A in the compounds of the invention.

5'- T,G.^mC,t,a,c,a,a,a_ta,c,^mC,^mC^-3' (SEQ ID NO 1 1 )

Wherein capital letters are beta-D-oxy LNA units (nucleosides), lower case letters are DNA units, subscript s is a phosphorothioate linkage, and a superscript m before the capital C illustrates that all LNA cytosines are 5-methyl cytosine. Compounds of the invention targeting PCSK9 may be conjugated to a conjugate which targets the oligomer to the liver, as disclosed herein, such as a carbohydrate or lipophilic conjugate, such as a GalNac conjugate or a sterol conjugate (e.g. cholesterol or tocopherol). The conjugate may be, for example, at the 5' end or the 3' end of the oligomer compound (suitably via region B).

Other oligomers which target PCSK9 are disclosed in WO2008/043753, WO201 1/009697, WO08/066776, WO07/090071 , WO07/14651 1 , WO07/143315, WO09/148605,

W011/123621 , and W011 133871 , which are hereby incorporated by reference.

miR-122

In some embodiments, the first region (or first and second region) form a single contiguous nucleobase sequence which is complementary, to a corresponding region of a microRNA- 122 such as miR-122a (i.e. targets), such as the has-miR-122 sequences (miRBase release 20: I0000442), such as:

>hsa^»mir-122 MI0000442

CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCU ACUGCUAGGC (SEQ ID NO 12)

>hsa-miR -122 -5p MIMAT0000421 UGGAGUGUGACAAUGGUGUUUG ( 5EQ ID HO 13 )

miR-122 has been indicated in HCV infection, where it is an essential host factor required for maintenance of the infection. Inhibitors of miR-122 may therefore be used in the treatment of hepatitis C infection,

Compounds of the invention which target miR-122 may be used in the treatment of HCV infection. The invention therefore provides for the oligomer according to the invention which targets miR-122 for use in the treatment of HCV infection. The invention further provides for a method of treatment of HCV infection, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

The invention provides for the use of an oligomer of the invention which targets miR-

122, for the manufacture of a medicament for the treatment of HCV infection.

The invention provides for a method of treating HCV infection, said method comprising administering an effective amount of an oligomer according to the invention which targets miR-122, to a patient suffering from HCV infection.

The invention provides for an in vivo or in vitro method for the inhibition of miR-122 in a cell which is expressing miR-122, such as an HCV infected cell or a HCV repiicon expressing cell, said method comprising administering an oligomer or conjugate or pharmaceutical composition according to the invention to said cell so as to inhibit miR-122 in said cell.

miR-122 has also been indicated in cholesterol metabolism, and it has been suggested that inhibition of miR-122 may be used for a treatment to reduce plasma cholesterol levels (Esau, Cell Metab. 2006 Feb;3(2):87-98.)

Inhibitors of miR-122 may therefore be used in a treatment to reduce plasma cholesterol levels, or in the treatment of a metabolic disease associated with elevated levels of cholesterol (related disorders), such as indications selected from the group consisting of atherosclerosis, hyperlipidaemia, hypercholesterolemia, familiar hypercholesterolemia, dyslipidemias, coronary artery disease (CAD), and coronary heart disease (CHD)

Compounds of the invention which target miR-122 may be used in the treatment of elevated cholesterol levels or related disorders. The invention therefore provides for the oligomer according to the invention which targets miR-122 for use in the treatment of elevated cholesterol levels or related disorders. The invention further provides for a method of treatment of elevated cholesterol levels or related disorders, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

The invention provides for the use of an oligomer of the invention which targets miR- 122, for the manufacture of a medicament for the treatment of elevated cholesterol levels or related disorders. The invention provides for a method of treating elevated cholesterol levels or related disorders, said method comprising administering an effective amount of an oligomer according to the invention which targets miR-122, to a patient suffering from said disorder.

The invention provides for an in vivo or in vitro method for the inhibition of miR-122 in a cell which is expressing miR-122, such as an HCV infected cell or a HCV replicon expressing cell, said method comprising administering an oligomer or conjugate or pharmaceutical composition according to the invention to said cell so as to inhibit miR-122 in said cell.

Oligomer's targeting miR-122 are disclosed in WO2007/1 12754, WO2007/1 12753, WO2009/043353, and may be mixmers, such as SPC3649, also referred to as miravirsen see below, or a tiny LNA, such as those disclosed in WO2009/043353 (e.g. 5 -ACACTCC- 3', 5'-CACACTCC-3', 5 -TCACACTCC-3', where capital letters are beta-D_oxy LNA, fully phosphorothioate and LNA C are 5-methyl cytosine). In some embodiments, the miR-122 targeting oligomers have a length of 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 (or 19, 20. 21 , 22 or 23 nucleotides) in length. In some embodiments, the miR-122 targeting oligomers a sequence which is fully complementary to miR-122 as measured across the length of the oligomer, and preferably include the sequence 5'-CACACTCC-3'. In some embodiments, the oligomer targeting a microRNA such as miR-122, is complementary to a corresponding region of the microRNA accorss the length of the oligomer and in some embodiments the 3' nucleoside of the oligomer is compelmentary to (i.e. aligns to) the first, second, third or fourth 5' nucleotides of the microRNA, such as miR-122, such as the second 5' nucleotide of the microRNA, such as miR-122.

The following is an oligomers which targets the has-miR-122 (human miR-122), and may be used as region A in the compounds of the invention.

Miravirsen: 5'- ^raC_sc_sA_st_st_sG_sT_sc_sa_s ^mC_sa_s ^mC_st_s ^mC_s ^mC -3" (SEQ ID NO 1 )

Other miR-122 targeting compounds which may be used in the context of the present invention (region A) are disclosed in WO2007/027894, WO2007/027775.

tGPAT: (NCBI gene ID 57678 - Chromosome:

10;NC_000010.10(1 13907971..113975153, complement) Mitochondrial glycerol-3- phosphate acyltransferase 1 (EC 2.3.1.15, also known as GPAT1 , mtGPATI , GPAM, mtGPAM) plays a major role in hepatic triglyceride formation, where high levels of mtGPATI activity results in fatty liver (hepatosteatosis) whereas the absence of mtGPATI results in low levels of liver triglycerides and stimulated fatty acid oxidation (see WO2010/000656 which discloses oligomers which target mtGPAT. Compounds of the invention which target MtGPAT may be used to treat conditions such as being overweight, obesity, fatty liver, hepatosteatosis, non alcoholic fatty liver disease (NAFLD), non alcoholic steatohepatitis (NASH), insulin resistance, diabetes such as non insulin dependent diabetes mellitus (NIDDM). The following oligomer targets human mtGPAT and may be used as an oligomer region in the compounds of the invention, for example in conjunction with one of the ApoB targeting compounds listed above (SEQ ID NO 9 or SEQ ID NO 10).

5·- A_S° T_S° T_s° c_s c_s c_s t_s g_{s ¾ ¾} t_s G,° T ° G° -3' (SEQ ID NO 14)

Compounds of the invention may therefore comprise a first oligomer region which comprises an mtGPAT targeting oligomer region, and a second oligomer region which targets an ApoB mRNA. FactorV!l {NCBI Gene ID 2155, NCBI J02933.1 GM 80333, or EU557239.1

Gl: 182257998). The oligomer or conjugate of the invention may target FactorVII, and thereby inhibit the production of Factor VII, a key component of the tissue factor coagulation pathway. Compounds of the invention which target FactorVII may be used for the treatment or prevention of thrombotic diseases (typically without causing bleeding) and as heart attack, stroke and blood clots, or inflammatory conditions. WO 2013/119979 and WO 2012/174154, hereby incorporated by reference disclose oligonucleotide compounds which target FVII which may be incorporated into the conjugates of the present invention.

Factor XI (NCBI Genbank BC122863.1 Gl:1 1410821 1 )- Factor Xi, a clotting factor that is produced in the liver. High levels of Factor XI are linked to heart attack, stroke and blood clots. WO 2013/070771 , hereby incorporated by reference, discloses oligonucleotide compounds which target XI which may be incorporated into the conjugates of the present invention. Compounds of the invention which target FactorXI may be used for the treatment or prevention of thrombotic diseases, and as heart attack, stroke and blood clots, or inflammatory conditions such as arthritis and colitis,

ApoClil (NCBI Genbank BC027977.1 G 1:20379764) a protein that regulates triglyceride metabolism in blood. High levels of apoC-lll are linked to inflammation, high triglycerides, atherosclerosis and metabolic syndrome. Compounds of the invention which target ApoClil may be used to reduce serum triglyceride levels or in the treatment of e.g. familial chylomicronemia syndrome and severely high triglycerides either as a single agent or in combination with other triglyceride-lowering agents. W011085271 hereby incorporated by reference, discloses oligonucleotide compounds which target ApoClil which may be incorporated into the conjugates of the present invention.

Apo(a) (NCBI Genbank NM_005577.2 Gl:1 16292749) inhibits the production of apo(a) in the liver and is designed to offer a direct approach to reducing Lp(a), an independent risk factor for cardiovascular disease. High levels of Lp(a) are associated with an increased risk of atherosclerosis, coronary heart disease, heart attack and stroke. Lp(a) promotes premature plaque buildup, or atherosclerosis, in arteries. Compounds of the invention which target Apo(a) may be used in the treatment of e.g. atherosclerosis and coronary heart disease. WO05000201 and WO03014307 hereby incorporated by reference, discloses oligonucleotide compounds which target apolipoprotein (a) which may be incorporated into the conjugates of the present invention.

Hepatitis B (HBV) (see for example NCBI D23884.1 Gl:560092; D23683.1 Gi: 560087; D23682.1 GI: 560082; D23681.1 Gi: 560077; D23680.1 Gi: 560072; D23679.1 GI: 560067; D23678.1 GI: 560062; D23677.1 GI: 560057; all of which are hereby incorporated by reference)

Oligomers which target HBV are well known in the art, for example see, WO96/03152, WO97/0321 1 , WO201 1/052911 , WO2012/145674, WO2012/145697, WO2013/003520 and WO2013/159109.

Compounds of the invention which target HBV may be used in the treatment HBV infection. The invention therefore provides for the oligomer according to the invention which targets HBV for use in the treatment of HBV. The invention further provides for a method of treatment of HBV infection, wherein said method comprises the administration of the oligomer of the invention to a subject in need to said treatment.

The invention provides for the oligomer or conjugate of the invention which targets hepatitis B (HBV) for use as a medicament, such as for the treatment hepatitis B infection or a related disorder.

The invention provides for the use of an oligomer or conjugate or pharmaceutical composition according to the invention which targets hepatitis B (HBV), for the manufacture of a medicament for the treatment of hepatitis B infection or a related disorder,

The invention provides for a method of treating treatment hepatitis B infection or a related disorder, said method comprising administering an effective amount of an oligomer or conjugate of the invention which targets HBV, to a patient infected with Hepatitis B virus. The invention provides for an in vivo or in vitro method for the inhibition of HBV replication in a cell infected with HBV, said method comprising administering an oligomer or conjugate of the invention which targets HBV to said cell so as to inhibit HBV replication. An example of an LNA oligomer which target's HBV is (as is disclosed in WO2011/47312) which may be used as the oligomer (region A) of the invention 5'- G_sA_sG_eGgC_ea_st_sa»gsC,a_eg_s ^mC_eA_eG_sG - 3'. Further compounds are disclosed in table 1 of WO201 1/47312, and in WO201 1/05291 1 , WO2012/145674, WO2012/145697, WO2013/003520 and WO2013/159109, hereby incorporated by reference. RG-101 is a compound which targets miR-122 and comprises a GalNac conjugate, and is being developed for treatment of HCV by Regulus Therapeutics.

ANGPTL3, (e.g. NCBi BC007059.1 G!: 14712025 or BC058287.1 Gi: 34849466)

ANGIOPOIETIN-LIKE 3 - a protein that regulates lipid, glucose and energy metabolism, Humans with elevated levels of ANGPTL3 have hyperlipidemia associated with an increased risk of premature heart attacks, increased arterial wall thickness as well as multiple metabolic abnormalities, such as insulin resistance. In contrast, humans with lower levels of ANGPTL3 have lower LDL-C and triglyceride levels and a lower risk of cardiovascular disease. Compounds of the invention which target ANGPTL3 may be used in the treatment of e.g. hyperlipidemia and related disorders, metabolic disorder, atherosclerosis, coronary heart disease or insulin resistance, W0 1085271 hereby incorporated by reference, discloses oligonucleotide compounds which target ANGPTL3 which may be incorporated into the conjugates of the present invention.

Glucagon receptor, or GCGR (BC1 12041 .1 GI: 85567507; L20316.1 GI: 405189):

Glucagon is a hormone that opposes the action of insulin and stimulates the liver to produce glucose, particularly in type 2 diabetes. In patients with advanced diabetes, uncontrolled glucagon action leads to a significant increase in blood glucose levels. Therefore, attenuating glucagon action may have a significant glucose lowering effect in patients with severe diabetes. In addition, reducing GCGR produces more active glucagon-like peptide, or GLP-1 , a hormone that preserves pancreatic function and enhances insulin secretion.

Compounds of the invention which target GCGR may be used in the treatment of e.g. or insulin resistance, hyperglycemia, diabetes, such as type 1 or 2 diabetes, preservation of pancreatic function, and to control of blood glucose levels, WO2007/134014 discloses oligonucleotide compounds which target GCGR which may be incorporated into the conjugates of the present invention.

Fibroblast growth factor receptor 4, or FGFR4. (NCBI Gene 2264 - NC_000005.9 ( 176513906..176525143) FGFR4 is expressed in the liver and fat tissues, and is indicated in decreasing the body's ability to store fat while simultaneously increasing fat burning and energy expenditure. Many anti-obesity drugs act in the brain to suppress appetite, commonly resulting in CNS side effects. Compounds of the invention which target FGFR4 may be used in the treatment of e.g. or insulin resistance, hyperglycemia, diabetes, such as type 1 or 2 diabetes, preservation of obesity (e.g. when used in combination with an appetite- suppressing drug), reducing body weight, and improvement in insulin sensitivity, diabetes, such as type 1 or 2 diabetes and to control of blood glucose levels. W 009046141 and W012174476 hereby incorporated by reference disclose oligonucleotide compounds which target FGFR4 which may be incorporated into the conjugates of the present invention.

Diacylglycerol acyltransferase-2, or DGAT-2 (NCBI GENE ID 84649): A key component in the synthesis of triglycerides. The inhibition of DGAT may reduce liver fat in patients with Nonalcoholic Steatohepatitis (NASH), and may also be used to treat type 2 diabetes and insulin resistance. Compounds of the invention which target DGAT-2 may be used to treat NASH, to reduce liver fat, to treat diabetes, such as type 2 diabetes, and treat insulin resistance. WO05019418 and WO2007136989, hereby incorporated by reference disclose oligonucleotide compounds which target DGAT-2 which may be incorporated into the conjugates of the present invention.

Glucocorticoid receptor, or GCCR (BC150257.1 Gi: 152013043): Glucocorticoid hormones affect a variety of processes throughout the body, and excessive levels of glucocorticoid hormones can have a detrimental effect on many of the tissues and organs in the body, Cushing's Syndrome is an orphan disease caused by prolonged exposure to high levels of glucocorticoids. If untreated, patients with Cushing's Syndrome can develop hypertension, diabetes and impaired immune functions and have an increased risk of early death. Although there are approved treatments for Cushing's Syndrome, current medicines are associated with significant side effects, such as hypertension and diabetes, and there remains a high unmet medical need for new therapies for these patients. Compounds of the invention which target GCCR-2 may be used to treat Cushing's Syndrome and associated conditions (such as those listed above), WO07035759 and WO2007 36988, which are hereby incorporated by reference disclose oligonucleotide compounds which target GCCR which may be incorporated into the conjugates of the present invention.

Complement component C5 ( 57729.1 GI: 179982): The complement system plays a central role in immunity as a protective mechanism for host defense, but its dysregulation results in serious, life-threatening complications in a broad range of human diseases including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic-uremic syndrome (aHUS), myasthenia gravis, neuromyelitis optica, amongst others. Compounds of the invention which target complement component C5 may be used to treat one or more of these disorders. C5 is a genetically and clinically validated target; loss of function human mutations are associated with an attenuated immune defense against certain infections and intravenously administered anti-C5 monoclonal antibody therapy has demonstrated clinical activity and tolerability in a number of complement-mediated diseases, transmembrane protease, serine 6 (Tmprss6) for the treatment of beta-thalassemia and iron-overload disorders. Alpha-1 antitrypsin (AAT): ( 1 1465.1 Gl: 177826) Liver disease associated with - W013142514 which is hereby incorporated by reference disclose oligonucleotide compounds which target AAT which may be incorporated into the oligomers or conjugates of the present invention. Compounds of the invention which target AAT may be used in methods for decreasing AIAT mRNA and protein expression and treating, ameliorating, preventing, slowing progression, or stopping progression of fibrosis, such as, AIATD associated liver disease, and pulmonary disease, such as, AIATD associated pulmonary disease in an individual in need thereof.

Transthyretin - TTR (BC005310.1 Gl: 13529049) : The oligomers of the invention which target TTR may be used to treat transthyretin amyloidosis, or TTR amyloidosis, a severe and rare genetic disease in which the patient inherits a mutant gene that produces a misfolded form of TTR, which progressively accumulates in tissues. In patients with TTR amyloidosis, both the mutant and normal forms of TTR can build up as fibrils in tissues, including heart, peripheral nerves, and the gastrointestinal tract. The presence of TTR fibrils interferes with the normal functions of these tissues, and as the TTR protein fibrils enlarge more tissue damage occurs and the disease worsens, TTR is a carrier protein that transports a thyroid hormone and retinol in the blood. In patients with TTR amyloidosis, both the mutant and normal forms of TTR can build up as fibrils in tissue. The compounds of the invention may be used to treat TTR amyloidosis. See Benson et al., Amyloid. 2010 Jun;17(2):43-9, and Ackermann et al., Amyloid. 2012 Jun;19 Suppl 1 :43-4.). Antisense compounds targeting TTR which may be used in the oligomers or conjugates of the invention are disclosed in US8101743, W011 139917 and W010017509, which are hereby incorporated by reference.

Aminolevulinate synthase-1 (ALAS-1) (BC01 1798.2 G!: 33877783; AK312566.1 Gl:

164690365; NM_199166.2 Gl: 362999012; NM_000688.5 Gl: 36299901 1 ). ALAS1 is a validated target for the treatment of porphyria, such as the treatment of hepatic porphyrias including acute intermittent porphyria (AIP). Compounds of the invention which target ALAS-1 may be used in the treatment of these disorders.

Vascular endothelial growth factor, or VEGF (GENE ID 7422, human Sequence:

Chromosome: 6; NC_000006.1 1 (43737946 .43754224)). VEGF is indicated in cancers. Compounds of the invention which target VEGF may be used in the treatment of

hyperproliferative disorders, such as cancer, such as liver cancer.

Table 1 provides for a group of liver targets which may be targeted by the compounds of the invention, as well as the medical indication / disorder for which such compounds may be used to treat (such as a person suffering from the associated disorder) (See Sehgal et al., Liver as a target for oligonucleotide therapeutics, J. of Hepatology 2013, In Press). Table 1

Hepatic Glucose 6-Phosphate glucose homeostasis, diabetes, type

Transporter- 1 2 diabetes

Sequences

In some embodiments, the oligomers, or first region thereof, comprise a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence present in the target nucleic acid (i.e. the sequence which the oligomer targets).Table 3 provides a group of mRNA and miRNA targets which are in pre-clinical or clinical development using oligonucleotide compounds for the associated indication, and are therefore suitable for targeting with the compounds of the present invention.

In some embodiments the target is selected from the group consisting of; miR-122 ,ApoB-100 .ApoCIII ,PCSK9 ,CRP ,KSP, VEGF ,PLK1 ,miR-34 ,FGFR4 .Factor iXa .Factor Xi ,TTR ,GCCR ,ΡΤΡ-1 B ,GCGR, AAT .ALDH2 ,ΗΑΜΡ pathway, miR-33 ,Apo(a) ,miR-7

,miR-378 ,miR-21 ,Myc ,miR-122 , the HCV genome such as the HCV 5'UTR or HCV NS5B RNA or NS3 RNA .TMPRSS6 .Antithrombin 111 .ApoCIII .ANGPLT3 ,ΜΤΡ .DGAT2 ,ALAS1 .Antithrombin 111 .Serum amyloid A and Factor VII,

In some embodiments, the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence. Region B may however be non- complementary and may therefore be disregarded when determining the degree of complementarity.

In determining the degree of "complementarity" between oligomers of the invention (or regions thereof) and the target region of the nucleic acid, such as those disclosed herein, the degree of "complementarity^*' (also, "homology" or "identity") is expressed as the percentage identity (or percentage homology) between the sequence of the oligomer (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical between the 2 sequences, dividing by the total number of contiguous monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of monomers within the gap differs between the oligomer of the invention and the target region.

As used herein, the terms "homologous" and "homology" are interchangeable with the terms "identity" and "identical".

The terms "corresponding to" and "corresponds to" refer to the comparison between the nucleotide sequence of the oligomer (i.e. the nucleobase or base sequence) or contiguous nucleotide sequence (a first region) and the equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the nucleic acid target. Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides. A first sequence which corresponds to a further sequence under i) or ii) typically is identical to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence) or, as described herein may, in some embodiments, is at least 80% homologous to a corresponding sequence, such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, such as 100% homologous (identical).

The terms "corresponding nucleotide analogue" and "corresponding nucleotide" are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical. For example, when the 2-deoxyribose unit of the nucleotide is linked to an adenine, the "corresponding nucleotide analogue" contains a pentose unit {different from 2-deoxyribose) linked to an adenine.

The terms "reverse complement", "reverse complementary" and "reverse

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

The contiguous nucleobase sequence of the oligomer (first region or first and second region) may therefore be complementary to a target, such as those referred to herein.

In some embodiments, the first region or first and second region form a single contiguous nucleobase sequence which is complementary to a region of a mRNA target, such as those referred to herein, including, for example, ApoB-100 (NM_000384.2

Gl: 105990531 or PCSK9 (NM_174936.3 Gl:299523249).

Nucleosides and Nucleoside analogues

The term "nucleotide" as used herein, refers to a glycoside comprising a sugar moiety (or analogue thereof), a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucieotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as "nucleotide analogues" herein. Herein, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit

It will be recognized that in the context of the present invention the term nucleoside and nucleotide are used to refer to both naturally occurring nucleotides/sides, such as DNA and RNA, as well as nucleotide/side analogues. Thus, "nucleobase" covers not only the known purine and pyrimidine heterocycles but also heterocyclic analogues and tautomeres thereof. It will be recognised that the DNA or RNA nucleosides of region B may have a naturally occurring and/or non-naturally occurring nucleobase(s), such as DNA nucleobases independently selected from the group A, C, T and G, or the group C, T and G.

In field of biochemistry, the term "nucleoside" is commonly used to refer to a glycoside comprising a sugar moiety and a base moiety, and may therefore be used when referring to the nucleotide units, which are covalently linked by the internucleoside linkages between the nucleotides of the oligomer. In the field of biotechnology, the term "nucleotide" is often used to refer to a nucleic acid monomer or unit, and as such in the context of an oligonucleotide may refer to the base - such as the "nucleotide sequence", typically refer to the nucleobase sequence (i.e. the presence of the sugar backbone and internucleoside linkages are implicit). Likewise, particularly in the case of oligonucleotides where one or more of the internucleoside linkage groups are modified, the term "nucleotide" may refer to a

"nucleoside" for example the term "nucleotide" may be used, even when specifying the presence or nature of the linkages between the nucleosides.

As one of ordinary skill in the art would recognize, the 5' terminal nucleotide of an oligonucleotide does not comprise a 5' internucleoside linkage group, although may or may not comprise a 5' terminal group.

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

"Nucleotide analogues" are variants of natural nucleotides, such as DNA or RNA nucleotides, by virtue of modifications in the sugar and/or base moieties. Analogues could in principle be merely "silent" or "equivalent" to the natural nucleotides in the context of the oligonucleotide, i.e. have no functional effect on the way the oligonucleotide works to inhibit target gene expression. Such "equivalent" analogues may nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label. Preferably, however, the analogues will have a functional effect on the way in which the oligomer works to inhibit expression; for example by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the ceil. Specific examples of nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1 :

Phosphor* loate 2 -O-Methyl 2'-MOE I taoiu

Botanophosphafcs

Scheme 1

The oligomer may thus comprise or consist of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred here generally as "DNA"), but also possibly ribonucleotides (referred here generally as " NA"), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues. Such nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.

Examples of suitable and preferred nucleotide analogues are provided by

WO2007/031091 or are referenced therein. Other nucleotide analogues which may be used in the oligomer of the invention include tricyclic nucleic acids, for example please see WO2013154798 and WO2013154798 which are hereby incorporated by reference.

Incorporation of affinity-enhancing nucleotide analogues in the oligomer, such as LNA or 2'-substituted sugars, can allow the size of the specifically binding oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place.

Oligomeric compounds, such as antisense oligonucleotides, such as the compounds referred to herein, including region A, and in some optional embodiments, region B, may contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides (nucleoside analogues) may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In some embodiments, nucleosides comprise a chemically modified

ribofiiranose ring moiety.

In some embodiments, the oligomer, or first region thereof, comprises at least one , such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24 or 25 nucleoside analogues, such as sugar modified nucleoside analogues.

Bicyclic nucleoside analogues include nucleoside analogues which comprise a bridge (or biradical) linking the second and forth carbon of the ribose ring, (C4^*-C2^* bridge or biradical). The presence of the biradical between the 2^nd and 4* carbon locks the ribose into a 3' endo- (north) confirmation, and as such bicyclic nucleoside analogues with a C2^*-C4^* biradical are often referred to as Locked nucleic acid (LNA). In some embodiments the nucleoside analogues are (optionally independently selected from the group consisting of bicyclic nucleoside analogues (such as LNA), and/or 2^* substituted nucleoside analogues, such as (optionally independently) selected from the group consisting of 2'-0-alkyl-RNA units, 2 -OMe-RNA units, 2'-amino-DNA units, 2'-AP, 2'-FANA, 2'-(3-hydroxy)propyl, and 2'- fluoro-DNA units, and/or other (optionally) sugar modified nucleoside analogues such as morpholino, peptide nucleic acid (PNA), CeNA, unlinked nucleic acid (UNA), hexitol nucleoic acid (HNA). bicyclo-HNA (see e.g. WO2009/100320), In some embodiments, the nucleoside analogues increase the affinity of the first region for its target nucleic acid (or a

complementary DNA or RNA sequence).

In some embodiments, the oligomer comprises at least one bicyclic nucleotide analogue, such as LNA. In some embodiments, the first region comprises of at least one bicyclic nucleoside analogues (e.g. LNA) and/or 2'substituted nucleoside analogues. In some embodiments, the nucleoside analogues present in the oligomer all comprise the same sugar modification. In some embodiments, at least one nucleoside analogue present in the first region is a bicyclic nucleoside analogue, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, for example all nucleoside analogues (except the DNA and or RNA nucleosides of region B) are sugar modified nucleoside analogues, such as such as bicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA or alpha-L- X-LNA (wherein X is oxy, amino or thio), or other LNAs disclosed herein including, but not limited to, R/S) cET, cMOE or 5 -Me-LNA. Examples of chemically modified ribofiiranose rings include, without limitation, addition of substituent groups (including 5^* and 2' substituent groups); bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(Ri){R₂) (R = H, Ci -C₂ alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars include, 2'-F-5'-methyl substituted nucleoside (see, PCT International Application WO 2008/101 157, published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides), replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see, published U.S. Patent Application

US2005/0130923, published on June 16, 2005), or, alternatively, S'-substitution of a BNA (see, PCT International Application WO 2007/134181 , published on 1 1/22/07, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).

Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5'-vinyl, 5 -methyl (R or S), 4"-S, 2'-F, 2'-OCH₃, and 2'-0(CH₂)2 O CH₃ substituent groups. The substituent at the 2^* position can also be selected from allyl, amino, azido, thio, O-allyl, O-C Ci₀ alkyl, OCF₃, O (CH₂)₂SCH₃, O (CH₂)2- O -N(Rm)(Rn), and O -CH₂-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-C ₀ alkyl.

As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, without limitation, nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In some embodiments, compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4' to 2' bicyclic nucleoside. Examples of such 4" to 2' bicyclic nucleosides, include, but are not limited to, one of the formulae: 4"-(CH₂)- O -2" (LNA); 4'- (CH₂)-S-2'; 4'-(CH₂)₂- O -2' (ENA); 4'-CH(CH₃)- O -2' and 4'-CH(CH₂OCH₃)-0-2\ and analogs thereof (see, U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH₃)(CH₃)-0-2', and analogs thereof (see, published PCT International Application WO2009/008478, published January 8, 2009); 4'-CH₂-N(OCH₃)-2', and analogs thereof (see, published PCT International Application WO2008/150729, published December 1 1 , 2008); 4'-CH₂-0- N(CH₃)-2' (see, published U.S. Patent Application US2004/0171570, published September 2, 2004); 4^*-CH₂-N(R)- O -2^*. wherein R is H, C C₁₀ alkyl, or a protecting group (see, U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH₂-C(H)(CH₃)-2' (see,

Chattopadhyaya, et al, J. Org. Chem.,2009, 74, 1 18-134); and 4'-CH₂-C(=CH₂)-2\ and analogs thereof (see, published PCT International Application WO 2008/154401 , published on 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. U. S. A., 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. Ther., 2001 , 3, 239-243; U.S. Patent Nos U.S. 6,670,461 , 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034, 133, 6,525,191 , 7,399,845; published PCT International applications WO 2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181 ; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831 , and US2008/0039618; and U.S. Patent Serial Nos. 12/129, 154, 60/989,574, 61/026,995, 61/026,998, 61/056,564,

61/086,231 , 61/097,787, and 61/099,844; and PCT international Application Nos.

PCT/US2008/064591 , PCT/US2008/066154, and PCT/US2008/068922. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and beta -D-ribofuranose (see PCT international application PCT DK98/00393, published on March 25, 1999 as WO 99/14226).

In some embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from - [CiR_aXR_b)]„-, -C(R_a)=C(R_b)-, -C(R_a)=N-, - C{=NR_e)-, -C(=0)-, -C(=S)-, - O -, -Si(Ra)₂-, -S(=0)_x-, and -N(Ra)-; wherein: x is 0, 1 , or 2; n is 1 , 2, 3, or 4; each R_a and R_b is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C C ₂ alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C2₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ-,J₂, SJi, N₃, COOJ,, acyl (C(=0)- H), substituted acyl, CN, sulfonyl (S(=0)₂-Ji), or sulfoxyl (S(=0)-J₁); and each Ji and J₂ is, independently, H, C C₆ alkyl, substituted C1-C12 alkyl, C₂-Gi₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-Ci₂ alkynyl, C5-C20 aryl, substituted C₅-C o aryl, acyl (C(=0)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1 -C ₂ aminoalkyl, substituted C C₁₂ aminoalkyl, or a protecting group.

In some embodiments, the bridge of a bicyclic sugar moiety is, -[C(R_a)(Rb)]_n-, - [C(R_a)(R_b)]_n- O -, -C(R_aR_b)-N(R)- O - or, -C(R_aR_b)- O -N(R)-. In some embodiments, the bridge is 4'-ΟΗ₂-2·, 4'-(CH2)2-2', 4'- (CH₂)₃-2\ 4"-CH₂- O -2', 4*-(CH₂)2- O -2", 4"-CH₂- O - N(R)-2', and 4'-CH₂-N(R)- O -2'-, wherein each R is, independently, H, a protecting group, or C C₁₂ alkyl.

In some embodiments, bicyclic nucleosides are further defined by isomeric

configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the a-L configuration or in the beta - D configuration. Previously, a-L-methyleneoxy (4'-CH₂- 0-2") BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21 , 6365- 6372).

In some embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L- Methyleneoxy (4'-CH₂-0-2') BNA, (B) beta -D-Methyleneoxy (4'-CH₂-0-2') BNA, (C) Ethyleneoxy (4'-(CH₂)₂-0-2') BNA, (D) Aminooxy (4'-CH₂-0-N(R)-2') BNA, (E) Oxyamino (4'- CH₂-N(R)-0-2") BNA, (F), Methyl (methyleneoxy) (4'-CH{CH₃)-0-2') BNA, (G) methylene-thio (4 -CH2-S-2') BNA, (H) methylene- amino (4'-CH₂-N(R)-2') BNA, (I) methyl carbocyclic (4'- ) BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protecting group or Ci-C₂ alkyi. odiments, bicyclic nucleoside having Formula I:

I

wherein:

Bx is a heterocyclic base moiety;

-Q_a-Q_b-Q_c- is -CH₂-N(Rc)-CH₂-, -C(=0)-N(R_c)-CH₂-, -CH₂-0-N(Rc)-, -CH₂-N(Rc)-0-, or^■ N(Rc)-0-CH₂;

R_c is C₁-C₁₂ alkyi or an amino protecting group; and T_a and T_b are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium.

In some embodiments, bicyclic nucleoside having Formula II:

li

wherein:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; Z_a is C C₆ alkyl, C₂-C₆ alkenyi, C₂-C₆ alkynyl, substituted C C₆ alkyl, substituted C₂-C₆ alkenyi, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio.

In some embodiments, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_c, J _d, SJ_C, N₃, OC(=X)J_c, and NJ_eC(=X)NJ_cJ_d, wherein each J_c, J_d, and J_e is, independently, H, Ci-C₆ aikyl, or substituted d-C₆ alkyl and X is O or NJ_C.

In some embodiments, bicyclic nucleoside having Formula III:

III wherein:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; R_d is C C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyi, substituted C C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C_e alkynyi, or substituted acyl (C(=0)-).

In some embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covaient attachment to a support medium;

R_d is C,-C₆ alkyl, substituted C,-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyi, substituted C₂-C₆ alkynyi; each q_b, q_c and q_d is, independently, H, halogen, C C₆ alkyl, substituted C C₆ alkyl, C₂-Ce alkenyl, substituted C₂-C₆ alkenyl, C₂-C_e alkynyi, or substituted C₂-C6 alkynyi, C C₆ alkoxyi, substituted Q- C₆ alkoxyi, acyl, substituted acyl, C C₆ aminoalkyl, or substituted C Cg aminoalkyl;

In some embodiments, bicyclic nucleoside having Formula V:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covaient attachment to a support medium; q_a, q_b, q_c and q_f are each, independently, hydrogen, halogen, d-C ₂ alkyl, substituted C C ₂ alkyl, C₂- C ₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C ₂ alkynyi, substituted C₂-Ci₂ alkynyi, Ci-Ci₂ alkoxy, substituted C Ci₂ alkoxy, OJ_j, SJ_j, SOJ_Jf S0₂J_j, NJ_jJ_k, N₃, CN, C(=0)OJ,, C(=0)NJ,J_k, C(=0)J_j, 0-C(=0)IMJ_jJ_k, N(H)C(=NH)NJ_jJ_k,

N(H)C(=0)NJ_jJ_k or N(H)C(=S)NJ_jJ_k; or q@ and q_f together are =C(q_g)(q_h); q_g and q_h are each, independently, H, halogen, C C_i2 alkyl, or substituted Ci-C₁₂ alkyl. The synthesis and preparation of the methyleneoxy (4'-CH₂-0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4'-CH₂-0-2') BNA, methyleneoxy (4'-CH_rO-2') BNA, and 2'-thio- BNAs, have also been prepared {see, e.g., Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (see, e.g., Wengel et al., WO 99/14226), Furthermore, synthesis of 2'-amino-BNA, a novel

comformationally restricted high-affinity oligonucleotide analog, has been described in the art (see, e.g., Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'- amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In some embodiments, the bi c clic nucleoside has Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_a and T_b are each, independently, H, a hydroxy I protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; each qj, qj, q_k and ql is, independently, H, halogen, C C₁₂ alkyl, substituted C C₁₂ alkyl, C₂-C12 alkenyl, substituted C₂-Ci₂ alkenyl, C₂-Ci₂alkynyl, substituted C₂-C₁₂ alkynyl, Ci-C₁₂ alkoxyl, substituted C₂- C₁₂ alkoxyl, OJ_j( SJ_j, SOJ_jt S0₂J_j, NJ_jJ_kl N₃, CN, C(=0)OJ_jf

and qi and q_j or ql and q_k together are =C(q_g)(q_h), wherein q_g and q_h are each,

independently, H, halogen, C C ₂ alkyl, or substituted C C₆ alkyl.

One carbocyclic bi cyclic nucleoside having a 4'-(CH₂)₃-2" bridge and the alkenyl analog, bridge 4'- CH=CH-CH₂-2', have been described (see, e.g., 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 along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

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

As used herein, "monocylic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In some embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, "2'-modified sugar" means a furanosyl sugar modified at the 2" position. In some embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and

unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and

unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In some embodiments, 2^* modifications are selected from substituents including, but not limited to: 0[(CH₂)_nO]_mCH₃₎ 0(CH₂)„NH₂, 0(CH₂)„CH₃, 0{CH₂)„ONH₂₎

OCH₂C(=0)N(H)CH₃, and 0(CH2)_nON[(CH₂)_nCH₃]2, where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: C C₁₂ alkyl; substituted alkyl;

alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH₃; OCN; Ci; Br; CN; CF₃; OCF₃; SOCH₃; S0₂CH₃; ON0₂; N0₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;

aminoalkylamino; polyalkylamino; substituted silyl; an R; a cleaving group; a reporter group; an intercalator; a group for improving pharmacokinetic properties; and a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In some embodiments, modified nucleosides comprise a 2 -MOE side chain {see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 1 1944-12000). Such 2 -MOE substitution have been described as having improved binding affinity compared to

unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2 -MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use {see, e.g., Martin, P., He/v. Chim. 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, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified ?THP

nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) {see Leumann, CJ. Bioorg. and Med, Chem. (2002) 10:841 -854), fluoro HNA (F-HNA), or those compounds having

Formula X:

X wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the

tetrahydropyran nucleoside analog to the antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃ and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or S'-terminal group; q_t q₂ q₃ q₄ q₅, q₆ and q₇ are each, independently, H, C C₆ alkyl, substituted C C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C_e alkynyl; and one of R, and R₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ,J₂, SJ„ N₃,

NJ₃C(=X) HJi ₂, and CN, wherein X is O, S, or and each J-,, J₂, and J₃ is, independently, H or C C₆ alkyl.

In some embodiments, the modified THP nucleosides of Formula X are provided wherein q_m, q_n, q_p, q_r, q_s, q_t, and q_u are each H. In some embodiments, at least one of q_m, q_n, q_p, q_r, q_s, q_t and q_u is other than H. In some embodiments, at least one of q_m, q„, q_p, q_r, q„, q_t and q_u is methyl. In some embodiments, THP nucleosides of Formula X are provided wherein one of Ri and R₂ is F, In some embodiments, Riis fluoro and R₂ is H, Ri is methoxy and R₂ is H, and Ri is methoxyethoxy and R₂ is H.

As used herein, "2'-modified" or "2'-substituted" refers to a nucleoside comprising a sugar comprising 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 ailyl, amino, azido, thio, O-allyl, O-C Ci₀ alkyl, -OCF₃, 0-{CH₂)₂-0-CH₃, 2'- 0(CH₂)₂SCH₃, 0-(CH₂)₂-0- N{R_m)(R_n), or 0-CH₂-C(=0)-N(R_m)(R_n), where each R_m and R„ is, independently, H or substituted or unsubstituted C C ₀ alkyl. 2'-modifed nucleosides may further comprise other modifications, for example, at other positions of the sugar and/or at the nucleobase.

As used herein, "2'-F" refers to a sugar comprising a fluoro group at the 2' position. As used herein, "2'-OMe" or "2'-OCH₃" or "2'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH₃ group at the 2^* position of the sugar ring.

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

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

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds {see, e.g., 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 the preparations of modified sugars are well known to those skilled in the art. In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In some embodiments, antisense compounds comprise one or more nucleotides having modified sugar moieties. In some embodiments, the modified sugar moiety is 2'- MOE. In some embodiments, the 2'-MOE modified nucleotides are arranged in a gapmer motif. In some embodiments, the modified sugar moiety is a cEt. In some embodiments, the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.

In some embodiments, in the BNA (LNA), R^4" and R^2* together designate the biradical -0-CH(CH₂OCH₃)- (2'0-methoxyethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem) - in either the R- or S- configuration.

In some embodiments, in the BNA (LNA), R^4" and R^2* together designate the biradical -0-CH(CH₂CH₃)- (2'O-ethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem), - in either the R- or S- configuration.

In some embodiments, in the BNA (LNA), R^4* and R^2* together designate the biradical -0-CH(CH₃)-. - in either the R- or S- configuration. In some embodiments, R^4* and R^2* together designate the biradical -0-CH₂-0-CH₂- - (Seth at al., 2010, J. Org. Chem).

In some embodiments, in the BNA (LNA), R^4* and R^2* together designate the biradical -0-NR-CH₃- (Seth at al., 2010, J. Org. Chem) .

In some embodiments, the LNA units have a structure selected from the following group:

(R.S)-cEt (RSj-cMOE (RSJ-S'- e-LNA

The oligomer may thus comprise or consist of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred to here generally as "DNA"), but also possibly ribonucleotides (referred to here generally as "RNA"), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues. Such nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.

Incorporation of affinity-enhancing nucleotide analogues in the oligomer, such as BNA, (e.g.) LNA or 2^*-substituted sugars, can allow the size of the specifically binding oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before nonspecific or aberrant binding takes place.

In some embodiments, the oligomer comprises at least 1 nucleoside analogue. In some embodiments the oligomer comprises at least 2 nucleotide analogues. In some embodiments, the oligomer comprises from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In the by far most preferred embodiments, at least one of said nucleotide analogues is a BNA, such as 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 BNA, such as LNA. In some embodiments all the nucleotides analogues may be BNA, such as LNA.

It will be recognized that when referring to a preferred nucleotide sequence motif or nucleotide sequence, which consists of only nucleotides, the oligomers of the invention which are defined by that sequence may comprise a corresponding nucleotide analogue in place of one or more of the nucleotides present in said sequence, such as BNA units or other nucleotide analogues, which raise the duplex stability/T_m of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues).

A preferred nucleotide analogue is LNA, such as oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino- LNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA). Most preferred is beta-D-oxy-LNA.

In some embodiments the nucleotide analogues present within the oligomer of the invention are independently selected from, for example: 2 '-O-alkyl-RNA units, 2'-amino-DNA units, 2^*-fluoro-DNA units, BNA units, e.g. LNA units, arabino nucleic acid (ANA) units, 2^*- fluoro-ANA units, HNA units, INA (intercalating nucleic acid -Christensen, 2002. Nucl. Acids, Res. 2002 30: 4918-4925, hereby incorporated by reference) units and 2'MOE units. In some embodiments there is only one of the above types of nucleotide analogues present in the oligomer of the invention, such as the first region, or contiguous nucleotide sequence thereof.

In some embodiments the nucleotide analogues are 2'-0-methoxyethyl-RNA (2'MOE),

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

embodiments at least one of said nucleotide analogues is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nucleotide units.

In some embodiments, the oligomer according to the invention comprises at least one BNA, e.g. Locked Nucleic Acid (LNA) unit, such as 1 , 2, 3, 4, 5, 6, 7, or 8 BNA/LNA units, such as from 3 - 7 or 4 to 8 BNA/ LNA units, or 3, 4, 5, 6 or 7 BNA/LNA units. In some embodiments, all the nucleotide analogues are BNA, such as LNA. In some embodiments, the oligomer may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In some embodiments all BNA, such as LNA, cytosine units are 5'methyl-Cytosine. In some embodiments of the invention, the oligomer (such as the first and optionally second regions) may comprise both BNA and LNA and DNA units. In some embodiments, the combined total of LNA and DNA units is 10-25, such as 10 - 24, preferably 10-20, such as 10 - 18, such as 12-16. In some embodiments of the invention, the nucleotide sequence of the oligomer, of first region thereof, such as the contiguous nucleotide sequence consists of at least one BNA, e.g. LNA and the remaining nucleotide units are DNA units. In some embodiments the oligomer, or first region thereof, comprises only BNA, e.g. LNA, nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages such as phosphorothioate.

The term "nucleobase" refers to the base moiety of a nucleotide and covers both naturally occurring a well as non-naturally occurring variants. Thus, "nucleobase" covers not only the known purine and pyrimidine heterocycles but also heterocyclic analogues and tautomeres thereof. It will be recognized that the DNA or RNA nucleosides of region B may have a naturally occurring and/or non-naturally occurring nucleobase(s). Examples of nucleobases include, but are not limited to adenine, 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 the nucleobases may be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, 5- methylcytosine. In some embodiments the nucleobases may be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, and 5-methylcytosine.

In some embodiments, at least one of the nucleobases present in the oligomer is a modified nucleobase 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.

LNA

The term "LNA" refers to a bicyclic nucleoside analogue which comprises a C2^* - C4^* biradical (a bridge), and is known as "Locked Nucleic Acid". It may refer to an LNA monomer, or, when used in the context of an "LNA oligonucleotide", LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. In some aspects bicyclic nucleoside analogues are LNA nucleotides, and these terms may therefore be used interchangeably, and is such embodiments, both are be characterized by the presence of a linker group (such as a bridge) between C2" and C4^* of the ribose sugar ring.

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

wherein Y is selected from the group consisting of -0-, -CH₂0-, -S-, -NH-, N(R^e) and/or - CH₂-; Z and Z^* are independently selected among an internucleotide linkage, R^H, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and R^H is selected from hydrogen and C_M-alkyI; R^a, R^b R°, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen, optionally substituted C_1-12-alkyl, optionally substituted C₂.i₂-alkenyl, optionally substituted C₂.i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C_2-12-alkoxyalkyl, C_2-i2-alkenyloxy, carboxy, Ci.₁₂-alkoxycarbonyl, Ci_i₂- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy- carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C _-6-alkyl)amino, carbamoyl, mono- and

amino-Ci-6-alkyl-aminocarbonyl, mono- and di(C₁^-alkyl)amino-Ci-6-alkyl-aminocarbonyl, C _-6-alkyl-carbonylamino, carbarn ido, C_1-6- alkanoyioxy, sulphono, C^-alkylsulphonyloxy, nitro, azido, sulphanyi, Ci-e-alkylthio, halogen, DNA intercalators, photochemicaiiy active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminai substituents R* and R^b together may designate optionally substituted methylene (=CH₂); and R^H is selected from hydrogen and d- -alkyl. In some embodiments R^a, R^b R^c, R^d and R^e are, optionally independently, selected from the group consisting of hydrogen and C _-6 alkyl. such as methyl. For all chiral centers, asymmetric groups may be found in either R or S orientation, for example, two exemplary

stereochemical isomers include the beta-D and alpha-L isoforms, which may be illustrated as follows:

Specific exemplary LNA units are shown below:

β-D-oxy-LNA

β-D-thio-LNA

β-D-ENA

β-D-amino-LNA

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

The term "amino-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-_» CH₂-N(H)-, and -CH₂-N(R)- where R is selected from hydrogen and C_M-aIkyI. Amino-LNA can be in both beta-D and alpha-L- configuration.

The term "oxy-LNA" comprises a locked nucleotide in which Y in the general formula above represents -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term "EN A" comprises a locked nucleotide in which Y in the general formula above is -CH₂-0- (where the oxygen atom of -CH₂-0- is attached to the 2'-position relative to the base B), R^e is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.

RNAse recruitment

It is recognized that an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, In some embodiments, the oligomers of the invention are capable of recruiting an

endoribonuclease (RNase), such as RNase H.

It is preferable such oligomers, such as region A, or contiguous nucleotide sequence, comprises of a region of at least 6, such as at least 7 consecutive nucleotide units, such as at least 8 or at least 9 consecutive nucleotide units (residues), including 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase. The contiguous sequence which is capable of recruiting RNAse may be region Y' as referred to in the context of a gapmer as described herein. In some embodiments the size of the contiguous sequence which is capable of recruiting RNAse, such as region Y', may be higher, such as 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. A oligomer is deemed capable of recruiting Nase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5%, such as at least 10% or .more than 20% of the of the initial rate determined using DNA only oligonucleotide, having the same base sequence but containing only DNA monomers, with no 2'

substitutions, with phosphorothioate linkage groups between all monomers in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.

In some embodiments, an oligomer is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1%, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2^* substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40 %, such as at least 60 %, such as at least 80 % of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.

Typically the region of the oligomer which forms the consecutive nucleotide units which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase consists of nucleotide units which form a DNA/RNA like duplex with the RNA target. The oligomer of the invention, such as the first region, may comprise a nucleotide sequence which comprises both nucleotides and nucleotide analogues, and may be e.g. in the form of a gapmer, a headmer or a mixmer.

A "headmer" is defined as an oligomer that comprises a region X' and a region Y^* that is contiguous thereto, with the 5'-most monomer of region Y^* linked to the 3'-most monomer of region X', Region X^* comprises a contiguous stretch of non-RNase recruiting nucleoside analogues and region Y^* comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase.

A "tailmer" is defined as an oligomer that comprises a region X' and a region Y' that is contiguous thereto, with the 5'-most monomer of region Y^* linked to the 3'-most monomer of the region X'. Region X' comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase, and region X' comprises a contiguous stretch of non-RNase recruiting nucleoside analogues.

Other "chimeric" oligomers, called "mixmers", consist of an alternating composition of (i) DNA monomers or nucleoside analogue monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analogue monomers.

In some embodiments, in addition to enhancing affinity of the oligomer for the target region, some nucleoside analogues also mediate RNase (e.g., RNaseH) binding and cleavage. Since a-L-LNA (BNA) monomers recruit RNaseH activity to a certain extent, in some embodiments, gap regions (e.g., region Y' as referred to herein) of oligomers containing a-L-LNA monomers consist of fewer monomers recognizable and cleavable by the RNaseH, and more flexibility in the mixmer construction is introduced.

Gapmer Design

In some embodiments, one ore more, such as 2 or 3 oligomer regions (e.g. A, A and A", or A, A^* and A") in the compound of the invention, comprises or is a gapmer. A gapmer oligomer is an oligomer which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region Y^* (Υ'), wherein region Y' is flanked both 5^* and 3" by regions of affinity enhancing nucleotide analogues, such as from 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions X" (Χ') and Z" (Ζ') respectively. Examples of gapmers are disclosed in WO2004/046160, WO2008/1 13832, and WO2007/14651 1.

In some embodiments, the monomers which are capable of recruiting RNAse are selected from the group consisting of DNA monomers, alpha-L-LNA monomers, C4^* alkylayted DNA monomers (see PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, hereby incorporated by reference), and UNA (unlinked nucleic acid) nucleotides (see Flutter et al., Mol. Biosyst., 2009, 0, 1039 hereby incorporated by reference). UNA is unlocked nucleic acid, typically where the C2 - C3 C-C bond of the ribose has been removed, forming an unlocked "sugar" residue. Preferably the gapmer comprises a ( polynucleotide sequence of formula (5' to 3"), X'-Y'-Z', wherein; region X" (Χ') (5' region) consists or comprises of at least one nucleotide analogue, such as at least one BNA (e.g. LNA) unit, such as from 1 -6 nucleotide analogues, such as BNA (e.g. LNA) units, and; region Y^* (Υ') consists or comprises of at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region Z' (Ζ') (3'region) consists or comprises of at least one nucleotide analogue, such as at least one BNA (e.gLNA unit), such as from 1 -6 nucleotide analogues, such as BNA (e.g. LNA) units. In some embodiments, region X' consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as BNA (e.g. LNA) units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region Z' consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as BNA (e.g. LNA) units, such as from 2-5 nucleotide analogues, such as 2-5 BNA (e.g. LNA units), such as 3 or 4 nucleotide analogues, such as 3 or 4 BNA {e.g. LNA) units.

In some embodiments Y^* consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12

consecutive nucleotides which are capable of recruiting RNAse, or from 6-10, or from 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse. In some embodiments region Y' consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably from 4-12 DNA units, more preferably from 6-10 DNA units, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments region X^* consist of 3 or 4 nucleotide analogues, such as BNA (e.g. LNA), region X' consists of 7, 8, 9 or 10 DNA units, and region Z' consists of 3 or 4 nucleotide analogues, such as BNA (e.g. LNA). Such designs include (Χ'-Υ'-Ζ') 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.

Further gapmer designs are disclosed in WO2004/046160, which is hereby

incorporated by reference. WO2008/113832, which claims priority from US provisional application 60/977,409 hereby incorporated by reference, refers to 'shortmer^* gapmer oligomers. In some embodiments, oligomers presented here may be such shortmer gapmers.

In some embodiments the oligomer, e.g. region X', is consisting of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence comprises or is of formula (5' - 3'), X'-Y'-Z' wherein; X' consists of 1 , 2 or 3 nucleotide analogue units, such as BNA (e.g. LNA) units; Y^* consists of 7, 8 or 9 contiguous nucleotide units which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a nriRNA target); and Z' consists of 1 , 2 or 3 nucleotide analogue units, such as BNA (e.g. LNA) units.

In some embodiments X" consists of 1 BNA (e.g. LNA) unit. In some embodiments X' consists of 2 BNA (e.g. LNA) units. In some embodiments X' consists of 3 BNA (e.g. LNA) units. In some embodiments Z' consists of 1 BNA (e.g. LNA) units. In some embodiments Z' consists of 2 BNA (e.g. LNA) units. In some embodiments Z^* consists of 3 BNA (e.g. LNA) units. In some embodiments Y' consists of 7 nucleotide units. In some embodiments Y' consists of 8 nucleotide units. In some embodiments Y' consists of 9 nucleotide units. . In certain embodiments, region Y' consists of 10 nucleoside monomers. In certain

embodiments, region Y' consists or comprises 1 - 10 DNA monomers. In some embodiments Y^* comprises of from 1 - 9 DNA units, such as 2, 3, 4, 5, 6, 7 , 8 or 9 DNA units. In some embodiments Y^* consists of DNA units. In some embodiments Y' comprises of at least one BNA unit which is 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 Y' comprises of at least one alpha-L-oxy BNA/LNA unit or wherein all the LNA units in the alpha-L- configuration are alpha-L-oxy LNA units. In some embodiments the number of nucleotides present in X'-Y'-Z' are selected from the group consisting of (nucleotide analogue units - region Y' - nucleotide analogue 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 , In some embodiments the number of nucleotides in X'-Y'-Z' are 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 certain embodiments, each of regions X^* and Y' consists of three BNA (e.g. LNA) monomers, and region Y' consists of 8 or 9 or 10 nucleoside monomers, preferably DNA monomers. In some embodiments both X' and Z' consists of two BNA (e.g. LNA) units each, and Y' consists of 8 or 9 nucleotide units, preferably DNA units. In various embodiments, other gapmer designs include those where regions X" and/or Z' consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2'-0-methoxyethyl-ribose sugar (2 - MOE) or monomers containing a 2 -fluoro-deoxyribose sugar, and region Y' consists of 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where regions X'-Y'-Z' have 3-9-3, 3-10- 3, 5-10-5 or 4-12-4 monomers. Further gapmer designs are disclosed in WO

2007/14651 1A2, hereby incorporated by reference.

BNA and LNA Gapmers: The terms BNA and LNA are used interchangeably. A BNA gapmer is a gapmer oligomer (region A) which comprises at least one BNA nucleotide. A LNA gapmer is a gapmer oligomer (region A) which comprises at least one LNA nucleotide. Splice switching oligomers

In some embodiments, an oligomer region is an antisense oligonucleotide which is a splice switching oligomer - i.e. an oligomer which targets the pre-mRNA causing an alternative splicing of the pre-mRNA.

Targets for the splice switching oligomer may include TNF receptor, for example the SSO may be one or more of the TNFR SSOs disclosed in WO2007/058894, WO08051306 A1 and PCT/EP2007/06121 1 , hereby incorporated by reference.

Splice switching oligomers are typically(essentially) not capable of recruiting RNaseH and as such gapmer, tailmer or headmer designs are generally not desirable. However, mixmer and totalmers designs are suitable designs for SSOs.

Spice switching oligomers have also been used to target dystrophin deficiency in Duchenne muscular dystrophy. ixmers

Most antisense oligonucleotides are compounds which are designed to recruit RNase enzymes (such as RNaseH) to degrade their intended target. Such compounds include DNA phosphorothioate oligonucleotides and gapmer, headmers and tailmers. These compounds typically comprise a region of at least 5 or 6 DNA nucleotides, and in the case of gapmers are flanked on either side by affinity enhancing nucleotide analogues.

The oligomers of the present invention may operate via an RNase (such as RNaseH) independent mechanism. Examples of oligomers which operate via a non-RNaseH (or non- RNase) mechanism are mixmers and totalmers.

The term 'mixrner' refers to oligomers which comprise both naturally and non-naturally occurring nucleotides, where, as opposed to gapmers, tailmers, and headmers there is no contiguous sequence of more than 5, and in some embodiments no more than 4

consecutive, such as no more than three consecutive, naturally occurring nucleotides, such as DNA units. In some embodiments, the mixrner does not comprise more than 5 consecutive nucleoside analogues, such as BNA (LNA), and in some embodiments no more than 4 consecutive, such as no more than three consecutive, consecutive nucleoside analogues, such as BNA (LNA). In such mixmers the remaining nucleosides may, for example by DNA nucleosides, and/or in non-bicyclic nucleoside analogues, such as those referred to herein, for example, 2' substituted nucleoside analogues, such as 2 -O-MOE and or 2'fluoro.

The oligomer according to the invention maybe mixmers - indeed various mixrner designs are highly effective as oligomer or first region thereof, particularly when targeting microRNA (antimiRs), microRNA binding sites on mRNAs (Blockmirs) or as splice switching oligomers (SSOs). See for example WO2007/112754 (LNA-AntimiRs™), WO2008/131807 (LNA splice switching oligos),

In some embodiments, the oligomer or mixrner may comprise of BNA and 2^* substituted nucleoside analogues, optionally with DNA nucleosides - see for example see WO07027894 and WO2007/1 12754 which are hereby incorporated by reference. Specific examples include oligomers or first regions which comprise LNA, 2'-0- OE and DNA, LNA, 2'fluoro and 2'-0-MOE, 2'-0-MOE and 2'fluoro, 2'-0-MOE and 2'fluoro and LNA, or LNA and 2'-0-MOE and LNA and DNA.

In some embodiments, the oligomer or mixrner comprises or consists of a contiguous nucleotide sequence of repeating pattern of nucleotide analogue and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogues. The repeating pattern, may, for instance be every second or every third nucleotide is a nucleotide analogue, such as BNA (LNA), and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2'substituted nucleotide analogue such as 2'MOE of 2'fluoro analogues as referred to herein, or, in some embodiments selected form the groups of nucleotide analogues referred to herein. It is recognized that the repeating pattern of nucleotide analogues, such as LNA units, may be combined with nucleotide analogues at fixed positions - e.g. at the 5' or 3' termini.

In some embodiments the first nucleotide of the oligomer or mixmer, counting from the 3' end, is a nucleotide analogue, such as an LNA nucleotide.

In some embodiments, which maybe the same or different, the second nucleotide of oligomer or mixmer, counting from the 3' end, is a nucleotide analogue, such as an LNA nucleotide.

In some embodiments, which maybe the same or different, the seventh and/or eighth nucleotide of oligomer or mixmer, counting from the 3^* end, are nucleotide analogues, such as LNA nucleotides.

In some embodiments, which maybe the same or different, the ninth and/or the tenth nucleotides of the first and/or second oligomer, counting from the 3* end, are nucleotide analogues, such as LNA nucleotides.

In some embodiments, which maybe the same or different, the 5^* terminal of oligomer or mixmer is a nucleotide analogue, such as an LNA nucleotide.

The above design features may, in some embodiments be incorporated into the mixmer design, such as antimiR mixmers.

In some embodiments, the oligomer or mixmer does not comprise a region of more than 4 consecutive DNA nucleotide units or 3 consecutive DNA nucleotide units. In some embodiments, the mixmer does not comprise a region of more than 2 consecutive DNA nucleotide units.

In some embodiments, the oligomer or mixmer comprises at least a region consisting of at least two consecutive nucleotide analogue units, such as at least two consecutive LNA units.

In some embodiments, the oligomer or mixmer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNA units.

In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 7 consecutive nucleotide analogue units, such as LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 6 consecutive nucleotide analogue units, such as LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 5 consecutive nucleotide analogue units, such as LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 4 consecutive nucleotide analogue units, such as LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 3 consecutive nucleotide analogue units, such as LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 2 consecutive nucleotide analogue units, such as LNA units. A mixmer is a oligomer which may comprise one or more short regions of DNA of no more than 4 consecutive DNA nucleotides, and typically comprises alternating regions of a nucleotide analogue (such as LNA units) and DNA nucleotides, optionally regions of other nucleotide analogues (e.g. non-LNA nucleotide analogues). Totalmers comprise of no DNA or RNA nucleotides (although may comprise analogues or derivatives of DNA and RNA). In some embodiments, the oligomer (e.g. region A) of the invention may, in some

embodiments, comprise of no more than 4 consecutive DNA nucleotides, or no more than 3 consecutive DNA nucleotides.

The following embodiments may apply to mixmers or totalmer oligomers (e.g. as region A): The oligomer (e.g. region A) of the invention may, in some embodiments, comprise of at least two alternating regions of LNA and non-LNA nucleotides (such as DNA or 2^* substituted nucleotide analogues).

The oligomer of the invention may, in some embodiments, comprise a contiguous sequence of formula: 5' ([LNA nucleotides]-,^ and [non-LNA nucleotides]^^ _ 12. 3'.

In some embodiments, the 5^* nucleotide of the contiguous nucleotide sequence (or the oligomer) is an LNA nucleotide.

In some embodiments, the 3' nucleotide of the contiguous nucleotide sequence is a nucleotide analogue, such as LNA, or the 2, 3, 4, 5 3' nucleotides are nucleotide analogues, such as LNA nucleotides, or other nucleotide analogues which confer enhanced serum stability to the oligomer.

In some embodiments, the contiguous nucleotide sequence of the oligomer has a formula 5^* ([LNA nucleotides]-, .5 - [non-LNA nucleotides]-_tJ,)₂_ ₁ - [LNA nucleotides]-,^ 3'.

In some embodiments, the contiguous nucleotide sequence of the oligomer has 2, 3 or 4 contiguous regions of LNA and non-LNA nucleotides - e.g. comprises formula 5' ([LNA nucleotides] _-5 and [non-LNA nucleotides]^-^, optionally with a further 3^* LNA region [LNA nucleotides]^.

In some embodiments, the contiguous nucleotide sequence of the oligomer comprises 5^* ([LNA nucleotides]-,^ and [non-LNA nucleotides]^^ _₅, optionally with a further 3' LNA region [LNA nucleotides] i_-3. In some embodiments, the contiguous nucleotide sequence of the oligomer comprises 5^* ([LNA nucleotides]^ and [non-LNA nucleotides]i.₃)₃, optionally with a further 3^* LNA region [LNA nucleotides^.

In some embodiments the non-LNA nucleotides are all DNA nucleotides,

In some embodiments, the non-LNA nucleotides are independently or dependently selected from the group consisting of DNA units, RNA units, 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, and 2'-fluoro-DNA units.

In some embodiments the non-LNA nucleotides are (optionally independently selected from the group consisting of 2' substituted nucleoside analogues, such as (optionally

independently) selected from the group consisting of 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, 2'-AP, 2 -FANA, 2'-(3-hydroxy)propyl, and 2'-fluoro-DNA units, and/or other (optionally) sugar modified nucleoside analogues such as morpholino, peptide nucleic acid (PNA), CeNA, unlinked nucleic acid (UNA), hexitol nucleoic acid (HNA). bicyclo- HNA (see e.g. WO2009/100320), In some embodiments, the nucleoside analogues increase the affinity of the first region for its target nucleic acid (or a complementary DNA or RNA sequence). Various nucleoside analogues are disclosed in Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, hereby incorporated by reference.

In some embodiments, the non-LNA nucleotides are DNA nucleotides. In some

embodiments, the oligomer or contiguous nucleotide sequence comprises of LNA

nucleotides and optionally other nucleotide analogues (such as the nucleotide analogues listed under non-LNA nucleotides) which may be affinity enhancing nucleotide analogues and/or nucleotide analogues which enhance serum stability.

In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of a contiguous nucleotide sequence of said nucleotide analogues.

In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of a contiguous nucleotide sequence of LNA nucleotides.

In some embodiments, the oligomer or contiguous nucleotide sequence is 8 - 12, such as 8 - 10_» or 10 - 20, such as 12™ 18 or 14 - 16 nts in length.

In some embodiments, the oligomer or contiguous nucleotide sequence is capable of forming a duplex with a complementary single stranded RNA nucleic acid molecule with phosphodiester internucleoside linkages, wherein the duplex has a T_m of at least about 60°C, such as at least 65°C.

Example of a T_m Assay: The oligonucleotide: Oligonucleotide and RNA target (PO) duplexes are diluted to 3 mM in 500 ml RNase-free water and mixed with 500 ml 2x T_m- buffer (200mM NaCI, 0.2mM EDTA, 20mM Naphosphate, pH 7.0). The solution is heated to 95°C for 3 min and then allowed to anneal in room temperature for 30 min. The duplex melting temperatures (T_m) is measured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is ramped up from 20°C to 95°C and then down to 25°C, recording absorption at 280 nm. First derivative and the local maximums of both the melting and annealing are used to assess the duplex T_m.

Totalmers

A totalmer is a single stranded oligomer which only comprises non-naturally occurring nucleosides, such as sugar-modified nucleoside analogues.

The first region according to the invention maybe totalmers - indeed various totalmer designs are highly effective as oligomers or first region thereof, e.g. particularly when targeting microRNA (antimiRs) or as splice switching oligomers (SSOs). In some embodiments, the totalmer comprises or consists of at least one XYX or YXY sequence motif, such as a repeated sequence XYX or YXY, wherein X is LNA and Y is an alternative {i.e. non LNA) nucleotide analogue, such as a 2 -O-MOE RNA unit and 2 -fluoro DNA unit. The above sequence motif may, in some embodiments, be XX Y, XYX, YXY or YYX for example.

In some embodiments, the totalmer may comprise or consist of a contiguous nucleotide sequence of between 7 and 16 nucleotides, such as 9, 10, 11 , 12, 13, 14, or 15 nucleotides, such as between 7 and 12 nucleotides.

In some embodiments, the contiguous nucleotide sequence of the totalmer comprises of at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as 95%, such as 100% BNA (LNA) units. The remaining units may be selected from the non-LNA nucleotide analogues referred to herein in, such those selected from the group consisting of 2'-0_alkyl-RNA unit. 2'-OMe-RNA unit, 2 -amino-DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2'MOE RNA unit, or the group 2^*-OMe RNA unit and 2^*-fluoro DNA unit.

In some embodiments the totalmer consist or comprises of a contiguous nucleotide sequence which consists only of LNA units. In some embodiments, the totalmer, such as the LNA totalmer, is between 7 - 12 nucleoside units in length. In some embodiments, the totalmer (as the oligomer or first region thereof) may be targeted against a microRNA (i.e. be antimiRs) - as referred to WO2009/043353, which are hereby incorporated by reference. In some embodiments, the oligomer or contiguous nucleotide sequence comprises of LNA nucleotides and optionally other nucleotide analogues which may be affinity enhancing nucleotide analogues and/or nucleotide analogues which enhance serum stability. In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of a contiguous nucleotide sequence of said nucleotide analogues.

MicroRNA modulation via the oligomer or first region thereof.

In some embodiments, one or more of the oligomer regions (such as A, A and A^* and, A and A' and A") are antimiR(s), such as an LNA mixmer or toalmer, which comprises or consists of a contiguous nucleotide sequence which is corresponds to or is fully

complementary to a microRNA sequence, such as a mature microRNA sequence or part thereof. The use of the present invention in controlling the in vivo activity of microRNA is considered of primary importance due to the fact that microRNAs typically regulate numerous mRNAs in the subject. The ability to inactivate therapeutic antimiRs is therefore very desirable.

Numerous microRNAs are related to a number of diseases. For example: non-limiting examples of therapeutic indications which may be treated by the pharmaceutical

compositions of the invention:

Tumor suppressor gene tropomysin 1 (TPM1 ) mRNA has been indicated as a target o^' miR-21 . Myotrophin (mtpn) mRNA has been indicated as a target of miR 375. The oligomer or first region thereof may therefore be an antimir which targets (i.e. comprises or consists of a contiguous nucleotide sequence which is fully complementary to (a corresponding region of) one of the microRNAs listed above or comprises of no more than a single mismatch thereto.

Hence, some aspects of the invention relates to the treatment of a disease associated with the expression of microRNAs selected from the group consisting of infectious diseases such as viral diseases such as hepatitis C virus and HIV, fragile X mental retardation, inflammatory diseases, cancer, such as chronic lymphocytic leukemia, breast cancer, lung cancer and colon cancer.

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs. The mature miRNAs are processed sequentially from longer hairpin transcripts by the RNAse 111 ribonucleases Drosha. Mature microRNAs (miRs) typically between 20 and 25 contiguous RNA nucleotides. It is now widely established that several microRNAs are associated with medical conditions and disease, and several companies are developing therapeutics based on oligomers which either mimic microRNAs or specifically hybridse to specific microRNAs associated with disease phenotypes - such oligomers are referred to, herein, as microRNA mimics and antimiRs respectfully, and the oligomer or first region thereof, in some embodiments may be such microRNA modulating oligomers.

In some embodiments the oligomer or first region thereof according to the invention, consists or comprises of a contiguous nucleotide sequence which corresponds to or is fully complementary to a microRNA sequence, such as a mature microRNA sequence, such as the human microRNAs published in miRBase (http://microrna.sanger.ac.uk/cgi- bin/sequences/mirna_summary.pl?org=hsa). In some embodiment the microRNA is a viral microRNA. At the time of writing, in miRbase 19, there are 1600 precursors and 2042 mature human miRNA sequences in miRBase which are all hereby incorporated by reference, including the mature microRNA sequence of each human microRNA. Other human microRNAs which may be targeted by the oligomer or first region thereof include those disclosed in WO08040355A, hereby incorporated by reference. In some embodiments the oligomer or first region thereof according to the invention, consists or comprises of a contiguous nucleotide sequence which corresponds to or is fully complementary to a microRNA sequence selected from the group consisting of hsa-miR19b, hsa-miR21 , hsa- miR 122, hsa-miR 142 a7b, hsa-miR 155, and hsa-miR 375. In some embodiments the oligomer or first region thereof according to the invention, consists or comprises of a contiguous nucleotide sequence which corresponds to or is fully complementary to a microRNA sequence selected from the group consisting of hsa-miR221 and hsa-miR222. In some embodiments the oligomer or first region thereof according to the invention, consists or comprises of a contiguous nucleotide sequence which corresponds to or is fully complementary to hsa-miR122 (NR_029667.1 Gl:262205241 ), such as the mature has- miR-122.

In some embodiments when the oligomer or first region thereof targets miR-122, the oligomer is for the use in the treatment of hepatitis C infection.

AntimiR oligomers

Preferred oligomer or first region thereof 'antimiR' designs and oligomers are disclosed in WO2007/1 12754, WO2007/1 12753, PCT/DK2008/000344 and US provisional

applications 60/979217 and 61/028062, all of which are hereby incorporated by reference. In some embodiments, the oligomer or first region thereof is an antimiR which is a mixmer or a totalmer.

AntimiR oligomers are oligomers which consist or comprise of a contiguous nucleotide sequence which is fully complementary to, or essentially complementary to (i.e. may comprise one or two mismatches), to a microRNA sequence, or a corresponding subsequence thereof. In this regards it is considered that the antimiR may be comprise a contiguous nucleotide sequence which is complementary or essentially complementary to the entire mature microRNA, or the antimiR may be comprise a contiguous nucleotide sequence which is complementary or essentially complementary to a sub-sequence of the mature microRNA or pre-microRNA - such a sub-sequence (and therefore the

corresponding contiguous nucleotide sequence) is typically at least 8 nucleotides in length, such as between 8 and 25 nucleotides, such as 9, 10, 1 1 , 12, 13. 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 nucleotides in length, such as between 10-17 or 10-16 nucleotides, such as between 12 - 15 nucleotides.

Numerous designs of AntimiRs have been suggested, and typically antimiRs for therapeutic use, such as the contiguous nucleotide sequence thereof comprise one or more nucleotide analogues units.

In some embodiments the antimiR may have a gapmer structure as herein described. However, as explained in WO2007/1 12754 and WO2007/1 12753, other designs may be preferable, such as mixmers, or totalmers.

WO2007/1 12754 and WO2007/112753, both hereby incorporated by reference, provide antimiR oligomers and antimiR oligomer designs where the oligomers which are complementary to mature microRNA

In some embodiments, a subsequence of the antimiR corresponds to the miRNA seed region, in some embodiments, the first or second 3' nucleobase of the oligomer corresponds to the second 5' nucleotide of the microRNA sequence. In some antimiR embodiments, nucleobase units 1 to 6 (inclusive) of the oligomer as measured from the 3' end the region of the oligomer are complementary to the microRNA seed region sequence.

In some antimiR embodiments, nucleobase units i to 7 (inclusive) of the oligomer as measured from the 3' end the region of the oligomer are complementary to the microRNA seed region sequence.

In some e antimiR embodiments, nucleobase units 2 to 7 (inclusive) of the oligomer as measured from the 3' end the region of the oligomer are complementary to the microRNA seed region sequence,

In some embodiments, the antimiR oligomer comprises at least one nucleotide analogue unit, such as at least one LNA unit, in a position which is within the region complementary to the miRNA seed region. The antimiR oligomer may, in some

embodiments comprise at between one and 6 or between 1 and 7 nucleotide analogue units, such as between 1 and 6 and 1 and 7 LNA units, in a position which is within the region complementary to the miRNA seed region.

In some embodiments, the antimiR of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 80 %, such as 85%, such as 90%, such as 95%, such as 100% of the nucleotides are LNA.

In some embodiments, the antimiR of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 80 % of the nucleotides are LNA, and wherein at least 80%, such as 85%, such as 90%, such as 95%, such as 100% of the internucleotide bonds are phosphorothioate bonds,

In some embodiments, the antimiR comprises one or two LNA units in positions three to eight, counting from the 3' end. This is considered advantageous for the stability of the A- helix formed by the oligonucleotide:microRNA duplex, a duplex resembling an RNA:RNA duplex in structure.

The table on pages 48 line 15 to page 51 , line 9 of WO2007/1 12754 provides examples of anti microRNA oligomers (i.e. antimiRs which may be the oligomer or first region thereof) and is hereby specifically incorporated by reference.

Some Further Poly antimiR oligomer compounds and Conjugates thereof

In some embodiments two of the oligomer regions target a microRNA nucleic acid, such as region A and region A', and optionally, region A". The oligomer regins may target the same or different microRNA targets. By way of example, the oligomer regions may all target the same microRNA, such as microRNA-122, microRNA-221 , microRNA-33 or microRNA-21. Alternatively, one oligomer region may target a first microRNA target, and a further oligomer region may target a second microRNA target. The invention therefore provides for a method for concurrent inhibition of 2 or more different microRNAs, and may herefore be used to target multiple members of a microRNA family, or two microRNAs: An example is poly-oligo compounds which comprise a first oligomer region which is compelementaty to at least 7 nucleotides present in miR-21 , and a further oligomer region which is complementaty to at least 7 nucleotides present in miR-221. Both miR-21 and miR-221 are indicated in some forms of cancer, such as hepatocellular carcinoma.

Poly mRNA Targeting Compounds

In some embodiments 2 of the oligomer regions target a mRNA nucleic acid, such as region A and region A', and optionally, region A". The oligomer regins may target the same or different mRNA targets. By way of example, the oligomer regions may all target the same microRNA, such as those provided herein, such as ApoB, for example

(Trivalent GalNAc)- G_sT_st₈g₈a_sc_ea_sc_st_sg_sT_sCca - G_s ^mC_sa_st_st_sg_sg₈t_sa_st₈T_s ^mC_sA 3' (SEQ ID NO 15)

(Trivalent GalNAc)- G₈C₈a₈t_st_sg_sg_st₈a₈t₈T₈C₈A ca - G^C.a^t.g.g_eWJ^C.A 3^* (SEQ ID NO 16)

(Trivalent GalNAc)- G , Q^W^ e _<Pca - ( TAgAiwtf£_eT_eC 3^* (SEO ID NO 17) (Trivalent GalNAc)-

3' (SEQ ID NO 18) Capital letters are LNA such as beta-D-oxy-LNA, lower case letters are DNA, subscript s is phosphorothioate linkage, other internucieoside linkages are phosphodiester. LNA cytosines may be 5-methyl cytosine. The Trivalent GalNAc may for example be Conj 1 , 2, 3, 4, 1a, 2a, 3a, or 4a, such as conj2a. The conjugate group may be linked to the oligo via a PO linker, e.g. a region of 1 - 5 phosphodiester linked DNA nucleosides, e.g. the 5' Conj - ca - 3' dinucleotide as used in the examples.

Alternatively, one oligomer region may target a first mRNA target, and a further oligomer region may target a second mRNA target. The invention therefore provides for a method for concurrent inhibition of 2 or more different mRNAs. An example is poly-oligo compounds which comprise a first oligomer region which is compelementaty to at least 10 nucleotides present in an ApoB mRNA, and a further oligomer region which is complementaty to at least 10 nucleotides present in an mtGPAT mRNA. By utilising a first LNA oligomer region which targets a first target (e.g. a mRNA, a microRNA, or a viral sequence), and a second LNA oligomer region which targets a second target (e.g. a mRNA, a microRNA, or a viral sequence), single compounds can be made which target two distinct targets, for example, the first oligomer region may target ApoB, and the second oligomer region may target another mRNA, such as mtGPAT mRNA, for example: (Trivalent GalNAc)- GJ^gsa^a_eCs gJsCcaA_eTJsC-Cs^ gsC-c CTgG - 3' (SEQ ID NO

19)

(Trivalent GalNAc)-

- 3' {SEQ ID NO 20)

Capital letters are LNA such as beta-D-oxy-LNA, lower case letters are DNA, subscript s is phosphorothioate linkage, other intern ueleoside linkages are phosphodiester. LNA cytosines may be 5-methyl cytosine. The Trivalent GalNAc may for example be Conj 1 , 2, 3, 4, 1 a, 2a, 3a, or 4a, such as conj2a. The conjugate group may be linked to the oligo via a PO linker, e.g. a region of 1 - 5 phosphodiester linked DNA nucleosides, e.g. the 5' Conj - ca - 3' dinucleotide as used in the examples.

MicroRNA mimics

In some embodiments the oligomer or first region thereof is in the form of a miRNA mimic which can be introduced into a cell to repress the expression of one or more mRNA target(s). miRNA mimics are typically fully complementary to the full length miRNA sequence. miRNA mimics are compounds comprising a contiguous nucleotide sequence which are homologous to a corresponding region of one, or more, of the miRNA sequences provided or referenced to herein. The use of miRNA mimics or antimiRs can be used to (optionally) further repress the mRNA targets, or to silence (down- regulate) the miRNA, thereby inhibiting the function of the endogenous miRNA, causing derepression and increased expression of the mRNA target.

Aptamers

In some embodiments the oligomer or first region thereof may be a therapeutic aptamer, a spiegelmer. Please note that aptamers may also be ligands, such as receptor ligands, and may therefore be used as a targeting moiety (i.e. region 3). Aptamers (also referred to as Spiegelmers) in the context of the present invention as nucleic acids of between 20 and 50 nucleotides in length, which have been selected on the basis of their conformational structure rather than the sequence of nucleotides - they elicit their therapeutic effect by binding with a target protein directly in vivo and they do not, therefore, comprise of the reverse complement of their target - indeed their target is not a nucleic acid but a protein. Specific aptamers which may be the oligomer or first region thereof include Macugen (OSI Pharmaceuticals) or ARC1779, (Archemix, Cambridge, MA). In some embodiments, the oligomer or first region thereof is not an aptamer. In some embodiments the oligomer or first region thereof is not an aptamer or a spiegelmer. Internucleotide Linkages

The nucleoside monomers of the oligomers (e.g. first and second regions) described herein are coupled together via pnternucleoside] linkage groups. Suitably, each monomer is linked to the 3' adjacent monomer via a linkage group.

The person having ordinary skill in the art would understand that, in the context of the present invention, the 5^* monomer at the end of an oligomer does not comprise a 5' linkage group, although it may or may not comprise a 5^* terminal group.

The terms "linkage group" or "internucleotide linkage" are intended to mean a group capable of covalently coupling together two nucleotides. Specific and preferred examples include phosphate groups and phosphorothioate groups.

The nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups. Suitably each nucleotide is linked to the 3^* adjacent nucleotide via a linkage group.

Suitable internucleotide linkages include those listed within WO2007/031091 , for example the internucleotide linkages listed on the first paragraph of page 34 of

yy 02007/031091 (hereby incorporated by reference).

It is, in some embodiments, other than the phosphodiester linkage(s) or region B, the preferred to modify the internucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate - these two, being cleavable by RNase H, also allow that route of antisense inhibition in reducing the expression of the target gene.

Suitable sulphur (S) containing internucleotide linkages as provided herein may be preferred, such as phosphorothioate or phosphodithioate. Phosphorothioate internucleotide linkages are also preferred, particularly for the first region, such as in gapmers, mixmers, antimirs splice switching oligomers, and totalmers.

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

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

W 009124238 refers to oligomeric compounds having at least one bicyclic nucleoside attached to the 3' or 5" termini by a neutral internucleoside linkage. The oligomers of the invention may therefore have at least one bicyclic nucleoside attached to the 3' or 5' termini by a neutral internucleoside linkage, such as one or more phosphotriester,

methylphosphonate, MMI, amide-3, formacetal or thioformacetal. The remaining linkages may be phosphorothioate.

Conjugates, targeting moieties and blocking groups

The term "conjugate" is intended to indicate a heterogenous molecule formed by the covalent attachment ("conjugation") of the oligomer as described herein to one or more non- nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide

components, such as a conjugate component.

In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.

In various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non-polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.

In some embodiments, the conjugate may be a lipophilic conjugate or a proteins (e.g., antibodies, enzymes, serum proteins); peptides; vitamins (water-soluble or lipid-soluble); polymers (water-soluble or lipid-soluble); small molecules including drugs, toxins, reporter molecules, and receptor ligands; carbohydrate complexes; nucleic acid cleaving complexes; metal chelators (e.g., porphyrins, texaphyrins, crown ethers, etc.); intercalators including hybrid photonuclease/intercalators; crosslinking agents (e.g., photoactive, redox active), and combinations and derivatives thereof. Numerous suitable conjugate moieties, their preparation and linkage to oligomeric compounds are provided, for example, in WO

93/07883 and U.S. Pat No. 6,395,492, each of which is incorporated herein by reference in its entirety. Oligonucleotide conjugates and their syntheses are also reported in

comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S . 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. [0034]

Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-o- hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such as cholesterol.

In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example from 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference.

The use of a conjugate is often associated with enhanced pharmacokinetic or pharmeodynamic dynamic properties. However, the presence of a conjugate group may interfere with the activity of the oligonucleotide against its intended target, for example via steric hindrance preventing hybridization or nuclease recruitment (e.g. RNAseH or RISC recruitment). The use of a DNA and/or RNA phosphodiester region (region B) between the oligonucleotide (region A)and the conjugate moiety (X), as according to the present invention, allows for the improved properties due to the presence of the conjugate group, whilst ensuring that once at the target tissue, the conjugate group does not prevent effective activity of the oligonucleotide.

The oligomeric compound of the invention is, in some embodiments, covalently attached to one or more conjugate group, optionally through one or more linkers. The resulting conjugate compounds may, for example have modified enhanced properties, such as modified or enhanced pharmacokinetic, pharmeodynamic, and other properties compared with non-conjugated oligomeric compounds. A conjugate moiety that can modify or enhance the pharmacokinetic properties of an oligomeric compound can improve cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligomeric compound. A conjugate moiety that can modify or enhance pharmacodynamic properties of an oligomeric compound can improve activity, resistance to degradation, sequence-specific hybridization, uptake, and the like. In some embodiments, the conjugate group may reduce or prevent in appropriate activity of the oligonucleotide, e.g. off target activity or activity in non-target tissues or organs. This may be achieved by use of a blocking moiety, which may for example be a conjugate, the presence of the blocking group covalently attached to the oligonucleotide (optionally via a linker), may prevent or hinder oligonucleotide hybridization and/or activity. The cleavage of the DNA/RNA phosphodiester region (e.g.at the intended target site), removes the blocking group, allowing delivery of the active oligonucleotide at the intended site.

In some embodiments, the compound of the invention comprises a conjugate group.

It will be recognized that one conjugate group may be used, for example for targeting to a specific tissue, for example a lipophilic group for targeting to the liver, and a second conjugate group may be used to provide a further benefit, for example a blocking group or a further therapeutic entity. Suitable one or both of the conjugates/moieties may be linked to the oligonucleotide via the DNA/RNA phosphodiester region according to the present invention. In some embodiments, the conjugate is covalently bound to the oligonucleotide, optionally via a linker, at the 5^* and/or 3' termini of the oligonucleotide. In this respect, if two conjugate/moiety groups are used, one may be linked to the 5' termini and one to the 3^* termini.

Carbohydrate conjugates

In some embodiments, the conjugate group is selected from the group consisting of a carbohydrate, a lipophilic moiety, a polymer, a protein or peptide, a label or dye, a small molecule, such as a small molecule therapeutic moiety, a cell surface receptor ligand.

In some embodiments, the conjugate is or may comprise a carbohydrate or comprises a carbohydrate group. In some embodiments, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6- phosphate. In some embodiments, the conjugate group is or may comprise mannose or mannose-6-phosphate. Carbohydrate conjugates may be used to enhance delivery or activity in a range of tissues, such as liver and/or muscle. See, f or example, EP1495769, W099/65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1 (10): 1401-17.

In some embodiments, the conjugate group is a carbohydrate moiety. In addition, the oligomer may further comprise one or more additional conjugate moieties, of which lipophilic or hydrophobic moieties are particularly interesting. These may for example, act as pharmacokinetic modulators, and may be covalently linked to either the carbohydrate conjugate, a linker linking the carbohydrate conjugate to the oligomer or a linker linking multiple carbohydrate conjugates (multi-valent) conjugates, or to the oligomer, optionally via a linker, such as a bio cleavable linker. I

In some embodiments, the conjugate is or may comprise a carbohydrate or comprises a carbohydrate group. In some embodiments, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6- phosphate. In some embodiments, the conjugate group is or may comprise mannose or mannose-6-phosphate. Carbohydrate conjugates may be used to enhance delivery or activity in a range of tissues, such as liver and/or muscle. See, for example, EP1495769, W099/65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21 . Zatsepin & Oretskaya Chem Biodivers. (2004) 1 (10): 1401-17.

GalNAc Conjugates

The invention also provides oligonucleotides, such as LNA antisense oligomers, which are conjugated to an asialoglycoprotein receptor targeting moiety. In some embodiments, the conjugate moiety (such as the third region or region C) comprises an asialoglycoprotein receptor targeting moiety, such as galactose, galactosamine, N-formyl-galactosamine, Nacetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N- isobutanoylgalactos-amine. In some embodiments the conjugate comprises a galactose cluster, such as N-acetylgalactosamine t rimer. In some embodiments, the conjugate moiety comprises a GalNAc (N-acetylgalactosamine), such as a mono-valent, di-valent, tri-valent of tetra-valent GalNAc. Trivalent GalNAc conjugates may be used to target the compound to the liver. GalNAc conjugates have been used with methylphosphonate and PNA antisense oligonucleotides (e.g. US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov- Dec;6(6):695-701 ) and siRNAs (e.g. WO2009/126933, WO2012/089352 &

WO2012/083046). The GalNAc references and the specific conjugates used therein are hereby incorporated by reference. WO2012/083046 discloses siRNAs with GalNAc conjugate moieties which comprise cleavable pharmacokinetic modulators, which are suitable for use in the present invention, the preferred pharmacokinetic modulators are C16 hydrophobic groups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E)-octadeca-9, 12- dienoyl, dioctanoyl, and C16-C20 acyl. The Ό46 cleavable pharmacokinetic modulators may also be cholesterol.

The 'targeting moieties (conjugate moieties) may be selected from the group consisting of: galactose, galactosamine, N-foimyl-galactosamine, N-acetylgalactosamine, Npropionyl- galactosamine, N-n-butanoyl-galactosamine, N-iso-butanoylgalactos-amine, galactose duster, and N-acetylgalactosamine trimer and may have a pharmacokinetic modulator selected from the group consisting of: hydrophobic group having 16 or more carbon atoms, hydrophobic group having 16-20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyi, (9E, 12E)-octadeca-9, 12dienoyl, dioctanoyl, and C16-C20 acyl, and cholesterol.

Certain GalNac clusters disclosed in Ό46 include: (E)-hexadec-8-enoyl (C16), oleyl (C18), (9,E, 12E)-octadeca-9, 12-dienoyl (CI 8), octanoyl (C8), dodececanoyl (C12), C-20 acyl, C24 acyl, dioctanoyl (2xC8). The targeting moiety-pharmacokinetic modulator targeting moiety may be linked to the polynucleotide via a physiologically labile bond or, e.g. a disulfide bond, or a PEG linker. The invention also relates to the use of phospodiester linkers, such as DNA phosphodiester linkers, between the oligomer region and the conjugate group (these may be as defined as region B herein, and suitably are positioned between the oligomer region and the carbohydrate conjugate group).

For targeting hepatocytes in liver, a preferred targeting ligand is a galactose cluster. A galactose cluster comprises a molecule having e.g. comprising two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. A terminal galactose derivative is attached to a molecule through its C-l carbon. The asialoglycoprotein receptor (ASGPr) is unique to hepatocytes and binds branched galactose-terminal glycoproteins. A preferred galactose cluster has three terminal galactosamines or galactosamine derivatives each having affinity for the asialoglycoprotein receptor. A more preferred galactose cluster has three terminal N-acetyl- galactosamines. Other terms common in the art include tri-antennary galactose, tri-valent galactose and galactose trimer. It is known that tri-antennary galactose derivative clusters are bound to the ASGPr with greater affinity than bi-antennary or mono-antennary galactose derivative structures (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al„ 1982,1. Biol. Chern., 257,939-945). Multivalency is required to achieve nM affinity. According to WO 2012/083046 the attachment of a single galactose derivative having affinity for the asialoglycoprotein receptor does not enable functional delivery of the RNAi polynucleotide to hepatocytes in vivo when co-administered with the delivery polymer.

A galactose cluster may comprise two or preferably three galactose derivatives each linked to a central branch point. The galactose derivatives are attached to the central branch point through the C-l carbons of the saccharides. The galactose derivative is preferably linked to the branch point via linkers or spacers (which may be region Y). A preferred spacer is a flexible hydrophilic spacer (U.S. Patent 5885988; 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 PEGS spacer. The branch point can be any small molecule which permits attachment of the three galactose derivatives and further permits attachment of the branch point to the oligomer. An exemplary branch point group is a di-lysine. A di-lysine molecule contains three amine groups through which three galactose derivatives may be attached and a carboxyl reactive group through which the di-lysine may be attached to the oligomer, Attachment of the branch point to oligomer may 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 may be attached to the 3' or 5' end of the oligomer using methods known in the art.

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

One embodiment of a Galactose cluster

Galactose cluster with PEG spacer between, branch point and nucleic acid

A GalNac conjugate is illustrated in figure 1 , Further examples of the conjugate of the invention are illustrated below:

Region A may, for example, be the oligomer region, such as in a non-limiting example an LNA antisense oligonucleotide (shown).

As described herein, a carbohydrate conjugate (e.g. GalNAc) may therefore be linked to the oligomer via a biocleavable linker, such as region B as defined herein, and optionally region Y, which is illustrated as a di-lysine in the above diagrams.

Where at the hydrophobic or lipophilic (or further conjugate) moiety (i.e. pharmacokinetic modulator) in the above GalNac cluster conjugates is, when using BNA or LNA oligomers, such as LNA antisense oligonucleotides, optional.

See the figures for specific GalNac clusters used in the present study, Conj 1 , 2, 3, 4 and Conjl a, 2a, 3a and 4a (which are shown with an optional C6 linker which joins the GalNac cluster to the oligomer™ See Figures 12 and 17),

Each carbohydrate moiety of a GalNac cluster (e.g. GalNAc) may therefore be joined to the oligomer via a spacer, such as (poly)ethylene glycol linker (PEG), such as a di, tri, tetra, penta, hexa-ethylene glycol linker. As is shown above the PEG moiety forms a spacer between the galactose sugar moiety and a peptide (trilysine is shown) linker.

In some embodiments, the GalNac cluster comprises a peptide linker, e.g. a Tyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide, which is attached to the oligomer (or to region Y or region B) via a biradical linker, for example the GalNac cluster may comprise the following biradical linkers:

R¹ is a biradical preferably selected from -C₂H₄-, -C₃H₆-, -C₄H₈-, -C₅H₁₀-, -C₆H₁₂-, 1 ,4- cyclohexyi (-C6H10-), 1 ,4-phenyl (-C₆H₄-), -C₂H₄OC₂H4-, -C₂H4(OC₂H4)2- or -C₂H₄(OC₂H₄)3-, C(0)CH₂-, -C(0)C₂H₄-, - C(0)C₃H₆-. - C(0)C₄H₈-, - C(O)C₅H₁₀-, - C(0)C₆H_ir, 1 ,4-cyclohexyl (-C(O)C6H10-), 1 ,4-phenyl (-C(0)C₆H₄-), - C(0)C₂H₄OC₂H₄-, - C(0)C₂H₄(OC₂H₄)2- or - C(0)C₂H₄(OC₂H₄)₃- . In some emboidments, R¹ is a biradical preferably selected from - C₂H₄-, -C₃H₆-, -C₄H₈-, -C₅H₁₀-, -C₆H₁₂-, 1 ,4-cyclohexyl (-C6H10-), 1 ,4-phenyl (-C₆H₄-), - C₂H₄OC₂H₄-, -C₂H₄(OC₂H₄)2- or -C₂H₄(OC₂H₄)₃- .

In addition, the carbohydrate conjugate (e.g. GalNAc), or carbohydrate-linker moiety (e.g. carbohydrate-PEG moiety) may be covalently joined (linked) to the oligomer (or region B) via a branch point group such as, an amino acid, or peptide, which suitably comprises two or more amino groups (such as 3, 4, or5), such as lysine, di-lysine or tri-lysine or tetra-lysine. A tri-lysine molecule contains four amine groups through which three carbohydrate conjugate groups, such as galactose & derivatives (e.g. GalNAc) and a further conjugate such as a hydrophobic or lipophilic moiety/group may be attached and a carboxyl reactive group through which the tri-lysine may be attached to the oligomer. The further conjugate, such as lipophilic/hydrophobic moiety may be attached to the lysine residue that is attached to the oligomer. In some embodiments, the conjugate (C) is not a monovalent GalNac.

The invention also provides LNA antisense oligonucleotides which are conjugated to an asialoglycoprotein receptor targeting moiety. In some embodiments, the conjugate moiety (such as the third region or region C) comprises an asialoglycoprotein receptor targeting moiety, such as galactose, galactosamine, N-formyl-galactosamine, Nacetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-isobutanoylgalactos-amine. In some embodiments the conjugate comprises a galactose cluster, such as N- acetylgalactosamine trimer. In some embodiments, the conjugate moiety comprises a

GalNac (N-acetylgalactosamine), such as a mono-valent, di-valent, tri-valent of tetra-valent GalNac. Trivalent GalNac conjugates may be used to target the compound to the liver. GalNac conjugates have been used with methylphosphonate and PNA antisense

oligonucleotides (e.g. US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov- Dec;6(6):695-701 ) and siRNAs (e.g. WO2009/126933, WO2012/089352 &

WO2012/083046). The GalNac references and the specific conjugates used therein are hereby incorporated by reference. WO2012/083046 discloses GalNac conjugate moieties which comprise cleavable pharmacokinetic modulators, the preferred pharmacokinetic modulators are C16 hydrophobic groups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12 E )-octadeca-9 , 12-d ie noyl , dioctanoyl, and C16-C20 acyl. The '046 cleavable

pharmacokinetic modulators may also be cholesterol. The Ό46 targeting moieties may be selected from the group consisting of: galactose, galactosamine, N-formyl-galactosamine, N- acetylgalactosamine, Npropionyl- galactosamine, N-n-butanoyl-galactosamine, N-iso- butanoylgalactos-amine, galactose cluster, and N-acetylgalactosamine trimer and may have a pharmacokinetic modulator selected from the group consisting of: hydrophobic group having 16 or more carbon atoms, hydrophobic group having 16-20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E)-octadeca-9,12dienoyl. dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clusters disclosed in Ό46 include: (E)-hexadec-8-enoyl (C16). oleyl (C18), (9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl (C12), C- 20 acyl, C24 acyl, dioctanoyl (2xC8). According to Ό46, the targeting moiety- pharmacokinetic modulator targeting moiety may be linked to the polynucleotide via a physiologically labile bond or, e.g. a disulfide bond, or a PEG linker.

Other conjugate moieties can include, for example, oligosaccharides and carbohydrate clusters such as Tyr-Glu-Glu-(aminohexyl GalNAc)3 (YEE(ahGalNAc)3; a glycotri peptide that binds to Gal/GalNAc receptors on hepatocytes, see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (e.g., L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and cholane-based galactose clusters (e.g., carbohydrate recognition motif for asialoglycoprotein receptor). Further suitable conjugates can include

oligosaccharides that can bind to carbohydrate recognition domains (CRD) found on the asiologlycoprotein-receptor (ASGP-R). Example conjugate moieties containing

oligosaccharides and/or carbohydrate complexes are provided in U.S. Pat. No. 6,525,031 , which is incorporated herein by reference in its entirity.

Pharmacokinetic Modulators

The compound of the invention may further comprise one or more additional conjugate moieties, of which lipophilic or hydrophobic moieties are particularly interesting, such as when the conjugate group is a carbohydrate moiety. Such lipophilic or hydrophobic moieties may act as pharmacokinetic modulators, and may be covalently linked to either the carbohydrate conjugate, a linker linking the carbohydrate conjugate to the oligomer or a linker linking multiple carbohydrate conjugates (multi-valent) conjugates, or to the oligomer, optionally via a linker, such as a bio cleavable linker. The oligomer or conjugate moiety may therefore comprise a pharmacokinetic modulator, such as a lipophilic or hydrophobic moieties. Such moieties are disclosed within the context of siRNA conjugates in WO2012/082046. The hydrophobic moiety may comprise a C8 - C36 fatty acid, which may be saturated or un-saturated. In some

embodiments, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32 and C34 fatty acids may be used. The hydrophobic group may have 16 or more carbon atoms. Exemplary suitable hydrophobic groups may be selected from the group comprising: sterol, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E)-octadeca-9, 12-dienoyl, dioctanoyl, and C16- C20 acyl. According to WO'346, hydrophobic groups having fewer than 16 carbon atoms are less effective in enhancing polynucleotide targeting, but they may be used in multiple copies (e.g. 2x, such as 2x C8 or C10, C12 or C14) to enhance efficacy. Pharmacokinetic modulators useful as polynucleotide targeting moieties may be selected from the group consisting of: cholesterol, alkyl group, alkenyl group, alkynyl group, aryl group, aralkyi group, aralkenyl group, and aralkynyl group, each of which may be linear, branched, or cyclic.

Pharmacokinetic modulators are preferably hydrocarbons, containing only carbon and hydrogen atoms. However, substitutions or heteroatoms which maintain hydrophobicity, for example fluorine, may be permitted.

Surprisingly, the present inventors have found that GalNac conjugates for use with LNA oligomers do not require a pharmacokinetic modulator, and as such, in some embodiments, the GalNac conjugate is not covalently linked to a lipophilic or hydrophobic moiety, such as those described here in, e.g. do not comprise a C8 - C36 fatty acid or a sterol. The invention therefore also provides for LNA oligomer GalNac conjugates which do not comprise a lipophilic or hydrophobic pharmacokinetic modulator or conjugate

moiety/group. In some embodiments, the conjugate moiety is hydrophilic. In some embodiments, the conjugate group does not comprise a lipophilic substituent group, such as a fatty acid substituent group, such as a C8 - C26, such as a palmotyl substituent group, or does not comprise a sterol, e.g. a cholesterol subtituent group. In this regards, part of the invention is based on the suprising discovery that LNA oligomers GalNAC conjugates have remarkable pharmacokinetic properties even without the use of pharmacokinetic modulators, such as fatty acid substituent groups (e.g. >C8 or >C16 fatty acid groups).

Lipophilic conjugates

The compounds of the invention may be conjugates comprising of the oligomer (A) and a lipophilic conjugate (C). The biocleavable linker (B) has found to be particularly effective in maintaining or enhancing the activity of such oligomer conjugates. In some embodiments the conjugate group (C) and or linker group (Y) comprises a lipophilic group. Representative conjugate moieties can include lipophilic molecules (aromatic and non- aromatic) including sterol and steroid molecules. Lipophilic conjugate moieties can be used, for example, to counter the hydrophilic nature of an oligomeric compound and enhance cellular penetration. Lipophilic moieties include, for example, steroids and related

compounds such as cholesterol (U.S. 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, and the like.

Other lipophilic conjugate moieties include aliphatic groups, such as, for example, straight chain, branched, and cyclic alkyls, alkenyls, and alkynyls. The aliphatic groups can have, for example, 5 to about 50, 6 to about 50, 8 to about 50, or 10 to about 50 carbon atoms. Example aliphatic groups include undecyl, dodecyl, hexadecyi, heptadecyl, octadecyl, nonadecyl, terpenes, bornyl, adamantyl, derivatives thereof and the like. In some embodiments, one or more carbon atoms in the aliphatic group can be replaced by a heteroatom such as O, S, or N (e.g., geranyloxyhexyl). Further suitable lipophilic conjugate moieties include aliphatic derivatives of glycerols such as alkylglycerols, bis(alkyI)giycerols, tris(alkyl)glycerols, monoglycerides, diglycerides, and triglycerides. In some embodiments, the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1 ,2-di-O- hexyldecyl-rac-glycerol ( anoharan et al., Tetrahedron Lett, 1995, 36, 3651 ; Shea, et al., Nuc. Acids Res., 1990, 18, 3777) or phosphonates thereof. Saturated and unsaturated fatty functionalities, such as, for example, fatty acids, fatty alcohols, fatty esters, and fatty amines, can also serve as lipophilic conjugate moieties. In some embodiments, the fatty functionalities can contain from about 6 carbons to about 30 or about 8 to about 22 carbons. Example fatty acids include, capric, caprylic, lauric, palmitic, myristic, stearic, oleic, linoleic, linolenic, arachidonic, eicosenoic acids and the like.

In further embodiments, lipophilic conjugate groups can be polycyclic aromatic groups having from 6 to about 50, 10 to about 50, or 14 to about 40 carbon atoms. Example polycyclic aromatic groups include pyrenes, purines, acridines, xanthenes, fluorenes, phenanthrenes, anthracenes, quinolines, isoquinolines, naphthalenes, derivatives thereof and the like. [0037] Other suitable lipophilic conjugate moieties include menthols, trityls {e.g., dimethoxytrityl (DMT)), phenoxazines, lipoic acid, phospholipids, ethers, thioethers (e.g., hexyl-S-tritylthiol), derivatives thereof and the like. Preparation of lipophilic conjugates of oligomeric compounds are well-described in the art, such as in, for example, Saison- Behmoaras et al, EM BO J„ 1991 , 10, 11 11; Kabanov et al„ FEBSLett, 1990, 259, 327; Svinarchuk et at, 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) can help provide an effective targeted delivery system. High expression levels of receptors for LDL on tumor cells makes LDL an attractive carrier for selective delivery of drugs to these cells (Rump, et al., Bioconjugate Chem., 1998, 9, 341; Firestone,

Bioconjugate Chem., 1994, 5, 105; Mishra, et al., Biochim. Biophys. Acta, 1995, 1264, 229).

Moieties having affinity for LDL include many lipophilic groups such as steroids (e.g., cholesterol), fatty acids, derivatives thereof and combinations thereof. In some

embodiments, conjugate moieties having LDL affinity can be dioleyl esters of cholic acids such as chenodeoxycholic acid and lithocholic acid.

In some embodiments, the conjugate group is or may comprise a lipophilic moiety, such as a sterol (for example, cholesterol, cholesteryl, cholestanol, stigmasterol, cholanic acid and ergosterol). In some embodiments, the conjugate is or may comprise cholesterol. See for example, Soutschek et al„ Nature (2004) 432, 173; Krutzfeldt Nature 2005, NAR

2007.

In some embodiments, the conjugate is, or may comprise a lipid, a phospholipid or a lipophilic alcohol, such as a cationic lipids, a neutral lipids, sphingolipids, and fatty acids such as stearic, oleic, elaidic, linoleic, linoleaidic, linolenic, and myristic acids. In some embodiments the fatty acid comprises a C4 - C30 saturated or unsaturated alkyl chain. The alkyl chain may be linear or branched.

In some embodiments, the lipophilic conjugates may be or may comprise biotin. In some embodiments, the lipophilic conjugate may be or may comprise a glyceride or glyceride ester.

Lipophilic conjugates, such as cholesterol or as disclosed herein, may be used to enhance delivery of the oligonucleotide to, for example, the liver (typically hepatocytes).

The following references 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.

Polymer conjugates

Conjugate moieties can also include polymers. Polymers can provide added bulk and various functional groups to affect permeation, cellular transport, and localization of the conjugated oligomeric compound. For example, increased hydrodynamic radius caused by conjugation of an oligomeric compound with a polymer can help prevent entry into the nucleus and encourage localization in the cytoplasm. In some embodiments, the polymer does not substantially reduce cellular uptake or interfere with hybridization to a complementary strand or other target. In further embodiments, the conjugate polymer moiety has, for example, a molecular weight of less than about 40, less than about 30, or less than about 20 kDa. Additionally, polymer conjugate moieties can be water-soluble and optionally further comprise other conjugate moieties such as peptides, carbohydrates, drugs, reporter groups, or further conjugate moieties.

In some embodiments, polymer conjugates include polyethylene glycol (PEG) and copolymers and derivatives thereof. Conjugation to PEG has been shown to increase nuclease stability of an oligomeric compound. PEG conjugate moieties can be of any molecular weight including for example, about 100, about 500, about 1000, about 2000, about 5000, about 10,000 and higher. In some embodiments, the PEG conjugate moieties contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 ethylene glycol residues. In further embodiments, the PEG conjugate moiety contains from about 4 to about 10, about 4 to about 8, about 5 to about 7, or about 6 ethylene glycol residues. The PEG conjugate moiety can also be modified such that a terminal hydroxyl is replaced by alkoxy, carboxy, acyl, amido, or other functionality. Other conjugate moieties, such as reporter groups including, for example, biotin or fluorescein can also be attached to a PEG conjugate moiety. Copolymers of PEG are also suitable as conjugate moieties. [0047] Preparation and biological activity of polyethylene glycol conjugates of oligonucleotides are described, for example, in Bonora, et al.,

Nucleosides Nucleotides, 1999, 18, 1723; Bonora, et al., Farmaco, 1998, 53, 634; Efirnov, Bioorg. Khim. 1993, 19, 800; and Jaschke, et al, Nucleic Acids Res., 1994, 22, 4810. Further example PEG conjugate moieties and preparation of corresponding conjugated oligomeric compounds is described in, for example, U.S. Pat. Nos. 4,904,582 and 5,872,662, each of which is incorporated by reference herein in its entirety. Oligomeric compounds conjugated to one or more PEG moieties are available commercially.

Other polymers suitable as conjugate moieties include polyamines, polypeptides, polymethacrylates (e.g., hydroxylpropyl methacrylate (HPMA)), poly(L-lactide), poly(DL lactide-co-glycolide (PGLA), polyacrylic acids, polyethylenimines (PEl), polyalkylacrylic acids, polyurethanes, polyacrylamides, N- alkylacrylamides, polyspermine (PSP), polyethers, cyciodextrins, derivatives thereof and co-polymers thereof. Many polymers, such as PEG and polyamines have receptors present in certain cells, thereby facilitating cellular uptake, Polyamines and other amine-containing polymers can exist in protonated form at physiological pH, effectively countering an anionic backbone of some oligomeric

compounds, effectively enhancing cellular permeation. Some example polyamines include polypeptides (e.g., polylysine, polyornithine, polyhistadine, polyarginine, and copolymers thereof), triethylenetetraamine, spermine, polyspermine, spermidine, synnorspermidine, C- branched spermidine, and derivatives thereof. Preparation and biological activity of polyamine conjugates are described, for example, in Guzaev, et al, Bioorg. Med. Chem, Lett., 1998, 8, 3671 ; Corey, et al, j Am. Chem. Soc, 1995, 117, 9373; and Prakash, et al, Bioorg. Med. Chem. Lett. 1994, 4, 1733. Example polypeptide conjugates of

oligonucleotides are provided in, for example, Wei, et al., Nucleic Acids Res., 1996, 24, 655 and Zhu, et al., Antisense Res. Dev., 1993, 3, 265. Dendrimeric polymers can also be used as conjugate moieties, such as described in U.S. Pat. No, 5,714,166, which is incorporated herein by reference in its entirety. [0049] As discussed above for polyamines and related polymers, other amine-containing moieties can also serve as suitable conjugate moieties due to, for example, the formation of cationic species at physiological conditions. Example amine-containing moieties include 3-aminopropyl, 3-(N,N-dimethylamino)propyl, 2-(2-(N,N- dimethylamino)ethoxy)ethyl, 2-(N-(2-aminoethyl)-N- methylaminooxy)ethyl, 2-(l- imidazolyl)ethyl, and the like. The G-clamp moiety can also serve as an amine-containing conjugate moiety (Lin, et al., J. Am. Chem. Soc, 1998, 120, 8531 ).

In some embodiments, the conjugate may be, or may comprise a polymer, such as a polymer selected from the group consisting of polyethyleneglycol (PEG), polyamidoamine (PAA), polyethylene oxide and polyethylenimine (PEI). Galactose, lactose, n- acetylgalactosamine, mannose, mannose-6-phosphate n some embodiments, the polymer is a polycationic polymer. In some embodiments, conjugate moieties can be, or based on (include) cationic polymers. Numerous studies have demonstrated that cationic polymers such as cationic albumin can greatly enhance delivery to particular cell types and/or tissues (e.g. brain delivery, see Lu, W. et. al. (2005) J of Control Release 107:428-448). Given the benefits of these molecules, the conjugate moieties can be cationic polymers such as polyethyleneimine, dendrimers, poly(alkylpyridinium) salts, or cationic albumin. In some embodiments is a hydrophilic polymer. In some embodiments, the polymer is

Poly(vinylpyrrolidone) (PVP). In some embodiments, the polymer is a polyamine or polyamide (e.g. US7, 816,337 & US5525465. For polymer conjugates see for example, Zhao et al., Bioconjugate Chem 2005, 16, 758-766); Kim et al., J. Control Release (2006) 1 16; 123. Pettit et al., Ther. Deliv. (201 1 ) 2(7): 907-17. Yang et al., Bioconjug Chem (2009) 20(2): 213-21. Winkler et al (2009) Eur J Med Chem 44(2): 670-7. Zelikin et al,

Biomacromolecules (2007) 8(9): 2950-3. See also W012092373 which refers to enzyme cleavable polynucleotide delivery conjugates.

Protein and peptide conjugates

Other conjugate moieties can include proteins, subunits, or fragments thereof. Proteins include, for example, enzymes, reporter enzymes, antibodies, receptors, and the like. In some embodiments, protein conjugate moieties can be antibodies or fragments thereof (Kuijpers, et al, Bioconjugate Chem., 1993, 4, 94). Antibodies can be designed to bind to desired targets such as tumor and other disease-related antigens. In further embodiments, protein conjugate moieties can be serum proteins such as HAS or glycoproteins such as asialoglycoprotein (Rajur, et al., Bioconjugate Chem., 1997, 6, 935). In yet further embodiments, oligomeric compounds can be conjugated to RNAi-related proteins, RNAi- related protein complexes, subunits, and fragments thereof. For example, oligomeric compounds can be conjugated to Dicer or RISC. [0067] Intercalators and minor groove binders (MGBs) can also be suitable as conjugate moieties. In some embodiments, the MGB can contain repeating DPI (l,2-dihydro-3H-pyrrolo(2,3-e)indole-7-carboxylate) subunits or derivatives thereof (Lukhtanov, et al., Bioconjugate Chem., 1998, 7, 584 and Afonina, et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 3199). Suitable intercalators include, for example, polycyclic aromatics such as naphthalene, perylene, phenanthridine, benzophenanthridine, phenazine, anthraquinone, acridine, and derivatives thereof. Hybrid intercalator/ligands include the photonuclease/intercalator ligand 6-[[[9-[[6- (4- nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexan oyl- pentafluorophenyl ester. This compound is both an acridine moiety that is an intercalator and a p-nitro benzamido group that is a photonuclease. [0069] In further embodiments, cleaving agents can serve as conjugate moieties. Cleaving agents can facilitate degradation of target, such as target nucleic acids, by hydrolytic or redox cleavage mechanisms. Cleaving groups that can be suitable as conjugate moieties include, for example, metallocomplexes, peptides, amines, enzymes, and constructs containing constituents of the active sites of nucleases such as imidazole, guanidinium, carboxyl, amino groups, etc.). Example metallocomplexes include, for example, Cu-terpyridyl complexes, Fe-porphyrin complexes, Ru-complexes, and lanthanide complexes such as various Eu(ill) complexes (Hall, et al., Chem. Biol, 1994, 1, 185; Huang, et al., J. Biol. Inorg. Chem., 2000, 5, 85; and Baker, et al, Nucleic Acids Res., 1999, 27, 1547). Other metallocomplexes with cleaving properties include metalloporphyrins and derivatives thereof. Example peptides with target cleaving properties include zinc fingers (U.S. Pat. No. 6,365,379; Lima, et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 10010).

Example constructs containing nuclease active site constituents include bisimiazole and histamine.

Conjugate moieties can also include peptides. Suitable peptides can have from 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 amino acid residues. Amino acid residues can be naturally or non-naturally occurring, including both D and L isomers. In some embodiments, peptide conjugate moieties are pH sensitive peptides such as fusogenic peptides. Fusogenic peptides can facilitate endosomal release of agents such as oligomeric compounds to the cytoplasm. It is believed that fusogenic peptides change conformation in acidic pH, effectively destabilizing the endosomal membrane thereby enhancing cytoplasmic delivery of endosomal contents. Example fusogenic peptides include peptides derived from polymyxin B, influenza HA2, GALA, KALA, EALA, melittin-derived peptide, a-helical peptide or Alzheimer beta -amyloid peptide, and the like. Preparation and biological activity of oligonucleotides conjugated to fusogenic peptides are described in, for example, Bongartz, et at., Nucleic Acids Res., 1994, 22, 4681 and U.S. Pat. Nos. 6,559,279 and 6,344,436. Other peptides that can serve as conjugate moieties include delivery peptides which have the ability to transport relatively large, polar molecules (including peptides, oligonucleotides, and proteins) across cell membranes. Example delivery peptides include Tat peptide from HIV Tat protein and Ant peptide from Drosophila antenna protein. Conjugation of Tat and Ant with oligonucleotides is described in, for example, Astriab-Fisher, et al., Biochem.

Pharmacol, 2000, 60, 83. These and other delivery peptides that can be used as conjugate moieties are provided below in Table I:

Conjugated delivery peptides can help control localization of oligomeric compounds to specific regions of a cell, including, for example, the cytoplasm, nucleus, nucleolus, and endoplasmic reticulum (ER). Nuclear localization can be effected by conjugation of a nuclear localization signal (NLS). in contrast, cytoplasmic localization can be facilitated by conjugation of a nuclear export signal (NES). [0054] Peptides suitable for localization of conjugated oligomeric compounds in the nucleus include, for example, N,N-dipalmitylglycyl- apo E peptide or N,N- dipalmitylglycyl-apolipoprotein E peptide (dpGapoE) (Liu, et al, Arterioscler. Thromb. Vase. Biol, 1999, 19, 2207; Chaloin, et al., Biochem. Biophys. Res. Commun., 1998, 243, 601 ). Nucleus or nucleolar localization can also be facilitated by peptides having arginine and/or lysine rich motifs, such as in HIV-1 Tat, FXR2P, and angiogenin derived peptides (Lixin, et al, Biochem. Biophys. Res. Commun., 2001 , 284,

185). Additionally, the nuclear localization signal (NLS) peptide derived from SV40 antigen T (Branden, et al., Nature Biotech, 1999, 17, 784) can be used to deliver conjugated oligomeric compounds to the nucleus of a cell. Other suitable peptides with nuclear or nucleolar localization properties are described in, for example, Antopolsky, et al.,

Bioconjugate Chem., 1999, 10, 598; Zanta, et al., Proc. Natl. Acad. Sci. USA, 999 (simian virus 40 large tumor antigen); Hum. Mol. Genetics. 2000, 9, 1487; and FEBSLett., 2002, 532, 36).

In some embodiments, the delivery peptide for nucleus or nucleolar localization comprises at least three consecutive arginine residues or at least four consecutive arginine residues. Nuclear localization can also be facilitated by peptide conjugates containing RS, RE, or RD repeat motifs (Cazalla, et al., ol Ceil. Biol, 2002, 22, 6871 ). In some

embodiments, the peptide conjugate contains at least two RS, RE, or RD motifs.

Localization of oligomeric compounds to the ER can be effected by, for example, conjugation to the signal peptide KDEL (Arar, et al., Bioconjugate Chem., 1995, 6, 573; Pichon, et al., Mol. Pharmacol. 1997, 57, 431 ). [0057] Cytoplasmic localization of oligomeric compounds can be facilitated by conjugation to peptides having, for example, a nuclear export signal (NES) (Meunier, et al., Nucleic Acids Res., 1999, 27, 2730). NES peptides include the leuctne-rich NES peptides derived from HIV-1 Rev (Henderson, et al., Exp. Cell Res., 2000, 256, 213), transcription factor III A, MAPKK, PKI-alpha, cyclin Bl, and actin (Wada, et al.. EMBO J., 1998, 17, 1635) and related proteins. Antimicrobial peptides, such as dermaseptin derivatives, can also facilitate cytoplasmic localization (Hariton-Gazal, et al., Biochemistry, 2002, 41 , 9208). Peptides containing RG and/or KS repeat motifs can also be suitable for directing oligomeric compounds to the cytoplasm. In some embodiments, the peptide conjugate moieties contain at least two RG motifs, at least two KS motifs, or at least one RG and one KS motif. [0058] As used throughout, "peptide" includes not only the specific molecule or sequence recited herein (if present), but also includes fragments thereof and molecules comprising all or part of the recited sequence, where desired functionality is retained. In some embodiments, peptide fragments contain no fewer than 6 amino acids. Peptides can also contain conservative amino acid substitutions that do not substantially change its functional characteristics. Conservative substitution can be made among the following sets of functionally similar amino acids: neutral- weakly hydrophobic (A, G, P, S, T), hydrophilic-acid amine (N, D, Q, E), hydrophilic-basic (I, M, L, V), and hydrophobic- aromatic (F, W, Y). Peptides also include homologous peptides. Homology can be measured according to percent identify using, for example, the BLAST algorithm (default parameters for short sequences). For example, homologous peptides can have greater than 50, 60, 70, 80, 90, 95, or 99 percent identity. Methods for conjugating peptides to oligomeric

compounds such as oligonucleotides is described in, for example, U.S. Pat. No. 6,559,279, which is incorporated herein by reference in its entirety.

In some embodiments, the conjugate moiety is or comprises a protein or peptide. In some embodiments the peptide is a cell penetrating peptides, e.g. Penetratin, transportan, Peptaibol (e.g. trichorovin-Xlla (TV-Xlla)), TAT peptide (HIV). In some embodiments, the peptide is polyarginine (e.g. stearyl-(RxR)(4)). In some embodiments the peptide is N-(2- hydroxypropyl) methacrylamide (HPMA) containing tetrapeptide Gly-Phe-Leu-Gly (GFLG). In some embodiments, the peptide is a beta-amyloid peptide. In some embodiments the protein or peptide in an antibody or antigen binding site containing fragment thereof (epitope binding site). In some embodiments the conjugate is or comprises M6P-HPMA-GFLG (see Yang et al 2009). In some embodiments, the conjugate is or comprises arginine rich peptides (WO2005/115479) - see also WO09005793 RGD peptides. In some

embodiments, the conjugate is or comprises a protein carrier (e.g. albumin, albumin-PEG conjugate - RGD-PEG-albumin) (Kang et al) see also WO09045536. In some embodiments, the conjugate is or comprises histidylated oligolysine (e.g. WO0032764). In some embodiments, the conjugate is or comprises Glycoproteins: transferrin-polycation (e.g. US5354844, WO9217210, WO9213570). In some embodiments, the conjugate is or comprises asialoglycoprotein (US5346696). In some embodiments, the conjugate is or comprises a polycationic protein (e.g. US803095). In some embodiments, the conjugate is or comprises poly-pseudo-lysine conjugates (e.g. WO07113531).

Reporter and dye conjugate groups

Reporter groups that are suitable as conjugate moieties include any moiety that can be detected by, for example, spectroscopic means. Example reporter groups include dyes, flurophores, phosphors, radiolabels, and the like. In some embodiments, the reporter group is biotin, flourescein, rhodamine, coumarin, or related compounds. Reporter groups can also be attached to other conjugate moieties. In some embodiments, the conjugate is or comprises a label or dye, such as a fluorophore, such as FAM (Carboxyfluorescein).

Cross-linking agents can also serve as conjugate moieties. Cross- linking agents facilitate the covalent linkage of the conjugated oligomeric compounds with other

compounds. In some embodiments, cross-linking agents can covaiently link double-stranded nucleic acids, effectively increasing duplex stability and modulating pharmacokinetic properties. In some embodiments, cross-linking agents can be photoactive or redox active. Example cross-linking agents include psoralens which can facilitate interstrand cross-linking of nucleic acids by photoactivation (Lin, et al, Faseb J, 1995, 9, 1371 ). Other cross-linking agents include, for example, mitomycin C and analogs thereof (Maruenda, et al.,

Bioconjugate Chem., 1996, 7, 541 ; Maruenda, et al., Anti-Cancer Drug Des., 1997, 12, 473; and Huh, et al, Bioconjugate Chem., 1996, 7, 659). Cross-linking mediated by mitomycin C can be effected by reductive activation, such as, for example, with biological reductants (e.g., NADPH-cytochrome c reductase/N AD PH system). Further photo-crosslinking agents include aryl azides such as, for example, N-hydroxysucciniimidyl-4- azidobenzoate (HSAB) and N-succinimidyl-6(-4'-azido-2'-nitrophenyl- amino)hexanoate (SAN PAH). Aryl azides conjugated to oligonucleotides effect crosslinking with nucleic acids and proteins upon irradiation. They can also crosslink with earner proteins (such as KLH or BSA).

Various functional conjugate groups Other suitable conjugate moieties include, for example, polyboranes, carboranes, metallopolyboranes, metallocarborane, derivatives thereof and the like (see, e.g., U.S. Pat. No. 5,272,250, which is incorporated herein by reference in its entirety).

Many drugs, receptor ligands, toxins, reporter molecules, and other small molecules can serve as conjugate moieties. Small molecule conjugate moieties often have specific interactions with certain receptors or other biomolecules, thereby allowing targeting of conjugated oligomeric compounds to specific cells or tissues. Example small molecule conjugate moieties include mycophenolic acid (inhibitor of inosine-5'-monophosphate dihydrogenase; useful for treating psoriasis and other skin disorders), curcumin (has therapeutic applications to psoriasis, cancer, bacterial and viral diseases). In further embodiments, small molecule conjugate moieties can be ligands of serum proteins such as human serum albumin (HSA), Numerous ligands of HSA are known and include, for example, arylpropionic acids, ibuprofen, warfarin, phenylbutazone, suprofen, carprofen, fenfufen, ketoprofen, aspirin, indomethacin, (S)-(+)-pranoprofen, dansylsarcosine, 2,3,5- triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide. chlorothiazide, diazepines, indomethicin, barbituates, cephalosporins, sulfa drugs, antibacterials, antibiotics (e.g., puromycin and pamamycin), and the like. Oligonucleotide-drug conjugates and their preparation are described in, for example, WO 00/76554, which is incorporated herein by reference in its entirety.

In some embodiments, the conjugate may be or comprise a small molecule, such as a small molecule drug or pro-drug. Certain drugs are highly effective at targeting specific target tissue or cells, and as such they may be used to target an oligonucleotide to its intended site of action. Furthermore, the small molecule may in itself have a therapeutic activity, typically once cleaved from the oligonucleotide component of the conjugate.

Examples include bisphosphonates (widely used for the treatment of osteoporosis and effective in targeting bone tissues), anti-cancer drugs and chemotherapeutic agents (e.g. doxorubicin or mitomycein C - see US5776907). In some embodiments, the drug may be a nucleoside analogue, such as a nucleoside polymerase inhibitor .

In yet further embodiments, small molecule conjugates can target or bind certain receptors or cells. T-cells are known to have exposed amino groups that can form Schiff base complexes with appropriate molecules. Thus, small molecules containing functional groups such as aldehydes that can interact or react with exposed amino groups can also be suitable conjugate moieties. Tucaresol and related compounds can be conjugated to oligomeric compounds in such a way as to leave the aldehyde free to interact with T-cell targets. Interaction of tucaresol with T-cells in believed to result in therapeutic potentiation of the immune system by Schiff-base formation (Rhodes, et al., Nature, 1995, 377, 6544). In some embodiments, the conjugate is or comprises a (e.g. cell surface) receptor ligand. In some embodiments the conjugate is or comprises a folate receptor ligand, such as a folic acid group - see for example, EP1572067 or WO2005/069994, WO2010/045584). Other ceil surface receptor ligands include antibodies and fragments thereof, prostate-specific membrane antigen, neuron surface antigens (see WO2011/131693)

In some embodiments, the conjugate moieties are ligands for receptors or can associate with molecules that (in turn) associate with receptors. Included in this class are bile acids, small molecule drug ligands, vitamins, aptamers, carbohydrates, peptides (including but not limited to hormones, proteins, protein fragments, antibodies or antibody fragments), viral proteins (e.g. capsids), toxins (e.g. bacterial toxins), and more. Also included in this class are conjugates that are steroidal in nature e.g. cholesterol, cholestanol, cholanic acid, stigmasterols, pregnolones, progesterones, corticosterones, aldosterones, testosterones, estradiols, ergosterols, and more), Preferred conjugate moieties of the disclosure are cholesterol (CHOL), cholestanol (CHLN), cholanic acid (CHLA), stigmasterol (STIG), and ergosterol (ERGO). In certain preferred embodiments, the conjugate moiety is cholesterol.

In some embodiments the conjugate comprises a sterol .such as cholesterol or tocopherol, optionally including a linker, such as a fatty acid linker, e.g. a C6 linker. In some embodiments the conjugates comprise ConjSa or Conj 6a.

Conjugate moieties can also include vitamins. Vitamins are known to be transported into cells by numerous cellular transport systems. Typically, vitamins can be classified as water soluble or lipid soluble. Water soluble vitamins include thiamine, riboflavin, nicotinic acid or niacin, the vitamin B6 pyridoxal group, pantothenic acid, biotin, folic acid, the B]2 cobamide coenzymes, inositol, choline and ascorbic acid. Lipid soluble vitamins include the vitamin A family, vitamin D, the vitamin E tocopherol family and vitamin K (and phytols). Related compounds include retinoid derivatives such as tazarotene and etretinate, [0040] In some embodiments, the conjugate moiety includes folic acid folate) and/or one or more of its various forms, such as dihydrofolic acid, tetrahydrofolic acid, folinic acid, pteropolyglutamic acid, dihydrofolates, tetrahydrofolates, tetrahydropterins, 1-deaza, 3-deaza, 5-deaza, 8- deaza, 10-deaza, 1 ,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza folate analogs, and antifolates. Folate is involved in the biosynthesis of nucleic acids and therefore impacts the survival and proliferation of cells. Folate cofactors play a role in the one-carbon transfers that are needed for the biosynthesis of pyrimidine nucleosides. Cells therefore have a system of transporting folates into the cytoplasm. Folate receptors also tend to be

overexpressed in many human cancer cells, and folate- mediated targeting of

oligonucleotides to ovarian cancer cells has been reported (Li, et al, Pharm. Res. 1998, 15, 1540, which is incorporated herein by reference in its entirety). Preparation of folic acid conjugates of nucleic acids are described in, for example, U.S. Pat. No. 6,528,631 , which is incorporated herein by reference in its entirety.

Vitamin conjugate moieties include, for example, vitamin A (retinol) and/or related compounds. The vitamin A family (retinoids), including retinoic acid and retinol, are typically absorbed and transported to target tissues through their interaction with specific proteins such as cytosol retinol-binding protein type II (CRBP-II), retinol-binding protein (RBP), and cellular retinol-binding protein (CRBP). The vitamin A family of compounds can be attached to oligomeric compounds via acid or alcohol functionalities found in the various family members. For example, conjugation of an N-hydroxy succinimide ester of an acid moiety of retinoic acid to an amine function on a linker pendant to an oligonucleotide can result in linkage of vitamin A compound to the oligomeric compound via an amide bond. Also, retinol can be converted to its phosphoramidite, which is useful for 5' conjugation, alpha-

Tocopherol (vitamin E) and the other tocopherols (beta through zeta) can be conjugated to oligomeric compounds to enhance uptake because of their lipophilic character. Also, vitamin D, and its ergosterol precursors, can be conjugated to oligomeric compounds through their hydroxyl groups by first activating the hydroxyl groups to, for example, hemisuccinate esters. Conjugation can then be effected directly to the oligomeric compound or to an arninolinker pendant from the oligomeric compound. Other vitamins that can be conjugated to oligomeric compounds in a similar manner on include thiamine, riboflavin, pyridoxine, pyridoxamine, pyridoxal, deoxypyridoxine. Lipid soluble vitamin K's and related quinone-containing compounds can be conjugated via carbonyl groups on the quinone ring. The phytol moiety of vitamin K can also serve to enhance binding of the oligomeric compounds to cells. Other functional groups which may be used as conjugates in compounds of the invention, include imidazole conjugate - RNase A catalytic center mimics (polyamine-imidazole conjugates) - see Guerniou et al Nucleic Acids Res (2007); 35 (20): 6778-87,

Conjugates are typically non-nucleotide moieties. However, in the context of blocking groups or targeting groups, or nucleotide analog small therapeutics, it is recognized that the oligonucleotide may be covalently linked to a nucleotide moiety via the DNA RNA

phosphodiester region of the invention. Suitably, a nucleic acid group, as used in the context of the invention may, in some embodiments, lack complementarity to the target of the oligonucleotide (region A).

In some embodiments, the blocking or targeting moiety is an aptamer (see e.g. Meng et al., PLoS One (2012) 7(4): e33434, WO2005/111238 & W012078637).

A blocking group may also be or comprise a oligonucleotide region which is

complementary to, e.g. part of, the antisense oligonucleotide. In this regard the blocking oligonucleotide is covalently bound to an antisense oligonucleotide via the DNA/RNA phosphodiester region (region b), and optionally a linker. The blocking oligonucleotide is, in some embodiments, therefore able to form a duplex with the antisense oligonucleotide. Suitably the blocking nucleotide sequence (as third region or region C) is a short

oligonucleotide sequence of e.g. 3 - 10 nucleotides in length which forms a duplex (i.e. is complementary to) with an equivalent length of the first region. In some embodiments a linker is used between the second region and the blocking region.

Like delivery peptides, nucleic acids can also serve as conjugate like moieties that can affect localization of conjugated oligomeric compounds in a cell. For example, nucleic acid conjugate moieties can contain poly A, a motif recognized by poly A binding protein (PABP), which can localize poly A-containing molecules in the cytoplasm (Gorlach, et al., Exp, Cell Res., 1994, 211 , 400, In some embodiments, the nucleic acid conjugate moiety contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, and at least 25 consecutive A bases. The nucleic acid conjugate moiety can also contain one or more AU-rich sequence elements (AREs), AREs are recognized by ELAV family proteins which can facilitate localization to the cytoplasm (Bollig, et al, Biochem. Bioophys. Res, Commun., 2003, 301 , 665). Example AREs include UUAUUUAUU and sequences containing multiple repeats of this motif. In other embodiments, the nucleic acid conjugate moiety contains two or more AU or AUU motifs. Similarly, the nucleic acid conjugate moiety can also contain one or more CU-rich sequence elements (CREs) (Wein, et al, Eur, J. Biochem,, 2003, 270, 350) which can bind to proteins HuD and/or HuR of the ELAV family of proteins. As with AREs, CREs can help localize conjugated oligomeric compounds to the cytoplasm. In some embodiments, the nucleic acid conjugate moiety contains the motif (CUUU)n, wherein, for example, n can be 1 to about 20, 1 to about 15, or 1 to about 11. The (CUUU)n motif can optionally be followed or preceded by one or more U. In some embodiments, n is about 9 to about 12 or about 11. The nucleic acid conjugate moiety can also include substrates of hnRNP proteins (heterogeneous nuclear

ribonucleoprotein), some of which are involved in shuttling nucleic acids between the nucleus and cytoplasm, (e.g., nhRNP AI and nhRNP K; see, e.g., Mill, et al, Mol. Cell Biol, 2001 , 21 , 7307). Some example hnRNP substrates include nucleic acids containing the sequence UAGGA U or (GG)ACUAGC(A). Other nucleic acid conjugate moieties can include Y strings or other tracts that can bind to, for example, linRNP I. In some

embodiments, the nucleic acid conjugate can contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, and at least 25 consecutive pyrimidine bases. In other embodiments the nucleic acid conjugate can contain greater than 50, greater than 60, greater than 70, greater than 80, greater than 90, or greater than 95 percent pyrimidine bases.

Other nucleic acid conjugate-like moieties can include pumilio (puf protein) recognition sequences such as described in Wang, et al., Cell, 2002, 110, 501. Example pumilio recognition sequences can include UGUANAUR, where N can be any base and R can be a purine base. Localization to the cytoplasm can be facilitated by nucleic acid conjugate moieties containing AREs and/or CREs. Nucleic acid conjugate-like moieties serving as substrates of hnRNPs can facilitate localization of conjugated oligomeric compounds to the cytoplasm (e.g., hnRNP Al or K) or nucleus (e.g., hnRNP I). Additionally, nucleus localization can be facilitated by nucleic acid conjugate-like moieties containing polypyrimidine tracts.

A reactive group

A reactive group is a group which is used in chemical synthesis, which in the context of the present invention may be used "conjugate" the oligonucleotide, or otherwise covalently link the oligonucleotide to the third region (X), such as the conjugate, blocking group or targeting group, or optionally the linker (Y). An example of a reactive group is a phosphoramidite, which is widely used in oligonucleotide synthesis.

An activation group

An activation group is a group which may be activated to form a reactive group. In this respect, an activation group may be considered as a protected reactive group, which may be deprotected prior to enable use of the reactive group, for example in the methods of synthesis/manufacture disclosed herein.

Linkage group A nucleoside linkage is the linkage group either between nucleosides in the oligonucleotide, or, when present, may also describe the group which attaches the third region (X or C) or the linker (Y) to region B - for example this linker may be a phosphate (containing) linkage group or a triazol group.

Blocker group (also referred to as a blocking/blocker moiety)

In some aspects, the third region is a blocking region. A blocker is typically a conjugate or an oligonucleotide (typically not complementary to the target region), which, for example (but not limited to) either through steric hindrance, or through hybridization to the first region (or first and second regions), prevents or reduces activity of the oligomer. The (blocked) activity may be against its intended target (the target) or in some embodiments unintended targets (off-targets).

The oligomeric compound of the invention may therefore comprise a first region, such as a gapmer or LNA gaper oligonucleotide (such as a gapmer of formula X'Y'Z), a second region which is a biocleavable linker, such as region B as described herein, and a third region, region C, which comprises a region of at least 2 consecutive nucleosides, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 nucleotides which are complementary to a corresponding part of the first region. In some embodiments at least 2 nucleosides of region C, such as 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides are high affinity nucleoside analogues, such as LNA (BNA) - in some embodiments, these may form the distal part of region C. The high affinity nucleoside analogues of region C may form a contiguous sequence of high affinity nucleoside analogues, which may be flanked by other nucleosides, such as DNA

nucleosides (also part of region C, referred to as the proximal part of region C). In some embodiments, region C comprises between 2 - 8 {such as 3, 4, 5, 6, & 7 LNA (BNA) nucleotides, and in the same or in a different embodiment a region of between 2 - 16 DNA nucleotides (such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15). In some embodiments, the distal part of region B comprises a contiguous region of high affinity nucleotide analogues, for example a contiguous region of 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides. The proximal region may comprise a contiguous region of non-LNA nucleotides, such as those referred to herein, such as DNA nucleotides, such as a region of 2 - 16 non-LNA nucleotides. It is however also understood that the proximal region may comprise high affinity nucleotide analogues including LNA, but as contiguous regions of LNA can restrict the conformational flexibility of the proximal region (which is thought to act as a loop) it may, in some

embodiments be useful to limit the use of long stretches of LNA in the proximal (or loop forming part), such as no more than 4 consecutive LNAs, such as no more than 3 consecutive LNAs, or no more than 2 consecutive LNAs. In some embodiments, the region of other nucleotides in region C (such as DNA

nucleotides) forms a contiguous sequence with region B, i.e. is proximal to the terminal nucleotide of region B), so that the region of high affinity nucleotides is distal to region B. In such an embodiment, region B and the proximal part of region C (e.g. the region comprising DNA nucleotides) may form a flexible loop, which allows the distal part of region C to hybridize with the first region. The proximal part of region C may or may not be

complementary to a corresponding part of region A. In some embodiments, the distal part of region C is complementary to nucleotides which form a region which is capable of recruiting RNaseH, such as the gap region of a gapmer (referred to herein region Y'). In such an embodiment, the blocking region (region C) forms a duplex with the gap region, or part thereof, thereby blocking the availability of the central region of the gapmer to interact with other molecules or the target or off-targets. The invention therefore provides a solution to the inherent toxicity of DNA phosphorothioate oligonucleotides (which are typically used for the gap region of gapmers), as it allows for the controlled activation of gapmer oligomers (region A) within the target tissue or cells. In this respect, the use of a blocking region can act as a pro-drug. It is recognized that the blocking region (region C or distal part thereof), may also be directed towards other regions of an oligomer, including a mixmer or totalmer oligomer, or the flanking regions of a gapmer, or across the wing region and the gap region of a gapmer. In such an embodiment, the hybridization or region C (or distal part thereof) to region A (or part of region A), prevents the hybridization of the corresponding part of region A to biomolecules, and may therefore also be used to prevent unintended interaction with other biomolecules, enhancing specificity, tissue specific activity, and diminishing the risk of toxicity. The internucleoside linkages between the nucleotides of region C may be other than phosphodiester, such as may be phosphorothioate.

Targeting group (also referred to as a targeting moiety)

A targeting moiety is a group whose presence on the oligomeric compound causes a differential pattern of biodistribution and/or cellular uptake of the oligomeric compound. Targeting groups may be, for example, receptor ligands, antibodies, hormones or hormone analogues, aptamers etc. The examples show the use of cholesterol as a targeting group - cholesterol is recognized by the LDL receptor in the surface of hepatocytes, resulting in the preferential uptake of cholesterol conjugated oligonucleotides into the liver. The examples also illustrate the use of GalNac, tocopherol, and folic acid as targeting groups.

Oligomer linked biocleavable conjugates

The oligomeric compound comprises at least one region B ( second region (region B) which is positioned between at least two of the oligomer regions (referred to as region A and A^* and optionally A"). Further region B regions may be positioned, e.g. between regions A' and A", or between anoligomer region and a function group, such as the conjugate (referred to as region C). Region B (or region Bs) may be a linker such as a cleavable linker (also referred to as a physiologically labile linkage).

In some embodiments, the compound of the invention comprises a biocleavable linker {also referred to as the physiologically labile linker, Nuclease Susceptible Physiological Labile Linkages, or nuclease susceptible linker), for example the phosphate nucleotide linker (such as region B) or a peptide linker, which joins the oligomer regions (or contiguous nucleotide sequence or region A), and optionally joins a conjugate moiety (or region C) to one of the oligomers, optionally via a further linker (Y)..

Biocleavable linkers according to the present invention refers to linkers which are susceptible to cleavage in a target tissue (i.e. physiologically labile), for example liver and/or kidney. It is preferred that the cleavage rate seen in the target tissue is greater than that found in blood serum. Suitable methods for determining the level {%) of cleavage in tissue (e.g. liver or kidney) and in serum are found in example 6, In some embodiments, the biocleavable linker (also referred to as the physiologically labile linker, or nuclease susceptible linker), such as region B, in a compound of the invention, are at least about 20% cleaved, such as at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 80% cleaved, such as at least about 70% cleaved, such as at least about 75% cleaved, in the the liver or kidney homogenate assay of Example 6. In some embodiments, the cleavage (%) in serum, as used in the assay in Example 6, is less than about 20%, such as less than about 10%, such as less than 5%, scuh as less than about 1%.

Biocleavable linkers according to the present invention refers to linkers which are susceptible to cleavage in a target tissue (i.e. physiologically labile), for example liver and/or kidney. It is preferred that the cleavage rate seen in the target tissue is greater than that found in blood serum. Suitable methods for determining the level (%) of cleavage in tissue (e.g. liver or kidney) and in serum are found in example 6. In some embodiments, the biocleavable linker (also referred to as the physiologically labile linker, or nuclease susceptible linker), such as region B, in a compound of the invention, are at least about 20% cleaved, such as at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 80% cleaved, such as at least about 70% cleaved, such as at least about 75% cleaved, in the liver or kidney homogenate assay of Example 6. In some embodiments, the cleavage (%) in serum, as used in the assay in Example 6, is less than about 30%, is less than about 20%, such as less than about 10%, such as less than 5%, such as less than about 1%, In some embodiments, which may be the same of different, the biocleavable linker (also referred to as the physiologically labile linker, or nuclease susceptible linker), such as region B, in a compound of the invention, are susceptible to S1 nuclease cleavage. Susceptibility to S1 cleavage may be evaluated using the S1 nuclease assay shown in Example 6. In some embodiments, the biocleavable linker (also referred to as the physiologically labile linker, or nuclease susceptible linker), such as region B, in a compound of the invention, are at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 60% cleaved, such as at least about 70% cleaved, such as at least about 80% cleaved, such as at least about 90% cleaved, such as at least 95% cleaved after 120min incubation with S1 nuclease according to the assay used in Example 6.

Nuclease Susceptible Physiological Labile Linkages: In some embodiments, the oligomer (also referred to as oligomeric compound) of the invention (or conjugate) comprises:

i) a first region (region A), which comprises 7 - 18 contiguous nucleotides;

ii) a second region (region B) which comprises a biocleavable linker

iii) a further first region (region A'), which comprises 7 - 18 contiguous nucleotides iv) optionally a further region B

v) a third region (C) which comprises a conjugate moiety, a targeting moiety, an activation moiety, wherein the third region is covalent linked to the second region.

Suitably the first regions are phosphorothioate linked oligomer regions. In some

embodiments, region B may be a phosphate nucleotide linker. The third region may be a conjugate, e.g. a lipophilic conjugate, such as a lipid, a fatty acid, sterol, such as cholesterol or tocopherol. Phosphate nucleotide linkers may also be used for other conjugates, for example carbohydrate conjugates, such as GalNac.

Peptide and other Linkers

In some embodiments, the biocleavable linker (region B) is a peptide, such as a trilysine peptide linker which may be used in a polyGalNac conjugate, such a triGalNac conjugate. Other linkers known in the art which may be used, including disulfide linkers (also referred to as dithio or disulphide herein). Other peptide linkers include , e.g. a Tyr-Asp(Asp) tri peptide or Asp(Asp) dipeptide.

Other phosphate nucleotide linkers as region B

In some embodiments, region B (a second region) comprises between 1 - 6 nucleotides, which is covalently linked to the 5' or 3' nucleotide of the first region (an oligomer region), such as via a internucleoside linkage group such as a phosphodiester linkage. In some embodiments, region B may be physiologically labile internucleoside linkages for example, the internucleoside linkage between the first (A) and second oligomer region (Α') and optionally between the second oligomer region (Α') and a third oligomer region (A") may be phosphodiester linkage and, optionally the nucleoside of the second oligomer region [such as immediately] adjacent to the first region is either DNA or RNA (See Figure 1 ).

In some embodiments, region B may be a physiologically labile internucleoside linkages for example, the internucleoside linkage between the one or more (or each) oligomer region and a linkage group (see Figure 2) may be phosphodiester linkage and, optionally the nucleoside of the oligomer region [such as immediately] adjacent to linkage group is either DNA or RNA.

In some embodiments, one or more (or each) region A and region B form a single contiguous nucleotide sequence of 8 - 22, such as 10 - 20 nucleotides in length.

In some aspects the internucleoside linkage between a region A and a region B may be considered part of region B.

Linkers

A linkage 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 via one or more covalent bonds. Conjugate moieties (or targeting or blocking moieties) can be attached to the oiigomeric compound directly or through a linking moiety (linker or tether) - a linker. Linkers are bifunctional moieties that serve to covalently connect a third region, e.g. a conjugate moiety, to an oiigomeric compound {such as to region B). In some embodiments, the linker comprises a chain structure or an oligomer of repeating units such as ethylene glyol or amino acid units. The linker can have at least two functionalities, one for attaching to the oiigomeric compound and the other for attaching to the conjugate moiety. Example linker functionalities can be electrophilic for reacting with nucleophilic groups on the oligomer or conjugate moiety, or nucleophilic for reacting with electrophilic groups. In some

embodiments, linker functionalities include amino, hydroxy I, carboxylic add, thiol, phosphoramidate, phophate, phosphite, unsaturations (e.g., double or triple bonds), and the like. Some example linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4- (N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), 6- aminohexanoic acid (AH EX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4- aminocyclohexylcarboxylic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane- l-carboxy-(6-amido-caproate) (LCSMCC), succinimidyl m-maleimido-benzoylate (MBS), succinimidyl N-e-maleimido-caproylate (EMCS), succinimidyl 6-(beta - maleimido-propionamido) hexanoate (SMPH), succinimidyl N-(a-maleimido acetate) (AMAS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), beta -alanine (beta -ALA), phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC), beta - (cyclopropyl) alanine (beta -CYP ), amino dodecanoic acid (ADC), alylene diols,

polyethylene glycols, amino acids, and the like.

A wide variety of further linker groups are known in the art that can be useful in the attachment of conjugate moieties to oligomeric compounds. A review of many of the useful linker groups can be found in, for example, Antisense Research and Applications, S. T. Crooke and B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350, A disulfide linkage has been used to link the 3' terminus of an oligonucleotide to a 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, 11 1 , 8524). Nelson, et al,, Nuc. Acids Res. 1989, 17, 7187 describe a linking reagent for attaching biotin to the 3'-terminus 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 under the name 3'-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991 , 32, 879. A similar commercial reagent for linking to the 5 '-terminus of an oligonucleotide is 5'- Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, et al, Antisense Research and Development 1991 , 1 , 161 to link fluorescein to the 5"- terminus of an oligonucleotide. Other compounds such as acridine have been attached to the 3 '-terminal phosphate group of an oligonucleotide via a polymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci, USA 1984, 81 , 3297). [0074] Any of the above groups can be used as a single linker or in combination with one or more further linkers.

Linkers and their use in preparation of conjugates of oligomeric compounds are provided throughout the art such as in WO 96/11205 and WO 98/52614 and U.S. Pat. Nos. 4,948,882; 5,525,465; 5,541 ,313; 5,545,730; 5,552,538; 5,580,731 ; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469; 5,245,022; 5,112,963; 5,391,723; 5,510475; 5,512,667; 5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437, each of which is incorporated by reference in its entirety.

As used herein, a physiologically labile bond is a labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body (also referred to as a cleavable linker). Physiologically labile linkage groups are selected such that they undergo a chemical transformation (e.g., cleavage) when present in certain physiological conditions. Mammalian intracellular conditions include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic or hydrolytic enzymes. In some embodiments, the cleavable linker is susceptible 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 region (e.g. 1 - 10) phosphodiester linked nucleosides, such as DNA nucleosides,

Chemical transformation (cleavage of the labile bond) may be initiated by the addition of a pharmaceutically acceptable agent to the cell or may occur spontaneously when a molecule containing the labile bond reaches an appropriate intra-and/or extra-cellular environment. For example, a pH labile bond may be cleaved when the molecule enters an acidified endosome. Thus, a pH labile bond may be considered to be an endosomal cleavable bond. Enzyme cleavable bonds may be cleaved when exposed to enzymes such as those present in an endosome or lysosome or in the cytoplasm, A disulfide bond may be cleaved when the molecule enters the more reducing environment of the cell cytoplasm. Thus, a disulfide may be considered to be a cytoplasmic cleavable bond. As used herein, a pH-labile bond is a labile bond that is selectively broken under acidic conditions (pH<7). Such bonds may also be termed endosomally labile bonds, since cell endosomes and lysosomes have a pH less than 7.

Activated oligomers

In some embodiments, the invention provides an activated oligomer - i.e. an intermediate used in the synthesis of the oligomer of the invention - e.g. the conjugated oligomer. In this respect, the oligomer of the invention may, in some embodiments comprise region A and region B as described herein, and region B in covalently linked to an activation (or reactive) group, suitable for use in conjugation of the oligomer.

The term "activated oligomer," as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described.

Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH₂ group of the adenine base, a spacer that is preferably hydrophiiic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH₂ group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W

Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,

triphenylmethyl, benzyloxycarbonyl, tert-b utoxycarbony I , and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self- cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionaiized at the 5' end in order to allow covalent attachment of the conjugated moiety to the 5' end of the oligomer. In other embodiments, oligomers of the invention can be functionaiized at the 3^* end. In still other embodiments, oligomers of the invention can be functionaiized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionaiized at more than one position independently selected from the 5' end, the 3^* end, the backbone and the base.

In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionaiized, and the oligomer is functionaiized upon completion of synthesis. In some embodiments, the oligomers are functionaiized with a hindered ester containing an aminoalkyi linker, wherein the alkyi portion has the formula (CH₂)_W, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyi portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-O-C(O)- (CH₂)_WNH).

In other embodiments, the oligomers are functionaiized with a hindered ester containing a (CH₂)_w-sulfhydryI (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyi portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-0-C(0)-(CH₂)_wSH)

In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can be

synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety,

In still other embodiments, the oligomers of the invention are functionaiized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Patent Nos. 4,962,029 and

4,91 ,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a

functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other

embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such

functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Patent Nos. 4,962,029 and 4,914,210.

In some embodiments, the 5'-terminus of a solid-phase bound oligomer is

functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing 2 -sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(0-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2"-0-{e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'- N,N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991 , 34, 7171.

In still further embodiments, the oligomers of the invention may have amine-containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III.).

Other commercially available linking groups are 5'-Ami no-Modifier C6 and 3'-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5'-Amino- Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3'-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).

Methods of synthesis and manufacture

The invention also provides methods of synthesis or manufacture of the oligomer of the invention. The oligomer may be made using standard oligonucleotide synthesis, which is typically performed on a solid support, such as a universal support. The oligomer of the invention may be synthesized, for example, by the sequential synthesis of a oligomer region A', region B (B), and a second oligomer region (Α'), optionally followed by the addition (e.g. conjugation) of the third region (C) optionally via a linker (Y). In some embodiments, the oligomer of the invention may be synthesized, for example, by the sequential synthesis of a oligomer region A', region B (B), and a second oligomer region (A), a second region B (B^*) optionally followed by the addition (e.g. conjugation) of the third region (C) optionally via a linker (Y). In some embodiments, the oligomer of the invention may be synthesized, for example, by the sequential synthesis of a oligomer region A', region B (B), and a second oligomer region (A), a second region B (Β') and a third oligomer region (A") optionally followed by the addition (e.g. conjugation) of the third region (C) optionally via a linker (Y). In some embodiments, the oligomer of the invention may be synthesized, for example, by the sequential synthesis of a oligomer region A', region B (B), and a second oligomer region (A), a second region B (Β') and a third oligomer region (A") followed by a third region B (B"), followed by the addition (e.g. conjugation) of the third region (C) optionally via a linker (Y).

Region Y, when present may be joined to the region B, and region C subsequently added to region Y, or region Y and C may be added to region B in a single reaction step.

Alternatively, the oligomer synthesis my occur via the initial coupling of region C, or region C and Y to the oligonucleotide support column, followed by sequential oligonucleotide synthesis of the oligonucleotide part of the compound of the invention. .

Alternatively, the use of a cleavable bidirectional group attached to the oligonucleotide synthesis support (in an initial or pre-step), allows for a method where the oligonucleotide regions of the oligonucleotide are synthesized on one reactive group of the bifunctional group, and region C or region C and Y are synthesized on a second reactive group of the bifunctional group, wherein the oligonucleotide synthesis or addition of C (or C and Y) to the support may occur in any order or even together. The cleavage of the bifunctional group from the support then produces the oligomer of the invention. The bifunctional group may for example be a nucleoside, where one entity (e.g. region B or C or C-Y-) is attached to a phosphate containing group on the nucleoside (e.g. a 5' or 3' group), and the other (e.g. region B or C or C-Y-), is attached, for example to an reactive group present on the nucleobase. Alternatively region C or C-Y may be joined to the oligomer (region B) after oligonucleotide synthesis, such as after the cleavage step. The invention therefore also relates to the intermediate oligomer, which comprises the oligonucleotide part of the compound of the invention, and a reactive or activation group attached to region B, which is subsequently used to join region C or regions C and Y to region B.

Region Y or region C may be linked to a region B or an oligomer as a

phosphoramidite, for example - allowing for the formation of the oligomer in a single oligonucleotide synthesis, followed by cleavage of the oligomer from the oligonucleotide synthesis support (US). In this regard, in some embodiments, the linkage group between an oligomer region or a region B and region C or Y may be a phosphate containing group, such as a nucleoside linkage, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, methylphosphonate or others, such as those referred to herein.

Alternatively other chemical linkages may be used such as a triazol group.

In some embodiments, the third region (C) or C-Y- may be linked to a region B or an oligomer region via a group other than a 5' or 3' phosphate, for example via a reactive group at another position, for example a reactive group, such as an amine on the base of a nucleoside in region B.

Oligonucleotide synthesis may occur in the 5 ' - 3' direction, or, as is typical of most oligonucleotide synthesis, in the 3' - 5^* direction.

In some non-limiting examples, the oligonucleotide-conjugate construct can be assembled in different ways, e.g.

A) The B - A part of the construct can be made on an oligonucleotide synthesis machine capable of synthesizing both phosphorothioate and phosphorodiester linkages. B - A can then optionally be elongated by standard phosphoramidite chemistry using a building block C-A-P (e.g. conjugate moiety with linker attached) to create C-A-B-A or with building block C-P (conjugate moiety with no linker) to create C-B-A

X-A-P x-p B) The B-A part of the construct can be made on an oligonucleotide synthesis machine capable of synthesizing both phosphorthioate and phosphordiester linkages. B-A can then optionally be sequentially elongated by standard phosphoramidite chemistry using a building block DMTrO-A-P followed by building block C-P to create C-A-B-A with a PO or PS linkage between the C and A part.

DMTrO-A-P

The B-A part of the construct can be made on an oligonucleotide synthesis machine capable of synthesizing both phosphorthioate and phosphordiester linkages. B-A can then optionally be sequentially elongated by standard phosphoramidite chemistry using a building block PGN-A-P to create H₂N-A-B-A. After cleavage and deprotection of the

oligonucleotide the free amine of the oligonucleotide can be conjugated with moiety C in which a functional group of C has been activated in order to react with the terminal primary amine of the oligonucleotide.

Compositions

The oligomer of the invention may be used in pharmaceutical formulations and compositions. Suitably, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant. WO2007/031091 provides suitable and preferred pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in

WO2007/031091 - which are also hereby incorporated by reference.

Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other

oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof,

Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts, A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound. In this regard the prodrug may comprise region B and a conjugate, targeting or blocking moiety as according to the present invention. In some embodiments, the oligomer of the invention is a pro-drug.

The use of lipophilic conjugates according to the invention allows for the incorporation of the oligomer of the invention into lipidoids or liposomes, e.g. cationic liposomes (e.g. cationic liposome SNALPs (stable nucleic acid lipid particle), which are particularly useful for delivery of oligomers e.g. to the liver, e.g. si NAs.

Applications

The oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.

In research, in some embodiments, such oligomers may be used to specifically inhibit the synthesis of protein (typically by degrading or inhibiting the mRNA and thereby prevent protein formation) in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.

For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of the target is treated by administering oligomeric compounds in accordance with this invention. Further provided are methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of the target by administering a

therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

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

Medical Indications

In some embodiments, the disease is cancer. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a cardiovascular disease, such as

In some embodiments the disease or disorder is myocardial infarction (Ml).

In some embodiments, the disease or disorder is, or results in or is associated with fibrosis, such as liver-fibrosis, cardiac fibrosis or local fibrosis.

In some embodiments, the disease or disorder is blood clotting disorder.

In some embodiments the disease or disorder is or comprises (results in or is associated with) bone-lose.

In some embodiments, the disease or disorder is a liver disease or disorder.

In some embodiments the disease or disorder is a metabolic disorder, which may for example be a liver disease or disorder, and/or in some aspects a cardiovascular disease or disorder).

Cardiovascular/Metabolic diseases include, for examples, metabolic syndrome, obesity, hyperlipidemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant,

hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD)., atherosclerosis, heart disease, diabetes (I and/or II), NASH, acute coronary syndrome

(ACS), NASH, chronic heart failure, cardiovascular disease, cardie metabolic disease, hyperlipidaemia and related disorders, metabolic syndrome, atherosclerosis, chronic heart failure, vascular disease, peripheral arterial disease, heart disease, ischemia, type 2 diabetes, type 1 diabetes,

In some embodiments, the disease or disorder is selected from the group consisting of metabolic syndrome, obesity, hyperlipidemia , atherosclerosis, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant, hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).

In some embodiments, the disease or disorder is selected from the group consisting of chronic heart failure, cardiovascular disease, cardio metabolic disease, chronic heart failure, vascular disease, peripheral arterial disease, heart disease, ischemia, acute coronary syndrome (ACS). In some embodiments, the disease or disorder is type 2 diabetes, type 1 diabetes,

In some embodiments, the disease or disorder is a viral disease, such as

polycythemia, hepatitis C, hepatitis B, BKV, HIV.

In some embodiments, the disease or disorder is a severe and rare diseases (or genetic disorder).

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

Generally stated, some aspects of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of the target, comprising administering to the mammal and therapeutically effective amount of an oligomer targeted to the target that comprises one or more LNA units. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount,

An interesting aspect of the invention is directed to the use of the compound as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition as referred to herein.

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

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

In some embodiments, the term 'treatment' as used herein refers to both treatment of an existing disease {e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.

fasa-iet~?b* CUAUACAACCUACUGCCUUCCC 99 GGTTGTATA 1036 GTTGTATA 1973 TTGTATA 2910 hsa~let~7c UGAGGUAGUAGGUUGUAUGGUU 100 TACTACCTC 1037 ACTACCTC 1974 CTACCTC 2911 hsa-iet-?c* DAGAGDUACACCCOGGGAGDDA 101 TGTAACTCT 1038 GTAACTCT 1975 TAACTCT 2912 hsa-let-7d AGAGGOAGOAGGUOGCADAGDD' 102 TACTACCTC 1039 ACTACCTC 1976 CTACCTC 2913 fasa~iet-7d* CUAOACGACCOGCOGCCUUD'CU' 103 GGTCGTATA 1040 GTCGTATA 1977 TCGTATA 2914 hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU 104 TCCTACCTC 1041 CCTACCTC 1978 CTACCTC 2915 hsa-iet~7e* CUAUACGGCCUCCUAGCUUUCC 105 GGCCGTATA 1042 GCCGTATA 1979 CCGTATA 2916 hsa~let~7f OGAGGOAGOAGAIJUGDAOAGOO 106 TACTACCTC 1043 ACTACCTC 1980 CTACCTC 2917 hsa~iet~?f -1* CUAUACAAUCUAUUGCCUUCCC 107 GATTGTATA 1044 ATTGTATA 1981 TTGTATA 2918 hsa~iet-7f -2* COAOACAGOCOACDGOCUUOCC 108 GACTGTATA 1045 ACTGTATA 1982 CTGTATA 2919 hsa-iet-7g OGAGGOAGDAGDUOGDACAGOD' 109 TACTACCTC 1046 ACTACCTC 1983 CTACCTC 2920 hsa~let~7g* CUGOACAGGCCACUGCCITUGC 110 GCCTGTACA 1047 CCTGTACA 1984 CTGTACA 2921 hsa-let -7i UGAGGUAGUAGDUUGOGCDGOU 111 TACTACCTC 1048 ACTACCTC 1985 CTACCTC 2922 hsa-let-7i* CUGCGCAAGCOACnGCCaOGCO 112 GCTTGCGCA 1049 CTTGCGCA 1986 TTGCGCA 2923 hsa~miR~l DGGAADGUAAAGAAGDADGOAO 113 TTACATTCC 1050 TACATTCC 1987 ACATTCC 2924 hsa~miR~lQ0 AACCCGDAGAOCCGAACITUGUG 114 TCTACGGGT 1051 CTACGGGT 1988 TACGGGT 2925 hsa-miR-100* CAAGCDUGOAO'CDAOAGGDADG 115 TACAAGCTT 1052 ACAAGCTT 1989 CAAGCTT 292S hsa-miR-101 UACAGOACDGOGADAA.CDGAA 116 CAGTACTGT 1053 AGTACTGT 1990 GTACTGT 2927 hsa-miR-101* CAGUUAUCACAGUGCUQAUGCU 117 GTGATAACT 1054 TGATAACT 1991 GATAACT 2928 hsa-miR-103 AGCAGCAOOGOACAGGGCUAUGA 118 CAATGCTGC 1055 AATGCTGC 1992 ATGCTGC 2929 hsa-miR-103-as UCAOAGCCCUGOACAAUGCUGCU 119 AGGGCTATG 1056 GGGCTATG 1993 GGCTATG 2930 hsa-miR-105 UCAAAUGCUCAGACUCCUGUGGO 120 GAGCATTTG 1057 AGCATTTG 1994 GCATTTG 2931 hsa-miR-105* ACGGAUGUUUGAGCAUGUGCUA 121 AAACATCCG 1058 AACATCCG 1995 ACATCCG 2932 hsa-miR-106a AAAAGUGCOUACAGUGCAGGUAG 122 AAGCACTTT 1059 AGCACTTT 1996 GCACTTT 2933 hsa-miR-106a* CUGCAAUGOAAGCACUUCUOAC 123 TACATTGCA 1060 ACATTGCA 1997 CATTGCA 2934 hsa~miR~106b OAAAGOGCOGACAGUGCAGAU 124 CAGCACTTT 1061 AGCACTTT 1998 GCACTTT 2935 hsa~miR~106b* CCGCACDGUGGGUACOUGCUGC 12S CACAGTGCG 1062 ACAGTGCG 1999 CAGTGCG 2936 hsa-miR-107 AGCAGCAUUGUACAGGGCUAUCA 126 CAATGCTGC 1063 AATGCTGC 2000 ATGCTGC 2937 hsa-miR-lOa UACCCDGOAGAUCCGAAUUUGUG 127 CTACAGGGT 1064 TACAGGGT 2001 ACAGGGT 2938 hsa~miR-10a* CAAAOUCGUAUCUAGGGGAAUA 128 TACGAATTT 1065 ACGAATTT 2002 CGAATTT 2939 hsa-miR-lOb UACCCUGDAGAACCGAAUUUGUG 129 CTACAGGGT 1066 TACAGGGT 2003 ACAGGGT 2940 hsa-miR-lOb* ACAGAUUCGAUUCUAGGGGAAU 130 TCGAATCTG 10S7 CGAATCTG 2004 GAATCTG 2941 hsa-miR-1178 UUGCUCACUGUUCUUCCCUAG 131 CAGTGAGCA 1068 AGTGAGCA 2005 GTGAGCA 2942 hsa-miR-1179 AAGCAUUCaUUCAUUGGUUGG 132 AAGAATGCT 1069 AGAATGCT 2006 GAATGCT 2943 hsa-miR-1180 UUUCCGGCUCGCGUGGGUGUGU 133 GAGCCGGAA 1070 AGCCGGAA 2007 GCCGGAA 2944 hsa-miR-1181 CCGUCGCCGCCACCCGAGCCG 134 GCGGCGACG 1071 CGGCGACG 2008 GGCGACG 2945 hsa-miR-1182 GAGGGUCUDGGGAGGGAOGOGAC 135 CAAGACCCT 1072 AAGACCCT 2009 AGACCCT 2946 hsa-miR-1183 CACUGUAGGUGAUGGUGAGAGUGGGCA 136 ACCTACAGT 1073 CCTACAGT 2010 CTACAGT 2947 hsa-miR-1184 CCUGCAGCGACUUGAUGGCUUCC 137 TCGCTGCAG 1074 CGCTGCAG 2011 GCTGCAG 2948 hsa-miR-1185 AGAGGAUACCCOUUGUAUGOU 138 GGTATCCTC 1075 GTATCCTC 2012 TATCCTC 2949 hsa-miR-1197 UAGGACACA.UGGUCUACUUCU 139 ATGTGTCCT 1076 TGTGTCCT 2013 GTGTCCT 2950 hsa-miR-1200 CUCCUGAGCCAUUCUGAGCCUC 140 GGCTCAGGA 1077 GCTCAGGA 2014 CTCAGGA 2951 hsa-miR-1201 AGCCUGAUOAAACACAUGCUCUGA 141 TAATCAGGC 1078 AATCAGGC 2015 ATCAGGC 2952 hsa-miR-1202 GUGCCAGCUGCAGUGGGGGAG 142 CAGCTGGCA 1079 AGCTGGCA 201S GCTGGCA 2953 hsa-miR-1203 CCCGGAGCCAGGAUGCAGCUC 143 TGGCTCCGG 1080 GGCTCCGG 2017 GCTCCGG 2954 hsa-miR-1204 UCGUGGCCUGGUCUCCAOUAU 144 CAGGCCACG 1081 AGGCCACG 2018 GGCCACG 2955 hsa-miR-1205 UCUGCAGGGUUUGCUUUGAG 145 ACCCTGCAG 1082 CCCTGCAG 2019 CCTGCAG 2956 hsa-miR-1206 UGUUCAUGOAGAUGOUUAAGC 146 TACATGAAC 1083 ACATGAAC 2020 CATGAAC 2957 hsa-miR- 120 -3p UCAGCUGGCCCOCAUUUC 147 GGCCAGCTG 1084 GCCAGCTG 2021 CCAGCTG 2958 hsa~miR-1207~5p UGGCAGGGAGGCUGGGAGGGG 148 CTCCCTGCC 1085 TCCCTGCC 2022 CCCTGCC 2959 hsa-miR-1208 UCACUGUUCAGACAGGCGGA 149 TGAACAGTG 1086 GAACAGTG 2023 AACAGTG 2960 hsa-miR-122 UGGAGDGOGACAAUGGDGUUUG 150 TCACACTCC 1087 CACACTCC 2024 ACACTCC 2961 hsa-miR-122* AACGCCAUUAUCACACUAAAUA 151 TAATGGCGT 1088 AATGGCGT 2025 ATGGCGT 2962 hsa-miR-1224-3p CCCCACCUCCUCUCUCCUCAG 152 GGAGGTGGG 1089 GAGGTGGG 2026 AGGTGGG 2963 hsa~miR~1224~5p GOGAGGACUCGGGAGGUGG 153 GAGTCCTCA 1090 AGTCCTCA 2027 GTCCTCA 2964 hsa-miR~1225-3p UGAGCCCCUGUGCCGCCCCCAG 154 CAGGGGCTC 1091 AGGGGCTC 2028 GGGGCTC 2965 hsa-miR- 1225-5p GUGGGUACGGCCCAGUGGGGGG 155 CCGTACCCA 1092 CGTACCCA 2029 GTACCCA 2966 hsa-miR- 1226 UCACCAGCCCOGUGIIUeeCUAG 156 GGGCTGGTG 1093 GGCTGGTG 2030 GCTGGTG 2967 hsa-miR-1226* GOGAGGGCAUGCAGGCCOGGAUGGGG 157 ATGCCCTCA 1094 TGCCCTCA 2031 GCCCTCA 2968 hsa-miR- 1227 CGUGCCACCCUUUUCCCCAG 158 GGGTGGCAC 1095 GGTGGCAC 2032 GTGGCAC 2969 hsa-miR- 1228 OCACACCOGCCOCGCCCCCC 159 GCAGGTGTG 1096 CAGGTGTG 2033 AGGTGTG 2970 hsa-miR- 1228* GOGGGCGGGGGCAGGOGUGDG 160 CCCCGCCCA 1097 CCCGCCCA 2034 CCGCCCA 2971 hsa-miR- 1229 COCOCACCACOGCCCD'CCCACAG 161 GTGGTGAGA 1098 TGGTGAGA 2035 GGTGAGA 2972 hsa-miR-1231 GDGD'CUGGGCGGACAGCDGC 1S2 GCCCAGACA 1099 CCCAGACA 2036 CCAGACA 2973 hsa-miR- 1233 OGAGCCCUGOCCUCCCGCAG 163 A.CAGGGCTC 1100 CAGGGCTC 2037 AGGGCTC 2974 hsa-miR- 1234 OCGGCCOGACCACCCACCCCAC 164 GTCAGGCCG 1101 TCAGGCCG 2038 CAGGCCG 2975 hsa-miR- 1236 CCUCUUCCCCUUGUCUCUCCAG 165 GGGGAAGAG 1102 GGGAAGAG 2039 GGAAGAG 2976 hsa-miR- 1237 UCCOOCOGCUCCGOCCCCCAG 166 AGCAGAAGG 1103 GCAGAAGG 2040 CAGAAGG 2977 hsa-miR-1238 COUCCOCGOCOGOCUGCCCC 167 GACGAGGAA 1104 ACGAGGAA 2041 CGAGGAA 2978 hsa-miR- 124 UAAGGCACGCGGUGAAOGCC 168 GCGTGCCTT 1105 CGTGCCTT 2042 GTGCCTT 2979 hsa-miR- 124* CGUGUUCACAGCGGACCUUGAU 169 TGTGAACAC 1106 GTGAACAC 2043 TGAACAC 2980 hsa-miR- 1243 AACOGGAUCAAUOAUAGGAGDG 170 TGATCCAGT 1107 GATCCAGT 2044 ATCCAGT 2981 hsa-miR- 1244 AAGUAGOUGGUUUGUAUGAGAUGGUU 171 CCAACTACT 1108 CAACTACT 2045 AACTACT 2982 hsa-miR- 12 5 AAGUGADCOAAAGGCCOACAO 172 TAGATCACT 1109 AGATCACT 2046 GATCACT 2983 hsa-miR- 1246 AADGGAOUOUUGGAGCAGG 173 AAAATCCAT 1110 AAATCCAT 2047 AATCCAT 2984 hsa-miR- 1247 ACCCGUCCCGUUCGUCCCCGGA 174 CGGGACGGG 1111 GGGACGGG 2048 GGACGGG 2985 hsa-miR- 12 8 ACCUUCUUGUAUAAGCACUGUGCUAAA 175 ACAAGAAGG 1112 CAAGAAGG 2049 AAGAAGG 2986 hsa-miR-1249 ACGCCCUUCCCCCCCUUCUUCA 176 GGAAGGGCG 1113 GAAGGGCG 2050 AAGGGCG 2987 hsa-miR-1250 ACGGOGCOGGAOGUGGCCOUU 177 CCAGCACCG 1114 CAGCACCG 2051 AGCACCG 2988 hsa-miR- 1251 ACDCDAGCDGCCAAAGGCGCD' 178 CAGCTAGAG 1115 AGCTAGAG 2052 GCTAGAG 2989 hsa-miR- 1252 AGAAGGAAAIJUGAAOUCADUUA 179 ATTTCCTTC 1116 TTTCCTTC 2053 TTCCTTC 2990 hsa-miR- 1253 AGAGAAGAAGAUCAGCCUGCA 180 CTTCTTCTC 1117 TTCTTCTC 2054 TCTTCTC 2991 hsa-miR- 1254 AGCCUGGAAGCDGGAGCCOGCAGO 181 CTTCCAGGC 1118 TTCCAGGC 2055 TCCAGGC 2992 hsa-miR- 1255a AGGAUGAGCAAAGAAAGUAGAUU 182 TGCTCATCC 1119 GCTCATCC 2056 CTCATCC 2993 hsa-miR- 1255b CGGADGAGCAAAGAAAGUGGUU 183 TGCTCATCC 1120 GCTCATCC 2057 CTCATCC 2994 hsa-miR- 1256 AGGCAUUGACUUCUCACUAGCU 184 GTCAATGCC 1121 TCAATGCC 2058 CAATGCC 2995 hsa-miR- 1257 AGUGAAUGAUGGGUUCUGACC 185 ATCATTCAC 1122 TCATTCAC 2059 CATTCAC 2996 hsa-miR-1258 AGUUAGGAUUAGGUCGUGGAA 186 AATCCTAAC 1123 ATCCTAAC 2060 TCCTAAC 2997 hsa-miR- 1259 AUAUAUGAUGACUUAGCUUUU 187 CATCATATA 1124 ATCATATA 2061 TCATATA 2998 hsa-miR- 125a-3p ACAGGUGAGGUUCUUGGGAGCC 188 CCTCACCTG 1125 CTCACCTG 2062 TCACCTG 2999 hsa-miR- 125a~ 5p UCCCUGAGACCCUUUAACCUGUGA 189 GTCTCAGGG 1126 TCTCAGGG 2063 CTCAGGG 3000 hsa-miR- 125b OCCCOGAGACCCOAACnUGOGA 190 GTCTCAGGG 1127 TCTCAGGG 2064 CTCAGGG 3001 hsa-miR- 125b-l* ACGGGTJUAGGCUCDUGGGAGCU 191 CCTAACCCG 1128 CTAACCCG 2065 TAACCCG 3002 hsa-miR- 125b-2* OCACAAGOCAGGCD'COUGGGAC 192 TGACTTGTG 1129 GACTTGTG 2066 ACTTGTG 3003 hsa-miR- 126 DCGOACCGDGAGOAADAADGCG 193 CACGGTACG 1130 ACGGTACG 2067 CGGTACG 3004 hsa-miR- 126* CAUUAUUACUUUUGGUACGCG 194 AGTAATAAT 1131 GTAATAAT 2068 TAATAAT 3005 hsa-miR- 1260 AUCCCACCUCUGCCACCA 195 GAGGTGGGA 1132 AGGTGGGA 2069 GGTGGGA 3006 hsa-miR- 1261 AUGGAUAAGGCUUUGGCUU 196 CCTTATCCA 1133 CTTATCCA 2070 TTATCCA 3007 hsa-miR- 1262 AUGGGUGAAUUUGUAGAAGGAU 197 ATTCACCCA 1134 TTCACCCA 2071 TCACCCA 3008 hsa-miR- 1263 AOGGOACCCOGGCAOACUGAGO 198 AGGGTACCA 1135 GGGTACCA 2072 GGTACCA 3009 hsa-miR- 1264 CAAGUCUUAUUUGAGCACCUGUn 199 ATAAGACTT 1136 TAAGACTT 2073 AAGACTT 3010 hsa-miR- 1265 CAGGAUGUGGUCAAGUGUUGUU 200 CCACATCCT 1137 CACATCCT 2074 ACATCCT 3011 hsa-miR- 1266 CCUCAGGGCOGUAGAACAGGGCO 201 AGCCCTGAG 1138 GCCCTGAG 2075 CCCTGAG 3012 hsa-miR- 1267 CCUGUUGAAGUGUAAUCCCCA 202 CTTCAACAG 1139 TTCAACAG 2076 TCAACAG 3013 hsa-miR- 1268 CGGGCGOGGOGGUGGGGG 203 ACCACGCCC 1140 CCACGCCC 2077 CACGCCC 3014 hsa-miR- 1269 COGGACUGAGCCGUGCUACUGG 204 CTCAGTCCA 1141 TCAGTCCA 2078 CAGTCCA 3015 hsa-miR-12'7-3p UCGGAUCCGUCUGAGCUUGGCU 205 ACGGATCCG 1142 CGGATCCG 2079 GGATCCG 3016 hsa-miR- 127~5p CDGAAGCDCAGAGGGCDCUGAO 206 TGAGCTTCA 1143 GAGCTTCA 2080 AGCTTCA 3017 hsa-miR- 1270 CDGGAGADADGGAAGAGCDGUGO' 207 ATATCTCCA 1144 TATCTCCA 2081 ATCTCCA 3018 hsa-miR- 1271 CUUGGCACCDAGCAAGCACUCA 208 AGGTGCCAA 1145 GGTGCCAA 2082 GTGCCAA 3019 hsa-miR-1272 GAUGAUGAUGGCAGCAAAUUCUGAAA 209 CATCATCAT 1146 ATCATCAT 2083 TCATCAT 3020 hsa-miR- 1273 GGGCGACAAAGCAAGACUCUUUCUU 210 TTTGTCGCC 1147 TTGTCGCC 2084 TGTCGCC 3021 hsa-miR- 1274a GUCCCUGUUCAGGCGCCA 211 GAACAGGGA 1148 AACAGGGA 2085 ACAGGGA 3022 hsa-miR- 1274b UCCCOGUUCGGGCGCCA 212 CGAACAGGG 1149 GAACAGGG 2086 AACAGGG 3023 hsa-miR- 1275 GUGGGGGAGAGGCUGUC 213 TCTCCCCCA 1150 CTCCCCCA 2087 TCCCCCA 3024 hsa-miR- 1276 OAAAGAGCCCOGOGGAGACA 214 GGGCTCTTT 1151 GGCTCTTT 2088 GCTCTTT 3025 hsa-miR- 1277 UACGUAGAUAUAUAUGUAUUUU 215 TATCTACGT 1152 ATCTACGT 2089 TCTACGT 3026 hsa-miR- 1278 UAGUACUGUGCAUAUCAUCUAU 216 CACAGTACT 1153 ACAGTACT 2090 CAGTACT 3027 hsa-miR- 1279 nCAOAOUGCUUCOOUCO 217 AGCAATATG 1154 GCAATATG 2091 CAATATG 3028 hsa-miR- 128 nCACAGUGAACCGGUCUCUUU 218 TTCACTGTG 1155 TCACTGTG 2092 CACTGTG 3029 hsa-miR- 1280 D^'CCCACCGCOGCCACCC 219 AGCGGTGGG 1156 GCGGTGGG 2093 CGGTGGG 3030 hsa-miR- 1281 DCGCCD^'CCD^'CCOCUCCC 220 GAGGAGGCG 1157 AGGAGGCG 2094 GGAGGCG 3031 hsa-miR- 1282 UCGUUUGCCUUUUUCUGCUU 221 AGGCAAACG 1158 GGCAAACG 2095 GCAAACG 3032 hsa-miR- 1283 UCUACAAAGGAAAGCGCUUUCU 222 CCTTTGTAG 1159 CTTTGTAG 2096 TTTGTAG 3033 hsa-miR- 1284 OCUAUACAGACCCOGGCUTJUUC 223 TCTGTATAG 1160 CTGTATAG 2097 TGTATAG 3034 hsa-miR- 1285 UCUGGGCAACAAAGUGAGACCU 224 GTTGCCCAG 1161 TTGCCCAG 2098 TGCCCAG 3035 hsa-miR- 1286 UGCAGGACCAAGAUGAGCCCU 22B TGGTCCTGC 1162 GGTCCTGC 2099 GTCCTGC 3036 hsa-miR- 1287 OGCOGGAIICAGUGGUUCGAGOC 226 TGATCCAGC 1163 GATCCAGC 2100 ATCCAGC 3037 hsa-miR- 1288 UGGACUGCCCUGAUCUGGAGA 227 GGGCAGTCC 1164 GGCAGTCC 2101 GCAGTCC 3038 hsa-miR- 1289 UGGAGUCCAGGAAUCUGCAUUUU 228 CTGGACTCC 1165 TGGACTCC 2102 GGACTCC 3039 hsa-miR- 129* AAGCCCOUACCCCAAAAAGOAD 229 GTAAGGGCT 1166 TAAGGGCT 2103 AAGGGCT 3040 hsa-miR- 129-3p AAGCCCUUACCCCAAAAAGCAU 230 GTAAGGGCT 1167 TAAGGGCT 2104 AAGGGCT 3041 hsa-miR- 129- 5p CODOUDGCGGUCUGGGCnOGC 231 CCGCAAAAA 1168 CGCAAAAA 2105 GCAAAAA 3042 hsa-miR- 1290 UGGAUUUUUGGAUCAGGGA 232 CAAAAATCC 1169 AAAAATCC 2106 AAAATCC 3043 hsa-miR- 1291 UGGCCCOGACUGAAGA.CCAGCAGO 233 GTCAGGGCC 1170 TCAGGGCC 2107 CAGGGCC 3044 hsa-miR- 1292 DGGGAACGGGOUCCGGCAGACGCDG 234 CCCGTTCCC 1171 CCGTTCCC 2108 CGTTCCC 3045 hsa-miR- 1293 OGGGUGGDCDGGAGADO^'UGDGC 235 AGACCACCC 1172 GACCACCC 2109 ACCACCC 3046 hsa-miR- 1294 DGUGAGGOUGGCAUDGUIIGDCU 236 CAACCTCAC 1173 AACCTCAC 2110 ACCTCAC 3047 hsa-miR- 1295 UUAGGCCGCAGAUCUGGGUGA 237 TGCGGCCTA 1174 GCGGCCTA 2111 CGGCCTA 3048 hsa-miR- 1296 UUAGGGCCCUGGCUCCAUCUCC 238 AGGGCCCTA 1175 GGGCCCTA 2112 GGCCCTA 3049 hsa-miR- 1297 DUCAAGUAAUUCAGGDG 239 ATTACTTGA 1176 TTACTTGA 2113 TACTTGA 3050 hsa-miR- 1298 UUCAUUCGGCUGUCCAGAUGUA 240 GCCGAATGA 1177 CCGAATGA 2114 CGAATGA 3051 hsa-miR- 1299 UUCUGGAAUUCUGUGUGAGGGA 241 AATTCCAGA 1178 ATTCCAGA 2115 TTCCAGA 3052 hsa-miR- 1300 UUGAGAAGGAGGCUGCUG 242 TCCTTCTCA 1179 CCTTCTCA 2116 CTTCTCA 3053 hsa-miR- 1301 UUGCAGCUGCCUGGGAGUGACUUC 243 GCAGCTGCA 1180 CAGCTGCA 2117 AGCTGCA 3054 hsa-miR- 1302 UUGGGACAUACUUAUGCUAAA 244 TATGTCCCA 1181 ATGTCCCA 2118 TGTCCCA 3055 hsa-miR- 1303 OUUAGAGACGGGGUCOUGCUCO 245 CGTCTCTAA 1182 GTCTCTAA 2119 TCTCTAA 3056 hsa-miR- 1304 OUUGAGGCUACAGOGAGADGDG 246 TAGCCTCAA 1183 AGCCTCAA 2120 GCCTCAA 3057 hsa-miR- 1305 aUUUCAACnCUAAUGGGAGAGA 247 GAGTTGAAA 1184 AGTTGAAA 2121 GTTGAAA 3058 hsa-miR- 1306 ACGUUGGCUCOGGUGGUG 248 GAGCCAA.CG 1185 AGCCAACG 2122 GCCAACG 3059 hsa-miR- 1307 ACUCGGCGUGGCGUCGGUCGUG 249 CACGCCGAG 1186 ACGCCGAG 2123 CGCCGAG 3060 hsa-miR- 1308 GCAUGGGUGGUUCAGUGG 250 CCACCCATG 1187 CACCCATG 2124 ACCCATG 3061 hsa-miR- 130a CAGUGCAAUGUUAAAAGGGCAU 251 CATTGCACT 1188 ATTGCACT 2125 TTGCACT 3062 hsa-miR- 130a* UUCACAUUGUGCUACUGUCUGC 252 ACAATGTGA 1189 CAATGTGA 2126 AATGTGA 3063 hsa-miR- 130b CAGUGCAAUGAUGAAAGGGCAU 253 CATTGCACT 1190 ATTGCACT 2127 TTGCACT 3064 hsa-miR- 13 Ob* ACUCUUUCCCUGUUGCACUAC 254 GGGAAAGAG 1191 GGAAAGAG 2128 GAAAGAG 3065 hsa-miR- 132 UAACAGUCUACAGCCAUGGUCG 255 TAGACTGTT 1192 AGACTGTT 2129 GACTGTT 3066 hsa-miR- 132 * ACCGUGGCUUUCGAUUGUUACU 256 AAGCCACGG 1193 AGCCA.CGG 2130 GCCACGG 3067 hsa-miR- 1321 CAGGGAGGUGAAUGUGAU 257 CACCTCCCT 1194 ACCTCCCT 2131 CCTCCCT 3068 hsa-miR- 1322 GAUGAUGCUGCUGAUGCDG 258 CAGCATCAT 1195 AGCATCAT 2132 GCATCAT 3069 hsa-miR- 1323 OCAAAACUGAGGGGCAUUUOCD^' 259 TCAGTTTTG 1196 CAGTTTTG 2133 AGTTTTG 3070 hsa-miR- 1324 CCAGACAGAAOUCUAUGCACUUUC 260 TTCTGTCTG 1197 TCTGTCTG 2134 CTGTCTG 3071 hsa-miR- 133a UOUGGUCCCCUUCAACCAGCnG 261 GGGGACCAA 1198 GGGACCAA 2135 GGACCAA 3072 hsa-miR- 133b OUUGGUCCCCUIICAACCAGCOA 262 GGGGACCAA 1199 GGGACCAA 2136 GGACCAA 3073 hsa-miR- 134 UGOGACUGGUUGACCAGAGGGG 263 ACCAGTCAC 1200 CCAGTCAC 2137 CAGTCAC 3074 hsa-~miR~135a UAUGGCUUUUUAUUCCUAUGUGA 264 AAAAGCCAT 1201 AAAGCCAT 2138 AAGCCAT 3075 hsa-miR~135a* UAUAGGGAUUGGAGCCGUGGCG 265 AATCCCTAT 1202 ATCCCTAT 2139 TCCCTAT 3076 hsa-miR~135b OAUGGCUOUU'CAUUCCUAUGUGA 266 AAAAGCCAT 1203 AAAGCCAT 2140 AAGCCAT 3077 hsa-miR-135b* AUGOAGGGCUAAAAGCCAUGGG 267 AGCCCTACA 1204 GCCCTACA 2141 CCCTACA 3078 hsa-tniR-136 ACOCCAUUUGUUUUGAUGAUGGA 268 CAAATGGAG 1205 AAATGGAG 2142 AATGGAG 3079 hsa-miR-136* CAUCAUCGUCUCAAAUGAGOCU 269 GACGATGAT 1206 ACGATGAT 2143 CGATGAT 3080 hsa-miR-137 UUAUUGCUDAAGAAUACGCGUAG 270 TAAGCAATA 1207 AAGCAATA 2144 AGCAATA 3081 hsa-miR-138 AGCUGGUGUUGUGAAUCAGGCCG 271 AACACCAGC 1208 ACACCAGC 2145 CACCAGC 3082 hsa-miR-138-1* GCUACUUCACAA.CACCAGGGCC 272 GTGAAGTAG 1209 TGAAGTAG 2146 GAAGTAG 3083 hsa-miR-138-2* GCUAUUUCACGACACCAGGGUU 273 GTGAAATAG 1210 TGAAATAG 2147 GAAATAG 3084 hsa-miR-139~3p GGAGACGCGGCCCUGDUGGAGU 274 CCGCGTCTC 1211 CGCGTCTC 2148 GCGTCTC 3085 hsa-miR-139-Bp UCUACAGUGCACGUGOCUCCAG 275 GCACTGTAG 1212 CACTGTAG 2149 ACTGTAG 3086 hsa-miR-140-3p UACCACAGGGUAGAACCACGG 276 CCCTGTGGT 1213 CCTGTGGT 2150 CTGTGGT 3087 hsa-miR-140-5p CAGUGGUUUUACCCUAOGGUAG 277 AAAACCACT 1214 AAACCACT 2151 AACCACT 3088 hsa-miR-1 1 UAA.CACUGUCUGGUAAAGAUGG 278 GACAGTGTT 1215 ACAGTGTT 2152 CAGTGTT 3089 hsa-miR-141* CAUCUUCCAGUACAGUGUUGGA 279 CTGGAAGAT 1216 TGGAAGAT 2153 GGAAGAT 3090 hsa-miR-142-3p UGUAGUGUUUeCUACUUUAUGG 280 AAACACTAC 1217 AACACTAC 2154 ACACTAC 3091 hsa-miR-142-5p CAUAAAGUAGAAAGCACUACU 281 CTACTTTAT 1218 TACTTTAT 2155 ACTTTAT 3092 hsa-miR-143 UGAGAUGAAGCACUGUAGCUC 282 CTTCATCTC 1219 TTCATCTC 2156 TCATCTC 3093 hsa-miR-143* GGOGCAGUGCUGCAUCUCUGGU 283 GCACTGCAC 1220 CACTGCAC 2157 ACTGCAC 3094 hsa-tniR-144 UACAGUADAGADGAUGUACU 284 CTATA.CTGT 1221 TATACTGT 2158 ATACTGT 3095 hsa-miR-144* GGAUAOCAUCAUAUACUGUAAG 28S GATGATATC 1222 ATGATATC 2159 TGATATC 3096 hsa-miR-145 GOCCAGUUUUCCCAGGAAUCCCU 286 AAAACTGGA 1223 AAACTGGA 2160 AACTGGA 3097 hsa-miR-145* GGAUUCCUGGAAAUACUGUUCU 287 CCAGGAATC 1224 CAGGAATC 2161 AGGAATC 3098 hsa-miR-1468 CUCCGOUDGCCUGUUUCGCUG 288 GCAAACGGA 1225 CAAACGGA 2162 AAACGGA 3099 hsa-miR-1469 CUCGGCGCGGGGCGCGGGCUCC 289 CCGCGCCGA 1226 CGCGCCGA 2163 GCGCCGA 3100 hsa-miR-146a UGAGAACUGAAUUCCAUGGGUU 290 TCAGTTCTC 1227 CAGTTCTC 2164 AGTTCTC 3101 fasa~miR~146a* CCUeUGAAAUUCAGUUCUUCAG 291 ATTTCAGAG 1228 TTTCAGAG 2165 TTCAGAG 3102 hsa-miR-146b-3p UGCCCUGUGGACUCAGOUCUGG 292 CCACAGGGC 1229 CACAGGGC 2166 ACAGGGC 3103 hea-miR-146b-5p UGAGAACUGAAUUCCADAGGCU 293 TCAGTTCTC 1230 CAGTTCTC 2167 AGTTCTC 3104 hsa-miR-147 GOGUGUGGAAAUGCUUCUGC 294 TTCCACACA 1231 TCCACACA 2168 CCACACA 3105 hsa-miR-1470 GCCCnCCGCCCGUGCACeeCG 295 GGCGGAGGG 1232 GCGGAGGG 2169 CGGAGGG 3106 hsa-miR-1 71 GCCCGCGUGUGGAGCCAGGUGU 296 ACACGCGGG 1233 CACGCGGG 2170 ACGCGGG 3107 fasa~miR-147b GUGOGCGGAAAUGCUUCUGCUA 297 TTCCGCACA 1234 TCCGCACA 2171 CCGCACA 3108 hsa-miR-148a UCAGDGCACOACAGAACOUUGU 298 AGTGCACTG 1235 GTGCACTG 2172 TGCACTG 3109 hsa-miR-148a* AAAGUUCUGAGACACOCCGACU 299 TCAGAACTT 1236 CAGAACTT 2173 AGAACTT 3110 hsa-miR-148b UCAGOGCAUCACAGAACUUUGU 300 GATGCACTG 1237 ATGCACTG 2174 TGCACTG 3111 fasa-miR~148b* AAGUUCUGUUAUACACUCAGGC 301 AACAGAACT 1238 ACAGAACT 2175 CAGAACT 3112 hea-miR-149 UCUGGCD'CCGUGUCUUCACUCCC 302 CGGAGCCAG 1239 GGAGCCAG 2176 GAGCCAG 3113 hsa-miR-149* AGGGAGGGACGGGGGCUGUGC 303 GTCCCTCCC 1240 TCCCTCCC 2177 CCCTCCC 3114 hsa-miR-150 UCUCCCAACCCUUGUACCAGUG 304 GGTTGGGAG 1241 GTTGGGAG 2178 TTGGGAG 3115 hsa-miR-150* CUGGUACAGGCCUGGGGGACAG 305 CCTGTACCA 1242 CTGTACCA 2179 TGTACCA 3116 hsa-miR-I51-3p CUAGACUGAAGCUCCUUGAGG 306 TTCAGTCTA 1243 TCAGTCTA 2180 CAGTCTA 3117 hsa~miR~151-5p UCGAGGAGCDCACAGUCUAGU 307 AGCTCCTCG 1244 GCTCCTCG 2181 CTCCTCG 3118 hsa-miR-152 UCAGUGCAUGACAGAACUUGG 308 GATGCACTG 1245 ATGCACTG 2182 TGCACTG 3119 hsa-tniR-153 UUGCAUAGUCACAAAAGUGAUC 309 GACTATGCA 1246 ACTATGCA 2183 CTATGCA 3120 hsa-miR-1537 AAAACCGUCUAGUUACAGUUGU 310 AGACGGTTT 1247 GACGGTTT 2184 ACGGTTT 3121 hsa-tniR-1538 CGGCCCGGGCUGCUGCUGUUCCU 311 GCCCGGGCC 1248 CCCGGGCC 2185 CCGGGCC 3122 hsa-miR-1539 UCCOGCGCGUCCCAGAUGCCC 312 ACGCGCAGG 1249 CGCGCAGG 2186 GCGCAGG 3123 hsa-miR-154 OAGGUUAUCCGUGUUGCCUUCG 313 GGATAACCT 1250 GATAACCT 2187 ATAACCT 3124 hsa-miR-154* AAUCAUA.CACGGUUGACCOAUU 314 GTGTATGAT 1251 TGTATGAT 2188 GTATGAT 3125 hsa-miR-155 UUAAUGCUAAUCGUGAUAGGGGU 315 TTAGCATTA 1252 TAGCATTA 2189 AGCATTA 3126 hsa-miR-155* CUCCUACAUAUUAGCAUUAACA 316 TATGTAGGA 1253 ATGTAGGA 2190 TGTAGGA 3127 hsa-miR-15a DAGCAGCACAOAAUGGUUUGOG 317 TGTGCTGCT 1254 GTGCTGCT 2191 TGCTGCT 3128 hsa-miR-15a* CAGGCCAUAUUGUGCOGCCO'CA 318 ATATGGCCT 1255 TATGGCCT 2192 ATGGCCT 3129 hsa-miR~15b UAGCAGCACAUCAUGGUUUACA 319 TGTGCTGCT 1256 GTGCTGCT 2193 TGCTGCT 3130 hsa-miR~15b* CGAAUCAUIIAIIUUGCOGCUCOA 320 TAATGATTC 1257 AATGATTC 2194 ATGATTC 3131 hsa-miR-16 UAGCAGCACGDAAAOAUUGGCG 321 CGTGCTGCT 1258 GTGCTGCT 2195 TGCTGCT 3132 hsa-miR-lS-l* CCAGUAOOAACOGOGCOGCnGA 322 TTAATACTG 1259 TAATACTG 2196 AATACTG 3133 hsa~miR-lS~2* CCAAUADUACUGUGCUGCUUUA 323 GTAATATTG 1260 TAATATTG 2197 AATATTG 3134 fasa-miR-17 CAAAGOGCUOACAGUGCAGGOAG 324 AAGCACTTT 1261 AGCACTTT 2198 GCACTTT 3135 hsa-miR-17* ACUGCAGUGAAGGCACUUGUAG 325 TCACTGCAG 1262 CACTGCAG 2199 ACTGCAG 3136 hsa-miR-181a AACAUUCAACGCOGUCGGOGAGU 326 GTTGAATGT 1263 TTGAATGT 2200 TGAATGT 3137 hsa-miR~181a* ACCAOCGACCGUUGAODGOACC 327 GGTCGATGG 1264 GTCGATGG 2201 TCGATGG 3138 hsa-miR~181a~2* ACCACOGACCGOUGACTJGDACC 328 GGTCAGTGG 1265 GTCAGTGG 2202 TCAGTGG 3139 hsa-miR-lBlb AACAUUCAUUGCUGUCGGUGGGU 329 AATGAATGT 1266 ATGAATGT 2203 TGAATGT 3140 hsa-miR-lSle AACAIIUCAACCOGUCGGUGAGU 330 GTTGAATGT 1267 TTGAATGT 2204 TGAATGT 3141 hsa-miR-181c* AACCAOCGACCGUOGAGDGGAC 331 GTCGATGGT 1268 TCGATGGT 2205 CGATGGT 3142 hsa~miR-181d AACAUO'CAOUGDUGUCGGUGGGO 332 AATGAATGT 1269 ATGAATGT 2206 TGAATGT 3143 hsa-miR-182 UUUGGCAAUGGUAGAACUCACACU 333 CATTGCCAA 1270 ATTGCCAA 2207 TTGCCAA 3144 hsa-miR-182* UGGUUCUAGACUUGCCAACUA 334 TCTAGAACC 1271 CTAGAACC 2208 TAGAACC 3145 hsa-miR-1825 occAGUGceeoceueuce 335 GGGCACTGG 1272 GGCACTGG 2209 GCACTGG 3146 hsa-miR-1826 AOUGAOCAOCGACACUUCGAACGCAAO 336 GATGATCAA 1273 ATGATCAA 2210 TGATCAA 3147 hsa-miR-1827 UGAGGCAGUAGAUUGAAU 337 TACTGCCTC 1274 ACTGCCTC 2211 CTGCCTC 3148 hsa-miR-183 UAUGGCACUGGUAGAAUUCACU 338 CAGTGCCAT 1275 AGTGCCAT 2212 GTGCCAT 3149 hsa-miR-183* GUGAAUUACCGAAGGGCCAO'AA 339 GGTAATTCA 1276 GTAATTCA 2213 TAATTCA 3150 hsa-miR-184 UGGACGGAGAACUGAUAAGGGO 340 TCTCCGTCC 1277 CTCCGTCC 2214 TCCGTCC 3151 hsa-miR-185 UGGAGAGAAAGGCAGUUCCUGA 341 TTTCTCTCC 1278 TTCTCTCC 2215 TCTCTCC 3152 hsa-miR-185* AGGGGCUGGCUUUCCUCUGGUC 342 GCCAGCCCC 1279 CCAGCCCC 2216 CAGCCCC 3153 hsa-miR-186 CAAAGAAUUCnCCUUUUGGGCU 343 GAAT CTTT 1280 AATTCTTT 2217 ATTCTTT 3154 hsa-miR-186* GCCCAAAGGUGAAUUUUUUGGG 344 ACCTTTGGG 1281 CCTTTGGG 2218 CTTTGGG 3155 hsa-miR-187 UCGUGUCUUGUGUUGCAGCCGG 345 CAAGACACG 1282 AAGACACG 2219 AGACACG 3156 hsa-miR-187* GGCOACAACACAGGACCCGGGC 346 TGTTGTAGC 1283 GTTGTAGC 2220 TTGTAGC 3157 hsa-miR-188-3p COCCCACAOGCAGGGOUDGCA 347 CATGTGGGA 1284 ATGTGGGA 2221 TGTGGGA 3158 hsa-miR-188-5p CAUCCCUUGCAUGGUGGAGGG 348 GCAAGGGAT 1285 CAAGGGAT 2222 AAGGGAT 3159 hsa-miR-18a UAAGGUGCAUCUAGUGCAGAUAG 349 ATGCACCTT 1286 TGCACCTT 2223 GCACCT 3160 hsa~miR-18a* ACUGCCCUAAGOGCOCCOUCOGG 350 TTAGGGCAG 1287 TAGGGCAG 2224 AGGGCAG 3161 hsa-miR-18b UAAGGUGCAUCUAGUGCAGUUAG 351 ATGCACCTT 1288 TGCACCTT 2225 GCACCTT 3162 hsa-miR-I8b* DGCCCTJAAAOGCCCCUUCDGGC 352 ATTTAGGGC 1289 TTTAGGGC 2226 TTAGGGC 3163 hsa-miR-190 UGAUAUGUUUGAUAUAUUAGGU 353 AAACATATC 1290 AACATATC 2227 ACATATC 3164 hsa-tniR-1908 CGGCGGGGACGGCGAUUGGUC 354 GTCCCCGCC 1291 TCCCCGCC 2228 CCCCGCC 3165 hsa-miR-1909 CGCAGGGGCCGGGUGCD'CACCG 355 GGCCCCTGC 1292 GCCCCTGC 2229 CCCCTGC 3166 hsa-miR-1909* OGAGOGCCGGOGCCOGCCCUG 356 CCGGCACTC 1293 CGGCACTC 2230 GGCACTC 3167 hsa-miR-190b nGAUAUGUUUGAUAUUGGGUU 357 AAACATATC 1294 AACATATC 2231 ACATATC 3168 hsa-miR-191 CAACGGAAUCCCAAAAGCAGCDG 358 GATTCCGTT 1295 ATTCCGTT 2232 TTCCGTT 3169 hsa-miR-191* GCUGCGCUUGGAUUUCGUCCCC 359 CAAGCGCAG 1296 AAGCGCAG 2233 AGCGCAG 3170 hsa-miR-1910 CCAGOCCOGOGCCOGCCGCCU 360 ACAGGACTG 1297 CAGGACTG 2234 AGGACTG 3171 hsa-miR-1911 UGAGUACCGCCAUGUCUGUUGGG 361 GCGGTACTC 1298 CGGTACTC 2235 GGTACTC 3172 hsa-miR-1911* CACCAGGCAUDGOGGOCOCC 362 ATGCCTGGT 1299 TGCCTGGT 2236 GCCTGGT 3173 hsa-miR-1912 OACCCAGAGCADGCAGOGOGAA 363 GCTCTGGGT 1300 CTCTGGGT 2237 TCTGGGT 3174 hsa-miR-1913 UCUGCCCCCUCCGCUGCUGCCA 364 AGGGGGCAG 1301 GGGGGCAG 2238 GGGGCAG 3175 hsa-miR-1914 CCCUGUGCCCGGCCCACUUCUG 365 GGGCACAGG 1302 GGCACAGG 2239 GCACAGG 3176 hsa-miR-1914* GGAGGGGD'CCCGCACUGGGAGG 366 GGACCCCTC 1303 GACCCCTC 2240 ACCCCTC 3177 hsa-miR-1915 CCCCAGGGCGACGCGGCGGG 367 CGCCCTGGG 1304 GCCCTGGG 2241 CCCTGGG 3178 hsa-miR-1915* ACCUUGCCOUGCUGCCCGGGCC 368 AAGGCAAGG 1305 AGGCAAGG 2242 GGCAAGG 3179 hsa-miR-192 CUGACCUAUGAAUUGACAGCC 369 CATAGGTCA 1306 ATAGGTCA 2243 TAGGTCA 3180 hsa-miR-192* COGCCAAOUCCAOAGGOCACAG 370 GAATTGGCA 1307 AATTGGCA 2244 ATTGGCA 3181 hsa-miR-193a-3p AACOGGCCUACAAAGOCCCAGD 371 TAGGCCAGT 1308 AGGCCAGT 2245 GGCCAGT 3182 hsa-miR-193a-5p OGGGOCDUUGCGGGCGAGAOGA 372 CAAAGACCC 1309 AAAGACCC 2246 AAGACCC 3183 hsa-miR-193b AACUGGCCCUCAAAGUCCCGCU 373 AGGGCCAGT 1310 GGGCCAGT 2247 GGCCAGT 3184 hsa-miR-193b* CGGGGUUUUGAGGGCGAGAUGA 374 CAAAACCCC 1311 AAAACCCC 2248 AAACCCC 3185 hsa-miR- 194 UGOAACAGCAACUCCAUGUGGA 375 TGCTGTTAC 1312 GCTGTTAC 2249 CTGTTAC 3186 hsa-miR-194* CCAGUGGGGCUGCUGUUAUCUG 376 GCCCCACTG 1313 CCCCACTG 2250 CCCACTG 3187 hsa-miR-195 UAG AGCACAGAAAUAUUGGC 377 TGTGCTGCT 1314 GTGCTGCT 2251 TGCTGCT 3188 hsa-miR-195* CCAAUAUUGGCUGUGCUGCUCC 378 CCAATATTG 1315 CAATATTG 2252 AATATTG 3189 hsa-miR~196a UAGGUAGUUUCAUGUUGUUGGG 379 AAACTACCT 1316 AACTACCT 2253 ACTACCT 3190 hsa-miR~19Sa* CGGCAACAAGAAACDGCCIJGAG 380 CTTGTTGCC 1317 TTGTTGCC 2254 TGTTGCC 3191 hsa-miR- 19Sb OAGGOAGUUOCCOGITUGUUGGG 381 AAACTACCT 1318 AACTACCT 2255 ACTACCT 3192 hsa-miR-197 UUCACCACCUUCUCCACCCAGC 382 AGGTGGTGA 1319 GGTGGTGA 2256 GTGGTGA 3193 hsa-miR-198 GGUCCAGAGGGGAGAUAGGUUC 383 CCTCTGGAC 1320 CTCTGGAC 2257 TCTGGAC 3194 hsa-miR-199a~5p CCCAGUGUUCAGACUACCUGUUC 384 GAACACTGG 1321 AACACTGG 2258 ACACTGG 3195 hsa-miR-199b~3p ACAGUAGUCUGCACAUUGGUUA 385 AGACTACTG 1322 GACTACTG 2259 ACTACTG 3196 hsa-miR- 199b- 5p CCCAGUGUUUAGACUAnCUGUUC 386 AAACACTGG 1323 AACACTGG 2260 ACACTGG 3197 hsa-miR- 19a OGOGCAAAOCUAUGCAAAACOGA 387 GATTTGCAC 1324 ATTTGCAC 2261 TTTGCAC 3198 hsa-miR-19a* AGUUUUGCAUAGUUGCACUACA 388 ATGCAAAAC 1325 TGCAAAAC 2262 GCAAAAC 3199 hsa-miR- 19b OGDGCAAADCCADGCAAAACUGA 389 GATTTGCAC 1326 ATTTGCAC 2263 TTTGCAC 3200 fasa-miR- 19b~l* AGUUDUGCAGGDUDGCAUCCAGC 390 CTGCAAAAC 1327 TGCAAAAC 2264 GCAAAAC 3201 hsa-miR- 19b-2* AGUUUUGCAGGUUUGCAUUUCA 391 CTGCAAAAC 1328 TGCAAAAC 2265 GCAAAAC 3202 hsa-miR-2QOa UAA.CACOGUCDGGOAACGAOGU 392 GACAGTGTT 1329 ACAGTGTT 2266 CAGTGTT 3203 hsa-miR-200a* CAUCUUACCGGACAGUGCUGGA 393 CGGTAAGAT 1330 GGTAAGAT 2267 GTAAGAT 3204 hsa-miR-200b DAAUACOGCCUGGUAAUGAUGA 394 GGCAGTATT 1331 GCAGTATT 2268 CAGTATT 3205 hsa-miR-200b* CAUCDUACUGGGCAGCADUGGA 395 CAGTAAGAT 1332 AGTAAGAT 2269 GTAAGAT 3206 hsa-miR-200c UAAUACOGCCGGGOAAOGAOGGA 39S GGCAGTATT 1333 GCAGTATT 2270 CAGTATT 3207 hsa-miR-200c* CGUCUUACCCAGCAGUGUIIIIGG 397 GGGTAAGAC 1334 GGTAAGAC 2271 GTAAGAC 3208 hsa-miR-202 AGAGGUAUAGGGCAUGGGAA 398 CTATACCTC 1335 TATACCTC 2272 ATACCTC 3209 hsa-miR-202* UUCCOAUGCAUAUACUUCIllIUG 399 TGCATAGGA 1336 GCATAGGA 2273 CATAGGA 3210 hsa-miR-203 GDGAAADGUUUAGGACCACDAG 400 AACATTTCA 1337 ACATTTCA 2274 CATTTCA 3211 hsa-miR-204 UUCCCUUUGHCAUCCUAUGCCU 401 ACAAAGGGA 1338 CAAAGGGA 2275 AAAGGGA 3212 hsa-miR-205 UCCUUCAUUCCACCGGAGUCUG 402 GAATGAAGG 1339 AATGAAGG 2276 ATGAAGG 3213 hsa-miR-206 OGGAAOGOAAGGAAGOGDGOGG 403 TTACATTCC 1340 TACATTCC 2277 ACATTCC 3214 hsa-miR-208a ADAAGACGAGCAAAAAGCDUGO 404 CTCGTCTTA 1341 TCGTCTTA 2278 CGTCTTA 3215 hsa-miR-208b ADAAGACGAACAAAAGGOUUGIJ 405 TTCGTCTTA 1342 TCGTCTTA 2279 CGTCTTA 3216 hsa-miR-20a UAAAGUGCUUAUAGUGCAGGUAG 406 AAGCACTTT 1343 AGCACTTT 2280 GCACTTT 3217 hsa-miR-20a* ACDGCAUUADGAGCACDDAAAG 407 ATAATGCAG 1344 TAATGCAG 2281 AATGCAG 3218 hsa-miR-20b CAAAGUGCOCAOAGOGCAGGDAG 408 GAGCACTTT 1345 AGCACTTT 2282 GCACTTT 3219 hsa-miR-20b* ACUGUAGOAUGGGCACUUCCAG 409 ATACTACAG 1346 TACTACAG 2283 ACTACAG 3220 hsa-miR-21 OAGCOUAUCAGACUGADGUUGA 410 TGATAAGCT 1347 GATAAGCT 2284 ATAAGCT 3221 hsa-miR-21* CAACACCAGUCGAUGGGCUGU 411 ACTGGTGTT 1348 CTGGTGTT 2285 TGGTGTT 3222 hsa-miR-210 CUGDGCGUGUGACAGCGGCOGA 412 ACACGCACA 1349 CACGCACA 2286 ACGCACA 3223 hsa-miR-211 UUCCCUUUGUCAUCCUUCGCCU 413 ACAAAGGGA 1350 CAAAGGGA 2287 AAAGGGA 3224 hsa-miR-212 UAACAGDCUCCAGUCACGGCC 414 GAGACTGTT 1351 AGACTGTT 2288 GACTGTT 3225 hsa-miR-214 ACAGCAGGCACAGACAGGCAGD' 415 TGCCTGCTG 1352 GCCTGCTG 2289 CCTGCTG 3226 hsa-miR-214* UGCCUGOCUACACUUGCUGOGC 416 TAGACAGGC 1353 AGACAGGC 2290 GACAGGC 3227 hsa-miR-215 AOGACCOAOGAAODGACAGAC 417 CATAGGTCA 1354 ATAGGTCA 2291 TAGGTCA 3228 hsa-miR-216a OAAOCOCAGCOGGCAACOGOGA 418 GCTGAGATT 1355 CTGAGATT 2292 TGAGATT 3229 hsa-miR-216b AAAOCOCDGCAGGCAAADGDGA 419 GCAGAGATT 1356 CAGAGATT 2293 AGAGATT 3230 hsa-miR-217 UACUGCAUCAGGAACUGAUUGGA 420 TGATGCAGT 1357 GATGCAGT 2294 ATGCAGT 3231 hsa-miR-218 UUGUGCUUGAUCUAACCAUGU 421 TCAAGCACA 1358 CAAGCACA 2295 AAGCACA 3232 hsa-miR-218-1* AUGGUUCCGUCAAGCACCAUGG 422 ACGGAACCA 1359 CGGAACCA 2296 GGAACCA 3233 hsa-miR-218-2* CAOGGOUCOGUCAAGCACCGCG 423 CAGAACCAT 1360 AGAACCAT 2297 GAACCAT 3234 hsa-miR-219-l-3p AGAGUUGAGUCUGGACGUCCCG 424 ACTCAACTC 1361 CTCAACTC 2298 TCAACTC 3235 hsa-miR-219-2-3p AGAAUUGUGGCUGGACAUCUGU 425 CCACAATTC 1362 CACAA.TTC 2299 ACAATTC 3236 hsa-miR-219-5p OGAOOGOCCAAACGC OαCD^, 426 TGGACAATC 1363 GGACAATC 2300 GACAATC 3237 hsa-miR-22 AAGCUGCCAGUUGAAGAACUGU 427 CTGGCAGCT 1364 TGGCAGCT 2301 GGCAGCT 3238 hsa-miR-22* AGUUCUUCAGUGGCAAGCUUUA 428 CTGAAGAAC 1365 TGAAGAAC 2302 GAAGAAC 3239 hsa~miR~220a CCACACCGUAUCUGACACUUU 429 TACGGTGTG 1366 ACGGTGTG 2303 CGGTGTG 3240 hsa-miR~220b CCACCACCGUGDCUGACACUU 430 ACGGTGGTG 1367 CGGTGGTG 2304 GGTGGTG 3241 hsa~miR~220c ACACAGGGCOGOOGUGAAGACO 431 AGCCCTGTG 1368 GCCCTGTG 2305 CCCTGTG 3242 hsa-miR-221 AGCUACAUUGUCTJGOJGGGUUUC 432 CAATGTAGC 1369 AATGTAGC 2306 ATGTAGC 3243 hsa-tniR-221* ACCUGGCAOA.CAAOGOAGADUU 433 TATGCCAGG 1370 ATGCCAGG 2307 TGCCAGG 3244 hsa-miR-222 AGCUACAUCDGGCOACUGGGD^' 434 AGATGTAGC 1371 GATGTAGC 2308 ATGTAGC 3245 hsa-miR-222 * CO^'CAGOAGCCAGUGUAGAOCCD^' 435 GGCTACTGA 1372 GCTACTGA 2309 CTACTGA 3246 hsa-miR-223 UGUCAGOUUGOCAAAOACCCCA 436 CAAACTGAC 1373 AAACTGAC 2310 AACTGAC 3247 hsa-miR-223 * CGOGDADUDGACAAGCUGAGUU 437 CAAATACAC 1374 AAATACAC 2311 AATACAC 3248 hsa-miR-224 CAAGD^'CACUAGUGGDOCCGOO^' 438 TAGTGACTT 1375 AGTGACTT 2312 GTGACTT 3249 hsa-miR-23a AUCACAUUGCCAGGGAUUUCC 439 GCAATGTGA 1376 CAATGTGA 2313 AATGTGA 3250 hsa-miR-23a* GGGGUUCCUGGGGAUGGGAUUU 440 CAGGAACCC 1377 AGGAACCC 2314 GGAACCC 3251 hsa-miR-23b ADCA.CAUUGCCAGGGADUACC 441 GCAATGTGA 1378 CAATGTGA 2315 AATGTGA 3252 hsa~miR~23b* OGGGUUCCUGGCAOGCDGAUUO^' 442 CAGGAACCC 1379 AGGAACCC 2316 GGAACCC 3253 hsa-miR-24 DGGCDCAGOOCAGCAGGAACAG 443 AACTGAGCC 1380 ACTGAGCC 2317 CTGAGCC 3254 hsa-miR-24- 1* DGCCUACDGAGCUGAOAUCAGO 444 TCAGTAGGC 1381 CAGTAGGC 2318 AGTAGGC 3255 hsa-miR-2 -2 * OGCCOACOGAGCUGAAACACAG 445 TCAGTAGGC 1382 CAGTAGGC 2319 AGTAGGC 3256 hsa-miR-25 CAOUGCACUDGD^'COCGGUCUGA 446 AAGTGCAAT 1383 AGTGCAAT 2320 GTGCAAT 3257 hsa-miR-25* AGGCGGAGACnOGGGCAAUOG 447 GTCTCCGCC 1384 TCTCCGCC 2321 CTCCGCC 3258 hsa-miR-26a OOCAAGOAAUCCAGGAOAGGCU 448 ATTACTTGA 1385 TTACTTGA 2322 TACTTGA 3259 hsa~tniR-26a- l* CCUADUCOUGGOUACOUGCACG 449 CAAGAATAG 1386 AAGAATAG 2323 AGAATAG 3260 hsa~miR~26a~2* CCUAUUCUUGAUUACUUGUUUC 450 CAAGAATAG 1387 AAGAATAG 2324 AGAATAG 3261 hsa-miR-26b UUCAAGUAAUUCAGGAUAGGU 451 ATTACTTGA 1388 TTACTTGA 2325 TACTTGA 3262 hsa-miR-26b* CCOGODCOCCAUOACaUGGCaC 452 GGAGAACAG 1389 GAGAACAG 2326 AGAACAG 3263 hsa-miR-2?a UUCACAGUGGCUAAGUUCCGC 453 CCACTGTGA 1390 CACTGTGA 2327 ACTGTGA 3264 hsa-tniR-27a* AGGGCUUAGCUGCUUGUGAGCA 454 GCTAAGCCC 1391 CTAAGCCC 2328 TAAGCCC 3265 hsa-miR-27b UUCACAGUGGCTAAGUUCUGC 455 CCACTGTGA 1392 CACTGTGA 2329 ACTGTGA 3266 hsa-miR-27b* AGAGCUUAGCUGAUUGGUGAAC 456 GCTAAGCTC 1393 CTAAGCTC 2330 TAAGCTC 3267 hsa~miR~28~3p CACOAGAODGOGAGCOCCOGGA 457 CAATCTAGT 1394 AATCTAGT 2331 ATCTAGT 3268 hsa-miR-28 - 5p AAGGAGCDCACAGDCOAUUGAG 458 TGAGCTCCT 1395 GAGCTCCT 2332 AGCTCCT 3269 hsa-miR-296 - 3p GAGGGOOGGGUGGAGGCOCOCC 459 CCCAACCCT 1396 CCAACCCT 2333 CAACCCT 3270 hsa-miR-296- 5p AGGGCCCCCCCUCAAOCCOGU 460 GGGGGGCCC 1397 GGGGGCCC 2334 GGGGCCC 3271 hsa-miR-297 AUGUAUGUGUGCAUGUGCAUG 461 ACACATACA 1398 CACATACA 2335 ACATACA 3272 hsa-miR-298 AGCAGAAGCAGGGAGGUUCUCCCA 4S2 TGCTTCTGC 1399 GCTTCTGC 2336 CTTCTGC 3273 hsa~miR~299~3p UAUGUGGGAUGGUAAACCGCUU 463 ATCCCACAT 1400 TCCCACAT 2337 CCCACAT 3274 hsa-miR-299- 5p UGGUUUACCGUCCCACAUACAU 464 CGGTAAACC 1401 GGTAAACC 2338 GTAAACC 3275 fasa~miR~29a OAGCACCAOCUGAAAUCGGUUA 465 GATGGTGCT 1402 ATGGTGCT 2339 TGGTGCT 3276 hsa~miR~29a* ACUGAUUUCUUUUGGUGUUCAG 466 AGAAATCAG 1403 GAAATCAG 2340 AAATCAG 3277 hsa-miR-29b UAGCACCAUUUGAAAnCAGUGUU 467 AATGGTGCT 1404 ATGGTGCT 2341 TGGTGCT 3278 hsa-mlR-29b- l* GCUGGUUUCAUAUGGUGGUUUAGA 468 TGAAACCAG 1405 GAAACCAG 2342 AAACCAG 3279 hsa-miR-29b-2 * COGGODUCACAUGGOGGCUUAG 469 GTGAAACCA 1406 TGAAACCA 2343 GAAACCA 3280 hsa-miR-29c UAGCACCAUUUGAAAUCGGUUA 470 AATGGTGCT 1407 ATGGTGCT 2344 TGGTGCT 3281 hsa-miR-29c* OGACCGAOUUCOCCOGGOGOUC 471 AAATCGGTC 1408 AATCGGTC 2345 ATCGGTC 3282 fasa-miR- S QQ UAUACAAGGGCAGACUCUCUCU 472 CCCTTGTAT 1409 CCTTGTAT 2346 CTTGTAT 3283 fasa-miR~ 3 Qla CAGUGCAAUAGUAUUGUCAAAGC 473 TATTGCACT 1410 ATTGCACT 2347 TTGCACT 3284 hsa-miR- 301b CAGUGCAAUGAUAUUGnCAAAGC 474 CATTGCACT 1411 ATTGCACT 2348 TTGCACT 3285 hsa-miR-302a UAAGUGCUUCCAUGUUUUGGUGA 475 GAAGCACTT 1412 AAGCACTT 2349 AGCACTT 3286 hea-miR-302a* ACUOAAACGDGGADGDACDDGCD 476 ACGTTTAAG 1413 CGTTTAAG 2350 GTTTAAG 3287 hsa-miR-302b UAAGUGCUUCCAUGUUUUAGUAG 477 GAAGCACTT 1414 AAGCACTT 2351 AGCACTT 3288 hsa-miR-302b* ACOUOAACAUGGAAGOGCUUUC 478 ATGTTAAAG 1415 TGTTAAAG 2352 GTTAAAG 3289 hsa-miR-302c UAAGUGCUUCCAUGUUUCAGUGG 479 GAAGCACTT 1416 AAGCACTT 2353 AGCACTT 3290 hea-miR-302c* UUUAACAUGGGGGUACCUGCUG 480 CCATGTTAA 1417 CATGTTAA 2354 ATGTTAA 3291 hsa-miR-302d UAAGUGCUUCCAUGUUUGAGUGU 481 GAAGCACTT 1418 AAGCACTT 2355 AGCACTT 3292 hsa-miR- 302d* ACUUUAACAUGGAGGCACUUGC 482 ATGTTAAAG 1419 TGTTAAAG 2356 GTTAAAG 3293 hsa-miR-302e UAAGUGCOUCCAUGCUD 483 GAAGCACTT 1420 AAGCACTT 2357 AGCACTT 3294 hsa-miR-302f UAAUUGCUUCCAUGUUU 484 GAAGCAATT 1421 AAGCAATT 2358 AGCAATT 3295 hsa-miR~30a UGOAAACAUCCOCGACUGGAAG 485 GATGTTTAC 1422 ATGTTTAC 2359 TGTTTAC 3296 hsa-miR- 30a* CUUUCAGUCGQAUGUUUGCAGC 486 CGACTGAAA 1423 GACTGAAA 2360 ACTGAAA 3297 hsa-miR~3Qb OGOAAACAUCCUACACOCAGCO 487 GATGTTTAC 1424 ATGTTTAC 2361 TGTTTAC 3298 hsa~miR~3Qb* COGGGAGGOGGAOGOUUACOUC 488 CACCTCCCA 1425 ACCTCCCA 2362 CCTCCCA 3299 hsa-miR~3Qc OGOAAACAD'CCOACACUCOCAGC 489 GATGTTTAC 1426 ATGTTTAC 2363 TGTTTAC 3300 hsa-miR-30c~l* CUGGGAGAGGGUUGUUUACUCC 490 CCTCTCCCA 1427 CTCTCCCA 2364 TCTCCCA 3301 hsa~miR~30c-2* COGGGAGAAGGCOGOUUACOCU 491 CTTCTCCCA 1428 TTCTCCCA 2365 TCTCCCA 3302 hsa-miR~30d OGOAAACAOCCCCGACOGGAAG 492 GATGTTTAC 1429 ATGTTTAC 2366 TGTTTAC 3303 fasa-miR- 30d* CUUUCAGUCAGAUGUUUGCTJGC 493 TGACTGAAA 1430 GACTGAAA 2367 ACTGAAA 3304 hsa-miR~30e DGDAAACAO^'CCOOGACIJGGAAG 494 GATGTTTAC 1431 ATGTTTAC 2368 TGTTTAC 3305 hsa-miR- 3 Qe * CUUUCAGUCGGAUGUUUACAGC 495 CGACTGAAA 1432 GACTGAAA 2369 ACTGAAA 3306 hsa-miR-31 AGGCAAGAOGCUGGCAOAGCO 496 CATCTTGCC 1433 ATCTTGCC 2370 TCTTGCC 3307 hsa-miR-31* OGCUAOGCCAACAOAaUGCCAO 497 TGGCATAGC 1434 GGCATAGC 2371 GCATAGC 3308 hsa-miR-32 DAUOGCACAUOACUAAGUUGCA 498 ATGTGCAAT 1435 TGTGCAAT 2372 GTGCAAT 3309 hsa-miR-32* CAAUUUAGUGUGUGUGAUAUUU 499 CACTAAATT 1436 ACTAAATT 2373 CTAAATT 3310 hsa-miR-320a AAAAGCOGGGOUGAGAGGGCGA 500 CCCAGCTTT 1437 CCAGCTTT 2374 CAGCTTT 3311 hsa-miR-32 Ob AAAAGCUGGGOOGAGAGGGCAA 501 CCCAGCTTT 1438 CCAGCTTT 2375 CAGCTTT 3312 hsa~miR-320c AAAAGCUGGGUUGAGAGGGO 502 CCCAGCTTT 1439 CCAGCTTT 2376 CAGCTTT 3313 hsa-miR~32Qd AAAAGCUGGGUUGAGAGGA 503 CCCAGCTTT 1440 CCAGCTTT 2377 CAGCTTT 3314 hsa-miR-323-3p CACADDACACGGOCGACCOCO 504 GTGTAATGT 1441 TGTAATGT 2378 GTAATGT 3315 hsa-miR-323 - 5p AGGUGGUCCGUGGCGCGUUCGC 505 CGGACCACC 1442 GGACCACC 2379 GACCACC 3316 hsa-miR-324~ 3p ACOGCCCCAGGUGCOGCOGG 506 CTGGGGCAG 1443 TGGGGCAG 2380 GGGGCAG 3317 hsa-miR~324- 5p CGCAO'CCCCOAGGGCAOUGGOGD 507 AGGGGATGC 1444 GGGGATGC 2381 GGGATGC 3318 hsa-miR-325 CCDAGDAGGUGO'CCAGDAAGDGO 508 ACCTACTAG 1445 CCTACTAG 2382 CTACTAG 3319 hsa-miR-326 CCUCOGGGCCCOUCCD'CCAG 509 GGCCCAGAG 1446 GCCCAGAG 2383 CCCAGAG 3320 hsa-miR-328 COGGCCCOCUCOGCCCOUCCGD' 510 AGAGGGCCA 1447 GAGGGCCA 2384 AGGGCCA 3321 hsa-miR-329 AACACACCUGGUUAACCUCUUU 511 CAGGTGTGT 1448 AGGTGTGT 2385 GGTGTGT 3322 hsa-miR- 33 0 - 3p GCAAAGCACACGGCCDGCAGAGA 512 TGTGCTTTG 1449 GTGCTTTG 2386 TGCTTTG 3323 hsa-miR- 33 Q ~ 5p UCUCUGGGCCUGUGUCUUAGGC 513 GGCCCAGAG 1450 GCCCAGAG 2387 CCCAGAG 3 324 hsa~miR-331- 3p GCCCCUGGGCCOAOCCOAGAA 514 GCCCAGGGG 1451 CCCAGGGG 2388 CCAGGGG 3325 hsa-miR-331-5p COAGGOAOGGUCCCAGGGAOCC 515 CCATACCTA 1452 CATACCTA 2389 ATACCTA 3326 hsa-miR-335 OCAAGAGCAADAACGAAAAAOGU 516 TTGCTCTTG 1453 TGCTCTTG 2390 GCTCTTG 3327 hsa-miR- 33 5 * UUUUUCAUUAUUGCUCCUGACC 517 TAATGAAAA 1454 AATGAAAA 2391 ATGAAAA 3328 hsa-miR~337-3p CUCCUAUAUGAUGCCUOUCUUC 518 CATATAGGA 1455 ATATAGGA 2392 TATAGGA 3329 hsa-miR~ 33 7 ~ 5p GAACGGCUUCAUACAGGAGUU 519 GAAGCCGTT 1456 AAGCCGTT 2393 AGCCGTT 3330 hsa-miR-338~ 3p UCCAGCAOCAGUGAaUODGUUG 520 TGATGCTGG 1457 GATGCTGG 2394 ATGCTGG 3331 hsa-miR- 338~ 5p AACAAUAUCCUGGUGCUGAGUG 521 GGATATTGT 1458 GATATTGT 2395 ATATTGT 3332 hsa-miR-339- 3p UGAGCGCCOCGACGA.CAGAGCCG 522 GAGGCGCTC 1459 AGGCGCTC 2396 GGCGCTC 3 33 3 hsa-miR-339- 5p UCCCUGUCCUCCAGGAGCUCACG 523 AGGACAGGG 1460 GGACAGGG 2397 GACAGGG 3 3 34 hsa-miR-33a GUGCAUUGUAGUUGCAUUGCA 524 TACAATGCA 1461 ACAATGCA 2398 CAATGCA 33 35 hsa-miR- 33 a * CAAUGUUUCCACAGUGCAUCAC 525 GGAAACATT 1462 GAAACATT 2399 AAACATT 3336 hsa-miR- 33b GUGCAOOGCUGOUGCAOOGC 526 AGCAATGCA 1463 GCAATGCA 2400 CAATGCA 3 3 3 7 hsa-miR-33b* CAGUGCCOCGGCAGOGCAGCCC 527 CGAGGCACT 1464 GAGGCACT 2401 AGGCACT 3338 hsa-miR-340 DUADAAAGCAADGAGACUGAOU 528 TGCTTTATA 1465 GCTTTATA 2402 CTTTATA 3 3 39 hsa-miR-340* OCCGUCUCAGOOACUUDAUAGC 529 CTGAGACGG 1466 TGAGACGG 2403 GAGACGG 3340 hsa-miR-342-3p UCnCACACAGAAAUCGCACCCGU 530 CTGTGTGAG 1467 TGTGTGAG 2404 GTGTGAG 3341 hsa-miR-342- 5p AGGGGUGCUAUCUGUGAUUGA 531 TAGCACCCC 1468 AGCACCCC 2405 GCACCCC 3342 hsa-miR-345 GCUGACUCCUAGUCCAGGGCUC 532 AGGAGTCAG 1469 GGAGTCAG 2406 GAGTCAG 3343 hsa-miR-346 UGUCUGCCCGCAUGCCUGCCUCTJ 533 CGGGCAGAC 1470 GGGCAGAC 2407 GGCAGAC 3 344 hsa-miR~34a OGGCAGOGncaUAGCaGGUOGD' 534 GACACTGCC 1471 ACACTGCC 2408 CACTGCC 3345 hsa-miR- 34 a * CAAOCAGCAAGOAOACOGCCCD 53 5 TTGCTGATT 1472 TGCTGATT 2409 GCTGATT 3346 hsa-miR- 3 b CAAOCACOAACUCCACOGCCAO 536 TTAGTGATT 1473 TAGTGATT 2410 AGTGATT 3347 hsa-miR- 3 b* DAGGCAGUGDCAUUAGCUGAUDG 53 7 ACACTGCCT 1474 CACTGCCT 2411 ACTGCCT 3348 hsa~miR- 34c - 3p AAOCACUAACCACACGGCCAGG 538 GTTAGTGAT 1475 TTAGTGAT 2412 TAGTGAT 3349 fasa~miR~34c~5p AQGCAGUGUAGUUAGCUGAUUGC 539 TACACTGCC 1476 ACACTGCC 2413 CACTGCC 3350 hsa~miR-361~3p UCCCCCAGGUGUGAUUCUGAUUU 540 ACCTGGGGG 1477 CCTGGGGG 2414 CTGGGGG 3351 hsa~miR~361~5p UUAUCAGAAUCUCCAGGGGUAC 541 ATTCTGATA 1478 TTCTGATA 2415 TCTGATA 3352 hsa~miR~362~3p AACACACCUAUUCAAGGAUUCA 542 TAGGTGTGT 1479 AGGTGTGT 2416 GGTGTGT 3353 hsa-tniR-362 -5p AAUCCUUGGAACCUAGGUGUGAGU 543 TCCAAGGAT 1480 CCAAGGAT 2417 CAAGGAT 3354 hsa-miR-363 AAUUGCACGGUAUCCAUCUGHA 544 CCGTGCAAT 1481 CGTGCAAT 2418 GTGCAAT 3355 hsa-miR-363* CGGGUGGAUCACGAUGCAAUUU 545 GATCCACCC 1482 ATCCACCC 2419 TCCACCC 3356 hsa-miR-365 UAAUGCCCCUAAAAAUCCUUAU 546 AGGGGCATT 1483 GGGGCATT 2420 GGGCATT 3357 hsa-miR-367 AAUUGCACUUUAGCAAUGGUGA 547 AAGTGCAAT 1484 AGTGCAAT 2421 GTGCAAT 3358 hsa-miR-367* ACOGOUGCOAAOADGCAACOCO' 548 TAGCAACAG 1485 AGCAACAG 2422 GCAACAG 3359 hsa-miR-369-3p AADAADACAOGGODGAD'CDDD 549 ATGTATTAT 1486 TGTATTAT 2423 GTATTAT 3360 hsa~miR-369~5p AGADCGACCGDGUUAOAIJOCGC 550 CGGTCGATC 1487 GGTCGATC 2424 GTCGATC 3361 hsa-miR-370 GCCOGCOGGGGOGGAACCUGGO 551 CCCAGCAGG 1488 CCAGCAGG 2425 CAGCAGG 3362 hsa-miR~371~3p AAGUGCCGCCAUCUUUUGAGUGU 552 GGCGGCACT 1489 GCGGCACT 2426 CGGCACT 3363 hsa-miR~371~5p ACOCAAACOGOGGGGGCACO 553 CAGTTTGAG 1490 AGTTTGAG 2427 GTTTGAG 3364 hsa-miR-372 AAAGUGCUGCGACAUUUGAGCGU 554 GCAGCACTT 1491 CAGCACTT 2428 AGCACTT 3365 hsa-miR-373 GAAGUGCUUCGAUUUUGGGGUGU 555 GAAGCACTT 1492 AAGCACTT 2429 AGCACTT 3366 hsa-miR-373* ACD'CAAAADGGGGGCGCOUUCC 556 CATTTTGAG 1493 ATTTTGAG 2430 TTTTGAG 3367 hsa-miR-374a UOAUAADACAACCUGADAAGUG 557 TGTATTATA 1494 GTATTATA 2431 TATTATA 3368 hsa-miR-374a* CUUAUCAGAOOGOAUOGUAAUU 558 ATCTGATAA 1495 TCTGATAA 2432 CTGATAA 3369 hsa-miR-374b AOAOAAOACAACCUGCOAAGDG 559 TGTATTATA 1496 GTATTATA 2433 TATTATA 3370 fasa~miR~374b* CUUAGCAGGUUGUAUUAUCAUU 560 ACCTGCTAA 1497 CCTGCTAA 2434 CTGCTAA 3371 hsa-miR-375 UUUGOOCGOUCGGCOCGCGOGA 561 AACGAACAA. 1498 ACGAACAA 2435 CGAACAA 3372 hsa-miR-376a AO'CADAGAGGAAAADCCACGU 562 CCTCTATGA 1499 CTCTATGA 2436 TCTATGA 3373 hsa-miR-376a* GOAGAOUCOCCnOCOAOGAGOA 563 GAGAATCTA 1500 AGAATCTA 2437 GAATCTA 3374 hsa-miR-376b AUCAUAGAGGAAAAUCCAUGUU 564 CCTCTATGA 1501 CTCTATGA 2438 TCTATGA 3375 hsa-miR-376c AACAUAGAGGAAAUUCCACGU 565 CCTCTATGT 1502 CTCTATGT 2439 TCTATGT 3376 hsa-miR-377 AUCACACAAAGGCAACUUUUGU 566 TTTGTGTGA 1503 TTGTGTGA 2440 TGTGTGA 3377 hsa-miR-377* AGAGGOUGCCCOOGGOGAAUUC 567 GGCAACCTC 1504 GCAACCTC 2441 CAACCTC 3378 hsa-miR-378 ACOGGACUUGGAGOCAGAAGG 568 CAAGTCCAG 1505 AAGTCCAG 2442 AGTCCAG 3379 hsa-miR-378* CUCCUGACUCCAGGUCCUGUGU 569 GAGTCAGGA. 1506 AGTCAGGA 2443 GTCAGGA 3380 fasa-miR~379 OGGOAGACOAUGGAACGOAGG 570 TAGTCTACC 1507 AGTCTACC 2444 GTCTACC 3381 hsa-miR-379* DAUGOAACAOGGUCCACOAACO 571 ATGTTACAT 1508 TGTTACAT 2445 GTTACAT 3382 hsa-miR-380 OAUGOAAOAUGGD'CCACAOCOD 572 ATATTACAT 1509 TATTACAT 2446 ATTACAT 3383 hsa-miR-380* UGGUUGACCAUAGAACAUGCGC 573 TGGTCAACC 1510 GGTCAACC 2447 GTCAACC 3384 hsa-miR-381 UAUACAAGGGCAAGCUCUCUGU 574 CCCTTGTAT 1511 CCTTGTAT 2448 CTTGTAT 3385 hsa-miR-382 GAAGUUGOUCGUGGUGGAUUCG 575 GAACAACTT 1512 AACAACTT 2449 ACAACTT 3386 hsa-miR-383 AGAUCAGAAGGOGADDGDGGCD' 576 CTTCTGATC 1513 TTCTGATC 2450 TCTGATC 3387 hsa~miR-384 AUUCCUAGAAAUUGUUCAUA 577 TTCTAGGAA 1514 TCTAGGAA 2451 CTAGGAA 3388 hsa-miR-409-3p GAAOGOUGCOCGGOGAACCCCO' 578 AGCAACATT 1515 GCAACATT 2452 CAACATT 3389 hea-miR-409-5p AGGOUACCCGAGCAACDUUGCAD' 579 CGGGTAACC 1516 GGGTAACC 2453 GGTAACC 3390 hsa-miR-410 AAOAOAACACAGAOGGCCOGU 580 GTGTTATAT 1517 TGTTATAT 2454 GTTATAT 3391 hsa-miR-411 UAGUAGACCGUADAGCGDACG 581 CGGTCTACT 1518 GGTCTACT 2455 GTCTACT 3392 hsa-miR-411* UAUGOAACACGGOCCACOAACC 582 GTGTTACAT 1519 TGTTACAT 2456 GTTACAT 3393 hsa-miR-412 ACUUCACCUGGOCCACOAGCCGO' 583 CAGGTGAAG 1520 AGGTGAAG 2457 GGTGAAG 3394 hsa~miR~421 AOCAACAGACAIJUAAOUGGGCGC 584 GTCTGTTGA 1521 TCTGTTGA 2458 CTGTTGA 3395 hsa-miR-422a ACUGGACUUAGGGUCAGAAGGC 585 TAAGTCCAG 1522 AAGTCCAG 2459 AGTCCAG 3396 h<3a-miR-423 -3p AGCUCGGUCUGAGGCCCCUCAGU 586 AGACCGAGC 1523 GACCGAGC 2460 ACCGAGC 3397 hsa-miR-423-Bp UGAGGGGCAGAGAGCGAGACUUU 587 CTGCCCCTC 1524 TGCCCCTC 2461 GCCCCTC 3398 hsa-miR-424 CAGCAGCAAUUCAUGUUUUGAA 588 ATTGCTGCT 1525 TTGCTGCT 2462 TGCTGCT 3399 hsa-miR-424* CAAAACGDGAGGCGCDGCDAO 589 TCACGTTTT 1526 CACGTTTT 2463 ACGTTTT 3400 hsa-miR-425 AAUGACACGAOCACOCCCGOUGA 590 CGTGTCAT 1527 CGTGTCAT 2464 GTGTCAT 3401 hsa-miR-425* AUCGGGAAUGUCGUGUCCGCCC 591 CATTCCCGA 1528 ATTCCCGA 2465 TTCCCGA 3402 hsa-miR-429 OAADACOGOCOGGOAAAACCGU 592 GACAGTATT 1529 ACAGTATT 2466 CAGTATT 3403 hsa-miR-431 OGUCUUGCAGGCCGUCAOGCA 593 CTGCAAGAC 1530 TGCAAGAC 2467 GCAAGAC 3404 hsa~miR~431* CAGGUCGUCUUGCAGGGCUUCU 594 AGACGACCT 1531 GACGACCT 2468 ACGACCT 3405 hsa-miR-432 UCUUGGAGUAGGUCAUUGGGUGG 595 TACTCCAAG 1532 ACTCCAAG 2469 CTCCAAG 3406 hsa-miR~432* COGGAUGGCOCCOCCAOGOCO 596 AGCCATCCA 1533 GCCATCCA 2470 CCATCCA 3407 hsa-miR-433 ADCAUGAUGGGCOCCOCGGDGD' 597 CCATCATGA 1534 CATCATGA 2471 ATCATGA 3408 hsa-miR-448 OOGCADADGDAGGAOGUCCCAD 598 ACATATGCA 1535 CATATGCA 2472 ATATGCA 3409 hsa-miR-449a UQGCAGUGUAUUGUUAGCUGGU 599 TACACTGCC 1536 ACACTGCC 2473 CACTGCC 3410 hsa-miR-449b AGGCAGOGUAOUGOUAGCDGGC 600 TACACTGCC 1537 ACACTGCC 2474 CACTGCC 3411 hsa~miR~45Qa UUUUGCGAUGUGUUCCUAAUAU 601 CATCGCAAA 1538 ATCGCAAA 2475 TCGCAAA 3412 fasa~miR~45Qb--3p UUGGGAOCAODOUGCAUCCAUA 602 ATGATCCCA 1539 TGATCCCA 2476 GATCCCA 3413 fasa-miR~450b~5p UUUUGCAAUAUGUUCCnGAAUA 603 TATTGCAAA 1540 ATTGCAAA 2477 TTGCAAA 3414 hsa-miR-451 AAACCGUUACCAUUACUGAGUU 604 GTAACGGTT 1541 TAACGGTT 2478 AACGGTT 3415 hsa-miR-452 AACUGUUUGCAGAGGAAACUGA 605 GCAAACAGT 1542 CAAACAGT 2479 AAACAGT 3416 hsa-miR-452* CUCAUCUGCAAAGAAGUAAGUG 606 TGCAGATGA 1543 GCAGATGA 2480 CAGATGA 3417 hsa-miR-453 AGGUOGOCCGOGGUGAGOUCGCA 607 CGGACAACC 1544 GGACAACC 2481 GACAACC 3418 hsa-miR-454 UAGUGCAAUAUUGCUUAUAGGGU 608 TATTGCACT 1545 ATTGCACT 2482 TTGCACT 3419 hsa-miR-454* ACCCUAUCAAUAUUGUCUCUGC 609 TTGATAGGG 1546 TGATAGGG 2483 GATAGGG 3420 hsa-miR-455-3p GCAGOCCAUGGGCAOAUACAC 610 CATGGACTG 1547 ATGGACTG 2484 TGGACTG 3421 hsa~miR~455-5p UAUGOGCCUUOGGACUACAO'CG 611 AAGGCACAT 1548 AGGCACAT 2485 GGCACAT 3422 fasa-miR~483-3p OCACOCCD'COCCUCCCGUCOD 612 AGAGGAGTG 1549 GAGGAGTG 2486 AGGAGTG 3423 hsa-miR-483-5p AAGACGGGAGGAAAGAAGGGAG S13 CTCCCGTCT 1550 TCCCGTCT 2487 CCCGTCT 3424 hsa-miR-484 UCAGGCUCAGUCCCCUCCCGAU 614 CTGAGCCTG 1551 TGAGCCTG 2488 GAGCCTG 3425 hsa-miR-485-3p GUCAUACACGGCUCUCCncnCU 615 CGTGTATGA 1552 GTGTATGA 2489 TGTATGA 3426 hsa-miR~485~5p AGAGGCUGGCCGOGAUGAAUO'C 616 GCCAGCCTC 1553 CCAGCCTC 2490 CAGCCTC 3427 hsa-miR-486-3p CGGGGCAGCD'CAGUACAGGAD 617 AGCTGCCCC 1554 GCTGCCCC 2491 CTGCCCC 3428 hsa~miR~486-5p O'CCUGUACUGAGCOGCCCCGAG 618 CAGTACAGG 1555 AGTACAGG 2492 GTACAGG 3429 hsa-miR-487a AAUCAUACAGGGACAUCCAGUU 619 CTGTATGAT 1556 TGTATGAT 2493 GTATGAT 3430 hsa-miR-487b AAUCGUACAGGGUCAUCCACUU 620 CTGTA.CGAT 1557 TGTACGA.T 2494 GTACGAT 3431 hsa-miR-488 UUGAAAGGCUAUUUCUUGGUC 621 AGCCTTTCA 1558 GCCTTTCA 2495 CCTTTCA 3432 hsa-miR-488* CCCAGAUAAUGGCACnCUCAA 622 ATTATCTGG 1559 TTATCTGG 2496 TATCTGG 3433 hsa-miR-489 GUGACAUCACAUAUACGGCAGC 623 GTGATGTCA 1560 TGATGTCA 2497 GATGTCA 3434 hsa-miR-490-3p CAACCOGGAGGACUCCAOGCOG 624 CTCCAGGTT 1561 TCCAGGTT 2498 CCAGGTT 3435 hsa-miR-490-5p CCAUGGAUCOeCAGGUGGGU 625 AGATCCATG 1562 GATCCATG 2499 ATCCATG 343S hsa-miR-491-3p CUUAUGCAAGAUUCCCUUCUAC 626 CTTGCATAA 1563 TTGCATAA 2500 TGCATAA 3437 hsa-miR-491-5p AGUGGGGAACCCUOCCAOGAGG 627 GTTCCCCAC 1564 TTCCCCAC 2501 TCCCCAC 3438 hsa-miR-492 AGGACCUGCGGGACAAGAUOCOD 628 CGCAGGTCC 1565 GCAGGTCC 2502 CAGGTCC 3439 hsa-miR-493 UGAAGGDCDACDGUGUGCCAGG 629 TAGACCTTC 1566 AGA.CCTTC 2503 GACCTTC 3440 hsa-miR-493* UUGUACAUGGUAGGCUUUCAUU 630 CCATGTACA 1567 CATGTACA 2504 ATGTACA 3441 hsa-miR-494 OGAAACAOACACGGGAAACCOC 631 GTATGTTTC 1568 TATGTTTC 2505 ATGTTTC 3442 hea-miR-495 AAACAAACAUGGUGCACIIUCUU 632 ATGTTTGTT 1569 TGTTTGTT 2506 GTTTGTT 3443 hsa-miR-496 UGAGUAUUACAUGGCCAAUCUC 633 GTAATACTC 1570 TAATACTC 2507 AATACTC 3444 hsa-miR-497 CAGCAGCACACOGDGGDUOGO 634 TGTGCTGCT 1571 GTGCTGCT 2508 TGCTGCT 3445 hsa-miR-497* CAAACCACACUGUGGUGUUAGA 635 GTGTGGTTT 1572 TGTGGTTT 2509 GTGGTTT 3446 hsa-miR-498 UUUCAAGCCAGGGGGCGUUUUUC 636 TGGCTTGAA 1573 GGCTTGAA 2510 GCTTGAA 3447 hsa~miR-499-3p AACAUCACAGCAAGUCUGUGCU 637 CTGTGATGT 1574 TGTGATGT 2511 GTGATGT 3448 hsa-miR-499-5p UUAAGACUUGCAGUGAUGUUU 638 CAAGTCTTA 1575 AAGTCTTA 2512 AGTCTTA 3449 hsa-miR-500 UAAUCCUUGCUACCUGGGUGAGA 639 GCAAGGATT 1576 CAAGGATT 2513 AAGGATT 3450 hsa-miR-500* AUGCACCUGGGCAAGGAUUCUG 640 CCAGGTGCA 1577 CAGGTGCA 2514 AGGTGCA 3451 hsa~miR~5Ql~3p AAUGCACCCGGGCAAGGAUUCU 641 CGGGTGCAT 1578 GGGTGCAT 2515 GGTGCAT 3452 hsa-miR-501-5p AAUCCOUUGUCCCUGGGOGAGA 642 ACAAAGGAT 1573 CAAAGGAT 2516 AAAGGAT 3453 hsa-miR-502-3p AADGCACCDGGGCAAGGAUUCA 643 CAGGTGCAT 1580 AGGTGCAT 2517 GGTGCAT 3454 hsa~mlR-502-5p AD'CCUDGCDAD'CUGGGUGCDA. 644 TAGCAAGGA 1581 AGCAAGGA 2518 GCAAGGA 3455 hsa-miR-503 OAGCAGCGGGAACAGOUCDGCAG 645 CCCGCTGCT 1582 CCGCTGCT 2519 CGCTGCT 3456 hsa-miR-504 AGACCCDGGOCUGCACO'COAOC 646 ACCAGGGTC 1583 CCAGGGTC 2520 CAGGGTC 3457 hsa-mlR-505 CGUCAACACUUGCUGGUUUCCU 647 AGTGTTGAC 1584 GTGTTGAC 2521 TGTTGAC 3458 hsa-miR-505* GGGAGCCAGGAAGUAUUGAUGU 648 CCTGGCTCC 1585 CTGGCTCC 2522 TGGCTCC 3459 hsa-miR-506 UAAQGCACCCUUCUGAGUAGA 649 GGGTGCCTT 1586 GGTGCCTT 2523 GTGCCTT 3460 hsa-miR-507 OUUUGCACCOUUUGGAGOGAA 650 AGGTGCAAA 1587 GGTGCAAA 2524 GTGCAAA 3461 hsa-miR-508-3p DGAOUGOAGCCUUUUGGAGOAGA 651 GCTACAATC 1588 CTACAATC 2525 TACAATC 3462 hsa-miR- 508 ~5p UACUCCAGAGGGCGUCACUCAUG 652 CTCTGGAGT 1589 TCTGGAGT 2526 CTGGAGT 3463 hsa-miR-5Q9-3-5p OACOGCAGACGOGGCAAD'CAOG 653 GTCTGCAGT 1590 TCTGCAGT 2527 CTGCAGT 3464 hsa-miR- 509- p DGAUDGGDACGDCOGOGGGUAG 654 GTACCAATC 1591 TACCAATC 2528 ACCAATC 3465 hsa-miR-509-5p DACOGCAGACAGOGGCAAOCA 655 GTCTGCAGT 1592 TCTGCAGT 2529 CTGCAGT 3466 hsa-miR-510 OACOCAGGAGAGUGGCAAOCAC 656 CTCCTGAGT 1593 TCCTGAGT 2530 CCTGAGT 3467 hsa-miR-511 GOGOCUOUUGCUCOGCAGOCA 657 CAAAAGACA 1594 AAAAGACA 2531 AAAGACA 3468 hsa-miR-512-3p AAGUGCUGDCAUAGCUGAGGUC 658 GACAGCACT 1595 ACAGCACT 2532 CAGCACT 3469 hsa-miR-512-5p CACUCAGCCUUGAGGGCACUUUC 659 AGGCTGAGT 1596 GGCTGAGT 2533 GCTGAGT 3470 hsa-miR-513a-3p UAAAUUUCACCUUUCOGAGAAGG 660 GTGAAATTT 1597 TGAAATTT 2534 GAAATTT 3471 hsa-miR-513a-5p UUCACAGGGAGGUGUCAU 661 TCCCTGTGA 1598 CCCTGTGA 2535 CCTGTGA 3472 hsa-miR-513b UOCACAAGGAGGUGD'CAODIIAO 662 TCCTTGTGA 1599 CCTTGTGA 2536 CTTGTGA 3473 hsa-miR-513e UUCUCAAGGAGGUGUCGUUUAU 663 TCCTTGAGA 1600 CCTTGAGA 2537 CTTGAGA 3474 hsa-miR- 514 AUUGACACUUCUGUGAGUAGA 664 AAGTGTCAA 1601 AGTGTCAA 2538 GTGTCAA 3475 hsa-miR~515-3p GAGUGCCUUCUUUUGGAGCGOU 665 GAAGGCACT 1602 AAGGCACT 2539 AGGCACT 3476 hsa-miR-515-5p UUCUCCAAAAGAAAGCACUUUCUG 666 TTTTGGAGA 1603 TTTGGAGA 2540 TTGGAGA 3477 hsa-miR-516a~3p OGCUllCCUUDCAGAGGGU 667 AAAGGAAGC 1604 AAGGAAGC 2541 AGGAAGC 3478 hsa-miR-516 ~5p UUCUCGAGGAAAGAAGCACUUUC 668 TCCTCGAGA 1605 CCTCGAGA 2542 CTCGAGA 3479 hsa-miR- 516b AUCUGGAGGUAAGAAGCACUUU 669 ACCTCCAGA 1606 CCTCCAGA 2543 CTCCAGA 3480 hsa-miR-517* CCOCUAGAOGGAAGCACUGOCO 670 CATCTAGAG 1607 ATCTAGAG 2544 TCTAGAG 3481 hsa~miR~517a AUCGUGCAUCCCUUUAGAGnGU 671 GATGCACGA 1608 ATGCACGA 2545 TGCACGA 3482 hsa-miR-517b UCGUGCAUCCCUUUAGAGUGUU 672 GGATGCACG 1609 GATGCACG 2546 ATGCACG 3483 hsa-miR-517c AUCGUGCAUCCUUUUAGAGUGU 673 GATGCACGA 1610 ATGCACGA 2547 TGCACGA 3484 hsa-miR-518a~3p GAAAGCGCUUCCCnUUGCOGGA 674 AAGCGCTTT 1611 AGCGCTTT 2548 GCGCTTT 3485 hsa-miR-518b CAAAGCGCUCCCCUUUAGAGGU 675 GAGCGCTTT 1612 AGCGCTTT 2549 GCGCTTT 3486 hsa-miR-518c CAAAGCGCnO'COCaOUAGAGOGO' 676 AAGCGCTTT 1613 AGCGCTTT 2550 GCGCTTT 3487 hsa~miR-518c* UCOCUGGAGGGAAGCACnUOCOG 677 CCTCCAGAG 1614 CTCCAGAG 2551 TCCAGAG 3488 hsa~miR~518d~3p CAAAGCGCUOCCCOUUGGAGC 678 AAGCGCTTT 1615 AGCGCTTT 2552 GCGCTTT 3489 hsa-miR- 518d-5p CUCUAGAGGGAAGCACUUUCUG 679 CCCTCTAGA 1616 CCTCTAGA 2553 CTCTAGA 3490 hsa-miR~518e AAAGCGCUUCCCUUCAGAGUG 680 GAAGCGCTT 1617 AAGCGCTT 2554 AGCGCTT 3491 hsa-miR- 518f GAAAGCGCUUCUCUUUAGAGG 681 AAGCGCTTT 1618 AGCGCTTT 2555 GCGCTTT 3492 hsa-miR- 518£* CUCUAGAGGGAAGCACUUUCUC 682 CCCTCTAGA 1619 CCTCTAGA 2556 CTCTAGA 3493 hsa-miR-519a AAAGOGCAOCCUUUOAGAGOGD 683 GATGCACTT 1620 ATGCACTT 2557 TGCACTT 3494 hsa-miR-519a* CDCDAGAGGGAAGCGCOUOCOG 684 CCCTCTAGA 1621 CCTCTAGA 2558 CTCTAGA 3495 hsa-miR-519b~3p AAAGUGCAUCCUUUUAGAGGUU 685 GATGCACTT 1622 ATGCACTT 2559 TGCACTT 3496 hsa-miR-519c-3p AAAGUGCAUCUUUUUAGAGGAU 686 GATGCACTT 1623 ATGCACTT 2560 TGCACTT 3497 hsa-miR-519d CAAAGUGCCUCCCUUUAGAGUG 687 AGGCACTTT 1624 GGCACTTT 2561 GCACTTT 3498 hsa-miR-519e AAGUGCCDCCOUUUAGAGOGOU 688 GGAGGCACT 1625 GAGGCACT 2562 AGGCACT 3499 hsa-miR-519e* UUCUCCAAAAGGGAGCACUUUC 689 TTTTGGAGA 1626 TTTGGAGA 2563 TTGGAGA 3500 hsa-miR- 520a-3p AAAGUGCUUCCCUUUGGACUGU 690 GAAGCACTT 1627 AAGCACTT 2564 AGCACTT 3501 hsa-miR- 520a-5p CUCCAGAGGGAAGUACUUD'CD' 691 CCCTCTGGA 1628 CCTCTGGA 2565 CTCTGGA 3502 hsa-miR- 52 Ob AAAGUGCUUCCUUUUAGAGGG 692 GAAGCACTT 1629 AAGCACTT 2566 AGCACTT 3503 hsa-miR- 520c-3p AAAGUGCUUCCUUUUAGAGGGU 693 GAAGCACTT 1630 AAGCACTT 2567 AGCACTT 3504 hsa-miR-520d~3p AAAGUGCUUCOCUUDGGOGGGn 694 GAAGCACTT 1631 AAGCACTT 2568 AGCACTT 3505 hsa-miR-52 Od- 5p COACAAAGGGAAGCCCUUOC 695 CCCTTTGTA 1632 CCTTTGTA 2569 CTTTGTA 3506 hsa-miR-520e AAAGUGCUUCCUUUUUGAGGG 696 GAAGCACTT 1633 AAGCACTT 2570 AGCACTT 3507 hsa-miR-520f AAGUGCUUCCUUUUAGAGGGUU 697 GGAAGCACT 1634 GAAGCACT 2571 AAGCACT 3508 hsa-miR- 52 Og ACAAAGUGCUUCCCUUUAGAGUGU 698 AGCACTTTG 1635 GCACTTTG 2572 CACTTTG 3509 hsa-miR- 520h ACAAAGOGCODCCCOUUAGAGO 699 AGCACTTTG 1636 GCACTTTG 2573 CACTTTG 3510 hsa-miR-521 AACGCACUUCCCaUUAGAGUGO 700 GAAGTGCGT 1637 AAGTGCGT 2574 AGTGCGT 3511 hsa-miR- 522 AAAAUGGUUCCCUUUAGAGUGU 701 GAACCATTT 1638 AACCATTT 2575 ACCATTT 3512 hsa-miR-523 GAACGCGCUUCCCUAUAGAGGGU 702 AAGCGCGTT 1639 AGCGCGTT 2576 GCGCGTT 3513 hsa-miR- 52 -3p GAAGGCGCITUCCCOUUGGAGU 703 AAGCGCCTT 1640 AGCGCCTT 2577 GCGCCTT 3514 hsa-miR- 524-5p CUACAAAGGGAAGCACUUUCUC 704 CCCTTTGTA 1641 CCTTTGTA 2578 CTTTGTA 3515 hsa-miR-525-3p GAAGGCGCUUCCCUUDAGAGCG 705 AAGCGCCTT 1642 AGCGCCTT 2579 GCGCCTT 3516 hsa-miR-525~5p CDCCAGAGGGADGCACUIIUCU 706 CCCTCTGGA 1S43 CCTCTGGA 2580 CTCTGGA 3517 hsa-miR-526b CDCUUGAGGGAAGCACD'DO'COGU 707 CCCTCAAGA 1644 CCTCAAGA 2581 CTCAAGA 3518 hsa~miR~ 526b* GAAAGUGCUUCCUUUUAGAGGC 708 AAGCACTTT 1645 AGCACTTT 2582 GCACTTT 3519 hsa-miR- 527 CDGCAAAGGGAAGCCCOUUC 709 CCCTTTGCA 1646 CCTTTGCA 2583 CTTTGCA 3520 hsa-miR~532~3p CCUCCCACACCCAAGGCUOGCA 710 GTGTGGGAG 1647 TGTGGGAG 2584 GTGGGAG 3521 hsa~miR-532 ~5p CADGCCUUGAGUGUAGGACCGU 711 TCAAGGCAT 1S 8 CAAGGCAT 2585 AAGGCAT 3522 hsa-miR-539 GGAGAAAUUAUCCUUGGUGUGU 712 TAATTTCTC 1649 AATTTCTC 2586 ATTTCTC 3523 hsa-miR-541 OGGUGGGCACAGAAOCOGGACD' 713 GTGCCCACC 1S50 TGCCCACC 2587 GCCCACC 3524 hsa-miR-541* AAAGGAOUCUGCOGOCGGUCCCACU 714 AGAATCCTT 1651 GAATCCTT 2588 AATCCTT 3525 hsa-miR-542-3p OGOGACAGAODGAUAACOGAAA 715 ATCTGTCAC 1S52 TCTGTCAC 2589 CTGTCAC 3526 hsa-miR- 542 ~5p OCGGGGAUCAOCAUGOCACGAGA 716 TGATCCCCG 1653 GATCCCCG 2590 ATCCCCG 3527 hsa-miR-543 AAACAUD'CGCGGDGCACOUCUU 717 GCGAATGTT 1654 CGAATGTT 2591 GAATGTT 3528 hsa-miR- 544 AUUCUGCAUUUUUAGCAAGUUC 718 AATGCAGAA 1655 ATGCAGAA 2592 TGCAGAA 3529 hsa-miR-545 OCAGCAAACAUUUAUIJGUGDGC 719 TGTTTGCTG 1656 GTTTGCTG 2593 TTTGCTG 3530 hsa-miR-545* UCAGUAAAUGUUUAUUAGAUGA 720 CATTTACTG 1657 ATTTACTG 2594 TTTACTG 3531 hsa-miR- 54Ba-3p CAAAACOGGCAAOUACOUUDGC 721 GCCAGTTTT 1658 CCAGTTTT 2595 CAGTTTT 3532 hsa-miR-5 8a- 5p AAAAGUAAUUGCGAGUUUUACC 722 AATTACTTT 1659 ATTACTTT 2596 TTACTTT 3533 hsa-miR-548b-3p CAAGAACCUCAGUUGCUUUUGU 723 GAGGTTCTT 1660 AGGTTCTT 2597 GGTTCTT 3534 hsa-miR- 548b- 5p AAAAGUAAUDGDGGUUUUGGCC 724 AATTACTTT 1661 ATTACTTT 2598 TTACTTT 3535 hsa-miR-548e-3p CAAAAAUCUCAAUUACDUUUGC 725 GAGATTTTT 16S2 AGATTTTT 2599 GATTTTT 3536 hsa-miR-548c-5p AAAAGUAADUGCGGDUUDIJGCC 726 AATTACTTT 1663 ATTACTTT 2600 TTACTTT 3537 hsa-miR-548d-3p CAAAAACCACAGUUUCUUUUGC 727 GTGGTTTTT 1664 TGGTTTTT 2601 GGTTTTT 3538 hsa-miR- 548d-5p AAAAGUAAUUGUGGUUUUUGCC 728 AATTACTTT 1S65 ATTACTTT 2602 TTACTTT 3539 hsa-miR-548e AAAAACUGAGACUACtJtlUtlGCA 729 CTCAGTTTT 1666 TCAGTTTT 2603 CAGTTTT 3540 hsa-miR-548f AAAAACUGUAAUUACUUUU 730 TACAGTTTT 1667 ACAGTTTT 2604 CAGTTTT 3541 hsa-miR- 548g AAAACUGUAAUUACUUUUGUAC 731 TTACAGTTT 1668 TACAGTTT 2605 ACAGTTT 3542 hsa-miR- 548h AAAAGUAAUCGCGGUUUUUGUC 732 GATTACTTT 1669 ATTACTTT 2606 TTACTTT 3543 hsa-miR-548i AAAAGUAAITUGCGGAIIUIJOGCC 733 AATTACTTT 1670 ATTACTTT 2607 TTACTTT 3544 hsa-miR-548 j AAAAGUAAUUGCGGUCUUUGGU 734 AATTACTTT 1671 ATTACTTT 2608 TTACTTT 3545 hsa-miR-548fc AAAAGUACUUGCGGAUUUDGCU 735 AAGTACTTT 1672 AGTACTTT 2609 GTACTTT 3546 hsa-miR-5 81 AAAAGUAUUUGCGGGUUUUGUC 736 AAATACTTT 1673 AATACTTT 2610 ATACTTT 3547 hsa-miR- 548m CAAAGGUAUUUGUGGUUUUUG 737 AATACCTTT 1674 ATACCTTT 2611 TACCTTT 3548 hsa-miR- 548n CAAAAGUAAUUGUGGAUUUUGU 738 ATTACTTTT 1675 TTACTTTT 2612 TACTTTT 3549 hsa-miR- 548o CCAAAACUGCAGIIOACUIIUDGC 739 GCAGTTTTG 1676 CAGTTTTG 2613 AGTTTTG 3550 hsa-miR- 548p UAGCAAAAACUGCAGUUACUUU 740 GTTTTTGCT 1677 TTTTTGCT 2614 TTTTGCT 3551 hsa-miR-549 UGACAACUAUGGAUGAGCUCU 741 ATAGTTGTC 1678 TAGTTGTC 2615 AGTTGTC 3552 hsa-miR- 550 AGUGCCUGAGGGAGUAAGAGCCC 742 CTCAGGCAC 1679 TCAGGCAC 2616 CAGGCAC 3553 hsa-miR- 550* UGO'CUOACIICCCUCAGGCACAU 743 GAGTAAGAC 1680 AGTAAGAC 2617 GTAAGAC 3554 hsa-miR-551a GCGACCCACUCUOGGUlItJCCA 744 AGTGGGTCG 1681 GTGGGTCG 2618 TGGGTCG 3555 hsa-miR- 551b GCGACCCAUACUUGGUUUCAG 745 TATGGGTCG 1682 ATGGGTCG 2619 TGGGTCG 3556 hsa-miR- 551b* GAAAO'CAAGCGDGGGOGAGACC 746 GCTTGATTT 1683 CTTGATTT 2620 TTGATTT 3557 hsa-miR-552 AACAGGUGACOGGOUAGACAA 7 7 GTCACCTGT 1684 TCACCTGT 2621 CACCTGT 3558 hsa-miR-553 AAAACGGUGAGAUUUUGUUUO 748 TCACCGTTT 1685 CACCGTTT 2622 ACCGTTT 3559 hsa-miR-554 GCOAGOCCUGACUCAGCCAGO 749 CAGGACTAG 1686 AGGACTAG 2623 GGACTAG 3560 hsa-miR-555 AGGGUAAGCUGAACCUCUGAU 750 AGCTTACCC 1687 GCTTACCC 2624 CTTACCC 3561 hsa-miR-556-3p AUAUUACCAUUAGCUCAUCUUU 751 ATGGTAATA 1688 TGGTAATA 2625 GGTAATA 3562 hsa-miR-55S-5p GAUGAGCUCAUUGUAAUAUGAG 752 TGAGCTCAT 1689 GAGCTCAT 2626 AGCTCAT 3563 hsa-miR-557 GUUUGCACGGGUGGGCCUUGUCU 753 CCGTGCAAA 1690 CGTGCAAA 2627 GTGCAAA 3564 hsa-miR-558 DGAGCOGCUGOACCAAAAO 754 CAGCAGCTC 1691 AGCAGCTC 2628 GCAGCTC 3565 hsa-miR-559 UAAAGUAAAUAUGCACCAAAA 755 ATTTACTTT 1692 TTTACTTT 2629 TTACTTT 3566 hsa-miR-561 CAAAGDUDAAGAOCCOUGAAGO' 756 TTAAACTTT 1693 TAAACTTT 2630 AAACTTT 3567 hsa-miR- 562 AAAGDAGCDGUACCAIJUDGC 757 CAGCTACTT 1694 AGCTACTT 2631 GCTACTT 3568 hsa-miR-563 AGGUUGACAUACGUUUCCC 758 ATGTCAACC 1695 TGTCAACC 2632 GTCAACC 3569 hsa-mi -564 AGGCACGQUGUCAGCAGGC 759 CACCGTGCC 1696 ACCGTGCC 2633 CCGTGCC 3570 hsa-miR-566 GGGCGCCOGUGAUCCCAAC 760 ACAGGCGCC 1697 CAGGCGCC 2634 AGGCGCC ₃₅₇₁ hsa-miR-567 AGUAUGUUCUUCCAGGACAGAAC 761 AGAACATAC 1698 GAACATAC 2635 AACATAC 3572 hsa-miR-568 AOGDAOAAADGDADACACAC 762 ATTTATACA 1699 TTTATACA 2636 TTATACA 3573 hsa-miR-569 AGODAAOGAAOCCUGGAAAGU 763 TTCATTAAC 1700 TCATTAAC 2637 CATTAAC 3574 hsa-miR-570 CGAAAACAGCAAOUACCUUOGC 764 GCTGTTTTC 1701 CTGTTTTC 2638 TGTTTTC ₃₅₇₅ hsa-miR-571 OGAGUOGGCCAOCUGAGDGAG 765 GGCCAACTC 1702 GCCAACTC 2639 CCAACTC 3576 hsa-miR-572 GDCCGCOCGGCGGDGGCCCA 766 CCGAGCGGA 1703 CGAGCGGA 2640 GAGCGGA 35₇₇ fasa~miR~573 CDGAAGDGADGOGDAACOGADCAG 767 ATCACTTCA 1704 TCACTTCA 2641 CACTTCA 3578 hsa-miR-574-3p CACGCD'CAOGCACACACCCACA 768 CATGAGCGT 1705 ATGAGCGT 2642 TGAGCGT 3579 hsa-miR-574~5p UGAGUGDGDGUGUGUGAGDGUGO 769 CACACACTC 1706 ACACACTC 2643 CACACTC 3580 hsa-miR-575 GAGCCAGOUGGACAGGAGC 770 CAACTGGCT 1707 AACTGGCT 2644 ACTGGCT 3581 fasa-miR~576-3p AAGAUGDGGAAAAAUUGGAAD'C 771 TCCACATCT 1708 CCACATCT 2645 CACATCT 3582 hsa~miR-576~5p AUUCUAAUUUCUCCACGUCUUU 772 AAA TAGAA 1709 AATTAGAA 2646 ATTAGAA 3583 hsa-miR-577 OAGAUAAAAUAOUGGOACCDG 773 ATTTTA.TCT 1710 TTTTATCT 2647 TTTATCT 3584 hsa~miR~578 CUUCUUGUGCUCUAGGAUUGU 774 GCACAAGAA 1711 CACAAGAA. 2648 ACAAGAA 3585 hsa-miR-579 UUCAUUUGGUAUAAACCGCGAUU 775 ACCAAATGA 1712 CCAAATGA 2649 CAAATGA 3586 hsa-miR-580 nOGAGAAOGAOGAAOCAaOAGG 776 TCATTCTCA 1713 CATTCTCA 2650 ATTCTCA 3587 hsa-miR-581 OCODGUGUOCD'CUAGAOCAGO 777 GAACACAAG 1714 AACACAAG 2651 ACACAAG 3588 hsa~miR~582-3p UAACUGGUUGAACAACUGAACC 778 CAACCAGTT 1715 AACCAGTT 2652 ACCAGTT 3589 hsa-miR~582-5p UUACAGUUGUUCAACCAGUUACU 779 ACAACTGTA 1716 CAACTGTA 2653 AACTGTA 3590 hsa-miR-583 CAAAGAGGAAGGUCCCAOUAC 780 TTCCTCTTT 1717 TCCTCTTT 2654 CCTCTTT 3591 hsa-miR-584 UUAUGGUUUGCCUGGGACUGAG 781 CAAACCATA 1718 AAACCATA 2655 AACCATA 3592 hea-miR-585 DGGGCGUAD'COGDAOGCDA 782 GATACGCCC 1719 ATACGCCC 2656 TACGCCC 3593 hsa-miR-586 UAUGCAOTJGUAUUUUUAGGUCC 783 ACAATGCAT 1720 CAATGCAT 2657 AATGCAT 3594 hea-miR-587 UUUCCAUAGGUGAUGAGUCAC 784 CCTATGGAA 1721 CTATGGAA 2658 TATGGAA 3595 hsa-miR-588 IJUGGCCACAAOGGGODAGAAC 785 TTGTGGCCA 1722 TGTGGCCA 2659 GTGGCCA 3596 hsa-miR-589 DGAGAACCACGOCOGCUCOGAG 786 GTGGTTCTC 1723 TGGTTCTC 2660 GGTTCTC 3597 fasa-miR-589* D'CAGAACAAAUGCCGGUUCCCAGA 787 TTTGTTCTG 1724 TTGTTCTG 2661 TGTTCTG 3598 hsa-miR-590-3p UAAUUUUAUGUAUAAGCUAGU 788 CATAAAATT 1725 ATAAAATT 2662 TAAAATT 3599 hsa-miR-590-5p GAGCUUAUUCAUAAAAGUGCAG 789 GAATAAGCT 1726 AATAAGCT 2663 ATAAGCT 3600 hsa-miR-591 AGACCAUGGGUUCUCAUUGU 790 CCCATGGTC 1727 CCATGGTC 2664 CATGGTC 3601 hsa-miR-592 UOGOGUCAADAOGCGAOGADGIJ 791 ATTGACACA 1728 TTGACACA 2665 TGACACA 3602 hsa-miR-593 UGUCUCUGCUGGGGUUUCU 792 AGCAGAGAC 1729 GCAGAGAC 2666 CAGAGAC 3603 hsa-miR-593* AGGCACCAGCCAGGCAUUGCD'CAGC 793 GCTGGTGCC 1730 CTGGTGCC 2667 TGGTGCC 3604 hsa-miR-595 GAAGOGDGCCGDGGUGDGD'CO' 794 GGCACACTT 1731 GCACACTT 2668 CACACTT 3605 hsa-miR-596 AAGCCUGCCCGGCUCCUCGGG 795 GGGCAGGCT 1732 GGCAGGCT 2669 GCAGGCT 3606 fasa-miR-597 UGDGUCACOCGAUGACCACOGO 796 GAGTGACAC 1733 AGTGACAC 2670 GTGACAC 3607 hsa-miR-598 OACGOCAOCGUUGUCAUCGOCA 797 CGATGACGT 1734 GATGACGT 2671 ATGACGT 3608 hsa-miR-599 GUUGUGUCAGUUUAUCAAAC 798 CTGACACAA 1735 TGACACAA 2672 GACACAA 3609 hsa-miR-600 ACUUACAGACAAGAGCCUUGCUC 799 GTCTGTAAG 1736 TCTGTAAG 2673 CTGTAAG 3610 hsa-miR-601 OGGUCOAGGAUUGUOGGAGGAG 800 TCCTAGACC 1737 CCTAGACC 2674 CTAGACC 3611 hsa-miR-602 GACACGGGCGACAGCUGCGGCCC 801 CGCCCGTGT 1738 GCCCGTGT 2675 CCCGTGT 3S12 hsa-miR-603 CACACACnGCAAUUACUUUUGC 802 GCAGTGTGT 1739 CAGTGTGT 2676 AGTGTGT 3613 hsa-miR-604 AGGCOGCGGAAODCAGGAC 803 TCCGCAGCC 1740 CCGCAGCC 2677 CGCAGCC 3614 hsa-miR-605 UAAAUCCCAOGGOGCCUD'COCCD' 804 ATGGGATTT 1741 TGGGATTT 2678 GGGATTT 3615 hsa-miR-606 AAACUACUGAAAAD'CAAAGAU 805 TCAGTAGTT 1742 CAGTAGTT 2679 AGTAGTT 3616 hsa-miR-607 GOOCAAAOCCAGAOCOAUAAC 806 GGATTTGAA 1743 GATTTGAA 2680 ATTTGAA 3617 hsa-miR-608 AGGGGUGGUGUUGGGACAGCUCCGU 807 CACCACCCC 1744 ACCACCCC 2681 CCACCCC 3618 hsa-miR-609 AGGGUGUUUCUCUCAUCUCU 808 GAAACACCC 1745 AAACACCC 2682 AACACCC 3619 hsa-miR-610 UGAGCUAAAUGUGUGCUGGGA 809 ATTTAGCTC 1746 TTTAGCTC 2683 TTAGCTC 3620 hsa-miR-611 GCGAGGACCCCDCGGGGOCOGAC 810 GGGTCCTCG 1747 GGTCCTCG 2684 GTCCTCG 3621 hsa-miR-612 GCUGGGCAGGGCUUCDGAGCUCCOO' 811 CCTGCCCAG 1748 CTGCCCAG 2685 TGCCCAG 3622 fasa~miR-613 AGGAAUGUOCCUUCUnOGCC 812 GAACATTCC 1749 AACATTCC 2686 ACATTCC 3623 hsa-miR-614 GAACGCCUGUnCUUGCCAGGUGG 813 ACAGGCGTT 1750 CAGGCGTT 2687 AGGCGTT 3624 fasa~miR~6i5~3p UCCGAGCCUGGGUCUCCCUCUU 814 CAGGCTCGG 1751 AGGCTCGG 2688 GGCTCGG 3625 hsa~miR-615~5p GGGGGUCCCCGGOGCUCGGADC 815 GGGGACCCC 1752 GGGACCCC 2689 GGACCCC 3626 hsa-miR-616 AGUCAUUGGAGGGUUUGAGCAG 816 TCCAATGAC 1753 CCAATGAC 2690 CAATGAC 3627 hsa-miR-616* ACUCAAAACCCUUCAGUGACUU 817 GGTTTTGAG 1754 GTTTTGAG 2691 TTTTGAG 3628 hsa-miR-617 AGACUUCCCAUUUGAAGGUGGC 818 TGGGAAGTC 1755 GGGAAGTC 2692 GGAAGTC 3629 hsa-miR-618 AAACDCDACOOGDCCOOCDGAGU 819 AGTAGAGTT 1756 GTAGAGTT 2693 TAGAGTT 3630 hsa-miR-619 GACCUGGACAUGUUUGUGCCCAGU 820 TGTCCAGGT 1757 GTCCAGGT 2694 TCCAGGT 3631 hsa-miR-620 AOGGAGAUAGADAOAGAAAO 821 CTATCTCCA 1758 TATCTCCA 2695 ATCTCCA 3632 hsa-miR-621 GGCUAGCAACAGCGCUUACCO 822 GTTGCTAGC 1759 TTGCTAGC 2696 TGCTAGC 3633 hsa-miR-622 ACAGOCUGCUGAGGDUGGAGC 823 AGCAGACTG 1760 GCAGACTG 2697 CAGACTG 3634 hsa-miR-623 AUCCCUUGCAGGGGCUGUUGGGU 824 TGCAAGGGA 1761 GCAAGGGA 2698 CAAGGGA 3635 hsa-miR-624 CACAAGGDAOUGGIJADUACCD 825 ATACCTTGT 1762 TACCTTGT 2699 ACCTTGT 3636 hsa-miR-624* UAGUACCAGOACCODGUGUUCA 826 ACTGGTACT 1763 CTGGTACT 2700 TGGTACT 3637 hsa-miR-625 AGGGGGAAAGUUCUAUAGOCC 827 CTTTCCCCC 1764 TTTCCCCC 2701 TTCCCCC 3638 hsa-miR-625* GACUAUAGAACUUUCCCCCUCA 828 TTCTATAGT 1765 TCTATAGT 2702 CTATAGT 3639 hsa-miR-626 AGCUGDCDGAAAADGOCOU 829 TCAGACAGC 1766 CAGACAGC 2703 AGACAGC 3640 hsa-miR-627 GDGAGDCD'CUAAGAAAAGAGGA 830 AGAGACTCA 1767 GAGACTCA 2704 AGACTCA 3641 hsa-miR-628~3p D'CDAGOAAGAGDGGCAGOCGA 831 TCTTACTAG 1768 CTTACTAG 2705 TTACTAG 3642 hsa-miR-628-5p AOGCOGACAOAOUOACOAGAGG 832 ATGTCAGCA 1769 TGTCAGCA 2706 GTCAGCA 3643 hsa-miR-629 DGGGIJ!JUACGUOGGGAGAACD 833 CGTAAACCC 1770 GTAAACCC 2707 TAAACCC 3644 hsa~miR~629* GUUCOCCCAACGDAAGCCCAGC 834 TTGGGAGAA 1771 TGGGAGAA 2708 GGGAGAA 3645 hsa-miR-630 AGDAOOCUGOA.CCAGGGAAGGD' 835 ACAGAATAC 1772 CAGAATAC 2709 AGAATAC 3646 hsa-miR-631 AGACCUGGCCCAGACCOCAGC 836 GGCCAGGTC 1773 GCCAGGTC 2710 CCAGGTC 3647 hsa-miR-632 GUGOCUGCOUCCOGUGGGA 837 AAGCAGACA 1774 AGCAGACA 2711 GCAGACA 3648 hsa-miR-633 COAADAGUADCDACCACAADAAA 838 ATACTATTA 1775 TACTATTA 2712 ACTATTA 3649 hsa-miR-634 AACCAGCACCCCAACOUDGGAC 839 GGTGCTGGT 1776 GTGCTGGT 2713 TGCTGGT 3650 hsa-mIR-635 ACUUGGGCACOGAAACAAOGUCC 840 GTGCCCAAG 1777 TGCCCAAG 2714 GCCCAAG 3651 hsa-miR-636 UGUGCUUGCUCGUCCCGCCCGCA 841 AGCAAGCAC 1778 GCAAGCAC 2715 CAAGCAC 3652 hsa-miR-637 ACUGGGGGCUUUCGGGCUCUGCGU 842 AGCCCCCAG 1779 GCCCCCAG 2716 CCCCCAG 3653 hsa-miR-638 AGGGAUCGCGGGCGGGUGGCGGCCU 843 CGCGATCCC 1780 GCGATCCC 2717 CGATCCC 3654 hsa-miR-639 AUCGCUGCGGUUGCGAGCGCUGU 844 CCGCAGCGA 1781 CGCAGCGA 2718 GCAGCGA 3655 hsa-miR-640 AOGAUCCAGGAACCUGCCOCD' 845 CCTGGATCA 1782 CTGGATCA 2719 TGGATCA 3656 hsa-miR-641 AAAGACAUAGGAUAGAGUCACCUC 846 CTATGTCTT 1783 TATGTCTT 2720 ATGTCTT 3657 hsa-miR-642 GUCCCUCUCCAAAUGUGUCUUG 847 GGAGAGGGA 1784 GAGAGGGA 2721 AGAGGGA 3658 hsa-miR-643 ACOUGUAUGCOAGCUCAGGIJAG 848 GCATACAAG 1785 CATACAAG 2722 ATACAAG 3659 hsa-miR-644 AGUGOGGCUUUCOUAGAGC 849 AAGCCACAC 1786 AGCCACAC 2723 GCCACAC 3660 hsa-miR-645 UCUAGGCUGGUACUGCUGA 850 CCAGCCTAG 1787 CAGCCTAG 2724 AGCCTAG 3661 hsa-mlR-646 AAGCAGCUGCCOCUGAGGC 851 GCAGCTGCT 1788 CAGCTGCT 2725 AGCTGCT 3662 hsa-miR-647 GUGGCOGCACUCACUUCCUUC 852 GTGCAGCCA 1789 TGCAGCCA 2726 GCAGCCA 3663 hsa-miR-648 AAGUGOGCAGGGCACUGGU 853 CTGCACACT 1790 TGCACACT 2727 GCACACT 3664 hsa-miR-649 AAACCUGUGUUGUUCAAGAGUC 854 ACACAGGTT 1791 CACAGGTT 2728 ACAGGTT 3665 hsa-miR-650 AGGAGGCAGCGCOCUCAGGAC 855 GCTGCCTCC 1792 CTGCCTCC 2729 TGCCTCC 3666 hsa-miR-651 UUUAGGAUAAGCUUGACUUUUG 856 TTATCCTAA 1793 TATCCTAA 2730 ATCCTAA 3667 hsa-miR-652 AAUGGCGCCACUAGGGODGUG 857 TGGCGCCAT 1794 GGCGCCAT 2731 GCGCCAT 3668 hsa-miR-653 GUGUUGAAACAAUCUCUACUG 858 GTTTCAACA 1795 TTTCAACA 2732 TTCAACA 3669 hsa~miR~654~3p UAUGUCUGCUGACCAUCACCUU 859 AGCAGACAT 1796 GCAGACAT 2733 CAGACAT 3670 hsa-miR-654 -5p UGGUGGGCCGCAGAACAUGUGC 860 CGGCCCACC 1797 GGCCCACC 2734 GCCCACC 3671 hsa-miR-655 AUAAUACAUGGUUAACCUCUUU 861 CATGTATTA 1798 ATGTATTA 2735 TGTATTA 3672 hsa-miR-656 AAUAUUAUACAGUCAACCUCU 862 GTATAATAT 1799 TATAATAT 2736 ATAATAT 3673 hsa-miR-657 GGCAGGUUCUCACCCUCUCUAGG 863 AGAACCTGC 1800 GAACCTGC 2737 AACCTGC 3674 fasa~miR~658 GGCGGAGGGAAGUAGGUCCGUUGGU 864 TCCCTCCGC 1801 CCCTCCGC 2738 CCTCCGC 3675 hea-miR-659 CUUGGUUCAGGGAGGGOCCCCA 865 CTGAACCAA 1802 TGAACCAA 2739 GAACCAA 3676 hsa-miR-660 OACCCAUUGCAOAUCGGAGUOG 866 GCAATGGGT 1803 CAATGGGT 2740 AATGGGT 3677 hsa-miR-661 UGCCUGGGUCUCUGGCCUGCGCGU 867 GACCCAGGC 1804 ACCCAGGC 2741 CCCAGGC 3678 hsa-miR-662 UCCCACGUUGUGGCCCAGCAG 868 CAACGTGGG 1805 AACGTGGG 2742 ACGTGGG 3679 hsa~miR~663 AGGCGGGGCGCCGCGGGACCGC 869 CGCCCCGCC 1806 GCCCCGCC 2743 CCCCGCC 3680 hsa-miR~S63b GGUGGCCCGGCCGUGCCOGAGG 870 CCGGGCCAC 1807 CGGGCCAC 2744 GGGCCAC 3681 hsa-miR-664 UAUUCAUUUAUCCCCAGCCUACA 871 TAAATGAAT 1808 AAATGAAT 2745 AATGAAT 3682 hsa-miR-664* ACDGGCDAGGGAAAAUGAOUGGAU 872 CCTAGCCAG 1809 CTAGCCAG 2746 TAGCCAG 3683 fasa-miR-665 ACCAGGAGGCUGAGGCCCCU 873 GCCTCCTGG 1810 CCTCCTGG 2747 CTCCTGG 3684 hsa-miR-668 UGUCACUCGGCOCGGCCCACOAC 874 CCGAGTGAC 1811 CGAGTGAC 2748 GAGTGAC 3685 hsa-miR-671-3p OCCGGUUCOCAGGGCOCCACC 87S 6AGAACCGG 1812 AGAACCGG 2749 GAACCGG 3686 hsa~miR~671-5p AGGAAGCCCOGGAGGGGCOGGAG 876 AGGGCTTCC 1813 GGGCTTCC 2750 GGCTTCC 3687 fasa~miR~675 UGGOGCGGAGAGGGCCCACAGOG 877 CTCCGCACC 1814 TCCGCACC 2751 CCGCACC 3688 fasa~miR-675b CUGUAOGCCCO'CACCGCaCA 878 GGGCATACA 1815 GGCATACA 2752 GCATACA 3689 hsa-tniR-7 UGGAAGACUAGDGAOUUOGOIJGD 879 TAGTCTTCC 1816 AGTCTTCC 2753 GTCTTCC 3690 hsa~miR-7-l* CAACAAAUCACAGOCOGCCAOA 880 TGATTTGTT 1817 GATTTGTT 2754 ATTTGTT 3691 fasa-miR~7~2* CAACAAAOCCCAGOCOACCOAA 881 GGATTTGTT 1818 GATTTGTT 2755 ATTTGTT 3692 hsa-miR-708 AAGGAGCUOACAAOCOAGCOGGG 882 TAAGCTCCT 1819 AAGCTCCT 2756 AGCTCCT 3693 hsa-miR-708* CAACOAGACOGOGAGCOOCOAG 883 AGTCTAGTT 1820 GTCTAGTT 2757 TCTAGTT 3694 hsa-miR-720 aCOCGCUGGGGCCUCCA 884 CCCAGCGAG 1821 CCAGCGAG 2758 CAGCGAG 3S95 hsa-miR-744 OGCGGGGCOAGGGCOAACAGCA 885 TAGCCCCGC 1822 AGCCCCGC 2759 GCCCCGC 3696 hsa-miR-744* COGOUGCCACOAACCaCAACCU 886 GTGGCAACA 1823 TGGCAACA 2760 GGCAACA 3697 hsa-miR-758 OUDGOGACCUGGD'CCACaAACC 887 AGGTCACAA 1824 GGTCACAA 2761 GTCACAA. 3698 hsa-miR-760 CGGCDCUGGGDCDGDGGGGA 888 CCCAGAGCC 1825 CCAGAGCC 2762 CAGAGCC 3699 hsa-miR-765 OGGAGGAGAAGGAAGGOGAOG 889 TTCTCCTCC 1826 TCTCCTCC 2763 CTCCTCC 3700 hsa-miR-766 ACUCCAGCCCCACAGCCUCAGC 890 GGGCTGGAG 1827 GGCTGGAG 2764 GCTGGAG 3701 hsa-miR-767~3p UCUGCUCAUACCCCAUGGUUUCU 891 TATGAGCAG 1828 ATGAGCAG 2765 TGAGCAG 3702 fasa-miR-767~5p OGCACCAOGGOOGOCUGAGCAUG 892 CCATGGTGC 1829 CATGGTGC 2766 ATGGTGC 3703 hsa-miR-769~3p COGGGAOCOCCGGGGOCOUGGUO 893 GAGATCCCA 1830 A6ATCCCA 2767 GATCCCA 3704 fasa-miR-769-5p UGAGACCnCOGGGOUCUGAGCO 894 AGAGGTCTC 1831 GAGGTCTC 2768 AGGTCTC 3705 hsa-miR-77Q-5p OCCAGOACCACGOGOCAGGGCCA 895 TGGTACTGG 1832 GGTACTGG 2769 GTACTGG 3706 hsa-miR-802 CAGOAACAAAGAUO'CADCCODGO' 896 TTTGTTACT 1833 TTGTTACT 2770 TGTTACT 3707 hsa-miR-873 GCAGGAACUUGUGAGUCUCCU 897 AAGTTCCTG 1834 AGTTCCTG 2771 GTTCCTG 3708 hsa-miR-874 COGCCCUGGCCCGAGGGACCGA 898 GCCAGGGCA 1835 CCAGGGCA 2772 CAGGGCA 3709 hsa-miR-875~3p CCOGGAAA.CACDGAGGOOGDG 899 TGTTTCCAG 1836 GTTTCCAG 2773 TTTCCAG 3710 hsa-miR~875~5p OAUACCUCAGUUUUAOCAGGUG 900 CTGAGGTAT 1837 TGAGGTAT 2774 GAGGTAT 3711 hsa-miR-876-3p UGGUGGUUUACAAAGUAAUUCA 901 TAAACCACC 1838 AAACCACC 2775 AACCACC 3712 hsa-miR-876~5p UGGAUUUCUUUGUGAAUCACCA 902 AAGAAATCC 1839 AGAAATCC 2776 GAAATCC 3713 fasa-miR-877 GUAGAGGAGAOGGCGCAGGG 903 TCTCCTCTA 1840 CTCCTCTA 2777 TCCTCTA 3714 fasa-miR-877* OCCOCUUCUCCCOCCOCCCAG 904 GAGAAGAGG 1841 AGAAGAGG 2778 GAAGAGG 3715 hsa-miR~885-3p AGGCAGCGGGGOGDAGOGGAUA 905 CCCGCTGCC 1842 CCGCTGCC 2779 CGCTGCC 3716 hsa~miR-885-5p UCCAUUACACOACCCOGCCOCO 906 GTGTAATGG 1843 TGTAATGG 2780 GTAATGG 3717 bsa-miR-886-3p CGCGGGOGCUOACaGACCCUU 907 AGCACCCGC 1844 GCACCCGC 2781 CACCCGC 3718 hsa~miR-886~5p CGGGUCGGAGUUAGCD'CAAGCGG 908 CTCCGACCC 1845 TCCGACCC 2782 CCGACCC 3719 hsa-miR-887 GOGAACGGGCGCCAO'CCCGAGG 909 GCCCGTTCA 1846 CCCGTTCA 2783 CCGTTCA 3720 bsa-miR-888 OACO'CAAAAAGCOGOCAGUCA 910 TTTTTGAGT 1847 TTTTGAGT 2784 TTTGAGT 3721 hsa-miR-888* GACUGACACCUCUUUGGGUGAA 911 GGTGTCAGT 1848 GTGTCAGT 2785 TGTCAGT 3722 lisa-miR-883 UOAAOAUCGGACAACCAUOGO 912 CCGATATTA 1849 CGATATTA 2786 GATATTA 3723 hsa-miR-890 DACDUGGAAAGGCAOCAGDDG 913 TTTCCAAGT 1850 TTCCAAGT 2787 TCCAAGT 3724 fasa~miR-891a UGCAACGAACCUGAGCCACUGA 914 GTTCGTTGC 1851 TTCGTTGC 2788 TCGTTGC 3725 hsa-miR-891b aGCAACOUACCOGAGOCAUOGA 915 GTAAGTTGC 1852 TAAGTTGC 2789 AAGTTGC 3726 hsa-miR-892a CACOGOGUCCUUUCOGCGOAG 916 GGACACAGT 1853 GACACAGT 2790 ACACAGT 3727 hsa~miR-892b CACUGGCUCCUUUCUGGGUAGA 917 GGAGCCAGT 1854 GAGCCAGT 2791 AGCCAGT 3728 hsa-tniR-9 UCUUUGGUUAUCUAGCUGUAUGA 918 TAACCAAAG 1855 AACCAAAG 2792 ACCAAAG 3729 hsa-miR-9* AUAAAGCOAGAOAACCGAAAGU 919 CTAGCTTTA 1856 TAGCTTTA 2793 AGCTTTA 3730 hsa-miR-920 GGGGAGCUGUGGAAGCAGUA 920 ACAGCTCCC 1857 CAGCTCCC 2794 AGCTCCC 3731 hsa-miR-921 CUAGUGAGGGACAGAACCAGGAUUC 921 CCCTCACTA 1858 CCTCACTA 2795 CTCACTA 3732 hsa-miR-922 GCAGCAGAGAAUAGGACOACGOC 922 TCTCTGCTG 1859 CTCTGCTG 2796 TCTGCTG 3733 hsa-miR-923 GUCAGCGGAGGAAAAGAAACO' 923 CTCCGCTGA 1860 TCCGCTGA 2797 CCGCTGA 3734

The above 7 - 9mer compounds (or oligomer regions) may be fully LNA modified, fully phosphorothioate, and LNA cytosine may be 5-methyl cytosine. The LNA may in some emboidments be beta-D-oxy LNA or for example, (S)cET.

In an independent embodiment, the invention provides for an oligomer comprising 7 - 12, such as 7 - 10, such as 7, 8 or 9 consecutive LNA nucleosides (see Table 2 for examples), conjugated to a GalNAc conjugate, such as a trivalent GalNAc conjugate, such as a GalNAc conjugate selected from the group consisting of any one of Conj 1 , 2, 3, 4, 1a, 2a, 3a, 4a, such as conj2a. Such oligomers may, in some emboidments be one of the oligomer regions as described herein. The above Table 2 or 7 - 12mer compounds (or oligomer regions) may be fully LNA modified, fully phosphorothioate, and LNA cytosine may be 5-methyl cytosine. The LNA may in some embodiments be beta-D-oxy LNA or for example, (S)cET. Beta-Doxy LNA is a preferred nucleoside. The 7 - 12 mer compounds may, for example, be capable of inhibiting a microRNA in a ceil expressing said microRNA.

In an independent embodiment, the invention provides for an oligomer comprising one (or more, such as to or three) of the compounds or table 2, and a GalNAc conjugate, such as a trivalent GalNAc conjugate such as a GalNAc conjugate selected from the group consisting of any one of Conjl , 2, 3, 4, 1a, 2a, 3a, 4a, such as conj2a.

EMBODIMENTS

The following aspects of the invention mya be combined with other aspects herein described,

1. An oligomeric compound comprising i) a first region of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; ii) a second region of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides; wherein the first and the second regions are covalently linked via iii) a region of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA or RNA nucleosides.

2, The oligomeric compound according to embodiment 1 , wherein the first region (i) and second region (ii) are positioned 5^* and 3^* respectively to the region of physiologically labile nucleotides (iii),

3. The oligomeric compound according to embodiment 1 or 2, wherein the first region or second region are covalently linked to a functional group (iv), such as a conjugate moiety.

4, The oligomeric compound according to embodiment 2 or 3, wherein the functional group is covalently linked to the first or the second region via a bio-cleavable linker (v). 5, The oligomeric compound according to embodiment 4, wherein the bio cleavable linker (v) comprises a region of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked nucleotides, such as DNA [or RNA] nucleosides,

8, The oligomeric compound according to any one of embodiments 3 - 5, wherein the conjugate moiety comprises a sterol, for example cholesterol, or a carbohydrate, such as GalNac/GalNac cluster.

7. The oligomeric compound complex according to any one of embodiments 3 - 5,

wherein the conjugate moiety comprises a lipophilic group (e.g. a lipid, a fatty acid, a sterol), a protein, a peptide, an antibody or fragment thereof, a polymer, a reporter group, a dye, a receptor ligand, a small molecule drug, a prodrug, or a vitamin.

8. The oligomeric compound according to any one of embodiments 1 - 7 wherein the contiguous sequence of nucleobases of the first and the second region are identical.

9. The oligomeric compound according to any one of embodiments 1 - 7 wherein the contiguous sequence of nucleobases of the first and the second region are different. 10. The oligomeric compound according to any one of embodiments 1 - 9 wherein the first and the second regions are targeted to the same nucleic acid target.

11. The oligomeric compound according to any one of embodiments 1 - 10 wherein the first and the second regions each comprise at least 1 LNA nucleoside.

12. The oligomeric compound according to any one of embodiments 1 - 1 1 wherein the first and/or second regions are gapmer oligomers, such as LNA gapmers.

13. The oligomeric compound according to any one of embodiments 1 - 12 wherein the first and/or second region target mRNA targets.

14. The oligomeric compound according to any one of embodiments 1 - 13 wherein the first and/or second regions target microRNA targets.

15. The oligomeric compound according to any one of embodiments 1 - 14 wherein the first and/or second regions are mixmer or totalmer oligomers.

18. The oligomeric compound according to any one of embodiments 1 - 15 wherein the first and second regions are 10 - 16 nucleotides in length.

17. The oligomeric compound according to any one of embodiments 1 - 15 wherein the first and second regions are 8 - 10 nucleotides in length.

18. The oligomeric compound according to any one of embodiments 1 - 16 wherein the first region is complementary to a [human] ApoB-100 mRNA nucleotide sequence and/or wherein the second region is complementary to a [human] mtGPAT mRNA nucleotide sequence.

19. The oligomeric compound according to any one of embodiments 1 - 18, wherein the first sequence is positioned 5' of the second sequence. 20. The oligomeric compound according to any one of embodiments 1 - 18, wherein the first sequence is positioned 3' of the second sequence.

21. A pharmaceutical composition comprising the oligomeric compound of any one of the preceding embodiments, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

22. The oligomeric compound according to any one of the preceding embodiments for use in the inhibition of a nucleic acid target in a cell.

23. The oligomeric compound according to any one of the preceding embodiments for use in medicine.

24. The oligomeric compound according to any one of the preceding embodiments for use in the treatment of a medical disease or disorder.

25. The use of the oligomeric compound according to any one of the preceding

embodiments for the preparation of a medicament for the treatment of a disease or disorder, such as a metabolic disease or disorder.

26. A method of treatment of a disease or disorder in a subject in need of treatment, said method comprising the steps of administering a pharmaceutical composition comprising the oligomeric compound of any one of the preceding embodiments to said subject in a therapeutically effective amount,

27. A method of inhibiting the expression of a target gene in a cell, said method

comprising administering the oligomeric compound according to any one of the preceding embodiments to a cell which is expressing said target gene, suitably in an amount effective to reduce the expression of the target gene in said cell.

EXAMPLES

Oligonucleotide List

In the following list, Capital letters represent LNA nucleosides, such as beta-D-oxy LNA, lower case letters represent DNA nucleosides. Capital L is a LNA, such as beta-D-oxy, and lower case d is a DNA nucleoside. LNA cytosines are optionally 5'methyl cytosine. The internucleosides within region A are phosphorothioate, and within region B are

phosphodiester (as shown). The internucleoside linkage between region A and B is phoshodiester, but where region B is >1 DNA nucleotide, may optionally be other than phosphodiester (e.g. may be phosphorothioate). There is, optionally a further linker (Y), between region B and region C, such as a C6 linker. # refers to SEQ ID No. ApoB Targeting Compounds with FAM label conjugates

#c Seq (5'-3') Cleavable linker Conjugate SEQ ID NO

Examples 1 - 5 amd 7 - 1 1 - see PCT/EP2013/073858 Examples 1 - 1 1 .

Example 6. In vitro cleavage of different DNA/PO-linkers

FAM-labeled ASOs with different DNA PO-linkers (PO linkers) were subjected to in vitro cleavage either in S1 nuclease extract (Fig. 6A), Liver or kidney homogenates or Serum FAM-labeled ASOs 100 μΜ with different DNA/PO-linkers were subjected to in vitro cleavage by S1 nuclease in nuclease buffer (60 U pr. 100 pL) for 20 and 120 minutes (A). The enzymatic activity was stopped by adding EDTA to the buffer solution. The solutions were then subjected to AIE HPLC analyses on a Dionex Ultimate 3000 using an Dionex DNApac p-100 column and a gradient ranging from 10mM - 1 M sodium perchlorate at pH 7.5. The content of cleaved and non cleaved oligonucleotide were determinded against a standard using both a fluoresense detector at 615 nm and a uv detector at 260 nm.

Conclusion: The PO linkers (or region B as referred to herein) results in the conjugate (or group C) being cleaved off, and both the length and/or the sequence composition of the linker can be used tomodulate susceptibility to nucleolytic cleavage of region B. The

Sequence of DNA/PO-linkers can modulate the cleavage rate as seen after 20 min in Nuclease S1 extract Sequence selection for region B (e.g. for the DNA PO-linker) can therefore also be used to modulate the level of cleavage in serum and in cells of target tissues.

Liver, kidney and Serum (B) were spiked with oligonucleotide NO 16 to concentrations of 200 pg/g tissue. Liver and kidney samples collected from NMRI mice were homogenized in a homogenisation buffer (0,5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCI, pH 8.0 (adjusted with 1 N NaOH). The homogenates were incubated for 24 hours at 37° and thereafter the homogenates were extracted with phenol - chloroform. The content of cleaved and non cleaved oligonucleotide in the extract from liver and kidney and from the serum were determinded against a standard using the above HPLC method.

Conclusion: The PO linkers (or region B as referred to herein) results in cleavage of the conjugate (or group C) from the oligonucleotide, in liver or kidney homogenate, but not in serum.

Note: cleavage in the above assays refers to the cleavage of the cleavable linker, the oligomer or region A should remain functionally intact. The susceptibility to cleavage in the above assays can be used to determine whether a linker is biocleavable or physiologically labile.

Example 12: LNA antimiRs GalNac Poly-Oligo Conjugates Compounds

Capital letters are LNA, such as beta-D-oxy LNA. Lower case letters are DNA. Subscript s is a phosphorothioate linkage. Other internucleoside linkages are phosphodiester

(phosphate) linkages. Superscript m before a C represents LNA 5-methyl cytosine. In some embodiments, the compounds may also be made with LNA cytosine. In some embodiments, the Conjla group may be another GalNAc conjugate group, such as those disclosed herein, for example Conj2a. miR-21 (Tiny) 5'- ^mC₈A«^mC«A.^mCT_e ^mC_a ^mC -3' (Comp NO 51 )

GalNAc-tiny 5 - Conjla ^mCA^mCA^mCJ,^mC_s ^mC -3' (Comp NO 52)

tiny-linker-tiny5'- ^mCA^mCA^mCJ_s ^mC₈ ^mCca^mCA^mCA^mCJ₈ ^mC.^mC -3' (Comp NO 53) SEQ ID No 26

GalNac-tiny-linker-tiny 5 - Conj 1 a ^mCA^mCA^mCJ_g ^mC_g ^mCca^mCA^mCA^mC,T₈ ^mC_s ^mC -3' (Comp NO 54) SEQ ID NO 27

An in vivo mouse study was performed using a total of 9 groups of mice (n=5). Each mouse was dosed i.v. on days 0, 2 and 4 with either 0,5 mg/kg or 2 mg/kg or equimolar doses of the GalNAc conjugated LNA's compared to parent LNA compound and equimolar active compound after cleavage of the double 8mer compared to the single 8mer compound. A saline control group was included (see study set up below). Serum samples were taken 4 days pre administration, interim at day 4 and at endpoint day 7. Liver and kidney samples were stored in RNA later. Validation of miR122 knock down of miR122 was done as described in Obad Nat Genet. 201 1 Mar 20:43(4):371 -8 (Fig.17). The cholesterol level in serum were analyzed as described in Elmen J, et al. LNA-mediated microRNA silencing in non-human primates. Nature. 2008;452:896-899. (Fig.18) and mRNA levels of two miR122 down regulated genes (Aldo A and Bckdk) were analyzed using standard QPCR assay techniques (Fig.19). ALT was measured to assess tolerability of the compounds (Fig.20).

Study Set-up

Conclusions: Conjugation of GalNAc to anti-miR122 (Comp ID 52 and 54) showed a remarkable improvement of miR122 knock down in the liver indicated by decreased total cholesterol levels (Fig.2) and up regulation of Aldo A and Bckdk mRNA already in the low dose group (Fig.3, 3 x 0,5 mg/kg). No effect of the anti- miR122 oligonucleotide was seen in the kidney. A very slight increase in ALT was measured for comp ID 52 which showed a tendency to improve by conjugation of 2 oligonucleotides to one GalNAc (comp ID 54). No major difference could be observed in activity of the GalNAc conjugated single Tiny LNA compound compared to the poly-oligo compound comp ID 52 compared to 54, FIG 18 and 19) which might be due to overdosing. The unconjugated Tiny LNA and poly-oligo compound showed no activity due to the low dose range. To allow better comparison of the single and poly-oligo concept a dose range finding study was conducted (Example 13).

Example 13: LNA antimiRs GalNac Poly-Oligo Conjugates- Dose range finding Compounds: as in Example 12.

An in vivo mouse study was performed using a total of 11 groups of mice (n=5). Each mouse was dosed i.v. on day 0 with either 0,05 mg/kg, 0,1 mg/kg, 0,2 mg/kg or 0,5 mg/kg of the GalNAc conjugated Tiny LNA (comp ID 52) or equimolar active compound after cleavage of the poly-oiigo compound (comp ID 54). For comparison two groups were dosed with 20 mg/kg of the unconjugated parent Tiny LNA (comp ID 51 ) and equimolar active compound after cleavage of the poly-oligo compound (comp ID 53). A saline control group was included (see study set up).

Samples were taken and analyzed as described in Example 12. mRNA levels of two miR122 down regulated genes (Aldo A and Bckdk) were analyzed using standard QPCR assay techniques (Fig. 21 ). ALT measurement showed no significant rise in ALT compared over compounds. Study Set-up

Conclusions: Activity of the GalNAc conjugated compounds, measured as increase of the 2 marker genes ALDO A and BCKDK, showed dose dependency and comparable activity for the Tiny LNA and the poly-oligonucleotide compound. The unconjugated parent compounds (single Tiny and poly-oligonucleotide) also showed comparable activity dosed at 20 mg/kg (Figure 21 ). The poly-oligonucleotide concept therefore offers improved cost of goods at same activity. It is likely that a poly-oligonucleotide approach could be beneficial when using a conjugate binding to a receptor class with limited expression or slower turnover than the Asialoglycoprotein-Receptor or when using a conjugate or receptor with impact on tolerability. Example 14: GalNAc conjugated LNA antisense oligonucleotides

Comp ID NO 55 is shown in Figure 10, and has the structure: Trivalent GalNAc -5'- G^T^L _sT_sG_sA_sCAC_sT_sG_sT^L _s ^MeC^L _sC₀A₀A^L _sT^L _sT^L _sC_sC_sC₈T_sG_sC_sC_sT_sG^L _sT^L _sG^L-3^* (SEQ ID NO 28). In this example GalNAd conjuagte was used, but other conjugates as described herein may be used. Note a capital L superscript after a capital A, T, C or G represents a LNA nucleoside, otherwise nucleosides are DNA, subscript s is a phosphorothioate linkage, and a subscript O is a phosphodiester linkage. An in vivo mouse study was performed using a total of 3 groups of mice (n=5, C57/bl). Each mouse was dosed i.v. on day 0 with either 0.25 mg/kg, or 0.5 mg/kg of the GalNAc conjugated poly LNA (Seq ID 55). A saline control group was included (see study set up in Table below). Liver and kidney samples were stored in RNA later. mRNA levels of the two targeted genes (ApoB and mtGPAT) were analyzed using standard QPCR assay techniques (Fig. 11 ).

Study Set-up:

Conclusions: GalNAc-poly LNA compound induced a down regulation of both targeted mRNA (ApoB and mtGPAT). Moreover, a dose dependency was found. The poly- oligonucleotide concept therefore offers the possibility to attach multiple LNA's to one conjugate linked together via a cleavable linker and have activity on intended targets from all the LNA parent compounds linked together in the original construct. It is likely that a poly- oligonucleotide approach could be beneficial when using a conjugate binding to a receptor class with limited expression or slower turnover than the Asialoglycoprotein-Receptor or when using a conjugate or receptor with impact on tolerability.

Claims

1. An oligomeric compound comprising

a. a first region of a contiguous sequence of 7 - 26 phosphorothioate linked

nucleosides (A);

b. a second region of a contiguous sequence of 7 - 26 phosphorothioate linked nucleosides (A);

c. a region of 1 - 5 physiologically labile nucleotides (B) which covalently links region A and A'; and

d. a functional group, such as a conjugate moiety (functional group (C) covalently linked to region A or A' via a biocleavable linker (Β'), such as a biocleavable linker comprising either a region of 1 - 5 physiologically labile nucleotides or a peptide linker, e.g. a lysine linker.

The oligomeric compound according to claim 1, wherein the first region (i) and second region (ii) are positioned 5' and 3' respectively to the region of physiologically labile nucleotides (iii).

The oligomeric compound according to any one of claims 1 or 2, wherein the conjugate moiety comprises a sterol, for example cholesterol, or a carbohydrate, such as GalNAc/GalNAc cluster.

The oligomeric compound complex according to any one of claims 1 - 3, wherein the conjugate moiety comprises a lipophilic group (e.g. a lipid, a fatty acid, a sterol), a protein, a peptide, an antibody or fragment thereof, a polymer, a reporter group, a dye, a receptor ligand, a small molecule drug, a prodrug, or a vitamin.

The oligomeric compound according to any one of claims 1 - 4, wherein the functional group (C) is covalently linked to region A or A' via a region of 1 - 5 physiologically labile nucleotides, such as 1 - 5 phosphodiester linked DNA nucleotides.

The oligomeric compound according to any one of claims 1 - 4, wherein the functional group (C) is covalently linked to region A or A' via a peptide region, such as a lysine linker.

The oligomeric compound according to any one of claims 1 - 6, wherein the functional group (C) is a carbohydrate conjugate such as a GaiNAc conjugate such as a trivalent GaiNAc conjugate.

The oligomeric compound according to any one of claims 1 - 6, wherein the functional group (C) is a lipophillic conjugate such as a sterol, such a a cholesterol conjugate. The oligomeric compound according to any one of claims 1 - 8 wherein the contiguous sequence of nucleobases of the first and the second region are identical.

10. The oligomeric compound according to any one of claims 1 - 8 wherein the contiguous sequence of nucleobases of the first and the second region are different.

11. The oligomeric compound according to any one of claims 1 - 10 wherein the first and the second regions are targeted to the same nucleic acid target.

12. The oligomeric compound according to any one of claims 1 - 10 wherein the first and the second regions are targeted to different nucleic acid targets,

13. The oligomeric compound according to any one of claims 1 - 12 wherein the first and the second regions each comprise at least 1 LNA nucleoside.

14. The oligomeric compound according to any one of claims 1 - 13 wherein the first and/or second regions are gapmer oligomers, such as LNA gapmers.

15. The oligomeric compound according to any one of claims 1 - 14 wherein the first and/or second region target mRNA targets,

16. The oligomeric compound according to any one of claims 1 - 15 wherein the first and/or second regions target microRNA targets,

17. The oligomeric compound according to any one of claims 1 - 16 wherein the first and/or second regions are mixmer or totalmer oligomers.

18. The oligomeric compound according to any one of claims 1 - 17 wherein the first and second regions are 10 - 16 nucleotides in length.

19. The oligomeric compound according to any one of claims 1 - 18 wherein the first and second regions are 8 - 10 nucleotides in length.

20. The oligomeric compound according to any one of claims 1 - 19, wherein at least one or both of the first and second regions target a microRNA selected from the group consisting of miR ID NO 40 - 976.

21. The oligomeric compound according to any one of claims 1 - 20 wherein the first region is complementary to a [e.g. human] ApoB-100 mRNA nucleotide sequence and/or wherein the second region is complementary to a [e.g. human] mtGPAT mRNA nucleotide sequence.

22. The oligomeric compound according to any one of claims 1 - 21 , wherein the first sequence is positioned 5' of the second sequence,

23. The oligomeric compound according to any one of claims 1 - 22, wherein the first sequence is positioned 3' of the second sequence.

24. A pharmaceutical composition comprising the oligomeric compound of any one of the preceding claims, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

25. The oligomeric compound according to any one of the preceding claims for use in the inhibition of a nucleic acid target in a cell.

26. The oligomeric compound according to any one of the preceding claims for use in the inhibition of two different nucleic acid targets in a cell,

27. The oligomeric compound according to any one of the preceding claims for use in medicine.

28. The oligomeric compound according to any one of the preceding claims for use in the treatment of a medical disease or disorder.

29. The use of the oligomeric compound according to any one of the preceding claims for the preparation of a medicament for the treatment of a disease or disorder, such as a metabolic disease or disorder.

30. A method of treatment of a disease or disorder in a subject in need of treatment, said method comprising the steps of administering a pharmaceutical composition comprising the oligomeric compound of any one of the preceding claims to said subject in a therapeutically effective amount.

31. A method of inhibiting the expression of a target gene in a cell, said method

comprising administering the oligomeric compound according to any one of the preceding claims to a ceil which is expressing said target gene, suitably in an amount effective to reduce the expression of the target gene in said cell.