Targeted lipid nanoparticles

Abstract

Lipid nanoparticles are provided which comprise a lipid matrix and a GalNAc-containing compound. The nanoparticles are capable of binding to asialoglycoprotein receptors and may thus be used, for example, in the targeted delivery of nucleic acid cargos to cells such as liver cells. Also provided are pharmaceutical compositions comprising the lipid nanoparticles, medical uses thereof, GalNAc-containing compounds per se, and processes for preparing the lipid nanoparticles and GalNAc compounds of the disclosure.

Description

[0001] ASG-105-PCT01-NP

[0002] TARGETED LIPID NANOPARTICLES

[0003] Cross-Reference to a Related Patent Application

[0004] This specification claims the benefit of priority to United States Provisional Application US 63 / 748,525 (filed 23 January 2025). The entire text of the above-referenced patent application is incorporated by reference into this specification.

[0005] Provided are GalNAc-containing lipid nanoparticles which may be useful, for example, in targeting therapeutic nucleic acids to specific cell and / or tissue types in vivo. In particular, lipid nanoparticles are provided which comprise a lipid matrix, a nucleic acid cargo, and a GalNAc-containing compound as defined herein. The compounds defined herein are capable of binding to asialoglycoprotein receptors (ASGPRs) and the nanoparticles which contain them may be used, for example, to target cells such as liver cells. Also provided are pharmaceutical compositions comprising the lipid nanoparticles and medical uses thereof, as well as GalNAc compounds as defined herein and processes for preparing the lipid nanoparticles and GalNAc compounds.

[0006] SUMMARY

[0007] Nucleic acids such as RNA (for example, messenger RNA [mRNA] and small interfering RNA [siRNA]) have broad potential as therapeutic treatments for a variety of diseases and disorders. However, challenges remain in administering oligonucleotide therapeutics. One route for formulating nucleic acids for delivery includes encapsulating the oligonucleotide within a lipid nanoparticle. Such lipid nanoparticle formulations may include (A) an ionizable or cationic lipid or polymeric material bearing a tertiary or quaternary amine to encapsulate the polyanionic mRNA; (B) a zwitterionic lipid that resembles the lipids in a cell membrane; (C) cholesterol to stabilize the lipid bilayer of the LNP; and (D) a polyethylene glycol (PEG)-lipid to give the nanoparticle a hydrating layer, improve colloidal stability and reduce protein absorption, (see, e.g., Kowalski etal., Molecular Therapy, 27(4), (2019), 710-728). Recently, lipid nanoparticle technology has successfully been applied to generate the first approved mRNA products for prophylactic vaccination against SARS-COV-2 virus (see, e.g., Shoenmaker et al. , International Journal of Pharmaceutics, 601, (2021), 120586).ASG-105-PCT01-NP

[0008] However, there remains a need to develop new lipid nanoparticle formulations for delivery of oligonucleotide therapeutics, and in particular therapies that target specific tissues or organs. In addition, there is a need to develop ways of modifying existing lipid nanoparticles to promote increased uptake of a nucleic acid into an organ or tissue. For example, it would be desirable to be able to modify the lipid nanoparticles described in WO 2023 / 089522 Al so that they are able to target specific organs or tissues.

[0009] Asialoglycoprotein receptors (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 (see Figure 2). As such, ASGPRs represent a promising target for hepatic delivery of therapeutic nucleic acid cargos (see, e.g., D’ Souza et al., J. Control Release, 203, (2015), 126-139). Ligands which can be used for targeting ASGPRs include monosaccharides such as A-acetyl galactosamine (GalNAc).

[0010] Target-binding complexes often contain more than one ligand. This functionality can be achieved by the use of branched core structures with linkers being used to couple the ligands 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);

[0011] WO 2017 / 156012 Al (Arrowhead Pharmaceuticals, Inc.); WO 2016 / 100401 Al (Dicerna Pharmaceuticals, Inc.); WO 2017 / 174657 Al (Silence Therapeutics GmbH); and

[0012] WO 2019 / 092280 Al (Silence Therapeutics GmbH). Complexes of the aforementioned types make use of different coupling chemistries to link ligands and cargo moieties to a branched core structure, and also make use of different core structures. There is, however, a need for alternative chemical functionalities to couple ligands in order to deliver cargo moieties to cells. The present disclosure seeks to address this need by providing novel lipid nanoparticles and compounds which may be used in said lipid nanoparticles e.g. for use in targeting therapeutic nucleic acids to cells such as liver cells.

[0013] In brief, the compounds of the present disclosure (which are formulated into the nanoparticles of the disclosure) contain three GalNAc ligands. These are joined together using a multidentate ‘splitter’ which is derived from shikimic acid. The use of shikimic acid and analogues thereof in preparing the branching unit provides a novel class of targeted lipidASG-105-PCT01-NP

[0014] nanoparticles which may have advantages over known methods, including for example the use of simple, natural precursors. The lipid nanoparticles of the disclosure can exhibit useful properties such as, e.g., improved targeting, internalisation, activity and / or pharmacokinetic properties in vivo. Further, the use of shikimic acid permits the efficient chemical synthesis of the compounds of the present disclosure. Processes for preparation of targeted nanoparticles can thus be performed more efficiently, e.g. using less solvent and other fewer reactants. Moreover, shikimic acid is a natural product and it and its derivatives are biocompatible. The use of a shikimic acid splitter in the compounds and lipid nanoparticles disclosed herein can therefore result in biocompatible metabolites, which decrease side effects, off-site effects and other toxicities associated with known GalNAc-containing compounds. Still further, the use of a splitter derived from shikimic acid does not compromise the stability of the lipid nanoparticles.

[0015] Summary of the Disclosure

[0016] The present disclosure includes the following aspects and embodiments which are presented as numbered clauses 1 to 42:

[0017] 1. A lipid nanoparticle comprising:

[0018] (i) a lipid matrix;

[0019] (ii) a nucleic acid cargo; and

[0020] (iii) a compound of Formula (I):

[0021]

[0022] or a pharmaceutically acceptable salt thereof, wherein:

[0023] X is -O- or -NR-;

[0024] wherein R is selected from -H and -(Ci-Ce)alkyl;ASG-105-PCT01-NP

[0025] Y is a linker comprising a linear chain of about 50 to about 400 atoms (e.g., selected from C, N, and O);

[0026] Z is a lipidic anchor;

[0027] represents a carbon-carbon single bond or double bond; and spacer in each case independently represents a moiety comprising a linear chain of 4 to 20 atoms (e.g., selected from C, N, and O).

[0028] 2. The lipid nanoparticle of clause 1, wherein represents a double bond.

[0029] 3. The lipid nanoparticle of clause 1 or clause 2, wherein the compound of Formula (I) has the structure of F ormul a (la) :

[0030]

[0031] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined in any of the preceding clause.

[0032] 4. The lipid nanoparticle of clause 3, wherein the compound of Formula (I) has the structure of F ormula (lb), F ormula (Ic), or F ormula (Id) :ASG-105-PCT01-NP

[0033]

[0034] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined in any of the preceding clauses.

[0035] 5. The lipid nanoparticle of any one of the preceding clauses, wherein each spacer is independently selected from a linear alkylene, which may optionally be interrupted by one or more amide groups.

[0036] 6. The lipid nanoparticle of any one of the preceding clauses, wherein X is -NR-, wherein R is -H or -CH3.ASG-105-PCT01-NP

[0037] 7. The lipid nanoparticle of any one of the preceding clauses, wherein the compound of Formula (I) has the structure of Formula (II):

[0038]

[0039] or a pharmaceutically acceptable salt thereof, wherein R, Y, and Z are as defined in any of the preceding clauses, and wherein:

[0040] A in each case independently represents a moiety comprising a linear chain of 1 to 16 atoms (e.g., selected from C, N, and O);

[0041] m in each case is independently selected from the integers from 1 to 6; and represents a carbon-carbon single bond or double bond.

[0042] 8. The lipid nanoparticle of clause 7, wherein the compound of Formula (II) has the structure of Formula (Ila):

[0043]

[0044] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined in any of the preceding clauses.

[0045] 9. The lipid nanoparticle of clause 8, wherein the compound of Formula (II) has the structure of Formula (lib), Formula (lie), or Formula (lid) :ASG-105-PCT01-NP

[0046]

[0047] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined in any of the preceding clauses.

[0048] 10. The lipid nanoparticle of any one of clauses 7 to 9, wherein each A is independently selected from -(Ci-Cie)alkylene-, optionally wherein each A is -(CH2)e-.ASG-105-PCT01-NP

[0049] 11. The lipid nanoparticle of any one of clauses 7 to 10, wherein m in each case is 2.

[0050] 12. The lipid nanoparticle of any one of the preceding clauses, wherein Y is a hydrophilic linker comprising a linear chain of about 50 to about 400 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O.

[0051] 13. The lipid nanoparticle of any one of the preceding clauses, wherein Y comprises a linear polymer of polyethylene glycol moi eties, e.g. a linear polymer of between 24 and 86 polyethylene glycol moieties.

[0052] 14. The lipid nanoparticle of any one of the preceding clauses, wherein Y has the structure:

[0053] a)

[0054] " "

[0055]

[0056] in which * denotes the point of attachment to Z, wherein:

[0057] n, p, and r are integers independently selected from 1 to 31;

[0058] q is an integer selected from 10 to 110;

[0059] R’ is selected from -H and -(Ci-Ce)alkyl;

[0060] R” is selected from -H and -(Ci-Ce)alkyl; and

[0061] R’” is selected from -H and -(Ci-Ce)alkyl.

[0062] 15. The lipid nanoparticle of clause 14, wherein:

[0063] the lipid nanoparticle is defined in part (a) of clause 14 and n is 4; p is 1; and q is an integer selected from 30 to 85 (e.g. from 35 to 45, from 40 to 50, or from 65 to 75); or the lipid nanoparticle is defined in part (b) of clause 14 and n is 1; p is 1; q is an integer selected from 30 to 55 or from 51 to 84; and r is 5.

[0064] 16. The lipid nanoparticle of clause 14 or clause 15, wherein R’ is -H, and / or wherein R” is -H, and / or wherein R’” is -H.ASG-105-PCT01-NP

[0065] 17. The lipid nanoparticle of any one of the preceding clauses, wherein Y has the structure:

[0066]

[0067] in which * denotes the point of attachment to Z, wherein q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70).

[0068] 18. The lipid nanoparticle of any one of the preceding clauses, wherein Z is a hydrophobic lipidic anchor selected from a sterol and a lipid (e.g. an ether lipid, an ester lipid, or a phospholipid).

[0069] 19. The lipid nanoparticle of clause 18, wherein Z is an ether lipid having the structure

[0070]

[0071] ORa, e.g. having the structureORawherein each Ra is independently a -(Ce-C24)alkyl group, e.g. a linear -(Ce-C24)alkyl group.

[0072] 20. The lipid nanoparticle of clause 19, wherein each Rais n-octadecyl.

[0073] 21. The lipid nanoparticle of any one of the preceding clauses, wherein Y and Z together have the structure:

[0074]

[0075] wherein q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70), and each Rais n-octadecyl.ASG-105-PCT01-NP

[0076] 22. The lipid nanoparticle of any one of the preceding clauses, wherein the compound of

[0077]

[0078] ASG-105-PCT01-NP

[0079]

[0080] ASG-105-PCT01-NP

[0081]

[0082] and the pharmaceutically acceptable salts thereof.

[0083] 23. The lipid nanoparticle of any one of the preceding clauses, wherein the lipid matrix comprises:

[0084] (a) ionizable lipid;

[0085] (b) neutral lipid;

[0086] (c) sterol; and

[0087] (d) polymer-conjugated lipid.

[0088] 24. The lipid nanoparticle of clause 23, wherein the ionizable lipid is selected from 1-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, bis(3-pentyloctyl) 9-((2-oxaspiro[3.3]heptan-6-yl)amino)heptadecanedioate, bis(3 -pentyloctyl) 9-((tetrahydro-2H-pyran-4-yl)amino)heptadecanedioate, bi s(3 -pentyl octyl) 9-(((tetrahydrofuran-3-yl)methyl)amino)heptadecanedioate, bis(3 -pentyloctyl) 9-(((tetrahydro-2H-pyran-4-yl)methyl)amino)heptadecanedioate, bi s(3 -pentyl octyl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, and combinations thereof.

[0089] 25. The lipid nanoparticle of clause 23 or clause 24, wherein the neutral lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and combinations thereof.ASG-105-PCT01-NP

[0090] 26. The lipid nanoparticle of any one of clauses 23 to 25, wherein the sterol comprises (e.g. is) cholesterol.

[0091] 27. The lipid nanoparticle of any one of clauses 23 to 26, wherein the polymer-conjugated lipid is selected from DMPE-PEG2000, DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-C-DOMG, PEG-C-DOPG, and combinations thereof.

[0092] 28. The lipid nanoparticle of any one of the preceding clauses, wherein the lipid matrix comprises l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000.

[0093] 29. The lipid nanoparticle of any one of clauses 1 to 28, wherein the lipid nanoparticle comprises:

[0094] a) from about 30 to about 75 mol% of ionizable lipid;

[0095] b) from about 5 to about 20 mol% of neutral lipid;

[0096] c) from about 20 to about 60 mol% of sterol;

[0097] d) from 0 to about 5 mol% of polymer-conjugated lipid; and

[0098] e) up to about 5 mol% of a compound of Formula (I) as defined in any one of clauses 1 to 21, wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo).

[0099] 30. The lipid nanoparticle of clause 29, wherein the lipid nanoparticle comprises:

[0100] a) about 50 mol% of l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate;

[0101] b) about 10 mol% of distearoyl phosphatidylcholine (DSPC);

[0102] c) about 38.5 mol% of cholesterol;

[0103] d) about (1.5 - x) mol% of DMG-PEG2000; and

[0104] e) about x mol% of a compound of Formula (I) as defined in any one of clauses 1 to 21, wherein x is less than or equal to about 1.0, and wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle.

[0105] 31. The lipid nanoparticle of clause 30, wherein x is between about 0.01 and about 0.5, e.g. between about 0.025 and about 0.15, optionally wherein x is about 0.05.ASG-105-PCT01-NP

[0106] 32. The lipid nanoparticle of any one of the preceding clauses, wherein the nucleic acid cargo comprises RNA, e.g. a mRNA.

[0107] 33. The lipid nanoparticle of any one of the preceding clauses, wherein:

[0108] a) the z-average particle diameter of the lipid nanoparticles is from about 40 nm to about 90 nm, e.g. from about 50 nm to about 80 nm, from about 60 nm to about 70, such as about 64 nm to about 70 nm, as measured by dynamic light scattering;

[0109] b) the poly dispersity of the lipid nanoparticle is less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1, such as between about 0.03 and about 0.08; and / or

[0110] c) the encapsulation efficiency (EE) of the lipid nanoparticle is at least about 90%, e.g. at least about 95% such as between about 95% to about 97%.

[0111] 34. A pharmaceutical composition comprising a plurality of the lipid nanoparticles of any one of the preceding clauses.

[0112] 35. The lipid nanoparticles of any one of clauses 1 to 33, or the pharmaceutical composition of clause 34, for use in treating a disease or disorder in a subject in need thereof.

[0113] 36. A method for increasing the uptake of a nucleic acid into an organ or tissue, wherein said organ or tissue expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising the use of a lipid nanoparticle of any one of clauses 1 to 33, or the pharmaceutical composition of clause 34.

[0114] 37. A process for the preparation of a lipid nanoparticle as defined in any one of clauses 1 to 33, the process comprising the steps of:

[0115] A) providing a first solution comprising the lipid matrix and the compound of Formula (I);

[0116] B) providing a second solution comprising the nucleic acid cargo in water or an aqueous buffer; and

[0117] C) combining the solution of (A) with the solution of (B) thereby to prepare the lipid nanoparticle.

[0118] 38. A method for improving the ability of a lipid nanoparticle to be targeted to an organ or tissue which expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising incorporating into the lipid nanoparticle a compound of Formula (I) as defined in any one of clauses 1 to 22 (e.g. by a process as defined in clause 37).ASG-105-PCT01-NP

[0119] 39. A compound of Formula (I) as defined in any one of clauses 1 to 22, or a pharmaceutically acceptable salt thereof.

[0120] 40. The compound of clause 39, selected from:

[0121]

[0122] ASG-105-PCT01-NP

[0123]

[0124] ASG-105-PCT01-NP

[0125]

[0126] and the pharmaceutically acceptable salts thereof.

[0127] 41. A process for the preparation of a compound of Formula (I) as defined in clause 39 or clause 40, or a pharmaceutically acceptable salt thereof, wherein the process comprises reacting a compound of Formula A’

[0128]

[0129] wherein X and spacer are as defined in any one of clauses 1 to 11, and wherein:

[0130] Y’ is a linker precursor comprising a linear chain of about 2 to about 100 atoms (e.g., selected from C, N, and O) and a first reactive group; and

[0131] Q is a hydroxyl protecting group or H,

[0132] with a compound of Formula B’

[0133] Y”-Z,

[0134] wherein:ASG-105-PCT01-NP

[0135] Y” is a second linker precursor comprising a linear chain of about 48 to about 398 atoms (e.g., selected from C, N, and O) and a second reactive group,

[0136] the process optionally further comprising a step of removing the hydroxyl protecting groups.

[0137] 42. A compound of Formula A’ as defined in clause 41, or a pharmaceutically acceptable salt thereof.

[0138] BRIEF DESCRIPTION OF THE FIGURES

[0139] FIG. 1 shows a schematic of a lipid nanoparticle of the disclosure. Each “Ligand” is GalNAc, the “Splitter” is a shikimic acid-derived moiety, and the “Lipidic anchor” denotes a lipophilic group which can insert into the matrix of the nanoparticle. The “Spacer” and “Linker” moieties denote linear chains which attach the components of the compounds together.

[0140] FIG. 2 shows a reverse phase chromatogram of Compound 1 that was obtained in Example 1. Compound 1 was obtained in >98% purity as determined by LCMS.

[0141] FIG. 3 shows a raw LCMS spectrum of Compound 1.

[0142] FIG. 4 shows a deconvoluted LCMS spectrum of Compound 1. The distribution of PEG moeities in the linker part of the molecule can clearly be seen in this trace.

[0143] FIG. 5 shows a deconvoluted LCMS spectrum of Compound 1.

[0144] FIG. 6 shows a schematic depicting the processes followed in Examples 2 and 3, relating to the formation of lipid nanoparticles of the disclosure, followed by IV administration to LDLr knockout mice (homozygous), and ex vivo imaging of organs for luciferase expression.

[0145] FIG. 7 shows individual average radiance (liver luciferase signal) for a range of different lipid nanoparticles used in the in vivo study of Example 3 in male LDLr homozygous knockout mice (B6-Ldlrtml.lAztc). The 0% control formulation is Z1 as shown in Table 2; the 0.01-1% test formulations are A1-A4, respectively (left to right).

[0146] FIG. 8 shows individual and average radiance (liver luciferase signal) for different lipid nanoparticles used in the in vivo study of Example 3 in male LDLr homozygous knockout mice (B6-Ldlrtml.lAztc). The 0% control formulation is Z1 as shown in Table 2; the 0.05% test formulations are A5 and Bl, respectively (left to right).ASG-105-PCT01-NP

[0147] FIG. 9 shows individual and average radiance (liver luciferase signal) for different lipid nanoparticles used in the in vivo study of Example 3 evaluated in male LDLr homozygous knockout mice (B6.129S7-LdlrtmlHer / J). The 0% control formulation is Z2 as shown in Table 2; the 0.025-0.15% “v2” test formulations are C1-C3, respectively; and the 0.05% “vl” formulation is A6 (left to right).

[0148] DETAILED DESCRIPTION

[0149] 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.

[0150] Definitions

[0151] 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.

[0152] 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.);

[0153] 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)ASG-105-PCT01-NP

[0154] 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).

[0155] 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.

[0156] 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”.

[0157] 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 stepsASG-105-PCT01-NP

[0158] 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’.

[0159] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as A-acetyl galactosamine. Reference to "GalNAc" or "A-acetyl galactosamine" herein denotes the P form, i.e., 2-(acetylamino)-2-deoxy-P-D-galactopyranose.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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, farmASG-105-PCT01-NP

[0164] 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.

[0165] “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.

[0166] “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.

[0167] 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 ofASG-105-PCT01-NP

[0168] 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.

[0169] 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).

[0170] 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.

[0171] 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 “(CiC3)alkyl”, “(CiC6)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(Ci-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.ASG-105-PCT01-NP

[0172] 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-l-enyl, prop-2-enyl, isopropenyl, but-l-enyl, 2-methyl-prop-l-enyl, and 2-methyl-prop-2-enyl. The terms “(C2-Ce)alkenyr, 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.

[0173] 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 naphthalenyl. 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.

[0174] 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 bicyclofl.1.1 ]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.

[0175] 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 selectedASG-105-PCT01-NP

[0176] 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).

[0177] 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.

[0178] 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 independently selected from the group consisting of: halogen (e.g., -F, -Cl, -Br

[0179] or -I); -OH; -SH; -NH2; -NHMe; -NMe2; -(Ci-C3)alkyl (e.g., -Me or -Et); and 3- or

[0180] 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.

[0181] 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. The skilled reader 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.

[0182] 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).ASG-105-PCT01-NP

[0183] 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).

[0184] As used herein, the term “lipid nanoparticle” means a nanoparticle comprised of one or more lipid components which typically includes an electron dense nanostructural core produced by microfluidic mixing of lipid-containing solutions in ethanol with aqueous solutions.

[0185] As used herein, the term “lipid matrix” means the assembly of lipids and lipid-like compounds which make up the core structure of the nanoparticle. The lipid matrix is typically comprises of a plutrality of different types of lipids and lipid-like compounds, including for example ionizable lipids, neutral lipids, sterols and polymer-conjugated lipids.

[0186] As used herein, the term “ionizable lipid” includes lipids containing a positive charge at the acidic scale of physiological pH range, for example 1, 2 -dil inoleyl oxy -3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, (see e.g., U.S. Patent No. 8,158,601), 2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see e.g., WO 2012 / 018754), Acuitas-5 (see e.g., WO 2015 / 199952), KL-10 (see e.g., U.S. Patent Application Publication 2012 / 0295832), C12-200 (see e.g., Love, KT et al., PNAS, 107: 1864 (2009)), and the like. Ionizable lipids are also described in WO 2023 / 089522 Al.

[0187] As used herein, the term “neutral lipid” includes lipids that have a zero-net charge at physiological pH, for example, lipids that exist in an uncharged form or neutral zwitterionic form at physiological pH, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and the like, and combinations thereof.

[0188] As used herein, the term “sterol” has its conventional meaning and includes cholesterol, and the like.

[0189] As used herein, the term “polymer-conjugated lipid” includes lipids that comprise a lipid portion and a polymer portion, such as pegylated lipids comprising both a lipid portion and a polyethylene glycol portion. Non-limiting examples include dimyristoyl phosphatidyl ethanolamine-poly(ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-C-DOMG, and PEG2000-C-DOPG.ASG-105-PCT01-NP

[0190] As used herein, the term “encapsulation efficiency” refers to the ratio of encapsulated cargo (e.g. nucleic acid segment) in the lipid nanoparticles to total cargo (e.g. nucleic acid segment) content in the lipid nanoparticle composition measured by lysis of the lipid nanoparticles using a detergent, e.g., Triton X-100.

[0191] As used herein, the term “lipidic anchor” means a lipophilic moiety which can embed itself in the matrix of the lipid nanoparticle. In this way, it ‘anchors’ the compound in the lipid matrix keeping the GalNAc-containing compound associated with the nanoparticle.

[0192] The term "z-average" as used herein refers to the z-average particle diameter, i.e. the intensity weighted mean hydrodynamic size of the ensemble collection of particles of a sample, as determined by dynamic light scattering. References to "z-average" and “z-average particle diameter” as used herein refer to z-average particle diameter as determined by dynamic light scattering (DLS). For example, z-average particle diameter can be determined by DLS analysis of aqueous suspensions performed at 25°C using a Malvern Zetasizer Ultra instrument. Samples can be prepared by mixing 10 pL of lipid nanoparticle dispersions with the appropriate dispersant (1 mL). Measurements can be performed, for example, using polystyrene cuvettes at 25 °C, measuring the scattered light at an angle of 173°.

[0193] 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 herein, 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, withASG-105-PCT01-NP

[0194] 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.

[0195] 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 etal., JPharm 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.

[0196] 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.

[0197] Compounds, compositions and methods provided herein may be combined with one or more of any of the other compounds, compositions and methods provided herein.

[0198] The following abbreviations and empirical formulae are used herein:

[0199] Ac acetyl

[0200] ASGPR asialoglycoprotein receptor

[0201] ASO antisense oligonucleotide

[0202] Boc tert-butyloxycarbonyl

[0203] BOP benzotriazole-1 -yl-oxy-tris-(dimethylamino)

[0204] CAM ceric ammonium molybdate

[0205] Cbz carboxybenzyl

[0206] CRISPR clustered regularly interspaced short palindromic repeats

[0207] crRNA CRISPR RNA

[0208] DCC N,N' -Dicyclohexylcarbodiimide

[0209] DCM dichloromethane

[0210] DIEA diethylamineASG-105-PCT01-NP

[0211] DIPEA N,N-diisopropylethylamine

[0212] DLS Dynamic Light Scattering

[0213] DMAP 4-dimethylaminopyridine

[0214] DMF dimethylformamide

[0215] DMPC dimyristoyl phosphatidylcholine

[0216] DMPE dimyristoyl phosphatidyl ethanolamine

[0217] DMSO dimethylsulfoxide

[0218] DNA deoxyribonucleic acid

[0219] DOPE dioleoyl phosphatidylethanolamine

[0220] DPPC dipalmitoyl phosphatidylcholine

[0221] DSPC distearoyl phosphatidylcholine

[0222] EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDTA ethylenediaminetetraacetic acid

[0223] EE encapsulation efficiency

[0224] ELS evaporative light scattering

[0225] ESI electrospray ionization

[0226] GalNAc 2-(acetylamino)-2-deoxy-D-galactopyranose

[0227] HPLC high performance liquid chromatography

[0228] HOBt 1 -hydroxybenzotriazole

[0229] LCMS liquid chromatography mass spectrometry

[0230] LDLr low density lipoprotein receptor

[0231] LNA locked nucleic acid

[0232] LNP lipid nanoparticle

[0233] MPA Mobile Phase A (water with 50 mM NELOAc + 0.5% HOAc) m / z mass to charge ratio

[0234] mRNA messenger RNA

[0235] MS mass spectrometry

[0236] NMR nuclear magnetic resonance

[0237] PCR polymerase chain reaction

[0238] PDA photodiode array

[0239] PEG polyethyleneglycol

[0240] PFP pentafluorophenyl

[0241] PNA peptide nucleic acid

[0242] PyBOP benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphateASG-105-PCT01-NP

[0243] RNA ribonucleic acid

[0244] RNase ribonuclease

[0245] RT room temperature

[0246] sgRNA single guide RNA

[0247] shRNA short hairpin RNA

[0248] siNA small interfering nucleic acid siRNA small interfering RNA TBAF tetrabutylammonium fluoride TBS tert-butyldimethylsilyl TEA triethylamine

[0249] TFA trifluoroacetic acid

[0250] THF tetrahydrofuran

[0251] TIC total ion chromatogram TIPS triisopropyl silane

[0252] TLC thin layer chromatography TMS-I Trimethyl silyl iodide tracrRNA trans-activating CRISPR RNAASG-105-PCT01-NP

[0253] Lipid nanoparticles

[0254] The present disclosure provides GalNAc-carrying lipid nanoparticles which are useful, inter alia, for targeting nucleic acid cargo moieties to specific locations (e.g., cell and / or tissue types) in vivo. The present disclosure also provides GalNAc-containing compounds which are useful, inter alia, in preparing lipid nanoparticles which can be targeted to specific locations in vivo. As illustrated schematically in Figure 1 (not shown to scale), the compounds (and hence the lipid nanoparticles which comprise them) have several ‘arms’ which carry the GalNAc ligands and a lipidic anchor. 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 lipid nanoparticles comprise said compounds which are embedded in the lipid matrix via the lipidic anchor. The present disclosure relates, in particular, to lipid nanoparticles comprising compounds in which the splitter is derived from shikimic acid, in which the ligand-containing arms are attached to the hydroxyl groups on the cyclohexane ring. The present disclosure also relates to the GalNAc-containing compounds per se. The use of shikimic acid and analogues thereof in preparing the branching unit provides a novel class of targeted lipid nanoparticles which may have advantages over known nanoparticles, including a simplified synthesis and the use of simple, natural precursors. The lipid nanoparticles of the disclosure can exhibit useful properties such as, e.g. improved targeting, uptake, activity and / or pharmacokinetic properties.

[0255] In more detail, the present disclosure provides a lipid nanoparticle comprising a lipid matrix and a GalNAc-containing compound as defined herein (i.e., a compound of Formula (I) or a pharmaceutically acceptable salt thereof).

[0256] Thus, in a first aspect, the disclosure provides a lipid nanoparticle comprising:

[0257] (i) a lipid matrix;

[0258] (ii) a nucleic acid cargo; and

[0259] (iii) a compound of Formula (I):ASG-105-PCT01-NP

[0260]

[0261] or a pharmaceutically acceptable salt thereof, wherein:

[0262] X is -O- or -NR-;

[0263] wherein R is selected from -H and -(Ci-Ce)alkyl;

[0264] Y is a linker comprising a linear chain of about 50 to about 400 atoms (e.g., selected from C, N, and O);

[0265] Z is a lipidic anchor;

[0266] represents a carbon-carbon single bond or double bond; and spacer in each case independently represents a moiety comprising a linear chain of 4 to 20 atoms (e.g., selected from C, N, and O).

[0267] In embodiments, represents a double bond. In other embodiments, represents a single bond.

[0268] In embodiments, X is -NR-. In embodiments, X is -NR- wherein R is selected from -H and -(Ci-C3)alkyl. In embodiments, R is -H. In embodiments, R is -CH3. In embodiments, X is -NH-. In embodiments, X is -N(CH3)-.

[0269] In embodiments, represents a single bond and X is -N(CH3)-. In embodiments, represents a double bond and X is -NH.

[0270] In embodiments, the compound of Formula (I) has the structure of Formula (la):ASG-105-PCT01-NP

[0271]

[0272] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein and represents a carbon-carbon single bond or double bond.

[0273] In embodiments, the compound of Formula (I) has the structure of Formula (lb):

[0274]

[0275] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein.

[0276] In embodiments, the compound of Formula (I) has the structure of Formula (Ic):

[0277]

[0278] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein.ASG-105-PCT01-NP

[0279] In embodiments, the compound of Formula (I) has the structure of Formula (Id):

[0280]

[0281] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein.

[0282] GalNAc ligands

[0283] The GalNAc ligands present in the compounds of the disclosure (and hence the lipid nanoparticles of the disclosure) have an affinity for the hepatic asialoglycoprotein receptor (ASGPR). In embodiments, the target ASGPR is on the surface of a mammalian liver cell. The compounds of the disclosure comprise three GalNAc ligands, e.g. as shown for Formula (I). The shikimate splitter provides an elegant solution to the problem of coupling three GalNAc ligands to the lipidic anchor. Shikimic acid has three adjacent alcohol groups that may each be conjugated to a GalNAc ligand via a spacer moiety. Binding to asialoglycoprotein receptors (ASGPRs) has been demonstrated herein to be effective when the lipid nanoparticles comprise compounds of Formula (I) in which all three of the shikimate alcohol groups are conjugated to a GalNAc ligand via a spacer moiety as defined herein. The spacer

[0284] The “spacer” comprises a chain of atoms, typically carbon atoms with one or more heteroatoms (e.g. selected independently from N and O) optionally intervening that serves to link the ligand to the splitter moiety (i.e., the shikimic acid-derived core). In the compounds of the present disclosure, the spacer comprises a linear chain of 4 to 20 atoms (e.g., a chain of 7-14 atoms) selected from C, N, P and O (e.g., selected from C, N, and O). Exemplary spacers include linear alkylenes (which may optionally be interrupted by one or more amide groups, e.g. a single amide group). Other exemplary spacers comprise short polyethyleneglycol chains. The spacer may usefully comprise one or more heteroatomsASG-105-PCT01-NP

[0285] within the linear chain or bonded to the linear chain, thereby increasing the hydrophilic nature of the spacer and reducing its tendency to associate with the nanoparticle matrix or the lipid anchor. 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.

[0286] In embodiments, a spacer (e.g., each spacer) has the formula:

[0287]

[0288] wherein: A in each case independently represents a moiety comprising a linear chain of 1 to 16 atoms (e.g., selected from C, N, O, S and P); and m in each case is independently selected from the integers from 1 to 6, wherein * denotes the point of attachment of the spacer to the GalNAc ligand (i.e. the oxygen atom of the GalNAc ligand) and f denotes the point of attachment to the splitter moiety (i.e., the shikimic acid-derived core).

[0289] In embodiments, m in each case is independently selected from the integers from 1 to 5. In embodiments, m in each case is independently selected from 1, 2, 3 and 4. In embodiments, m in each case is independently selected from 2, 3, and 4. In embodiments, each m is the same. In embodiments, m is 2. In embodiments, each m is 2.

[0290] In embodiments, m is independently selected from the integers from 1 to 4, e.g. from 1 to 3. In embodiments, m is independently 1 or 2. In embodiments, m is 2. In embodiments, A is n-hexylene (i.e. -(CH2)e-) and m is 2. In embodiments, each A is n-hexylene (i.e. -(CH2)e-) and each m is 2.

[0291] In embodiments, A in each case is independently selected from -(Ci-Cie)alkylene- and -(C2-Qalkenylene-. In embodiments, A in each case is independently selected

[0292] from -(Ci-Ci2)alkylene-, such as -(C2-Cio)alkylene- or -(C4-Cs)alkylene-, e.g. wherein A is n-hexylene (i.e. -(CH2)e-). In embodiments, each A is the same. In embodiments, each A is n-hexylene.

[0293] In particular embodiments, the lipidic anchor-containing arm is attached to the splitter via an amide, and the spacer includes an amide. Viewed from this aspect, the disclosure provides aASG-105-PCT01-NP

[0294] lipid nanoparticle as defined herein, wherein the compound of Formula (I) has the structure of Formula (II):

[0295]

[0296] or a pharmaceutically acceptable salt thereof, wherein R, Y, and Z are as defined herein, and wherein:

[0297] A in each case independently represents a moiety comprising a linear chain of 1 to 16 atoms (e.g., selected from C, N, and O);

[0298] m in each case is independently selected from the integers from 1 to 6; and represents a carbon-carbon single bond or double bond.

[0299] In embodiments, the compound of Formula (II) has the structure of Formula (Ila):

[0300]

[0301] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined herein.

[0302] In embodiments, the compound of Formula (II) has the structure of Formula (lib):ASG-105-PCT01-NP

[0303]

[0304] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined herein.

[0305] In embodiments, the compound of Formula (II) has the structure of Formula (lie):

[0306]

[0307] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined herein.

[0308] In embodiments, the compound of Formula (II) has the structure of Formula (lid):ASG-105-PCT01-NP

[0309]

[0310] or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined herein.

[0311] In embodiments, m is 2. In embodiments, A is n-hexylene (i.e. -(CH2)e-). In embodiments, A is n-hexylene and m is 2. In embodiments, each A is n-hexylene and each m is 2.

[0312] The linker

[0313] The “linker” group, Y, acts to join the lipidic anchor, Z, to the rest of the molecule (e.g. the compound of Formula (I)) via the splitter. Suitable linkers will be apparent to the skilled person based on the current description and its Examples.

[0314] In embodiments, the linker moiety is created by reacting a linker precursor (Y’) with a lipidic anchor containing precursor (Y”-Z). Suitable methods for reacting the linker and lipidic anchor precursors are described in the following Examples and would be apparent to the skilled person. Examples of such methods include amide coupling reactions between an amine (e.g. of the linker precursor) and an activated carbonate (also see, e.g., Fantoni et al., Chem. Rev. (2021) 121(12):7122- 7154). In other embodiments, the linker moiety (Y) is introduced into the complex without the lipidic anchor attached and the lipidic anchor is subsequently bound directly to the linker. The complex may conveniently be coupled with a lipidic anchor in the penultimate step of a synthesis (i.e. before a final deprotection step) to yield the final compound (see, e.g., Beaucage, Curr Opin Drug Discov Dev (2008) 11(2):203 -216). Other examples of linkers which might be used in accordance with the present disclosure are found, e.g., in international patent publications WO 2014 / 179620,

[0315] WO 2015 / 177668, WO 2009 / 073809, WO 2012 / 083046, WO 2017 / 156012,ASG-105-PCT01-NP

[0316] WO 2016 / 100401, WO 2017 / 174657 and WO 2019 / 092280, the contents of each of which are incorporated herein in their entirety.

[0317] In the compounds of formula (I) described herein, Y is a linker comprising a linear chain of about 50 to about 400 atoms which links the lipidic anchor to the rest of the molecule. In embodiments, Y is a linker comprising an optionally substituted linear chain of about 50 to about 400 atoms selected from C, N, and O, e.g. a chain of about 311 to about 371 atoms, about 165 to about 265 atoms, or about 105 to about 175 atoms. In embodiments, Y comprises one or more amide groups (e.g., obtained by reaction of a carboxylic acid or acid derivative with an amine).

[0318] In embodiments, Y is a hydrophilic linker. This is advantageous because the hydrophilic nature of the linker is more favourably solvated by water molecules, e.g. in aqueous physiological environments. This helps to ensure that the lipidic anchor, Z (which is attached to one end of linker), is incorporated within the lipid matrix of the nanoparticle, while the hydrophilic linker, Y, extends outwards from the lipid matrix, so that it may i) be solvated, and / or ii) avoid unfavourable hydrophilic-hydrophobic interactions with the lipid matrix. Since the other end of Y is attached to the shikimic acid-derived splitter (which is itself attached via the spacers to the GalNAc ligands), this can result in an optimal presentation and orientation of the GalNAc ligands for binding to ASGPRs.

[0319] In embodiments, Y is a hydrophilic linker comprising a linear chain of about 50 to about 400 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O. In embodiments, the hydrophilic linker comprises a linear chain of about 311 to about 371 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O, optionally wherein Y comprises PEG5000. In embodiments, the hydrophilic linker comprises a linear chain of about 165 to about 265 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O, optionally wherein Y comprises PEG3400. In embodiments, the hydrophilic linker comprises a linear chain of about 165 to about 225 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O, optionally wherein Y comprises PEG3000. In embodiments, the hydrophilic linker comprises a linear chain of about 105 to about 175 atoms, wherein the linear chain comprises carbon atoms and one or moreASG-105-PCT01-NP

[0320] heteroatoms independently selected from N and O, optionally wherein Y comprises PEG2000.

[0321] In embodiments, the hydrophilic linker comprises a linear chain of about 341 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O. In embodiments, the hydrophilic linker comprises a linear chain of about 220 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O. In embodiments, the hydrophilic linker comprises a linear chain of about 130 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O. In embodiments, the hydrophilic linker comprises a linear chain of about 150 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O.

[0322] In embodiments, Y comprises a linear polymer of polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of between 24 and 132 polyethylene glycol moieties, e.g. between 50 and 130, between 75 and 128, between 100 and 126, or between 104 and 124 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of between 104 and 124 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 114 polyethylene glycol moieties. In embodiments, Y comprises PEG5000.

[0323] In embodiments, Y comprises a linear polymer of between 24 and 86 polyethylene glycol moieties, e.g. between 51 and 84, between 52 and 78, or between 55 and 75 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of between 51 and 84 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 67 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 68 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 69 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 70 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 71 polyethylene glycol moieties. In embodiments, Y comprises PEG3400.

[0324] In embodiments, Y comprises a linear polymer of between 26 and 60 polyethylene glycol moieties, e.g. between 32 and 53, or between 35 and 55 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of between 38 and 53 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 44 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 45 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 46 polyethylene glycolASG-105-PCT01-NP

[0325] moieties. In embodiments, Y comprises a linear polymer of about 47 polyethylene glycol moi eties. In embodiments, Y comprises a linear polymer of between 24 and 86 polyethylene glycol moieties, e.g. between 26 and 78, between 28 and 70, between 30 and 62, between 32 and 54, or between 35 and 45 polyethylene glycol moieties. In other embodiments, Y comprises a linear polymer of between 32 and 51 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 37 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 38 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 39 polyethylene glycol moieties. In embodiments, Y comprises a linear polymer of about 40 polyethylene glycol moieties.

[0326] Where a set number of polyethylene glycol moieties is referenced herein, it will be appreciated that this may be, for example, the median number of PEG moieties within the linker.

[0327] In embodiments, Y comprises an amide, a carbamate, and / or a linear polymer of polyethylene glycol moieties. In embodiments, Y has the structure:

[0328]

[0329] "

[0330] in which * denotes the point of attachment to Z, wherein:

[0331] n and p are both integers independently selected from 1 to 31;

[0332] q is an integer selected from 10 to 110;

[0333] R’ is selected from -H and -(Ci-Ce)alkyl; and

[0334] R” is selected from -H and -(Ci-Ce)alkyl.

[0335] In embodiments, n is an integer selected from 1 to 30, e.g. from 1 to 28, from 1 to 26, from 1 to 24, from 1 to 22, from 1 to 20, from 1 to 18, from 1 to 16, from 1 to 14, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 7, from 2 to 6, or from 3 to 5. In embodiments, n is an integer selected from 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 3, 2, and 1. In embodiments, n is 4.

[0336] In embodiments, p is an integer selected from 1 to 30, from 1 to 28, from 1 to 26, from 1 to 24, from 1 to 22, from 1 to 20, from 1 to 18, from 1 to 16, from 1 to 14, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 4, or from 1 to 3. In embodiments, p is an integer selected from 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 3, 2, and 1. In embodiments, p is 1.ASG-105-PCT01-NP

[0337] In embodiments, q is an integer selected from 30 to 85, e.g. from 32 to 55 (such as from 32 to 51, or from 38 to 53), or from 51 to 84. In embodiments, q is an integer selected from 31 to 77, from 32 to 69, from 33 to 61, from 34 to 53, or from 35 to 45. In embodiments, q is an integer selected from 35 to 45. In embodiments, q is an integer selected from 32 to 82, from 33 to 78, from 34 to 74, from 35 to 70, from 36 to 66, from 37 to 62, from 38 to 58, from 39 to 54, or from 40 to 50. In embodiments, q is an integer selected from 40 to 50. In embodiments, q is an integer selected from 37 to 81, from 42 to 80, from 47 to 79, from 52 to 78, from 57 to 77, from 62 to 76, or from 65 to 75. In embodiments, q is an integer selected from 65 to 75.

[0338] In embodiments, R’ is selected from -H and -(Ci-C3)alkyl. In embodiments, R’ is -H. In embodiments, R’ is -CH3.

[0339] In embodiments, R” is selected from -H and -(Ci-C3)alkyl. In embodiments, R” is -H. In embodiments, R” is -CH3.

[0340] In embodiments, R’ is -H, and / or R” is -H. In embodiments, R’ and R” are both -H.

[0341] In embodiments, n is 4; p is 1; and q is an integer selected from 30 to 85 (e.g. from 35 to 45, from 40 to 50, or from 65 to 75).

[0342] In embodiments, Y has the structure:

[0343]

[0344] in which * denotes the point of attachment to Z, wherein q is an integer selected from 30 to 55 (e.g. 39, or 46) or from 50 to 85 (e.g., 68, or 70).

[0345] In other embodiments, Y has the structure:

[0346]

[0347] in which * denotes the point of attachment to Z, wherein:

[0348] n, p, and r are integers independently selected from 1 to 31;

[0349] q is an integer selected from 10 to 110;

[0350] R’ is selected from -H and -(Ci-Ce)alkyl;

[0351] R” is selected from -H and -(Ci-Ce)alkyl; andASG-105-PCT01-NP

[0352] R’” is selected from -H and -(Ci-C6)alkyl.

[0353] In embodiments, n is an integer as defined herein. In embodiments, n is 1. In embodiments, p is an integer as defined herein. In embodiments, p is 1. In embodiments, n and p are both 1.

[0354] In embodiments, r is an integer selected from 1 to 30, e.g. from 1 to 28, from 1 to 26, from 1 to 24, from 1 to 22, from 1 to 20, from 1 to 18, from 1 to 16, from 1 to 14, from 1 to 12, from 2 to 10, from 3 to 8, or from 4 to 6. In embodiments, r is an integer selected from 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, and 1. In embodiments, r is 5.

[0355] In embodiments, R’” is selected from -H and -(Ci-C3)alkyl. In embodiments, R’” is -H. In embodiments, R’” is -CH3. In embedments, R’ and R” are as defined herein. In embodiments, R’ is -H, and / or R” is -H, and / or R’” is -H. In embodiments, R’, R”, and R’” are all -H.

[0356] In embodiments, q is an integer as defined herein. In embodiments, q is an integer selected from 30 to 55, e.g. from 32 to 51 (such as 39), or from 38 to 53 (such as 46). In other embodiments, q is an integer selected from 51 to 84 (such as 68, or 70);

[0357] In embodiments, n is 1; p is 1; q is an integer selected from 30 to 55 (e.g. 39, or 46); and r is 5. In other embodiments, n is 1; p is 1; q is an integer selected from 51 to 84 (e.g. 68, or 70); and r is 5.

[0358] In embodiments, Y has the structure:

[0359]

[0360] in which * denotes the point of attachment to Z, wherein q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70).

[0361] The lipidic anchor

[0362] The lipidic anchor, Z, is attached to the rest of the molecule via the linker group, Y.

[0363] The function of the lipidic anchor, Z, is to anchor (i.e. embed or secure) the compound of Formula (I) in the lipid matrix of the lipid nanoparticle. The incorporation of a lipidic anchor in the structure of the compounds described herein is important because it enables the “labelling” or “functionalization” of nanoparticles of different compositions. The resulting lipid nanoparticles, which may comprise a nucleic acid cargo (e.g. a therapeutic RNA), canASG-105-PCT01-NP

[0364] target specific tissues or organs in a selective way. Processes are described herein that permit the “one pot” synthesis of lipid nanoparticles comprising compounds of Formula (I) as defined herein.

[0365] In embodiments, Z is a hydrophobic lipidic anchor selected from a sterol and a lipid (e.g. an ether lipid, an ester lipid, or a phospholipid). In embodiments, Z is a lipid. In embodiments, Z is an ether lipid.

[0366] In embodiments, Z is an ether lipid having the structure

[0367] 'x^^^ORa

[0368] ORa

[0369] wherein each Rais independently a -(Ce-C24)alkyl group, or a -(Ce-C24)alkenyl group. In 'x^xj^^ORa

[0370] embodiments, Z is an ether lipid having the structure ORa. In other embodiments, Z >0>^^ORa

[0371] is an ether lipid having the structureORa

[0372] In embodiments, each Rais independently a linear or a branched -(Ce-C24)alkyl group. In embodiments, each Rais independently a linear -(Ce-C24)alkyl group.

[0373] In embodiments, each Rais independently a -(Ce)alkyl, -(C?)alkyl, -(Cs)alkyl, -(C9)alkyl, -(Cio)alkyl, -(Cn)alkyl, -(Ci2)alkyl, -(Ci3)alkyl, -(Ci4)alkyl, -(Cis)alkyl, -(Cie)alkyl, -(Ci?)alkyl, -(Cis)alkyl, -(Ci9)alkyl, -(C2o)alkyl, -(C2i)alkyl, -(C22)alkyl, -(C23)alkyl, or -(C24)alkyl group. In embodiments, each Rais a -(Cs)alkyl group (i.e. an octadecyl group). In embodiments, each Rais a linear -(Cs)alkyl group (i.e. an n-octadecyl group). In embodiments, each Rais n-octadecyl.

[0374] In embodiments, each Rais the same.

[0375] In embodiments, Y and Z together have the structure:

[0376]

[0377] wherein q is an integer as defined herein, and each Rais n-octadecyl. In embodiments, q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70).ASG-105-PCT01-NP

[0378] In embodiments, Y and Z together have the structure:

[0379]

[0380] wherein q is an integer as defined herein, and each Rais n-octadecyl. In embodiments, q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70).

[0381] In embodiments, Y and Z together have the structure:

[0382]

[0383] In embodiments, Y and Z together have the structure:

[0384]

[0385] In embodiments, Y and Z together have the structure:

[0386]

[0387] In embodiments, Y and Z together have the structure:

[0388]

[0389] In embodiments, Y and Z together have the structure:

[0390]

[0391] In embodiments, Y and Z together have the structure:

[0392]

[0393] ASG-105-PCT01-NP

[0394] In embodiments, Y and Z together have the structure:

[0395]

[0396] In embodiments, Y and Z together have the structure:

[0397]

[0398] In embodiments, Y and Z together have the structure:

[0399]

[0400] In embodiments, the compound of Formula (I) is selected from:

[0401]

[0402] ASG-105-PCT01-NP

[0403]

[0404] ASG-105-PCT01-NP

[0405]

[0406] In embodiments, the compound of Formula (I) is selected from:

[0407]

[0408] ASG-105-PCT01-NP

[0409]

[0410] ASG-105-PCT01-NP

[0411]

[0412] and the pharmaceutically acceptable salts thereof.

[0413] Compounds

[0414] Disclosed herein are compounds of Formula (I) per se. One of ordinary skill in the art will appreciate that statements and embodiments relating to compounds of Formula (I) in the context of lipid nanoparticles (e.g. as set out above) will also apply to the compounds per se, and vice versa.

[0415] Thus, in one aspect, the disclosure provides a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof. In embodiments, the compound is a compound of formula (la), formula (lb), formula (Ic) or formula (Id), or a pharmaceutically acceptable salt thereof. In embodiments, the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof. In embodiments, the compound is a compound of formula (Ila), formula (lib), formula (lie) or formula (lid), or a pharmaceutically acceptable salt thereof.

[0416] In embodiments, the compound is a compound as illustrated in any one of the Examples described below, e.g. a compound obtainable by (or obtained by) a process as described inASG-105-PCT01-NP

[0417] Example 1. In embodiments, the compound is selected from Compounds 1 to 6, and the pharmaceutically acceptable salts thereof. Viewed from this aspect the disclosure provides a compound selected from:

[0418]

[0419] ASG-105-PCT01-NP

[0420]

[0421] ASG-105-PCT01-NP

[0422]

[0423] 10 and the pharmaceutically acceptable salts thereof.

[0424] In embodiments, the compound is selected from:

[0425]

[0426] ASG-105-PCT01-NP

[0427]

[0428] ASG-105-PCT01-NP

[0429]

[0430] Lipid matrix

[0431] A lipid nanoparticle of the present disclosure comprises a lipid matrix. The lipid matrix has several functions. For example, the lipid matrix acts as a “body” or “medium” for the other components of the lipid nanoparticle to associate with. The lipid matrix enables the encapsulation and / or adsorption of the nucleic acid cargo. Attractive intermolecular forces (e.g. Van der Waals’ or hydrophobic interactions) between the lipidic anchor, Z, and the lipid matrix, result in the compound of Formula (I) becoming secured or embedded within the lipid matrix. This advantageously enables the lipid nanoparticles of the disclosure to be targeted to specific tissues and / or cells in vivo. Typically, the lipid matrix provides major portion of the lipid nanoparticles of the disclosure, e.g. at least 50% by weight.

[0432] As described herein, the lipid nanoparticles of the disclosure typically comprise: (i) a lipid matrix; (ii) a nucleic acid cargo; and (iii) a compound of Formula (I).ASG-105-PCT01-NP

[0433] The lipid nanoparticles disclosed herein may comprise any components used in conventional nanoparticle technology, e.g., an ionizable lipid, a neutral lipid, a sterol, and / or a polymer -conjugated lipid. Advantageously, the physical properties of the lipid nanoparticles of the disclosure may be optimised by the incorporation of additional components. For example, changing the formulation of the lipid nanoparticles may result in e.g., improved bioavailability, cell permeability, safety, etc.

[0434] In embodiments, the lipid matrix comprises: (a) ionizable lipid; (b) neutral lipid; (c) sterol; and (d) polymer-conjugated lipid.

[0435] The core of the nanoparticle may comprise the ionizable lipid and a sterol, and one or more layers comprising neutral lipids and / or polymer-conjugated lipids may subsequently surround the core. For instance, in embodiments, the core of the lipid nanoparticle may comprise an ionizable lipid and a sterol (e.g., cholesterol) in any particular ratio, surrounded by a neutral lipid monolayer (e.g., DSPC) of any particular thickness, further surrounded by an outer polymer-conjugated lipid monolayer of any particular thickness. In such embodiments, the nucleic acid cargo may be incorporated into any one of the core or subsequent layers depending upon the nature of the intended target cells, and the characteristics of the nucleic acid cargo to be delivered. The core and outer layers may further comprise other components typically incorporated into lipid nanoparticles known in the art. Furthermore, it is understood by one skilled in the art that liposomes are delivery vehicles that possess a vesicular structure distinct from the lipid nanoparticles as disclosed herein. The liposome vesicles are composed of a lipid bilayer that forms in the shape of a hollow sphere encompassing an aqueous phase. For example, liposomes contain the lamellar phase while the lipid nanoparticles have non-lamellar structures.

[0436] In addition, the molar percent of the components of the lipid nanoparticle (e.g., the ionizable lipids, neutral lipids, sterols, and / or polymer-conjugated lipids) may be selected in order to provide a particular physical parameter of the overall lipid nanoparticle, such as the surface area of one or more of the lipids. For example, the molar percent of the ionizable lipids, neutral lipids, sterols, and / or polymer-conjugated lipids that comprise the lipid nanoparticles may be selected to yield a surface area per neutral lipid, for example, DSPC. By way of nonlimiting example, the molar percent of the ionizable lipids, neutral lipids, sterols, and / or polymer-conjugated lipids may be determined to yield a surface area per DSPC of about 1.0ASG-105-PCT01-NP

[0437] nm2to about 2.0 nm2, for example about 1.2 nm2. Exemplary mol% values for the various components of the lipid nanoparticle are given below.

[0438] Ionizable lipid

[0439] In embodiments, the lipid matrix comprises an ionizable lipid. The ionizable lipid can allow the formation of a stable lipid matrix that is capable of encapsulating a nucleic acid cargo. Such a lipid matrix permits the incorporation of other components, e.g. a GalNAc-containing compound as defined herein, which in turn allows the lipid nanoparticles to be used in the targeted delivery of cargos to cells such as liver cells.

[0440] In embodiments, the ionizable lipid is selected from any ionizable lipid, and combination thereof, described by International patent publication WO 2023 / 089522 Al, which is incorporated herein by reference in its entirety.

[0441] In embodiments, the ionizable lipid is selected from l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, bi s(3 -pentyl octyl) 9-((2-oxaspiro[3.3]heptan-6-yl)amino)heptadecanedioate, bis(3-pentyloctyl) 9-((tetrahydro-2H-pyran-4-yl)amino)heptadecanedioate, bis(3-pentyloctyl) 9-(((tetrahydrofuran-3-yl)methyl)amino)heptadecanedioate, bi s(3 -pentyloctyl) 9-(((tetrahydro-2H-pyran-4-yl)methyl)amino)heptadecanedioate, bis(3 -pentyloctyl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, and combinations thereof. In embodiments, the ionizable lipid is l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate.

[0442] Other non-limiting examples of ionizable lipids that may be combined with the compound of Formula (I), Formula (II), or any subgenus or species thereof in a lipid nanoparticle include, e.g., lipids containing a positive charge at the acidic scale of physiological pH range, such as l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, (see e.g., U.S. Patent No. 8,158,601), 2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see e.g., WO 2012 / 018754), Acuitas-5 (see e.g., WO 2015 / 199952), KL-10 (see e.g., U.S. Patent Application Publication 2012 / 0295832), C12-200 (see e.g., Love, KT et al., PNAS, 107: 1864 (2009)), and the like.

[0443] In embodiments, the lipid nanoparticle comprises from about 5 mol% to about 90 mol% of ionizable lipid, such as from about 10 mol% to about 80 mol%, for instance from about 25ASG-105-PCT01-NP

[0444] mol% to about 75 mol%, for example, from about 40 mol% to about 60 mol%, from about 40 mol% to about 50 mol%, such as about 45 mol% or about 50 mol%, of ionizable lipid, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo).

[0445] In embodiments, the lipid nanoparticle comprises from about 30 to about 75 mol% of ionizable lipid, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo). In embodiments, the lipid nanoparticle comprises about 50 mol% of ionizable lipid. In embodiments, the lipid nanoparticle comprises about 50 mol% of ionizable lipid, wherein the ionizable lipid is l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate.

[0446] Neutral lipid

[0447] In embodiments, the lipid matrix comprises a neutral lipid. In embodiments the lipid nanoparticles comprise a neutral lipid. The term “neutral lipid” includes lipids that have a zero-net charge at physiological pH, for example, lipids that exist in an uncharged form or neutral zwitterionic form at physiological pH.

[0448] In embodiments, the neutral lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and combinations thereof. In embodiments, the neutral lipid is DSPC. In embodiments, the neutral lipid is DOPE. In embodiments, the neutral lipid is DPPC. In embodiments, the neutral lipid is DMPC. -In embodiments, the lipid nanoparticle comprises from about 1 mol% to about 50 mol% of neutral lipid, such as from about 5 mol% to about 20 mol%, for example, 7.5 mol% to about 12.5 mol%, for instance, about 10 mol%, of neutral lipid, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo). In embodiments, the lipid nanoparticle comprises from about 5 to about 20 mol% of neutral lipid. In embodiments, the lipid nanoparticle comprises about 10 mol% of neutral lipid. In embodiments, the lipid nanoparticle comprises about 10 mol% of neutral lipid, wherein the neutral lipid is DSPC.

[0449] SterolASG-105-PCT01-NP

[0450] In embodiments, the lipid matrix comprises a sterol. The sterol can act to stabilize the lipid structure of the lipid nanoparticle.

[0451] In embodiments, the sterol is cholesterol or a derivative thereof. In embodiments, the cholesterol or derivative thereof is selected from 5 -heptadecylresorcinol, cholesterol hemi succinate, or combinations thereof. In embodiments, the cholesterol or a derivative thereof is cholesterol. In embodiments, the derivative of cholesterol is a polar cholesterol analogue. In embodiments, the polar cholesterol analogue is selected from 5a-cholestanol, 5P-coprostanol, cholesteryl(2'-hydroxy)-ethyl ether, cholesteryl-( 4'-hydroxy)-butyl ether, 6-ketocholestanol, or combinations thereof. In embodiments, the derivative of cholesterol is a non-polar cholesterol analogue. In embodiments, the non-polar cholesterol analogue is selected from 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, cholesteryl decanoate, or combinations thereof.

[0452] In embodiments, the lipid nanoparticle comprises from 0 to about 60 mol% of sterol, e.g. an amount ranging from about 10 to about 55 mol%, about 20 to about 50 mol%, about 30 to about 45 mol%, about 36 to about 41 mol%, or about 38 to about 39 mol%, of sterol, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo). In embodiments, the lipid nanoparticle comprises from about 20 to about 60 mol% of sterol. In embodiments, the lipid nanoparticle comprises about 38.5 mol% of sterol. In embodiments, the lipid nanoparticle comprises about 38.5 mol% of sterol, wherein the sterol is cholesterol or a derivative thereof. In embodiments, the lipid nanoparticle comprises about 38.5 mol% of sterol, wherein the sterol is cholesterol.

[0453] Polymer-conjugated lipid

[0454] In embodiments, the lipid matrix comprises a polymer-conjugated lipid. The polymer-conjugated lipid can act to give the nanoparticle a hydrating layer and improve colloidal stability.

[0455] In embodiments, the polymer-conjugated lipid is selected from DMPE-PEG2000, DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-C-DOMG, PEG-C-DOPG and combinations thereof. In embodiments, the polymer-conjugated lipid is DMG-PEG2000. In embodiments, the PEG moiety is an optionally substituted linear or branched polymer ofASG-105-PCT01-NP

[0456] ethylene glycol or ethylene oxide. In embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups.

[0457] A wide range of molecular weights of PEG moi eties may be used in the polymer-conjugated lipids of the disclosure. The PEG moi eties may range from about 500 to about 10,000 Da, or from about 1,000 to about 5,000 Da.

[0458] In embodiments, the lipid nanoparticle comprises from about 0 to about 10 mol% of polymer-conjugated lipid, e.g. an amount ranging from about 0 to about 9 mol%, about 0 to about 8 mol%, about 0 to about 7 mol%, about 0 to about 6 mol%, about 0 to about 5 mol%, about 0 to about 4 mol%, about 0 to about 3 mol%, about 0 to about 2 mol%, about 0.5% to about 1.5 mol%, about 1% to about 1.49 mol%, about 1.35% to about 1.475 mol%, or about 1.45 mol% of polymer-conjugated lipid, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo). In embodiments, the lipid nanoparticle comprises up to about 1.5 mol% of polymer-conjugated lipid, e.g. up to about 1.49, 1.48, 1.45, 1.43, 1.40, 1.35, 1.30, 1.25, 1.20, 1.10, 1.00, 0.75, or 0.50 mol%, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo).

[0459] In embodiments, the lipid nanoparticle comprises from about 0 to about 5 mol% of polymer-conjugated lipid, wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo). In embodiments, the lipid nanoparticle comprises up to about 1.5 mol% of polymer-conjugated lipid, e.g. about (1.5-x) mol% of polymer-conjugated lipid, wherein x is less than or equal to about 1.0. In embodiments, the lipid nanoparticle comprises up to about 1.5 mol% of polymer-conjugated lipid, e.g. about (1.5-x) mol% of polymer-conjugated lipid, wherein x is less than or equal to about 1.0, and wherein the polymer-conjugated lipid is DMG-PEG2000.

[0460] Compounds of Formula (I) anchored in the lipid matrix

[0461] In embodiments, the lipid nanoparticle comprises about x mol% of a compound of Formula (I) as defined herein, wherein x is less than or equal to about 1.0, and wherein mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipidASG-105-PCT01-NP

[0462] nanoparticle. In embodiments, x is about 0.05, about 0.08, about 0.10, about 0.13, about 0.15, about 0.20, about 0.30, about 0.40, or about 0.50.

[0463] The lipid nanoparticles of the disclosure can be effectively targeted when they comprise compounds of Formula (I) in small amounts (i.e. having a low load of the compound). For example, it was surprisingly found that a loading of 0.025 mol% of GalNAc-containing compound resulted in greater targeting than a higher loading of 0.5 or 1.0 mol% (see, e.g., Example 3 and Figure 7). This particularly advantageous because it reduces cost of goods, simplifies the processes described herein, and results in decreased wastage. In embodiments, x is between about 0.01 and about 0.5, e.g. between about 0.025 and about 0.15, optionally wherein x is about 0.05.

[0464] In embodiments, the lipid matrix comprises l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000.

[0465] In embodiments, the lipid nanoparticle comprises:

[0466] a) from about 30 to about 75 mol% of ionizable lipid;

[0467] b) from about 5 to about 20 mol% of neutral lipid;

[0468] c) from about 20 to about 60 mol% of sterol;

[0469] d) from 0 to about 5 mol% of polymer-conjugated lipid; and

[0470] e) up to about 5 mol% of a compound of Formula (I) as defined herein, wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo).

[0471] In embodiments, the lipid nanoparticle comprises:

[0472] a) about 50 mol% of l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate;

[0473] b) about 10 mol% of distearoyl phosphatidylcholine (DSPC);

[0474] c) about 38.5 mol% of cholesterol;

[0475] d) about (1.5 - x) mol% of DMG-PEG2000; and

[0476] e) about x mol% of a compound of Formula (I) as defined herein, wherein x is less than or equal to about 1.0, and wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle.

[0477] PropertiesASG-105-PCT01-NP

[0478] As defined herein, the lipid nanoparticles of the disclosure are particularly suitable for delivering nucleic acid cargos to cells and tissues. Advantageously, the properties of the lipid nanoparticles may be varied or optimised for a specific application. Some illustrative examples of the properties of the lipid nanoparticles that may be varied are provided below.

[0479] a) Z-average particle diameter

[0480] The size (z-average particle diameter) of the lipid nanoparticles of the disclosure is an important physical property that influences other important properties. Non-limiting examples of such properties include nucleic acid cargo encapsulation, biological half life (it is generally accepted that nanoparticles <10 nm are readily filtered from the blood by the kidneys and excreted), and suitability for parenteral administration (it is generally accepted that nanoparticles for parenteral administration are optimally <100 nm).

[0481] The particle size distribution of lipid nanoparticles of the present disclosure can be controlled using processes such as extrusion, sonication, homogenization, and microfluidic methods.

[0482] In embodiments, the lipid nanoparticles have a z-average particle diameter (<d>z) of about 200 nm or less, for example, less than or equal to about 100 nm, or, for instance, less than or equal to about 75 nm. In embodiments, the lipid nanoparticles have a z-average particle diameter ranging from about 40 nm to about 90 nm, e.g. from about 50 nm to about 80 nm, from about 60 nm to about 70, such as about 64 nm to about 70 nm.

[0483] b) Polydispersity

[0484] Advantageously, the lipid nanoparticle of the disclosure have low poly dispersity parameters. A low poly dispersity means that the lipid nanoparticles are highly uniform in size. The low poly dispersity is advantageous because each lipid nanoparticle comprising a nucleic acid cargo agent will have similar properties. In embodiments, the poly dispersity of the nanoparticle composition is less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1, such as between about 0.03 and about 0.08. In embodiments, the poly dispersity of the nanoparticle composition is less than about 0.1, such as between about 0.03 and about 0.08.

[0485] c) Encapsulation efficiency (EE)

[0486] The spherical shape of the lipid nanoparticles of the disclosure contributes towards many of their advantageous properties. Spherical lipid nanoparticles have many different applicationsASG-105-PCT01-NP

[0487] due to their high surface area to volume ratio. The spherical shape of the lipid nanoparticles of the disclosure is a significant advantage because a greater proportion of the nucleic acid cargo is encapsulated by the lipid nanoparticles. The lipid nanoparticles of the disclosure therefore have high encapsulation efficiencies. A high encapsulation efficiency is beneficial because this results in a stronger therapeutic effect with reduced side effects. In embodiments, the encapsulation efficiency of the lipid nanoparticle is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In embodiments, the encapsulation efficiency of the lipid nanoparticles is at least about 70%, e.g. at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In embodiments, the encapsulation efficiency of the lipid nanoparticles is at least about 90%, e.g. at least about 95% such as between about 95% to about 97%.

[0488] d) Weight ratios and molar ratios

[0489] The relative proportions of (a) a lipid matrix and a compound of Formula (I) to (b) a nucleic acid cargo, may be varied depending on the desired application. For the avoidance of doubt, the term “total lipid phase” is intended to mean the sum total of a lipid matrix and a compound of Formula (I) or any subgenus or species thereof. As will be appreciated, varying the weight ratio will result in an inherent change in the molar ratio of the compound of Formula (I) or any subgenus or species thereof, or a pharmaceutically acceptable salt thereof, to nucleic acid.

[0490] In embodiments, the weight ratio of total lipid phase to nucleic acid ranges from about 40: 1 to about 1 : 1, such as about 10:1. In yet another example, the weight ratio of total lipid phase to nucleic acid ranges from about 30:1 to about 1:1, such as about 22:1 to about 20:1.

[0491] However, the molar ratio of lipid phase and / or lipid phase components to the nucleic acid may be determined by the nature of the intended target cells and characteristics of nucleic acid and thus, are not limited in scope to the above-identified embodiments.

[0492] Nucleic acid cargo

[0493] As defined herein, the lipid nanoparticles of the disclosure are particularly suitable for delivering nucleic acid cargos to cells and tissues. The “cargo” groups of the lipid nanoparticles and pharmaceutical compositions thereof disclosed herein are typicallyASG-105-PCT01-NP

[0494] polyanionic nucleic acids, especially RNA or DNA polynucleotides. The lipid nanoparticles of the disclosure may also be used for delivering oligonucleotides or other anionic macromolecules.

[0495] The nucleic acid may be a functional nucleic acid, e.g. 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 cargo comprises a functional nucleic acid selected from the group consisting of mRNA, and combinations of RNA and DNA. In embodiments, the nucleic acid cargo comprises RNA, e.g. a mRNA such as a modified mRNA. In embodiments, the nucleic acid is mRNA.

[0496] The nucleic acids herein may be wild type or modified. In embodiments, the lipid nanoparticles may comprise a plurality of different nucleic acids. In embodiments, the nucleic acid (wild-type or modified), encodes a polypeptide of interest. A modified nucleic acid includes nucleic acid with chemical modifications to any part of the structure such that the nucleic acid is not naturally occurring. In embodiments, the nucleic acid is an RNA. In embodiments, the nucleic acid is an mRNA. In embodiments, the nucleic acid is a modified mRNA.

[0497] The nucleic acids may be of any length and can have any number of nucleotides such that they are effective for the intended purpose. In embodiments, the functional nucleic acid is an RNA vaccine, e.g. an mRNA vaccine. In other embodiments, the cargo comprises a CRISPR guide RNA such as a sgRNA (e.g. comprising a crRNA sequence fused to a tracrRNA sequence).

[0498] 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.

[0499] The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogous. The nucleic acid may further comprise aASG-105-PCT01-NP

[0500] 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.

[0501] 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.

[0502] In embodiments, the nucleic acid cargo is encapsulated by the lipid nanoparticle (e.g. it is encapsulated by the lipid matrix). In embodiments, the nucleic acid cargo is adsorbed onto the lipid nanoparticle (e.g. it is adsorbed onto the lipid matrix). In embodiments, the nucleic acid cargo is encapsulated by and / or adsorbed onto the lipid nanoparticle (e.g. it is encapsulated by and / or adsorbed onto the lipid matrix).

[0503] Populations of lipid nanoparticles and compounds

[0504] As will be appreciated by one of ordinary skill in the art, on a macroscopic scale, lipid nanoparticles can be considered as populations or ensembles of (almost inevitably different) discrete lipid nanoparticles as defined herein. The present disclosure thus provides a population of lipid nanoparticles, wherein the population comprises lipid nanoparticles of the present disclosure. The discrete lipid nanoparticles of the disclosure may differ, for example, in their size, poly dispersity, encapsulation efficiency, weight ratio and molar ratio, C,-potential, and / or the type and / or amount of nucleic acid cargo.

[0505] The nucleic acid may be comprised by some of the lipid nanoparticles in a sample, and absent from others. Populations of lipid nanoparticle of the disclosure are thus provided herein. In embodiments, the nucleic acid cargo is encapsulated by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of the lipid nanoparticle population. In embodiments, the nucleic acid cargo is adsorbed onto at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, orASG-105-PCT01-NP

[0506] at least 99.5% of the lipid nanoparticle population. In embodiments, the nucleic acid cargo is encapsulated by and / or adsorbed onto at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of the lipid nanoparticle population.

[0507] The composition of discrete lipid nanoparticles that together form the lipid nanoparticle populations of the disclosure may vary. For example, one lipid nanoparticle may comprise different numbers of particular compounds or nucleic acid cargos, or they may be present in different proportions. In embodiments, the lipid nanoparticle population comprises lipid nanoparticles that do not comprise a nucleic acid cargo.

[0508] Differences may exist between the chemical structures of discrete lipid nanoparticles due to the nature of the components used in the processes defined herein to prepare the lipid nanoparticles. For example, in embodiments in which Y comprises a linear polymer of polyethylene glycol moi eties, e.g. a linear polymer of between 24 and 86 polyethylene glycol moieties, the polyethylene glycol (PEG) source often comprises a distribution of different PEG chain lengths. Although it can be helpful to characterise a PEG chain length by an average (e.g., modal) value, if the PEG source comprises a range of different chain lengths, this will result in the compounds of Formula (I) defined herein possessing a range of PEG chain lengths. Thus, for example, a PEG having an average of 46 PEG units may have a distribution of lengths between 38 and 53 PEG units, and a group Y which incorporates this distribution will result in a family of compounds of Formula (I) which have Y groups of a different length. Lipid nanoparticles formed from such processes (including those defined herein) will comprise a distribution of said compounds of Formula (I). It will be appreciated that the range of values used to describe a particular distribution of chain lengths will not necessarily cover the PEGs at the very edges of the distribution. Thus, the range of values listed typically covers the majority of the distribution and the modal value. Such a distribution may be observed, e.g., in Figure 5.

[0509] For example, in embodiments in which Y comprises a PEG2000 moiety (e.g. as described in the following Examples) the use of a PEG reagent having a range of chain lengths may lead to a distribution of compounds of Formula (I) which contain between 32 and 51 PEG moieties or between 38 and 53 PEG moieties. In embodiments in which Y comprises a PEG3400 moiety (e.g. as described in the following Examples) the use of a PEG reagentASG-105-PCT01-NP

[0510] having a range of chain lengths may lead to a distribution of compounds of Formula (I) which contain between 51 and 84 PEG moieties.

[0511] Thus, in embodiments a lipid nanoparticle of the disclosure comprises a plurality of compounds of Formula (I). In embodiments, the population of lipid nanoparticles comprises a plurality of compounds of Formula (I).

[0512] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I) having the following structure:

[0513]

[0514] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 PEG moieties. In embodiments, the modal average number of PEG moieties is 39.

[0515] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I) having the following structure:ASG-105-PCT01-NP

[0516]

[0517] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 PEG moieties. In embodiments, the modal average number of PEG moieties is 70. In embodiments, the modal average number of PEG moieties is 68.

[0518] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I) having the following structure:

[0519]

[0520] ASG-105-PCT01-NP

[0521] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 PEG moieties. In embodiments, the modal average number of PEG moieties is 46.

[0522] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I)

[0523]

[0524] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 PEG moieties. In embodiments, the modal average number of PEG moieties is 39. In embodiments, the modal average number of PEG moieties is 46.

[0525] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I) having the following structure:

[0526]

[0527] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 PEG moieties. In embodiments, the modal average number of PEG moieties is 70. In embodiments, the modal average number of PEG moieties is 68.

[0528] In embodiments the lipid nanoparticle comprises a plurality of compounds of Formula (I) having the following structure:ASG-105-PCT01-NP

[0529]

[0530] or a pharmaceutically acceptable salt thereof. In embodiments, each Formula (I) molecule independently comprises 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 PEG moieties. In embodiments, the modal average number of PEG moieties is 70. In embodiments, the modal average number of PEG moieties is 68.

[0531] The use of PEG sources comprising a distribution of PEG chain lengths can yield advantages in terms of availability and cost. The compounds and lipid nanoparticles of the present disclosure can tolerate such variability.

[0532] Pharmaceutical compositions

[0533] In one aspect, the disclosure provides a pharmaceutical composition comprising a plurality of lipid nanoparticles described herein. In embodiments, the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising (i) a lipid matrix; (ii) a nucleic acid cargo; and (iii) a compound of Formula (I) or a pharmaceutically acceptable salt thereof as described herein, and at least one pharmaceutically acceptable carrier.

[0534] In embodiments, the disclosure provides a pharmaceutical composition comprising a plurality of lipid nanoparticles comprising (i) a lipid matrix; (ii) a nucleic acid cargo; and (iii) a compound of Formula (I) (e.g., Formula (la), Formula (lb), Formula (Ic), or Formula (Id)) or a pharmaceutically acceptable salt thereof. In embodiments, the disclosure provides a pharmaceutical composition comprising a plurality of lipid nanoparticles comprising (i) aASG-105-PCT01-NP

[0535] lipid matrix; (ii) a nucleic acid cargo; and (iii) a compound of Formula (II) (e.g., Formula (Ila), Formula (lib), Formula (lie), or Formula (lid)) or a pharmaceutically acceptable salt thereof.

[0536] In another aspect, the disclosure provides a pharmaceutical composition comprising a population of lipid nanoparticles as described herein and at least one pharmaceutically acceptable carrier.

[0537] Lipid nanoparticles comprising a nucleic acid cargo 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. The lipid nanoparticles may be locally delivered in vivo by direct injection or by use of an infusion pump.

[0538] The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0539] The pharmaceutical compositions may be in a form suitable for parenteral administration. The pharmaceutical compositions may be in a form suitable for intratracheal instillation, bronchial instillation, and / or inhalation. Pharmaceutical liquid compositions can be nebulized by use of inert gases. Nebulized suspensions may be breathed directly from the nebulizing device or the nebulizing device can be attached to face masks tent, or intermittent positive pressure breathing machine.

[0540] The amount of nucleic acid cargo that is combined with one or more pharmaceutically acceptable carriers to produce a single dosage form will necessarily vary depending upon the subject treated and the particular route of administration. For further information on routes of administration and dosage regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0541] Medical usesASG-105-PCT01-NP

[0542] Lipid nanoparticles of the present disclosure can deliver therapeutic nucleic acids in vivo, which gives them utility in the treatment of numerous disorders and conditions.

[0543] Viewed from this aspect, the disclosure provides a lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein) for use in therapy. In a related aspect is provided the use of a lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein) 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 lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein) to the subject. In another related aspect is provided the use of a lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein) in the manufacture of a medicament.

[0544] In particular, lipid nanoparticles 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 lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein) 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.

[0545] 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 using the therapeutic acid as the nucleic acid cargo to form a lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein).

[0546] 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 lipid nanoparticle of the disclosure (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same as described herein).ASG-105-PCT01-NP

[0547] Delivery of nucleic acids to cells

[0548] In a further aspect, the disclosure provides a method of delivering a nucleic acid cargo to a cell using a lipid nanoparticle according to the present disclosure, wherein the cell carries (e.g., expresses) a binding partner for a ligand of the lipid nanoparticle (e.g. the compound of Formula (I)). The method comprises the steps of contacting the cell with the lipid nanoparticle. 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.

[0549] A further aspect provides a method for increasing the uptake of a nucleic acid into an organ or tissue, wherein said organ or tissue expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising the use of a lipid nanoparticle (or a population of lipid nanoparticles, or a pharmaceutical composition comprising the same) as described herein.

[0550] A further aspect provides a method for improving the ability of a lipid nanoparticle to be targeted to an organ or tissue which expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising incorporating into the lipid nanoparticle a compound of Formula (I) described herein (e.g. by a process as described herein). In embodiments, the compound is a compound of Formula (II).

[0551] In embodiments, the binding partner is ASGPR (e.g., human ASGPR). 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).

[0552] In embodiments, the method comprises contacting a cell which carries (e.g., expresses) ASGPR (e.g., a hepatocyte cell) with a lipid nanoparticle which carries at least one GalNAc moiety, e.g. wherein the lipid nanoparticle comprises a nucleic acid cargo and a compound Formula (I) or Formula (II). In embodiments, the method comprises contacting a cell which carries (e.g., expresses) ASGPR (e.g., a hepatocyte cell) with a lipid nanoparticle which comprises a compound of Formula (I) or Formula (II).

[0553] Processes for making compounds and intermediates

[0554] The disclosure also provides processes for the preparation of compounds as described herein, as well as intermediates useful in the preparation of said compounds.ASG-105-PCT01-NP

[0555] Viewed from this aspect, the disclosure provides a process for the preparation of a compound of Formula (I) as defined herein

[0556]

[0557] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein,

[0558] wherein the process comprises reacting a compound of Formula A’

[0559] >

[0560]

[0561] wherein:

[0562] Y’ is a linker precursor comprising a linear chain of about 2 to about 100 atoms (e.g., selected from C, N, and O) and a first reactive group; and

[0563] Q is a hydroxyl protecting group or H,

[0564] with a compound of Formula B’

[0565] Y”-Z,

[0566] wherein:

[0567] Y” is a second linker precursor comprising a linear chain of about 48 to about 398 atoms (e.g., selected from C, N, and O) and a second reactive group,ASG-105-PCT01-NP

[0568] the process optionally further comprising a step of removing the hydroxyl protecting groups.

[0569] It will be appreciated that Y’ and Y’ ’ should be capable of reacting together (either directly or indirectly via their reactive groups) to form a group Y as defined herein. As such, the sum of (i) the atoms in the linear chain of Y’ which are retained in Y and (ii) the atoms in the linear chain of Y” which are retained in Y (i.e., (i) plus (ii)) should not exceed 400.

[0570] In embodiments, Y’ has the structure:

[0571]

[0572] wherein n is as defined herein.

[0573] In embodiments, Y’ has the structure:

[0574] "

[0575]

[0576] wherein n, r, and R’ ’ ’ are as defined herein.

[0577] In embodiments, Y’ has the structure:

[0578]

[0579] wherein n is as defined herein.

[0580] In embodiments, Y’ has the structure:

[0581]

[0582] wherein n, r, and R’ ’ ’ are as defined herein.

[0583] In embodiments, the process of reacting Compound A’ with Compound B’ comprises the use of coupling conditions such as amide coupling conditions. A range of coupling conditions will be known to a person of ordinary skill in the art, e.g. PyBOP / EbN / DMF or DCC / DMAP / DCM. The skilled reader will appreciate that amide coupling conditions which employ an activating agent such as, e.g., PyBOP or DCC, may be appropriate whenASG-105-PCT01-NP

[0584] Compound A’ is not activated, for example when it comprises a carboxylic acid. It will also be appreciated that in embodiments in which Compound A’ is activated (e.g., when it comprises a pentafluorophenyl ester), an activating agent may not be required. In these embodiments, Compound A’ may simply be contacted with Compound B’ in a solvent such as, e.g., DCM. Optionally, a base such as DIPEA or pyridine may be used.

[0585] In embodiments, the compound of Formula A’ has the structure of Formula A’-l:

[0586]

[0587] wherein A, m, and R are as described herein, and Q is a hydroxyl protecting group.

[0588] In embodiments, Q is acetate, i.e. -C(O)CH3. In embodiments, R is methyl.

[0589] In embodiments, the compound of Formula A’ has the structure of Formula A’-2:

[0590]

[0591] wherein A, m, and R are as described herein, and Q is a hydroxyl protecting group.

[0592] In embodiments, Q is acetate i.e. -C(O)CH3. In embodiments, R is H.ASG-105-PCT01-NP

[0593] In embodiments, the compound of Formula A’ has the structure of Formula A’-3:

[0594]

[0595] wherein A, m, R, and R’” are as described herein, and Q is a hydroxyl protecting group. In embodiments, Q is acetate i.e. -C(O)CH3. In embodiments, R and / or R’” are H.

[0596] In embodiments, the compound of Formula A’ has the structure of Formula A’-4:

[0597]

[0598] wherein A, m, R, and R’” are as described herein, and Q is a hydroxyl protecting group. In embodiments, Q is acetate i.e. -C(O)CH3. In embodiments, R and / or R’” are H.

[0599] In embodiments, the compound of Formula B’ is a compound of Formula B’-l:

[0600]

[0601] ASG-105-PCT01-NP

[0602] wherein p, q, R’, R”, and Z are as defined herein.

[0603] In embodiments, the process for the preparation of a compound of Formula (I) as defined herein comprises a deprotection step. In embodiments, the deprotection step comprises a step of deacetylation using a base such as NaOMe. In embodiments, the deprotection step comprises the deprotection of a compound of Formula C’

[0604]

[0605] wherein X, Y, Z, and spacer are as defined herein.

[0606] In embodiments, the compound of Formula C’ is a compound of Formula C’-l:

[0607]

[0608] wherein A, m, q, R, and Raare as defined herein. In embodiments, R is methyl.

[0609] In embodiments, the compound of Formula C’ is a compound of Formula C’-2:ASG-105-PCT01-NP

[0610]

[0611] wherein A, m, q, R, and Raare as defined herein. In embodiments, R is H.

[0612] In embodiments, the compound of Formula C’ is a compound of Formula C’-3:

[0613]

[0614] wherein A, m, q, R, and Raare as defined herein. In embodiments, R is H.

[0615] The present disclosure is also directed to intermediates useful in the preparation of the compounds of the disclosure. The skilled reader will appreciate that the processes described herein in the context of preparing compounds of the disclosure can be used to provide such intermediates.

[0616] Viewed from this aspect, the disclosure provides a compound of Formula A’ASG-105-PCT01-NP

[0617]

[0618] wherein =, X, Y’, Q and spacer are as defined herein.

[0619] In embodiments, the compound of Formula A’ has the structure of Formula A’-l:

[0620]

[0621] wherein A, m, Q, and R are as described herein.

[0622] In embodiments, the compound of Formula A’ has the structure of Formula A’-2:ASG-105-PCT01-NP

[0623]

[0624] wherein A, m, Q, and R are as described herein.

[0625] In embodiments, the compound of Formula A’ has the structure of Formula A’-3:

[0626]

[0627] wherein A, m, Q, R, and R’” are as described herein.

[0628] In embodiments, the compound of Formula A’ has the structure of Formula A’-4:ASG-105-PCT01-NP

[0629]

[0630] wherein A, m, Q, R, and R’” are as described herein.

[0631] The disclosure also provides a compound of Formula B’ as defined herein.

[0632] In embodiments, the compound of Formula B’ is a compound of Formula B’-l:

[0633]

[0634] B'-1

[0635] wherein p, q, R’, R”, and Z are as defined herein.

[0636] The disclosure also provides a compound of Formula C’

[0637]

[0638] wherein X, Y, Z, and spacer are as defined herein.

[0639] In embodiments, the compound of Formula C’ is a compound of Formula C’-l:ASG-105-PCT01-NP

[0640]

[0641] wherein A, m, q, R, and Raare as defined herein.

[0642] In embodiments, the compound of Formula C’ is a compound of Formula C’-2:

[0643]

[0644] C-2

[0645] , wherein A, m, q, R, and Raare as defined herein.

[0646] In embodiments, the compound of Formula C’ is a compound of Formula C’-3:

[0647]

[0648] wherein A, m, q, R, and Raare as defined herein.ASG-105-PCT01-NP

[0649] Processes for preparing lipid nanoparticles

[0650] The disclosure also provides processes for the preparation of lipid nanoparticles as described herein. Such processes will typically result in the preparation of lipid nanoparticle compositions (e.g., as described herein) an ensemble or population of nanoparticles of the disclosure. Thus, references to the preparation of lipid nanoparticles herein also refer to the preparation of lipid nanoparticle compositions.

[0651] Viewed from this aspect, the disclosure provides a process for the preparation of a lipid nanoparticle comprising:

[0652] (i) a lipid matrix;

[0653] (ii) a nucleic acid cargo; and

[0654] (iii) a compound of Formula (I):

[0655]

[0656] or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined herein,

[0657] wherein the process comprises the steps of:

[0658] A) providing a first solution comprising the lipid matrix and the compound of Formula (I);

[0659] B) providing a second solution comprising the nucleic acid cargo in water or an aqueous buffer; and

[0660] C) combining the solution of (A) with the solution of (B) thereby to prepare the lipid nanoparticle.

[0661] In embodiments, the lipid nanoparticle is a lipid nanoparticle as defined herein and / or produced by a process as defined herein.ASG-105-PCT01-NP

[0662] In embodiments, the lipid matrix of step (A) is provided in an organic solvent. In embodiments, the organic solvent is a protic organic solvent such as EtOH (e.g. 99.5% EtOH).

[0663] In embodiments, the lipid matrix is as defined herein. For example, the lipid matrix may comprise:

[0664] (a) ionizable lipid;

[0665] (b) neutral lipid;

[0666] (c) sterol; and

[0667] (d) polymer-conjugated lipid.

[0668] In embodiments, the lipid matrix comprises (a) ionizable lipid; (b) neutral lipid; (c) sterol; and (d) polymer-conjugated lipid.

[0669] In embodiments, the first solution comprises ionizable lipid, neutral lipid, sterol, polymer-conjugated lipid, and compound of Formula (I) in a molar ratio of about 50:10:38.5:(1.5-x):x, wherein x is less than or equal to about 1.0. In embodiments, the ionisable lipid is 1-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, the neutral lipid is DSPC, the sterol is cholesterol, and the polymer-conjugated lipid is DMG-PEG2000.

[0670] In embodiments, the total concentration of lipids (i.e., lipid matrix and compound of Formula (I)) in the first solution is from about 0.1 mM to about 100 mM, e.g. from about 1 mM to about 50 mM, from about 5 mM to about 25 mM, from about 10 mM to about 15 mM, or from about 12 mM to about 13 mM. In embodiments, the total concentration of lipid matrix components in the first solution is about 12.5 mM.

[0671] In embodiments, the second solution is provided in water. In embodiments, the second solution is provided in an aqueous buffer. In embodiments, the aqueous buffer is a salt prepared from an organic acid or base. In embodiments, the aqueous buffer is a citrate buffer. In embodiments, the aqueous buffer is RNase-free. In embodiments, the aqueous buffer has a pH of about 2 to about 4, about 2.5 to about 3.5, e.g. about 3. In embodiments, the aqueous buffer is an RNase-free citrate buffer with a pH of 3.

[0672] In embodiments, the concentration of the nucleic acid cargo in the second solution is from about 0.01 mg / mL to about 10 mg / mL, e.g. from about 0.02 mg / mL to about 5 mg / mL, from about 0.04 mg / mL to about 1 mg / mL, from about 0.06 mg / mL to about 0.5 mg / mL, fromASG-105-PCT01-NP

[0673] about 0.08 mg / mL to about 0.25 mg / mL, from about 0.1 mg / mL to about 0.15 mg / mL, or from about 0.12 mg / mL to about 0.14 mg / mL, e.g. about 0.13 mg / mL. In embodiments, the concentration of the nucleic acid cargo in the second solution is about 0.13 mg / mL.

[0674] In embodiments, step (C) is performed by microfluidic mixing, such as rapid microfluidic mixing. In embodiments, step (C) is performed using a NanoAssemblr™ Ignite™. In embodiments, the solutions are mixed in step (C) at a flow rate ratio of from about 1 : 1 to about 1 : 10, from about 1 :2 to about 1:5, or from about 1 :2 to about 1 :4, such as about 1 :3. In embodiments, the flow rate in step (C) is about 50 mL / min, about 40 mL / min, about 30 mL / min, about 30 mL / min, about 20 mL / min, about 18 mL / min, about 16 mL / min, about 14 mL / min, about 12 mL / min, about 10 mL / min, about 8 mL / min, about 6 mL / min, about 4 mL / min, about 2 mL / min, or about 1 mL / min. In embodiments, the flow rate in step (C) is about 12 mL / min.

[0675] In embodiments, the process comprises an additional step (D), wherein step (D) comprises dialysis, e.g. dialysis overnight in a pH 7.4 phosphate buffered saline.

[0676] Having been generally described herein, the following non-limiting examples are provided to further illustrate this disclosure.

[0677] EXAMPLES

[0678] General synthetic schemes

[0679] The following scheme, Scheme 1, illustrates an exemplary way of preparing compounds in accordance with the present disclosure and examples:ASG-105-PCT01-NP

[0680]

[0681] SCHEME 1

[0682] According to Scheme 1 (in which A, q, R, Ra, and Q may be e.g. as described above, and m may be 2), Compound B can be obtained in STEP 1 by the nucleophilic substitution of Compound A by an amine, RNH2. Compound B can react with shikimic acid in STEP 2 to form Compound C, using BOP and DMAP. Compound C can then be reacted with an acrylate (e.g. tert-butyl acrylate) in STEP 3 in the presence of a base such as CS2CO3, to form Compound D. Compound D may then be reduced in STEP 4 to form Compound E under hydrogenation conditions. Compound E may then be reacted with TFA in STEP 5 to form Compound F. Separately, Compound G may be deprotected under hydrogenation conditions in STEP 6 to form Compound H. Compound H may then be reacted with Compound F under amide coupling conditions (e.g. using PyBOP, DIPEA and DMAP) in STEP 7 to form Compound J. Compound J may then be oxidised under Jones’ conditions in STEP 8 to form Compound K. Separately, Compound L (a derivative of the lipidic anchor, Z), may be reactedASG-105-PCT01-NP

[0683] with a chloroformate (e.g. 4-nitrophenyl carb onochlori date) under basic conditions (e.g.

[0684] EtsN) in STEP 9 to form Compound M. Compound M may then be reacted with Compound N using DMAP in STEP 10 to form Compound O. Compound O may be deprotected to form Compound P using TIPS and TFA in STEP 11. Compound P and Compound K may then be coupled in an amide coupling reaction using e.g. PyBOP and EtsN in STEP 12 to form Compound Q. Compound Q may then be deprotected (deacetylated) using a base such as NaOMe to form Compound R in STEP 13.

[0685] As will be appreciated by a person of ordinary skill in the art, various steps in the above scheme may be achieved using alternative reagents and conditions. For example, STEP 10 may alternatively be performed using conditions of pyridine, THF, and RT. STEP 11 may alternatively be performed using TFA, DCM, and RT. STEP 12 may alternatively be performed using DCC, DMAP, DCM, and RT, etc.

[0686] One of ordinary skill in the art will appreciate that compounds of the disclosure may be prepared comprising a carbon-carbon double bond in the shikimic acid portion by following a similar route to the synthesis shown in Scheme 1 but omitting a reduction step (STEP 4). An illustrative example is shown in Scheme 2.

[0687]

[0688] SCHEME 2

[0689] According to Scheme 2 (in which A, q, R, and Ramay be e.g. as described above, and m mayASG-105-PCT01-NP

[0690] be 2), Compound T can be obtained in STEP 1 by coupling of Compound S with shikimic acid using EDC, HOBt, and DIEA. Compound T can react with an acrylate (e.g. tert-butyl acrylate) in the presence of a base such as CS2CO3 in STEP 2, to form Compound U.

[0691] Compound U may then be deprotected using TBAF in STEP 3 to form Compound V.

[0692] Compound V may then be reacted with TFA in STEP 4 to form Compound W. Compound W may be reacted with an activated ester to form Compound X using DIEA in STEP 5.

[0693] Compound X may then be coupled with Compound H using DIEA in STEP 6. STEPs 7-9 may then be performed as described in STEP 8, alternative STEP 12, and STEP 13, of Scheme 1, to afford Compound Y as shown above.

[0694] As a person of ordinary skill in the art will appreciate from the disclosure, a variety of different groups, Y and Z (e.g. as depicted in Formula (I)), may be incorporated into the structures of the compounds described herein. Scheme 3 illustrates the incorporation of an alternative linker group Y.ASG-105-PCT01-NP

[0695]

[0696] SCHEME 3

[0697] According to Scheme 3 (in which A, q, Ra, and Q may be e.g. as described above, and m may be 2), Compound Z can be obtained in STEP 1 by coupling of a Cbz-protected amine with shikimic acid using EDC, HOBt, and DIEA. Compound Z can react with an acrylate (e.g. tert-butyl acrylate) in the presence of a base such as CS2CO3 in STEP 2, to form Compound AA. Compound AA may then be deprotected using TFA in STEP 3 to form Compound AB. Compound AB may be coupled with Compound H in STEP 6 (as in STEP 7 in Scheme 1) to form Compound AC. Compound AC may then be reacted with TMSI to form Compound AD in STEP 5. Compound AD may then be reacted with dihydrofuran-2, 5-dione in the presence of DMAP and TEA, in STEP 6, to form Compound AE. Compound AE may then be reacted with reacted with pentafluorophenyl trifluoroacetate in STEP 7 to form Compound AF. Two example routes to Compound AH are then illustrated. In the first approach, Compound AE is reacted with Compound P in STEP 8 (as in STEP 10 in Scheme 1) to form Compound AG.ASG-105-PCT01-NP

[0698] Compound AG can also be formed in STEP 8 from Compound AF in the presence of Compound P. Compound AF may then be deprotected using a base such as NaOMe to form Compound AG in STEP 8.

[0699] Example 1 - Synthesis of GalNAc-containing compounds

[0700] Compound 1

[0701]

[0702] Compound 1 was prepared according to the method illustrated in the scheme above.

[0703] Synthesis of 1-2: A solution of (((6-bromohexyl)oxy)methyl)benzene (1-1) (100 g, 184.37 mmol) in 2M MeNFE in ethanol (3000 mL) was stirred at RT for 70 hours. The solvent was removed under reduced pressure to afford product 2-2 (70.0 g, 85 %) as a white solid. The product was used in the next step directly without further purification. 1H-NMR (500 MHz, DMSO, 24°C) 5 1.32 (dp, J = 11.1, 7.0, 5.6 Hz, 4H), 1.55 (dq, J = 13.3, 7.0, 6.5 Hz, 4H), 2.54 (s, 3H), 2.77-2.92 (m, 2H), 3.42 (t, J = 6.5 Hz, 2H), 4.45 (s, 2H), 7.18-7.43 (m, 5H), 8.38 (s, 1H). LCMS Expected 221.3, Observed 222.2 ([M+H]+).ASG-105-PCT01-NP

[0704] Synthesis of 1-3: 1-2 (35 g, 158.12 mmol) was added to a solution of N-ethyl-N-isopropylpropan-2-amine (83 mL, 474.37 mmol), shikimic acid (30.3 g, 173.94 mmol), BOP (105 g, 237.19 mmol) and DMAP (19.32 g, 158.12 mmol) in DMF (700 mL). The resulting mixture was stirred at RT for 1 hour. The reaction mixture was poured into water (1 L), extracted with EtOAc (2 x 1 L), the organic layer was washed with saturated brine (2 x 1 L), the organic layer was dried over Na2SO4, filtered and evaporated to afford brown gum. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% MeOH in DCM. Pure fractions were evaporated to dryness to afford product 1-3 (50.0 g, 84 %) as a yellow oil. 1H-NMR (500 MHz, DMSO, 26°C) 5 1.21-1.25 (m, 2H), 1.33 (s, 2H), 1.41-1.56 (m, 4H), 1.88-1.97 (m, 1H), 2.33-2.42 (m, 1H), 2.81 (s, 2H), 2.92 (s, 1H), 3.25 (d, J = 26.1 Hz, 3H), 3.41 (t, J = 6.5 Hz, 2H), 3.50 (dd, J = 7.1, 4.0 Hz, 1H), 3.83 (dt, J = 7.1, 5.1 Hz, 1H), 4.13 (s, 1H), 4.45 (s, 2H), 5.48 (t, J = 2.2 Hz, 1H), 6.73-6.74 (m, 1H), 7.25-7.38 (m, 5H), 8.15 (d, J = 5.2 Hz, 1H). LCMS Expected 377.2, Observed 378.2 ([M+H]+).

[0705] Synthesis of 1-4: CS2CO3 (85 g, 262.26 mmol) was added to 1-3 (30 g, 79.47 mmol) and tertbutyl acrylate (698 mL, 4768.45 mmol) in t-BuOH (1500 mL). The resulting mixture was stirred by mechanical stirring at RT for 3 days. The reaction mixture was diluted with EtOAc (3000 ml). The solutions was filtered through celite. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 45% EtOAc in petroleum ether. Fractions containing product were evaporated to dryness. The unpure product was further purified by flash silica (330g silicone column) chromatography, elution gradient 0 to 35.5% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford product 1-4 (31.5 g, 52.0 %) as a colorless oil. 1H-NMR (300 MHz, DMSO, 22°C) 5 1.39 (d, J = 1.8 Hz, 34H), 1.99 (s, 2H), 2.40 (dq, J = 7.4, 4.5, 3.9 Hz, 7H), 2.83 (d, J = 24.1 Hz, 3H), 3.25 (s, 2H), 3.41 (t, J = 6.4 Hz, 2H), 3.58-3.83 (m, 8H), 3.97-4.06 (m, 1H), 4.44 (s, 2H), 5.51 (s, 1H), 7.21-7.41 (m, 5H). LCMS Expected 761.4, Observed 762.6 ([M+H]+).

[0706] Synthesis of 1-5: Pd / C (10%, 50 w / w% water) (2.79 g, 2.62 mmol) was added to 1-4 in MeOH (200 mL), the mixture was stirred under an atmosphere of hydrogen (20 atm) at 50 °C for 18 hours. The reaction mixture was filtered through the Buchner funnel. The solvent was removed under reduced pressure, the resulting oil was dissolved in acetonitrile then concentrated to dryness to afford product 1-5 (8.50 g, 96 %) as a colourless oil. The product was used in the next step directly without further purification. (Compound 1-5 was isolated as a single diastereoisomer) 1H-NMR (300 MHz, DMSO, 23°C) 5 1.09-1.29 (m, 4H), 1.34-1.45 (m,ASG-105-PCT01-NP

[0707] 27H), 1.56 (t, J= 11.5 Hz, 7H), 2.21-2.47 (m, 6H), 2.75 (d, J = 9.4 Hz, 2H), 2.95 (s, 2H), 3.25 (q, J = 8.3, 7.2 Hz, 2H), 3.33-3.51 (m, 4H), 3.65 (dt, J = 16.2, 9.7 Hz, 6H), 3.79 (dt, J = 10.9, 5.6 Hz, 2H), 4.33 (td, J = 5.2, 2.2 Hz, 1H). LCMS Expected 673.4, Observed 674.4 ([M+H]+).

[0708] Synthesis of 1-6: TFA (22.87 mL, 296.79 mmol) was added to 2-5 (2.0 g, 2.97 mmol) in DCM (20 ml) at RT. The resulting mixture was stirred at RT for 3 hours. The solvent was removed under reduced pressure to afford product 1-6 (1.5 g, 84 %) as a colourless oil. The product was used in the next step directly without further purification. 1H-NMR (300 MHz, DMSO, 25 °C) 5 1.29-1.39 (m, 4H), 1.54 (d, J = 8.8 Hz, 4H), 1.68 (p, J = 6.6 Hz, 4H), 2.43 (d, J = 6.3 Hz, 6H), 2.95 (s, 3H), 3.23 (t, J = 7.4 Hz, 3H), 3.63 (dqt, J = 15.5, 9.6, 4.9 Hz, 11H). LCMS Expected 601.2, Observed 602.3 ([M+H]+).

[0709] Synthesis of 1-8: Pd-C (10%, 50 w / w% water) (0.641 g, 0.60 mmol) was added to (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-(((benzyloxy)carbonyl)amino) hexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (1-7) (5 g, 8.61 mmol) in EtOH (100 mL) at RT. The resulting solution was stirred under an atmosphere of hydrogen at RT for 16 hours. The reaction mixture was filtered through celite to afford crude product, washed with MeOH (3 x 150 ml). The solvent was removed under reduced pressure to afford product 1-8 (3.6 g, 94 %) as a colourless oil, which was used without further purification. 1H-NMR (300 MHz, DMSO, 24°C) 5 1.29 (d, J = 16.4 Hz, 6H), 1.46 (s, 2H), 1.78 (d, J = 1.2 Hz, 3H), 1.90 (s, 3H), 2.00 (s, 3H), 2.09 (d, J = 9.1 Hz, 8H), 3.34-3.49 (m, 1H), 3.70 (dt, J = 9.7, 6.1 Hz, 1H), 3.78-3.93 (m, 1H), 4.03 (s, 2H), 4.49 (d, J = 8.5 Hz, 1H), 4.97 (dd, J = 11.2, 3.4 Hz, 1H), 5.22 (d, J = 3.4 Hz, 1H), 7.83 (d, J = 9.2 Hz, 1H). LCMS Expected 446.2, Observed 447.2 ([M+H]+).

[0710] Synthesis of 1-9: 1-6 (0.5 g, 0.83 mmol) was added to a solution of 1-8 (1.299 g, 2.91 mmol), N-ethyl-N-isopropylpropan-2-amine (1.452 mL, 8.31 mmol), PyBOP (1.838 g, 4.16 mmol), and DMAP (0.305 g, 2.49 mmol) in DMF (25 mL). The resulting mixture was stirred at RT for 16 hours. The solvent was removed under reduced pressure to afford the crude product. The product was purified by flash C18-flash chromatography, elution gradient 0 to 38% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford a yellow oil. The oil was dissolved in acetonitrile and water then dried by lyophilization to afford product 1-9 (1.0 g, 67.2 %) as a yellow solid. 1H-NMR (300 MHz, DMSO, 25 °C) 5 1.11-1.31 (m, 17H), 1.32-1.58 (m, 21H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 9H), 2.11 (s, 8H), 2.17-2.38 (m, 6H), 2.75 (d, J = 10.8 Hz, 3H), 2.97 (d, J = 9.9 Hz, 2H), 3-3.13 (m, 5H), 3.22 (d, J = 10.4 Hz, 3H), 3.35-3.42 (m, 5H), 3.5-3.78 (m, 12H), 3.87 (dt, J = 11.2, 8.8 Hz, 3H), 4.03 (s, 8H), 4.35 (dt, J =ASG-105-PCT01-NP

[0711] 15.4, 5.3 Hz, 1H), 4.49 (d, J = 8.4 Hz, 3H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 5.22 (d, J = 3.4 Hz, 3H), 7.45-8.24 (m, 6H). LCMS Expected 1789.9, Observed 1790.7 ([M+H]+).

[0712] Synthesis of 1-10: Chromosulfuric acid (1.228 mL, 2.46 mmol) was added to 1-9 (2.2 g, 1.23 mmol) in acetone (25 mL) at 0°C. The resulting mixture was stirred at RT for 16 hours. The reaction was quenched with i-PrOH (50 ml), the solvent was removed under reduced pressure. The crude product was diluted by DCM (100 ml), washed sequentially with water (100 mL), saturated NaHCO3 (2 x 100 mL), and saturated brine (100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 40% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford a colourless oil, which was dried by lyophilization to afford product 1-10 (2.3 g, 82 %) as a white solid. 1H-NMR (500 MHz, DMSO, 25 °C) 5 1.17-1.31 (m, 15H), 1.32-1.35 (m, 7H), 1.37-1.41 (m, 7H), 1.49-1.67 (m, 7H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 8H), 2.11 (s, 8H), 2.20 (dt, J= 15.0, 7.5 Hz, 2H), 2.25-2.34 (m, 5H), 2.66-2.84 (m, 3H), 2.95 (s, 2H), 2.98-3.14 (m, 6H), 3.17-3.33 (m, 3H), 3.33-3.46 (m, 4H), 3.5-3.59 (m, 5H), 3.6-3.79 (m, 7H), 3.87 (dt, J = 11.5, 8.9 Hz, 3H), 4.03 (q, J = 4.0 Hz, 9H), 4.49 (dd, J = 8.4, 1.4 Hz, 3H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 5.22 (d, J = 3.4 Hz, 3H), 7.6-8.04 (m, 6H). LCMS Expected 1803.8, Observed 903.4 (z = 2 ([M+H]+).

[0713] Synthesis of 1-12: (S)-2,3-bis(octadecyloxy)propan-l-ol (1-11) (1.906 g, 3.19 mmol) and 4-nitrophenyl carb onochlori date (0.965 g, 4.79 mmol) was added to a dry flask under Argon. Anhydrous DCM (20.39 ml) was added and the resulting mixture was sonicated to dissolve the solids. Cooled to 0°C, then triethylamine (0.890 ml, 6.38 mmol) was added dropwise. The reaction was stirred at RT for 23 h, then quenched with NH4CI and extracted with EtOAc. Dried over MgSO4, filtered, and concentrated to a white solid. Purified on a silica column (50 g) with a gradient 0-10% EtOAc in Heptane, Product 1-12 (1.8 g, 74 %) was isolated as a clear oil that foamed to a white solid under vacuum. 1H-NMR (500 MHz, CDC13, 25°C) 5 0.88 (t, J = 6.9 Hz, 6H), 1.25 (d, J = 3.9 Hz, 60H), 1.57 (q, J = 7.1 Hz, 4H), 3.43-3.62 (m, 6H), 3.72 (t, J = 4.8 Hz, 1H), 4.33 (dd, J= 11.3, 6.0 Hz, 1H), 4.45 (dd, J= 11.3, 3.7 Hz, 1H), 7.36-7.41 (m, 2H), 8.25-8.31 (m, 2H). LCMS (not observed - the compound did not ionize).

[0714] Synthesis of 1-13: NH2-PEG2000-NHBoc (1 g, 0.48 mmol), 1-12 (0.436 g, 0.57 mmol), pyridine (0.385 mL, 4.77 mmol), DMAP (0.058 g, 0.48 mmol), and THF (4 mL) were combined in a microwave vial under nitrogen with stirring at 40°C for 6 hours. The reaction was cooled and stirred overnight. The solvent was evaporated and the crude material wasASG-105-PCT01-NP

[0715] purified by flash silica chromatography, elution gradient 0-20% MeOH in DCM. Product 1-13 (1.280 g, 99 %) was isolated as a white solid. 1H NMR (500 MHz, MeOD, 25°C) 50.87 - 0.94 (m, 6H), 1.30 (s, 60H), 1.44 (s, 9H), 1.55 (dt, J = 12.8, 6.5 Hz, 4H), 3.22 - 3.57 (m, 10H), 3.64 (s, 176H), 3.75 - 3.81 (m, 1H), 4.03-4.16 (m, 2H). LCMS (PEG n: 46) Expected: 2851.9. Observed: 2851.9

[0716] Synthesis of 1-14: 1-13 (1.27 g, 0.47 mmol), triisopropylsilane (0.191 mL, 0.93 mmol), TFA (3 mL), and DCM (3.00 mL) were combined in a vial under nitrogen with stirring on ice. The reaction was allowed to warm to room temperature and stirred for 2 hours. The solvent was removed by rotovap and the residue was purified by flash silica chromatography, elution gradient 0-20% MeOH in DCM (1% NH4OH). Product 1-14 (0.977 g, 80 %) was isolated as a white solid. 1H NMR 1H NMR (500 MHz, CDC13, 25°C) 5 0.88 (t, J = 6.9 Hz, 6H), 1.25 (s, 60H), 1.51-1.57 (m, 4H), 3.17-3.21 (m, 2H), 3.37-3.55 (m, 10H), 3.64 (s, 176H), 3.77-3.83 (m, 1H), 4.03-4.17 (m, 2H). 1H NMR (500 MHz, CD3OD) 0.87 - 0.97 (m, 6H), 1.24 - 1.43 (m, 60H), 1.53 - 1.65 (m, 4H), 3.20 - 3.42 (m, 10H), 3.45 - 3.76 (m, 176H), 3.77 - 3.83 (m, 1H), 4.03 - 4.17 (m, 2H). LCMS Expected (PEGn: 46): 2751.9. Observed: 2751.9

[0717] Synthesis of 1-15: 1-14 (100 mg, 0.04 mmol), 1-10 (83 mg, 0.05 mmol), PyBOP (27.8 mg, 0.05 mmol), triethylamine (0.053 mL, 0.38 mmol), and DMF (2 mL) were added in a microwave vial and stirred under a nitrogen atmosphere for 16 hours. The solvent was evaporated under reduced pressure and the residue was dissolved in 20 mL of DCM and the solution was washed with saturated sodium bicarbonate, saturated ammonium chloride, and brine. The organic phase was dried over magnesium sulfate. The crude material was purified by flash silica chromatography, elution gradient 0-20% MeOH in DCM (1% NH4OH) / DCM. Product 1-15 (88 mg, 52 %) was isolated as a white solid. 1H NMR (500 MHz, CD3OD ) 0.92 (t, J = 6.9 Hz, 6H), 1.25 - 1.46 (m, 74H), 1.48 - 1.75 (m, 22H), 1.96 (d, J= 11.3 Hz, 18H), 2.05 (s, 9H), 2.16 (s, 9H), 2.20 - 2.30 (m, 2H), 2.32 - 2.42 (m, 2H), 2.42 - 2.49 (m, 5H), 2.89 - 2.94 (m, 2H), 3.10 (s, 2H), 3.15 - 3.24 (m, 6H), 3.25 - 3.30 (m, 2H), 3.36 - 3.44 (m, 4H), 3.45 - 3.93 (m, 176H), 3.97 - 4.24 (m, 15H), 4.58 (d, J = 8.4 Hz, 3H), 5.09 (dd, J = 11.5, 2.8 Hz, 3H), 5.30 - 5.39 (m, 3H). LCMS Expected (PEGn: 46): 4537.7. Observed: 4537.7

[0718] Synthesis of 1: 1-15 (88 mg, 0.02 mmol), MeOH (1.8 mL), and sodium methanolate (0.5M, 0.120 mL, 0.06 mmol) were combined in a vial under nitrogen atmosphere at room temperature and stirred for 2 hours. The resulting solution was neutralized using DOWEX50 / H+ beads until the pH reached about 7 based on spotting on pH test paper. The mixture was filtered through aASG-105-PCT01-NP

[0719] syringe filter and the solvent evaporated. The crude material was dissolved in 1.2 mL 40:60 EtOH:MPA (MPA: 50 mM NH4OAc + 0.5% HO Ac). Approximately 400 uL of the solution was injected on a Water Fractionlynx system with a SQD2 mass spectrometer and a PDA detector set to 230 nm. Chromatographic separation was achieved using a Waters XSelect CSH Fluoro Phenyl ODB 5p 19x150mm column. The gradient (35% B for 0.5 min, 35-35% B in 1.5 min, 35-70% B in 14 min, 70-99% B in 0.1 min) was applied at room temperature with a flow rate of 30 mL / min. The fractions were collected manually every 5 seconds (2.5 mL fractions) by observing the TIC trace. Product 1 was isolated as a white solid (10.6 mg, 13%). Stored under Argon at -201H (500 MHz, MeOD, 25°C) 5 0.87-0.94 (m, 6H), 1.30 (d, J = 4.0 Hz, 74H), 1.48-1.57 (m, 14H), 1.64 (d, J = 8.1 Hz, 2H), 1.98 (d, J = 1.0 Hz, 9H), 2.18-2.27 (m, 2H), 2.34-2.49 (m, 6H), 3.07-3.29 (m, 8H), 3.46-3.49 (m, 6H), 3.51-3.56 (m, 6H), 3.64 (s, 176H), 3.71-3.94 (m, 22H), 4.37 (ddd, J = 8.4, 2.1, 1.2 Hz, 2H). LCMS Expected (PEGn: 46): 4159.6. Observed: 4159.6. (The PEG chain of the polymeric product ranged from 38 to 53 PEG units, with the main fraction being 46 PEG units long.)

[0720] Compound 2

[0721]

[0722] Compound 2 was prepared according to the method illustrated in the scheme above.

[0723] Synthesis of 2-1: 1-12 (0.273 g, 0.36 mmol) and NH2-PEG3400-NHBoc (1.075 g, 0.30 mmol) was added to a microwave vial and evacuated and backfilled with Argon. THF (5.86 ml) and pyridine (0.121 ml, 1.49 mmol) was added. Dissolved by sonication. Stirred overnight at RT, the reaction progression was monitored by TLC (10% MeOH in DCM, CAM stain). The solvent was evaporated. Purified on a 10 g silica column (gradient 0-10% MeOH in DCM using ELS detector). Isolated product 2-1 (0.96 g, 76 %) as a white solid. Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 5 0.87-0.94 (m, 6H), 1.30 (s, 60H), 1.44 (s, 9H), 1.55 (dt, J = 12.8, 6.5 Hz, 4H), 3.22 (t, J = 5.6 Hz, 2H), 3.43-3.57 (m, 10H), 3.64 (s, 264H),ASG-105-PCT01-NP

[0724] 3.75-3.81 (m, 1H), 4.03-4.16 (m, 2H). LCMS (PEG n: 70) Expected: 3908.6. Observed: 3908.6.

[0725] Synthesis of 2-2: 2-1 (346 mg, 0.08 mmol) was dissolved in DCM (1324 pl). TFA (316 pl, 4.10 mmol) was added. Stirred overnight at RT, the reaction progression was monitored by TLC (10% MeOH in DCM, CAM stain). The solvent was removed under reduced pressure, the resulting oil was mixed with 1 mL water and sonicated. The water was evaporated and the residue was dried under high vacuum to a white solid. Purified sequentially on a 5 g kp-amino column and a 5 g silica column using a gradient 0-10% MeOH in DCM and ELS detector. Isolated product 2-2 as a white solid (228 mg, 81%). Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 50.88-0.93 (m, 6H), 1.30 (s, 60H), 1.56 (q, J = 6.6 Hz, 4H), 2.86 (t, J = 5.2 Hz, 2H), 3.28 (d, J = 5.6 Hz, 2H), 3.43-3.58 (m, 10H), 3.64 (s, 272H), 3.75-3.8 (m, 1H), 4.02-4.16 (m, 2H). 1H NMR (500 MHz, CDC13, 25°C) 50.85-0.9 (m, 6H), 1.25 (s, 60H), 1.55 (t, J = 6.7 Hz, 4H), 3.12 - 3.58 (m, 10H), 3.64 (s, 264H), 3.71-3.81 (m, 1H), 3.87 - 4.17 (m, 2H). LCMS Expected (PEGn: 70): 3808.5. Observed: 3808.5

[0726] Synthesis of 2-3: A microwave vial was heated under vacuum, backfilled with Argon. Charged with 2-2 (104 mg, 0.03 mmol), 1-10 (50.1 mg, 0.03 mmol), DMAP (0.617 mg, 5.05 pmol) and DCC (5.01 pl, 0.03 mmol). The vial was evacuated and backfilled with Argon. DCM (493 pl) and triethylamine (7.04 pl, 0.05 mmol) was added. Stirred overnight at RT. The reaction progression was monitored by TLC (9:1 DCM:MeOH, CAM stain). The reaction mixture was diluted with 2 mL DCM and filtered through a 0.45 pm syringe filter. Concentrated to a colorless oil. Purified on a 5g silica column with a 15 cv gradient 0-10% MeOH in DCM using ELS detector. Isolated Product 2-3 (90 mg, 60 %) as a white solid. Stored under Argon at -20°C. (The polymeric product was characterized with LCMS and it was found that the PEG chain ranged from 51 to 84 PEG units with the main fraction being 70 PEG units long.) 1H NMR (500 MHz, CDC13, 25°C) 50.87 (t, J = 6.9 Hz, 6H), 1.15-1.39 (m, 74H), 1.39-1.66 (m, 22H), 1.89-2.19 (m, 36H), 2.39 (d, J= 14.5 Hz, 4H), 2.88 (s, 1H), 3.02 (s, 1H), 3.05-3.57 (m, 24H), 3.63 (s, 264H), 3.7-3.82 (m, 6H), 3.82-3.93 (m, 4H), 3.99-4.25 (m, 6H), 4.62-4.79 (m, 1H), 5.2-5.41 (m, 4H). LCMS Expected (PEGn: 70): 5594.4. Observed: 5594.4.

[0727] Synthesis of 2: 2-3 (105 mg, 0.02 mmol) was dissolved in MeOH (1906 pl) and sodium methanolate (3.09 mg, 0.06 mmol) was added. The reaction was stirred at RT for 2h, monitored reaction progression with TLC (10% MeOH in DCM, CAM stain), after 3h the reaction was neutralized with DOWEX50 / H+ beads until the pH reached about 7 based on spotting on pHASG-105-PCT01-NP

[0728] test paper. The mixture was filtered through a syringe filter and the solvent evaporated. The crude material was dissolved in 40:60 EtOH:MPA (MPA: 50 mM NT OAc + 0.5% HOAc). Approximately 400 uL of the solution was injected on a Water Fractionlynx system with a SQD2 mass spectrometer and a PDA detector set to 230 nm. Chromatographic separation was achieved using a Waters XSelect CSH Fluoro Phenyl ODB 5p 19x150mm column. The gradient (35% B for 0.5 min, 35-35% B in 1.5 min, 35-70% B in 14 min, 70-99% B in 0.1 min) was applied at room temperature with a flow rate of 30 mL / min. The fractions were collected manually every 5 seconds (2.5 mL fractions) by observing the TIC trace. Compound 2 (20.8 mg, 21%) was isolated as a white solid. Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 5 0.87-0.94 (m, 6H), 1.30 (s, 74H), 1.47-1.70 (m, 22H), 1.89 (s, 3H), 1.98 (s, 9H), 2.17-2.28 (m, 2H), 2.33-2.48 (m, 6H), 2.90 (s, 2H), 3.07 (s, 1H), 3.09-3.24 (m, 5H), 3.27- 3.30 (m, 2H), 3.33-3.39 (m, 4H), 3.44-3.57 (m, 16H), 3.64 (s, 272H), 3.68-3.94 (m, 27H), 4.02-4.16 (m, 2H), 4.37 (ddd, J = 8.4, 2.0, 1.2 Hz, 3H). 1H NMR (500 MHz, CDC13, 25°C) 5 0.88 (t, J = 6.9 Hz, 6H), 1.25 (m, 74H), 1.44 (m, 18H), 3.26-3.59 (m, 22H), 3.64 (m, 308H).

[0729] LCMS Expected (PEG n: 70): 5216.3. Observed: 5216.3. (The PEG chain of the polymeric product ranged from 51 to 84 PEG units, with the main fraction being 70 PEG units long.) Compound 3

[0730]

[0731] Compound 3 was prepared according to the method illustrated in the scheme above.

[0732] Synthesis of 3-2: 3-1 (5 g, 21.60 mmol) was added to (3R,4S,5R)-3,4,5-trihydroxycyclohex-l- ene-1 -carboxylic acid (4.14 g, 23.76 mmol), EDC (8.70 g, 45.37 mmol), HOBt (6.62 g, 43.20ASG-105-PCT01-NP

[0733] mmol) and DIEA (11.32 mL, 64.81 mmol) in DMF (50 mL). The resulting mixture was stirred at RT for 1 hour. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash silica chromatography, elution gradient 0-5 % MeOH in DCM. Pure fractions were evaporated to dryness to afford product 3-2 (4.40 g, 72 %) as a yellow liquid. 1H-NMR (400 MHz, DMSO, 24°C) 50.10 (d, J = 1.7 Hz, 6H), 0.85 (d, J = 6.3 Hz, 9H), 1.24 (s, 2H), 1.33-1.49 (m, 6H), 1.94-2.02 (m, 2H), 3.07-3.09 (m, 2H), 3.56 (m, 4H), 4.03 (q, J = 7.1 Hz, 1H), 4.17 (d, J = 3.8 Hz, 1H), 6.27 (dd, J = 3.4, 1.7 Hz, 1H), 7.23-7.32 (m, 2H), 7.80 (q, J = 1.8, 1.4 Hz, 1H). LCMS Expected 387.2, Observed 388.4 ([M+H+]).

[0734] Synthesis of 3-3: CS2CO3 (24.13 g, 74.07 mmol) was added to tert-Butyl acrylate (197 mL, 1346.78 mmol) and 3-2 (8.7 g, 22.45 mmol) in t-BuOH (800 mL). The resulting mixture was stirred at RT for 3 days. The reaction mixture was diluted with EtOAc (1 L), the mixture was filtered through a Celite pad. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford product 3-3 (8.30 g, 47.9 %) as a yellow oil. The crude product was used in the next step directly without further purification. 1H-NMR (400 MHz, DMSO, 24°C) 50.86 (s, 9H), 1.12 (s, 2H), 1.40 (dd, J = 4.3, 1.3 Hz, 31H), 1.47-1.58 (m, 2H), 2.36-2.43 (m, 8H), 3.06 (d, J = 6.6 Hz, 2H), 3.49 (t, J = 6.2 Hz, 1H), 3.65-3.77 (m, 9H), 3.92 (dt, J = 21.1, 4.6 Hz, 1H), 6.26-6.31 (m, 1H), 7.87 (tdd, J = 8.2, 5.6, 2.8 Hz, 1H). Three protons were exchanged. LCMS Expected 771.5, Observed 772.4 ([M+H+]).

[0735] Synthesis of 3-4: TBAF (IM in THF) (9.45 mL, 9.45 mmol) was added to 3-3 (7.3 g, 9.45 mmol) in THF (80 mL). The resulting mixture was stirred at RT for 16 hours. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 80% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford product 3-4 (1.50 g, 24 %) as a colorless oil.

[0736] 1H-NMR (400 MHz, DMSO, 23°C) 5 1.18-1.31 (m, 4H), 1.37-1.45 (m, 29H), 2.16 (dd, J = 18.2, 3.6 Hz, 1H), 2.32-2.47 (m, 7H), 3.06 (q, J = 6.6 Hz, 2H), 3.17 (d, J = 5.2 Hz, 3H), 3.37 (m, 2H), 3.73 (m, 6H), 4.10 (q, J = 5.2 Hz, 1H), 4.33 (t, J = 5.1 Hz, 1H), 6.29 (d, J = 2.5 Hz, 1H), 7.87 (q, J = 5.8, 4.5 Hz, 1H). One proton was exchanged. LCMS Expected 657.4, Observed 658.5 ([M+H+]).

[0737] Synthesis of 3-5: TFA (9.22 mL, 119.71 mmol) was added to 3-4 (3.15 g, 4.79 mmol) in DCM (20 mL) at 0 °C. The resulting mixture was stirred at RT for 2 hours. The solvent was removedASG-105-PCT01-NP

[0738] under reduced pressure. DCM (3 x 50 mL) was added to the residue and concentrated to dryness to afford product 3-5 (2.58 g, 92%) as a colorless oil. The product was used in the next step directly without further purification. 1H-NMR (500 MHz, DMSO, 25 °C) 5 1.27-1.30 (m, 6H), 1.40-1.43 (m, 2H), 1.51-1.62 (m, 1H), 1.62-1.75 (m, 2H), 2.14 (dd, J = 18.3, 3.7 Hz, 1H), 2.31-2.49 (m, 6H), 2.98-3.26 (m, 2H), 3.57-3.85 (m, 7H), 4.37 (t, J = 6.6 Hz, 2H), 6.26 (d, J = 2.7 Hz, 1H), 7.88 (q, J = 5.7 Hz, 1H). Three protons have been exchanged. LCMS Expected 585.2, Observed 586.2 ([M+H+]).

[0739] Synthesis of 3-6: Perfluorophenyl 2,2,2-trifluoroacetate (3.37 mL, 19.60 mmol) was added to 3-5 (2.55 g, 4.36 mmol) and DIEA (5.70 mL, 32.66 mmol) in DCM (50 mL) at RT. The resulting mixture was stirred at RT for 1 hour. The reaction mixture was diluted with DCM (40 ml) and washed sequentially with IM NaHSO4 (40 ml), saturated NaHCO3 (50 ml), and saturated brine (50 ml). The organic layer was dried over Na2SO4, filtered and evaporated to afford product 3-6 (3.65 g, 77%) as a yellow oil. The product was used in the next step directly without further purification. 1H-NMR (500 MHz, DMSO, 25°C) 5 1.32-1.35 (m, 4H), 1.42-1.45 (m, 3H), 1.59-1.73 (m, 3H), 2.14-2.27 (m, 1H), 2.38-2.47 (m, 1H), 3.05-3.21 (m, 4H), 3.67-4.03 (m, 10H), 4.36 (t, J = 6.6 Hz, 3H), 6.34 (s, 1H), 7.88 (t, J = 5.8 Hz, 1H). LCMS Expected 1083.1, Observed 1084.2 ([M+H+]).

[0740] Synthesis of 3-7: DIEA (5.63 mL, 32.24 mmol) was added to 1-8 (5.76 g, 12.89 mmol) and 3-6 (3.5 g, 3.22 mmol) in DMF (100 mL) at 0°C. The resulting mixture was stirred at RT for 2 hours. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0-50% MeCN in water (0.05% TFA). Pure fractions were evaporated to dryness to afford product 3-7 (2.70 g, 47%) as a white solid. 1H-NMR (500 MHz, DMSO, 25 °C) 5 1.14-1.31 (m, 17H), 1.31-1.52 (m, 17H), 1.77 (s, 9H), 1.89 (s, 9H), 1.99 (s, 9H), 2.10 (s, 10H), 2.18-2.43 (m, 7H), 3.03-3.22 (m, 8H), 3.29-3.45 (m, 6H), 3.74 (d, J = 6.9 Hz, 8H), 3.87 (dt, J = 11.3, 8.8 Hz, 4H), 4.02 (q, J = 3.9 Hz, 10H), 4.48 (d, J = 8.5 Hz, 3H), 4.97 (dd, J = 11.3, 3.5 Hz, 3H), 5.21 (d, J = 3.4 Hz, 3H), 6.27 (s, 1H), 7.67-7.98 (m, 7H). LCMS Expected 1773.8, Observed 888.3 (z=2, ([M+H+]).

[0741] Synthesis of 3-8: Chromosulfuric acid (0.732 mL, 1.46 mmol) was added to 3-7 (1.3 g, 0.73 mmol) in acetone (35 mL) at 0°C. The resulting mixture was stirred at RT for 1 hour. The reaction mixture was quenched with i-PrOH (20 ml). The mixture was diluted with DCM (30 ml), after 10 min, saturated NaHCOs (50 ml) was added. The solution was evaporated toASG-105-PCT01-NP

[0742] dryness to afford the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0-30% MeCN in water (0.05% TFA). Pure fractions were evaporated to dryness to afford product 3-8 (0.308 g, 23%) as a yellow solid. 1H-NMR (500 MHz, DMSO, 25 °C) 5 1.25-1.27 (m, 17H), 1.28-1.32 (m, 6H), 1.38-1.52 (m, 9H), 1.77 (s, 9H), 1.89 (s, 9H), 1.99 (s, 9H), 2.10 (s, 9H), 2.18-2.42 (m, 8H), 3.02-3.12 (m, 8H), 3.28-3.42 (m, 5H), 3.70-3.82 (m, 10H), 3.86 (q, J = 9.6 Hz, 3H), 4.02 (q, J = 6.3, 5.3 Hz, 9H), 4.48 (d, J = 8.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 5.21 (d, J = 3.4 Hz, 3H), 6.27 (s, 1H), 7.68-7.82 (mz, 6H), 11.95 (brs, 1H). One proton has been exchanged. LCMS Expected 1787.8, Observed 895.1 (z=2 ([M+H+]).

[0743] Synthesis of 3-9: 1-12 (385 mg, 0.50 mmol) and BocNH-PEG2k-NH2 (901 mg, 0.42 mmol) was added to a microwave vial which was evacuated and backfilled with Argon. Anhydrous THF (2.63 ml) was added, dissolved by sonication and vortex. Anhydrous Pyridine (0.170 ml, 2.10 mmol) was added and the reaction stirred atRT overnight. The reaction progression was monitored by TLC (10% MeOH in DCM, CAM stain). The solvent was evaporated. Purified on a 25 g silica column with a gradient 0-10% MeOH in DCM using ELS detector. Isolated product 3-9 (1.1 g, 91 %) as a white solid. Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 50.87-0.94 (m, 6H), 1.30 (s, 60H), 1.44 (s, 9H), 1.51-1.60 (m, 4H), 3.22 (t, J = 5.6 Hz, 2H), 3.28 (s, 2H), 3.43-3.58 (m, 10H), 3.64 (s, 148H), 3.75-3.81 (m, 1H), 4.01-4.18 (m, 2H). 1H (500 MHz, CDC13, 25°C) 5 0.88 (6H, t), 1.25 (60H, m), 1.44 (s, 9H), 1.55 (4H, m), 3.36 (2H, m), 3.41-3.58 (10H, m), 3.64 (148H, m), 3.78 (1H, m), 4.06-4.22 (2H, m).

[0744] LCMS (PEGn: 39) Expected: 2543.8. Observed: 2543.8.

[0745] Synthesis of 3-10: 3-9 (494 mg, 0,18 mmol) was dissolved in DCM (2.9 ml) in a microwave vial. TFA (689 pl, 8.93 mmol) was added and the reaction was stirred overnight at RT. The reaction progression was monitored by TLC (10% MeOH in DCM, CAM stain). After complete conversion the solvent was evaporated and the residue was purified on a 5 g Biotage KP -Amino column with a gradient 0-10% MeOH in DCM using ELS detector. Product 3-10 (420 mg, 88%) was isolated as a white solid. Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 5 0.88-0.93 (m, 6H), 1.29 (s, 60H), 1.56 (q, J = 6.5 Hz, 4H), 2.83 (t, J = 5.3 Hz, 2H), 3.26-3.3 (m, 2H), 3.43-3.58 (m, 10H), 3.64 (s, 148H), 3.75-3.80 (m, 1H), 4.02-4.15 (m, 2H). 1H NMR (500 MHz, CDC13, 25°C) 5 0.88 (t, J = 6.8 Hz, 6H), 1.25 (s, 60H), 1.55 (s, 4H), 3.41-3.58 (m, 1 OH), 3.58-3.68 (m, 148H), 3.78 (s, 1H), 4.06-4.22 (m, 2H). LCMS Expected (PEGn: 39): 2443.7. Observed: 2443.7ASG-105-PCT01-NP

[0746] Synthesis of 3-11: A microwave vial was dried under vacuum and backfilled with Argon. Charged with 3-10 (0.2 g, 0.07 mmol), 3-8 (0.132 g, 0.07 mmol), DMAP (5.41 mg, 0.04 mmol) and DCC (0.015 ml, 0.09 mmol). The vial was capped and purged. Anhydrous DCM (1.462 ml) was added, and the mixture was stirred at RT for 24 hours. The reaction progression was monitored by TLC (10% MeOH in DCM, CAM stain). The suspension was filtered through a syringe filter and washed once with DCM. Solvent was evaporated and the product purified on a 10 g silica column with a gradient 0-10% MeOH in DCM using a ELS detector. The product was re-purified on a 5g silica column. Eluted with 2cv 100% EtOAc, 2cv gradient to 100% DCM then 10 cv gradient to 10% MeOH in DCM followed by isocratic elution of the compound (streaked a lot). Isolated product 3-11 as a light yellow oily solid (73 mg, 0.016 mmol, 22%). 1H NMR (500 MHz, CDC13, 25°C) 5 0.88 (t, J = 6.9 Hz, 6H), 1.25 (s, 74H), 1.40-1.69 (m, 22H), 1.99 - 2.16 (m, 36H), 3.37 (s, 2H), 3.41-3.57 (m, 12H), 3.64 (s, 148H), 3.78 (1H, d), 3.87-4.00 (m, 4H), 4.01-4.28 (m, 8H), 4.80 (bs, 2H), 5.24 (bs, 2H), 5.36 (bs, 2H), 6.55 (bs, 1H). LCMS Expected (PEGn: 39): 4213.5. Found: 4213.5.

[0747] Synthesis of 3: 3-11 (73 mg, 0.016 mmol) was dissolved in MeOH (1642 pl) and sodium methanolate (5.4 M, 9.18 pl, 0.05 mmol) was added. The reaction was stirred at RT for 2 hours, the reaction progression was monitored with TLC (10% MeOH in DCM, CAM stain). The reaction was neutralized with DOWEX50 / H+ beads until the pH reached about 7 based on spotting on pH test paper, filtered through a 0.45 pm syringe filter and concentrated to a light yellow oil. The residue was suspended in 0.3 mL DCM by sonication and the solvent evaporated to yield a light yellow wax. The crude material was dissolved in 40:60 EtOH:MPA (MPA: 50 mM NH4OAc + 0.5% HO Ac). Approximately 400 uL of the solution was injected on a Water Fractionlynx system with a SQD2 mass spectrometer and a PDA detector set to 230 nm. Chromatographic separation was achieved using a Waters XSelect CSH Fluoro Phenyl ODB 5p 19x150mm column. The gradient (35% B for 0.5 min, 35-35% B in 1.5 min, 35-70% B in 14 min, 70-99% B in 0.1 min) was applied at room temperature with a flow rate of 30 mL / min. The fractions were collected manually every 5 seconds (2.5 mL fractions) by observing the TIC trace. Product 3 (6.2 mg, 9%) was isolated as a white solid. Stored under Argon at -20°C. 1H NMR (500 MHz, MeOD, 25°C) 50.90 (t, J = 6.8 Hz, 6H), 1.30 (s, 74H), 1.49-1.67 (m, 14H), 1.98 (s, 9H), 2.15-2.27 (m, 2H), 2.34-2.53 (m, 4H), 3.06-3.3 (m, 8H), 3.41-3.59 (m, 16H), 3.64 (s, 148H), 3.75-3.92 (m, 15H), 4.02-4.22 (m, 3H), 4.37 (d, J = 8.4 Hz, 2H), 6.39-6.45 (m, 1H). LCMS Expected (PEG n: 39): 3835.4. Observed: 3835.4. (TheASG-105-PCT01-NP

[0748] PEG chain of the polymeric product ranged from 32 to 51 PEG units, with the main fraction being 39 PEG units long.)

[0749] Alternative Synthesis of 3-11

[0750]

[0751] Alternative synthesis of 3-11: A microwave vial was charged with 3-10 (153 mg, 0.06 mmol) and GalNac-PFP ester 6-1 (obtainable as described in PCT / EP2024 / 070929, 122 mg, 0.06 mmol). DCM (1.2 ml) was added, and the mixture was stirred at RT for 2 hours at which point product completely and cleanly formed. Reaction progression was monitored with TLC (10% MeOH in DCM, CAM stain). The solvent was evaporated, and the residue was dried under high vacuum. Isolated crude product 3-11 as a light yellow sticky solid (275 mg, 0.06 mmol, 109%).

[0752] Compound 4

[0753]

[0754] ASG-105-PCT01-NP

[0755]

[0756] Compound 4 may be prepared according to the method illustrated in the scheme above.

[0757] Synthesis of 4-1: DIEA (70.2 mL, 401.95 mmol) was added to N-l-Z-l,6-diaminohexane,HCl (39.5 g, 137.81 mmol), EDC (33.0 g, 172.26 mmol), HOBt (35.2 g, 229.68 mmol) and (3R,4S,5R)-3,4,5-trihydroxycyclohex-l-ene-l -carboxylic acid (20 g, 114.84 mmol) in DMF (300 mL) at RT. The resulting mixture was stirred at RT for 1 hour. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 10% MeCN in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford benzyl (6-((3R,4S,5R)-3,4,5-trihydroxycyclohex-l-ene-l-carboxamido)hexyl)carbamate (4-1) (35.0 g, 75.0 %) as a white solid. 1H NMR(500 MHz, DMSO, 25°C) 5 1.17-1.28 (4H, m), 1.34-1.45 (4H, m), 1.99 (1H, dd), 2.49-2.55 (1H, m), 2.98 (2H, q), 3.08 (2H, hept), 3.51 (1H, dd), 3.83 (1H, q), 4.19 (1H, s), 4.57 (1H, s), 4.73-4.77 (1H, m), 4.86 (1H, s), 5.00 (2H, s), 6.28 (1H, d), 7.22 (1H, t), 7.27-7.39 (5H, m), 7.82 (1H, t). LCMS m / z Expected 585.2, Observed [M+H]+586.2.

[0758] Synthesis of 4-2: CS2CO3 (91 g, 280.09 mmol) was added to 4-1 (34.5 g, 84.88 mmol) and tertbutyl acrylate (746 mL, 5092.51 mmol) in t-BuOH (750 mL) at RT. The resulting mixture was stirred at 40 °C for 4 days. The reaction mixture was filtered through celite. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford tri -tert-butyl 3,3',3"-(((lR,2S,3R)-5-((6-ASG-105-PCT01-NP

[0759] (((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-l,2,3-triyl)tris(oxy))tripropionate (4-2) (25.00 g, 37.2 %) as a colorless oil. 1H NMR (500 MHz, DMSO, 25°C) 5 1.21-1.27 (4H, m), 1.34-1.4 (22H, m), 1.41 (9H, s), 2.12-2.21 (1H, m), 2.32-2.42 (5H, m), 2.44 (2H, t), 2.98 (2H, q), 3.06 (2H, q), 3.63-3.81 (8H, m), 4.05 (1H, q), 5.01 (2H, s), 6.29 (1H, d), 7.23 (1H, t), 7.27-7.4 (5H, m), 7.86 (1H, t). LCMS m / z Expected 790.4, Observed [M+H]+791.4.

[0760] Synthesis of 4-3: A solution of tri -tert-butyl 3,3',3"-(((lR,2S,3R)-5-((6- (((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-l,2,3-triyl)tris(oxy))tripropionate (24.5 g, 30.97 mmol) in formic acid (240 mL) was stirred at RT for 2.5 hours. The solvent was removed under reduced pressure to afford 3,3',3"-(((lR,2S,3R)-5-((6-(((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-l,2,3-triyl)tris(oxy))tripropionic acid (4-3) (17.00 g, 88 %) as a colorless oil. The product was used in the next step directly without further purification.1H NMR (500 MHz, DMSO, 25°C) 5 1.22-1.27 (4H, m), 1.35-1.45 (4H, m), 2.1-2.18 (1H, m), 2.36-2.5 (7H, m), 2.98 (2H, q), 3.06 (2H, q), 3.64-3.84 (8H, m), 4.08 (1H, dd), 5.01 (2H, s), 6.27 (1H, s), 7.22 (1H, t), 7.27-7.4 (5H, m), 7.89 (1H, t), 12.23 (3H, s). LCMS m / z Expected 622.3, Observed [M+H]+623.2.

[0761] Synthesis of 4-4: (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, HC1 (47.3 g, 106.00 mmol) was added to 4-3 (16.5 g, 26.50 mmol), N-ethyl-N-isopropylpropan-2-amine (46.3 mL, 264.99 mmol), ((lH-benzo[d][l,2,3]triazol-l-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (51.6 g, 116.60 mmol), and DMAP (9.71 g, 79.50 mmol) in DMF (300 mL) at RT under N2. The resulting mixture was stirred at RT for 16 hours. The solvent was removed under reduced pressure to afford the crude product. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 35% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford [(2A,3A,4A,5A,6A)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(lA,5A,6S)-5,6-bis[3-[6-[(2A,3A,4A,5A,6A)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyhexylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-l-yl]oxypropanoylamino]hexoxy]tetrahydropyran-2-yl]methyl acetate (4-4) (27.0 g, 53.4 %) as a yellow solid. 1H-NMR (500 MHz, DMSO, 25°C) 5 1.15-1.31 (16H, m), 1.31-1.42 (10H, m), 1.42-1.52 (6H, m), 1.78 (9H, s), 1.90 (10H, s), 2.00 (9H, s), 2.11 (10H, s), 2.12-2.18 (1H, m), 2.26 (4H, qt), 2.32 (2H, q), 2.38 (1H, d), 2.94-3.08 (10H, m), 3.41 (3H, dt), 3.63-3.8 (9H, m), 3.83-3.92 (3H, m), 3.98-4.07 (10H, m), 4.49 (3H, d), 4.97 (3H, dd), 5.00 (2H, s), 5.22 (3H,ASG-105-PCT01-NP

[0762] d), 6.24-6.31 (1H, m), 7.22 (1H, q), 7.27-7.4 (5H, m), 7.74-7.87 (7H, m). LCMS m / z Expected 1906.9, Observed [M+H]+1907.9.

[0763] Synthesis of 4-5: lodotrimethylsilane (3.15 g, 15.72 mmol) was added to 4-4 (3 g, 1.57 mmol) in MeCN (60 mL) at RT under nitrogen. The resulting mixture was stirred at RT for 15 minutes. The reaction went to completion by LC-MS. A solution of succinic anhydride (0.787 g, 7.86 mmol) and DMAP (1.345 g, 11.01 mmol) in MeCN (15 mL) was added slowly at 0° C. After stirring at 0° C for 5 minutes, Et3N (3.18 mL, 31.44 mmol) was added. The resulting mixture was stirred at RT for 5 hours. The solvent was removed under reduced pressure to afford the crude product. Six duplicated reactions were conducted in parallel, and the crude product was combined for further purification. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 30% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford 4-oxo-4-[6-[[(3R,4S,5R)-3,4,5-tris[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyhexylamino]-3-oxo-propoxy]cyclohexene-l-carbonyl]amino]hexylamino]butanoic acid (4-5) (12.30 g, 69.6 %) as a white solid. 1H-NMR (500 MHz, DMSO, 25°C) 5 1.12-1.3 (16H, m), 1.3-1.53 (16H, m), 1.78 (9H, s), 1.90 (10H, s), 2.00 (9H, s), 2.11 (10H, s), 2.23-2.38 (8H, m), 2.41 (2H, t), 2.97-3.1 (11H, m), 3.41 (3H, dt), 3.63-3.79 (10H, m), 3.83-3.92 (3H, m), 3.98-4.07 (10H, m), 4.49 (3H, d), 4.97 (3H, dd), 5.22 (3H, d), 6.28 (1H, s), 7.73-7.88 (8H, m), 12.53 (1H, s). LCMS m / z Expected 1873.0, Observed [M+H]+ 1874.4.

[0764] Synthesis of 4-6:Perfluorophenyl 2,2,2-trifluoroacetate (4.83 mL, 28.09 mmol) was added to DIEA (7.09 mL, 40.58 mmol) and 4-5 (11.7 g, 6.24 mmol) in DCM (250 mL) at RT. The resulting mixture was stirred at RT for 2 hours. The reaction mixture was diluted with DCM (50 ml), and washed sequentially with 1 M NaHSO4 (250 ml), saturated NaHCO3 (250 ml), and saturated brine (250 ml). The organic layer was dried over Na2SO4, filtered and evaporated to dryness to afford the crude product. The crude product was suspended in Et2O (150 ml) and the suspension was stirred for one hour. The solid was collected by filtration, washed with Et2O (2 x 50 ml) and dried under vacuum to afford (2,3,4,5,6-pentafhiorophenyl) 4-oxo-4-[6-[[(3A,45,5A)-3,4,5-tris[3-[6-[(2A,3A,4A,5A,6A)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyhexylamino]-3-oxo-propoxy]cyclohexene-l-carbonyl]amino]hexylamino]butanoate 4-6 (11.01 g, 86 %) as a white solid.1H-NMR (500 MHz, DMSO, 25°C) 5 1.2-1.3 (16H, m), 1.31-1.43 (10H, m), 1.43-1.5 (6H, m), 1.78 (9H, s), 1.90 (9H, s), 2.00 (9H, s), 2.11 (9H, s), 2.12-2.18 (1H, m), 2.2-2.38 (8H, m), 2.38-2.44 (1H, m), 2.95 (1H, t), 2.98-3.17 (11H, m), 3.36-3.49 (3H, m), 3.55-3.81 (11H, m), 3.82-3.93 (3H,ASG-105-PCT01-NP

[0765] m), 3.94-4.12 (10H, m), 4.49 (3H, d), 4.97 (3H, dd), 5.22 (3H, d), 6.28 (1H, s), 7.67-8.05 (8H, m). LCMS m / z Expected 2039.9, Observed [M / 2+H]+ 1021.1.

[0766] Synthesis of 4: 4-7 is synthesised from 4-5 and 3-10 following the procedure described above to prepare 3-11 from 3-8 and 3-10. Alternatively, 4-7 is synthesised from 4-6 and 3-10.

[0767] Compound 4 is synthesised from 4-7 following the procedure described above to prepare 3 from 3-11.

[0768] Synthesis of 4: 4-6 (97 mg, 0.02 mmol) was dissolved in MeOH (2243 pl) and sodium methoxide (12.53 pl, 0.07 mmol, 5.4 M) was added. The reaction was stirred at RT for Ih, at which point the SM was consumed and a baseline spot was present on TLC (10% MeOH in DCM, CAM stain). The reaction was neutralized with Dowex 50, filtered through a syringe filter and concentrated to yield product 4 (86 mg, 97%) as a light yellow wax. Stored at -20°C.

[0769] IH NMR (500 MHz, MeOD, 25°C) 5 0.87-0.94 (m, 6H), 1.29 (s, 74H), 1.45-1.6 (m, 22H), 1.98 (s, 9H), 2.2-2.31 (m, IH), 2.36-2.61 (m, 11H), 3.08-3.25 (m, 11H), 3.27-3.29 (m, 2H), 3.42-3.56 (m, 20H), 3.64 (s, 148H), 3.72-3.95 (m, 27H), 4.01-4.15 (m, 3H), 4.37 (dd, J = 8.4, 0.9 Hz, 3H), 6.41 (s, IH). LCMS Expected (PEG n: 39): 3920.5 Found: 3920.5.

[0770] Compound 5

[0771]

[0772] Compound 5 may be prepared according to the method illustrated in the scheme above.ASG-105-PCT01-NP

[0773] Synthesis of 5: 5-1 is synthesised from 4-5 and 2-2 following the procedure described above in Example 3 to prepare 3-11 from 3-8 and 3-10. Alternatively, 5-1 is synthesised from 4-6 and 2-2. 5 is synthesised from 5-1 following the procedure described above in Example 3 to prepare 3 from 3-11.

[0774] Synthesis of 5-1: A microwave vial was dried under vacuum and backfilled with Argon. Charged with 2-2 (111 mg, 0.03 mmol), 4-5 (55.2 mg, 0.03 mmol), DMAP (2.160 mg, 0.02 mmol) and DCC (7.30 mg, 0.04 mmol). Vial was capped and purged. Anhydrous DCM (589 pl) was added and the mixture stirred at RT for 20h at which point the SMs were consumed and a new less polar spot was present on TLC (10% MeOH in DCM, CAM stain). The suspension was filtered through a syringe filter and washed once with DCM. Concentrated to to yield product 5-1 (133 mg, 80%) as a light beige oil that foamed to solid under vacuum (product was contaminated with DMAP). 1H NMR (500 MHz, MeOD, 25°C) 50.87-0.94 (m, 6H), 1.30 (d, J = 4.0 Hz, 74H), 1.45-1.62 (m, 22H), 1.91-1.97 (m, 18H), 2.03 (d, J = 0.9 Hz, 9H), 2.15 (s, 9H), 2.22-2.27 (m, 1H), 2.36-2.61 (m, 11H), 3.1-3.25 (m, 11H), 3.27-3.29 (m, 2H), 3.42-3.55 (m, 20H), 3.64 (s, 272H), 3.7-3.94 (m, 14H), 3.94-4.21 (m, 15H), 4.56 (dd, J = 8.4, 1.3 Hz, 3H), 5.07 (ddd, J = 11.3, 3.4, 0.9 Hz, 3H), 5.34 (dd, J = 3.5, 1.2 Hz, 3H), 6.41 (s, 1H). LCMS Expected (PEGn: 70): 5663.4 Found: 5663.4.

[0775] Synthesis of 5: 5-1 (133 mg, 0.02 mmol) was dissolved in MeOH (2352 pl) and sodium methoxide (13.14 pl, 0.07 mmol, 5.4 M) was added. The reaction was stirred at RT for Ih, at which point the SM was consumed and a baseline spot was present on TLC (10% MeOH in DCM, CAM stain). The reaction was neutralized with Dowex 50, filtered through a syringe filter and concentrated to yield product 4 (123 mg, 99%) as a light yellow wax (product was contaminated with DMAP). Stored at -20°C. IH NMR (500 MHz, MeOD, 25°C) 50.87-0.94 (m, 6H), 1.29 (s, 74H), 1.45-1.61 (m, 22H), 1.98 (s, 9H), 2.21-2.30 (m, IH), 2.37-2.62 (m, 11H), 3.08-3.24 (m, 11H), 3.26-3.29 (m, 2H), 3.42-3.56 (m, 20H), 3.64 (s, 272H), 3.71-3.94 (m, 27H), 4.02-4.15 (m, 3H), 4.37 (dd, J = 8.3, 0.9 Hz, 3H), 6.41 (s, IH). LCMS Expected (PEGn: 70): 5285.3 Found: 5285.3.

[0776] Compound 6ASG-105-PCT01-NP

[0777]

[0778] Compound 6 may be prepared according to the method illustrated in the scheme above.

[0779] Synthesis of 6: 6-1 is synthesised from 3-8 following the procedure described above in Example 4 to prepare 4-7 from 4-6. Synthesis of 6-2: 6-2 is synthesised from 3-8 and 2-2 following the procedure described above in Example 3 to prepare 3-11 from 3-8 and 3-10.

[0780] Alternatively, 6-2 is synthesised from 6-1 and 2-2. Synthesis of 6: 6 is synthesised from 6-2 following the procedure described above in Example 3 to prepare 3 from 3-11.

[0781] Example 2 - Preparation of Lipid Nanoparticles (ENPs)

[0782] Lipid nanoparticles were formulated by rapid microfluidic mixing of a lipid-ethanol solution and an aqueous solution of mRNA in 50 mM RNase-free citrate buffer, pH 3 as illustrated in Figure 6. The lipid mixture containing the ionizable lipid, sterol, neutral and polymer conjugated lipid was prepared in 99.5% ethanol at a molar ratio of 50:38.5:10:1.5 respectively. Further details of the lipid nanoparticle and components are given in Table 1 below. The final total lipid concentration in the ethanolic solution was 12.5 mM. The concentration of mRNA in the citrate buffer was 0.13 mg / ml.ASG-105-PCT01-NP

[0783] Table 1: Composition of LNP formulations

[0784]

[0785] Lipid nanoparticles were prepared using aNanoAssemblr™ Ignite™ (Precision Nanosystems, Vancouver, Canada). The lipid and mRNA solutions were mixed at a flow rate ratio of 1:3 at a rate of 12 ml / minute to give a final lipid:mRNA ratio of~20: 1. The LNPs were then dialysed overnight using a Slider-A-LyzerlOK molecular weight cut-off dialysis cassettes (Thermo Scientific) in a pH 7.4 phosphate buffered saline. The lipid nanoparticle formulations were concentrated to the desired concentration using centrifugal filters (3 OK molecular weight cut-off, Amicon), sterile filtered (0.2 pm) and then diluted to the required concentration of 0.06 mg / ml mRNA for in vivo dosing.

[0786] For particle characterization, the lipid nanoparticles were diluted in pH 7.4 phosphate buffered saline and particle size (z-average) and poly dispersity measured using a Zetasizer Nano ZS (Malvern). The mRNA content was analysed using the Ribogreen assay (ThermoFisher Scientific) according to manufacturer’s guidelines following dilution with Tris-EDTA / Triton X buffer to determine encapsulated mRNA by interpolation against a relevant mRNA standard curve.

[0787] Table 2 below summarizes the characterization of LNP formulations comprising GalNAc conjugates as described herein.

[0788] Table 2: Characterization of LNPs

[0789]

[0790] ASG-105-PCT01-NP

[0791]

[0792] Example 3 - In vivo biological assays

[0793] In vivo evaluation of the lipid nanoparticles was conducted using male, low density lipoprotein receptor (LDLr), homozygous, knockout mice (approximately 9-12 weeks old). Mice were dosed (tail vein injection) with lipid nanoparticles at 0.3 mg / kg luciferase mRNA with a dosing volume of 5 ml / kg while under light anesthesia (isoflurane). Six hours after dose administration, the mice were given a 150 mg / kg of luciferin (IVISbrite D-luciferin bioluminescent substrate in RediJect solution (Revvity)) administered by subcutaneous injection at a dosing volume of 5 ml / kg. The mice were euthanised 20 minutes after the luciferin administration and liver, lung, kidney heart and spleen dissected. The extracted organs were immediately scanned in an IVIS Spectrum™ in vivo imaging system (Revvity). The total individual and average radiance from each organ of each individual animal was quantified using Living Image™ software from Revvity. Pairwise two-sample equal variance t-test were used for statistical analysis.

[0794] Assessment of formulations Al to A4 as compared to Z1 (0% control) in B6-Ldlrtml.lAztc mice showed that luciferase signal was significantly enhanced at all concentrations of GalNAc compounds (Figure 7). Maximum signal was observed around 0.025 mol% of GalNAc compound, with the signal dropping off at higher concentrations.

[0795] Comparison of formulations A5 and Bl (both 0.05 mol% of GalNAc compound) in B6-Ldlrtml.lAztc mice showed that compounds having 2 kDa PEG and 3.4 kDa PEG in the linker both provided a significant increase over control (Figure 8).

[0796] Assessment of formulations C1-C3 as compared to Z2 (0% control) in B6.129S7-LdlrtmlHer / J mice showed that luciferase signal was significantly enhanced at all concentrations of GalNAc compounds (Figure 9). Maximum signal was observed around

[0797] - Ill -ASG-105-PCT01-NP

[0798] 0.05 mol% of GalNAc compound. Signal enhancement was not significantly different between formulations C2 and A6 (both having 0.05 mol% of GalNAc compound).

[0799] 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.

[0800] 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.

[0801] 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

ASG-105-PCT01-NPCLAIMS1. A lipid nanoparticle comprising:(i) a lipid matrix;(ii) a nucleic acid cargo; and(iii) a compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein:X is -O- or -NR-;wherein R is selected from -H and -(Ci-Ce)alkyl;Y is a linker comprising a linear chain of about 50 to about 400 atoms (e.g., selected from C, N, and O);Z is a lipidic anchor;represents a carbon-carbon single bond or double bond; and spacer in each case independently represents a moiety comprising a linear chain of 4 to 20 atoms (e.g., selected from C, N, and O).

2. The lipid nanoparticle of claim 1, wherein represents a double bond.

3. The lipid nanoparticle of claim 1 or claim 2, wherein the compound of Formula (I) has the structure of Formula (la):ASG-105-PCT01-NPor a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined in any of the preceding claims.

4. The lipid nanoparticle of claim 3, wherein the compound of Formula (I) has the structure of Formula (lb), Formula (Ic), or Formula (Id):"ASG-105-PCT01-NPor a pharmaceutically acceptable salt thereof, wherein X, Y, Z, and spacer are as defined in any of the preceding claims.

5. The lipid nanoparticle of any one of the preceding claims, wherein each spacer is independently selected from a linear alkylene, which may optionally be interrupted by one or more amide groups.

6. The lipid nanoparticle of any one of the preceding claims, wherein X is -NR-, wherein R is -H or -CH3.

7. The lipid nanoparticle of any one of the preceding claims, wherein the compound of Formula (I) has the structure of Formula (II):or a pharmaceutically acceptable salt thereof, wherein R, Y, and Z are as defined in any of the preceding claims, and wherein:A in each case independently represents a moiety comprising a linear chain of 1 to 16 atoms (e.g., selected from C, N, and O);ASG-105-PCT01-NPm in each case is independently selected from the integers from 1 to 6; and represents a carbon-carbon single bond or double bond.

8. The lipid nanoparticle of claim 7, wherein the compound of Formula (II) has the structure of Formula (Ila):or a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined in any of the preceding claims.

9. The lipid nanoparticle of claim 8, wherein the compound of Formula (II) has the structure of Formula (lib), Formula (lie), or Formula (lid) :"ASG-105-PCT01-NPor a pharmaceutically acceptable salt thereof, wherein A, m, R, Y, and Z are as defined in any of the preceding claims.

10. The lipid nanoparticle of any one of claims 7 to 9, wherein each A is independently selected from -(Ci-Cie)alkylene-, optionally wherein each A is -(CH2)e-.

11. The lipid nanoparticle of any one of claims 7 to 10, wherein m in each case is 2.

12. The lipid nanoparticle of any one of the preceding claims, wherein Y is a hydrophilic linker comprising a linear chain of about 50 to about 400 atoms, wherein the linear chain comprises carbon atoms and one or more heteroatoms independently selected from N and O.

13. The lipid nanoparticle of any one of the preceding claims, wherein Y comprises a linear polymer of polyethylene glycol moi eties, e.g. a linear polymer of between 24 and 86 polyethylene glycol moieties.ASG-105-PCT01-NP14. The lipid nanoparticle of any one of the preceding claims, wherein Y has the structure:a)in which * denotes the point of attachment to Z, wherein:n, p, and r are integers independently selected from 1 to 31;q is an integer selected from 10 to 110;R’ is selected from -H and -(Ci-Ce)alkyl;R” is selected from -H and -(Ci-Ce)alkyl; andR’” is selected from -H and -(Ci-Ce)alkyl.

15. The lipid nanoparticle of claim 14, wherein:the lipid nanoparticle is defined in part (a) of claim 14 and n is 4; p is 1; and q is an integer selected from 30 to 85 (e.g. from 35 to 45, from 40 to 50, or from 65 to 75); or the lipid nanoparticle is defined in part (b) of claim 14 and n is 1; p is 1; q is an integer selected from 30 to 55 or from 51 to 84; and r is 5.

16. The lipid nanoparticle of claim 14 or claim 15, wherein R’ is -H, and / or wherein R” is -H, and / or wherein R’” is -H.

17. The lipid nanoparticle of any one of the preceding claims, wherein Y has the structure:a)b)ASG-105-PCT01-NPin which * denotes the point of attachment to Z, wherein q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70).

18. The lipid nanoparticle of any one of the preceding claims, wherein Z is a hydrophobic lipidic anchor selected from a sterol and a lipid (e.g. an ether lipid, an ester lipid, or a phospholipid).

19. The lipid nanoparticle of claim 18, wherein Z is an ether lipid having the structureORa, e.g. having the structure ORawherein each Ra is independently a -(Ce-C24)alkyl group, e.g. a linear -(Ce-C24)alkyl group.

20. The lipid nanoparticle of claim 19, wherein each Rais n-octadecyl.

21. The lipid nanoparticle of any one of the preceding claims, wherein Y and Z together have the structure:wherein q is an integer selected from 30 to 85 (e.g. 39, 46, 68, or 70), and each Rais n-octadecyl.

22. The lipid nanoparticle of any one of the preceding claims, wherein the compound of Formula (I) is selected from:ASG-105-PCT01-NPASG-105-PCT01-NPASG-105-PCT01-NPand the pharmaceutically acceptable salts thereof.

23. The lipid nanoparticle of any one of the preceding claims, wherein the lipid matrix comprises:(a) ionizable lipid;(b) neutral lipid;(c) sterol; and(d) polymer-conjugated lipid.

24. The lipid nanoparticle of claim 23, wherein the ionizable lipid is selected from 1-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, bis(3-pentyloctyl) 9-((2-oxaspiro[3.3]heptan-6-yl)amino)heptadecanedioate, bis(3 -pentyloctyl) 9-((tetrahydro-2H-pyran-4-yl)amino)heptadecanedioate, bi s(3 -pentyl octyl) 9-(((tetrahydrofuran-3-yl)methyl)amino)heptadecanedioate, bis(3 -pentyloctyl) 9-(((tetrahydro-2H-pyran-4-yl)methyl)amino)heptadecanedioate, bi s(3 -pentyl octyl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, and combinations thereof.

25. The lipid nanoparticle of claim 23 or claim 24, wherein the neutral lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), and combinations thereof.ASG-105-PCT01-NP26. The lipid nanoparticle of any one of claims 23 to 25, wherein the sterol comprises (e.g. is) cholesterol.

27. The lipid nanoparticle of any one of claims 23 to 26, wherein the polymer-conjugated lipid is selected from DMPE-PEG2000, DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-C-DOMG, PEG-C-DOPG, and combinations thereof.

28. The lipid nanoparticle of any one of the preceding claims, wherein the lipid matrix comprises l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate, distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000.

29. The lipid nanoparticle of any one of claims 1 to 28, wherein the lipid nanoparticle comprises:a) from about 30 to about 75 mol% of ionizable lipid;b) from about 5 to about 20 mol% of neutral lipid;c) from about 20 to about 60 mol% of sterol;d) from 0 to about 5 mol% of polymer-conjugated lipid; ande) up to about 5 mol% of a compound of Formula (I) as defined in any one of claims 1 to 21,wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle (i.e. excluding the amount of nucleic acid cargo).

30. The lipid nanoparticle of claim 29, wherein the lipid nanoparticle comprises:a) about 50 mol% of l-(heptadecan-9-yl) 17-(undecan-3-yl) 9-((oxetan-3-ylmethyl)amino)heptadecanedioate;b) about 10 mol% of distearoyl phosphatidylcholine (DSPC);c) about 38.5 mol% of cholesterol;d) about (1.5 - x) mol% of DMG-PEG2000; ande) about x mol% of a compound of Formula (I) as defined in any one of claims 1 to 21,wherein x is less than or equal to about 1.0, and wherein each mol% is calculated based on the total amount of lipid matrix and compound of Formula (I) in the lipid nanoparticle.ASG-105-PCT01-NP31. The lipid nanoparticle of claim 30, wherein x is between about 0.01 and about 0.5, e.g. between about 0.025 and about 0.15, optionally wherein x is about 0.05.

32. The lipid nanoparticle of any one of the preceding claims, wherein the nucleic acid cargo comprises RNA, e.g. a mRNA.

33. The lipid nanoparticle of any one of the preceding claims, wherein:a) the z-average particle diameter of the lipid nanoparticles is from about 40 nm to about 90 nm, e.g. from about 50 nm to about 80 nm, from about 60 nm to about 70, such as about 64 nm to about 70 nm, as measured by dynamic light scattering;b) the poly dispersity of the lipid nanoparticle is less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1, such as between about 0.03 and about 0.08; and / orc) the encapsulation efficiency (EE) of the lipid nanoparticle is at least about 90%, e.g. at least about 95% such as between about 95% to about 97%.

34. A pharmaceutical composition comprising a plurality of the lipid nanoparticles of any one of the preceding claims.

35. The lipid nanoparticles of any one of claims 1 to 33, or the pharmaceutical composition of claim 34, for use in treating a disease or disorder in a subject in need thereof.

36. A method for increasing the uptake of a nucleic acid into an organ or tissue, wherein said organ or tissue expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising the use of a lipid nanoparticle of any one of claims 1 to 33, or the pharmaceutical composition of claim 34.

37. A process for the preparation of a lipid nanoparticle as defined in any one of claims 1 to 33, the process comprising the steps of:A) providing a first solution comprising the lipid matrix and the compound of Formula (I);B) providing a second solution comprising the nucleic acid cargo in water or an aqueous buffer; andASG-105-PCT01-NPC) combining the solution of (A) with the solution of (B) thereby to prepare the lipid nanoparticle.

38. A method for improving the ability of a lipid nanoparticle to be targeted to an organ or tissue which expresses or comprises the asialoglycoprotein receptor (ASGPR), the method comprising incorporating into the lipid nanoparticle a compound of Formula (I) as defined in any one of claims 1 to 22 (e.g. by a process as claimed in claim 37).

39. A compound of Formula (I) as defined in any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof.

40. The compound of claim 39, selected from:ASG-105-PCT01-NPASG-105-PCT01-NP41. A process for the preparation of a compound of Formula (I) as defined in claim 39 or claim 40, or a pharmaceutically acceptable salt thereof, wherein the process comprises reacting a compound of Formula A’wherein X and spacer are as defined in any one of claims 1 to 11, and wherein:ASG-105-PCT01-NPY’ is a linker precursor comprising a linear chain of about 2 to about 100 atoms (e.g., selected from C, N, and O) and a first reactive group; andQ is a hydroxyl protecting group or H,with a compound of Formula B’Y”-Z,wherein:Y” is a second linker precursor comprising a linear chain of about 48 to about 398 atoms (e.g., selected from C, N, and O) and a second reactive group,the process optionally further comprising a step of removing the hydroxyl protecting groups.

42. A compound of Formula A’ as defined in claim 41, or a pharmaceutically acceptable salt thereof.

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