US20150291958A1 - Anti apob antisense conjugate compounds - Google Patents

Anti apob antisense conjugate compounds Download PDF

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US20150291958A1
US20150291958A1 US14/443,369 US201314443369A US2015291958A1 US 20150291958 A1 US20150291958 A1 US 20150291958A1 US 201314443369 A US201314443369 A US 201314443369A US 2015291958 A1 US2015291958 A1 US 2015291958A1
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region
conjugate
group
oligomer
lna
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Nanna Albaek
Henrik Frydenlund Hansen
Susanne Kammler
Jacob Ravn
Henrik Orum
Mark Turner
Marie Lindholm
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Roche Innovation Center Copenhagen AS
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Roche Innovation Center Copenhagen AS
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Definitions

  • the present invention relates to conjugates of LNA antisense oligonucleotides (oligomers) that target ApoB.
  • SPC3833 and SPC4955 are two LNA compounds which have been previously identified as potent compounds which target human ApoB mRNA.
  • WO2007/146511 reports on short bicyclic (LNA) gapmer antisense oligonucleotides which apparently are more potent and less toxic than longer compounds.
  • the exemplified compounds appear to be 14 nts in length.
  • EP 1 984 381 B1 Seth et al., Nucleic Acids Symposium Series 2008 No. 52 553-554 and Swayze et al., Nucleic Acid Research 2007, vol 35, pp 687-700, LNA oligonucleotides cause significant hepatotoxicity in animals. According to WO2007/146511, the toxicity of LNA oligonucleotides may be avoided by using LNA gapmers as short as 12-14 nucleotides in length. EP 1 984 381 B1 recommends using 6′ substituted bicyclic nucleotides to decrease the hepatotoxicity potential of LNA oligonucleotides. According to Hagedorn et al., Nucleic Acid Therapeutics 2013, the hepatotoxic potential of antisense oligonucleotide may be predicted from their sequence and modification pattern.
  • Oligonucleotide conjugates have been extensively evaluated for use in siRNAs, where they are considered essential in order to obtain sufficient in vivo potency.
  • WO2004/044141 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.
  • WO2012/083046 reports on a galactose cluster-pharmacokinetic modulator targeting moiety for siRNAs.
  • 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.
  • 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 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.
  • the invention provides for an antisense oligonucleotide conjugate (the compound of the invention) comprising a first region of an oligomer (region A—such as an LNA oligomer, a gapmer oligomer or an LNA gapmer oligomer), targeting an ApoB nucleic acid, covalently joined to a further region (region C) comprising a conjugate moiety selected from the group consisting of an asialoglycoprotein receptor targeting conjugate and a lipophilic conjugate, wherein the lipophilic conjugate, and optionally the asialoglycoprotein receptor targeting conjugate, is joined to the LNA oligomer via biocleavable linker.
  • region A such as an LNA oligomer, a gapmer oligomer or an LNA gapmer oligomer
  • region C comprising a conjugate moiety selected from the group consisting of an asialoglycoprotein receptor targeting conjugate and a lipophilic conjugate, wherein the lipophilic conjugate, and optionally
  • the invention provides for a conjugate comprising an LNA antisense oligomer (the compound of the invention) targeting to a ApoB nucleic acid (A) and at least one non-nucleotide or non-polynucleotide moiety (C) covalently attached to said oligomer (A), wherein the non-polynucleotide moiety is selected from the group consisting of an asialoglycoprotein receptor targeting conjugate and a lipophilic conjugate, wherein the lipophilic conjugate, and optionally the asialoglycoprotein receptor targeting conjugate, is covalently joined to the LNA antisense oligomer via a biocleavable linker (region B)
  • the invention provides for an oligomeric compound (the compound of the invention), which targets an ApoB nucleic acid target, which comprises three regions:
  • region A and region B form a single contiguous nucleotide sequence of 8-35 nucleotides in length.
  • the internucleoside linkage between the first and second regions may be considered part of the second region.
  • a phosphorus containing linkage group between the second and third region.
  • the phosphorus linkage group may, for example, be a phosphate (phosphodiester), a phosphorothioate, a phosphorodithioate or a boranophosphate group.
  • this phosphorus containing linkage group is positioned between the second region and a linker region which is attached to the third region.
  • the phosphate group is a phosphodiester.
  • the oligomeric compound comprises at least two phosphodiester groups, wherein at least one is as according to the above statement of invention, and the other is positioned between the second and third regions, optionally between a linker group and the second region.
  • the third region is an activation group, such as an activation group for use in conjugation.
  • the invention also provides activated oligomers comprising region A and B and a activation group, e.g an intermediate which is suitable for subsequent linking to the third region, such as suitable for conjugation.
  • the third region is a reactive group, such as a reactive group for use in conjugation.
  • the invention also provides oligomers comprising region A and B and a reactive group, e.g an intermediate which is suitable for subsequent linking to the third region, such as suitable for conjugation.
  • the reactive group may, in some embodiments comprise an amine of alcohol group, such as an amine group.
  • region A comprises at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 internucleoside linkages other than phosphodiester, such as internucleoside linkages which are (optionally independently] selected from the group consisting of phosphorothioate, phosphorodithioate, and boranophosphate, and methylphosphonate, such as phosphorothioate.
  • region A comprises at least one phosphorothioate linkage.
  • At least 50%, such as at least 75%, such as at least 90% of the internucleoside linkages, such as all the internucleoside linkages within region A are other than phosphodiester, for example are phosphorothioate linkages. In some embodiments, all the internucleoside linkages in region A are other than phosphodiester.
  • the oligomeric compound comprises an antisense oligonucleotide, such as an antisense oligonucleotide conjugate.
  • the antisense oligonucleotide may be or may comprise the first region, and optionally the second region.
  • region B may form part of a contiguous nucleobase sequence which is complementary to the (nucleic acid) target. In other embodiments, region B may lack complementarity to the target.
  • the invention provides a non-phosphodiester linked, such as a phosphorothioate linked, oligonucleotide (e.g. an antisense oligonucleotide) which has at least one terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacent nucleoside of the oligonucleotide via a phosphodiester linkage, wherein the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting moiety or a blocking moiety, optionally via a linker moiety.
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting
  • the invention provides for pharmaceutical composition
  • a pharmaceutically acceptable diluent, carrier, salt or adjuvant comprising the compound of the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
  • the invention provides for the compound or pharmaceutical composition of the invention, for use as a medicament, such as for the treatment of acute coronary syndrome, or hypercholesterolemia or related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).
  • FCHL familial hyperlipidemia
  • CAD coronary artery disease
  • CHD coronary heart disease
  • the invention provides for the use of the compound or pharmaceutical composition of the invention, for the manufacture of a medicament for the treatment of acute coronary syndrome, or hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting of atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).
  • FCHL familial hyperlipidemia
  • CAD coronary artery disease
  • CHD coronary heart disease
  • the invention provides for a method of treating acute coronary syndrome, or hypercholesterolemia or a related disorder, such as a disorder selected from the group consisting atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD), said method comprising administering an effective amount of the compound or pharmaceutical composition according to the invention, to a patient suffering from, or likely to suffer from hypercholesterolemia or a related disorder.
  • a disorder selected from the group consisting atherosclerosis, hyperlipidemia, hypercholesterolemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD) said method comprising administering an effective amount of the compound or
  • 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 the compound of the invention to said cell so as to inhibit ApoB in said cell.
  • the invention provides for the compound of the invention for use in medicine, such as for use as a medicament.
  • the invention also provides for an LNA oligomer, comprising a contiguous region of 12-24 phosphorothioate linked nucleosides which are complementary to a corresponding region of a ApoB nucleic acid target, and further comprising between 1 and 6 DNA nucleosides which are contiguous with the LNA oligomer, wherein the internucleoside linkages between the DNA, and/or adjacent to the DNA nucleoside(s), is physiologically labile, such as is/are phosphodiester linkages.
  • an LNA oligomer may be in the form of a conjugate, as described herein, or may, for example be an intermediate to be used in a subsequent conjugation step.
  • 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.
  • a sterol such as cholesterol or tocopherol
  • a (non-nucleotide) carbohydrate such as a GalNac conjugate, such as a GalNac cluster, e.g. triGalNac, or another conjugate as described herein.
  • the invention provides for an LNA antisense oligomer (which may be referred to as region A herein) comprising an antisense oligomer comprising a contiguous region of 12-24 phosphorothioate linked nucleosides which are complementary to a corresponding region of a ApoB nucleic acid target, and 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).
  • the LNA antisense oligomer may be 12-24, and may be in the form of a gapmer oligomer.
  • FIG. 1 Non-limiting illustration of oligomers of the invention attached to an activation group (i.e. a protected reactive group—as the third region).
  • the internucleoside linkage L may be, for example phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, such as phosphodiester.
  • PO is a phosphodiester linkage.
  • Compound a) has a region B with a single DNA or RNA, the linkage between the second and the first region is PO.
  • Compound b) has two DNA/RNA (such as DNA) nucleosides linked by a phosphodiester linkage.
  • Compound c) has three DNA/RNA (such as DNA) nucleosides linked by a phosphodiester linkages.
  • Region B may be further extended by further phosphodiester DNA/RNA (such as DNA nucleosides).
  • the activation group is illustrated on the left side of each compound, and may, optionally be linked to the terminal nucleoside of region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or in some embodiments a triazole linkage.
  • Compounds d), e), & f) further comprise a linker (Y) between region B and the activation group, and region Y may be linked to region B via, for example, a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or in some embodiments a triazole linkage.
  • a linker (Y) between region B and the activation group
  • region Y may be linked to region B via, for example, a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or in some embodiments a triazole linkage.
  • FIG. 2 Equivalent compounds as shown in FIG. 1 , however a reactive group is used in place of the activation group.
  • the reactive group may, in some embodiments be the result of activation of the activation group (e.g. deprotection).
  • the reactive group may, in non-limiting examples, be an amine or alcohol.
  • FIG. 3 Non-limiting Illustration of compounds of the invention. Same nomenclature as FIG. 1 .
  • X may in some embodiments be a conjugate, such as a lipophilic conjugate such as cholesterol, or another conjugate such as those described herein.
  • X may be a targeting group or a blocking group.
  • X may be an activation group (see FIG. 1 ), or a reactive group (see FIG. 2 ).
  • X may be covalently attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or may be linked via via an alternative linkage, e.g. a triazol linkage (see L in compounds d), e), and f)).
  • FIG. 4 Non-limiting Illustration of compounds of the invention, where the compounds comprise the optional linker between the third region (X) and the second region (region B). Same nomenclature as FIG. 1 .
  • Suitable linkers are disclosed herein, and include, for example alkyl linkers, for example C6 linkers.
  • the linker between X and region B is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or may be linked via an alternative linkage eg. a triazol linkage (Li).
  • Lii represents the internucleoside linkage between the first (A) and second regions (B).
  • FIGS. 5 a and b. 5 b shows a non-limiting example of a method of synthesis of compounds of the invention.
  • US represent a oligonucleotide synthesis support, which may be a solid support.
  • X is the third region, such as a conjugate, a targeting group, a blocking group etc.
  • X is added to the oligonucleotide synthesis support. Otherwise the support with X already attached may be obtained (i).
  • region B is synthesized (ii), followed by region A (iii), and subsequently the cleavage of the oligomeric compound of the invention from the oligonucleotide synthesis support (iv).
  • the pre-step involves the provision of a oligonucleotide synthesis support with a region X and a linker group (Y) attached (see FIG. 5 a ).
  • a linker group Y
  • FIG. 6 A non-limiting example of a method of synthesis of compounds of the invention which comprise a linker (Y) between the third region (X) and the second region (B).
  • US represents a oligonucleotide synthesis support, which may be a solid support.
  • X is the third region, such as a conjugate, a targeting group, a blocking group etc.
  • Y is added to the oligonucleotide synthesis support. Otherwise the support with Y already attached may be obtained (i).
  • region B is synthesized (ii), followed by region A (iii), and subsequently the cleavage of the oligomeric compound of the invention from the oligonucleotide synthesis support (iv).
  • region X may be added to the linker (Y) after the cleavage step (v).
  • Y is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • FIG. 7 A non-limiting example of a method of synthesis of compounds of the invention which utilize an activation group.
  • the activation group is attached the oligonucleotide synthesis support (i), or the oligonucleotide synthesis support with activation group is otherwise obtained.
  • region B is synthesized, followed by region A (iii).
  • the oligomer is then cleaved from the oligonucleotide synthesis support (iv).
  • the intermediate oligomer (comprising an activation group) may then be activated (vl) or (viii) and a third region (X) added (vi), optionally via a linker (Y) (ix).
  • X (or Y when present) is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • a phosphorus nucleoside linkage group such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • FIG. 8 A non-limiting example of a method of synthesis of compounds of the invention, wherein a bifunctional oligonucleotide synthesis support is used (i).
  • a bifunctional oligonucleotide synthesis support is used (i).
  • either the oligonucleotide is synthesized in an initial series of steps (ii)-(iii), followed by the attachment of the third region (optionally via a linker group Y), the oligomeric compound of the invention may then be cleaved (v).
  • the third region (optionally with a linker group (Y) is attached to the oligonucleotide synthesis support (this may be an optional pre-step)—or a oligonucleotide synthesis support with the third region (optionally with Y) is otherwise provided, the oligonucleotide is then synthesized (vii-viii).
  • the oligomeric compound of the invention may then be cleaved (ix).
  • X (or Y when present) is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • the US may in some embodiment, prior to the method (such as the pre-step) comprise a step of adding a bidirectional (bifunctional) group which allows the independent synthesis of the oligonucleotide and the covalent attachment of group X, Y (or X and Y) to support (as shown)—this may for example be achieved using a triazol or of nucleoside group.
  • the bidirectional (bifunctional) group, with the oligomer attached may then be cleaved from the support.
  • FIG. 9 A non-limiting example of a method of synthesis of compounds of the invention:
  • the first region (A) is synthesized (ii), followed by region B.
  • the third region is then attached to region B (iii), optionally via a phosphate nucleoside linkage (or e.g. a triazol linkage).
  • the oligomeric compound of the invention may then be cleaved (iv).
  • a linker (Y) is used, in some embodiments the steps (v)-(viii) may be followed: after synthesis of region B, the linker group (Y) is added, and then either attached to (Y) or in a subsequent step, region X is added (vi).
  • oligomeric compound of the invention may then be cleaved (vii).
  • X (or Y when present) is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • FIG. 10 A non-limiting example of a method of synthesis of compounds of the invention: In this method an activation group is used: Steps (i)-(iii) are as per FIG. 9 . However after the oligonucleotide synthesis (step iii), an activation group (or a reactive group) is added to region B, optionally via a phosphate nucleoside linkage. The oligonucleotide is then cleaved from the support (v).
  • the activation group may be subsequently activated to produce a reactive group, and then the third region (X), such as the conjugate, blocking group or targeting group, is added to the reactive group (which may be the activated activation group or the reactive group), to produce the oligomer (vi).
  • the third region (X) such as the conjugate, blocking group or targeting group
  • the reactive group which may be the activated activation group or the reactive group
  • region X is added to produce the oligomer (viii).
  • the reactive group or activation group is attached to region B via a phosphorus nucleoside linkage group, such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, or an alternative linkage, such as a triazol linkage.
  • a phosphorus nucleoside linkage group such as phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate
  • an alternative linkage such as a triazol linkage.
  • FIG. 11 Silencing of ApoB mRNA with Cholesterol-conjugates in vivo. Mice were injected with a single dose of 1 mg/kg unconjugated LNA-antisense oligonucleotide (#3833) or equimolar amounts of LNA antisense oligonucleotides conjugated to Cholesterol with different linkers (Tab. 3) and sacrificed at days 1, 3, 7 and 10 after dosing. RNA was isolated from liver and kidney and subjected to ApoB specific RT-qPCR A. Quantification of ApoB mRNA from liver samples normalized to GAPDH and shown as percentage of the average of equivalent saline controls B. Quantification of ApoB mRNA from kidney samples normalized to GAPDH and shown as percentage of the average of equivalent saline controls.
  • FIG. 12 Shows the cholesterol C6 conjugate which may be used as X-Y- in compounds of the invention, as well as specific compounds used in the examples, include specific compounds of the invention.
  • FIG. 13 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.
  • FIG. 14 Examples of cholesterol and tocopherol conjugate moieties.
  • the wavy line represents the covalent link to the oligomer.
  • FIG. 16 Example 8—ApoB mRNA expression
  • FIG. 17 Example 8—Total cholesterol in serum
  • FIG. 18 Example 8—Oligonucleotide content in liver and kidney
  • the invention relates to oligomeric compounds which targets an ApoB nucleic acid, such as LNA antisense oligonucleotides, which are covalently linked to a conjugate group, a targeting group, a reactive group, an activation group, or a blocking group, via a short region comprising (e.g. 1-10) of phosphodiester linked DNA or RNA nucleoside(s).
  • ApoB nucleic acid such as LNA antisense oligonucleotides
  • 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).
  • a single nucleotide (unit) may also be referred to as a monomer or unit.
  • 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.
  • the oligomer of the invention may be an LNA oligomer, i.e. comprises at least one LNA nucleoside unit, or a gapmer, such as an LNA gapmer.
  • the oligomer of the invention may comprise between 10-22, such as 12-22 nucleotides in length.
  • the oligomer of the invention may comprise a contiguous sequence of 10-20 nucleotides which are complementary, such as fully complementary, to a corresponding length of the ApoB nucleic acid target (such as NM — 000384 or genbank accession No: NG — 011793, NM — 000384.2 GI:105990531 and NG — 011793.1 GI:226442987 are hereby incorporated by reference).
  • the contiguous sequence of 10-20 nucleotides may linked, for example, by phosphorothioate linkages.
  • the oligomer of the invention may comprise the sequence of nucleobases shown in SEQ ID NO 1 or SEQ ID No 2.
  • the compound (e.g. oligomer or conjugate) of the invention targets ApoB, and as such is capable of inhibiting ApoB, such as human ApoB, in a cell expressing said ApoB.
  • the internucleoside linkages of the contiguous sequence may be phosphorothioate linkages, and may comprise affinity enhancing nucleotide analogues.
  • the nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides independently or dependently selected from the group consisting of: Locked Nucleic Acid (LNA or BNA) units; 2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, and 2′-fluoro-DNA units.
  • LNA Locked Nucleic Acid
  • BNA Locked Nucleic Acid
  • the nucleotide analogues comprise or are Locked Nucleic Acid (LNA, also known as BNA) units.
  • LNA Locked Nucleic Acid
  • the oligomer of the invention comprises or is a gapmer, such as a LNA gapmer oligonucleotide.
  • the oligomer of the invention comprises a contiguous sequence of 13, 14, 15 or 16 nucleotides which are complementary to a corresponding length of the ApoB nucleic acid target, and may optionally comprise a further 1-10, for example 1-6 nucleotides, which may form or comprise a biocleavable nucleotide region, such as a phosphate nucleotide linker.
  • the biocleavable nucleotide region is formed of a short stretch (eg. 1, 2, 3, 4, 5 or 6) of nucleotides which are physiologically labile. This may be achieved by using phosphodiester linkages with DNA/RNA nucleosides, or if physiological liability can be maintained, other nucleosides may be used.
  • the oligomer of the invention may therefore comprise of a contiguous nucleotide sequence of 10-20 nts in length which is complementary to a corresponding length of the ApoB nucleic acid target (A first region, or region A).
  • the oligomer of the invention may comprise a further nucleotide region.
  • the further nucleotide region comprises a biocleavable nucleotide region, such as a phosphate nucleotide sequence (a second region, region B), which may covalently link region A to a non-nucleotide moiety, such as a conjugate group, (a third region, or region C).
  • the contiguous nucleotide sequence of the oligomer of the invention (region A) is directly covalently linked to region C.
  • region C is biocleavable.
  • the may oligomer consists or comprises of a contiguous nucleotide sequence of from 10-22, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, nucleotides in length, such as 13-16, or 13 or 14, or 15 or 16 nucleotides in length.
  • the oligomer may therefore refer to the combined length of region A and region B, e.g. (Region A 10-16 nt) and region B (1-6 nt).
  • the compound of the invention does not comprise RNA (units).
  • the compound according to the invention, the first region, or the first and second regions together e.g. as a single contiguous sequence
  • the oligomer may therefore be single stranded molecule.
  • 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 oligomer (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.
  • the oligomer of the invention is capable of down-regulating expression of the APO-B gene, such as ApoB-100 or ApoB-48 (APOB).
  • the oligomer of the invention can affect the inhibition of APOB, typically in a mammalian such as a human cell, such as liver cells.
  • the oligomers of the invention bind to the target nucleic acid and effect 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 modulation is seen when using between 0.04 and 25 nM, such as between 0.8 and 20 nM concentration of the compound of the invention.
  • the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition.
  • Modulation of expression 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.
  • the level of down-regulation when using an appropriate dosage is, In some embodiments, typically to a level of between 10-20% the normal levels in the absence of the compound of the invention.
  • the invention therefore provides a method of down-regulating or inhibiting the expression of APO-B protein and/or mRNA in a cell which is expressing APO-B protein and/or mRNA, said method comprising administering the compound of the invention to the invention to said cell to down-regulating or inhibiting the expression of APO-B protein and/or mRNA in said cell.
  • 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.
  • target nucleic acid refers to the DNA or RNA encoding mammalian APO-B polypeptide, such as human APO-B100, such as human APO-B100 mRNA.
  • APO-B100 encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA.
  • the “target nucleic acid” may be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets.
  • the oligomer according to the invention is preferably capable of hybridising to the target nucleic acid. It will be recognised that human APO-B mRNA is a cDNA sequence, and as such, corresponds to the mature mRNA target sequence, although uracil is replaced with thymidine in the cDNA sequences.
  • naturally occurring variant thereof refers to variants of the APO-B1 polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and preferably human.
  • the term also may encompass any allelic variant of the APO-B encoding genomic DNA by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom.
  • “Naturally occurring variants” may also include variants derived from alternative splicing of the APO-B100 mRNA.
  • the term when referenced to a specific polypeptide sequence, e.g., the term also includes naturally occurring forms of the protein which may therefore be processed, e.g. by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.
  • the oligomers (region A) may comprise or consist of a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence present in e.g. the human APO-B mRNA.
  • the oligomer (region A) may comprise or consist of a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian APO-B (e.g., human APO-B100 mRNA).
  • the oligomer (region A) can comprise or consist of an antisense nucleotide sequence.
  • the oligomer may tolerate 1 or 2 mismatches, when hybridising to the target sequence and still sufficiently bind to the target to show the desired effect, i.e. down-regulation of the target.
  • Mismatches may, for example, be compensated by increased length of the oligomer nucleotide sequence and/or an increased number of nucleotide analogues, such as LNA, present within the nucleotide sequence.
  • the nucleotide sequence of the oligomer may comprise additional 5′ or 3′ nucleotides, such as, independently, 1, 2, 3, 4, 5 or 6 additional nucleotides 5′ and/or 3′, which are non-complementary to the target sequence—such non-complementary oligonucleotides may form region B.
  • the oligomer of the invention may, in some embodiments, comprise a contiguous nucleotide sequence which is flanked 5′ and or 3′ by additional nucleotides.
  • the additional 5′ or 3′ nucleotides are naturally occurring nucleotides, such as DNA or RNA.
  • the additional 5′ or 3′ nucleotides may represent region D as referred to in the context of gapmer oligomers herein.
  • the internucleoside linkages between the additional nucleotides, and optionally between the additional nucleotides and the oligomer are phosphodiester linkages.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO:1, or a sub-sequence of at least 10 or 12 nucleobases thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO:2, or a sub-sequence of at least 10 or 12 nucleobases thereof.
  • the GalNac conjugate itself is biocleavable, utilizing a peptide linker in the GalNac cluster, and as such a further biocleavable linker (B) may or may not be used.
  • a biocleavable linker (B) such as the phosphate nucleotide linkers disclosed herein may enhance activity of such GalNac cluster oligomer conjugates.
  • the use of a biocleavable linker greatly enhances compound activity inclusion of a biocleavable linker (B), such as the phosphate nucleotide linkers disclosed herein is recommended.
  • the conjugate moiety (and region B or region Y or B and Y, may be positioned, e.g. 5′ or 3′ to the SEQ ID, such as 5′ to region A.
  • 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/region A) and the reverse complement of the nucleic acid target, or sub-region thereof.
  • Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides.
  • the oligomers (or first region thereof) are complementary to the target region or sub-region, such as fully complementary.
  • nucleotide analogue and “corresponding nucleotide” are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical.
  • the “corresponding nucleotide analogue” contains a pentose unit (different from 2-deoxyribose) linked to an adenine.
  • 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 recognised that the DNA or RNA nucleosides of region B may have a naturally occurring and/or non-naturally occurring nucleobase(s).
  • 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.
  • the nucleobases may be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, 5-methylcytosine.
  • nucleobases may be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, and 5-methylcytosine.
  • 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.
  • the oligomers may comprise or consist of a contiguous nucleotide sequence of a total of between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides in length. Lengths may include region A or region A and B for example.
  • the oligomers comprise or consist of a contiguous nucleotide sequence of a total of between 10-22, such as 12-18, such as 13-17 or 12-16, such as 13, 14, 15, 16 contiguous nucleotides in length.
  • the oligomer according to the invention consists of no more than 22 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments the oligomer of the invention comprises less than 20 nucleotides.
  • nucleotide refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group, such as a phosphate or phosphorothioate internucleotide 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.
  • a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit.
  • 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 internucleotide linkages between the nucleotides of the oligomer.
  • the 5′ nucleotide of an oligonucleotide does not comprise a 5′ internucleotide 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.
  • 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 cell.
  • 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:
  • 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 “RNA”), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues.
  • DNA 2′-deoxynucleotides
  • RNA ribonucleotides
  • nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.
  • nucleotide analogues examples include tricyclic nucleic acids, for example please see WO2013154798 and WO2013154798 which are hereby incorporated by reference.
  • affinity-enhancing nucleotide analogues in the oligomer 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.
  • 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 locked nucleic acid (LNA); for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleotide analogues may be LNA. In some embodiments all the nucleotides analogues may be LNA.
  • LNA locked nucleic acid
  • 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 LNA units or other nucleotide analogues, which raise the duplex stability/T m of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues).
  • 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 2 ⁇ T m -buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM 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 260 nm. First derivative and the local maximums of both the melting and annealing are used to assess the duplex T m .
  • 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.
  • 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.
  • At least one nucleoside analogue present in the first region (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, (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.
  • Y is selected from the group consisting of —O—, —CH 2 O—, —S—, —NH—, N(R e ) and/or —CH 2 —;
  • Z and Z* are independently selected 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 1-4 -alkyl;
  • R a , R b R c , R d and R e are, optionally independently, selected from the group consisting of hydrogen, optionally substituted C 1-12 -alkyl, optionally substituted C 2-12 -alkenyl, optionally substituted C 2-12 -alkynyl, hydroxy, C 1-12 -alkoxy, C 2-12 -alkoxyalkyl, C 2-12 -alkenyloxy, carboxy, C 1-12 -alkoxy
  • R a , R b R c , R d and R e are, optionally independently, selected from the group consisting of hydrogen and C 1-6 alkyl, such as methyl.
  • R a , R b R c , R d and R e are, optionally independently, selected from the group consisting of hydrogen and C 1-6 alkyl, such as methyl.
  • C 1-6 alkyl such as methyl.
  • 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:
  • thio-LNA comprises a locked nucleotide in which Y in the general formula above is selected from S or —CH 2 —S—.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which Y in the general formula above is selected from —N(H)—, N(R)—, CH 2 —N(H)—, and —CH 2 —N(R)— where R is selected from hydrogen and C 1-4 -alkyl.
  • Amino-LNA can be in both beta-D and alpha-L-configuration.
  • Oxy-LNA comprises a locked nucleotide in which Y in the general formula above represents —O—. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ENA comprises a locked nucleotide in which Y in the general formula above is —CH 2 —O— (where the oxygen atom of —CH 2 —O— is attached to the 2′-position relative to the base B).
  • R e is hydrogen or methyl.
  • 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.
  • 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.
  • compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4′ to 2′ bicyclic nucleoside.
  • 4′ to 2′ bicyclic nucleosides include, but are not limited to, one of the formulae: 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2′; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-CH(CH 3 )—O-2′ and 4′-CH(CH 2 OCH 3 )—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH 3 )(CH 3 )—O-2′, and analogs thereof (see, published PCT International Application WO2009/006478, published Jan.
  • 4′-CH 2 —N(OCH 3 )-2′ and analogs thereof (see, published PCT International Application WO2008/150729, published Dec. 11, 2008); 4′-CH 2 —O—N(CH 3 )-2′ (see, published U.S. Patent Application US2004/0171570, published Sep. 2, 2004); 4′-CH 2 —N(R)—O-2′, wherein R is H, C 1 -C 10 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH 2 —C(H)(CH 3 )-2′ (see, Chattopadhyaya, et al, J. Org.
  • 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 Mar. 25, 1999 as WO 99/14226).
  • 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 a XR b )]—, —C(R a ) ⁇ C(R b )—, —C(R a ) ⁇ N—, —C( ⁇ NR a )—, —C( ⁇ O)—, —C( ⁇ S)—, —O—, —Si(R a ) 2 —, —S( ⁇ O) x —, and —N(Ra)—; 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, C 1 -C 12 alkyl, substituted C 1 -C 12
  • the bridge of a bicyclic sugar moiety is, —[C(R a )(Rb)] n —, —[C(R a )(R b )] n —O—, —C(R a R b )—N(R)—O— or, —C(R a R b )—O—N(R)—.
  • the bridge is 4′-CH 2 -2′, 4′-(CH 2 ) 2 -2′, 4′-(CH 2 ) 3 -2′, 4′-CH 2 —O-2′, 4*-(CH 2 )2-O-2′, 4′- CH 2 —O—N(R)-2′, and 4′-CH 2 —N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C 1 -C 12 alkyl.
  • bicyclic nucleosides are further defined by isomeric configuration.
  • a nucleoside comprising a 4′-2′ methylene-oxy bridge may be in the a-L configuration or in the beta-D configuration.
  • a-L-methyleneoxy (4′-CH 2 —O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).
  • bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4′-CH 2 —O-2′) BNA, (B) beta-D-Methyleneoxy (4′-CH 2 —O-2′) BNA, (C) Ethyleneoxy (4′-(CH 2 ) 2 —O-2′) BNA, (D) Aminooxy (4′-CH 2 —O—N(R)-2′) BNA, (E) Oxyamino (4′-CH 2 —N(R)—O-2′) BNA, (F), Methyl(methyleneoxy) (4′-CH(CH 3 )—O-2′) BNA, (G) methylene-thio (4′-CH 2 —S-2′) BNA, (H) methylene-amino (4′-CH 2 —N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH 2 —CH(CH 3 )-2′) BNA
  • Bx is the base moiety and R is, independently, H, a protecting group or C 1 -C 2 alkyl.
  • Bx is a heterocyclic base moiety
  • -Q a -Q b -Q c - is —CH 2 —N(Rc)-CH 2 —, —C( ⁇ O)—N(R c )—CH 2 —, —CH 2 —O—N(Rc)-, —CH 2 —N(Rc)-O—, or —N(Rc)-O—CH 2 ;
  • R c is C 1 -C 12 alkyl or an amino protecting group
  • 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.
  • bicyclic nucleoside having Formula II having Formula II:
  • 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 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio.
  • each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ c , NJ d , SJ C , N 3 , OC( ⁇ X)J c , and NJ e C( ⁇ X)NJ c J d , wherein each J c , J d , and J e is, independently, H, C 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl and X is O or NJ C .
  • bicyclic nucleoside having Formula III having Formula III:
  • 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 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, or substituted acyl (C( ⁇ O)—).
  • bicyclic nucleoside having Formula IV having Formula IV:
  • 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 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl; each q b , q c and q d is, independently, H, halogen, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -Ce alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, or substituted C 2 -C6 alkynyl, C 1 -C 6 alkoxyl, substituted Q-C 6 alkoxyl, acyl, substituted acyl, C 1 -C 6 aminoalkyl, or substituted C 1 -C 6 aminoalkyl;
  • bicyclic nucleoside having Formula V 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 covalent attachment to a support medium;
  • q a , q b , q c and q f are each, independently, hydrogen, halogen, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 1 -C 12 alkoxy, substituted C 1 -C 12 alkoxy, OJ j , SJ j , SOJ j , SO 2 J j , NJ j J k , N 3 , CN, C( ⁇ O)OJ j , C( ⁇ O)NJ j J k , C( ⁇ O)
  • BNA methyleneoxy (4′-CH 2 —O-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.
  • 2′-amino-BNA a novel comformationally restricted high-affinity oligonucleotide analog
  • synthesis of 2′-amino-BNA has been described in the art (see, e.g., Singh et al., J. Org. Chem., 1998, 63, 10035-10039).
  • 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.
  • the bicyclic nucleoside has Formula VI:
  • 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; each qj, qj, q k and ql is, independently, H, halogen, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 1 -C 12 alkoxyl, substituted C 2 -C 12 alkoxyl, OJ j , SJ j , SOJ j , SO 2 J j , NJ j J k , N 3 , CN, C( ⁇ O)OJ j , C( ⁇ O)NJ j J k , C( ⁇ O)J j ,
  • 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.
  • nucleosides refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties.
  • sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
  • 2′-modified sugar means a furanosyl sugar modified at the 2′ position.
  • 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.
  • 2′ modifications are selected from substituents including, but not limited to: O[(CH 2 ) n O] m CH 3 , O(CH 2 ),NH 2 , O(CH 2 ),CH 3 , O(CH 2 ),ONH 2 , OCH 2 C( ⁇ O)N(H)CH 3 , and O(CH2) n ON[(CH 2 ) n CH 3 ]2, where n and m are from 1 to about 10.
  • 2′-substituent groups can also be selected from: C 1 -C 12 alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH 3 ; OCN; Cl; Br; CN; CF 3 ; OCF 3 ; SOCH 3 ; S0 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; 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.
  • modified nucleosides comprise a 2′-MOE side chain ⁇ see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 11944-12000).
  • 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.
  • 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, C J. Bioorg. and Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), or those compounds having Formula X:
  • Bx is a heterocyclic base moiety
  • 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 n , q p , q r , q s , q t and q u is methyl.
  • THP nucleosides of Formula X are provided wherein one of R 1 and R 2 is F.
  • R 1 is fluoro and R 2 is H
  • R 1 is methoxy and R 2 is H
  • R 1 is methoxyethoxy and R 2 is H.
  • 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 allyl, amino, azido, thio, O-allyl, O—C 1 -C 10 alkyl, —OCF 3 , O—(CH 2 ) 2 —O—CH 3 , 2′-O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ), or O—CH 2 —C( ⁇ O)—N(R m )(R n ), where each R m and R, is, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
  • 2′-modified nucleosides may
  • 2′-F refers to a sugar comprising a fluoro group at the 2′ position.
  • 2′-OMe or “2′-OCH 3 ” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH 3 group at the 2′ position of the sugar ring.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
  • 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.
  • antisense compounds comprise one or more nucleotides having modified sugar moieties.
  • the modified sugar moiety is 2′-MOE.
  • the 2′-MOE modified nucleotides are arranged in a gapmer motif.
  • the modified sugar moiety is a cEt.
  • the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.
  • R 4 * and R 2 * together designate the biradical —O—CH(CH 2 OCH 3 )— (2′O-methoxyethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem)—in either the R- or S-configuration.
  • R 4 * and R 2 * together designate the biradical —O—CH(CH 2 CH 3 )— (2′O-ethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem).—in either the R- or S-configuration.
  • R 4 * and R 2 * together designate the biradical —O—CH(CH 3 )—.—in either the R- or S-configuration. In some embodiments, R 4 * and R 2 * together designate the biradical —O—CH 2 —O—CH 2 ——(Seth at al., 2010, J. Org. Chem).
  • R 4 * and R 2 * together designate the biradical —O—NR—CH 3 ——(Seth at al., 2010, J. Org. Chem).
  • the LNA units have a structure selected from the following group:
  • 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 non-specific or aberrant binding takes place.
  • BNA e.g.
  • LNA low-density nucleic acid
  • 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.
  • 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.
  • LNA locked nucleic acid
  • 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).
  • 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-EN
  • the oligomer of the invention may comprise BNA or LNA units and other nucleotide analogues.
  • Further 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.
  • 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.
  • the oligomer according to the invention may therefore 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.
  • BNA Locked Nucleic Acid
  • all the nucleotide analogues are BNA, such as LNA.
  • 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.
  • the oligomer (such as the first and optionally second regions) may comprise both BNA and LNA and DNA units.
  • 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.
  • 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.
  • 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.
  • BNA e.g. LNA
  • nucleotide analogues such as RNA or DNA, most preferably DNA nucleotides
  • naturally occurring nucleotides such as RNA or DNA, most preferably DNA nucleotides
  • modified internucleotide linkages such as phosphorothioate.
  • an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods.
  • the oligomers of the invention are capable of recruiting an endoribonuclease (RNase), such as RNase H.
  • RNase endoribonuclease
  • 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 (such as DNA units).
  • 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.
  • the size of the contiguous sequence which is capable of recruiting RNAse, such as region Y′ may be higher, such as 10, 11, 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 RNase 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.
  • 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.
  • 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.
  • 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.
  • the oligomer 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′ (Y′), 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′ (X′) and Z′ (Z′) respectively. Examples of gapmers are disclosed in WO2004/046160, WO2008/113832, and WO2007/146511.
  • the monomers which are capable of recruiting RNAse are selected from the group consisting of DNA monomers, alpha-L-LNA monomers, C4′ alkylated 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 Fluiter et al., Mol. Biosyst., 2009, 10, 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.
  • the gapmer comprises a (poly)nucleotide sequence of formula (5′ to 3′), X′-Y′-Z′, wherein; region X′ (X′) (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.
  • BNA e.g. LNA
  • region Y′ (Y′) 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
  • region Z′ (Z′) (3′ 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.
  • 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.
  • BNA e.g. LNA
  • 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
  • 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.
  • 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.
  • 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
  • region Z′ consists of 3 or 4 nucleotide analogues, such as BNA (e.g. LNA).
  • BNA e.g. LNA
  • Such designs include (X′-Y′-Z′) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3.
  • oligomers presented here may be such shortmer gapmers.
  • 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 LNA
  • 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 mRNA target); and Z′ consists of 1, 2 or 3 nucleotide analogue units, such as BNA (e.g. LNA) units.
  • BNA e.g. LNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • gapsmer designs include those where regions X′ and/or Z′ consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2′-O-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.
  • regions X′ and/or Z′ consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or monomers containing a 2′-fluoro-deoxyribose sugar
  • region Y′ consists of 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where regions X
  • 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.
  • SEQ ID NO 2 and 3 are LNA gapmer oligomers.
  • the oligomers with a contiguous sequence of 10-16 nucleotides which are complementary to a corresponding length of SEQ ID NO 33 or 34 may also be gapmer oligomers such as BNA gapmers or LNA gapmers.
  • nucleoside monomers of the oligomers e.g. first and second regions
  • oligomers e.g. first and second regions
  • linkage groups e.g. first and second linkage groups.
  • each monomer is linked to the 3′ adjacent monomer via a linkage group.
  • 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.
  • 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.
  • nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups.
  • 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 WO2007/031091 (hereby incorporated by reference).
  • 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.
  • 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.
  • the nucleotides and/or nucleotide analogues are linked to each other by means of phosphorothioate groups.
  • 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.
  • 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.
  • WO09124238 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.
  • conjugate moieties which have been used with oligonucleotides can include lipophilic molecules (aromatic and non-aromatic) including steroid molecules; 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.
  • lipophilic molecules aromatic and non-aromatic
  • proteins e.g., antibodies, enzymes, serum proteins
  • peptides e.g., antibodies, enzymes, serum proteins
  • vitamins water-soluble or lipid-soluble
  • the oligomer of the invention is targeted to the liver—i.e. after systemic administration the compound accumulates in the liver cells (such as hepatocytes).
  • Targeting to the liver can be greatly enhanced by the addition of a conjugate moiety (C).
  • C conjugate moiety
  • B biocleavable linker
  • B nucleotide phosphate linker
  • the oligomeric compound is a BNA or LNA oligomer, such as a gapmer, or for example an LNA antisense oligomer, (which may be referred to as region A herein) comprising an antisense oligomer, optionally a biocleavable linker, such as region B, and a carbohydrate conjugate (which may be referred to as region C).
  • the LNA antisense oligomer may be 7-30, such as 8-26 nucleosides in length and it comprises at least one LNA unit (nucleoside).
  • the carbohydrate moiety is not a linear carbohydrate polymer.
  • the oligomeric compound is a LNA oligomer, for example an LNA antisense oligomer, (which may be referred to as region A herein) comprising an antisense oligomer, region B as defined herein, and an asialoglycoprotein receptor targeting moiety conjugate moiety, such as a GalNAc moiety (which may be referred to as 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).
  • 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 a conjugate comprising the oligomer of the invention and a carbohydrate conjugate moiety.
  • the invention provides a conjugate comprising the oligomer of the invention and 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).
  • the invention also provides LNA antisense oligonucleotides which are conjugated to an asialoglycoprotein receptor targeting moiety.
  • 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.
  • the conjugate comprises a galactose cluster, such as N-acetylgalactosamine trimer.
  • the conjugate moiety comprises an 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. U.S. Pat. No. 5,994517 and Hangeland et al., Bioconjug Chem. 1995 November-December; 6(6):695-701) and siRNAs (e.g. WO2009/126933, WO2012/089352 & WO2012/083046).
  • 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 '046 cleavable pharmacokinetic modulators may also be cholesterol.
  • 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.
  • a pharmacokinetic modulator selected from the group consisting of: hydrophobic group having 16 or
  • GalNac clusters disclosed in '046 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 (2 ⁇ C8).
  • 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 phosphodiester linkers between the oligomer and the conjugate group (these are referred to as region B herein, and suitably are positioned between the LNA oligomer and the carbohydrate conjugate group).
  • a preferred targeting ligand is a galactose cluster.
  • a galactose cluster comprises a molecule having e.g. comprising two to four terminal galactose derivatives.
  • 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—I 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. Chem., 257,939-945). Multivalency is required to achieve nM affinity.
  • RNAi polynucleotide 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—I carbons of the saccharides.
  • the galactose derivative is preferably linked to the branch point via linkers or spacers.
  • a preferred spacer is a flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546).
  • a preferred flexible hydrophilic spacer is a PEG spacer.
  • a preferred PEG spacer is a PEG3 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.
  • hydrophobic or lipophilic (or further conjugate) moiety i.e. pharmacokinetic modulator
  • BNA or LNA oligomers such as LNA antisense oligonucleotides, optional.
  • Each carbohydrate moiety of a Galnac cluster 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.
  • PEG polyethylene glycol linker
  • the PEG moiety forms a spacer between the galactose sugar moiety and a peptide (trilysine is shown) linker.
  • 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:
  • R 1 is a biradical preferably selected from —C 2 H 4 —, —C 3 H 6 —, —C 4 H 8 —, —C 5 H 10 —, —C 6 H 12 —, 1,4- cyclohexyl (—C 6 H 10 —), 1,4-phenyl (—C 6 H 4 —), —C 2 H 4 OC 2 H 4 —, —C 2 H 4 (OC 2 H 4 ) 2 — or —C 2 H 4 (OC 2 H 4 ) 3 —.
  • the carbohydrate conjugate e.g. GalNAc
  • carbohydrate-linker moiety e.g. carbohydrate-PEG moiety
  • a branch point group such as, an amino acid, or peptide, which suitably comprises two or more amino groups (such as 3, 4, or 5), 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 GalNAc
  • 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.
  • 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.
  • 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.
  • 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. 2 ⁇ , such as 2 ⁇ 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, aralkyl 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.
  • the conjugate is or may comprise a carbohydrate or comprises a carbohydrate group.
  • the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate.
  • 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, WO99/65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17.
  • 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.
  • the conjugate group is or may comprise a lipophilic moiety, such as a sterol (for example, cholesterol, cholesteryl, cholestanol, stigmasterol, cholanic acid and ergosterol).
  • a sterol for example, cholesterol, cholesteryl, cholestanol, stigmasterol, cholanic acid and ergosterol.
  • the conjugate is or comprises tocopherol.
  • the conjugate is or may comprise cholesterol.
  • 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.
  • the fatty acid comprises a C4-C30 saturated or unsaturated alkyl chain. The alkyl chain may be linear or branched.
  • 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, sterols stanols, and steroids and related compounds such as cholesterol (U.S. Pat. No. 4,958,013 and Letsinger et al., Proc. Natl. Acad. Sci.
  • 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.
  • the conjugate may be selected from the group consisting of cholesterol, thiocholesterol, lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone, and 17-hydroxycorticosterone.
  • Other lipophilic conjugate moieties include aliphatic groups, such as, for example, straight chain, branched, and cyclic alkyls, alkenyls, and alkynyls.
  • 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, hexadecyl, heptadecyl, octadecyl, nonadecyl, terpenes, bornyl, adamantyl, derivatives thereof and the like.
  • one or more carbon atoms in the aliphatic group can be replaced by a heteroatom such as O, S, or N (e.g., geranyloxyhexyl).
  • lipophilic conjugate moieties include aliphatic derivatives of glycerols such as alkylglycerols, bis(alkyl)glycerols, tris(alkyl)glycerols, monoglycerides, diglycerides, and triglycerides.
  • the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-O-hexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea, et al., Nuc. Acids Res., 1990, 18, 3777) or phosphonates thereof.
  • Saturated and unsaturated fatty functionalities can also serve as lipophilic conjugate moieties.
  • 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.
  • 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.
  • 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.
  • trityls e.g., dimethoxytrityl (DMT)
  • phenoxazines e.g., lipoic acid
  • phospholipids e.g., phospholipids
  • ethers e.g., thioethers (e.g., hexyl-S-tritylthiol), derivatives thereof and the like.
  • thioethers e.g., hexyl-S-tritylthiol
  • 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.
  • conjugate moieties having LDL affinity can be dioleyl esters of cholic acids such as chenodeoxycholic acid and lithocholic acid.
  • 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 sterols, stanols, and stains, such as cholesterol or as disclosed herein, may be used to enhance delivery of the oligonucleotide to, for example, the liver (typically hepatocytes).
  • 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 can be attached to the oligomeric 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 oligomeric compound (such as to region B).
  • 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 oligomeric 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.
  • linker functionalities include amino, hydroxyl, carboxylic acid, thiol, phosphoramidate, phosphorothioate, phosphate, phosphite, unsaturations (e.g., double or triple bonds), and the like.
  • linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-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), succini
  • 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.
  • 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.
  • 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,Wo2012/083046 each of which is incorporated by reference in its entirety.
  • 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.
  • 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 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.
  • a pH labile bond may be cleaved when the molecule enters an acidified endosome.
  • 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.
  • a disulfide may be considered to be a cytoplasmic cleavable bond.
  • 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.
  • the oligomeric compound may optionally, comprise a second region (region B) which is positioned between the oligomer (referred to as region A) and the conjugate (referred to as region C).
  • Region B may be a linker such as a cleavable linker (also referred to as a physiologically labile linkage). (see Example 7)
  • the oligomer (also referred to as oligomeric compound) of the invention (or conjugate) comprises three regions:
  • region B may be a phosphate nucleotide linker.
  • linkers may be used when the conjugate is 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.
  • 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, include disulfide linkers.
  • region B comprises between 1-6 nucleotides, which is covalently linked to the 5′ or 3′ nucleotide of the first region, such as via a internucleoside linkage group such as a phosphodiester linkage, wherein either
  • region A and region B form a single contiguous nucleotide sequence of 12-22 nucleotides in length.
  • the internucleoside linkage between the first and second regions may be considered part of the second region.
  • a phosphorus containing linkage group between the second and third region.
  • the phosphorus linkage group may, for example, be a phosphate (phosphodiester), a phosphorothioate, a phosphorodithioate or a boranophosphate group.
  • this phosphorus containing linkage group is positioned between the second region and a linker region which is attached to the third region.
  • the phosphate group is a phosphodiester.
  • the oligomeric compound comprises at least two phosphodiester groups, wherein at least one is as according to the above statement of invention, and the other is positioned between the second and third regions, optionally between a linker group and the second region.
  • the third region is an activation group, such as an activation group for use in conjugation.
  • the invention also provides activated oligomers comprising region A and B and a activation group, e.g an intermediate which is suitable for subsequent linking to the third region, such as suitable for conjugation.
  • the third region is a reactive group, such as a reactive group for use in conjugation.
  • the invention also provides oligomers comprising region A and B and a reactive group, e.g an intermediate which is suitable for subsequent linking to the third region, such as suitable for conjugation.
  • the reactive group may, in some embodiments comprise an amine of alcohol group, such as an amine group.
  • region A comprises at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 internucleoside linkages other than phosphodiester, such as internucleoside linkages which are (optionally independently] selected from the group consisting of phosphorothioate, phosphorodithioate, and boranophosphate, and methylphosphonate, such as phosphorothioate.
  • region A comprises at least one phosphorothioate linkage.
  • At least 50%, such as at least 75%, such as at least 90% of the internucleoside linkages, such as all the internucleoside linkages within region A are other than phosphodiester, for example are phosphorothioate linkages. In some embodiments, all the internucleoside linkages in region A are other than phosphodiester.
  • the oligomeric compound comprises an antisense oligonucleotide, such as an antisense oligonucleotide conjugate.
  • the antisense oligonucleotide may be or may comprise the first region, and optionally the second region.
  • region B may form part of a contiguous nucleobase sequence which is complementary to the (nucleic acid) target. In other embodiments, region B may lack complementarity to the target.
  • the invention provides a non-phosphodiester linked, such as a phosphorothioate linked, oligonucleotide (e.g. an antisense oligonucleotide) which has at least one terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacent nucleoside of the oligonucleotide via a phosphodiester linkage, wherein the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting moiety or a blocking moiety, optionally via a linker moiety.
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting
  • the oligomeric compound comprises an antisense oligonucleotide, such as an antisense oligonucleotide conjugate.
  • the antisense oligonucleotide may be or may comprise the first region, and optionally the second region.
  • region B may form part of a contiguous nucleobase sequence which is complementary to the (nucleic acid) target. In other embodiments, region B may lack complementarity to the target.
  • At least two consecutive nucleosides of the second region are DNA nucleosides (such as at least 3 or 4 or 5 consecutive DNA nucleotides).
  • oligonucleotide of the invention may be described according to the following formula:
  • A is region A
  • PO is a phosphodiester linkage
  • B is region B
  • Y is an optional linkage group
  • X is a conjugate, a targeting, a blocking group or a reactive or activation group.
  • region B comprises 3′-5′ or 5′-3′: i) a phosphodiester linkage to the 5′ nucleoside of region A, ii) a DNA or RNA nucleoside, such as a DNA nucleoside, and iii) a further phosphodiester linkage
  • the further phosphodiester linkage link the region B nucleoside with one or more further nucleoside, such as one or more DNA or RNA nucleosides, or may link to X (is a conjugate, a targeting or a blocking group or a reactive or activation group) optionally via a linkage group (Y).
  • region B comprises 3′-5′ or 5′-3′: i) a phosphodiester linkage to the 5′ nucleoside of region A, ii) between 2-10 DNA or RNA phosphodiester linked nucleosides, such as a DNA nucleoside, and optionally iii) a further phosphodiester linkage:
  • [PO-B]n represents region B, wherein n is 1-10, such as 1, 2, 3,4, 5, 6, 7, 8, 9 or 10, PO is an optional phosphodiester linkage group between region B and X (or Y if present).
  • the invention provides compounds according to (or comprising) one of the following formula:
  • Region B may for example comprise or consist of:
  • phosphate linked biocleavable linkers may employ nucleosides other than DNA and RNA.
  • Bio cleavable nucleotide linkers may, for example, be identified using the assays in Example 7.
  • 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 (or contiguous nucleotide sequence or region A), to a conjugate moiety (or region C).
  • a biocleavable linker also referred to as the physiologically labile linker, Nuclease Susceptible Physiological Labile Linkages, or nuclease susceptible linker
  • the phosphate nucleotide linker such as region B
  • a peptide linker which joins the oligomer (or contiguous nucleotide sequence or region A), to a conjugate moiety (or region C).
  • the susceptibility to cleavage in the assays shown in Example 7 can be used to determine whether a linker is biocleavable or physiologically labile.
  • 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.
  • 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 60% cleaved, such as at least about 70% cleaved, such as at least about 75% cleaved, in the liver or kidney homogenate assay of Example 7.
  • the cleavage (%) in serum, as used in the assay in Example 7 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%.
  • the biocleavable linker also referred to as the physiologically labile linker, or nuclease susceptible linker
  • 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 7.
  • 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 120 min incubation with S1 nuclease according to the assay used in Example 7.
  • region B does not form a complementary sequence when the oligonucleotide region A and B is aligned to the complementary target sequence.
  • region B does form a complementary sequence when the oligonucleotide region A and B is aligned to the complementary target sequence.
  • region A and B together may form a single contiguous sequence which is complementary to the target sequence.
  • 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.
  • 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).
  • the potency of the compound for target down-regulation may be optimized for the desired tissue/cell.
  • region B comprises a dinucleotide of sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein C may be 5-methylcysteine, and/or T may be replaced with U.
  • region B comprises a trinucleotide of sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG wherein C may be 5-methylcysteine and/or T may be replaced with U.
  • region B comprises a trinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, GATX, CALX, CAGX, CTAX, CTGX, CTCX, CTTX, COAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCG
  • 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).
  • the invention further provides for the LNA oligomer intermediates which comprise an antisense LNA oligomer which comprises an (e.g. terminal, 5′ or 3′) amino alkyl, such as a C2-C36 amino alkyl group, including, for example C6 and C12 amino alkyl groups.
  • the amino alkyl group may be added to the LNA oligomer as part of standard oligonucleotide synthesis, for example using a (e.g. protected) amino alkyl phosphoramidite.
  • the linkage group between the amino alkyl and the LNA oligomer may for example be a phosphorothioate or a phosphodiester, or one of the other nucleoside linkage groups referred to herein, for example.
  • the amino alkyl group may be covalently linked to, for example, the 5′ or 3′ of the LNA oligomer, such as by the nucleoside linkage group, such as phosphorothioate or phosphodiester
  • the invention also provides a method of synthesis of the LNA oligomer comprising the sequential synthesis of the LNA oligomer, such as solid phase oligonucleotide synthesis, comprising the step of adding a amino alkyl group to the oligomer, such as e.g. during the first or last round of oligonucleotide synthesis.
  • the method of synthesis my further comprise the step of reacting the a conjugate to the amino alkyl-LNA oligomer (the conjugation step).
  • the a conjugate may comprise suitable linkers and/or branch point groups, and optionally further conjugate groups, such as hydrophobic or lipophilic groups, as described herein.
  • the conjugation step may be performed whilst the oligomer is bound to the solid support (e.g. after oligonucleotide synthesis, but prior to elution of the oligomer from the solid support), or subsequently (i.e. after elution).
  • the invention provides for the use of an amino alkyl linker in the synthesis of the oligomer of the invention.
  • the invention provides for a method of synthesizing (or manufacture) of an oligomeric compound, such as the oligomeric compound of the invention, said method comprising either:
  • steps f), g) or h) are performed either prior to or subsequent to cleavage of the oligomeric compound from the oligonucleotide synthesis support.
  • the method may be performed using standard phosphoramidite chemistry, and as such the region X and/or region X or region X and Y may be provided, prior to incorporation into the oligomer, as a phosphoramidite. Please see FIGS. 5-10 which illustrate non-limiting aspects of the method of the invention.
  • the invention provides for a method of synthesizing (or manufacture) of an oligomeric compound, such as the oligomeric compound of the invention, said method comprising a step of [sequential] oligonucleotide synthesis of a first region (A) and optionally a second region (B), wherein the synthesis step is followed by a step of adding a third region [phosphoramidite comprising] region X (also referred to as region C) or Y, such as a region comprising a group selected from the group consisting of a conjugate, a targeting group, a blocking group, a functional group, a reactive group [e.g. an amine or an alcohol] or an activation group (X), or an -Y-X group followed by the cleavage of the oligomeric compound from the [solid phase] support.
  • a third region [phosphoramidite comprising] region X also referred to as region C
  • Y such as a region comprising a group selected from the group consist
  • the region X or X-Y may be added after the cleavage from the solid support.
  • the method of synthesis may comprise the steps of synthesizing a first (A), and optionally second region (B), followed by the cleavage of the oligomer from the support, with a subsequent step of adding a third region, such as X or X-Y group to the oligomer.
  • the addition of the third region may be achieved, by example, by adding an amino phosphoramidite unit in the final step of oligomer synthesis (on the support), which can, after cleavage from the support, be used to join to the X or X-Y group, optionally via an activation group on the X or Y (when present) group.
  • region B may be a non-nucleotide cleavable linker for example a peptide linker, which may form part of region X (also referred to as region C) or be region Y (or part thereof).
  • region X such as C
  • X-Y such as the conjugate (e.g. a GalNAc conjugate) comprises an activation group, (an activated functional group) and in the method of synthesis the activated conjugate (or region x, or X-Y) is added to the first and second regions, such as an amino linked oligomer.
  • the amino group may be added to the oligomer by standard phosphoramidite chemistry, for example as the final step of oligomer synthesis (which typically will result in amino group at the 5′ end of the oligomer).
  • a protected amino-alkyl phosphoramidite is used, for example a TFA-aminoC6 phosphoramidite (6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite).
  • Region X or region C as referred to herein
  • the conjugate e.g. a GalNac conjugate
  • the conjugate e.g. a GalNac conjugate
  • N-hydroxysuccinimide may be used as activating group for region X (or region C, such as a conjugate, such as a GalNac conjugate moiety.
  • the invention provides an oligomer prepared by the method of the invention.
  • region X and/or region X or region X and Y may be covalently 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.
  • a phosphate nucleoside linkage such as those described herein, including phosphodiester or phosphorothioate
  • an alternative group such as a triazol group.
  • the internucleoside linkage between the first and second region is a phosphodiester linked to the first (or only) DNA or RNA nucleoside of the second region, or region B comprises at least one phosphodiester linked DNA or RNA nucleoside.
  • the second region may, in some embodiments, comprise further DNA or RNA nucleosides which may be phosphodiester linked.
  • the second region 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.
  • 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.
  • the oligomeric compound comprises a cleavable (labile) linker.
  • the cleavable linker is preferably present in region B (or in some embodiments, between region A and B).
  • the invention provides a non-phosphodiester linked, such as a phosphorothioate linked, oligonucleotide (e.g. an antisense oligonucleotide) which has at least one terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacent nucleoside of the oligonucleotide via a phosphodiester linkage, wherein the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting moiety or a blocking moiety, optionally via a linker moiety.
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • a phosphorothioate linked, oligonucleotide e.g. an antisense oligonucleotide
  • the terminal DNA or RNA nucleoside is further covalently linked to a conjugate moiety, a targeting
  • the oligomer of the invention may be used in pharmaceutical formulations and compositions.
  • 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.
  • the oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
  • such oligomers may be used to specifically inhibit the synthesis of APOB 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.
  • APOB protein typically by degrading or inhibiting the mRNA and thereby prevent protein formation
  • the oligomers may be used to detect and quantitate APOB expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
  • an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of APOB is treated by administering oligomeric compounds in accordance with this invention.
  • oligomeric compounds in accordance with this invention.
  • 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 APOB 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.
  • oligomers and other compositions according to the invention can be used for the treatment of conditions associated with over expression or expression of mutated version of the ApoB.
  • 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 as referred to herein.
  • one aspect of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels and/or activity of APOB, comprising administering to the mammal and therapeutically effective amount of an oligomer targeted to APOB 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.
  • the disease or disorder may, in some embodiments be associated with a mutation in the APOB gene or a gene whose protein product is associated with or interacts with APOB. Therefore, in some embodiments, the target mRNA is a mutated form of the APOB sequence.
  • An interesting aspect of the invention is directed to the use of an oligomer (compound) as defined herein or a conjugate as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition as referred to herein.
  • the methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of APOB.
  • the invention is furthermore directed to a method for treating abnormal levels and/or activity of APOB, said method comprising administering a oligomer of the invention, or a conjugate of the invention or a pharmaceutical composition of the invention to a patient in need thereof.
  • the invention also relates to an oligomer, a composition or a conjugate as defined herein for use as a medicament.
  • the invention further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of APOB or expression of mutant forms of APOB (such as allelic variants, such as those associated with one of the diseases referred to herein).
  • 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.
  • treatment 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 recognised that treatment as referred to herein may, In some embodiments, be prophylactic.
  • the invention relates to compounds or compositions comprising compounds for treatment of hypercholesterolemia and related disorders, or methods of treatment using such compounds or compositions for treating hypercholesterolemia and related disorders, wherein the term “related disorders” when referring to hypercholesterolemia refers to one or more of the conditions selected from the group consisting of: atherosclerosis, hyperlipidemia, hypercholesterolemia, familiar hypercholesterolemia e.g. gain of function mutations in APOB, HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD.
  • FCHL familial hyperlipidemia
  • CAD coronary artery disease
  • CHD coronary heart disease
  • mouse experiments may be performed as follows:
  • mice 7-10 week old C57Bl6-N mice were used, animals were age and sex matched (females for study 1, 2 and 4, males in study 3). Compounds were injected i.v. into the tail vein. For intermediate serum sampling, 2-3 drops of blood were collected by puncture of the vena facialis, final bleeds were taken from the vena cava inferior. Serum was collected in gel-containing serum-separation tubes (Greiner) and kept frozen until analysis.
  • Gel-containing serum-separation tubes Gel-containing serum-separation tubes (Greiner) and kept frozen until analysis.
  • RNA isolation and mRNA analysis mRNA analysis from tissue was performed using the Qantigene mRNA quantification kit (“bDNA-assay”, Panomics/Affimetrix), following the manufacturers protocol. For tissue lysates, 50-80 mg of tissue was lysed by sonication in 1 ml lysis-buffer containing Proteinase K. Lysates were used directly for bDNA-assay without RNA extraction. Probesets for the target and GAPDH were obtained custom designed from Panomics. For analysis, luminescence units obtained for target genes were normalized to the housekeeper GAPDH.
  • Serum analysis for ALT, AST and cholesterol was performed on the “Cobas INTEGRA 400 plus” clinical chemistry platform (Roche Diagnostics), using 10 ⁇ l of serum.
  • BIOPHEN FVII enzyme activity kit (#221304, Hyphen BioMed) was used according to the manufacturer's protocol.
  • a fluorescently-labeled PNA probe is hybridized to the oligo of interest in the tissue lysate.
  • the same lysates are used as for bDNA-assays, just with exactly weighted amounts of tissue.
  • the heteroduplex is quantified using AEX-HPLC and fluorescent detection.
  • Oligonucleotides were synthesized on uridine universal supports using the phosphoramidite approach on an Expedite 8900/MOSS synthesizer (Multiple Oligonucleotide Synthesis System) at 4 ⁇ mol scale. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using aqueous ammonia for 1-2 hours at room temperature, and further deprotected for 16 hours at 65° C. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC) and characterized by UPLC, and the molecular mass was further confirmed by ESI-MS. See below for more details.
  • RP-HPLC reverse phase HPLC
  • UPLC UPLC
  • the coupling of ⁇ -cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-T or C6-S—S linker) is performed by using a solution of 0.1 M of the 5′-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator.
  • DCI 4,5-dicyanoimidazole
  • Thiolation for introduction of phosphorthioate linkages is carried out by using xanthane hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphordiester linkages are introduced using 0.02 M iodine in THF/Pyridine/water 7:2:1. The rest of the reagents are the ones typically used for oligonucleotide synthesis.
  • a commercially available C6 aminolinker phorphoramidite was used in the last cycle of the solid phase synthesis and after deprotection and cleavage from the solid support the aminolinked deprotected oligonucleotide was isolated.
  • the conjugates was introduced via activation of the functional group using standard synthesis methods.
  • the crude compounds were purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10 ⁇ 150 ⁇ 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile was used as buffers at a flowrate of 5 mL/min. The collected fractions were lyophilized to give the purified compound typically as a white solid.
  • RNAlater Sampling of liver and kidney tissue. The animals were anaesthetised with 70% CO 2 -30% O 2 and sacrificed by cervical dislocation according to the table above. One half of the large liver lobe and one kidney were minced and submerged in RNAlater.
  • Total RNA Isolation and First strand synthesis Total RNA was extracted from maximum 30 mg of tissue homogenized by bead-milling in the presence of RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen cat. no. 74106) according to the manufacturer's instructions. First strand synthesis was performed using Reverse Transcriptase reagents from Ambion according to the manufacturer's instructions.
  • RNA 0.5 ⁇ g total RNA was adjusted to (10.8 ⁇ l) with RNase free H 2 O and mixed with 2 ⁇ l random decamers (50 ⁇ M) and 4 ⁇ l dNTP mix (2.5 mM each dNTP) and heated to 70° C. for 3 min after which the samples were rapidly cooled on ice.
  • 2 ⁇ l 10 ⁇ Buffer RT, 1 ⁇ l MMLV Reverse Transcriptase (100 U/ ⁇ l) and 0.25 ⁇ l RNase inhibitor (10 U/ ⁇ l) were added to each sample, followed by incubation at 42° C. for 60 min, heat inactivation of the enzyme at 95° C. for 10 min and then the sample was cooled to 4° C.
  • cDNA samples were diluted 1:5 and subjected to RT-QPCR using Taqman Fast Universal PCR Master Mix 2 ⁇ (Applied Biosystems Cat #4364103) and Taqman gene expression assay (mApoB, Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers protocol and processed in an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7) in fast mode.
  • Seq ID #3 was conjugated to either monoGalNAc, Folic acid, FAM or Tocopherol using a non-cleavable linker or biocleavable linker (Dithio (SS) or 2 DNA nucleotides with Phosphodiester backbone (PO)). Additionally the monoGalNAc was compared to a GalNAc cluster (Conjugate 2a). C57BL6In mice were treated i.v. with saline control or with a single dose of 1 or 0.25 mg/kg of ASO conjugates. After 7 days the animals were sacrificed and RNA was isolated from liver and kidney samples and analysed for ApoB mRNA expression ( FIG. 15 ).
  • Tocopherol conjugated to the ApoB compound with a DNA/PO-linker increased ApoB knock down in the liver compared to the unconjugated ApoB compound (#3) while decreasing activity in kidney (compare FIGS. 15A and B). This points towards an ability of the Tocopherol to redirect the ApoB compound from kidney to liver.
  • the non-cleavable (#24) and SS-linker (#25) were inactive in both tissues.
  • Mono-GalNAc conjugates with a non-cleavable (#17) and with bio-cleavable DNA/PO linker (#19) show a tendency to preserve the activity of the unconjugated compound (#3) in kidney while improving activity in the Liver.
  • FIGS. 15A and B Introduction of a SS-linker decreased activity in both tissues (compare FIGS. 15A and B). Conjugation of different GalNAc conjugates e.g. mono GalNAcPO (#19) and a GalNAc cluster (#20) also allows fine tuning of the compound activity with focus on either liver or kidney ( FIG. 15C ). Folic acid and FAM conjugates with the cleavable DNA/PO-linker (SEQ ID Nos16 and 23) behave comparable to the unconjugated compound (3).
  • the introduction of a non-cleavable (14 and 21) or SS-linker (15 and 22) decreases compound activity in both tissues (compare FIGS. 15 a and 15 b ).
  • RNA isolation and mRNA analysis Total RNA was extracted from liver and kidney samples and ApoB mRNA levels were analysed using a branched DNA assay
  • the primary objective for this study is to investigate selected lipid markers over 7 weeks after a single slow bolus injection of anti-ApoB LNA conjugated compounds to cynomolgus monkeys and assess the potential toxicity of compounds in monkey.
  • the compounds used in this study are SEQ ID NOs 7, 20, 28 & 29, prepared in sterile saline (0.9%) at an initial concentration of 0.625 and 2.5 mg/ml).
  • mice of at least 24 months old are used, and given free access to tap water and 180 g of OWM(E) SQC SHORT expanded diet (Dietex France, SDS, Saint Gratien, France) will be distributed daily per animal.
  • the total quantity of food distributed in each cage will be calculated according to the number of animals in the cage on that day.
  • fruit or vegetables will be given daily to each animal.
  • the animals will be acclimated to the study conditions for a period of at least 14 days before the beginning of the treatment period. During this period, pre-treatment investigations will be performed.
  • the animals are dosed i.v. at a dose if, for example, 0.25 mg/kg or 1 mg/kg.
  • the dose volume will be 0.4 mL/kg. 2 animals are used per group.
  • the data will be analyzed and a second group of animals using a higher or lower dosing regimen may be initiated—preliminary dose setting is 0.5 mg/kg and 1 mg/kg, or lower than that based on the first data set.
  • the dose formulations will be administered once on Day 1. Animals will be observed for a period of 7 weeks following treatment, and will be released from the study on Day 51. Day 1 corresponds to the first day of the treatment period. Clinical observations and body weight and food intake (per group) will be recorded prior to and during the study.
  • Blood (approximately 1.0 mL) is taken into lithium heparin tubes (using the ADVIA 1650 blood biochemistry analyzer): Apo-B, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea, creatinine, total bilirubin (TBIL), total cholesterol, triglycerides, alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT),creatine kinase, gamma-glutamyl transferase (GGT), lactate dehydrogenase, total protein, albumin, albumin/globulin ratio.
  • Apo-B sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea, creatinine, total bilirubin (TBIL), total cholesterol, triglycerides, alkaline phosphata
  • Blood samples for ApoB analysis will be collected from Group 1-16 animals only (i.e. animals treated with anti-PCSK9 compounds) on Days-8, -1, 4, 8, 15, 22, 29, 36, 43 and 50.
  • Venous blood (approximately 2 mL) will be collected from an appropriate vein in each animal into a Serum Separating Tube (SST) and allowed to clot for at least 60 ⁇ 30 minutes at room temperature.
  • Blood will be centrifuged at 1000 g for 10 minutes under refrigerated conditions (set to maintain +4° C.). The serum will be transferred into 3 individual tubes and stored at ⁇ 80° C. until analyzed at CitoxLAB France using an ELISA method (Circulex Human PCSK9 ELISA kit, CY-8079, validated for samples from cynomolgus monkey).
  • WO2010142805 provides the methods for the following analysis: qPCR, ApoB mRNA analysis.
  • Other analysis includes ApoB protein ELISA, serum Lp(a) analysis with ELISA (Mercodia No. 10-1106-01), tissue and plasma oligonucleotide analysis (drug content), Extraction of samples, standard- and QC-samples, Oligonucleotide content determination by ELISA.
  • Compounds of the invention can be evaluated for their toxicity profile in rodents, such as in mice or rats.
  • rodents such as in mice or rats.
  • the following protocol may be used: Wistar Han Crl:Wl(Han) are used at an age of approximately 8 weeks old. At this age, the males should weigh approximately 250 g. All animals have free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiuschen GmbH, Soest, Germany) and to tap water (filtered with a 0.22 ⁇ m filter) contained in bottles. The dose level of 10 and 40 mg/kg/dose is used (sub-cutaneous administration) and dosed on days 1 and 8. The animals are euthanized on Day 15. Urine and blood samples are collected on day 7 and 14. A clinical pathology assessment is made on day 14.
  • Body weight is determined prior to the study, on the first day of administration, and 1 week prior to necropsy. Food consumption per group will be assessed daily. Blood samples are taken via the tail vein after 6 hours of fasting. The following blood serum analysis is performed: erythrocyte count mean cell volume packed cell volume hemoglobin mean cell hemoglobin concentration mean cell hemoglobin thrombocyte count leucocyte count differential white cell count with cell morphology reticulocyte count, sodium potassium chloride calcium inorganic phosphorus glucose urea creatinine total bilirubin total cholesterol triglycerides alkaline phosphatase alanine aminotransferase aspartate aminotransferase total protein albumin albumin/globulin ratio.
  • Urinalysis are performed ⁇ -GST, ⁇ -2 Microglobulin, Calbindin, Clusterin, Cystatin C, KIM-1, Osteopontin, TIMP-1, VEGF, and NGAL. Seven analytes (Calbindin, Clusterin, GST- ⁇ , KIM-1, Osteopontin, TIMP-1, VEGF) will be quantified under Panel 1 (MILLIPLEX® MAP Rat Kidney Toxicity Magnetic Bead Panel 1, RKTX1MAG-37K).
  • ⁇ -2 Microglobulin, Cystatin C, Lipocalin-2/NGAL Three analytes ( ⁇ -2 Microglobulin, Cystatin C, Lipocalin-2/NGAL) will be quantified under Panel 2 (MILLIPLEX® MAP Rat Kidney Toxicity Magnetic Bead Panel 2, RKTX2MAG-37K).
  • the assay for the determination of these biomarkers' concentration in rat urines is based on the Luminex xMAP® technology.
  • Microspheres coated with anti- ⁇ -GST/ ⁇ -2 microglobulin/calbindin/clusterin/cystacin C/KIM-1/osteopontin/TIMP-1/VEGF/NGAL antibodies are color-coded with two different fluorescent dyes. The following parameters are determined (Urine using the ADVIA 1650): Urine protein, urine creatinine.
  • Quantitative parameters volume, pH (using 10-Multistix SG test strips/Clinitek 500 urine analyzer), specific gravity (using a refractometer).
  • Semi-quantitative parameters using 10-Multistix SG test strips/Clinitek 500 urine analyzer: proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment (by microscopic examination).
  • Qualitative parameters Appearance, color. After sacrifice, the body weight and kidney, liver and spleen weight are determined and organ to body weight ratio calculated. Kidney and liver samples will be taken and either frozen or stored in formalin. Microscopic analysis is performed.
  • LNA nucleosides such as beta-D-oxy LNA
  • DNA nucleosides lower case letters are DNA nucleosides.
  • Subscript s represents a phosphorothioate internucleoside linkages.
  • LNA cytosines are optionally 5-methyl cytosine.
  • FAM-labelled ASOs with different DNA/PO-linkers were subjected to in vitro cleavage either in S1 nuclease extract, Liver or kidney homogenates or Serum.
  • FAM-labeled ASOs 100 ⁇ M with different DNA/PO-linkers were subjected to in vitro cleavage by S1 nuclease in nuclease buffer (60 U pr. 100 ⁇ L) for 20 and 120 minutes (see table below). 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 10 mM-1 M sodium perchlorate at pH 7.5. The content of cleaved and non cleaved oligonucleotide were determined against a standard using both a fluoresense detector at 615 nm and a uv detector at 260 nm.
  • the PO linkers 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 to modulate 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 were spiked with oligonucleotide SEQ ID NO 32 to concentrations of 200 ⁇ g/g tissue (see table below).
  • 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 NaCl, 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 determined against a standard using the above HPLC method.
  • the PO linkers 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 assays shown in Example 7 may be used to determine whether a linker is biocleavable or physiologically labile.
  • LNA nucleosides such as beta-D-oxy LNA
  • lower case letters are a DNA nucleoside.
  • Subscript s represents a phosphorothioate internucleoside linkage (region A).
  • LNA cytosines are optionally 5-methyl cytosine.
  • the 2PO linker (region B) is 5′ to the sequence region A, and comprises of two DNA nucleosides linked by phosphodiester linkage, with the internucleoside linkage between the 3′ DNA nucleoside of region A and the 5′ LNA nucleoside of region A also being phosphodiester.
  • a linkage group (Y) may be used to link the conjugate group, when present, to region B, or A (SEQ ID NO 7, 20 and 30).
  • C57BL6/J mice were injected either iv or sc with a single dose saline or 0.25 mg/kg unconjugated LNA-antisense oligonucleotide (SEQ ID NO3) or equimolar amounts of LNA antisense oligonucleotides conjugated to GalNAc1, GalNAc2, or cholesterol (2PO) and sacrificed at days 1-7 according to the table below (experimental design).
  • GalNAc1 and GalNAc2 conjugated to an ApoB LNA antisense oligonucleotide showed knock down of ApoB mRNA better than the unconjugated ApoB LNA ( FIG. 16 ).
  • GalNAc 1 conjugate SEQ ID NO 30
  • iv dosing is better than sc dosing which is surprising since the opposite has been reported for another GalNAc clusters (Alnylam, 8th Annual Meeting of the Oligonucleotide Therapeutics Society).
  • the total cholesterol data show how the GalNAc cluster conjugates (SEQ ID NO 30 and 20) gives better effect than the unconjugated and the cholesterol conjugated compounds (SEQ ID NO 7) both at iv and sc administration ( FIG. 17 , a and b).
  • the tissue content of the oligonucleotides shows how the conjugates enhances the uptake in liver while giving less uptake in kidney compared to the parent compound. This holds for both iv and sc administration.
  • GalNAc 1 SEQ ID NO 30
  • GalNAc 2 SEQ ID NO 20
  • activity is good for both compounds the GalNAc 2 conjugate appears to induce a higher specific activity than GalNAc 1 conjugate indicating that GalNAc conjugates without the pharmacokinetic modulator may be particularly useful with LNA antisense oligonucleotides.
  • liver and kidney tissue Sampling of liver and kidney tissue. The animals were anaesthetised with 70% CO 2 -30% O 2 and sacrificed by cervical dislocation according to the above table. One half of the large liver lobe and one kidney were minced and submerged in RNAlater. The other half of liver and the other kidney was frozen and used for tissue analysis.
  • Total RNA Isolation and First strand synthesis Total RNA was extracted from maximum 30 mg of tissue homogenized by bead-milling in the presence of RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen cat. no. 74106) according to the manufacturer's instructions. First strand synthesis was performed using Reverse Transcriptase reagents from Ambion according to the manufacturer's instructions.
  • RNA 0.5 ⁇ g total RNA was adjusted to (10.8 ⁇ l) with RNase free H 2 O and mixed with 2 ⁇ l random decamers (50 ⁇ M) and 4 ⁇ l dNTP mix (2.5 mM each dNTP) and heated to 70° C. for 3 min after which the samples were rapidly cooled on ice.
  • 2 ⁇ l 10 ⁇ Buffer RT, 1 ⁇ l MMLV Reverse Transcriptase (100 U/ ⁇ l) and 0.25 ⁇ l RNase inhibitor (10 U/ ⁇ l) were added to each sample, followed by incubation at 42° C. for 60 min, heat inactivation of the enzyme at 95° C. for 10 min and then the sample was cooled to 4° C.
  • cDNA samples were diluted 1:5 and subjected to RT-QPCR using Taqman Fast Universal PCR Master Mix 2 ⁇ (Applied Biosystems Cat #4364103) and Taqman gene expression assay (mApoB, Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers protocol and processed in an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7) in fast mode. Oligonucleotide content in liver and kidney was measured by sandwich ELISA method.
  • Serum cholesterol analysis Immediately before sacrifice retro-orbital sinus blood was collected using S-monovette Serum-Gel vials (Sarstedt, Nümbrecht, Germany) for serum preparation. Serum was analyzed for total cholesterol using ABX Pentra Cholesterol CP (Triolab, Brondby, Denmark) according to the manufacturer's instructions.

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