Multivalent cargo-carrying complexes and uses thereof

Abstract

Provided are multivalent saccharide-containing compounds which are bonded to nucleic acids. These compounds contain a cysteine-derived core and are useful for delivering nucleic acids to cells or tissues, e.g. for use in therapeutic treatments. Also provided are pharmaceutical compositions comprising the aforementioned compounds and medical uses of the same, including their use in treating or preventing conditions such as liver diseases.

Description

[0001] MULTIVALENT CARGO-CARRYING COMPLEXES AND USES THEREOF

[0002] Multivalent saccharide-containing compounds are provided which are bonded to nucleic acids. These compounds are useful for delivering nucleic acids to cells or tissues, e.g. for use in therapeutic treatments. In particular, the compounds comprise specific monosaccharides which are capable of binding to asialoglycoprotein receptors (ASGPRs). Such compounds may be used, for example, in the targeted delivery of therapeutic oligonucleotides to cells such as liver cells. Also provided are pharmaceutical compositions comprising the aforementioned compounds and medical uses of the same, including their use in treating or preventing conditions such as liver diseases.

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This specification claims the benefit of priority to GB Patent Application No. 2311324.4 (filed 24 July 2023). The entire text of the above-referenced patent application is incorporated by reference into this specification.

[0005] SUMMARY

[0006] There are numerous ways to target the delivery of cargo moieties, including therapeutic nucleic acids, to cells. One approach is to tether the cargo to one or more saccharide ligands to take advantage of saccharide-binding proteins associated with the target cells. Exemplary saccharide-binding proteins include cell surface receptors such as glycoprotein receptors, including asialoglycoprotein receptors (ASGPRs). ASGPRs have been shown to be highly expressed on the surface of mammalian hepatocytes, as well as carcinoma cell lines; they are also expressed on other cell types, albeit generally at a lower level. As such, ASGPRs represent a promising target for hepatic delivery of cargo, including therapeutic nucleic acid agents (see, e.g., D’Souza et al., J. Control Release, (2015) 203:126-139). Ligands which can be used for targeting ASGPRs include monosaccharides such as JV-acetyl galactosamine (GalNAc).

[0007] Target-binding complexes often contain more than one ligand and / or more than one cargo moiety. This functionality can be achieved by the use of branched core structures with linkers being used to couple the ligands and / or the cargo moieties to the core. In this way, a complex can be prepared which has the appropriate number and arrangement of functional parts for the desired use. Examples of multivalent, e.g. branched, complexes are described in International Patent Publications: WO 2014 / 179620 (Isis Pharmaceuticals, Inc.); WO 2015 / 177668 Al (Pfizer Inc.); WO 2009 / 073809 A2 (Alnylam Pharmaceuticals, Inc.); WO 2012 / 083046 A2 (Arrowhead Research Corporation); WO 2017 / 156012 Al (Arrowhead Pharmaceuticals, Inc.); WO 2016 / 100401 Al (Dicema Pharmaceuticals, Inc.); WO 2017 / 174657 Al (Silence Therapeutics GmbH); and WO 2019 / 092280 Al (Silence Therapeutics GmbH). Complexes of the aforementioned types make use of different coupling chemistries to link the ligands and cargo moieties to the branched core structure, and also make use of different core structures. There is, however, a need for alternative chemical functionalities to couple cargo moieties and ligands in order to prepare complexes useful in delivering cargo moieties to cells. The present disclosure seeks to address this need by providing novel compounds and complexes, e.g. for use in targeting therapeutic nucleic acids to cells such as liver cells.

[0008] In brief, the compounds and complexes of the present disclosure contain one or more monosaccharide ligands and one or more nucleic acids. These are joined together using a multi dentate ‘splitter’ which is derived from, or chemically related to, the amino acid cysteine. The use of cysteine, and analogues thereof, in preparing the branching unit provides a novel way of coupling targeting and cargo moieties which may have advantages over known methods, including for example the use of simple, natural precursors. The compounds of the invention exhibit useful properties such as, e.g., improved targeting, increased activity and / or improved pharmacokinetic properties in vivo.

[0009] Summary of the Disclosure

[0010] The present disclosure includes the following aspects and embodiments which are presented as numbered clauses 1 to 31.

[0011] 1. A compound having the structure of F ormula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0012] X is selected from

[0013] R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2; and

[0014] G1to G4each independently represents a -[spacer]-[ligand] moiety or a -[tether] -[linker] -[cargo] moiety, wherein each ligand is independently a monosaccharide and each cargo is independently a nucleic acid, with the proviso that at least one of G1to G4represents a -[spacer]-[ligand] moiety and at least one of G1to G4represents a -[tether]-[linker]-[cargo] moiety.

[0015] 2. The compound or pharmaceutically acceptable salt thereof of clause 1, wherein X is -S-.

[0016] 3. The compound or pharmaceutically acceptable salt thereof of clause 1 or clause 2, wherein each R is -H.

[0017] 4. The compound or pharmaceutically acceptable salt thereof of any one of clauses 1-3, wherein m is 1 , and n is i.

[0018] 5. The compound or pharmaceutically acceptable salt thereof of any one of clauses 1-4, wherein each cargo is independently selected from an antisense oligonucleotide (ASO), an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice altering oligonucleotide, a splice-switching oligonucleotide, a triplex forming oligonucleotide, a siRNA, a saRNA, a microRNA, a microRNA mimic, an anti-miR, a double stranded RNA, a single stranded RNA, a ribozyme, an aptamer, a spiegelmer, a CRISPR oligonucleotide and a G-quadruplex.

[0019] 6. The compound or pharmaceutically acceptable salt thereof of any one of clauses 1-5, wherein each spacer independently comprises a chain of 2-20 atoms selected from C, N, O, S and P, e.g., wherein each spacer is independently selected from a linear alkylene (which may optionally be interrupted by one or more amide and / or phosphate groups) and a polyethylene glycol.

[0020] 7. The compound or pharmaceutically acceptable salt thereof of any one of clauses 1-6, wherein tether and linker together in each case independently represents a moiety comprising a linear chain of 8 to 30 atoms.

[0021] 8. The compound or pharmaceutically acceptable salt thereof of any one of clauses 1-7, wherein the compound has the structure of Formula (la): wherein X, R, m, n, G1, G2, G3and G4are as defined in any of the preceding clauses.

[0022] 9. A compound having the structure of F ormula (II) :

[0023] (II) or a pharmaceutically acceptable salt thereof, wherein:

[0024] X is selected from

[0025] R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2;

[0026] A1to A3in each case is independently a moiety which spaces the ligand from the rest of the molecule, e.g. comprising a linear chain of between 2 and 16 atoms;

[0027] Z is a cargo-containing moiety, which may comprise a linker and a tether, and wherein the cargo is a nucleic acid; and

[0028] [ligand] in each case is independently a monosaccharide.

[0029] 10. The compound or pharmaceutically acceptable salt thereof of clause 9, wherein Z represents -[tether]-[linker]-[cargo], in which [tether] -[linker] represents a moiety comprising a linear chain of 8 to 30 atoms; and [cargo] is a nucleic acid.

[0030] 11. The compound or pharmaceutically acceptable salt thereof of clause 9 or clause 10, wherein one or more of A1to A3is independently selected from *-(C1-C12)alkylene- (where * denotes the point of attachment to the ligand).

[0031] 12. The compound or pharmaceutically acceptable salt thereof of clause 9 or clause 10, wherein one or more of A1to A3is independently a moiety selected from -(CH2CH2O)p(CH2)2-* and -(CH2)oCONH(CH2CH2O)P(CH2)2-* (where * in each case denotes the point of attachment to the ligand), wherein o is an integer selected from 1, 2, 3 and 4; and p is an integer selected from 1, 2, 3 and 4.

[0032] 13. The compound or pharmaceutically acceptable salt thereof of any one of clauses 9-12, wherein the compound has the structure of Formula (Ila): (Ila) wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined in any of the preceding clauses.

[0033] 14. A compound having the structure of F ormula (III) : or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined in any of the preceding clauses.

[0034] 15. The compound or pharmaceutically acceptable salt thereof of clause 14, wherein:

[0035] X is selected from R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2;

[0036] A1to A3in each case is independently a moiety comprising a linear chain of between

[0037] 2 and 16 atoms; and Z represents -[tether] -[linker] -[cargo], in which [tether] -[linker] represents a moiety comprising a linear chain of 8 to 30 atoms; and [cargo] is a nucleic acid.

[0038] 16. The compound or pharmaceutically acceptable salt thereof of clause 14 or clause 15, wherein R in each case is -H, m is 1, and n is 1. 17. The compound or pharmaceutically acceptable salt thereof of any one of clauses 14-

[0039] 16, wherein the compound has the structure of Formula (Illa): wherein X, R, m, n, A1, A2, A3and Z are as defined in any of the preceding clauses.

[0040] 18. A compound selected from:

[0041] and the pharmaceutically acceptable salts thereof, wherein nucleic acid denotes a cargo as defined in any of the preceding clauses.

[0042] 19. The compound or pharmaceutically acceptable salt thereof of clause 18, wherein nucleic acid denotes an ASO or siRNA. 20. A compound as defined in any one of clauses 1-19, or a pharmaceutically acceptable salt thereof, having binding affinity for ASGPR characterised by an ICso value of less than about 10 nM (e.g., an ICso value of less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM).

[0043] 21. A compound as defined in any one of clauses 1-19, or a pharmaceutically acceptable salt thereof, having binding kinetics for ASGPR characterised by a Kd value of less than about 8 nM (e.g., a Kd value of less than about 1.0 nM).

[0044] 22. A compound as defined in any one of clauses 1-19, or a pharmaceutically acceptable salt thereof, having an activity in knocking down gene expression in HEK293 cells characterised by an ICso value of less than about 400 nM (e.g., an ICso value of less than about 200 nM, 100 or 50 nM). 23. A pharmaceutical composition comprising a compound as defined in any one of clauses 1-22, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

[0045] 24. A compound or pharmaceutically acceptable salt thereof according to any one of clauses 1-22, or a pharmaceutical composition according to clause 23, for use in therapy.

[0046] 25. A compound or pharmaceutically acceptable salt thereof according to any one of clauses 1-22, or a pharmaceutical composition according to clause 23, for use in the treatment of a condition selected from liver disease (e.g., liver cancer), genetic disease, haemophilia and bleeding disorders, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g. acromegaly), metabolic disease, cardiovascular disease, obesity, thalassemia, liver injury (e.g., drug induced liver injury), hemochromatosis, alcoholic liver disease, alcohol dependence, anaemia, and anaemia of chronic disease.

[0047] 26. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of clause 25, wherein the condition is selected from NASH, NAFLD, a metabolic disease and a cardiovascular disease.

[0048] 27. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of clause 25, wherein the condition is NASH.

[0049] 28. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of clause 25, wherein the condition is a metabolic disease selected from hypercholesterolemia, dyslipidaemia, and hypertriglyceridemia.

[0050] 29. A compound having the structure of Formula (V):

[0051] (V) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand] and [tether] are as defined in any of the preceding clauses; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or a phosphoramidite. 30. A process for the preparation of a compound as defined in any one of clauses 9-22, characterised in that a compound of Formula (V) as defined in clause 29 is reacted with a compound having the structure

[0052] Q- [cargo] wherein cargo is as defined in any of the preceding clauses, and Q denotes a group which is reactive to group J as defined in clause 29.

[0053] 31. The process of clause 30, wherein Q denotes a BCN-containing group and J is -Ns.

[0054] BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1 shows a schematic example of compounds of the disclosure. The cargo nucleic acid is attached to the rest of the molecule via a linker and tether. The splitter moiety is bound to the tether on one side and to multiple ligands on the other, in each case with a spacer in between the ligand and the splitter. The complex shown in the figure carries three ligands, but other numbers of ligands are possible. In operation, the ligands (which may be, e.g., GalNAc moieties) typically bind to a receptor or other biomolecule which is associated with a cell or tissue; this facilitates association with and / or uptake into the cell or tissue where the cargo (e.g., a siRNA) can exert a biological effect.

[0056] FIG. 2 shows the results of an in vivo assay assessing knockdown of MALAT-1 mRNA in liver cells after a single dose of 50 nmol / kg (Example 5). The relative level of MALAT-1 mRNA in liver is shown compared to the PBS control (as 100%, column 1, left hand side). Column 2 shows mRNA levels after treatment with a naked MALAT-1 ASO. Columns 3 and 4 show mRNA levels after treatment with the compounds of Examples 1 and 2, respectively.

[0057] FIG. 3 shows the pharmacokinetics of ASO exposure after a single dose of 50 nmol / kg (Example 5). Column 1 (left hand side) shows exposure in liver (nmol / kg) after treatment with a naked MALAT-1 ASO. Columns 2 and 3 show exposure in liver after treatment with the compounds of Examples 1 and 2, respectively.

[0058] FIG. 4 shows the results of an in vivo assay assessing knockdown of MALAT-1 mRNA in kidney cells after a single dose of 50 nmol / kg (Example 5). The relative level of MALAT-1 mRNA in kidney is shown compared to the PBS control (as 100%, column 1, left hand side). Column 2 shows mRNA levels after treatment with a naked MALAT-1 ASO. Columns 3 and 4 show mRNA levels after treatment with the compounds of Examples 1 and 2, respectively. FIG. 5 shows the pharmacokinetics of ASO exposure after a single dose of 50 nmol / kg (Example 5). Column 1 (left hand side) shows exposure in kidney (nmol / kg) after treatment with a naked MALAT-1 ASO. Columns 2 and 3 show exposure in kidney after treatment with the compounds of Examples 1 and 2, respectively.

[0059] FIG. 6 shows the ANGPTL3 protein concentration measured pre-subcutaneous injection in the in vivo assay of Example 5. The level of ANGPTL3 in ng / ml is shown for the PBS control (column 1, left side), the Control siRNA cluster (column 2, middle), and Compound 3 (column 3, right side). No statistical significance is observed between groups.

[0060] FIG. 7 shows the ANGPTL3 protein concentration in plasma at Day 28 (Example 5). Column 1 (left hand side) shows results after administration of PBS control. Columns 2 and 3 show exposure after treatment with the Control siRNA cluster and Compound 3, respectively. Two way ANOVA is used for statistical analysis.

[0061] FIG. 8 shows the ANGPTL3 protein concentration in plasma corrected to PBS control over time (Example 5). The black line and circles (top) show the PBS control values. The light grey line and circles (middle) show the results for Compound 3, and the dark grey line and circles (bottom) show the results for the Control siRNA cluster.

[0062] FIG. 9 shows hepatic Angptl3 mRNA levels from the in vivo study of Example 5. Values are normalised to PBS control (column 1, left side). Columns 2 and 3 show relative exposure after treatment with the Control siRNA cluster and with Compound 3, respectively.

[0063] DETAILED DESCRIPTION

[0064] Although specific embodiments of the present disclosure will now be described with reference to the description and examples, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present disclosure. Various changes and modifications will be obvious to those of skill in the art given the benefit of the present disclosure and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0065] Definitions

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0067] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of chemical synthesis, tissue culture, immunology, molecular biology, microbiology, cell biology, recombinant DNA, etc., which are within the skill of the art. See, e.g., Michael R. Green and Joseph Sambrook, Molecular Cloning (4thed., Cold Spring Harbor Laboratory Press 2012); the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0068] All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges, are approximations which are varied ( + ) or ( - ) by increments of, e.g., 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”, which is used to denote a conventional level of variability. For example, a numerical designation which is “about” a given value may vary by ± 10% of said value; alternatively, the variation may be ± 5%, ± 2%, or ± 1% of the value. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0069] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, without excluding other elements. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. Use of the term “comprising” herein is intended to encompass, and to disclose, the corresponding statements in which the term “comprising” is replaced by “consisting essentially of’ or “consisting of’.

[0070] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N- acetyl galactosamine. Reference to "GalNAc" or "N- acetyl galactosamine" herein denotes the form, i.e., 2-(acetylamino)-2-deoxy- -D-galactopyranose.

[0071] The term “cargo” as used herein refers to a chemical or biological entity which is suitable for targeting to and / or delivery to a cell or tissue, e.g. as part of a compound of the disclosure. The cargo in accordance with the present disclosure is a nucleic acid.

[0072] The term “nucleic acid” as used herein includes nucleic acids selected from the group consisting of DNA, RNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA). The nucleic acid may be a functional nucleic acid, e.g., whereby the functional nucleic acid is selected from the group consisting of mRNA, micro-RNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acid (e.g., antisense oligonucleotide (ASO)), ribozymes, aptamers and spiegelmers. A “peptide nucleic acid” is a polymer which is similar to DNA or RNA in which the backbone is composed of repeating amino acid (typically N-(2- aminoethyl)-glycine) units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge and a carbonyl group. A “locked nucleic acid” is a nucleic acid in which the 2’ hydroxyl is connected, e.g., by a methylene bridge, to the 4’ carbon of the same ribose sugar.

[0073] By “duplex region” 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. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another. 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.

[0074] The term "abasic" as used herein in connection with nucleotides, refers to moieties lacking a base or having other chemical groups in place of a base at the 1' position, for example a 3', 3'- linked or 5',5'-linked deoxyabasic ribose derivative.

[0075] The term “alternating” as used herein in connection with nucleic acids 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.

[0076] 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 disclosure; 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.

[0077] 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.

[0078] A "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions of the disclosure is contemplated.

[0079] A “subject,” “individual”, or “patient” is used interchangeably herein, and typically refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, farm animals, sport animals, pets, and primates; for example murines, rats, rabbit, simians, bovines, ovines, porcines, canines, felines, equines, and humans. In a particular embodiment, the mammal is a human.

[0080] “Administering” is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being contacted with (e.g., being inside) the subject’s body. Such an administration can be by any route including, without limitation, oral, transdermal (e.g., by the vagina, rectum, or oral mucosa), by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, or into the central nervous system), or by inhalation (e.g., oral or nasal). Administration may also involve providing a substance or composition to a part of the surface of the subject’s body, for example by topical administration to the skin. Pharmaceutical preparations are, of course, given by forms suitable for each administration route.

[0081] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; and / or (3) relieving the disease, i.e. causing regression of the disease or its clinical symptoms. A patient or individual may be predisposed to the disease because of the presence of genetic mutations associated with the disease.

[0082] An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including, for example, the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, the severity of the particular disorder being treated and the form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to treat (e.g., improve) one or more symptoms associated with the condition. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.

[0083] The term “delivering” when used in connection with the nucleic acid-containing compounds and compositions of the disclosure typically denotes some active targeting of the nucleic acid to the target cell or tissue. Thus, delivery of a nucleic acid using the compounds and compositions of the disclosure typically results in exposure of the target cell or tissue to the nucleic acid at a level which is greater than the exposure following administration of the same nucleic acid without the rest of the complex (e.g., when administered as ‘naked’ nucleic acid). As used herein, the term “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical ingredients, for example as described in Remington’s Pharmaceutical Sciences (20thed., Mack Publishing Co. 2000). Such excipients include carriers such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. Pharmaceutical compositions also can include stabilizers, preservatives, adjuvants, fillers, binders, lubricants, and the like.

[0084] As used herein, the term “alkyl” means a saturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms. The term “alkylene” has the corresponding meaning in connection with the divalent free radical. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, etc. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. The terms “(C1-C3)alkyl”, “(C1-C6)alkyl”, etc., have equivalent meanings, i.e., a saturated linear or branched free radical consisting essentially of 1 to 3 (or 1 to 6) carbon atoms and a corresponding number of hydrogen atoms. The definition of “alkyl” also applies in the context of other groups which comprise alkyl groups, such as “-O(C1- C3)alkyl”. The term “haloalkyl” means an alkyl group which is substituted by one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, trifluoroethyl, difluoroethyl, pentafluoroethyl, chloromethyl, etc. One or more carbon atoms in the backbone of the alkyl group may be substituted by (or bonded to) a heteroatom by a multiple bond (e.g., a double bond); for example, a carbon atom of the alkyl group may be bonded to oxygen via a double bond (i.e., substituted by oxo to provide a carbonyl function). The presence of such a substituent does not prevent the carbon backbone of the free radical being considered as an alkyl group. In embodiments, the alkyl group is linear. Alkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0085] As used herein, the term “alkenyl” means an unsaturated linear or branched free radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms, which free radical comprises at least one carbon-carbon double bond. The term “alkenylene” has the corresponding meaning in connection with the divalent free radical. Exemplary alkenyl groups include ethenyl, prop-1 -enyl, prop-2-enyl, isopropenyl, but-l-enyl, 2-methyl-prop-l- enyl, and 2-methyl-prop-2-enyl. The terms “(C2-C6)alkenyl”, etc., have equivalent meanings, i.e., an unsaturated linear or branched free radical consisting essentially of 2 to 6 carbon atoms and a corresponding number of hydrogen atoms. Alkenyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0086] As used herein, the term “aryl” means an aromatic free radical having at least 6 carbon atoms (i.e., ring atoms) that form a ring. It will be appreciated that the aryl group may be monocyclic or multicyclic (e.g., fused). In the case of multicyclic aryl groups, there are further rings, e.g. 1 or more further rings, all of which contain at least 3 carbon atoms (i.e., ring atoms). Examples of aryl groups include phenyl and naphthal enyl. The aryl group may contain from 6 to 10 carbon atoms in the ring portion of the group, which may be monocyclic or multicyclic (e.g., fused). In embodiments, aryl is phenyl. Aryl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0087] As used herein, the term “cycloalkyl” means a saturated free radical having at least 3 to 9 carbon atoms (i.e., ring atoms) that form a ring. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. It will be appreciated that the cycloalkyl group may be monocyclic or multicyclic (e.g., fused, bridged, or spirocyclic). In the case of multicyclic cycloalkyl groups, there are further rings, e.g. 1 or more further rings, all of which contain from 3 to 7 carbon atoms (i.e., ring atoms). Exemplary cycloalkyl groups having such further rings include bicyclo[l.l.l]pentanyl. One or more ring atoms of the cycloalkyl group may be substituted by (i.e., bonded to) a heteroatom by a double bond (e.g., cycloalkyl substituted by oxo). The presence of such a substituent does not prevent the carbon backbone of the free radical being considered as a cycloalkyl group. Cycloalkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0088] As used herein, the term “heterocycloalkyl” means a saturated free radical having at least 3 to 10 atoms (i.e., ring atoms) that form a ring, wherein at least 1 to 9 of said ring atoms are carbon and the remaining at least 1 to 9 ring atom(s) (i.e., hetero ring atom(s)) are selected independently from the group consisting of nitrogen, sulphur, and oxygen. Heterocycloalkyl rings may have oxo substituents, typically adjacent to a heteroatom (e.g., 2-oxopyrrolidinyl), but the oxygen atom does not form part of the ring and is excluded from the number of ring atoms. The presence of such a substituent does not prevent the ring (or rings) of the free radical being considered as a heterocycloalkyl group. Exemplary heterocycloalkyl groups include tetrahydrofuranyl, piperidinyl, morpholinyl and piperazinyl. In the case of multicyclic heterocyclic groups, there are further rings, e.g. 1 or more further rings, all of which contain from 3 to 7 ring atoms selected from carbon, nitrogen, sulphur, and oxygen. The further rings may be saturated, or partially or fully unsaturated (e.g., having aromatic character). Multicyclic heterocyclic groups include fused, bridged and spirocyclic ring systems. Where a multicyclic heterocycloalkyl group contains an unsaturated fused ring, the group is typically not bonded to the rest of the molecule via that fused ring. Heterocycloalkyl groups of the present disclosure may be substituted with one or more optional substituents as defined herein.

[0089] The term "phosphate" is typically used herein to denote a radical (or diradical) group which comprises a central phosphorus atom bonded to four oxygen atoms (one via a double bond). The oxygen atoms which are bonded to phosphorus via single bonds may represent points of attachment to the rest of the molecule, or may carry hydrogen atoms, or may be considered to carry a negative charge (e.g., in the case of salts). The term “thiophosphate” is typically used herein to denote a phosphate analogue in which at least one oxygen atom is replaced by sulphur; such groups are also called “phosphorothioates” herein.

[0090] An “optional substituent” is a group which is covalently attached to a moiety (generally via a carbon atom of the moiety, and typically in place of a hydrogen atom on said carbon atom). The optional substituent may be chosen to be a group which does not significantly alter the steric and / or electronic properties of the molecule. In embodiments, each optional substituent is independently selected from the group consisting of: halogen (e.g., -F, -Cl, -Br or -I); -OH; -SH; -NH2; -NHMe; -NMe2; -(Ci-C3)alkyl (e.g., -Me or -Et); and 3- or 4-membered cycloalkyl or heterocycloalkyl group (e.g., cyclopropyl or epoxide), which may optionally be substituted with one or more halo. A group defined as “optionally substituted” may be either unsubstituted, or substituted with one or more substituents, e.g. 1, 2, 3, 4, 5, 6, or more substituents. In embodiments, a substituted group has 1 to 4 substituents, e.g. 1, 2, or 3 substituents. In embodiments, a substituted group has 1 or 2 substituents. In embodiments, a substituted group has 3 substituents.

[0091] As used herein, the terms “halo” and “halogen” mean fluorine, chlorine, bromine, or iodine. These terms are used interchangeably and may refer to a halogen free radical group or to a halogen atom as such. Those of skill in the art will readily be able to ascertain the identification of which in view of the context in which this term is used in the present disclosure. In embodiments, the halogen is fluorine. The compounds of the present disclosure are described, inter alia, by way of structural formulae. It will be appreciated that these formulae typically show only one form (e.g., resonance form, tautomeric form, etc.) of the compound, whereas certain compounds may exist in more than one such form. This will be readily apparent to the skilled reader. The present disclosure includes all possible tautomers of the compounds characterised by the structural formulae herein, including as single tautomers, or as any mixture of tautomers in any ratio. It will also be appreciated that certain of the present compounds may exist in one or more isomeric (e.g., stereoisomeric) forms. The present disclosure includes all possible stereoisomers, enantiomers, diastereomers, etc. of the compounds described hereinbefore and below, as well as cis- and trans- forms and conformers of the same. The purification and the separation of isomers may be accomplished by methods described hereinafter, as well as by techniques known in the art. For example, optical isomers of the compounds can be obtained by resolution of the racemic mixture of diastereoisomeric salts thereof (e.g., using an optically active acid or base, or by the formation of covalent diastereomers). A different process for separation of optical isomers involves the use of chiral chromatography (e.g., HPLC columns using a chiral phase), with or without conventional derivatization. Enzymatic separation, with or without derivatisation, may also be useful, and optically active compounds of the present disclosure can likewise be obtained by chiral syntheses utilizing optically active starting materials. The present disclosure includes all possible stereoisomers of the compounds described herein as single stereoisomers, or as any mixture of said stereoisomers, e.g. (R)- or (S)- isomers, in any ratio.

[0092] The compounds of the disclosure may exist in the form of free acids or bases, or may exist as addition salts with suitable acids or bases. Methods for forming salts are described below and are also known in the art (see, e.g., Berge et al., J Pharm Sci. (1977) 66:1-19). As used herein, the term “pharmaceutically acceptable” when used in connection with salts means a salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained.

[0093] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Compounds, compositions and methods provided herein may be combined with one or more of any of the other compounds, compositions and methods provided herein.

[0094] The following abbreviations and empirical formulae are used herein:

[0095] Ac acetyl

[0096] ACN acetonitrile

[0097] ANGPTL3 angiopoietin-like 3 anti-miR anti-microRNA

[0098] Appp 5 '-adenosine cap

[0099] ASGPR asialoglycoprotein receptor

[0100] ASO antisense oligonucleotide

[0101] BCN bicyclo[6.1.0]non-4-yne group

[0102] Boc tert-butyloxy carbonyl cDNA complementary DNA cEt constrained ethyl

[0103] CRISPR clustered regularly interspaced short palindromic repeats dA deoxyadenosine dC deoxycytidine

[0104] DCM dichloromethane dG 2'-deoxyguanosine

[0105] DIPEA diisopropylethylamine

[0106] DMEM Dulbecco's modified eagle medium

[0107] DMF A AMimethy I formamide

[0108] DMTr 4,4'-dimethoxytrityl

[0109] DNA deoxyribonucleic acid

[0110] Ds 7-(2-thienyl)-imidazo[4,5-b]pyridine dT thymidine

[0111] ELISA enzyme-linked immunosorbent assay

[0112] ESI electrospray ionization

[0113] GalNAc 2-(acetylamino)-2-deoxy-D-galactopyranose

[0114] HATU hexafluorophosphate azabenzotriazole tetramethyl uronium

[0115] HBTU JVJVJV',iV'-T etramethy I -< -( I / / -benzotriazol - 1 -yl)uronium hexafluorophosphate

[0116] HEK293 human embryonic kidney 293 Hept heptane

[0117] HPLC high performance liquid chromatography invAb inverted abasic deoxyribose

[0118] LCMS liquid chromatography mass spectrometry

[0119] LNA locked nucleic acid m / z mass to charge ratio m6A N6-methyladenosine

[0120] MALAT1 metastasis associated lung adenocarcinoma transcript 1 mC 5 ’ -methylcytidine

[0121] MMT 5’-monomethoxytrityl

[0122] MOE methoxyethyl

[0123] MPEG poly(ethylene glycol) methyl ether

[0124] MPS mononuclear phagocyte system mQ Milli-Q® mRNA messenger RNA

[0125] MS mass spectrometry

[0126] NAFLD non-alcoholic fatty liver disease

[0127] NASH non-alcoholic steatohepatitis

[0128] NMM V-methylmorpholine

[0129] Pa pyrrole 2-carbaldehyde

[0130] PBS phosphate-buffered saline

[0131] PCR polymerase chain reaction

[0132] PEG polyethyleneglycol

[0133] PHH primary human hepatocytes

[0134] PNA peptide nucleic acid

[0135] PO phosphodiester (linkage) pol III RNA polymerase III

[0136] PS phosphorothioate (linkage) qPCR real-time polymerase chain reaction

[0137] RES reticuloendothelial system

[0138] RISC RNA-induced silencing complex

[0139] RNA ribonucleic acid

[0140] RNAi RNA interference saRNA small activating ribonucleic acid SEQ ID sequence identifier shRNA short hairpin RNA siNA small interfering nucleic acid siRNA small interfering RNA

[0141] TAMRA carboxytetramethylrhodamine

[0142] TFA trifluoroacetic acid

[0143] THF tetrahydrofuran

[0144] TOF time of flight

[0145] Tos toluenesulfonyl

[0146] TPPC4 trafficking protein particle complex subunit 4

[0147] U uracil

[0148] UV ultraviolet

[0149] Compounds

[0150] The present disclosure provides monosaccharide-containing compounds which are useful, inter alia, for targeting nucleic acid cargo moieties to specific locations (e.g., cell and / or tissue types) in vivo. As illustrated schematically in Figure 1, the compounds have several ‘arms’ which carry the targeting ligands and cargo moieties. These, in turn, are typically bound together by a ‘splitter’ moiety (also called a “branching unit”), which has the appropriate functionality to join the arms of the compound together. The present disclosure relates, in particular, to compounds in which the splitter is derived from, or chemically related to, the amino acid cysteine (or homocysteine) in which the sulphur atom forms a central point on the splitter moiety. The use of cysteine and analogues thereof in preparing the branching unit provides a novel way of coupling targeting and cargo moieties which may have advantages over known methods, including for example the use of simple, natural precursors, and the ability to use shorter and / or asymmetrical linkers and spacers. Moreover, the degree of oxidation of the central sulphur atom can allow for a tailoring of the properties of the compound, e.g. pharmacokinetic properties such as extent and speed of elimination in vivo.

[0151] Thus, viewed from a first aspect the present disclosure provides a compound, or a pharmaceutically acceptable salt thereof, the compound having the structure of Formula (I): wherein:

[0152] X is selected from

[0153] R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2; and

[0154] G1to G4each independently represents a -[spacer] -[ligand] moiety or a -[tether]-[linker]-[cargo] moiety, with the proviso that at least one of G1to G4represents a -[spacer] -[ligand] moiety and at least one of G1to G4represents a -[tether] -[linker] -[cargo] moiety.

[0155] In embodiments, X is selected from -S-, -S(O)- and -S(O)2-. In embodiments, X is -S-. In embodiments, X is -S(O)-. In embodiments, X is -S(O)2-.

[0156] In embodiments, each R is -H.

[0157] In embodiments, m is 1. In other embodiments, m is 2. In embodiments, n is 1. In other embodiments, n is 2. In embodiments, m is 1, and n is 1.

[0158] In embodiments, X is -S-, m is 1, n is 1 and R is -H.

[0159] In embodiments, the compound is a compound having the structure of Formula (la): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, G1, G2, G3and G4are as defined herein.

[0160] In embodiments, the compound is a compound having the structure of Formula (lb):

[0161] (lb) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, G1, G2, G3and G4are as defined herein.

[0162] In embodiments, the compound is a compound having the structure of Formula (Ic):

[0163] (Ic) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, G1, G2, G3and G4are as defined herein.

[0164] In embodiments, the compound is a compound having the structure of Formula (Id):

[0165] (Id) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, G1, G2, G3and G4are as defined herein.

[0166] The “spacer” comprises a chain of atoms, typically carbon atoms with one or more heteroatoms (e.g. selected independently fromN, O, S, and P) optionally intervening that serves to link the ligand to the splitter (i.e., the cysteine-derived core). In embodiments, the spacer comprises a chain of 2-20 atoms selected from C, N, O, S and P (e.g., a chain of 7-14 atoms). Exemplary spacers include linear alkylenes (which may optionally be interrupted by one or more amide and / or phosphate groups) and polyethylene glycols. Suitable spacers will be apparent to the skilled person based on the current description and its Examples. Other examples of spacers which might be used in accordance with the present disclosure are found, e.g., in international patent publications WO 2014 / 179620, WO 2015 / 177668, WO 2009 / 073809, WO 2012 / 083046, WO 2017 / 156012, WO 2016 / 100401, WO 2017 / 174657 and WO 2019 / 092280, the contents of each of which are incorporated herein in their entirety.

[0167] Each “ligand” which is present in the compounds of the disclosure is a monosaccharide. The monosaccharide may be selected to have an affinity for the hepatic asialoglycoprotein receptor (ASGPR). In embodiments, the target ASGPR is on the surface of a mammalian liver cell (e.g., a human ASGPR). In embodiments, the ligand is GalNAc. In embodiments, the compounds of the disclosure have 1, 2 or 3 (e.g., 3) ligands which are all GalNAc.

[0168] The “tether” and “linker” groups taken together act to join the cargo moiety (or moieties) to the rest of the molecule, i.e. to the splitter. Suitable tethers and linkers will be apparent to the skilled person based on the current description and its Examples. In embodiments, the [tether] -[linker] moiety is obtained by reacting a tether precursor (which may be bonded to the rest of the molecule, i.e. to the spacer / ligand construct) with a linker precursor (which may be bonded to the cargo) to join the two together. Suitable methods for reacting the tether and linker precursors are described in the following Examples and would be known to the skilled person. Examples of such methods include ‘click’ chemistry reactions such as copper catalysed cycloadditions between an azide and an alkyne (see, e.g., Fantoni et al., Chem. Rev. (2021) 121 (12): 7122— 7154). In other embodiments, the [tether] -[linker] moiety is introduced into the complex without the cargo attached and the cargo is subsequently bound to the linker. The complex may conveniently be coupled with a cargo in a final step of a solid-phase synthesis, e.g. where the [tether] -[linker] moiety is initially introduced with a phosphate group at its distal end that can be reacted with a cargo nucleic acid on solid phase to yield the final compound (see, e.g., Beaucage, Curr Opin Drug Discov Dev (2008) 11 (2):203-216). Other examples of tethers and / or linkers which might be used in accordance with the present disclosure are found, e.g., in international patent publications WO 2014 / 179620, WO 2015 / 177668, WO 2009 / 073809, WO 2012 / 083046, WO 2017 / 156012,

[0169] WO 2016 / 100401, WO 2017 / 174657 and WO 2019 / 092280, the contents of each of which are incorporated herein in their entirety. In embodiments, the tether and linker together represent a moiety comprising a linear chain of 8 to 30 atoms which links the cargo to the rest of the molecule. In embodiments, the tether and linker together represent an optionally substituted linear chain of 8 to 30 atoms selected from C, N, O , S and P, e.g. a chain of 9 to 24 atoms, 10 to 20 atoms, or 12 to 18 atoms. In embodiments, the tether and linker together comprise one or more groups selected from PEG, an amide (e.g., obtained by reaction of a carboxylic acid or acid derivative with an amine), an ester (e.g., obtained by reaction of a carboxylic acid or acid derivative with an alcohol, such as an aliphatic or aromatic alcohol), or a 1,2,3-triazole (e.g., obtained by reaction of an azide with an alkyne). In embodiments, the tether and linker together comprise (e.g., consist of) a group represented by:

[0170] -(CH2)q-D-(CH2)s-* or -[CH2CH2O]rCH2CH2-D-(CH2)s-* wherein D is selected from (i) a direct bond, (ii) -C(O)NH-, and (ii) a 1,2,3-triazole containing group; q is an integer from 1 to 12 (e.g., from 2 to 8, such as 6); r is an integer from 1 to 3 (e.g., 2); and s is an integer from 0 to 12 (e.g., from 2 to 8, such as 6). The group may be optionally substituted and the * denotes the point of attachment to the cargo. It will be appreciated that the [tether] -[linker] group illustrated above will typically be connected to the cargo via a phosphate group. Thus, in embodiments tether and linker together comprise a group as shown above which further comprises a phosphate group (e.g., -OP(=O)(OH)O-) at the position marked with *.

[0171] In embodiments, D is a direct bond. The tether and linker together may thus comprise (e.g., consist of) a group represented by -(CH2)q(CH2)s-* or -[CH2CH2O]rCH2CH2(CH2)s-*, wherein q is an integer from 1 to 12 (e.g., 6), r is an integer from 1 to 3 (e.g., 2), and s is an integer from 0 to 12 (e.g., 6). In embodiments, s is 0, in which case the tether and linker together comprise (e.g., consist of) a group represented by -(CH2)q- or -[CH2CH2O]rCH2CH2-* wherein q is an integer from 1 to 12 (e.g., 6) and r is an integer from 1 to 3 (e.g., 2).

[0172] In other embodiments, D is -C(O)NH-. The tether and linker together may thus comprise (e.g., consist of) a group represented by -(CH2)q-C(O)NH-(CH2)s-* or -[CH2CH2O]rCH2CH2-C(O)NH-(CH2)s-*, wherein q is an integer from 1 to 12 (e.g., 6), r is an integer from 1 to 3 (e.g., 2), and s is an integer from 0 to 12 (e.g., 6). In embodiments, q is 6 and s is 6. In other embodiments, r is 2 and s is 6.

[0173] In yet further embodiments, D is a 1,2,3-triazole containing group, e.g. a group which is obtained by the reaction of BCN with an azide. Thus, in embodiments D comprises (e.g., consists of) a group wherein f denotes the point of attachment to -(CH2)S-. In embodiments, tether and linker together comprise (e.g., consist of) a group represented by wherein r and s are defined herein (e.g., wherein r is 2 and s is 6).

[0174] As will be appreciated, the [tether] -[linker] moiety can conceptually be ‘split’ in a number of ways to yield the individual tether and linker components. In embodiments, the [tether] -[linker] moiety is generated by the reaction of a cargo-containing moiety (that comprises the linker, or a portion thereof) with a ligand-containing precursor (that comprises the tether, or a portion thereof). Thus, in embodiments the tether is represented by a group -(CH2)q-, -(CH2)q-C(O)-, or -[CH2CH2O]rCH2CH2-, wherein q and r are as defined herein. Likewise, in embodiments the linker is represented by a group -D-(CH2)s-* or -NH-(CH2)S-*, wherein D and s are as defined herein. In embodiments, the tether is -(CH2)q-C(O)- wherein q is 6, and the linker is -NH-(CH2)s-* wherein s is 6. In other embodiments, the tether is -[CH2CH2O]rCH2CH2- wherein r is 2, and the linker is -D-(CH2)S-* wherein D is a group and s is 6.

[0175] The “cargo” groups of the compounds disclosed herein are nucleic acids. These may be single stranded or double stranded. In embodiments, the cargo is an oligonucleotide. In embodiments, the cargo is selected from an antisense oligonucleotide (ASO), an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice altering oligonucleotide, a splice-switching oligonucleotide, a triplex forming oligonucleotide, a siRNA, a saRNA, a microRNA, a microRNA mimic, an anti-miR, a double stranded RNA, a single stranded RNA, a ribozyme, an aptamer, a spiegelmer, a CRISPR oligonucleotide and a G-quadruplex. In one embodiment, the cargo is an antisense oligonucleotide (ASO). In another embodiment, the cargo is a siRNA.

[0176] In embodiments, G1to G3independently represent a -[spacer]-[ligand] moiety, and G4represents a -[tether]-[linker]-[cargo] moiety, e.g. moieties as described herein. Viewed from this aspect, the disclosure provides a compound, or a pharmaceutically acceptable salt thereof, the compound having the structure of Formula (II):

[0177] (II) wherein:

[0178] X is selected from

[0179] R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2;

[0180] A1to A3in each case is independently a moiety which spaces the ligand from the rest of the molecule, e.g. comprising a linear chain of between 2 and 16 atoms;

[0181] Z is a cargo-containing moiety, which may comprise a linker and a tether (e.g. as defined herein), and wherein the cargo is a nucleic acid; and

[0182] [ligand] in each case is independently a monosaccharide (e.g., GalNAc).

[0183] In embodiments, Z represents -[tether]-[linker]-[cargo], in which [tether] -[linker] represents a moiety as defined herein (e.g., comprising a linear chain of 8 to 30 atoms); and [cargo] is a nucleic acid (e.g., as defined herein).

[0184] In embodiments, one or more of A1to A3is independently selected from *-(C1-C16)alkylene-, *-(C2-C16)alkenylene-, *-(C1-C12)alkylene-C(O)-, *-(C2-Ci2)alkenylene-C(O)-, *-(C1-C8)alkylene-C(O)NR’-(C1-C6)alkylene-, *-(C2-C8)alkenylene-C(O)NR’-(C1-C6)alkylene-, *-(C1-C8)alkylene-C(O)NR’-(C2-C6)alkenylene- and *-(C2-C8)alkenylene-C(O)NR’-(C2-C6)alkenylene- (where * in each case denotes the point of attachment to the ligand), wherein R’ in each case is independently selected from -H and -(C1-C6)alkyl. In embodiments, one or more of A1to A3is independently selected from *-(C1-C12)alkylene- and *-(C1-C6)alkylene-C(O)NR’-(C1-C4)alkylene-, wherein R’ in each case is independently selected from -H and -(C1-C6)alkyl. In embodiments, one or more of A1to A3is independently selected from *-(C1-C12)alkylene-, such as *-(C2-C8)alkylene- or *-(C3-C6)alkylene-, e.g. wherein A1, A2and / or A3is n-butylene. In other embodiments, one or more of A1to A3is independently selected from *-(C1-C6)alkylene-C(O)NR’-(Ci-C4)alkylene- (wherein R’ in each case is independently selected from -H and -(C1-C6)alkyl), such as *-(C2-C5)alkylene-C(O)NR’-(C2-C4)alkylene-, e.g. wherein A1, A2and / or A3is *-(C4)alkylene-C(O)NH-(C3)alkylene-. In embodiments, each of A1, A2and A3is the same.

[0185] In embodiments, one or more of A1to A3is independently a moiety comprising a polyethylene glycol, e.g. selected from -(CH2CH2O)P(CH2)2-* and -(CH2)OCONH(CH2CH2O)P(CH2)2-* (where * in each case denotes the point of attachment to the ligand), wherein o is an integer selected from 1, 2, 3 and 4 (e.g., 2); and p is an integer selected from 1, 2, 3 and 4 (e.g., 2 or 3). In embodiments, each of A1to A3is independently selected from -(CH2CH2O)P(CH2)2-*, wherein p is an integer selected from 1, 2, 3 and 4 (e.g., 3). In embodiments, each of A1to A3is independently selected from -(CH2)OCONH(CH2CH2O)P(CH2)2-*, wherein o is an integer selected from 1, 2, 3 and 4 (e.g., 2); and p is an integer selected from 1, 2, 3 and 4 (e.g., 3). In embodiments, each of A1, A2and A3is the same.

[0186] In embodiments, the compound is a compound having the structure of Formula (Ila):

[0187] („a) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined herein.

[0188] In embodiments, the compound is a compound having the structure of Formula (lib):

[0189] (lib) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined herein.

[0190] In embodiments, the compound is a compound having the structure of Formula (He):

[0191] (He) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined herein.

[0192] In embodiments, the compound is a compound having the structure of Formula (lid):

[0193] (lid) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined herein.

[0194] In particular embodiments, the ligand in each case is N-acetylglucosamine (“GalNAc”) which may facilitate or promote binding of the compound to biological components such as ASGRPs. This may target the delivery of cargo molecules to cells and tissues which express such receptors. Viewed from this aspect, the disclosure provides a compound, or a pharmaceutically acceptable salt thereof, the compound having the structure of Formula (HI) wherein X, R, m, n, A1, A2, A3and Z are as defined herein.

[0195] In embodiments: X is selected from

[0196] R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2;

[0197] A1to A3in each case is independently a moiety which spaces the ligand from the rest of the molecule, e.g. comprising a linear chain of between 2 and 16 atoms; and

[0198] Z is a cargo-containing moiety, which may comprise a linker and a tether (e.g. as defined herein), and wherein the cargo is a nucleic acid.

[0199] In embodiments: X is selected from R in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2; A1to A3in each case is independently a moiety comprising a linear chain of between 2 and 16 atoms; and Z represents -[tether] -[linker] -[cargo], in which [tether] -[linker] represents a moiety comprising a linear chain of 8 to 30 atoms; and [cargo] is a nucleic acid.

[0200] In embodiments, R in each case is -H, m is 1, and n is 1.

[0201] In embodiments, the compound is a compound having the structure of Formula (Illa): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined herein.

[0202] In embodiments, the compound is a compound having the structure of Formula (Illb):

[0203] or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined herein.

[0204] In embodiments, the compound is a compound having the structure of Formula (IIIc): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined herein.

[0205] In embodiments, the compound is a compound having the structure of Formula (IId): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined herein.

[0206] In embodiments, the compound is a compound as illustrated in any one of the Examples described below. Viewed from this aspect the disclosure provides a compound selected from:

[0207] and the pharmaceutically acceptable salts thereof, wherein “nucleic acid” denotes a cargo nucleic acid as defined herein. In embodiments, the nucleic acid is an ASO (e.g., a MALAT1 ASO having a sequence comprising SEQ ID NO: 1). In other embodiments, the nucleic acid is a siRNA (e.g., an ANGPTL3 siRNA having a sequence comprising SEQ ID NO: 2 and / or SEQ ID NO: 3).

[0208] In embodiments, the compound is a compound of any one of Examples 1 to 4 (Compounds 1 to 5) or a pharmaceutically acceptable salt thereof. In embodiments, the compound is Compound 1 or a pharmaceutically acceptable salt thereof. In embodiments, the compound is Compound 2 or a pharmaceutically acceptable salt thereof. In embodiments, the compound is Compound 3 or a pharmaceutically acceptable salt thereof. In embodiments, the compound is Compound 4 or a pharmaceutically acceptable salt thereof. In embodiments, the compound is Compound 5 or a pharmaceutically acceptable salt thereof.

[0209] In embodiments, the compound of the disclosure is characterised according to its binding affinity for ASGPR, e.g., human ASGPR (e.g., as measured according to the HTRF assay described in Example 5 below). In embodiments, the compound has an ICso value of less than about 10 nM, e.g., an ICso value of less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM. In embodiments, the compound has an ICso value of less than about 2.0 nM, e.g. less than about 1.5 nM, 1.0 nM or 0.5 nM.

[0210] In embodiments, the compound of the disclosure is characterised according to its binding kinetics for ASGPR, e.g., human ASGPR (e.g., as measured according to the SPR assay described in Example 5 below). In embodiments, the compound has a Kd value of less than about 8 nM, e.g., a Kd value of less than about 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In embodiments, the compound has a Kd value of less than about 1.0 nM, e.g. less than about 0.5 nM.

[0211] In embodiments, the compound of the disclosure is characterised according to its binding affinity for native ASGPR, e.g., murine ASGPR (e.g., as measured according to the fluorescence polarization assay described in Example 5 below). In embodiments, the compound has an ICso value of less than about 10 nM, e.g., an ICso value of less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM. In embodiments, the compound has an ICso value of less than about 2.0 nM, e.g. less than about 1.0 nM. In other embodiments, the compound has an ICso value of less than about 100 nM, e.g., an ICso value of less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM or 20 nM.

[0212] In embodiments, the compound of the disclosure is characterised according to its activity in knocking down gene expression in HEK293 cells, e.g. in HEK293 cells overexpressing ASGPR (e.g., as measured according to the qPCR described Example 5 below). In embodiments, the compound has an ICso value of less than about 1000 nM, e.g., an ICso value of less than about 800 nM, 700 nM, 600 nM or 500 nM. In embodiments, the compound has an ICso value of less than about 400 nM, e.g. an ICso value of less than about 200 nM, 100 nM or 50 nM. In embodiments, the compound of the disclosure is characterised according to its activity in knocking down gene expression in PHH cells, e.g. in PHH cells overexpressing ASGPR (e.g., as measured according to the qPCR described Example 5 below). In embodiments, the compound has an ICso value of less than about 1000 nM, e.g., an ICso value of less than about 800 nM, 700 nM, 600 nM or 500 nM. In embodiments, the compound has an ICso value of less than about 400 nM, e.g. an ICso value of less than about 200 nM or 100 nM.

[0213] In embodiments, the compound of the disclosure is characterised according to its activity in knocking down gene expression in tissues such as liver in vivo (e.g., as measured according to the assay described Example 6 below). In embodiments, the compound can knockdown levels of the target mRNA by at least about 40%, e.g. by at least about 45% or 50%. In embodiments, the knockdown of at least about 40% is achieved in liver but not in one or more other tissues (e.g., not in kidney).

[0214] Nucleic acids

[0215] As defined herein, the compounds of the disclosure are particularly suitable for delivering nucleic acids to cells and tissues. The “cargo” groups of the compounds disclosed herein are thus nucleic acids. The term “nucleic acid” as used herein includes nucleic acids selected from the group consisting of DNA, RNA, PNA and LNA. The nucleic acid may be a functional nucleic acid, e.g., whereby the functional nucleic acid is selected from the group consisting of mRNA, micro-RNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acid (e.g., antisense oligonucleotide (ASO)), ribozymes, aptamers and spiegelmers. In embodiments, the nucleic acid is selected from siRNA and ASO. In embodiments, the nucleic acid is siRNA. In other embodiments, the nucleic acid is ASO.

[0216] The nucleic acids may be of any length and can have any number of nucleotides such that they are effective for the intended purpose (e.g., RNAi). In embodiments, a siRNA ranges 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, 30 base pairs. In embodiments, the nucleic acid has 19 to 23 base pairs. For example, the nucleic acid may be 19, 20, 21, 22 or 23 base pairs in length. A double stranded RNAi may be blunt ended at one end or on both ends. A double stranded RNAi may have overhangs of 1 or more nucleotides 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. 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. For example, 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. 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. The nucleic acid of the disclosure 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.

[0217] A nucleic acid of the disclosure may comprise two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and can result in downregulation of gene expression in a targeted manner. The nucleic acid may comprise 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. The first strand may also be referred to as an antisense strand. The second strand may also be referred to as a sense strand.

[0218] The 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.

[0219] 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 hybridise under physiological conditions. 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%. 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.

[0220] Unmodified polynucleotides, particularly ribonucleotides, may be prone to degradation by cellular nucleases, and, as such, modification and / or modified nucleotides may be included in the nucleic acid of the disclosure. One or more nucleotides on the second and / or first strand of the nucleic acid of the disclosure may be modified. Modifications of the nucleic acid of the present disclosure 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 disclosure may be modified by chemical modifications. Modified nucleic acid can also minimise 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 disclosure 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.

[0221] One or more nucleotides of a nucleic acid of the present disclosure 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. A nucleic acid of the disclosure may have 1 modified nucleotide or a nucleic acid of the disclosure 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

[0222] 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.

[0223] The nucleic acid may comprise 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-methyl cytidine), 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-acetyl cytidine, 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 (m6A), 7-methylguanosine, 5- methoxyaminomethyl-2-thiouridine, 5 -methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5 -methyloxy uridine, 5-methyl-2 -thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2- thiocytidine.

[0224] Nucleic acids discussed herein include unmodified RNA as well as RNA which has 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.

[0225] Many of the modifications described herein 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 the anon-linking oxygen 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 terminal regions, 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 disclosure or may only occur in a single strand region of a nucleic acid of the disclosure. A phosphorothioate modification at a non-linking oxygen 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.

[0226] Stability of a nucleic acid of the disclosure 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 deoxy ribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

[0227] Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and can render oligoribonucleotides more stable to nucleases. Modified nucleic acids, as used herein, can include one or more of:

[0228] (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 disclosure);

[0229] (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

[0230] (iii) replacement of the phosphate moiety with "dephospho" linkers;

[0231] (iv) modification or replacement of a naturally occurring base;

[0232] (v) replacement or modification of the ribose-phosphate backbone;

[0233] (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 labelled moiety, to either the 3’ or 5' end of RNA.

[0234] The terms replacement, modification, alteration, indicate a difference from a naturally occurring molecule.

[0235] 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). The phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulphur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

[0236] 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. 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, 0(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). "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), -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 substituents of certain embodiments include 2'- methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0237] 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 anucleobase at C-T. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.

[0238] The 2' modifications may be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The phosphate group can be replaced by nonphosphorus containing connectors. 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.

[0239] The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides. Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0240] 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 labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based, e.g., on sulphur, silicon, boron or an 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)-NH-, -(CH2)nO-, -(CH2)nS-, O(CH2CH2O)nCH2CH2OH (e.g., where n=3 or 6), abasic sugars, amide, carboxy, amine, oxy amine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' end can be an -OH group.

[0241] 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, radiolabelled 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, acridineimidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0242] 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 disclosure, 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-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O- (HO)(O)P-O-P(HO)(O)-O-5'); 5 '-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate); (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxy gen / sulphur replaced monophosphate, diphosphate and triphosphates (e.g., 5 '-alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(0H)(0)-0-5'-, (OH)2(O)P-5'-CH2-), 5'-vinylphosphonate, 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(0H)(0)-0-5'-).

[0243] The nucleic acid of the present disclosure 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.

[0244] 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.

[0245] 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. For example, 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 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 pyrrolidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynyl cytosine, 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-methyl uracil, 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, 3-(3- amino-3-carboxypropyl)uracil, 3 -methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine, 2- thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-mcthylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

[0246] 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' OMe nucleotide; and nucleotide analogs including 4',5'- methylene nucleotide; l-(P-D-erythrofuranosyl)nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5 '-amino-alkyl phosphate; l,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-dihydroxybutyl 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. The nucleic acids of the disclosure may include one or more inverted nucleotides, for example inverted thymidine or inverted adenine (see, e.g., Takei et al., J Biol Chem (2002) 277(26):23800-23806).

[0247] The nucleic acid of the present disclosure may comprise an abasic nucleotide. 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. In alternating nucleotides, 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.

[0248] / ?A74 Modifications

[0249] Modifications of the siRNA molecules of the present disclosure generally provides 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 siRNA according to the disclosure may be modified by chemical modifications. Modified siRNA can also minimize the possibility of activating interferon activity in humans. Modification can further enhance the functional delivery of a siRNA to a target cell. The modified siRNA of the present disclosure may comprise one or more chemically modified ribonucleotides of either or both of the antisense strand or the sense 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.

[0250] One or more nucleotides of a siRNA of the present disclosure may comprise a modified base. In one aspect, the siRNA comprises at least one nucleotide comprising a modified base. In one embodiment, the modified base in on the antisense strand. In another embodiment, the modified base in on the sense strand. In another embodiment, the modified base is in the duplex region. In another embodiment, the modified base is outside the duplex region, i.e., in a single stranded region. In another embodiment, the modified base is on the antisense strand and is outside the duplex region. In another embodiment, the modified base is on the sense strand and is outside the duplex region. In another embodiment, the 3'-terminal nucleotide of the antisense strand is a nucleotide with a modified base. In another embodiment, the 3'- terminal nucleotide of the sense strand is nucleotide with a modified base. In another embodiment, the 5'-terminal nucleotide of the antisense strand is nucleotide with a modified base. In another embodiment, the 5'-terminal nucleotide of the sense strand is nucleotide with a modified base.

[0251] The modified base may be a purine or a pyrimidine. In another embodiment, at least half of the purines are modified. In another embodiment, at least half of the pyrimidines are modified. In another embodiment, all of the purines are modified. In another embodiment, all of the pyrimidines are modified. In another embodiment, the siRNA may comprise a nucleotide comprising a modified base, 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.

[0252] In another aspect, a siRNA of the present disclosure comprises an abasic nucleotide. As used herein, a nucleotide with a modified base does not include abasic nucleotides.

[0253] Modifications to sugar moiety

[0254] Another aspect relates to modifications to a sugar moiety. One or more nucleotides of a siRNA of the present disclosure may comprise a modified ribose moiety. Modifications at the 2'-position where the 2'-OH is substituted include the non-limiting examples selected from alkyl, substituted alkyl, alkaryl-, arylalkyl-, -F, -Cl, -Br, -CN, -CF3, -OCF3, -OCN, -O-alkyl, - S-alkyl, HS-alkyl-O, -O-alkenyl, -S-alkenyl, -N-alkenyl, -SO-alkyl, -alkyl-OSH, -alkyl-OH, - O-alkyl-OH, -O-alkyl-SH, -S-alkyl-OH, -S-alkyl-SH, -alkyl-S-alkyl, -alkyl-O-alkyl, -ONO2, - NO2, -N3, -NH2, alkylamino, dialkylamino-, aminoalkyl-, aminoalkoxy, aminoacid, aminoacyl-, -ONH2, -O-aminoalkyl, -O-aminoacid, -O-aminoacyl, heterocycloalkyl-, heterocycloalkaryl-, aminoalkylamino-, polyalklylamino-, substituted silyl-, methoxyethyl- (MOE), alkenyl and alkynyl. "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 is further included as a 2' modification of the present disclosure. In embodiments, substituents are 2'-methoxy ethyl, 2'-O-CH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro (2’-F).

[0255] Modified groups

[0256] In one aspect, the antisense duplex region comprises a plurality of groups of modified nucleotides, referred to herein as "modified groups", wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a second group of nucleotides, referred to herein as "flanking groups", wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense duplex region is identical, i.e., each modified group consists of an equal number of identically modified nucleotides. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the antisense duplex region comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2' position.

[0257] In another aspect, the sense duplex region comprises a plurality of groups of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the sense duplex region is identical. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of said modified groups in the sense duplex region comprise a modified base. In another embodiment, the nucleotides of said modified groups comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups comprise a modified 2' position.

[0258] In another aspect, the antisense duplex region and the sense duplex region each comprise a plurality of modified groups, wherein each modified group consists of one or more identically modified nucleotides, wherein each modified group is flanked on one or both sides by a flanking group, wherein each said flanking group consists of one or more nucleotides that are either unmodified or modified in a manner different from the nucleotides of said modified group. In one embodiment, each modified group in the antisense duplex region and the sense duplex region are identical. In another embodiment, each flanking group in the antisense duplex region and the sense duplex region each have an equal number of nucleotides. In another embodiment, each flanking group in the antisense duplex region and in the sense duplex region are identical. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise the same modified groups and the same flanking groups. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified base. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified phosphate backbone. In another embodiment, the nucleotides of said modified groups in the antisense duplex region and the sense duplex region each comprise a modified 2' position.

[0259] Modifications to the phosphate backbone

[0260] Another aspect relates to modifications to a phosphate backbone. All or a portion of the nucleotides of the siRNA of the disclosure may be linked through phosphodiester bonds, as found in unmodified nucleic acid. A siRNA of the present disclosure however, may comprise a modified phosphodiester linkage. The phosphodiester linkages of either the antisense stand or the sense strand may be modified to independently include at least one heteroatom selected from nitrogen and sulphur. In one embodiment, a phosphoester group connecting a ribonucleotide to an adjacent ribonucleotide is replaced by a modified group. In one embodiment, the modified group replacing the phosphoester group is selected from phosphorothioate, methylphosphonate, phosphorodithioate or phosphoramidate group.

[0261] 5 ' and 3 ' end modifications

[0262] The siRNA of the present disclosure may include nucleic acid molecules comprising one or more modified nucleotides, abasic nucleotides, acyclic or deoxyribonucleotide at the terminal 5'- or 3'-end on either or both of the sense or antisense strands. The 5'-end nucleotide of the antisense and / or strand may be phosphorylated. In another embodiment, the 5'-end nucleotide of the antisense strand is phosphorylated and the 5'-end nucleotide of the sense strand has a free hydroxyl group (5'-OH). In another embodiment, the 5'-end nucleotide of the antisense strand is phosphorylated and the 5'-end nucleotide of the sense strand is modified. In another embodiment the 5'-end nucleotide of the antisense strand carries a 5'E vinylphosphonate.

[0263] Modifications to the 5'- and 3 '-end nucleotides are not limited to the 5' and 3' positions on these terminal nucleotides. Examples of modifications to end nucleotides include, but are not limited to, biotin, inverted (deoxy) abasics, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, Ci-Cio lower alkyl, substituted lower alkyl, alkaryl or arylalkyl, OCF3, OCN, O-, S-, or N-alkyl; O-, S-, orN-alkenyl; SO-CH3; SO2CH3; ONO2; N02, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described, e.g., in PCT patent publication WO 99 / 54459, or in European patent EP 0 586 520 Bl or EP 0 618 925 Bl, each of which is incorporated by reference in its entirety.

[0264] In another embodiment, the terminal 3' nucleotide or two terminal 3 '-nucleotides on either or both of the antisense strand or sense strand is a 2'-deoxynucleotide. In another embodiment, the 2'-deoxynucleotide is a 2'-deoxy-pyrimidine. In another embodiment, the 2'- deoxynucleotide is a 2' deoxy-thymidine. shRNA (short hairpin loop RNA) and linked siRNA

[0265] Another aspect relates to shRNA and linked siRNA. The antisense strand and the sense strand may be covalently linked to each other. Such linkage may occur between any of the nucleotides forming the antisense strand and sense strand, respectively and can be formed by covalent or non-covalent linkages. Covalent linkage may be formed by linking both strands one or several times and at one or several positions, respectively, by a compound selected from the group comprising methylene blue and bifunctional groups. In embodiments, bifunctional groups are selected from the group comprising bis(2-chloroethyl)amine, N- acetyl-NV'-(p-glyoxylbenzoyl)cystamine, 4-thiouracil and psoralene.

[0266] Further, the antisense strand and the sense strand may be linked by a loop structure. The loop structure may be comprised of a non-nucleic acid polymer such as polyethylene glycol. The 5'-end of the antisense strand may be linked to the 3 '-terminus of the sense strand or the 3'- end of the antisense strand may be linked to the 5'-end of the sense strand. The loop may consist of a nucleic acid, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or the loop may be formed by polymers. The length of the loop may be sufficient for linking the two strands covalently in a manner that a back folding can occur through a loop structure or similar structure.

[0267] The ribonucleic acid constructs may be incorporated into suitable vector systems. In embodiments, the vector comprises a promoter for the expression of RNAi. The promoter may be selected from any known in the art such as, e.g., pol III, U6, Hl or 7SK.

[0268] The nucleic acids according to the disclosure may comprise one or more phosphorothioate intemucleotide linkages. The phosphorothioate intemucleotide linkages may be distributed across the entire nucleotide sequences and may occur in any number at any position. The nucleic acids can comprise between one to ten phosphorothioate intemucleotide linkages. The antisense strand may have at least 1 phosphorothioate modification at each end. The antisense strand may have 1-3 phosphorothioate modifications at each end. For example, the antisense strand may have 2 phosphorothioate modifications at each end. The sense strand may have at least 1 phosphorothioate modification at the 3' end. The sense strand may have 1-3 phosphorothioate modification at the 3' end. For example, the sense strand may have 2 phosphorothioate modifications at the 3 ' end. siRNA with overhangs

[0269] An overhang at the 3'-end or 5' end of the sense strand or the antisense strand may be selected from the group consisting of 1, 2, 3, 4 and 5 nucleotides in length. Alternatively, the siRNA molecule may be blunt-ended on both ends and may have a length of 16 to 29 consecutive nucleotides. In one embodiment, the siRNA molecule is blunt-ended on one end and the double stranded or duplex portion of the siRNA molecule has a length selected from 16 to 29 consecutive nucleotides. In one embodiment, the siRNA molecule has overhangs on both ends on either strand and the double stranded or duplex portion of the siRNA molecule has a length of 16 to 29 consecutive nucleotides. The overhang may comprise at least one deoxy ribonucleotides and / or a TT dinucleotide.

[0270] It will be appreciated by one skilled in the art that the modification, modifications of the sugar moiety, pattern, 5' and 3' end modifications, overhangs, formulations, delivery, dosage and routes of delivery as described above may equally be applied to any type of RNAi molecule and is not limited to siRNAs.

[0271] The nucleic acid of the present disclosure can be produced using routine methods in the art including chemically synthesis or expressing the nucleic acid either in vitro (e.g., run off transcription) or in vivo. For example, using solid phase chemical synthesis or using an expression vector. In one embodiment, the expression vector can produce the nucleic acid of the disclosure in a target cell. Methods for the synthesis and purification of the nucleic acid molecules described herein are known to persons skilled in the art.

[0272] Pharmaceutical compositions

[0273] In one aspect, the disclosure provides a pharmaceutical composition comprising a compound described herein (e.g., a compound of Formula (I)) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier. In embodiments, the pharmaceutical composition comprises a compound of Formula (I) (e.g., a compound of Formula (la)) or a pharmaceutically acceptable salt thereof. In embodiments, the pharmaceutical composition comprises a compound of Formula (II) (e.g., a compound of Formula (Ila)) or a pharmaceutically acceptable salt thereof. In embodiments, the pharmaceutical composition comprises a compound of Formula (III) (e.g., a compound of Formula (Illa)) or a pharmaceutically acceptable salt thereof.

[0274] Nucleic acids and conjugated compounds can be delivered to cells, both in vitro and in vivo, by a variety of methods known to those skilled in the art, including direct contact with cells or by combination with one or more agents that facilitate targeting and / or delivery into cells. Such agents and methods include lipoplexes, liposomes, iontophoresis, hydrogels, cyclodextrins, nanocapsules, micro- and nanospheres and proteinaceous vectors. The nucleic acid / vehicle combination may be locally delivered in vivo by direct injection or by use of an infusion pump.

[0275] The composition of the disclosure may comprise surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing stability of a liposome or lipoplex solutions by preventing their aggregation and fusion. The formulations also have the added benefit in vivo of resisting opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug. Such liposomes may accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues. The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (see, e.g., Liu et al, J. Biol. Chem. (1995) 42:24864-24780; and PCT publication Nos. WO 96 / 10391; WO 96 / 10390; and WO 96 / 10392, the contents of each of which are incorporated herein in their entirety). Long- circulating liposomes may also protect siRNA from nuclease degradation.

[0276] The pharmaceutical compositions of the present disclosure may be used as medicaments or as diagnostic agents. For example, one or more compounds (e.g., siRNA conjugates) of the disclosure can be combined with a delivery vehicle (e.g., liposomes) and excipients, such as carriers or diluents. Other agents such as preservatives and stabilizers can also be added. Methods for the delivery of nucleic acid-containing molecules are known in the art and within the knowledge of the person skilled in the art. The compounds (e.g., siRNA conjugates) of the present disclosure can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose.

[0277] The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form. In one embodiment, the pharmaceutical composition comprises lyophilized lipoplexes or an aqueous suspension of lipoplexes. In embodiments, the lipoplexes comprise a compound of the disclosure. Such lipoplexes may be used to deliver the compounds of the disclosure to a target cell either in vitro or in vivo.

[0278] The pharmaceutical compositions and medicaments of the present disclosure may be administered to a subject (e.g., a mammal) in a pharmaceutically effective dose. The mammal may be selected from humans, dogs, cats, horses, cattle, pig, goat, sheep, mouse, rat, hamster and guinea pig. In embodiments, the subject is human.

[0279] A compound or composition of the disclosure (e.g., a composition that includes a double stranded siRNA) can be delivered to a subject by a variety of routes. Exemplary routes include: subcutaneous, intramuscular, intradermal, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. In embodiments, a compound of the disclosure is delivered in vivo by means selected from intravenous, subcutaneous, intramuscular or intradermal injection, or by inhalation. In one embodiment, the compound of the disclosure is delivered by intravenous injection or infusion.

[0280] The route and / or site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the composition in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the compound or composition and mechanically introducing the nucleic acid.

[0281] The pharmaceutical compositions of the disclosure may be formulated for administration in solid or liquid form, e.g., using conventional carriers or excipients. Compositions may be adapted for, e.g., oral administration (e.g., as a solution, suspension, tablet, or capsule), parenteral administration (e.g., as a solution, dispersion, suspension, or emulsion, or as a dry powder for reconstitution), or topical application (e.g., as a cream, ointment, patch, or spray to be applied to the skin) using techniques known in the art.

[0282] Medical Uses

[0283] Compounds of the present disclosure act as modulators of nucleic acids in vivo, which gives them utility in the treatment of numerous disorders and conditions.

[0284] Viewed from this aspect, the disclosure provides a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) for use in therapy. In a related aspect is provided the use of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) as a medicament. In another related aspect is provided a method of treating a subject in need thereof, the method comprising administering an effective amount of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) to the subject. In another related aspect is provided the use of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) in the manufacture of a medicament.

[0285] In particular, compounds of the disclosure can target cells and / or tissues which carry an ASGP receptor and are useful in the treatment of such cells and tissues by delivery of therapeutic nucleic acids. View from this aspect, the disclosure provides a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) for use in the treatment of a condition in a subject, which condition is treatable by delivery of a therapeutic nucleic acid to cells and / or tissues of the subject which express ASGPR. In a related aspect, the disclosure provides a method for improving the therapeutic activity of a therapeutic nucleic acid in treating a condition in a subject, which condition is treatable by delivery of the therapeutic nucleic acid to cells and / or tissues of the subject which express ASGPR, the method comprising coupling the therapeutic nucleic acid with one or more molecules to form a compound of the present disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt). In another related aspect, the disclosure provides a method for improving the treatment of a condition in a subject, which condition is treatable by delivery of a therapeutic nucleic acid to cells and / or tissues of the subject which express ASGPR, wherein the method comprises delivering the therapeutic nucleic acid as part of a compound of the present disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt).

[0286] Examples of conditions which may be treatable by delivery of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) include liver diseases (e.g., liver cancer, such as hepatocellular carcinoma), genetic diseases, haemophilia and bleeding disorders, liver fibrosis, nonalcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g., acromegaly), metabolic diseases (e.g. hypercholesterolemia, dyslipidaemia, hypertriglyceridemia), cardiovascular diseases, obesity, thalassemia, liver injury (e.g., drug induced liver injury), hemochromatosis, alcoholic liver diseases, alcohol dependence, anaemia, and anaemia of chronic diseases. In embodiments, the condition is selected from NASH, NAFLD, a metabolic disease and a cardiovascular disease. In embodiments, the condition is NASH.

[0287] Viewed from another aspect, the disclosure provides a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) for use in the treatment of a condition as defined herein. In a related aspect is provided the use of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) in the treatment of a condition as defined herein. In another related aspect is provided a method of treating a condition in a subject in need thereof, the method comprising administering an effective amount of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) to the subject, wherein the condition is as defined herein. In a further related aspect is provided the use of a compound of the disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt) in the manufacture of a medicament for the treatment of a condition as defined herein. In embodiments of the above aspects, the condition is selected from NASH, NAFLD, a metabolic disease (e.g., selected from hypercholesterolemia, dyslipidaemia, and hypertriglyceridemia) and a cardiovascular disease. In embodiments, the condition is NASH. Delivery of nucleic acids to cells

[0288] A further aspect provides a method of delivering a nucleic acid to a cell using a compound according to the present disclosure (or a pharmaceutically acceptable salt thereof), wherein the cell carries (e.g., expresses) a binding partner for a ligand of the compound. The method comprises the steps of contacting the cell with the compound. The method may be used in vitro (e.g., for diagnostic or research purposes) or in vivo (e.g., for diagnostic or therapeutic purposes). In embodiments, the method is an in vitro method.

[0289] In embodiments, the binding partner is ASGPR (e.g., human ASGPR) and the compound carries at least one monosaccharide ligand (e.g., the compound carries at least one GalNAc moiety). In embodiments, the cell is a hepatocyte, e.g. a mammalian hepatocyte such as a human hepatocyte. In embodiments, the cell is a malignant hepatocyte (e.g., a hepatocellular carcinoma cell).

[0290] In embodiments, the method comprises contacting a cell which carries (e.g., expresses) ASGPR (e.g., a hepatocyte cell) with a compound which carries at least one GalNAc moiety, e.g. wherein the compound is a compound of Formula (I) (e.g., Formula (la)), Formula (II) (e.g., Formula (Ila)) or Formula (III) (e.g., Formula (Illa)), or a pharmaceutically acceptable salt of the foregoing.

[0291] Processes and intermediates

[0292] The disclosure also provides process for the preparation of compounds as described herein, as well as intermediates useful in the preparation of said compounds.

[0293] Viewed from this aspect, the disclosure provides a compound having the structure of

[0294] Formula (IV): or a pharmaceutically acceptable salt thereof, wherein X, R, m, and n are as defined herein; and wherein Z1to Z4each represents a group independently selected from: (i) G1to G4as defined herein; and (ii) -[tether]-J, wherein J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), N3 or a phosphorami dite, and wherein [tether] is as defined herein, with the proviso that at least one of Z1to Z4represents a group independently selected from G1to G4, and at least one of Z1to Z4represents a group independently selected from -[tether] -J.

[0295] In embodiments, the compound is a compound having the structure of Formula (IVa): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, and Z1to Z4are as defined herein.

[0296] In embodiments, the compound is a compound having the structure of Formula (IVb): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, and Z1to Z4are as defined herein.

[0297] In embodiments, the compound is a compound having the structure of Formula (IVc): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, and Z1to Z4are as defined herein.

[0298] In embodiments, the compound is a compound having the structure of Formula (IVd): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, and Z1to Z4are as defined herein. The disclosure also provides a compound having the structure of Formula (V):

[0299] (V) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand], [tether] and J are as defined herein.

[0300] In embodiments, the compound is a compound having the structure of Formula (Va):

[0301] (Va) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand], [tether] and J are as defined herein.

[0302] In embodiments, the compound is a compound having the structure of Formula (Vb):

[0303] (Vb) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand], [tether] and J are as defined herein.

[0304] In embodiments, the compound is a compound having the structure of Formula (Vc):

[0305] (Vc) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand], [tether] and J are as defined herein. In embodiments, the compound is a compound having the structure of Formula (Vd):

[0306] (Vd) or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand], [tether] and J are as defined herein. The disclosure also provides a compound having the structure of Formula (VI): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [tether] and J are as defined herein.

[0307] In embodiments, the compound is a compound having the structure of Formula (Via): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [tether] and J are as defined herein.

[0308] In embodiments, the compound is a compound having the structure of Formula (VIb):

[0309] or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [tether] and J are as defined herein.

[0310] In embodiments, the compound is a compound having the structure of Formula (Vic): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [tether] and J are as defined herein.

[0311] In embodiments, the compound is a compound having the structure of Formula (Vid): or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [tether] and J are as defined herein.

[0312] In a related aspect is provided a process for the preparation of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, characterised in that a compound of Formula (IV) is reacted with a compound having the structure

[0313] Q- [cargo] wherein cargo is as defined herein, and Q denotes a group which is reactive to group J as defined herein (e.g., wherein Q denotes a BCN-containing group). In embodiments, the compound of Formula (I) is a compound of Formula (la) and the compound of Formula (IV) is a compound of Formula (IV a). In embodiments, the compound of Formula (I) is a compound of Formula (lb) and the compound of Formula (IV) is a compound of Formula (IVb). In embodiments, the compound of Formula (I) is a compound of Formula (Ic) and the compound of Formula (IV) is a compound of Formula (IV c). In embodiments, the compound of Formula (I) is a compound of Formula (Id) and the compound of Formula (IV) is a compound of Formula (IV d).

[0314] In another related aspect is provided a process for the preparation of a compound of Formula

[0315] (II), or a pharmaceutically acceptable salt thereof, characterised in that a compound of Formula (V) is reacted with a compound having the structure

[0316] Q- [cargo] wherein cargo and Q are as defined herein. In embodiments, the compound of Formula (II) is a compound of Formula (Ila) and the compound of Formula (V) is a compound of Formula (Va). In embodiments, the compound of Formula (II) is a compound of Formula (lib) and the compound of Formula (V) is a compound of Formula (Vb). In embodiments, the compound of Formula (II) is a compound of Formula (lie) and the compound of Formula (V) is a compound of Formula (Vc). In embodiments, the compound of Formula (II) is a compound of Formula (lid) and the compound of Formula (V) is a compound of Formula (Vd).

[0317] In another related aspect is provided a process for the preparation of a compound of Formula

[0318] (III), or a pharmaceutically acceptable salt thereof, characterised in that a compound of Formula (VI) is reacted with a compound having the structure

[0319] Q- [cargo] wherein cargo and Q are as defined herein. In embodiments, the compound of Formula (III) is a compound of Formula (Illa) and the compound of Formula (VI) is a compound of Formula (Via). In embodiments, the compound of Formula (III) is a compound of Formula (Illb) and the compound of Formula (VI) is a compound of Formula (VIb). In embodiments, the compound of Formula (III) is a compound of Formula (IIIc) and the compound of Formula (VI) is a compound of Formula (Vic). In embodiments, the compound of Formula (III) is a compound of Formula (Hid) and the compound of Formula (VI) is a compound of Formula (Vid).

[0320] In embodiments of the above processes, Q denotes a BCN-containing group and J is -Ns.

[0321] Having been generally described herein, the follow non-limiting examples are provided to further illustrate this disclosure.

[0322] EXAMPLES

[0323] The synthesis of compounds of the disclosure will be apparent to the skilled reader, based on the following exemplary protocols and methods.

[0324] Synthesis of precursors and cargo molecules

[0325] Synthesis of common intermediate (9)

[0326] Methyl (S)-2,3-bis((tert-butoxycarbonyl)amino)propanoate (2)

[0327] To a suspension of H-Dap(Boc)-OMe HC1 (5.3 g, 20.81 mmol) in dichloromethane (60 ml) was added triethylamine (8.68 ml, 62.4 mmol) at room temperature, followed by di-tert-butyl dicarbonate (6.81 g, 31.2 mmol). Reaction mixture was stirred for 2 h. LCMS showed completion of reaction. Reaction mixture was washed with 0.5M aq. KHSO4 (2x 50 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (120g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 -> 1 : 1). Fractions were combined and concentrated under reduced pressure to afford 6.16 gm of compound 2.

[0328] 'H NMR (400 MHz, CDCh) 8 5.43 (s, 1H), 4.86 (s, 1H), 4.35 (s, 1H), 3.76 (s, 3H), 3.60 - 3.42 (m, 2H), 1.45 (s, 9H), 1.43 (s, 9H). LCMS m / z ESI Expected 318.2, observed 341.1 ([M+Na]+). di -tert-butyl (3-hydroxypropane-l,2-diyl)(S)-dicarbamate (3)

[0329] Ethanol (Abs) (10 ml) was added dropwise under nitrogen atmosphere to a solution of Lithium borohydride, 4M (10 wt.%) solution in THF, AcroSeal (12.09 ml, 48.4 mmol) in Tetrahydrofuran (dry) (35 ml) at 0 °C. After stirring for 10 min, a solution of methyl (<S)-2,3- bis((tert-butoxycarbonyl)amino)propanoate (6.16 g, 19.35 mmol) in Tetrahydrofuran (dry) (35 ml) was added dropwise and the mixture was stirred at 0 °C for Ih. The reaction mixture was quenched by addition of water (30 mL) at 0 °C and portionwise addition of aq. IM HC1 (40 mL). Mainly at the beginning a lot of effervescence was observed and a suspension was obtained at first. After full addition of aq. HC1 a solution was obtained which was extracted with ethyl acetate (3x50 mL). The combined organics were washed with brine (100 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The residue was purified by flash chromatography (120g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1 :0 -> 1:4). Pure fractions were combined and concentrated under reduced pressure to afford 5.09 g of compound 3 as a white solid. Formed product was confirmed by TLC and 'H NMR.

[0330] 'H NMR (400 MHz, CDCh) 8 5.19 - 4.90 (m, 2H), 3.76 - 3.62 (m, 2H), 3.61 - 3.47 (m, 2H), 3.39 - 3.12 (m, 2H), 1.45 (s, 9H), 1.44 (s, 9H). di -tert-butyl (3 -iodopropane- l,2-diyl)(S)-dicarbamate (4)

[0331] To an ice-bath cooled solution of di-tert-butyl (3-hydroxypropane-l,2-diyl)(S)-dicarbamate (5.09 g, 17.53 mmol) in 1,2-di chloroethane (88 ml) was added triphenylphosphine (5.52 g, 21.04 mmol), imidazole (3.58 g, 52.6 mmol) and iodine (5.78 g, 22.79 mmol) under nitrogen atmosphere. After addition the mixture was stirred for 5 min to obtain a yellow suspension at first, which quite quickly turned into a brown suspension. The ice-bath was removed and reaction mixture was stirred for 4 h. The reaction mixture was diluted with dichloromethane (75 mL) and washed with 1 / 2 saturated aqueous Na2S20s (100 mL). The aqueous phase was extracted with DCM (50 mL) and the combined organic layers were dried over Na2SO4(s) and concentrated under reduced pressure. The residue was purified by flash chromatography (120g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 -> 3:2). Pure fractions were combined and concentrated under reduced pressure to afford 4.78 g of compound 4.

[0332] 'H NMR (400 MHz, CDCh) 8 5.29 (s, 1H), 4.81 (s, 1H), 3.61 (s, 1H), 3.46 - 3.18 (m, 4H), 1.44 (s, 18H). LCMS m / z ESI Expected 400.1, observed 423.0 ([M+Na]+). diallyl 3,3'-disulfanediyl(2R,2'R)-bis(2-((tert-butoxycarbonyl)amino)propanoate) (6)

[0333] To a solution ofN,N'-di-Boc-L-Cysteine (10g, 22.70 mmol) in N,N-Dimethylformamide (dry) (100 ml) was added Cesium carbonate (7.99 g, 24.52 mmol) and Allyl bromide (4.35 ml, 49.9 mmol) under inert atmosphere. Reaction mixture was stirred for 48 h at rt. Reaction mixture was diluted with EtOAc (500 mL), washed with 80% sat. brine (3x 500 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (220g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 — > 7:3). Pure fractions were combined and concentrated under reduced pressure to afford 10.6 gm of compound 6 as a white solid.

[0334] 'H NMR (400 MHz, CDCh) 8 6.10 - 5.80 (m, 2H), 5.56 - 5.21 (m, 6H), 4.72 - 4.56 (m, 6H), 3.18 (d, J= 5.3 Hz, 4H), 1.45 (s, 18H). LCMS m / z ESI Expected 520.2, observed 543.2 ([M+Na]+). allyl (tert-butoxycarbonyl)-L-cysteinate (7) To a solution of diallyl 3,3'-disulfanediyl(2R,2'R)-bis(2-((tert-butoxycarbonyl)amino) propanoate) (6) (10.6 g, 20.36 mmol) in Tetrahydrofuran (dry) (150 ml) was added dropwise Tri-n-butylphosphine (7.63 ml, 30.5 mmol) at room temperature under nitrogen atmosphere. After stirring for 30 min, Water (25.00 ml) was added and stirred for additional Ih. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate (75 mL) and washed with 10% citric acid (75 mL) and water (75 mL). In both washes some brine was added to aid separation. The organic layer was washed with brine (75 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The residue was purified by flash chromatography (220g silica, wet loaded: in minimal amount of DCM, gradient (Hept / EtOAc): 1:0 3:1). Pure fractions were combined and concentrated under reduced pressure to afford

[0335] 10.09 g of compound 7 as a colourless oil.

[0336] 1H NMR (400 MHz, CDC1) 8 6.03 - 5.83 (m, IH), 5.51 - 5.22 (m, 3H), 4.82 - 4.58 (m, 3H), 3.14 - 2.87 (m, 2H), 1.46 (s, 10H). LCMS m / z ESI Expected 261.1, observed 284.0 ([M+Na]+). allyl S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert-butoxycarbonyl)-L- cysteinate (8)

[0337] First batch: A solution of Boc-Cys-OAllyl (7) (1.2g, 4.59 mmol) in N,N- Dimethylformamide (dry) (23 ml) was purged with argon for 5 min. Then, tri ethylamine (2.55 ml, 18.37 mmol) was added at room temperature under argon atmosphere, followed by di-tert- butyl (3 -iodopropane- 1 ,2-diyl)(S)-dicarbamate (4) (2.389 g, 5.97 mmol). The reaction flask was transferred into pre-heated reaction block (80 °C) and stirred for 4h. TLC analysis showed almost full conversion. The residue was diluted with ethyl acetate (50 mL) and washed with 0.1M HC1 (50 mL) and brine (40 mL). The separated organic layer was dried over Na2SO4(s), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 0% to 40% ethyl acetate in heptane) to give allyl S-((S)-2,3-bis((tert- butoxycarbonyl)amino)propyl)-N-(tert-butoxycarbonyl)-L-cysteinate (8) (1.76 g, 3.30 mmol, 71.8 % yield) as colourless residue.

[0338] Second batch: A solution of Boc-Cys-OAllyl (7) (3.12 g, 11.94 mmol) in N,N- Dimethylformamide (dry) (60 ml) was purged with argon for 5 min. Then, tri ethylamine (6.64 ml, 47.8 mmol) was added at room temperature, followed by di-tert-butyl (3 -iodopropane- 1,2- diyl)(S)-dicarbamate (4.78 g, 11.94 mmol). Reaction mixture was heated to 80 °C for 6 h. Heating was stopped and left for cooling overnight in a slowly cooling oil bath. To reaction mixture was added 0.5M KHSO4 (150 mL) and brine (150 mL) and the mixture was extracted with EtOAc (3x 300 mL). The combined organic layers were washed with sat. aq. Na2S20s (500 mL) and 80% sat., brine (3x 500 mL), dried over NA2SO4(s) and concentrated under reduce pressure. The crude product was purified using FCC (220g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1: — > 3:2). Pure fractions were combined and concentrated under reduced pressure to afford 4.05 g of compound 8 as a yellowish oil.

[0339] 1H NMR (400 MHz, CDCk) 86.03 - 5.84 (m, 1H), 5.58 - 5.15 (m, 4H), 5.00 (s, 1H), 4.66 (dd, J= 5.8, 1.4 Hz, 2H), 4.55 (q, J= 6.1 Hz, 1H), 3.82 - 3.65 (m, 1H), 3.46 - 3.15 (m, 2H), 3.14 - 2.89 (m, 2H), 2.83 - 2.56 (m, 2H), 1.55 - 1.39 (m, 27H). LCMS m / z ESI Expected 533.3, observed 556.2 ([M+Na]+).

[0340] S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert-butoxycarbonyl)-L- cysteine (9)

[0341] Batch 1 : To a solution of allyl S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert- butoxycarbonyl)-L-cysteinate (8) (4.05 g, 7.59 mmol) in Tetrahydrofuran (dry) (45 ml) was added Pd(PPhs)4 (1.754 g, 1.518 mmol) at room temperature under argon atmosphere, followed by morpholine (0.797 ml, 9.11 mmol) and reaction mixture was stirred for 30 min. Reaction mixture was concentrated under reduced pressure and purified using FCC (120g silica, dry loaded: isolute, gradient (DCM / MeOH)*: 1:0 — > 95:5). Pure fractions were combined and concentrated under reduced pressure to afford 3.59 g of compound 9 as a yellow collapsing foam.

[0342] To a solution of allyl S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert- butoxycarbonyl)-L-cysteinate (8) (1.63g, 3.05 mmol) in Tetrahydrofuran (dry) (30 ml) a Pd(PPhs)4 (0.706 g, 0.611 mmol) was added at room temperature under argon atmosphere, followed by morpholine (0.321 ml, 3.67 mmol). After stirring for 30 min, Sample-1 on TLC showed full conversion. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 0% to 10% methanol in dichloromethane) to give S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert- butoxycarbonyl)-L-cysteine (9, 1.36 g, 2.76 mmol, 90 % yield).

[0343] LCMS m / z ESI Expected 493.3, observed 492.2 ([M-H]'). Synthesis of common intermediate (12a, 12b)

[0344] Common intermediate acid 9 was used successfully in the amide-coupling towards various amides 10 containing conjugation handle like azide for Click chemistry (10a) or activated PFP-esters (10b) with different linkers. Deprotection of the Boc groups in compound 11 using TFA afforded TFA-salt 12 which was used crude in the next step without further purification.

[0345] - tri-tert-butyl ((1 lR,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9-azahexadecane-

[0346] 11,15,16-triyl)tricarbamate (1 la)

[0347] To a solution of S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert-butoxycarbonyl)- L-cysteine (9) (1.2 g, 2.431 mmol) in N,N-Dimethylformamide (dry) (20 ml) a HATU (0.924 g, 2.431 mmol) was added at 0 °C under argon atmosphere, followed by DIPEA (0.847 ml, 4.86 mmol). After stirring for 10 min, a 2-[2-(2-Azidoethoxy)ethoxy]ethanamine (10a) (0.466 g, 2.67 mmol) as a solution in 0.5 mL of DMF was added. After stirring for Ih, LCMS showed full conversion. The reaction mixture was diluted with ethyl acetate (30 mL), washed with IM HC1 (50 mL) and brine (50 mL). The separated organic layer was dried over NazSCh, filtered and concentrated under reduced pressure. Purification: flash chromatography (silica, 30% to 100% ethyl acetate in heptane) to give compound Ila (1.35 g) contains impurity from previous step. Acidic preparative MPLC was performed (Linear Gradient: t=0 min 5% A, t=l min 5% A; t=16 min 70% A; t=17 min 100%; t=22 min 100% A; detection: 220 / 254 nm) to give tri-tert-butyl ((1R,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9- azahexadecane-l l,15,16-triyl)tricarbamate (Ila) (0.855 g, 1.316 mmol, 54.1 % yield) after lyophilization. 1 H NMR (400 MHz, CDC1) 8 7.08 (s, IH), 5.48 (s, 2H), 5.12 (s, IH), 4.33 (d, J= 7.6 Hz, IH), 3.81 - 3.61 (m, 7H), 3.58 (t, J= 5.3 Hz, 2H), 3.53 - 3.44 (m, 2H), 3.44 - 3.32 (m, 3H),

[0348] 3.27 (s, IH), 2.93 (dd, J= 14.0, 5.8 Hz, IH), 2.88 - 2.75 (m, 2H), 2.70 - 2.55 (m, IH), 1.64 -

[0349] 1.27 (m, 27H). LCMS m / z ESI Expected 649.4, observed 650.3 ([M+H]+).

[0350] (1 lR,15S)-l-Azido-10-oxo-3,6-dioxa-13-thia-9-azahexadecane-l 1,15,16-triaminium 2,2,2-trifluoroacetate (12a)

[0351] To a solution of tri-tert-butyl ((1R,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9-azahexadecane- ll,15,16-triyl)tricarbamate (Ila) (0.855 g, 1.316 mmol) in Dichloromethane (10 ml) a Trifluoroacetic acid (2.500 ml) was added at room temperature. After stirring for 2h, LCMS showed desired mass. The reaction mixture was concentrated under reduced pressure and lyophilized from water (20 mL) to give (1 lR,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9- azahexadecane-l l,15,16-triaminium 2,2,2-trifluoroacetate (12a) (1.05 g, 1.518 mmol, 115 % yield) as white residue.

[0352] LCMS m / z ESI Expected 349.2 (free base), observed 350.4 ([M+H]+). benzyl (7S,11R)-7,1 l-bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3- oxa-9-thia-5 , 13 -diazanonadecan- 19-oate (1b)

[0353] To a solution of S-((S)-2,3-bis((tert-butoxycarbonyl)amino)propyl)-N-(tert-butoxycarbonyl)- L-cysteine (9) (3.00 g, 1 Eq, 6.08 mmol) in N,N-Dimethylformamide (30 mL) were added DIPEA (2.36 g, 3.18 mL, 3 Eq, 18.2 mmol) and HATU (2.31 g, 1 Eq, 6.08 mmol). After stirring for 10 minutes, benzyl 6-aminohexanoate 4-methylbenzenesulfonate (10b) (2.63 g, 1.1 Eq, 6.69 mmol) was added and the mixture was stirred for 3 h. Reaction mixture was diluted with EtOAc (150 mL) and 80% sat. brine (150 mL) was added. The layers were separated and the aqueous phase was extracted with EtOAc (2x 150 mL). The combined organic layers were washed with 80% sat. brine (3x 300 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (120g silica, wet loaded: DCM, Gradient (Hept / EtOAc): 1:0 -> 0:1. Pure fractions were combined and concentrated under reduced pressure to afford 2.05 g of compound 11b as a yellow foam.

[0354] 'H NMR (400 MHz, DMSO) 6 7.92 - 7.79 (m, 1H), 7.46 - 7.24 (m, 5H), 6.95 - 6.72 (m, 2H), 6.61 (t, J= 8.4 Hz, 1H), 5.08 (s, 2H), 4.09 - 3.92 (m, 1H), 3.71 - 3.48 (m, 1H), 3.11 - 2.91 (m, 4H), 2.66 - 2.53 (m, 2H), 2.49 - 2.40 (m, 1H), 2.33 (t, J= 7.4 Hz, 2H), 1.61 - 1.48 (m, 2H), 1.37 (s, 30H), 1.31 - 1.19 (m, 2H). LCMS m / z ESI Expected 696.4, observed 697.2 ([M+H]+). benzyl 6-((R)-3-(((S)-2,3-bis(2,2,2-trifluoroacetamido)propyl)thio)-2-(2,2,2- trifluoroacetamido)propanamido)hexanoate (12b)

[0355] To a solution of benzyl (7S,llR)-7,l l-bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12- dioxo-3-oxa-9-thia-5,13-diazanonadecan-19-oate (11b) (1.000 g, 1 Eq, 1.435 mmol) in Dichloromethane (6.6 mL) was added TFA (4.908 g, 3.316 mL, 30 Eq, 43.05 mmol) and reaction mixture was stirred for 1 h. Reaction mixture was concentrated under reduced pressure and coevaporated with DCM (3x) to afford 12b as a brown oil.

[0356] LCMS m / z ESI Expected 396.2 (free base), observed 397.2 ([M+H]+).

[0357] Synthesis of azide analogues of GalNAc trimer (15a, 15b)

[0358]

[0359] Coupling of amine 12a and GalNAc acid 13 (part (A) above) or 14 (part (B) above) furnished the azide analogue of GalNAc trimer. The compound thus obtained could be conjugated to various oligonucleotide cargo moieties to provide compounds according to the present disclosure.

[0360] 4-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl) tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)-4-oxobutanoic acid (14)

[0361] To a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (0.738 g, 1.542 mmol) in dichloromethane (10 ml) a triethylamine (0.214 ml, 1.542 mmol) was added at room temperature under argon atmosphere, followed by Succinic anhydride (0.154 g, 1.542 mmol). After stirring for Ih, LCMS showed full conversion. The reaction mixture was purified by flash chromatography (silica, 0% to 10% methanol in di chloromethane (1% AcOH)) to give triethylamine 4-((2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)-4-oxobutanoate (14) (0.835 g, 1.228 mmol, 80 % yield) after concentration and lyophilisation from water (8 mL) as yellow oily residue.

[0362] 'H NMR (400 MHz, CDCh) 8 12.12 (s, IH), 6.98 (s, IH), 6.49 (d, J= 8.9 Hz, IH), 5.34 (dd, .7= 3.4, 1.1 Hz, IH), 5.21 (dd, J= 11.2, 3.4 Hz, IH), 4.77 (dd, J= 8.3, 5.4 Hz, IH), 4.24 - 4.08 (m, 3H), 4.04 - 3.90 (m, 3H), 3.79 (dt, J= 11.4, 5.1 Hz, IH), 3.72 - 3.55 (m, 9H), 3.53 - 3.39 (m, 2H), 2.75 - 2.61 (m, 2H), 2.53 (t, J= 6.4 Hz, 2H), 2.16 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.01 (d, J= 1.7 Hz, 3H), 1.97 (s, 2H). impurities observed). LCMS m / z ESI Expected 578.2, observed 579.4 ([M+H]+).

[0363] GalNAc trimer 15a:

[0364] To a solution of (HR,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9-azahexadecane-l 1,15,16- triaminium 2,2,2-trifluoroacetate (12a) (75mg, 0.108 mmol) and 5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (146 mg, 0.325 mmol) in N,N-Dimethylformamide (dry) (1 ml) a DIPEA (0.113 ml, 0.651 mmol) was added at room temperature under argon atmosphere, followed by HBTU (123 mg, 0.325 mmol). After stirring for Ih, LCMS showed the desired mass. The reaction mixture was purified by acidic preparative MPLC (Linear Gradient: t=0 min 5% A, t=l min 5% A; t=l 6 min 50% A; t=l 7 min 100%; t=22 min 100% A; detection: 220 nm) to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(((llR)-l-azido-l l-((((2R)-2,3-bis(5- (((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)pentanamido)propyl)thio)methyl)-l 0,13-dioxo-3,6-dioxa-9, 12-diazaheptadecan-l 7- yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (15a) (0.134 g, 0.082 mmol, 75 % yield) as white foam after lyophilization.

[0365] LCMS m / z ESI+Expected 1638.7, observed 819.8 (z = 2). GalNAc trimer 15b:

[0366] To a solution of triethylamine 4-((2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)-4-oxobutanoate (442 mg, 0.651 mmol) and (HR,15S)-l-azido-10-oxo-3,6-dioxa-13-thia-9-azahexadecane-

[0367] 11,15,16-triaminium 2,2,2-trifluoroacetate (12a) (150mg, 0.217 mmol) in N,N- dimethylformamide (dry) (2 ml) a DIPEA (0.227 ml, 1.301 mmol) was added at room temperature under argon atmosphere, followed by HBTU (247 mg, 0.651 mmol). After stirring for Ih, LCMS showed full conversion. The reaction mixture was purified by purified by acidic preparative MPLC (Linear Gradient: t=0 min 5% A, t=l min 5% A; t=16 min 50% A; t=17 min 100%; t=22 min 100% A; detection: 220 nm) to give azido product (15b) (64 mg, 0.032 mmol, 14.53 % yield) as white solid after lyophilization.

[0368] LCMS m / z ESI+Expected 2029.9, observed 1016.4 (z = 2).

[0369] Synthesis of PFP analogues of GalNAc trimer (18)

[0370] Coupling of amine 12b and GalNAc acid 13 furnished 16 in 76% yield. Hydrogenolysis of benzyl ester 16 using palladium catalysis afforded 17 in 51% yield on scale-up. Obtained acid 17 was converted to the PFP analogue efficiently to furnish 18 which could be conjugated to various oligonucleotide cargo moieties to provide compounds according to the present disclosure. benzyl ester (16) To a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (1.926 g, 3 Eq, 4.305 mmol) and benzyl 6-((R)-2-amino-3-(((S)-2,3-diaminopropyl)thio)propanamido)hexanoate tris(2,2,2-trifluoroacetate) (1.060 g, 1 Eq, 1.435 mmol) in N,N-dimethylformamide (dry) (14 mL) was added DIPEA (1.113 g, E50 L, 6 Eq, 8.610 mmol) and HBTU (1.633 g, 3 Eq, 4.305 mmol). Reaction mixture was stirred for 1 h at rt. More 5-(((3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (0.642 g, 1 Eq, E435 mmol), DIPEA (0.371 g, 0.50 mL, 2 Eq, 2.870 mmol) and HBTU (0.544 g, 1 Eq, 1.435 mmol) were added and stirring was continued for 30 min. Stirring was continued for 15 min. Reaction mixture was diluted with 80% sat. brine (75 mL) and was extracted with EtOAc (3x 75 mL) The combined organic layers were washed with 80% sat. brine (3x 150 mL), dried over Na2SO4(s) (filter cake washed with DCM) and concentrated under reduced pressure. The crude product was purified using flash column chromatography (120g silica, wet loaded: DCM, First isocratic EtOAc, then gradient (DCM / MeOH): 1:0 — > 9:1). Pure fractions were combined and concentrated under reduced pressure to afford 1.84 g of (16) as an off-white solid. Performing FCC by first flushing HOBt residues from the column with EtOAc, then continuing with DCM / MeOH gradient yields the target compound in high purity.

[0371] 'H NMR (400 MHz, DMSO) 6 7.97 (t, J= 7.8 Hz, 2H), 7.88 - 7.76 (m, 3H), 7.73 (t, J= 5.8 Hz, 1H), 7.64 (d, J= 8.2 Hz, 1H), 7.44 - 7.26 (m, 5H), 5.21 (d, J= 3.4 Hz, 3H), 5.08 (s, 2H), 4.97 (dd, J= 11.2, 3.3 Hz, 3H), 4.49 (dd, J= 8.5, 1.5 Hz, 3H), 4.35 (q, J= 8.0 Hz, 1H), 4.14 - 3.96 (m, 9H), 3.87 (dt, J= 11.5, 8.8 Hz, 4H), 3.77 - 3.66 (m, 3H), 3.40 (d, J= 8.7 Hz, 3H), 3.28 - 3.15 (m, 1H), 3.14 - 2.96 (m, 3H), 2.78 (dd, J= 13.4, 5.9 Hz, 1H), 2.59 (dd, J= 13.1, 7.0 Hz, 1H), 2.34 (t, J = 7.4 Hz, 2H), 2.18 - 2.02 (m, 15H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.63 - 1.34 (m, 17H), 1.32 - 1.18 (m, 3H). LCMS m / z ESI Expected 1683.7, observed 1684.6 ([M+H]+).

[0372] 6-((R)-2-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)-3-(((S)-2,3-bis(5- (((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanamido)propyl)thio)propanamido)hexanoic acid (17)

[0373] Under nitrogen atmosphere, THF (100 mL) was added to benzyl ester 16 (1.700 g, 1 Eq,

[0374] I.009 mmol) and the suspension was heated to 50°C to obtain a solution. Pd / C (10% (w / w) 50% wet) (2.148 g, 5% Wt, 1 Eq, 1.009 mmol) was added and the atmosphere was replaced by hydrogen (balloon). Reaction mixture was stirred overnight at 50 °C. Reaction mixture was filtered over celite. Slow filtration. Solubility of product was poor in THF at RT. Filter cake was washed with DCM / MeOH (9:1) (fast filtration). The combined filtrate (black solution : : another celite filtration did not remove any of the black colour) was concentrated under reduced pressure to afford 1.68 g of Batch A as a black foam. Batch A was further purified using acidic Prep-LCMS. The product fractions were combined and concentrated using lyophilization to afford 0.925 g of title product 17 as a white solid.

[0375] 'H NMR (400 MHz, DMSO) 6 8.09 - 7.78 (m, 7H), 5.21 (d, J= 3.4 Hz, 3H), 4.97 (dd, J =

[0376] II.3, 3.3 Hz, 3H), 4.49 (dd, J= 8.4, 2.8 Hz, 3H), 4.35 (q, J= 7.6 Hz, 1H), 4.10 - 3.96 (m, 9H), 3.87 (q, J= 9.4 Hz, 4H), 3.78 - 3.64 (m, 3H), 3.51 - 3.38 (m, 2H), 3.26 - 3.15 (m, 2H), 3.15 - 2.96 (m, 3H), 2.79 (dd, J= 13.7, 6.1 Hz, 1H), 2.66 - 2.53 (m, 2H), 2.19 - 2.02 (m, 18H), 2.00 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.63 - 1.32 (m, 17H), 1.27 (d, J= 7.8 Hz, 2H). LCMS m / z ESI Expected 1593.7, observed 1594.6 ([M+H]+).

[0377] - (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((((S)-3-(((R)-2-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)pentanamido)-3-oxo-3-((6-oxo-6-(perfluorophenoxy)hexyl)amino)propyl) thio)propane-l,2-diyl)bis(azanediyl))bis(5-oxopentane-5,l-diyl))bis(oxy))bis(5- acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate (18)

[0378] To a solution of 6-((R)-2-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)-3-(((S)-2,3-bis(5- (((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)pentanamido)propyl)thio)propanamido)hexanoic acid (17) (1.184 g, 1 Eq, 0.743 mmol) in N,N-Dimethylformamide (dry) (15.0 mL) were added DIPEA (0.288 g, 0.388 mL, 3 Eq, 2.227 mmol) and perfluorophenyl 2,2,2-trifluoroacetate (PFPTFA) (0.312 g, 0.191 mL, 1.5 Eq, 1.114 mmol) and the reaction was stirred for 45 min. [sample, diluted withMeCN, si, LCMS (acid; 210nm): 39% target, 21%o SM, 33%> PFPTFA] More DIPEA (0.288 g, 0.388 mL, 3 Eq, 2.227 mmol) and perfluorophenyl2,2,2-trifluoroacetate (0.312 g, 0.191 mL, 1.5 Eq, 1.114 mmol) were added and stirring was continued for 10 min. [sample, diluted with MeCN, s2, LCMS (acid; 210nm): 42%> target, <0.5% SM, 45% PFPTFA]

[0379] Reaction mixture was purified using Prep-Reveleris (acid; 4 runs). Product fractions were combined, and the solvents were removed by lyophilisation to afford 611 mg of product [analysed: A, LCMS (acid; 210nm): >95% target; 1H-NMR (DMSO): in agreement with structure] LCMS indicated mixed fractions were combined and the solvents were removed by lyophilisation to afford 232 mg of 18 as a white solid, [analysed: B, LCMS (acid; 210nm): 51% target, 39%> PFPTFA] Batch B was further purifies using Prep-Reveleris (acid; 1 run). Fractions were combined and the solvents were removed by lyophilisation to afford 64 mg of C. [analysed: C, LCMS (acid; 210nm): >95% target; 1H-NMR (DMSO): in agreement with structure] Batches A, B and C were combined to give 0.769 g of title product 18 as a white solid.

[0380] 'H NMR (400 MHz, DMSO) 6 8.04 - 7.95 (m, 2H), 7.88 - 7.78 (m, 3H), 7.74 (t, 1H), 7.65 (d, J= 8.2 Hz, 1H), 5.22 (d, J= 3.4 Hz, 3H), 4.98 (dd, J= 11.3, 3.4 Hz, 3H), 4.49 (d, J= 8.5 Hz, 3H), 4.42 - 4.29 (m, 1H), 4.09 - 3.98 (m, 9H), 3.87 (q, J= 9.5 Hz, 4H), 3.76 - 3.66 (m, 3H), 3.40 (d, J= 12.3 Hz, 3H), 3.29 - 3.18 (m, 1H), 3.13 - 2.98 (m, 3H), 2.77 (t, J= 7.3 Hz, 3H), 2.60 (dd, J= 13.8, 6.5 Hz, 2H), 2.11 (s, 11H), 2.09 - 2.03 (m, 4H), 2.00 (s, 9H), 1.90 (s, 9H), 1.78 (s, 9H), 1.72 - 1.62 (m, 2H), 1.59 - 1.41 (m, 14H), 1.41 - 1.30 (m, 3H). LCMS m / z ESI Expected 1759.7, observed 1760.8 ([M+H]+).

[0381] Synthesis of oxidised analogues of the cysteine splitter (19, 20)

[0382] Benzyl ester 16 was oxidized at sulphur to furnish sulfoxide and sulfone analogues. Selective oxidation towards sulfoxide 19 was carried out with hydrogen peroxide in hexafluoroisopropanol as the solvent. Full oxidation towards sulfone 20 was achieved using 2.5 eq of mCPBA. The sulfoxide contained mixture of diastereomers due to the newly formed chiral centre.

[0383] - (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((((2S)-3-(((2R)-2-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)pentanamido)-3-((6-(benzyloxy)-6-oxohexyl)amino)-3- oxopropyl)sulfinyl)propane- 1 ,2-diyl)bis(azanediyl))bis(5-oxopentane-5, 1 - diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate (19)

[0384]

[0385] To a solution of benzyl ester 16 (60 mg, 1 Eq, 36 μmol) in 1,1,1,3,3,3-Hexafluoroisopropanol (1.0 mL) was added hydrogen peroxide (8. 1 mg, 7.3 pL, 30% Wt, 2 Eq, 71 pmol) and reaction mixture was stirred for 1 h. 0.5M aq. Na2SO3 (1 mL) was added and reaction mixture was stirred for 15 min. The layers were separated over a phase separator and the aqueous phase was further extracted with DCM (2x 1 mL). The combined organic layers were concentrated under reduced pressure. The crude product was purified using Prep-Reveleris (base; 1 run; loaded as a solution in MeOH). Pure product fractions were combined and concentrated by lyophilization to afford 70 mg of product 19 as a mixture of diastereomers (white solid), [diastereomers in ~3:5 ratio]

[0386] 'H NMR (400 MHz, DMSO) 6 8.29 - 8.12 (m, 1H), 8.09 - 7.92 (m, 1H), 7.92 - 7.75 (m, 5H), 7.45 - 7.28 (m, 5H), 5.21 (d, J= 3.3 Hz, 3H), 5.08 (s, 2H), 5.03 - 4.92 (m, 3H), 4.65 - 4.53 (m, 1H), 4.49 (dd, J= 8.4, 2.4 Hz, 3H), 4.13 (m, 1H), 4.02 (m, 9H), 3.87 (q, J= 9.5 Hz, 3H), 3.78 - 3.63 (m, 3H), 3.51 - 3.37 (m, 3H), 3.25 - 3.07 (m, 1H), 3.08 - 2.93 (m, 2H), 2.94 - 2.71 (m, 3H), 2.34 (t, J= 7.4 Hz, 2H), 2.18 - 2.01 (m, 15H), 1.99 (s, 9H), 1.89 (s, 9H), 1.78 (s, 9H), 1.62 - 1.32 (m, 17H), 1.30 - 1.17 (m, 3H). LCMS m / z ESI Expected 1699.7, observed 1700.8 ([M+H]+).

[0387] - (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((((S)-3-(((R)-2-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)pentanamido)-3-((6-(benzyloxy)-6-oxohexyl)amino)-3- oxopropy l)sulfonyl)propane- 1 ,2-diy l)bis(azanediy l))bis(5-oxopentane-5 , 1 - diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate (20)

[0388] To a solution of benzyl ester 16 (50 mg, 1 Eq, 30 pmol) in DCM (1 mL) was added a spatula point of mCPBA (17 mg, 75% Wt, 2.5 Eq, 74 pmol) and the mixture was stirred for 1 h. LCMS confirmed formation of product. Reaction mixture was diluted with DCM (2 mL) and was washed with aq. sat. NaHCCL (3x2 mL). The organic layer was dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using Prep-Reveleris (acid; 1 run; loaded as a solution in MeOH+DMSO). Pure fractions were combined and concentrated by lyophilization to afford 49 mg of product 20 as a white solid.

[0389] 'H NMR (400 MHz, DMSO) 6 8.22 (d, J= 8.4 Hz, 1H), 7.94 (t, J= 5.7 Hz, 1H), 7.90 - 7.77 (m, 5H), 7.45 - 7.28 (m, 5H), 5.21 (d, J= 3.4 Hz, 3H), 5.08 (s, 2H), 4.97 (ddd, J= 11.4, 3.6,

[0390] 2.1 Hz, 3H), 4.82 - 4.67 (m, 1H), 4.49 (dd, J= 8.4, 1.8 Hz, 3H), 4.38 - 4.20 (m, 1H), 4.10 - 3.95 (m, 9H), 3.94 - 3.81 (m, 3H), 3.78 - 3.62 (m, 3H), 3.51 (d, J= 11.1 Hz, 1H), 3.49 - 3.36 (m, 3H), 3.28 - 3.09 (m, 3H), 3.07 - 2.94 (m, 2H), 2.34 (t, J= 7.4 Hz, 2H), 2.15 - 2.01 (m, 15H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81 - 1.70 (m, 9H), 1.61 - 1.33 (m, 17H), 1.31 - 1.14 (m, 3H). LCMS m / z ESI Expected 1715.7, observed 1716.8 ([M+H]+).

[0391] Synthesis of azide analogue of GalNAc trimer (Precursor 34) tert-butyl (R)-3-((tert-butoxycarbonyl)amino)-4-hydroxybutanoate (22)

[0392] Under nitrogen a solution of (R)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4- oxobutanoic acid (5.00 g, 1 Eq, 17.3 mmol) in THF (50 mL) was cooled in an ice-bath. To this was added N-methylmorpholine (1.75 g, 1.90 mL, 1 Eq, 17.3 mmol) followed by dropwise addition of ethyl chloroformate (1.88 g, 1.66 mL, 1 Eq, 17.3 mmol). After complete addition the reaction mixture was stirred for 1 h while cooled in the ice-bath. The mixture was filtered over celite and the filtrate was concentrated. The oily residue was dissolved in Methanol (25 mL) and cooled in an ice-bath. To this was added sodium borohydride (1.31 g, 2 Eq, 34.6 mmol) (vigorous gas formation, next time add more slowly). After stirring for 1 h at 0 °C water and 0.5 M HC1 (aq) were added and the aqueous mixture was extracted with ethyl acetate twice. The organic layers were combined, washed with brine, dried and concentrated. The crude product was coated on isolute and purified over silica by flash column chromatography (80 g silica, 10 to 100% gradient of EtOAc in heptane). Pure fractions were combined, concentrated and coevaporated with ether. Yield: 4.3 g of a sticky colourless oil, 84% yield.

[0393] 1H NMR (400 MHz, CDC1) 8 5.22 (s, 1H), 4.04 - 3.88 (m, 1H), 3.69 (d, J= 4.8 Hz, 2H), 2.53 (t, J= 6.7 Hz, 2H), 1.45 (d, J= 4.5 Hz, 18H). tert-butyl (R)-3-((tert-butoxycarbonyl)amino)-4-iodobutanoate (23)

[0394] Under nitrogen a solution of tert-butyl (R)-3-((tert-butoxycarbonyl)amino)-4- hydroxybutanoate (22, 4.13 g, 1 Eq, 15.0 mmol), imidazole (1.63 g, 1.60 Eq, 24.0 mmol) and triphenylphosphine (6.29 g, 1.60 Eq, 24.0 mmol) in THF (35 mL) was cooled at 0 °C. To this was added portion-wise iodine (5.33 g, 1.40 Eq, 21.0 mmol) (reaction mixture turned brown rather quickly) and the reaction mixture was stirred at 0 °C for 2 h. The reaction mixture was quenched by addition of sat. NEUCl (aq). Water was added and the aqueous mixture was extracted with ethyl acetate. After separation the organic layer was washed with brine, dried and concentrated. The crude product was coated on isolute and purified over silica by flash column chromatography (80 g silica, 0 to 35% gradient of EtOAc in heptane). Pure were combined, concentrated and coevaporated with ether. Yield: 3.82 g (66%) of a white solid.

[0395] 'H NMR (400 MHz, CDCh) 8 5.08 (d, J= 8.6 Hz, 1H), 3.88 (s, 1H), 3.47 - 3.32 (m, 2H), 2.73 - 2.48 (m, 2H), 1.45 (d, J= 4.5 Hz, 18H).

[0396] - tert-butyl (R)-4-azido-3-((tert-butoxycarbonyl)amino)butanoate (24)

[0397] To a solution of tert-butyl (R)-3-((tert-butoxycarbonyl)amino)-4-iodobutanoate (23, 3.8 g, 1 Eq, 9.9 mmol) in DMF (10 mL) was added sodium azide (1.3 g, 2 Eq, 20 mmol) and the reaction mixture was stirred at room temp overnight. TLC showed incomplete conversion. The reaction mixture was heated at 80 °C for 1 h. TLC showed complete conversion of starting material. The reaction mixture was cooled to room temp, diluted with ethyl acetate and washed three times with water / brine (—1 / 3). The organic layer was dried and concentrated. The crude product was coated on isolute and purified over silica by flash column chromatography (40 g silica, 0 to 35% gradient of EtOAc in heptane). Pure fractions were combined, concentrated and coevaporated with pentane. Yield: 2.9 g (93%) of a colourless oil.

[0398] 'H NMR (400 MHz, CDCh) 8 5.11 (s, 1H), 4.07 (s, 1H), 3.61 - 3.38 (m, 2H), 2.49 (d, J= 6.0 Hz, 2H), 1.45 (d, J= 5.2 Hz, 18H).

[0399] - tert-butyl (R)-3,4-bis((tert-butoxycarbonyl)amino)butanoate (25)

[0400] A solution of tert-butyl (R)-4-azido-3-((tert-butoxycarbonyl)amino)butanoate (24, 2.50 g, 1 Eq, 8.32 mmol) in THF (35 mL) was flushed with nitrogen for 5 min. BOC2O (2.72 g, 2.87 mL, 1.5 Eq, 12.5 mmol) and Pd-C (10 wt%, 50% water) (0.90 g, 5% Wt, 0.051 Eq, 0.42 mmol) were added and the mixture was flushed with hydrogen for 5 min. The reaction mixture was vigorously stirred under hydrogen atmosphere for 1 h. The reaction mixture was again flushed with hydrogen for 5 min and vigorously stirred under hydrogen atmosphere for 1 h. TLC (hep / EtOAc, —4 / 1) showed full conversion into a slower moving spot. The reaction mixture was filtered over a plug of hyflo and the filtrate was concentrated. The crude product was coated on isolute and purified over silica by flash column chromatography (40 g silica, 0 to 40 % gradient of EtOAc in heptane). Pure fractions were combined and concentrated. Yield: 2.70 g (87%) of a white solid.

[0401] 'H NMR (400 MHz, CDCh) 85.25 (s, 1H), 4.86 (s, 1H), 4.11 - 3.79 (m, 1H), 3.26 (t, J= 6.4 Hz, 2H), 2.44 (d, J= 6.2 Hz, 2H), 1.44 (d, J= 5.4 Hz, 27H). methyl (tert-butoxycarbonyl)-L-homocysteinate (30)

[0402] To a solution of dimethyl 4,4'-disulfanediyl(2S,2'S)-bis(2-((tert- butoxycarbonyl)amino)butanoate) (29, 5.0 g, 1 Eq, 10 mmol) in THF (dry) (80 mL) was added drop wise tributyl phosphine (3.1 g, 3.8 mL, 1.5 Eq, 15 mmol) at rt. After stirring for 30 min Water (13 mL) was added and stirring was continued for 1 h.

[0403] The reaction mixture was diluted with ethyl acetate (75 mL) and washed with 10% citric acid (75 mL + 25mL brine). The aqueous phase was extracted with EtOAc (2 x 75 mL) and the combined organic layers were washed with brine (250 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The residue was purified by flash chromatography (80g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 -> 3:2. Pure fractions were combined and concentrated under reduced pressure to afford 4.78 g of 30 as a colourless oil, (98% yield).

[0404] 'H NMR (400 MHz, CDCh) 8 5.09 (d, J= 8.6 Hz, 1H), 4.47 (td, J= 8.6, 4.8 Hz, 1H), 3.76 (s, 3H), 2.71 - 2.48 (m, 2H), 2.19 - 2.04 (m, 1H), 2.01 - 1.85 (m, 1H), 1.59 (t, J= 8.3 Hz, 1H), 1.45 (s, 9H). di -tert-butyl (4-hydroxy butane- l,2-diyl)(R)-dicarbamate (26) Under nitrogen, to a solution of tert-butyl (R)-3,4-bis((tert-butoxycarbonyl)amino)butanoate (25, 2.800 g, 1 Eq, 7.477 mmol) in THF (35 mL) was added dropwise lithium borohydride (4 M in THF) (651.4 mg, 7.477 mL, 4.0 molar, 4 Eq, 29.91 mmol). After complete addition the reaction mixture was heated at 50 °C for 90 min. [TLC (Hept / EtOAc 1:1) showed full conversion towards 1 spot].

[0405] The reaction mixture was cooled to room temp and poured into sat. aq NH4CI (35 mL). Some water was added followed by ethyl acetate (35 mL) and the mixture was stirred until gas evolution had ceased. The layers were separated and the aqueous layer was extracted with ethyl acetate (2 x 35 mL). The combined organic layers were dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (80g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 — > 0:1. Pure fractions were combined, concentrated under reduced pressure and coevaporated with Et20 (2x) to afford 2.02 g of 26 as a collapsing foam. (83% yield).

[0406] 'H NMR (400 MHz, CDCh) 8 5.30 - 5.16 (m, 1H), 5.01 - 4.81 (m, 1H), 3.91 - 3.73 (m, 1H), 3.75 - 3.58 (m, 2H), 3.61 - 3.47 (m, 1H), 3.34 - 3.11 (m, 2H), 1.83 - 1.71 (m, 1H), 1.44 (d, J = 4.3 Hz, 18H). di-tert-butyl (4-iodobutane-l,2-diyl)(R)-dicarbamate (27)

[0407] To an ice-bath cooled solution of di-tert-butyl (4-hydroxy butane- 1 ,2-diyl)(R)-di carbamate (26, 1.800 g, 1 Eq, 5.913 mmol) in DCM (30 mL) was added triphenylphosphine (1.861 g, 1.2 Eq, 7.096 mmol), imidazole (1.208 g, 3 Eq, 17.74 mmol) and iodine (1.951 g, 1.3 Eq, 7.688 mmol). The ice-bath was removed and reaction was stirred for 4 h.

[0408] The reaction mixture was diluted with dichloromethane (30 mL) and washed with 1 / 2 saturated aqueous Na2S20s (60 mL). The aqueous phase was extracted with DCM (2x 50 mL) and the combined organic layers were dried over Na2SO4(s) and concentrated under reduced pressure. The residue was purified by flash chromatography (80g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 -> 3:2). Pure fractions were combined and concentrated under reduced pressure to afford 1.0 g of 27 as a white solid (37% yield). 'H NMR (400 MHz, CDCk) δ 5.00 - 4.03 (m, 2H), 3.86 - 3.58 (m, 1H), 3.32 - 3.06 (m, 4H), 2.15 - 1.85 (m, 2H), 1.44 (s, 18H). methyl S-((R)-3,4-bis((tert-butoxycarbonyl)amino)butyl)-N-(tert-butoxycarbonyl)-L- homocysteinate (28)

[0409] A solution of di-tert-butyl (4-iodobutane-l,2-diyl)(R)-dicarbamate (27, 1.00 g, 1 Eq, 2.41 mmol), methyl (tert-butoxycarbonyl)-L-homocysteinate (30, 722 mg, 1.2 Eq, 2.90 mmol) and triethylamine (977 mg, 1.35 mL, 4 Eq, 9.66 mmol) in DMF (anhydrous) (12 mL) was purged with argon for a few minutes and was then heated to 80 °C for 3 h. [sample, diluted with MeCN, si, LCMS (acid; 210nm): 44% target, 29% thiol, 17% elimination of iodine, no more SM-iodine observed].

[0410] Reaction was cooled down to room temperature. 0.5M KHSO4 (30 mL) and brine (30 mL) were added and the mixture was extracted with EtOAc (3 x 60 mL). The combined organic layers were washed with 80% sat. brine (3 x 100 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (40g silica, wet loaded: DCM, gradient (Hept / EtOAc): 1:0 — > 3:2). Pure fractions were combined and concentrated under reduced pressure to afford 530 mg of 28 as a white solid (41% yield).

[0411] 'H NMR (400 MHz, CDC1) 8 5.24 - 5.12 (m, 1H), 4.94 - 4.67 (m, 2H), 4.49 - 4.33 (m, 1H),

[0412] 3.83 - 3.64 (m, 4H), 3.27 - 3.07 (m, 2H), 2.75 - 2.44 (m, 4H), 2.21 - 2.02 (m, 1H), 1.99 -

[0413] 1.83 (m, 1H), 1.81 - 1.55 (m, 2H), 1.44 (d, J= 4.0 Hz, 27H). LCMS m / z ESI Expected 535.3, observed 558.2 ([M+Na]+). lithium S-((R)-3,4-bis((tert-butoxycarbonyl)amino)butyl)-N-(tert-butoxycarbonyl)-L- homocysteinate (31) To a solution of methyl S-((R)-3,4-bis((tert-butoxycarbonyl)amino)butyl)-N-(tert- butoxycarbonyl)-L-homocysteinate (28, 470 mg, 1 Eq, 877 pmol) in MeOH (3.0 mL) / THF (3.0 mL) was added a solution of Lithium hydroxide monohydrate (40.5 mg, 26.8 pL. 1.1 Eq, 965 pmol) in Water (3.0 mL) and reaction was stirred for 1.5 h. [sample, diluted with MeOH, si, LCMS (acid; 210nm): 92% target, 4% SM], Reaction was concentrated under reduced pressure and coevaporated with MeCN (3x) to afford white solid (92% Yield).

[0414] - tri-tert-butyl ((1S,17R)-l-azido-10-oxo-3,6-dioxa-l 4-thia-9-azaoctadecane- 11,17,18-triyl)tricarbamate (32)

[0415] S-((R)-3,4-bis((tert-butoxycarbonyl)amino)butyl)-N-(tert-butoxycarbonyl)-L-homocysteinate (31, 463 mg, 1 Eq, 878 pmol) in DMF (dry) (8.0 mL) were added DIPEA (227 mg, 306 pL, 2 Eq, 1.76 mmol) and HATU (334 mg, 1 Eq, 878 pmol). The mixture was stirred for 15 min, after which 2-(2-(2-azidoethoxy)ethoxy)ethan-l -amine (168 mg, 1.1 Eq, 965 pmol) was added. Stirring was continued for 1 h. [sample, diluted with MeCN, si, LCMS (acid; 210nm): full conversion towards target]. Reaction was purified using Prep-Rev el eris (acid; 1 run). Pure fractions were combined, concentrated under reduced pressure and coevaporated with MeCN (3x) to afford 421 mg of 32 as a glass (71% Yield).

[0416] 'H NMR (400 MHz, CDCh) 6 1.3 - 1.55 (m, 27H), 1.55 - 2.2 (m, 4H), 2.4 -2.6 (m, 4H), 3.1 -3.25 (m, 2H), 3.30 - 3.75 (m, 13H), 4.25 -4.27 (d, 1H), 4.7 -5.15 (bs, 2H), 5.32 (bs, 1H), 6.63 (bs, 1H). LCMS m / z ESI Expected 677.9, observed 678.2 ([M+]).

[0417] (S)-N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-(((R)-3,4-bis((2,2,2-trifluoroacetyl)-14- azaneyl)butyl)thio)-2-((2,2,2-trifluoroacetyl)-14-azaneyl)butanamide (33)

[0418] To a solution of tri-tert-butyl ((1S,17R)-l-azido-10-oxo-3,6-dioxa-14-thia-9-azaoctadecane- ll,17,18-triyl)tricarbamate (32, 313 mg, 1 Eq, 462 pmol) in DCM (4.0 mL) was added TFA (3.0 g, 2.0 mL, 56 Eq, 26 mmol) and reaction was stirred for 1 h. LCMS showed full conversion. Reaction was concentrated under reduced pressure and coevaporated with DCM (3x) to afford 33 which was used as such in next reaction.

[0419] (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(((l lS)-l-azido-ll-(2-(((3R)-3,4- bis(5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H- py ran-2-yl)oxy)pentanamido)buty l)thio)ethy 1)- 10,13 -dioxo-3 ,6-dioxa-9, 12- diazaheptadecan-17-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (Precursor 34)

[0420] To a solution of 5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro- 2H-pyran-2-yl)oxy)pentanoic acid (620 mg, 3 Eq, 1.39 mmol) and (S)-2-amino-N-(2-(2-(2- azidoethoxy)ethoxy)ethyl)-4-(((R)-3,4-diaminobutyl)thio)butanamide tris(2,2,2- trifluoroacetate) (33, 332 mg, 1 Eq, 462 pmol) in N,N-Dimethylformamide (dry) (4.5 mL) was added DIPEA (358 mg, 483 pL, 6 Eq, 2.77 mmol) and HBTU (526 mg, 3 Eq, 1.39 mmol). Reaction was stirred for 1 h at rt. [sample, diluted with MeCN, si, LCMS (acid; 210nm): 24% target, 45% bis-coupled, 12% acid]. More DIPEA (358 mg, 483 pL, 6 Eq, 2.77 mmol), 5-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanoic acid (620 mg, 3 Eq, 1.39 mmol) and HBTU (210 mg, 1.2 Eq, 554 pmol) were added and stirring was continued for 1 h. [sample, diluted with MeCN, s2, LCMS (acid; 210nm): 65% target, 9%> bi s -coupled] .

[0421] Reaction was diluted with 80% sat. brine (25 mL) and was extracted with EtOAc (3x 25 mL) The combined organic layers were washed with 80% sat. brine (3x 75 mL), dried over Na2SO4(s) and concentrated under reduced pressure. The crude product was purified using FCC (80g silica, dry loaded: isolute, gradient (DCM / MeOH): 1:0 -> 9:1). Pure fractions were combined and concentrated under reduced pressure to afford 0.59 g of 34 as a colourless glass. Batch was further purified using Prep-Reveleris (2 runs, acid). The product fractions were combined and concentrated by lyophilization to afford 174 mg as a white solid. (17% yield). 'H NMR (400 MHz, DMSO) 6 7.97 - 7.87 (m, 2H), 7.86 - 7.79 (m, 3H), 7.73 (t, J = 6.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 5.22 (d, J = 3.5 Hz, 3H), 4.98 (dd, J = 11.2, 3.4 Hz, 3H), 4.49 (d, J = 8.5 Hz, 3H), 4.36 - 4.24 (m, 1H), 4.02 (d, J = 5.0 Hz, 9H), 3.93 - 3.77 (m, 4H), 3.76 - 3.66 (m, 3H), 3.65 - 3.58 (m, 2H), 3.58 - 3.48 (m, 4H), 3.48 - 3.38 (m, 8H), 3.26 - 3.15 (m, 2H), 3.15 - 2.97 (m, 2H), 2.48 - 2.30 (m, 4H), 2.11 (s, 11H), 2.08 - 2.02 (m, 4H), 2.00 (s, 9H), 1.90 (s, 9H), 1.78 (d, J = 1.5 Hz, 9H), 1.74 - 1.57 (m, 2H), 1.56 - 1.38 (m, 13H). LCMS m / z ESI Expected 1664.7, observed 1665.6 ([M+H]+).

[0422] Experimental techniques

[0423] 1H NMR Spectra at 400 and 500 MHz were performed on a Bruker Avance DRX-400 and Bruker Avance DPX-500 spectrometer, respectively, with the chemical shifts (6 in ppm) in the solvent dimethyl sulfoxide-d6 (DMSO-d6) referenced at 2.5 ppm or CDCh referenced at 7.24 ppm at the quoted temperatures. Coupling constants (J) are given in Hertz.

[0424] The liquid chromatography / mass spectra (LC / MS) were obtained on a UPLC Acquity Waters instrument, light scattering detector Sedere and SQD Waters mass spectrometer using UV detection DAD 210<l<400 nm and column Acquity UPLC CSH C18 1.7 pm, dimension 2.1x30 mm, mobile phase H2O + 0.1% HCO2H / CH3CN + 0.1% HCO2H.

[0425] All synthetic reactions were performed under an inert atmosphere, unless otherwise stated. In the following examples, when the source of the starting products is not specified, it should be understood that said products are known compounds (e.g., commercially available compounds from suppliers such as Sigma-Aldrich).

[0426] Oligonucleotide synthesis

[0427] MALAT1 ASO was purchased from Axo lab (full PS, PO between ASO, and HA (hexyl amine)). Otherwise, oligonucleotides were synthesized on a 10 pmol scale on a K&A system using CUTAG CPG support (Sigma-Aldrich, 25-35 pmol / g). Nucleotide phosphoramidites were purchased from Sigma-Aldrich or WuXi. Linker phosphoramidites were purchased from Glen Research or WuXi. All cEt phosphoramidites were obtained from Pharmaron. The 5'-amino-modifier C6 was obtained from GlenResearch. UV purities were determined using ion-pairing LCMS and are stated at 260 nm. Yields are given based on the initial resin loading and oligonucleotide content of the final product, as calculated from UV absorption. The sequences of the synthesized oligonucleotides representing an ASO (MALAT1) cargo or an siRNA (ANGPTL3) cargo are shown in the table below:

[0428] HA denotes hexylamine invAb denotes inverted abasic * denotes phosphorothioate linkage CAPS denotes 2'-F for SEQ ID NOs: 2 and 3, and DNA for SEQ ID NO: 1 BOLD denotes 2'-O,4'-C-methylene locked nucleic acid lower case denotes 2'-0Me

[0429] General synthetic procedure

[0430] This procedure was used unless otherwise indicated.

[0431] Phosphoramidites were dissolved to a final concentration to 0.1 M (3 equivalents) in DNA- grade acetonitrile (ACN) prior to use. Detritylation was performed using 3 vol-% dichloroacetic acid in DCM (contact time 5 x 35 s). Di- and tri-antennary linkers were deprotected using double detritylation. Activator 42 was used as activating agent (0.25 M in ACN) for the couplings. Recirculation times of phosphoramidites were 4 min for DNA building blocks, 10 min for all 2’ -modified building blocks. Triple 10 min coupling was used for linker phosphoramidites and quadruple coupling for GalNAc phosphoramidite. Xanthane hydride was dissolved in pyridine (0.2 M) and used as thiolation reagent with a contact time of 5 min. Oxidizer solution was purchased from Sigma- Aldrich and used as such with a contact time of 9 s. Equal volumes of Cap A (9.1 vol-% acetic anhydride in tetrahydrofuran (THF)) and Cap B (THF / JV-methylimidazole / pyridine 80:10:10 vol-%) were mixed in situ for capping (contact time 50 s). Cyanoethyl backbone removal was performed with 20 vol-% di ethylamine in ACN (contact time 7 x 1 min) after a final 5 ’-detritylation where required. Oligonucleotides were cleaved from the solid support and further deprotected by treatment with methanolic ammonia (3 M) at 55 °C for 15-20 h and were subsequently purified using ion-pairing HPLC on reverse phase columns. For siRNA compounds, single strands were separately prepared as mQ water solutions of equal concentrations, mixed in equal volumes, warmed to 950C for 5 min and then allowed to cool down to room temperature over 1 h. Oligonucleotide was synthesized following general procedure with the exception that 5'-amino- modifier C6 (hexyl amine) was introduced as last coupling (0.2M, 10 min contact time, double coupling) as last step. The oligonucleotide was purified with the 5 ’-monomethoxy trityl (MMT) protecting group on. MMT group was removed by treatment with aqueous acetic acid solution (pH 4.5). Thus formed 5’-hexyl amine functionality containing oligonucleotide was conjugated to GalNAc constructs containing suitable conjugation handles like BCN for Click reaction or hexyl amine for PFP-esters containing GalNAc.

[0432] For siRNA synthesis, passenger strand (PO to HA) was synthesized using standard oligonucleotide synthesis procedure. Guide strand was synthesized following general synthetic protocol. For annealing, passenger and guide strands were dissolved in PBS buffer separately to make equimolar solutions of each strands. Both the solutions were mixed. Next, following standard conditions (5 min at 95 °C, then slow cooling down to 20 °C for 1 h with gentle shaking) annealing was performed. QC gel was performed for analysis. Product solution was adjusted to 1 mM in PBS buffer.

[0433] For preparing complexes containing siRNA, passenger strand containing 5 ’-hexyl amine after MMT deprotection was reacted with the pentafluorophenyl ester (PFP-ester) of the GalNAc moiety. PFP-ester were dissolved in ACN / THF / DMF and added to a solution of the oligonucleotide containing hexyl amine in borate buffer (pH 9). Conjugate products were subsequently purified using HPLC. Once passenger strand containing GalNAc conjugate and guide strands were synthesized, standard annealing procedure was followed for siRNA synthesis. Strand solutions in PBS buffer were mixed and heated for 5 min at 95 °C, then slowly cooled down to 20 °C (Ih) with gentle shaking. The QC gel was performed, concentrations were adjusted and reannealed if needed. Compounds were stored as 1 mm PBS buffer solutions.

[0434] Synthesis ofBCN-modified hexyl amine (HA) MALAT1 ASO (21): MALAT1 -hexylamine modified ASO (50 mg) was first dissolved in 500 mL BBS solvent. NHS-activated BCN (1.5 eq, 4 mg) was dissolved separately in 200 mL THF and added to MALAT1 solution. Reaction mixture became turbid. Reaction mixture was sonicated for couple of minutes and kept under stirring. LCMS showed conversion after 2h. Continued stirring for overnight. Oligonucleotide was precipitated by adding 2 mL of Absolute EtOH and 0.2 mL 3M Sodium Acetate (9:1 V / V). The white precipitate was centrifuged for 15 minutes at 4 °C. Removed supernatant layer and repeated washing once more time by 2 mL ethanol. The precipitate after solubilization in 5 mL water was lyophilized overnight to furnish white solid. The obtained product was dissolved in 1 mL deionized water and directly used in the next step.

[0435] LCMS m / z ES" Expected 5681.704, observed 1894.9 (z = 3), 1420.7 (z = 4), 1136.34 (z = 5).

[0436] Examples 1 to 4 - Synthesis of compounds

[0437] Example 1: Cysteine splitter, C4 spacer, BCN tether

[0438] BCN-modified HA MALAT1 ASO (21) (25 mg, 1 equiv.) was dissolved in deionized water (0.5 mL), Cysteine GalNAc (15a) (10.80 mg, 1.5 equiv.) was dissolved in 0.2 mL acetonitrile. Acetonitrile solution of 15a was added to ASO solution under stirring. Clear solution was seen. Reaction mixture was stirred at RT for 20 h. LCMS after 20 h showed main peak of expected product (acetyl protected). The mixture was diluted with 700p.L of 25% aq. NH3 and left under stirring at RT for 5h during which time acetyl groups hydrolyzed. The crude product was dissolved in Water: Acetonitrile (1:1, 2 mL) and freeze dried. Obtained white solid was purified by Prep HPLC. Pure fractions were freeze dried to obtain white solid (12 mg, 38% yield).

[0439] LCMS m / z ES" Expected 6940.289, observed 1736.0813 (z = 4), 1389.0670 (z = 5).

[0440] Compound 1 ASO (MALAT1) Example 2: Cysteine splitter, PEG spacer, BCN tether

[0441] BCN-modified HA MALAT1 ASO (21) (25 mg, 1 equiv.) was dissolved in deionized water (0.5 mL), Cysteine GalNAc (15b) (13.40 mg, 1.5 equiv.) was dissolved in 0.2 mL Acetonitrile. Acetonitrile solution of 15b was added to ASO solution under stirring. Clear solution was seen. Reaction mixture was stirred at RT for 20 h. LCMS after 20 h showed main peak of expected product (acetyl protected). The mixture was diluted with 700p.L of 25% aq. NH3 and left under stirring at RT for 5h during which time acetyl groups hydrolyzed. The crude product was dissolved in Water: Acetonitrile (1:1, 2 mL) and freeze dried. Obtained white solid was purified by Prep HPLC. Pure fractions were freeze dried to obtain white solid (13 mg, 39% yield).

[0442] LCMS m / z ES’ Expected 7333.463, observed 1834.3780 (z = 4), 1467.7040 (z = 5).

[0443] Compound 2 ASO (MALAT1) Example 3: Cysteine splitter, C4 spacer, hexamide tether

[0444] ANGPTL3 conjugate

[0445] Passenger and guide strands were synthesized as described above. Passenger strand was conjugated with GalNAc to form the GalNac(Cysteine)-HA- invAb*gcucaacaUAUuugaucagua*invAb (PO to HA) complex.

[0446] Passenger strand (PO to HA) (60 mg, 1 equiv.) was dissolved in borate buffer (pH 9, 1ml). Cysteine GalNAc PFP ester (19.63 mg, 1.5 equiv.) was dissolved in ACN (0.2 mL). Added ACN solution to borate buffer solution gradually and stirred the reaction mixture. After 2h full conversion was seen by LCMS. Next, aq. ammonia (1 mL) was added to the reaction mixture and shaken overnight to hydrolyze acetyl groups. The solvent was evaporated and compound was purified by HPLC. Pure fractions were freeze dried. Obtained white solid was dissolved in 0.3 mL water and product was precipitated by using aq. NaOAc and ethanol. Centrifuged product was freeze dried to furnish sodium salt of passenger strand as a white solid.

[0447] LCMS m / z ES" Expected 8665.785, Observed 2165.4087 (z = 4)

[0448] Compound 3 siRNA (ANGPTL3) MALAT1 conjugate

[0449] MALAT1 ASO (PO to HA) (30 mg, 1 equiv.) was dissolved in borate buffer (pH 8.7, 0.5 mL). GalNAc PFP ester, Precursor 18 (9.9 mg, 1.5 equiv.), was dissolved in ACN (0.2 mL). This ACN solution was added to the borate buffer solution gradually and the reaction mixture was stirred. After 20 h, full conversion was seen by LCMS. Next, aq. ammonia (1 mL) was added to the reaction mixture and shaken overnight to hydrolyze acetyl groups. The solvent was evaporated and the compound was purified by HPLC. Pure fractions were freeze dried to furnish a white solid (10 mg, 38% UV Purity = 89 Area %).

[0450] LCMS m / z ES" Expected 6703.177, observed 1674.794 (z = 4). Compound 4 ASO (M AL ATI)

[0451] Example 4: Cysteine splitter, C4 spacer, BCN tether

[0452] BCN-modified HA MALAT1 ASO (21) (40 mg, 1 equiv.) was dissolved in borate buffer (pH 9, 0.5 mL). GalNAc azide precursor 34 (9 mg, 1 equiv.), was dissolved in ACN (0.2 mL). This ACN solution was added to the borate buffer solution gradually and the reaction mixture was stirred. After 20 h, full conversion was seen by LCMS. Next, aq. ammonia (1 mL) was added to the reaction mixture and shaken overnight to hydrolyze acetyl groups. The solvent was evaporated and the compound was purified by HPLC. Pure fractions were freeze dried to furnish a white solid (11 mg, 22% UV Purity = 93 Area %).

[0453] LCMS m / z ES" Expected 6968.320, observed 1741.077 (z = 4).

[0454] Compound 5 ASO (MALAT1) Control siRNA cluster: triantennary / amide spacers and prolinol linker

[0455] This reference compound (control cluster coupled to ANGPTL3 siRNA) was synthesized by an analogous protocol to that described above.

[0456] Control siRNA cluster siRNA (ANGPTL3) Example 5 - In vitro biological assays

[0457] Several assays were performed to assess the activity of compounds of the Examples in vitro as compared to control compounds (e.g., naked MALAT1 ASO).

[0458] Human ASGPR1 Homogeneous Time Resolved Fluorescence Assay (“HTRF IC so assay”)

[0459] A binding assay was performed to determine the binding affinities of ASGPR ligands. The Homogeneous Time Resolved Fluorescence (HTRF) assay is a no-wash technology using standard FRET technology with time-resolved measurement of fluorescence. In the human ASGPR1 HTRF assay, HIS-tagged human trimeric ASGPR1 protein and a tri-antennary Alexa-647 labelled probe were used. Upon adding detection reagents containing Terbium- labelled anti-HIS-antibody, a TR-FRET signal was generated and measured. An ASGPR1 ligand can compete with Alexa-647 labelled probe for binding to the ASGPR1 protein and thus lead to decreased TR-FRET signal ratio (Em665 / Em620).

[0460] The human ASGPR1 HTRF binding assay was performed in 384-well microplates (Greiner, #784075). Recombinant human ASGPR1 protein with a HIS-tag was expressed in mammalian cells and purified via chromatography. Anti-HIS-antibody was coupled to a donor (Terbium in this case) and Alexa-647 fluorophore-labelled trivalent GalNAc - tool compound was used as an acceptor. In the human ASGPR1 HTRF binding experiments, 100 nL / well of compounds were dispensed to 384 well assay plates, 5 pL of solution 1 containing 20 nM of HIS-tagged recombinant human trimeric ASGPR1 protein was added to wells and mixed well. After 15 min incubation at room temperature, 5 pL of solution 2 containing 10 nM of the Alexa-647 - labelled trivalent GalNAc and 0.2 pg / mL of Terbium-labelled anti- HIS-antibody were added. After 60 min incubation at room temperature, the HTRF signals were measured in a PHERAstar FSX plate reader with a HTRF module. The optimal HTRF binding assay buffer was 50 mM Tris-Cl (pH7.5), 20 mM CaC12, 0.01% Pluronic F-127. IC50 (Half maximal inhibitory concentration) values were calculated based on 10 concentration response curves.

[0461] Human ASGPR1 Surface plasmon resonance assay

[0462] A Human ASGPR1 Surface plasmon resonance (SPR) binding assay was performed to determine the binding affinities and kinetics of ASGPR ligands.

[0463] The SPR binding assay used surface immobilized Asialoglycoprotein Receptor Protein, ASGPR1(148-291). Immobilization of the biotinylated6His-Avi-TEV-ASGPRl(148-291) was performed by direct capture onto a streptavidin-derivatized dextran sensor chip. Typical immobilization level was 4000-8000 RU. Reference surface was left untreated. Test compounds were diluted in assay buffer (50mM Tris pH 7.4, 150mM NaCl, 50mM CaCh, 0- 1% DMSO) and injected over the sensor surface to generate concentration response curves. Sensorgrams were fitted to a 1:1 binding model to determine affinities and binding kinetics. All data were generated using an 8K or S200 Biacore (Cytiva). Mouse ASGPR fluorescent polarization assay (“Microsome FPIC50” assay)

[0464] A mouse ASGPR Fluorescent Polarization (FP) assay was used to measure the binding affinities of ASGPR ligands to native ASGPR in mouse hepatocytes membrane. The assay was performed in 384-well microplates (Greiner, #781076). The membrane prepared from C57BL / 6 mice livers was used as a source of ASGPR and Alexa-647 fluorophore-labelled trivalent GalNAc compound (supra) was used as tracer.

[0465] In the FP binding experiments, 100 nL of compound was mixed with 5 pL of solution A containing 4 pg of mouse liver membrane. After 15 min incubation at room temperature, 5 pL of solution B containing Alexa-647 - labelled trivalent GalNAc (final concentration 0.5 nM) were added. After 60 min incubation at room temperature, the FP signals were measured in a PHERAstar FSX plate reader with a FP optical module (FP 590 / 575 / 575). The optimal FP assay buffer was 50 mM Tris-Cl of pH7.5, 3 mM CaC12, 0.04% Triton X-100. ICso (Half maximal inhibitory concentration) values were calculated based on 10 concentration response curves.

[0466] Human ASGPR qPCR assay

[0467] An assay was performed to determine the knockdown efficiency of ASGPR conjugates in HEK293 cells and human hepatocytes.

[0468] The qPCR (quantitative polymerase chain reaction) assay was applied to measure mRNA levels of the target sequence Malatl, which is in conjugate with the ASGPR1 tri-antennary ligand. This was done in HEK293 cells overexpressing the human ASGPR1 subunit. The assay was performed in PDL-coated 384-well microplates, prepared with conjugates (lOpM final top concentration with a 1:3 dilution in 10-point dose response) using Echo dispensing. Cells in DMEM GlutMax media (#31966, supplemented with 10% FBS) were seeded on top for 48h incubation. Medium was removed, cells were lysed and cDNA was prepared from the resulting lysates by reverse transcription. cDNA was diluted 1:4 and finally quantitative PCR (qPCR) by Malatl Taqman assay was run to analyse target expression. A Rackl Taqman assay was included as an internal control of housekeeping gene expression. Dose response was normalized to 100% inhibition control: naked Malatl (lOpM) and 0% control: water.

[0469] Human hepatocytes were obtained from a commercial supplier (BioIVT, QNT lot) and cultured in rat tail collagen I coated plates. The hepatocytes were maintained in William's E Medium, supplemented with hepatocyte supplemented media and 5C supplements (DAPT, SB431542, Forskolin, IWP2 and LDN193189). The cells were cultured at 37°C in a humidified incubator with 5% CO2. Next after plating (Day 1), cells were treated with different siRNA conjugates (concentrations ranging from 10 pM to 0.01 nM) and PBS in maintenance media. After 24 h of treatment, media was changed and cells were harvested on Day 4 (72 hr from start of the treatment) for qPCR. Total RNA was extracted according to standard protocol setup in the lab. cDNA synthesis and qPCR were also run according to standard validated protocol. MALAT1 and ANGPTL3 TaqMan primers were purchased from Thermo Fischer. The half maximal inhibitory concentration (IC50) for each sequence was calculated using GraphPad Prism 8 software.

[0470] The results of the above assays for exemplary compounds are shown in Tables 1 and 2 below:

[0471] Table 1: Assay results for exemplary ASO compounds

[0472] ” denotes that the Compound was not tested

[0473] Table 2: Assay results for exemplary siRNA compounds

[0474] ” denotes that the Compound was not tested

[0475] PHH denotes primary human hepatocytesThe data in Tables 1 and 2 indicate that the tested compounds are active, having favourable IC50 and Kd values, and being more active than control (non-complexed) nucleic acid.

[0476] Example 6 -In vivo biological assays

[0477] ASO knockdown of MALAT1 in mice

[0478] Compounds were tested in vivo for knockdown of MALAT-1. Animals were housed in microisolator cages on a constant 12-hour light / dark cycle with controlled temperature and humidity and with access to normal chow and water ad libitum. All in vivo experiments were performed in accordance with AstraZeneca Institutional Animal Care and Use Committee guidelines. C57BL / 6 mice (male 25-27g; Taconic or Charles River) were treated subcutaneously or intravenously with a single dose of O.Olumol / kg, 0.05umol / kg, or 0.25umol / kg, ASO or PBS (mock control) in groups of n=3. Blood samples were drawn 72h post-dosing and animals terminated. The inhibition of ASO target MALAT1 gene expression was quantified by real-time PCR in liver and kidney tissue samples. In addition, plasma samples to measure ALT were performed by biochemical analyzer.

[0479] Total RNA was extracted from cells or tissue samples using the RNeasy 96 QIAcube HT Kit (Qiagen). Complementary DNA (cDNA) was generated using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Reverse transcription qPCR analysis was performed with a QuantStudio 7 Flex using MALAT1 TaqMan assays (Mouse Mm01227912, Applied Biosystems) following the manufacturer’s instructions.

[0480] The relative mRNA levels in liver and kidney after treatment are shown in Figs. 2 and 4 (normalized to the PBS control as 100%; column 1). The naked ASO (column 2) did not produce any significant change in mRNA levels, whereas Compounds 1 and 2 each yielded a significant knockdown in mRNA levels in liver (to around 50% in each case, columns 3 and 4 of Fig. 2, respectively) but not in kidney (columns 3 and 4 of Fig. 4). PK data also showed significant exposure of compounds in liver (Fig. 3) with minimum accumulation in kidney (Fig. 5). siRNA knockdown of ANGPTL3 in mice

[0481] Compound 3 and the Control siRNA cluster, targeting Angptl3, were investigated in vivo in male C57BL6 / J mice (10-11 weeks old with body weight 25-30 g) using a single subcutaneous dose of 0.5 mg / kg (n=5 per group). An additional control group dosed with phosphate-buffered saline (PBS) was also included in the study (n=5).

[0482] Blood samples were drawn pre-dose (Fig. 6) and 7, 14, and 21 days after dosing, as well as after animals were euthanized on day 28 after dosing (Figs. 7 and 8). The ANGPTL3 protein concentration in circulation was measured by enzyme-linked immunosorbent assay ELISA (Catalog Number MANL30; R&D Systems). Liver tissues were sampled on day 28 for measurement of Angptl3 mRNA levels by qPCR. Total RNA was extracted from cells or tissue samples using the RNeasy 96 QIAcube HT Kit (Qiagen). Complementary DNA (cDNA) was generated using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Reverse transcription qPCR analysis was performed with a QuantStudio 7 Flex using ANGPTL3 TaqMan assays (Mouse Mm00803820, Applied Biosystems) following the manufacturer’s instructions. Hepatic Angptl3 mRNA levels were normalized over the mean of RPS16 mRNA levels and then normalized to PBS control (Fig. 9).

[0483] The primary readout in form of ANGPTL3 protein data indicated no statistical difference in baseline levels between groups, with baseline ANGPTL3 protein around 200 ng / mL corresponding to the typical level observed in historical data for this in vivo model. By Day 28, we observed a statistically significant reduction in ANGPTL3 protein concentrations with both the Control siRNA cluster and Compound 3 groups relative to PBS. The baseline and control corrected data indicate around a 50% reduction in ANGPTL3 protein after one week with this reduction sustained up to Day 21 with both groups. A 40% reduction in ANGPTL3 protein after Day 28 would indicate reduced potency of the compounds and potentially the slow return to baseline concentrations. For the secondary readout, expression level AAngptl3 mRNA in liver homogenate, data indicated no significant knockdown of the mRNA levels for the treated groups compared to the control group. While this observation was unexpected, this may indicate a recovery in hepatic mRNA production with a premature effect on protein levels (given the residual reduction of circulating ANGPTL3 protein at Day 28.)

[0484] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0485] In addition, where features or aspects are described in terms of Markush groups, those skilled in the art will recognize that such features or aspects are also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0486] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

CLAIMSA compound having the structure of Formula (I):or a pharmaceutically acceptable salt thereof, wherein:X is selected fromR in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2; andG1to G4each independently represents a -[spacer]-[ligand] moiety or a -[tether] -[linker] -[cargo] moiety, wherein each ligand is independently a monosaccharide and each cargo is independently a nucleic acid, with the proviso that at least one of G1to G4represents a -[spacer]-[ligand] moiety and at least one of G1to G4represents a -[tether]-[linker]-[cargo] moiety.

2. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein X is -S-.

3. The compound or pharmaceutically acceptable salt thereof of claim 1 or claim 2, wherein each R is -H.

4. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein m is 1 , and n is i.

5. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-4, wherein each cargo is independently selected from an antisense oligonucleotide (ASO), an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice altering oligonucleotide, a splice-switching oligonucleotide, a triplex forming oligonucleotide, asiRNA, a saRNA, a microRNA, a microRNA mimic, an anti-miR, a double stranded RNA, a single stranded RNA, a ribozyme, an aptamer, a spiegelmer, a CRISPR oligonucleotide and a G-quadruplex.

6. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-5, wherein each spacer independently comprises a chain of 2-20 atoms selected from C, N, O, S and P, e.g., wherein each spacer is independently selected from a linear alkylene (which may optionally be interrupted by one or more amide and / or phosphate groups) and a polyethylene glycol.

7. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein linker and tether together in each case independently represents a moiety comprising a linear chain of 8 to 30 atoms.

8. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-7, wherein the compound has the structure of Formula (la):wherein X, R, m, n, G1, G2, G3and G4are as defined in any of the preceding claims.

9. A compound having the structure of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:X is selected fromR in each case is independently selected from -H and -Me;m is 1 or 2; n is 1 or 2;A1to A3in each case is independently a moiety which spaces the ligand from the rest of the molecule, e.g. comprising a linear chain of between 2 and 16 atoms;Z is a cargo-containing moiety, which may comprise a linker and a tether, and wherein the cargo is a nucleic acid; and[ligand] in each case is independently a monosaccharide.

10. The compound or pharmaceutically acceptable salt thereof of claim 9, wherein Z represents -[tether]-[linker]-[cargo], in which [tether] -[linker] represents a moiety comprising a linear chain of 8 to 30 atoms; and [cargo] is a nucleic acid.

11. The compound or pharmaceutically acceptable salt thereof of claim 9 or claim 10, wherein one or more of A1to A3is independently selected from *-(Ci-Ci2)alkylene- (where * denotes the point of attachment to the ligand).

12. The compound or pharmaceutically acceptable salt thereof of claim 9 or claim 10, wherein one or more of A1to A3is independently a moiety selected from -(CH2CH2O)p(CH2)2-* and -(CH2)oCONH(CH2CH2O)p(CH2)2-* (where * in each case denotes the point of attachment to the ligand), wherein o is an integer selected from 1, 2, 3 and 4; and p is an integer selected from 1, 2, 3 and 4.

13. The compound or pharmaceutically acceptable salt thereof of any one of claims 9-12, wherein the compound has the structure of Formula (Ila):wherein X, R, m, n, A1, A2, A3, Z and [ligand] are as defined in any of the preceding claims.

14. A compound having the structure of F ormula (III) :or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1, A2, A3and Z are as defined in any of the preceding claims.

15. The compound or pharmaceutically acceptable salt thereof of claim 14, wherein:X is selected fromR in each case is independently selected from -H and -Me; m is 1 or 2; n is 1 or 2;A1to A3in each case is independently a moiety comprising a linear chain of between 2 and 16 atoms; andZ represents -[tether] -[linker] -[cargo], in which [tether] -[linker] represents a moiety comprising a linear chain of 8 to 30 atoms; and [cargo] is a nucleic acid.

16. The compound or pharmaceutically acceptable salt thereof of claim 14 or claim 15, wherein R in each case is -H, m is 1, and n is 1.

17. The compound or pharmaceutically acceptable salt thereof of any one of claims 14-16, wherein the compound has the structure of Formula (Illa):wherein X, R, m, n, A1, A2, A3and Z are as defined in any of the preceding claims.and the pharmaceutically acceptable salts thereof, wherein [nucleic acid] denotes a cargo as defined in any of the preceding claims.

19. The compound or pharmaceutically acceptable salt thereof of claim 18, wherein nucleic acid denotes an ASO or siRNA.

20. A compound as claimed or defined in any one of claims 1-19, or a pharmaceutically acceptable salt thereof, having binding affinity for ASGPR characterised by an ICso value of less than about 10 nM (e.g., an ICso value of less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM).

21. A compound as claimed or defined in any one of claims 1-19, or a pharmaceutically acceptable salt thereof, having binding kinetics for ASGPR characterised by a Kd value of less than about 8 nM (e.g., a Kd value of less than about 1.0 nM).

22. A compound as claimed or defined in any one of claims 1-19, or a pharmaceutically acceptable salt thereof, having an activity in knocking down gene expression in HEK293 cells characterised by an ICso value of less than about 400 nM (e.g., an ICso value of less than about 200 nM, 100 or 50 nM).

23. A pharmaceutical composition comprising a compound as claimed in any one of claims 1-22, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

24. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-22, or a pharmaceutical composition according to claim 23, for use in therapy.

25. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-22, or a pharmaceutical composition according to claim 23, for use in the treatment of a condition selected from liver disease (e.g., liver cancer), genetic disease, haemophilia and bleeding disorders, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g. acromegaly), metabolic disease, cardiovascular disease, obesity, thalassemia, liver injury (e.g., drug induced liver injury), hemochromatosis, alcoholic liver disease, alcohol dependence, anaemia, and anaemia of chronic disease.

26. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of claim 25, wherein the condition is selected from NASH, NAFLD, a metabolic disease and a cardiovascular disease.

27. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of claim 25, wherein the condition is NASH.

28. The compound, pharmaceutically acceptable salt or pharmaceutical composition for use of claim 25, wherein the condition is a metabolic disease selected from hypercholesterolemia, dyslipidaemia, and hypertriglyceridemia.

29. A compound having the structure of Formula (V):or a pharmaceutically acceptable salt thereof, wherein X, R, m, n, A1to A3, [ligand] and [tether] are as defined in any of the preceding claims; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or a phosphoramidite.

30. A process for the preparation of a compound as claimed in any one of claims 9-22, characterised in that a compound of Formula (V) as claimed in claim 29 is reacted with a compound having the structureQ- [cargo] wherein cargo is as defined in any of the preceding claims, and Q denotes a group which is reactive to group J as defined in claim 29.

31. The process of claim 30, wherein Q denotes a BCN-containing group and J is -N3.- Il l -

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