Galnac phosphoramidites and their uses

Abstract

The invention generally relates to the field of phosphoramidite derivatives. In particular, the invention relates to N-Acetylgalactosamine phosphoramidite molecules and to conjugates of nucleic acid molecules with N-Acetylgalactosamine phosphoramidite containing molecules, and more specifically, to variants of N-Acetylgalactosamine phosphoramidite molecules and to conjugates of nucleic acid molecules with said variants of N-Acetylgalactosamine phosphoramidite containing molecules. Further, the invention provides associated methods of producing and using these molecules, in particular, in the field of medicine. More precisely, the N-Acetylgalactosamine derivatives of the present invention bear in the position 6' a protecting group selected from TMTr, DMTr, MMTr, Fmoc, Px, DMPx.

Description

[0001] GalNAc Phosphoramidites and Their Uses

[0002] Field of the invention

[0003] The invention generally relates to the field of phosphoramidite derivatives. In particular, the invention relates to N-Acetylgalactosamine phosphoramidite molecules and to conjugates of nucleic acid molecules with N-Acetylgalactosamine phosphoramidite containing molecules, and more specifically, to variants of N-Acetylgalactosamine phosphoramidite molecules and to conjugates of nucleic acid molecules with said variants of N-Acetylgalactosamine phosphoramidite containing molecules. Further, the invention provides associated methods of producing and using these molecules, in particular, in the field of medicine.

[0004] Background

[0005] Nucleic acid molecules in therapy are gaining more and more attention in recent years, with several new therapeutic modalities having been developed. Nucleic acid molecules have been conjugated to a variety of ligands including, inter alia, peptides, lipids, sterols, and carbohydrates. In particular, small interfering RNAs (siRNAs), which are of increasing importance in the context of RNA interference (RNAi) therapies, have been evaluated extensively. One currently known approach is the attachment to the nucleic acid of a conjugate moiety containing terminal N-acetyl galactosamine (GalNAc) or a derivative thereof, thereby targeting the nucleic acid molecule to hepatocytes via specific binding of GalNAc to cell surface asialoglycoprotein receptors (ASGPR).

[0006] The Asialoglycoprotein receptor (ASGP-R) is a high capacity receptor which is highly abundant on hepatocytes. One of the first disclosures of triantennary cluster glycosides was in US patent number US 5,885,968. Conjugates having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (2003). The ASGP-R shows a 50-fold higher affinity for N-Acetyl-D-Galactosamine (GalNAc) than D-Gal. Hepatocytes expressing the lectin asialoglycoprotein receptor (ASGPR) which recognizes specifically terminal 0- galactosyl subunits of glycosylated proteins or other oligosaccharides (P. H. Weigel et. al., 2002,) can be used for targeting a drug to the liver by covalent coupling of galactose or galactosamine to the drug substance (S. Ishibashi, et. al. 1994). Furthermore, the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting unit (E. A. L. Biessen et. al., 1995). The ASGPR is a mediator for an active endosomal transport of terminal p-galactosyl containing glycoproteins, thus ASGPR is highly suitable for targeted delivery of drug candidates like siRNA, which have to be delivered into a cell (Akinc et al.).

[0007] US. Pat. No. 11 ,174,483 describes a nucleic acid that interferes with TMPRSS6 gene expression or inhibits its expression and its therapeutic use, wherein said nucleic acid is conjugated to a ligand which comprises one or more of the aforementioned GalNAc building blocks. In particular, the ligand comprising one or more GalNAc moieties and / or derivatives thereof is conjugated through a linker to the TMPRSS6 directed nucleic acid.

[0008] GalNAc conjugation to siRNA can be achieved in three major ways. Application of 1) preformed trivalent or even higher valency cluster building blocks (Nair, J.K., Willoughby, J.L.S., Chan, A., Charisse, K., Alam, M.R., Wang, Q., Hoekstra, M., Kandasamy, P., Kel’in, A.V., Milstein, S., et al. (2014). Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136, 16958-16961.), 2) consecutive and serially used mono valent GalNAc nucleosidic (Matsuda, S., Keiser, K., Nair, J.K., Charisse, K., Manoharan, R.M., Kretschmer, P., Peng, C.G., V Kel’in, A., Kandasamy, P., Willoughby, J.L., et al. (2015). ACS Chem. Biol. 10, 1181-1187.) or non-nucleosidic building blocks (Matulic-Adamic, J., Serebryany, V., Haeberli, P., Mokler, V.R., and Beigelman, L. (2002). Bioconjug. Chem. 13, 1071-1078.), and 3) a combination of brancher building block and monovalent GalNAc building block that is simultaneously added to each arm of the brancher (Dubber, M., and Frechet, J.M.J. (2003). Bioconjug. Chem. 14,239-246).

[0009] To use such building blocks in state-of-the-art solid phase synthesis of oligonucleotides such building blocks are for example prepared as phosphoramidites. Phosphoramidites are generally stable and storable compounds. A GalNAc phosphoramidite building block that is used as described above in 3) would consist of the GalNAc equipped with suitable protective groups at the hydroxyl groups (e.g. acetyl) linked through the oxygen atom at the anomeric centre to a linker arm of variable length and structure that again is connected via an oxygen to the P(lll) atom equipped with the O-cyanoethyl group and the diisopropyl amine (or similar) as activatable leaving group for coupling in solid phase synthesis.

[0010] The N-Acetylgalactosamine phosphoramidite for the use as described above must be synthesized yearly at the kilogram scale to supply siRNA production for the mentioned therapies. However, production of these phosphoramidites and their subsequent use in siRNA synthesis is made difficult due to its physical properties, which are isolated as resinous oils. In particular, fully acetylated C4-GalNAc phosphoramidite oil typically forms a foam during the final concentration and aliquoting steps, limiting the amount of material that can be dispensed into a single bottle. As a result, the phosphoramidite must be shipped to oligonucleotide production facilities in a large number of bottles at significant expense. Handling and reconstitution of high numbers of individual containers at the location of use bears a high risk for the purity and quality of the raw material. Impaired quality and purity have a direct impact on the yield, quality and purity of the final siRNA product. Furthermore, purification and analysis of the monomer by chromatographic methods is hampered because of the phosphoramidite’s lack of absorption in the UV region, which is a major drawback.

[0011] It is therefore an object of the invention to provide novel N-Acetylgalactosamine phosphoramidite molecules which are easy to produce and have favorable physical properties, facilitating handling, purification and analysis.

[0012] Summary of the invention

[0013] In one aspect, the present invention relates to variants of N-acetyl galactosamine (GalNAc) phosphoramidites.

[0014] In one aspect, the invention relates to a compound having the formula (I) wherein

[0015] A’ is selected from and and

[0016] R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx.

[0017] In one aspect, the invention relates to a compound selected from the group consisting of: wherein R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx.

[0018] In one aspect, the invention relates to a compound selected from the group consisting of: wherein R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx, and wherein Z is a nucleic acid.

[0019] In one aspect, the invention relates to a method of producing any of the above-mentioned compounds.

[0020] Detailed description of the invention

[0021] The definitions and explanations below are for the terms as used throughout this entire document including both the specification and the claims.

[0022] Unless specified otherwise, the following terms have the following meanings:

[0023] “Conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamics, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties.

[0024] “GalNAc” means N-acetyl galactosamine. The term “variants of N-acetylgalactosamine (GalNAc)” as used herein means any GalNAc having different protecting groups and / or linkers at the positions further described in detail in the description.

[0025] The term “phosphoramidite” as used herein means any compound containing a phosphorous atom in oxidation state III which is bound covalently to at least one nitrogen atom. Preferred phosphoramidites of the invention are compounds containing a phosphorous atom in oxidation state III which is bound covalently to at least one nitrogen atom and two oxygen atoms. Alternative phosphoramidites of the invention are compounds containing a phosphorous atom in oxidation state III which is bound covalently to at least one nitrogen atom and one oxygen atom and has a sulphur atom or a C, -CO- alkyl group instead of a second oxygen atom.

[0026] Cx-Cy alkyl refers to a saturated aliphatic hydrocarbon group having x-y carbon atoms which may be linear or branched. For example, C1-C6 alkyl and includes Ci, C2, C3, C4, Cs and Ce. “Branched” means that at least one carbon branch point is present in the group. For example, tert-butyl and isopropyl are both branched groups. Examples of C1-C6 alkyl groups include methyl, ethyl, propyl, butyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1 -butyl, 3 methyl-1- butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-pentyl, 3-methyl-1-pentyl, 4-methyl-1- pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3- dimethyl-1-butyl, 2-ethyl-1 -butyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl and n-hexyl. This also applies for C1-C6 alkylene.

[0027] Cx-Cy alkoxy refers to a group or part of a group having an -O-Cx-Cy alkyl group according to the definition of Cx-Cy alkyl above. C1-C3 alkoxy contains from 1 to 3 carbon atoms and includes Ci, C2 and C3. Examples of C1-C3 alkoxy include methoxy, ethoxy, propoxy and isopropoxy. Alkoxy as employed herein also extends to embodiments in which the O, an oxygen atom (e.g., a single oxygen atom) is located within the alkyl chain, for example CH2CH2OCH3 or CH2OCH3. Thus, the alkoxy may be linked through carbon to the remainder of the molecule, for example, -CH2CH2OCH3, or alternatively, the alkoxy is linked through oxygen to the remainder of the molecule, for example -OC1-3 alkyl. In certain instances, the alkoxy may be linked through oxygen to the remainder of the molecule but the alkoxy group contains a further oxygen atom, for example -OCH2CH2OCH3. The term “nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA), double-stranded nucleic acids (dsDNA), small interfering ribonucleic acids (siRNA) and microRNAs (miRNA). A nucleic acid may also comprise any combination of these elements in a single molecule.

[0028] The term "treat" or "treating" or “treatment” may include prophylaxis and means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition. The compounds of the invention are useful in the treatment of humans and nonhuman animals.

[0029] By "effective amount" or "therapeutically effective amount" or "effective dose" is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder. Prevention of the disorder is manifested by delaying the onset of the symptoms of the disorder to a medically significant extent. T reatment of the disorder is manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.

[0030] A “pharmaceutical composition” or “composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a pharmaceutical carrier e.g., a sterile aqueous solution.

[0031] In one aspect, the present invention relates to N-acetyl galactosamine (GalNAc) phosphoramidites and variants thereof. Accordingly, the invention provides a compound having the general formula (I): wherein A’ is a linker group consisting of an oxygen atom (O) directly linked to a hydrocarbon chain with a length of 2-18 carbon atoms, wherein one or more of the carbon atoms in the chain may each independently be replaced by -NHCO, CONH- and / or a heteroatom, particularly O. In some aspects, the linker group A’ is selected from the group consisting of C1-C30- alkenylene, -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2)1O-, -(CH2)11-, -(CH2)I2-, -CH2CH2OCH2CH2-, -CH2CH2OCH2CH2OCH2CH2-, and - CH2CH2(OCH2CH2)2OCH2CH2-, more preferably from -(CH2)4-, -(CH2)6-, and -(CH2)8-.

[0032] The linker group A’ is selected according to the desired application of the GalNAc phosphoramidite. In some aspects, one or more of the carbon atoms in the chain may each independently be replaced by -NH-CO-, -CO-NH- and / or a heteroatom, particularly O.

[0033] In some aspects, A’ is selected from 2-hydroxy-ethyl, 3-hydroxy-propyl, 4-hydroxy-butyl, 5- hydroxy-pentyl, 6-hydroxy-hexyl, 7-hydroxy-heptyl, 8-hydroxy-octyl, 9-hydroxy-nonyl, 10- hydroxy-decyl, 11 -hydroxy-undecyl, 12-hydroxy-dodecyl;1-Hydroxy-3-oxapentyl, 1-hydroxy- 3,6- dioxaoctyl, 1-hydroxy-3,6,9-trioxaundecanyl; 2-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) ethyl, 3-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) propyl, 4- (((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) butyl, 5-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) pentyl, 6-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) hexyl, 7-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) heptyl, 8- (((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) octyl, 9-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) nonyl, 10-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) decyl, 11-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) undecyl, 12-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) dodecyl; and

[0034] 3-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) 3-oxapentyl, 3-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy) 3,6-dioxaoctyl, 3-(((2-cyanoethoxy) (diisopropylamino) phosphaneyl)oxy)-3,6,9-trioxaundecanyl;

[0035] R is a protective group selected from the group consisting of 4,4',4"-trimethoxytrityl (TMTr), 4,4'-dimethoxytrity (DMTr), 4-monomethoxyltrityl (MMTr), fluorenylmethyloxycarbonyl (Fmoc), 9-phenylxanthyl (Px), or 5-0-2, 7-dimethylpixyl (DMPx). In one aspect, the protective group is DMTr or Px.

[0036] Preferably the resulting compound is a non-oily compound. As used herein, non-oily means not oily in substance or appearance, for example a solid under normal ambient conditions (e.g., 20°C, 101.3 kPa). As used here, a solid refers to matter in solid state that can be ground or otherwise broken down to a powder. In certain aspects, the compound may be an amorphous solid, polycrystalline or crystalline.

[0037] In one aspect, the present invention relates to certain compounds and their precursors. The provided compounds are as follows: wherein R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px, or DMPx.

[0038] In one aspect the protective group is DMTr or Px.

[0039] The compounds of formulas (ll)-(VII) are non-oily compounds. Preferably the compounds are solid or crystalline. In one aspect the compounds are solid.

[0040] In one aspect the invention provides non-oily compounds of the following formulas:

[0041] The compounds of formulas (VIII)-(XIII) are non-oily compounds. Preferably the compounds are solid or crystalline. In one aspect the compounds are solid. In one aspect the invention provides a method of producing compound (III) of (II), (V) of (IV), and (VII) of (VI), which equally corresponds to formulas (IX) of (VIII), (XI) of (X) and (XIII) of (XII). In all cases, the method involves the reaction of an unprotected hydroxyl group in the precursor with a phosphitylation reagent to generate the functional building block. This conversion can be achieved through the use of phosphitylation reagents such as 2-cyanoethyl diisopropylchlorophosphoramidite (PCI), 2-cyanoethyl N,N,N',N'- tetraisopropylphosphorodiamidite (PN), or others. precursor phosphitylation functional with free OH-group reagent building block e.g.: VIII, X, XII e.g.: PN e.g.: VIII, X, XII

[0042] A detailed example of the mentioned method is described below in Example 2. The solid GalNAc building blocks of formulas (I) to (XIII) may be used in a method of producing a conjugate compound, according to the structures (XIV) to (XXIII). The method comprises adding together several components, comprising at least one GalNAc building block, at least one branching unit, at least one bridging unit and at least one nucleic acid, wherein the at least one branching unit and the at least one bringing unit may be present in a single structure. Thus, the method comprises adding together one or more of the GalNAc building blocks to a branching unit, a bridging unit and a nucleic acid, wherein the linkage between the bridging unit and the nucleic acid may be a phosphate or thiophosphate. Preferably three GalNAc building blocks are added to a branching unit, a bridging unit and a nucleic acid. The GalNAc building blocks may be the same or different, preferably the GalNAc building blocks are the same. The branching unit provides three reactive sites in order to accommodate conjugation of the three saccharide ligands. The branching unit is covalently attached to the tethered ligands and the nucleic acid. The branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups. The branching unit may comprise groups selected from alkyl and ether groups.

[0043] The bridging unit may also be referred to as the conjugate linker. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid. It may be selected from - C1-C20 alkylene-, -C2-C20 alkenylene-, an alkylene ether of formula -(C1-C20 alkylene)-O-(Ci- C20 alkylene)-, -C(0)-C1-C20 alkylene-, -C0-C4 alkylene(Cy)Co-C4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C1-C4 alkylene-NHC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)NH-C1-C4 alkylene-, -C1-C4 alkylene-SC(O)-C1-C4 alkylene-, -C1-C4 alkylene- C(O)S-C1-C4 alkylene-, -C1-C4 alkylene-OC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)O-Ci-C4 alkylene-, and -C1-C6 alkylene-S-S-C1-C6 alkylene-. The bridging unit may be an alkylene ether of formula -(C1-C20 alkylene)-0-(C1-C20 alkylene)-. The bridging unit may be an alkylene ether of formula -(C1-C20 alkylene)-0-(C4-C2o alkylene)-, wherein said (C4-C20 alkylene) is linked to Z. The bridging unit may be selected from the group consisting of -CH2-O-C3He-, -CH2-O-C4H8- , -CH2-O-C6H12- and -CH2-O-C8H16-, especially -CH2-O-C4H8-, -CH2-O-C6H12- and -CH2-O- C8Hi6-, wherein in each case the -CH2- group is linked to the branching unit. Preferably, the bridging unit is selected from the group consisting of -C3H6-, -C4H8-, -CeHi2- and -C8H16-, especially -C4H8-, -C6H12- and -C8H16-. In one aspect the invention provides conjugate compounds having the structure: wherein Z is a nucleic acid and R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or

[0044] (XlVa)

[0045] In one aspect, the present invention provides conjugate compounds having the structure:

[0046] (XV) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0047] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or

[0048] (XVa) wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure:

[0049] (XVI) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0050] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or wherein Z is a nucleic acid.

[0051] In one aspect, the present invention provides conjugate compounds having the structure:

[0052] (XVII) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0053] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure: (XVIII) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or

[0054] (XVI 11 a) wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure:

[0055] (XIX) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0056] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure:

[0057] (XX) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or (XXa) wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure: (XXI) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0058] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or

[0059] (XXI a) wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure:

[0060] (XXII) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; or (XXI I a) wherein Z is a nucleic acid. In one aspect, the present invention provides conjugate compounds having the structure: (XXIII) wherein Z is a nucleic acid and R is a protective group selected from the group consisting of

[0061] TMTr, DMTr, MMTr, Fmoc, Px, and DMPx, wherein R is preferably DMTr or Px; wherein Z is a nucleic acid.

[0062] In all cases described herein, the nucleic acid comprises nucleic acid residues or building blocks that may be selected from the group consisting of DNA, RNA, PNA and LNA.

[0063] The nucleic acid may be a functional nucleic acid, whereby preferably the functional nucleic acid is selected from the group consisting of an oligonucleotide, mRNA, micro-RNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acid, ribozymes, aptamers and spiegelmers. In particular, the nucleic acid may be siRNA.

[0064] The term "oligonucleotide" (or simply "oligo") refers, in the context of the present invention, to a molecule formed by covalent linkage of two or more nucleobases. When used in the context of the oligonucleotide of the invention. The term ‘nucleobase’ refers to nucleotides, such as DNA and RNA, and nucleotide analogues. The term "microRNA" or "miRNA.", in the context of the present invention, means an RNA oligonucleotide consisting of between 18 to 25 nucleotides. In functional terms miRNAs are typically regulatory endogenous RNA molecules.

[0065] The nucleic acid may be selected from RNAi, siRNA, antisense nucleic acid, sgRNA, shRNA, ribozymes, aptamers and spiegelmers.

[0066] The nucleic acids of the invention may be of any length and can have any number of nucleotides such that they are therapeutically effective. The nucleic acid of the invention may be an oligonucleotide for RNAi, which may be in a length of from 8 to 26 nucleobases, 10 to 26, or 12 to 26. The oligonucleotide may have a length of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 nucleobases. In a preferred example, the oligonucleotide of the invention has complementarity to a human microRNA sequence, preferably it is 100% complementary to a human microRNA sequence.

[0067] When the nucleic acid of the invention is siRNA for RNAi, preferably, the siRNA will be of a length of from 15 to 30 nucleotides. The duplex region of a double stranded RNA may range from 15 to 30 nucleotide base pairs using the Watson-crick base pairing. The duplex region may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs.

[0068] Preferably, the nucleic acid of the invention, e.g., siRNA, has a length of 19 to 23 bases. For example, the nucleic acid may be 19, 20, 21 , 22 or 23 bases in length.

[0069] Double stranded siRNAs of the invention may be blunt ended at one end or on both ends. The double stranded siRNAs may have overhangs of 1 or more nucleotides on one or both strands at one or both ends. The overhangs may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0070] For any of the above aspects, the nucleic acid may be a modified nucleic acid. The modification may be selected from substitutions or insertions with analogues of nucleic acids or bases and chemical modification of the base, sugar or phosphate moieties. The nucleic acid may: a) be blunt ended at both ends; b) have an overhang at one end and a blunt end at the other; or c) have an overhang at both ends.

[0071] One or more nucleotides on the first and / or second strand may be modified, to form modified nucleotides. One or more of the odd numbered nucleotides of the first strand may be modified. One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more add nucleotides. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.

[0072] A plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification.

[0073] One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and / or one or more of the even numbered nucleotides of the second strand may be by the same modification of the odd numbered nucleotides of the first strand. At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides. A plurality of odd numbered nucleotides of the second strand may be modified by a common modification and / or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first strand odd numbered nucleotides. A plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.

[0074] The second strand comprises adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.

[0075] In the nucleic acid of the invention, each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with a second different modification.

[0076] The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0077] The modification and I or modifications may each and individually be selected from the group consisting of 3' terminal deoxy thymine, 2' O methyl, a 2' deoxy modification, a 2' amino modification, a 2' alkyl modification, a morpholino modification, a phosphoramidate modification, 5'-phosphorothioate group modification, a 5' phosphate or 5' phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and / or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide. At least one modification may be 2'- O-methyl and / or at least one modification may be 2'-F.

[0078] By nucleic acid it is meant a nucleic acid, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner. The nucleic acid may be single stranded or double stranded. When the nucleic acid of the invention is siRNA for RNAi the nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand. The first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which 'folds' to form a double stranded molecule. The nucleic acid may be an siRNA molecule.

[0079] The first strand may also be referred to as an antisense strand. The second strand may also be referred to as a sense strand.

[0080] Said nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogous. The nucleic acid may further comprise a double stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand). The duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive. By duplex region, it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may exist as 5' and 3' overhangs, or as single stranded regions. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. T echniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

[0081] The portion of the first strand and second strand that form at least one duplex region may be fully complementary and are at least partially complementary to each other.

[0082] Depending on the length of a nucleic acid, a perfect match in terms of base complementarity between the first strand and second strand is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions.

[0083] The complementarity between the first strand and second strand in the at least one duplex region may be perfect in that there are no nucleotide mismatches or additional / deleted nucleotides in either strand. Alternatively, the complementarity may not be perfect. The complementarity may be at least 70%, 75%, 80%, 85%, 90% or 95%.

[0084] The first strand and the second strand may each comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides.

[0085] An “overhang” as used herein has its normal and customary meaning in the art, i.e., a single stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double strand nucleic acid. The term “blunt end” includes double stranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may be base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may not be paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may be base paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.

[0086] The nucleic acid may have an overhang at one end and a blunt end at the other. The nucleic acid may have an overhang at both ends. The nucleic acid may be blunt ended at both ends. The nucleic acid may be blunt ended at the end with the 5' end of the first strand and the 3' end of the second strand or at the 3’-end of the first strand and the 5' end of the second strand.

[0087] The nucleic acid may comprise an overhang at a 3’ or 5' end. The nucleic acid may have a 3' overhang on the first strand. The nucleic acid may have a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand. The nucleic acid may have a 5' overhang on the second strand. The nucleic acid may have an overhang at both the 5' end and 3' end of the first strand. The nucleic acid may have an overhang at both the 5' end and 3' end of the second strand. The nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.

[0088] An overhang at the 3’-end or 5’ end of the second strand or the first strand may be selected from consisting of 1 , 2, 3, 4 and 5 nucleotides in length. Optionally, an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.

[0089] Unmodified polynucleotides, particularly ribonucleotides, may be prone to degradation by cellular nucleases, and, as such, modification / modified nucleotides may be included in the nucleic acid of the invention.

[0090] One or more nucleotides the nucleic acid of the invention may be modified. When the nucleic acid of the invention is siRNA for RNAi, one or more nucleotides on the second and / or first strand of the nucleic acid may be modified.

[0091] Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules. The nucleic acid according to the invention may be modified by chemical modifications. Modified nucleic acid can also minimize the possibility of inducing interferon activity in humans. Modification can further enhance the functional delivery of a nucleic acid to a target cell. The modified nucleic acid of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand. A ribonucleotide may comprise a chemical modification of the base, sugar, or phosphate moieties. The ribonucleic acid may be modified by substitution or insertion with analogues of nucleic acids or bases.

[0092] One or more nucleotides of a nucleic acid of the present invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be on the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in a single stranded region. The modified nucleotide may be on the first strand and may be outside the duplex region. The modified nucleotide may be on the second strand and may be outside the duplex region. The 3’-terminal nucleotide of the first strand may be a modified nucleotide. The 3’-terminal nucleotide of the second strand may be a modified nucleotide. The 5’-terminal nucleotide of the first strand may be a modified nucleotide. The 5’- terminal nucleotide of the second strand may be a modified nucleotide.

[0093] An nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, 24-26 modified nucleotides or about 26-28 modified nucleotides. In each case the nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or above of its activity as compared to the same nucleic acid but without said modified nucleotides.

[0094] The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotides may be selected from the group consisting of a 3' terminal deoxy thymine (dT) nucleotide, a 2' O methyl modified nucleotide, a 2’ modified nucleotide, a 2' deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2' amino modified nucleotide, a 2' alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0095] The nucleic acid may comprise a nucleotide comprising a modified nucleotide, wherein the base is selected from 2-aminoadenosine, 2, 6-diaminopurine, inosine, pyridin-4-one, pyridin- 2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. 6- methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4- acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

[0096] Nucleic acids discussed herein include unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, for example as occur naturally in the human body. Modified nucleotide as used herein refers to a nucleotide in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature. While they are referred to as modified nucleotides they will of course, because of the modification, include molecules which are not nucleotides, for example a polynucleotide molecule in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows hybridisation between strands i.e., the modified nucleotides mimic the ribophosphate backbone.

[0097] Many of the modifications described below that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the possible positions / nucleotides in the polynucleotide but in many cases it will not. A modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a nucleic acid of the invention or may only occur in a single strand region of an nucleic acid of the invention. A phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and / or in single strand regions, particularly at termini. The 5' end or 3’ ends may be phosphorylated.

[0098] Stability of a nucleic acid of the invention may be increased by including particular bases in overhangs, or to include modified nucleotides, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. Purine nucleotides may be included in overhangs. All or some of the bases in a 3' or 5' overhang may be modified. Modifications can include the use of modifications at the 2' OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphothioate modifications. Overhangs need not be homologous with the target sequence.

[0099] Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render oligoribonucleotides more stable to nucleases.

[0100] Modified nucleic acids, as used herein, can include one or more of:

[0101] (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens (referred to as linking even if at the 5' and 3' terminus of the nucleic acid of the invention);

[0102] (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

[0103] (iii) replacement of the phosphate moiety with “dephospho” linkers;

[0104] (iv) modification or replacement of a naturally occurring base;

[0105] (v) replacement or modification of the ribose-phosphate backbone;

[0106] (vi) modification of the 3' end or 5' end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., a fluorescently labeled moiety, to either the 3' or 5' end of RNA.

[0107] The terms replacement, modification, alteration, indicates a difference from a naturally occurring molecule.

[0108] Specific modifications are discussed in more detail below. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulphur. One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0109] The phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

[0110] A modified nucleotide can include modification of the sugar groups. The 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.

[0111] Examples of “oxy”-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).

[0112] “Deoxy” modifications include hydrogen halo; amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino),

[0113] — NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio- alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substitutents of certain embodiments include 2'-methoxyethyl, 2 -OCH3, 2 '-O-allyl, 2 '-C-allyl, and 2'-fluoro.

[0114] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleotides may contain a sugar such as arabinose. Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C — I'. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.

[0115] The 2' modifications may be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate).

[0116] The phosphate group can be replaced by non-phosphorus containing connectors.

[0117] Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.

[0118] The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides.

[0119] Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0120] The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end or the 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g. on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and / or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., — (CH2)n — , — (CH2)nN — , — (CH2)nO — , — (CH2)nS — , O(CH2CH2O)nCH2CH2OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end can be an — OH group.

[0121] Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0122] Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs. Nucleic acids of the invention, on the first or second strand, may be 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P— 0-5'); 5'-diphosphate ((HO)2(O)P— O— P(HO)(O)— 0-5'); 5'-triphosphate ((HO)2(O)P — O — (HO)(O)P — O — P(HO)(O) — 0-5'); 5'-guanosine cap (7-methylated or nonmethylated) (7m-G-O-5'-(HO)(O)P— O— (HO)(O)P— O— P(HO)(O)— 0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N — 0-5'-(H0)(0)P — O — (HO)(O)P — O — P(HO)(O) — 0-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P — O- 5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P — 0-5'), 5'-phosphorothiolate ((HO)2(O)P — S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P — NH-5', (H0)(NH2)(0)P — 0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O) — 0-5'-, (OH)2(O)P-5'-CH2-), 5'vinylphosphonate, 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O) — 0-5'-).

[0123] The nucleic acid of the present invention may include one or more phosphorothioate modifications on one or more of the terminal ends of the first and / or the second strand. Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate modified nucleotides. Optionally, each or either end of the second strand may comprise one or two or three phosphorothioate modified nucleotides. Optionally, both ends of the first strand and the 5’ end of the second strand may comprise two phosphorothioate modified nucleotides. By phosphorothioate modified nucleotide it is meant that the linkage between the nucleotide and the adjacent nucleotide comprises a phosphorothioate group instead of a standard phosphate group.

[0124] Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.

[0125] Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” can be employed. Examples include

[0126] 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O- 6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7- alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1 ,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3- nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5- methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,

[0127] 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine, 2-th iocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

[0128] As used herein, the terms “non-pairing nucleotide analog” means a nucleotide analog which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo II, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In some embodiments the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments it is a deoxy ribonucleotide.

[0129] As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, ether groups.

[0130] Certain moieties may be linked to the 5' terminus of the first strand or the second strand and includes abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2' O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5'0Me nucleotide; and nucleotide analogs including 4', 5'- methylene nucleotide; 1-(P-D-erythrofuranosyl)nucleotide; 4 -thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1 ,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1 ,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3', 4 seco nucleotide; 3, 4-di hydroxy butyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted abasic moiety; 1 ,4-butanediol phosphate; 5'-amino; and bridging or non-bridging methylphosphonate and 5'-mercapto moieties.

[0131] The nucleic acids of the invention may be included one or more inverted nucleotides, for example inverted thymidine or inverted adenine (for example see Takei, et al., 2002. JBC 277 (26):23800-06).

[0132] As used herein, the term “inhibit”, “down-regulate”, or “reduce” with respect to gene expression means the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a nucleic acid of the invention; for example the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that observed in the absence of an inhibitor.

[0133] The nucleic acid of the present invention may comprise an abasic nucleotide. The term "abasic" as used herein, refers to moieties lacking a base or having other chemical groups in place of a base at the T position, for example a 3',3'-linked or 5',5'-linked deoxyabasic ribose derivative.

[0134] The nucleic acid may comprise one or more nucleotides on the second and / or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides. Alternating as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example, if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.

[0135] One or more of the odd numbered nucleotides of the first strand of the nucleic acid of the invention may be modified wherein the first strand is numbered 5’ to 3’. The term “odd numbered” as described herein means a number not divisible by two. Examples of odd numbers are 1 , 3, 5, 7, 9, 11 and so on. One or more of the even numbered nucleotides of the first strand of the nucleic acid of the invention may be modified, wherein the first strand is numbered 5’ to 3’. The term “even numbered” as described herein means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. One or more of the odd numbered nucleotides of the second strand of the nucleic acid of the invention may be modified wherein the second strand is numbered 3' to 5'. One or more of the even numbered nucleotides of the second strand of the nucleic acid of the invention may be modified, wherein the second strand is numbered 3' to 5'.

[0136] One or more nucleotides on the first and / or second strand may be modified, to form modified nucleotides. One or more of the odd numbered nucleotides of the first strand may be modified. One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more add nucleotides. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.

[0137] A plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification.

[0138] One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and / or one or more of the even numbered nucleotides of the second strand may be by the same modification of the odd numbered nucleotides of the first strand. At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides. A plurality of odd numbered nucleotides of the second strand may be modified by a common modification and / or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first stand odd numbered nucleotides. A plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.

[0139] The second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.

[0140] In the nucleic acid of the invention, each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with the second modification.

[0141] The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

[0142] One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more of the odd numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the odd numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. In one aspect the present invention provides a composition comprising a compound according to any of the above aspects and a suitable carrier or excipient.

[0143] In one aspect the present invention provides a compound or composition according to any of the above aspects for use in medicine.

[0144] The compound or composition may be for use in the treatment of liver diseases, genetic diseases, hemophilia and bleeding disorders, liver fibrosis, non-alcoholic steotohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g. acromegaly), metabolic diseases (e.g. hypercholesterolemia, dyslipidemia, hypertriglyceridemia), cardiovascular diseases, obesity, thalassemia, liver injury (e.g. drug induced liver injury), hemochromatosis, alcoholic liver diseases, alcohol dependence, anemia, and anemia of chronic diseases.

[0145] The compound or composition may be for use in the treatment of liver disease, anemia, chronic diseases, thalassemia; drug induced liver injury, hemochromatosis and anemia of chronic disease.

[0146] Brief Description of the Figures

[0147] Figure 1 : Synthetic scheme for C4-GalNAc amidite precursor (5).

[0148] Figure 2: Synthetic scheme for acetyl-protected C4-GalNAc amidite (7) from precursor (5).

[0149] Figure 3: Synthetic scheme for DMT-protected C4-GalNAc phosphoramidite (13) from precursor (5).

[0150] Examples

[0151] Example 1 - C4-GalNAc amidite precursor synthesis

[0152] (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]oxazole-6,7-diyl diacetate (2). To a suspension of Galactosamine pentaacetate (1 , 125 g, 321 mmol) in dichloromethane (870 mL) at room temperature was added, via drop wise addition, trimethylsilyltrifluoromethanesulfonate (107 g, 482 mmol, 87 mL, 1.5 equiv) over a period of 30 minutes. The reaction mixture was heated to 40 °C for a period of 2 hours, after which it was cooled back to room temperature and quenched by pouring in an ice-cold aqueous saturated NaHCOs solution (1000 mL). The layers were separated and the aqueous layer was extracted twice more with dichloromethane (2x 300 mL). The combined organic layers were washed with water (500 mL) and brine (800 mL), followed by drying over Na2SO4. After concentrating in vacuo 2 was obtained as a pale yellow oil (109 g, crude yield 103%).1H NMR (400 MHz, Chloroform-5) 5 6.00 (d, J = 6.8 Hz, 1 H), 5.47 (t, J = 3.0 Hz, 1 H), 4.91 (dd, J = 7.4, 3.3 Hz, 1 H), 4.29 - 4.06 (m, 3H), 4.03 - 3.97 (m, 1 H), 2.13 (s, 3H), 2.07 (d, J = 1.0 Hz, 6H), 2.06 (d, J = 1.3 Hz, 3H).

[0153] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(benzyloxy)butoxy)tetrahydro- 2H-pyran-3,4 diyl diacetate (4). To a solution of 2 (109 g, 331 mmol) in dichloromethane (1200 mL) were added powdered Molsieves 4A (75 g) followed by stirring for 15 minutes at room temperature. To the mixture was added 4-benzyloxy-1 -butanol (3, 89 g, 497 mmol, 87 mL, 1.5 equiv) and stirring was continued for another 15 minutes. Then, via drop wise addition, was added trimethylsilyltrifluoromethanesulfonate (44.1 g, 199 mmol, 36.0 mL, 0.6 equiv) over a period of 15 minutes. Stirring of the reaction mixture was continued for 2 hours. Filtration of the mixture was performed over a plug of kieselguhr followed by rinsing once with dichloromethane (200 mL). The filtrate was then quenched by pouring in an ice-cold saturated aqueous NaHCCh solution (1000 mL). The layers were separated followed by extracting the aqueous layer twice more with dichloromethane (2x 500 mL). The combined organic layers were washed with water (600 mL) and brine (600 mL) followed by drying over Na2SO4. After concentrating in vacuo, purification was performed by flash column chromatography on silica neutralized with 1% Et3N (20-80% EtOAc in heptane) to obtain 4 as a colourless oil which slowly crystalized (109 g, yield 65%).1H NMR (400 MHz, DMSO-δ6) 5 7.83 (d, J = 9.3 Hz, 1 H), 7.39 - 7.23 (m, 5H), 5.21 (d, J = 3.5 Hz, 1 H), 4.96 (dd, J = 11.2, 3.5 Hz, 1 H), 4.48 (d, J = 8.5 Hz, 1 H), 4.44 (s, 2H), 4.07 - 3.97 (m, 3H), 3.87 (dt, J = 11.2, 8.8 Hz, 1 H), 3.72 (p, J = 5.3 Hz, 1 H), 3.49 - 3.37 (m, 3H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 1.54 (qd, J = 8.0, 5.2, 4.6 Hz, 4H).

[0154] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-hydroxybutoxy)tetrahydro-2H- pyran-3,4-diyl diacetate (5). To a solution of 4 (109.6 g, 215 mmol) in tetrahydrofuran (1000 mL) and 2-propanol (1000 mL) was added 10% palladium on carbon (17.17 g, 16.13 mmol, 10 %, 0.075 equiv) and the flask was charged with hydrogen (atmospheric pressure). Stirring of the reaction mixture was continued overnight at room temperature. The mixture was filtered over a plug of kieselguhr and concentrated in vacuo. After stripping the material twice with toluene (2x 300 mL) and dichloromethane (2x 300 mL), 17 was obtained as a white sticky solid (87 g, yield 97%).1H NMR (400 MHz, Methanol-δ) δ 5.33 (dd, J = 3.5, 1 .0 Hz, 1 H), 5.05 (dd, J = 11.3, 3.3 Hz, 1 H), 4.55 (d, J = 8.5 Hz, 1 H), 4.20 - 3.97 (m, 4H), 3.87 (dt, J = 10.1 , 5.8 Hz, 1 H), 3.60 - 3.48 (m, 3H), 3.30 (p, J = 1.8 Hz, 1 H), 2.14 (s, 3H), 2.02 (s, 3H), 1.94 (s, 3H), 1.92 (s, 3H), 1.61 (dtd, J = 16.8, 11.0, 10.1, 3.6 Hz, 4H).

[0155] Synthetic scheme for C4-GalNAc amidite precursor (5) is shown in Figure 1.

[0156] Example 2 - Acetyl-protected C4-GalNAc amidite synthesis

[0157] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-(((2 cyanoethoxy)(diisopropylamino)phosphino)oxy)butoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (7). To a solution of 4,5-dicyanoimidazole (1.940 g, 16.43 mmol, 0.65 eq.) in dry acetonitrile (20 mL) and dry dichloromethane (20 mL), under an argon atmosphere, were added grinded Molsieves 4A (9 g). Then, 2-cyanoethyl tetraisopropylphosphoro-diamidite (6, 10.00 g, 33.2 mmol, 10.53 mL, 1.31 equiv) was added via a syringe and stirred at room temperature for 10 minutes. Via drop wise addition was then added a solution of 5 (10.6 g, 25.3 mmol) in dry dichloromethane (50 mL) over a period of 10 minutes. After stirring for an additional 30 minutes, the reaction mixture was filtered over a cotton plug and concentrated in vacuo.

[0158] Purification of the material was performed by multiple flash column chromatograph steps (0- 100% EtOAc in heptane with 5% EtsN) to obtain 7 as a pale yellow oil (11.75 g, yield 72%).1H NMR (400 MHz, DMSO-δ6) δ 7.82 (d, J = 9.2 Hz, 1 H), 5.21 (d, J = 3.4 Hz, 1 H), 4.96 (dd, J = 11 .2, 3.5 Hz, 1 H), 4.48 (d, J = 8.5 Hz, 1 H), 4.02 (s, 3H), 3.93 - 3.82 (m, 1 H), 3.78 - 3.65 (m, 3H), 3.64 - 3.49 (m, 4H), 3.48 - 3.40 (m, 1 H), 2.76 (t, J = 5.9 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.62 - 1.46 (m, 4H), 1.13 (dd, J = 6.8, 3.6 Hz, 12H).31P NMR (162 MHz, Chloroform-d) 147 (d, J = 8.6 Hz).

[0159] Synthetic scheme for acetyl-protected C4-GalNAc amidite (7) from precursor (5) is shown in Figure 2.

[0160] Example 3 - Building block synthesis

[0161] The synthetic scheme for DMT-protected C4-GalNAc phosphoramidite is shown in Figure 3. In short, starting material 5 was prepared as described in Example 1 and was first protected using TBDMSCI to yield 8. The acyl protecting groups on the hydroxyls were subsequently removed using NaOMe, and the DMT group was then installed on the free primary hydroxyl group to yield 10. The acyl protecting groups were reintroduced to the remaining free hydroxyls using (Ac)20, and then the TBDMS group was removed with TBAF to give 12. Finally, phosphorylating reagent 6 was used to convert 12 into the phosphoramidite product 13.

[0162] (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(4-((TBDMS)oxy)butoxy)tetrahydro- 2H-pyran-3,4-diyl diacetate (8). To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2- (acetoxymethyl)-6-(4-hydroxybutoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (5) (50 g, 119 mmol) in dichloromethane (300 ml) was added imidazole (12.17 g, 179 mmol) followed by dropwise addition of TBDMSCI (21.56 g, 143 mmol) dissolved in Dichloromethane (150 ml). The obtained suspension was stirred at RT. After 30 minutes LCMS indicated complete consumption of 5. Water (200ml) was added, and the layers were separated. The organic layer was washed with water (200 ml) and brine (150 ml), dried over Na2SO4 and finally concentrated in vacuo to obtain 68 g. The residue was purified by silica gel gravity column chromatography (heptane, 25%-100% EtOAc). The collected product was combined and concentrated in vacuo to obtain 54 g (yield: 85 %) of target compound 8, C24H43NOwSi (533.69 g mol'1); MS (ESI') (m / z) calculated: 532.3 ^FkNOwSi]', found: 532.4;1H-NMR (400MHz, DMSO-d6, δ 7.73 (d, 1 H), 5.19 (d, 1 H), 4.94 (dd, 1 H), 4.46 (d, 1 H), 4.03-3.96 (m, 3H), 3.85 (m, 1 H), 3.70 (m, 1 H), 3.54 (m, 2H), 3.40 (m, 1 H), 2.08 (s, 3H), 1.97 (s, 3H), 1.91 (s, 3H), 1.84 (s, 3H), 1.45 (m, 4H), 0.83 (s, 9H), 0.0 (s, 6H).

[0163] N-((2R,3R,4R,5R,6R)-2-(4-((TBDMS)oxy)butoxy)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (9). To a solution of 8 (54 g, 101 mmol, colorless oil) in methanol (600 ml) sodium methoxide, 5.4M (30 wt.%) solution in methanol (0.937 ml, 5.06 mmol) was added. The mixture was stirred for 120 min. After 2h LCMS indicated complete consumption of 8. The mixture was neutralized after 2 hours with acetic acid (0.584 ml, 10.12 mmol), concentrated in vacuo and co-evaporated with DCM (3x150 ml) to obtain 42.7g of 9 (colourless solid) (NMR: contains 5.1% (w / w) DCM) used as such in the next step.CisHsyNOySi (407.58 g mol-1); MS(ESI') (m / z) calculated: 406.2 [Ci8H36NO7Si]', found: 406.4;1H-NMR (400MHz, DMSO-d6, δ 7.58 (d, 1 H), 4.54 (m, 3H), 4.19 (d, 1 H), 3.69-3.62 (m, 3H), 3.55-3.49 (m, 3H), 3.47-3.24 (m, 4H), 1.76 (s, 3H), 1.43 (m, 4H), 0.76 (s, 9H), 0.00 (s, 6H).

[0164] (2R,3R,4R,5R,6R)-5-acetamido-2-(DMT-oxymethyl)-6-(4- ((TBDMS)oxy)butoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (11). To a solution of of N- ((2R,3R,4R,5R,6R)-2-(4-((TBDMS)oxy)butoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro- 2Hpyran-3-yl)acetamide (9, 42.7 g , 99 mmol) in abs. Pyridine (600 ml) was added DMTCI (38.7 g, 114 mmol) portion wise (20 portions) over a period of 10 min. The mixture was stirred for 1 hour at room temperature. After 30 min LCMS indicated 75% mono DMT-ether (10) formation and -10% double DMT-ether formation. Therefore, acetic anhydride (74.7 ml, 795 mmol) was added and the mixture was stirred overnight. LCMS indicated full conversion and -80% of the wanted product formed. The reaction mixture was concentrated and purified by gravity column chromatography (1.5 Kg, heptane + 1 % Et3N, 25%-100% EtOAc +1% Et3N). Product containing fractions were pooled and concentrated in vacuo to obtain 66 g of target compound 11. C43Hs9NOnSi (794.03 g mol-1); MS(ESI') (m / z) calculated: 792.39 [C43H58NOnSi]-, found: 792.4;1H-NMR (400 MHz, DMSO-d6, 5 7.83 (d, 1 H), 7.36 - 7.19 (m, 9H), 6.89 (m, 4H), 5.41 (d, 1 H), 5.01 (m, 1 H), 4.48 (d, 1 H), 4.04 (m, 1 H), 3.81 (m, 1 H), 3.76 (s, 6H), 3.67 (m, 1 H), 3.54 (2H, m), 3.40 (m, 1 H), 3.14 (m, 1 H), 2.81 (m, 1 H), 1.90 (s, 3H), 1.88 (s, 3H), 1.77 (s, 3H), 1.46 (m, 4H), 0.84 (s, 9H), 0.00 (s, 6H).

[0165] (2R,3R,4R,5R,6R)-5-acetamido-2-((DMT-oxy)methyl)-6-(4-hydroxybutoxy)tetrahydro-2H- pyran-3,4-diyl diacetate (12). To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-((DMT- oxy)methyl)-6-(4-((TBDMS)oxy)butoxy) tetrahydro-2H-pyran-3,4-diyl diacetate (11 , 66 g, 83 mmol) in abs. THF (1500 ml) under a nitrogen protective gas atmosphere was added TBAF (1.0M in THF, 91 ml, 91 mmol) dropwise added over a period of 10 minutes. The mixture was stirred overnight at RT. LCMS indicated full conversion of the starting material (11). The mixture was concentrated in vacuo and purified by gravity column chromatography (1.5 kg, DCM + 1 % Et3N), 1 %-3 % MeOH + 1 % Et3N). Product containing fractions were pooled and concentrated and co-evaporated with MeCN (3x) to obtain a colourless foam that was broken down to a colourless powder of 42.2 g of target compound 11. C37H45NO11 (679.76 g mol-1); MS(ESI-) (m / z) calculated: 678.30 [C37H44NO11]-, found: 678.4;1H-NMR (400 MHz, DMSO-d6, δ7 7.82 (d, 1 H), 7.35 - 7.17 (m, 9H), 6.89 (m, 4H), 5.39 (d, 1 H), 4.99 (m, 1 H), 4.47 (d, 1 H), 4.33 (m, 1 H), 4.05 (m, 1 H), 3.80 (m, 1 H), 3.74 (s, 6H), 3.66 (m, 1 H), 3.36 (m, 2H), 3.12 (m, 1 H), 2.80 (m, 1 H), 1.89 (s, 3H), 1.86 (s, 3H), 1.77 (s, 3H), 1.50-1.34 (m, 4H).

[0166] (2R,3R,4R,5R,6R)-5-acetamido-2-((DMT-oxy)methyl)-6-(4-(((2- cyanoethoxy)(diisopropylamino)phosphaneyl) oxy)butoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (13). The reaction was performed at 0 °C under an argon blanket (balloon). Into a 250 mL round bottom flask, N-Methylimidazolium triflate (2.464 g, 10.61 mmol) was charged and stripped with 2x 25 mL abs. acetonitrile. Then, a magnetic stirring bar was added to the reaction tube followed by a septum with an argon balloon. Thereafter, abs. acetonitrile (100 ml) was added (solution), followed by the dropwise addition of 2-cyanoethyl N,N,N,N- tetraisopropylphosphoramidite (6, 4.94 ml, 15.57 mmol) over a period of 5 min. (2R,3R,4R,5R,6R)-5-acetamido-2-((DMT-oxy)methyl)-6-(4-hydroxybutoxy)tetrahydro-2H- pyran-3,4-diyl diacetate (12, 10 g, 14.15 mmol) was stripped with 2x 25 mL abs. acetonitrile, dissolved in abs. acetonitrile (40 ml) and added dropwise over a period of 10 min. into the reaction mixture at 0 °C. The mixture was stirred for 60 min. LCMS indicated full conversion. Therefore, the reaction mixture was concentrated in vacuo at 30°C. The residue was dissolved in dichloromethane (20 ml) by purified by flash column chromatography (220g, heptane +1 % Et3N), 20%- 100% EtOAc + 1% Et3N). Product containing fractions were concentrated in vacuo and co-evaporated with EtOAc to obtain a colourless foam. The foam was broken down to yield 8.9 g of target compound 12. C46H62N3O12P (879.98 g mol-1); MS(ESI+) (m / z) calculated: 880.41 [C46H63N3Oi2P]+, found: 880.4;1H-NMR (400 MHz, DMSO-d6, δ 7.81 (d, 1 H), 7.35 - 7.18 (m, 9H), 6.89 (m, 4H), 5.39 (d, 1 H), 4.99 (m, 1 H), 4.46 (d, 1 H), 4.04 (m, 1 H), 3.79 (m, 1 H), 3.74 (s, 6H), 3.69 (m, 3H), 3.53 (m, 4H), 3.39 (m, 1 H), 3.12 (m, 1 H), 2.80 (m, 1 H), 2.73 (m, 2H), 1.89 (s, 3H), 1.86 (s, 3H), 1.76 (s, 3H), 1.51 (m, 4H), 1.10 (m, 12H).

[0167] Example 4: Building block synthesis with alternative protection groups

[0168] In a similar fashion to Examples 1 to 3, MMT (Monomethoxytrityl)- and TMT (Trimethoxytrityl)- protected GalNAc-building blocks can be prepared. For this purpose, compound 5 (Example 1) was reacted first with TBDMSCI to mask the free hydroxyl group at the C4 arm of 5. Successively the acetyl groups were cleaved with NaOMe top yield compound 14. In order to prepare the MMT-GalNAc-amidite, 14 was further reacted with MMTCI followed by acetic anhydride to yield 15. After cleavage of the TBDMS group by TBAF the free alcohol in 16 could be phosphitylated to give the solid target compound MMT-GalNAc-amidite (17).

[0169] In order to prepare the TMT-GalNAc-amidite, 14 was further reacted with TMTCI followed by acetic anhydride to yield 18. After cleavage of the TBDMS group by TBAF the free alcohol in 19 could be phosphitylated to give the solid target compound TMT-GalNAc-amidite (20).

[0170] Example 5: oligonucleotide synthesis

[0171] Example compounds were synthesised according to methods described below and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.

[0172] Building block synthesis

[0173] Synthesis of the phosphoramidite derivatives of ST41 (ST41-phos), ST43 (ST43-phos) as well as ST23 (ST23-phos) and their precursor compounds can be performed as described in WO2017 / 174657 and in here in Example 2 (compound 7):

[0174] ST41-phos: ST23-phos:

[0175] ST23-phos

[0176] Synthesis of Oligonucleotides

[0177] Oligonucleotides were synthesized on an AKTA oligopilot 100 and 10 synthesizers using standard phosphoramidite chemistry. Commercially available base loaded solid supports, 2'OMe nucleotide phosphoramidites and 2'F nucleotide phosphoramidites (all standard protection) were purchased from ChemGenes and used according to the manufacturers recommended procedures.

[0178] Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 10 min. A Cap / OX / Cap or Cap / Thio / Cap cycle was applied (Cap: Ac2O / NMI / Lutidine / Acetonitrile. Oxidizer: 0.1M I2 in pyridine / H2O). Phosphorothioates were introduced using 0.2M XH (0.2 M Xanthane hydride in pyridine). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All other reagents and solvents were commercially available and used in standard reagent quality.

[0179] All oligonucleotides were synthesized in DMT-off mode. The single strands were cleaved off the support by 40% aq. methylamine treatment (90min, RT). The resulting crude oligonucleotide was concentrated under reduced pressure to a concentration of ~10 mg / mL and then analyzed.

[0180] All final single stranded products were analyzed by AEX-HPLC (DNA Pac PA200 4.0x250mm & DNAPac PA200 Guard 4x50 mm at 80 °C, using a gradient of 25-70% B (10 % MeCN, 20 mM TRIS, 0.4 M LiCIO4 in water, pH =7.4) in A (10 % MeCN, 20 mM TRIS in water) over 10 min, flow rate 1 mL / min). Purity is given in %FLP (% full length product) which is the percentage of the UV-area under the assigned product signal in the UV-trace of the AEX-HPLC analysis of the product. Identity of the respective single stranded products was confirmed by UPLC-MS analysis (Acquity UPLC BEH C18 2.1x50mm, 1.7 pm Acquity BEH C18 1.7 pM Vanguard PreCol at 60°C, using a gradient of 10-22% B (100 mM HFIP, 15 mM TEA in MeOH) in A (100 mM HFIP, 15 mM TEA, 5% MeOH in water) over 5 min, flow rate 0.3 mL / min). Test oligonucleotide: 5’- (ST23)3*ST41* caguguUCUugcucuau*a*a - 3’ (SEQ ID NO: 1) where * denotes phosphorothioate backbone, CAPS denotes 2’-fluoro modification, Italics denotes 2’-0Me modification. Table 1: Identity and crude purity of oligonucleotides made by use of differently protected GalNAc building blocks.

Claims

CLAIMS:

1. A compound having the formula (I):whereinA’ is selected fromandR is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx.

2. A compound selected from the group consisting of:wherein R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx.

3. A compound selected from the group consisting of:andwherein R is a protective group selected from the group consisting of TMTr, DMTr, MMTr, Fmoc, Px and DMPx, and wherein Z is a nucleic acid.

4. A compound according to claim 3, wherein the nucleic acid is selected from RNAi, siRNA, antisense nucleic acid, sgRNA, shRNA, ribozymes, aptamers and spiegelmers.

5. A compound according to claim 3 or 4, wherein the nucleic acid is modified.

6. A compound according to claim 5, wherein the modification is selected from substitutions or insertions with analogues of nucleic acids or bases and chemical modification of the base, sugar or phosphate moieties.

7. A composition comprising a compound as defined in any preceding claim and a suitable carrier or excipient.

8. A method for making a compound of formula (III), (V) or (VII) as claimed in claim 2, the method comprising the step of reacting an unprotected hydroxyl group in the precursor (II), (IV), or (VI) with a phosphoramidite group to generate the functional building block of formula (III), (V) or (VII).

9. A method for making a compound as defined in any one of claims 3-6 or a composition as defined in claim 7, the method comprising adding together each component to form the compound of any one of claims 3-6 or composition of claim 7, wherein the components comprise three building blocks selected from the formulas (III), (V) and (VII), a branching unit, a bridging unit and at least one nucleic acid, the building blocks may be the same or different.

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