Lipid nanoparticles

Abstract

Provided is lipid nanoparticle functionalised with a glycopolymer. The glycopolymer comprises at least four carbohydrate units, wherein each carbohydrate unit is independently selected from a monosaccharide, a disaccharide and an oligosaccharide. The lipid nanoparticle can be used for inducing an immune response in a subject.

Description

[0001] LIPID NANOPARTICLES

[0002] This invention relates generally to lipid nanoparticles, pharmaceutical compositions comprising lipid nanoparticles and uses thereof.

[0003] The COVID-19 pandemic has brought attention to RNA vaccines as a rapid and flexible platform for controlling emerging infections. The accelerated development of RNA vaccines has demonstrated their promising potential against such infectious diseases.

[0004] Self-amplifying RNA (saRNA) lipid nanoparticles (LNPs) have emerged as a crucial platform for mRNA-based vaccines and have shown promise in reducing the dose needed to induce a protective immune response. However, the transition from pre-clinical models to clinical trials is challenging, with some individuals not responding to saRNA vaccines. Further, these vaccine technologies require optimization, such as ameliorating their specific delivery to different tissues and cell types.

[0005] Whilst the immune response triggered by RNA vaccines can facilitate the recruitment and activation of antigen-presenting cells and cells from the adaptive immune system, excessive inflammation can hinder the translation process. Understanding the factors influencing the immune response to RNA vaccines is therefore crucial for their effective deployment.

[0006] There is a need to provide a means of enhancing the precision of vaccine delivery, as well as in optimizing the efficiency and efficacy of the formulations. Further, there is a need to minimize the amount of cargo / encapsulated material whilst enhancing cellular uptake. Moreover, there is a need to enhance the recruitment and activation of the subsequent immune response while minimizing the negative impact on antigen expression.

[0007] It is therefore a first non-exclusive object of the invention to provide a lipid nanoparticle that overcomes one or more drawbacks of the prior art.

[0008] Accordingly, a first aspect of the invention provides a lipid nanoparticle functionalised with a glycopolymer, wherein the glycopolymer comprises at least 4 carbohydrate units, wherein each carbohydrate unit is independently selected from a monosaccharide, a disaccharide and an oligosaccharide. All of the carbohydrate units present in the glycopolymer may be the same, or they may be different.

[0009] Each monosaccharide unit may be independently selected from glucose, fucose, mannose, galactose, N-acetyl-galactosamine or N-acetyl-glucosamine.

[0010] In some embodiments, each carbohydrate unit is a monosaccharide. In some embodiments, each carbohydrate unit is mannose.

[0011] Each disaccharide unit may be independently selected from sucrose, lactose and maltose.

[0012] In some embodiments, each carbohydrate unit is a disaccharide. In some embodiments, each disaccharide is lactose.

[0013] In some embodiments, each carbohydrate unit is an oligosaccharide.

[0014] As used herein, the term “glycopolymer” should be understood to mean a synthetic polymer with pendant carbohydrate groups. The glycopolymer according to the invention comprises a polymer backbone with 4 or more pendant carbohydrate units.

[0015] As used herein, the term “oligosaccharide” should be understood to mean a carbohydrate chain formed from 3 to 10 monosaccharides. In some embodiments, the oligosaccharide comprises no more than 8, no more than 6, no more than 5, no more than 4 or no more than 3 monosaccharides. For example, the oligosaccharide may be formed from 3, 4, 5, 6, 7, 8, 9 or 10 monosaccharides, such as the monosaccharides described herein (e.g. mannose). The monosaccharides present in the oligosaccharide may all be the same, or they may be different.

[0016] The lipid nanoparticle may comprise a first molecule comprising a lipophilic moiety. The first molecule may be functionalised with the glycopolymer.

[0017] The first molecule may be incorporated into the lipid nanoparticle by virtue of its lipophilic moiety. The first molecule may serve to anchor the glycopolymer to the lipid nanoparticle. Thus, the first molecule may be described as an “anchor” or an “anchor molecule”. The term “lipophilic moiety”, as used herein, refers to a portion of the first molecule which has a tendency to combine with or dissolve in lipids, fats, oils and non-polar solvents (e.g. hexane or toluene). Lipophilic molecules, or lipophilic moieties of molecules, dissolve more easily in lipids than in water. Examples of lipophilic moieties include non-polar functional groups, e.g. aryl and alkyl groups (e.g. fatty acid chains and fused cycloalkyl groups).

[0018] The first molecule may be a sterol, a steroid or a phospholipid.

[0019] The sterol may be cholesterol (e.g. cholesterol chain transfer agent (CTA)), p-sitosterol, sigmasterol, campesterol, brassicasterol, cholestanol, a-tocopherol, fecosterol, sitosterol, ergosterol, tomatidine or ursolic acid.

[0020] The phospholipid may be phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA) or phosphatidylcholine (PC), 2- lysophosphatidyl choline or sphingomyelin.

[0021] The phospholipid may include one or more fatty acid moieties selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0022] The phospholipid may be selected from the group consisting of 1 ,2-dilinoleoyl-sn-glycero- 3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2- diundecanoyl-sn-glycero-phosphocholine (DLIPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl- sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1 ,2-dioleoyl-sn-glycero-3- phosphoethanola mine (DOPE), 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), and sphingomyelin.

[0023] The first molecule may be functionalised with a single glycopolymer. Alternatively, a single glycopolymer may functionalise two or more first molecules. For example, two first molecules may be connected by a glycopolymer. It may be that a first molecule is provided at each end of the glycopolymer chain.

[0024] In an embodiment of the invention there is provided a lipid nanoparticle comprising a sterol (e.g. cholesterol) functionalised with a glycopolymer, wherein the glycopolymer comprises at least 4 carbohydrate units (e.g. at least 4 monosaccharides).

[0025] Advantageously, the glycopolymer provides a synthetic multivalent carbohydrate presenting macromolecule that can be incorporated onto a lipid nanoparticle. The glycopolymer may function as a targeting ligand, for example a targeting ligand which is capable of binding to cell-surface receptors. Conveniently, the glycopolymer may facilitate delivery of molecules, e.g. nucleic acids, to cells.

[0026] The lipid nanoparticle may comprise an ionizable lipid, a helper lipid and / or a PEG lipid. In some embodiments, the lipid nanoparticle comprise an ionizable lipid, a helper lipid and a PEG lipid.

[0027] The ionizable lipid may be selected from 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1- (9Z, 12Z)-9, 12-octadecadien-1 -yl-10, 13-nonadecadien-1-yl ester (DLin-MC3-DMA), 1 , 1 '- [[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1- piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), dimethyldioctadecylammonium (DDA), dioleoyl-3-trimethylammonium propane (DOTAP) or cephalin.

[0028] The helper lipid may be selected from 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), hydrogenated soy phosphatidylcholine (HSPC), phosphatidylcholine (POPC), diacylglycerophosphoethanolamine (DAPE), 1 ,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE) or phosphatidylethanolamine (POPE).

[0029] The PEG lipid may have a chain length of from 1000 to 4000. The PEG lipid may be DMPE-PEG-Maleimide. The PEG lipid may be, for example, 1 ,2-dimyristoyl-rac-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), 1 , 2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG2000) or 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

[0030] The ionizable lipid, the helper lipid, the first molecule, e.g. sterol (for example cholesterol), and the PEG lipid may be present in the lipid nanoparticle at a molar ratio of: from 25 to 75 ionizable lipid: from 5 to 20 helper lipid: from 10 to 60 first molecule, e.g. sterol: from 0.5 to 20 PEG lipid.

[0031] In an embodiment, the ionizable lipid, the helper lipid, the first molecule, e.g. sterol (for example cholesterol), and the PEG lipid are present at a molar ratio of 50:10:38.5:1.5 or 35:16:46.5:2.5 or 56:12:22:10.

[0032] The weight ratio of PEG lipid:glycopolymer may be from 1 :1 to 1 :10, e.g. 1 :1, 1 :2, 1 :3, 1 :4 or 1 :5.

[0033] The lipid nanoparticle may have an average particle size in the range of from 2 to 400 nm, for example from 3 to 350 nm, from 4 to 300 nm, from 5 to 250 nm, from 10 to 200 nm, from 15 to 190 nm, from 20 to 180 nm, from 30 to 170 nm, from 40 to 165 nm, from 50 to 160 nm, from 60 to 155 nm or from 70 to 150 nm. The particle size can be determined using the methods described herein.

[0034] The lipid nanoparticle may have a substantially spherical morphology. Alternatively, the lipid nanoparticle may have a protrusion, or “bleb”. A lipid nanoparticle having a protrusion may be described as having “currant bun” morphology. The glycopolymer may comprise polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone (PVP), poly(2-oxazoline) or poly(2-oxazine), or mixtures thereof.

[0035] In other words, the glycopolymer may have a backbone formed from monomers of acrylate, methacrylate, acrylamide, methacrylamide, vinylpyrrolidone, 2-oxazoline or 2- oxazine , or mixtures thereof.

[0036] In some embodiments each monomer of the glycopolymer comprises a pendant carbohydrate unit. It will be appreciated that, in such embodiments, the spacing between adjacent carbohydrate units will depend on the nature of the glycopolymer backbone. In some embodiments, adjacent carbohydrate units are separated by 2, 3, 4 or more atoms. For example, the glycopolymer backbone may be formed from acrylate or methacrylate monomers, separating the carbohydrate units by two carbon atoms. In another example, the backbone may be formed from 2-oxazoline monomers, separating the carbohydrate units by two carbon atoms and one nitrogen atom. In a further example, the backbone may comprise 2-oxazine monomers, separating the carbohydrate units by three carbon atoms and one nitrogen atom.

[0037] The lipid nanoparticle may comprise an ionizable lipid, a helper lipid, a PEG lipid and a first molecule comprising a lipophilic moiety, wherein the first molecule is functionalised with a glycopolymer, wherein the glycopolymer comprises n carbohydrate units independently selected from a monosaccharide, a disaccharide or an oligosaccharide in accordance with any of the following embodiments:

[0038] (Ai) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DSPC, and n is 10;

[0039] (Aii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DSPC, and n is 20;

[0040] (Aiii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DSPC, and n is 30;

[0041] (Aiv) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DSPC, and n is 40;(Bi) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPC, and n is 10;

[0042] (Bii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPC, and n is 20;

[0043] (Biii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPC, and n is 30;

[0044] (Biv) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPC, and n is 40;(Ci) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPE, and n is 10;

[0045] (Cii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPE, and n is 20; (Ciii) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPE, and n is 30;

[0046] (Civ) the ionizable lipid is DLin-MC3-DMA, the helper lipid is DOPE, and n is 40;

[0047] (Di) the ionizable lipid is C12-200, the helper lipid is DSPC, and n is 10;

[0048] (Dii) the ionizable lipid is C12-200, the helper lipid is DSPC, and n is 20;

[0049] (Diii) the ionizable lipid is C12-200, the helper lipid is DSPC, and n is 30;

[0050] (Div) the ionizable lipid is C12-200, the helper lipid is DSPC, and n is 40;(Ei) the ionizable lipid is C12-200, the helper lipid is DOPC, and n is 10;

[0051] (Eii) the ionizable lipid is C12-200, the helper lipid is DOPC, and n is 20;

[0052] (Eiii) the ionizable lipid is C12-200, the helper lipid is DOPC, and n is 30;

[0053] (Eiv) the ionizable lipid is C12-200, the helper lipid is DOPC, and n is 40;(Ei) the ionizable lipid is C12-200, the helper lipid is DOPE, and n is 10;

[0054] (Eii) the ionizable lipid is C12-200, the helper lipid is DOPE, and n is 20;

[0055] (Eiii) the ionizable lipid is C12-200, the helper lipid is DOPE, and n is 30;

[0056] (Eiv) the ionizable lipid is C12-200, the helper lipid is DOPE, and n is 40.

[0057] In any of embodiments (Ai) to (Eiv) above, the first molecule may be a sterol, for example cholesterol.

[0058] In any of embodiments (Ai) to (Eiv) above, the first molecule may be a phospholipid, for example phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA) or phosphatidylcholine (PC).

[0059] In any of the embodiments (Ai) to (Eiv) above the carbohydrate unit may be a monosaccharide independently selected from glucose, fucose, mannose, galactose, N- acetyl-galactosamine, N-acetyl-glucosamine. In some embodiments of (Ai) to (Eiv) above the carbohydrate unit is mannose.

[0060] In any of the embodiments (Ai) to (Eiv) above the PEG lipid may be DMPE-PEG- Maleimide, e.g. DMPE-PEG2000, or DMG-PEG-2000 or DSPE-PEG-2000.

[0061] In some embodiments of (Ai) to (Eiv) above, the first molecule is a sterol, e.g. cholesterol, the carbohydrate unit is mannose and the PEG lipid is DMPE-PEG-Maleimide, e.g. DMPE-PEG2000. In some embodiments, the lipid nanoparticle comprises a sterol, e.g. cholesterol, the ionizable lipid is independently selected from MC3 and C12-200, the helper lipid is independently selected from DSPC, DOPC and DOPE, the PEG lipid is DMPE-PEG2000, the glycopolymer comprises 10, 20, 30 or 40 carbohydrate units and each carbohydrate unit is mannose,

[0062] The glycopolymer may comprise from 5 to 1000 carbohydrate units, for example from 6 to 900, from 7 to 800, from 8 to 700, from 9 to 600, from 10 to 500, from 12 to 400, from 15 to 300, from 20 to 200 or from 30 to 100 carbohydrate units, e.g. from 5 to 100, from 10 to 90, from 15 to 80, from 20 to 75, from 25 to 70, from 30 to 60 or from 40 to 50, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 carbohydrate units.

[0063] Advantageously, the presence of the glycopolymer may not substantially change the physicochemical properties, stability and / or toxicity of the lipid nanoparticle.

[0064] The lipid nanoparticle may encapsulate one or more molecules. The one or more molecules may be independently selected from a nucleic acid, a protein, a polysaccharide, a lipid, a radioactive substance, a therapeutic agent, a prodrug, a nutritional supplement, a biomarker, an enzyme (e.g. a restriction enzyme), a dye or any combination thereof.

[0065] The nucleic acid(s) may be selected from deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination thereof.

[0066] The DNA may be selected from plasmid DNA, vector DNA, A-DNA, Z-DNA, C-DNA ,D- DNA or E-DNA.

[0067] The RNA may be selected from messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), self-amplifying RNA (saRNA), small or short interefering RNA (siRNA), or guide RNA.

[0068] The lipid nanoparticle may encapsulate a zinc finger nuclease (ZFN), a CRISPR / Cas system, a transcription activator-like effector nuclease (TALEN), or a meganuclease. Advantageously, the functionalisation of the lipid nanoparticles with the glycopolymer enables increased uptake of the encapsulated molecule(s), e.g. genetic material, in desired organs in comparison to non-functionalized lipid nanoparticle formulations, i.e. lipid nanoparticles absent the glycopolymer.

[0069] According to a second aspect of the invention there is provided a pharmaceutical composition comprising a lipid nanoparticle according to the first aspect of the invention and a pharmaceutically acceptable excipient or carrier.

[0070] A “pharmaceutically acceptable excipient or carrier,” as used herein, refers to any ingredient other than lipid nanoparticle described herein (for example, a vehicle capable of suspending or dissolving the lipid nanoparticle having the properties of being nontoxic and non-inflammatory in a patient. Suitable pharmaceutically acceptable excipients will be known to those skilled in the art. The excipient or carrier may comprise one or more of: a stabiliser, a dye, a preservative, a suspending or dispersing agent, an ion exchanger an anti-oxidant, a solvent (e.g. water or saline) or a buffer.

[0071] The pharmaceutical composition may be formulated for oral or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, transdermal, inhaled / aerosolized, intraarterial, intrathecal, intratumoral, intracystic, peritumoral, intraperitomeal, intraluminal, intrapleural) administration.

[0072] The pharmaceutical composition may be formulated as a tablet, a solution, a suspension, a dispersion, a cream, a gel, a paste, a lyophilized powder or a suppository. When being administered orally, the pharmaceutical composition can be in the form of a pill, tablet, or capsule. A composition formulated for parenteral administration may include an aqueous or non-aqueous solution (e.g. a sterile solution). The solution may contain one or more anti-oxidants, buffers, bacteriostats, or solutes.

[0073] In some embodiments, the pharmaceutical composition is a vaccine composition. In such embodiments, the lipid nanoparticle may encapsulate a nucleic acid, for example an RNA molecule. In particular, the lipid nanoparticle may encapsulate an saRNA molecule. According to a further aspect of the invention there is provided the lipid nanoparticle according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for use as a medicament.

[0074] The lipid nanoparticle of the invention, or a pharmaceutical composition comprising the lipid nanoparticle, finds particular utility as a vaccine, and / or in gene therapy.

[0075] According to a further aspect of the invention there is provided the lipid nanoparticle according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for use in the treatment and / or prevention of a disease or disorder selected from the group consisting of: a genetic disease or disorder; an infectious disease or disorder; an autoimmune disease or disorder; a metabolic disease or disorder; or a cancerous disease or disorder.

[0076] For example, the lipid nanoparticle or pharmaceutical composition may be for use in the treatment and / or prevention of arterial dysfunction, heart disease, cystic fibrosis, alpha-1 antitrypsin deficiency, hemophilia, beta thalassemia, sickle cell disease, neuroendocrine tumours, pancreatic cancer, gastrointestinal neuroendocrine tumours, adrenocortical carcinoma or liver cancer.

[0077] The infectious disease may be caused by an infectious agent being selected from a virus, a bacterium, a fungus, or a protozoan.

[0078] In some embodiments, the infectious disease is a viral infection. The viral infection may be a pulmonary viral infection. In certain embodiments the viral infection may be caused by or associated with a virus selected from respiratory syncytial virus, influenza virus, parainfluenza virus, human metapneumovirus, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus OC43, Semliki Forest Virus, a human rhinovirus (HRVs) and human adenovirus (HAdV).

[0079] In certain embodiments the viral infection is caused by or associated with a Pneumoviridae virus, for example a Human respiratory syncytial virus (HRSV) (e.g. HRSV-A2, HRSV-B1 or HRSV-S2). In certain embodiments the viral infection is caused by or associated with a Coronaviridae virus. In certain embodiments the viral infection is caused by or associated with a virus is selected from Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Preferably the viral infection is caused by or associated with a Betacoronavirus. Thus is certain embodiments the viral infection is caused by or associated with a virus is selected from severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-229E, HCoV-NL63, HCoV- OC43 and HCoV-HKU1. In a particular embodiment the viral infection is caused by or associated with SARS-CoV-2. This may be that the viral infection is COVID-19.

[0080] In some embodiments the viral infection is caused by or associated with an influenza virus.

[0081] The viral infection may also be caused by or associated with a virus selected from Flaviviridae (e.g. Zika virus (ZIKV), dengue (e.g. DENV 1-4), West Nile virus (WNV), yellow fever virus (YFV, e.g. yellow fever 17D virus), and Japanese encephalitis virus (JEV), Hepatitis C virus (HCV), Filoviridae (e.g. Ebola virus)), Togaviridae (e.g. Alphaviruses such as Chikungunya virus (CHIKV), Sindbis virus and Ross River virus), Herpes (e.g. y-herpesvirus, Human herpesvirus 8, herpesvirus 1 and herpesvirus 2) and Adenoviridae (e.g. Human adenoviruses (HAdVs)).

[0082] According to a further aspect of the invention there is provided the lipid nanoparticle according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for use in the preparation of a gene therapy medicament or a vaccine.

[0083] In an embodiment, the gene therapy medicament or vaccine may be for use in the treatment of hypercholesterolemia, cancer (e.g. solid tumours, advanced or metastatic cancer e.g. advanced or metastatic pancreatic cancer), hepatitis B, advanced hepatic fibrosis, cystic fibrosis, hemophilia A and B or duchenne muscular dystrophy (DMD).

[0084] In an embodiment, the gene therapy medicament or vaccine may be administered in an amount effective to produce an antigen-specific immune response. According to a further aspect of the invention there is provided the lipid nanoparticle according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for use in a method of inducing an immune response in a subject.

[0085] Advantageously, the functionalisation of the lipid nanoparticle with a glycopolymer may provide enhanced cellular uptake (e.g. by immune system cells, dendritic cells, lymphocytes (T cells, B cells, and NK cells), neutrophils and / or monocytes / macrophages), compared to an equivalent lipid nanoparticle lacking the glycopolymer.

[0086] The term ‘subject’ as used herein may be for example a mammal (e.g. a mouse, rat, rabbit, cat, dog, sheep, horse, goat, pig, guinea pig or chimpanzee), preferably a human.

[0087] According to a further aspect of the invention there is provided the lipid nanoparticle according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for use in enhancing immunogenicity, modulating (e.g. enhancing) an inflammatory response, inducing antibody release or modulating (e.g. stimulating) cytokine release in a subject.

[0088] In an embodiment, modulating cytokine release may comprise stimulating (e.g. increasing) cytokine release. The cytokine may be an interferon, an interleukin, a chemokine, a mesenchymal growth factor, a tumor necrosis factor or an adipokine. The cytokine may be for example, ENA-78, GM-CSF, IFN-a, IFN-b, IL-1a, IL-1b, IL-6, IP-10, MCP-1, MDC, MIP-1 a, MIP-1 (3, Eotaxin-2, GRO-alpha, MCP-3, KC / GRO, IL-1 (subdivided in IL-1 a and IL-1 P), TNF-a, , IL-2, IL-12, IL-17, IL-18, IFN-y.

[0089] According to a yet further aspect of the invention there is provided a method of delivering an encapsulated molecule, e.g. a nucleic acid, to a subject, the method comprising administering to said subject an effective amount of a lipid nanoparticle according to the first aspect of the invention or the pharmaceutical preparation according to the second aspect of the invention.

[0090] According to a yet further aspect of the invention there is provided a method of inducing an immune response in a subject, the method comprising administering to the subject an effective amount (e.g. an immune stimulatory amount) of a lipid nanoparticle composition according to the first aspect of the invention or the pharmaceutical preparation according to the second aspect of the invention.

[0091] Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. For the avoidance of doubt, the terms “may”, “and / or”, “e.g.”, “for example” and any similar term as used herein should be interpreted as non-limiting such that any feature so-described need not be present. Indeed, any combination of optional features is expressly envisaged without departing from the scope of the invention, whether or not these are expressly claimed. The applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to amend any originally filed claim to depend from and / or incorporate any feature of any other claim although not originally claimed in that manner.

[0092] Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:

[0093] Figure 1 is a schematic representation for RAFT polymerization of a reference polymer;

[0094] Figure 2 is a schematic representation of the monomer compositions of homopolymers according to embodiments of the invention;

[0095] Figures 3A and 3B are UV-vis spectra showing the binding to a model lectin extracted by the jack-bean (ConcanavalinA) of the glycosylated LNPs;

[0096] Figures 4A-4L are graphs showing SPR dilutions of the reference polymers and glycopolymers of embodiments of the invention with three different human lectins;

[0097] Figures 5 to 7 show cryo-TEM images of LNPs produced with different lipid compositions;

[0098] Figures 8A-8F are graphs showing the physicochemical characteristics of LNPs with and without glycopolymers;

[0099] Figures 9-11 are graphs showing the effect of glycopolymer addition on saRNA expression in vitro; Figures 12A-12D are graphs showing the effect of glycopolymer addition on the levels of saRNA expression in vivo;

[0100] Figures 13A-13M show the cytokine profile induced by stimulation of human PBMCs with C12-200 DOPC LNPs with and without glycopolymers;

[0101] Figures 14A-14J show cytokine profile induced by vaccination in Balb / c mice with C12-200 DOPC LNPs with and with and without glycopolymers;

[0102] Figures 15A to 15M show the cytokine profile induced by stimulation of human PBMCs with MC3 DSPC LNPs with and without glycopolymers;

[0103] Figures 16A to 16M show the cytokine profile induced by stimulation of human PBMCs with C12-200 DSPC LNPs with and without glycopolymers;

[0104] Figure 17A shows in vivo saRNA expression levels in mice injected with injected with MC3 and C12-200 LNPs encapsulating firefly luciferase (fLuc) saRNA at a dose of 10 pg. Figures 17B and 17C are graphs showing the total expression signal and the expression in organs normalised by muscle expression at 24 hours;

[0105] Figure 18 is a graph showing the change in hydrodynamic diameter of glycosylated LNPs after incubation in cell culture medium; and

[0106] Figures 19A and 19B show saRNA expression levels in human skin explants transfected with mannosylated LNPs;

[0107] Figures 20A to 20D show binding and neutralizing antibody titres following immunization with MC3 and C12-200 LNPs with glycopolymers in vivo;

[0108] Figures 21 A to 21 D show the in vivo cytokine response to mannosylated saRNA LNP formulations at 4- and 24-hours post-immunization; and

[0109] Figures 22A to 22D show the effects of in vivo influenza challenge in animals vaccinated with mannosylated LNPs. Figures 22A-22B show the change in body weight. Figures 22C to 22D show lung viral load.

[0110] Example 1

[0111] Referring now to Figure 1 , there is shown a schematic representation for RAFT polymerization to produce a reference polymer R1 (see Table 1).

[0112] Referring now to Figure 2 there is shown a schematic representation of the monomer compositions of glycopolymers according to embodiments of the invention. Polymer Synthesis and Characterization. A library of four polymers (R1 , P1-P3) were synthesized using RAFT polymerization. Three of these (P1-P3) were prepared by varying the content of Acetylated Mannose Acrylamide (10, 20 and 30 units, Table 1), and subsequently deprotected to obtain the final polymers (D1-D3) used in this study. Furthermore, Chol-HEAA20 (R1) was prepared as control. Polymerizations were performed with the Cholesterol CTA at 70°C using thermal initiation with AIBN and the general conditions were selected as [CTA] / [initiator] =

[0010] / [1],

[0113] Table 1. Structure and characterization of the synthesized polymers. a Calculated using the Theoretical Molar Mass Calculation Equation 1 , as set out in the materials and methods. b Determined using DMF-GPC

[0114] 1H NMR spectrums were analysed and revealed successful polymer deacetylation with retention of the sugar moieties as well as the cholesterol end-group. The peaks observed at 5 1.75 - 2.25 for acetyl protons in the NMR spectra of protected polymers were completely absent in the case of deprotected polymers. DMF-GPC chromatograms were analysed and showed a clear shift of the retention time after deprotection, due to the elimination of protecting functional groups. The monomodal chromatograms and negligible increase of polydispersity index after deacetylation indicates the absence of any side-reactions during deprotection. The deprotected glycopolymers were readily soluble in water compared to their precursor protected ones, which were insoluble in water.

[0115] Turbidimetry assay using ConcanavalinA (ConA) and LNPs: Turbidimetry was used to determine the rate of lectin binding between the glycomaterial and the protein. All the LNPs formulations were diluted in freshly prepared HBS buffer solutions (0.25 mg / ml) and then added into a ConA solution (90 uM) and placed in the LIV spectrometer. The absorbance of the mixture was recorded at 420 nm for 15 min every 0.10 s (see Figure 3). The formulation containing polymer D3 showed the highest binding signal with both C12- 200 LNP and MC3 LNP (see Figures 3A and 3B). The controls comprising the LNP alone (i.e. no glycopolymer) and the LNP with reference polymer R1 showed no binding.

[0116] Surface Plasmon Resonance Analysis using MBL, DC-SIGN, L-SIGN and Cholesterol Functionalized Polymers: In the context of human gene delivery and therapeutic applications, the binding of the decorating glycopolymers with the C-type dendritic cell lectin (DC-SIGN and L-SIGN), and the C-type Mannose-binding lectin (MBL) was investigated. MBL belongs to a class of soluble collectins found in serum, playing an important role in innate immunity and binding to a number of pathogens in the body such as fungi, bacteria, and viruses. Moreover, MBL activates the complement lectin pathway upon binding to highly glycosylated SARS-CoV-2 spike protein. L-SIGN and DC-SIGN are presented at the cell surface as tetramers and they are well-known to play a key role in viral infections. DC-SIGN is a trans-membrane protein expressed on the surface of immature dendritic cells, responsible for antigen capture and presentation, uptake and intracellular signalling. L-SIGN, the liver / lymph node-specific homologue of DC-SIGN, has been shown to act as a SARS-COV-2 entry receptor and perform critical functions in the angiogenic responses of endothelial cells.

[0117] The three lectins, MBL (Figures 4A-4C), DC-SIGN (Figures 4D-4F) and L-SIGN (Figures 4G-4I), were immobilized on a gold chip and the binding assays were recorded in a concentration range from 40.0 to 1.25 pM.

[0118] The glycopolymers D1 (Figures 4A, 4D, 4G), D2 (Figures 4B, 4E, 4H) and D3 (Figures 4C, 4F, 4I) showed high binding affinity toward the three mannose specific lectins MBL, DC-SIGN and L-SIGN. Reference polymer R1 with hydroxyl groups instead of carbohydrate moieties was prepared and used as negative control (see Figures 4J, 4K and 4L).

[0119] Advantageously, the self-amplifying mRNA lipid nanoparticles vaccines decorated with carbohydrates can enhance cellular uptake toward immune system cells.

[0120] From the calculated kinetic values, the dissociation rates (kd) of polymers D1 , D2 and D3 are shown to decrease (see Table 2). Table 2. Kinetic values (ka, kd, Rmax, KA, KD) obtained from fitting of experimental SPR curves with 1 :1 Langmuir binding model.

[0121] Surprisingly, the association kinetics (ka) toward all the three lectins of the glycopolymer containing 20 sugar moieties (D2) was lower compared to that of glycopolymer with 10 mannose units (D1).

[0122] Without being bound by theory, it is suggested that the introduction of a lipophilic motif may lead to a coil conformation, at a certain polymer chain length (for example chain lengths in the range from 4 to 200, from 5 to 50 or from 10 to 30), which affects the binding affinity. To exclude any non-specific binding, reference polymer R1 with pendant hydroxyl groups was tested (see Figures 4J, 4K and 4L). Although some non-specific binding was observed, due to the presence of the hydroxyl groups and cholesterol, the response traces are significantly lower than those observed with the glycopolymers.

[0123] Cryo-TEM of LNPs

[0124] The morphology and size of the LNPs were assessed using cryo-TEM.

[0125] Figure 5 shows cryo-TEM images of LNP produced with lipid composition MC3 / DSPC / Chol / DMPE-PEG2000 (50:10:38.5:1.5; mol / mol), where 5A is a representative image of unmodified saRNA LNPs, 5B is R1 saRNA LNPs, 5C is D1 saRNA LNPs, 5D is D2 saRNA LNPs and 5E is D3 saRNA LNPs.

[0126] Figure 6 shows cryo-TEM images of LNP produced with lipid composition C12- 200 / DSPC / Chol / DMPE-PEG2000 (35:16:46.5:2.5; mol / mol), where 6A is a representative image of unmodified saRNA LNPs, 6B is R1 saRNA LNPs, 6C is D1 saRNA LNPs, 6D is D2 saRNA LNPs and 6E is D3 saRNA LNPs.

[0127] Figure 7 shows cryo-TEM images of LNP produced with lipid composition C12- 200 / DOPC / Chol / DMPE-PEG2000 (35:16:46.5:2.5; mol / mol), where 7A is a representative image of unmodified saRNA LNPs, 7B is R1 saRNA LNPs, 70 is D1 saRNA LNPs, 7D is D2 saRNA LNPs and 7E is D3 saRNA LNPs.

[0128] The scale bar in each of Figures 5 to 7 is 200 nm.

[0129] The particles all exhibited particle diameters equivalent to the DLS particle size characterization (70-150 nm). The morphology was observed to change as a function of the lipidic composition. Notably, the LNPs prepared with MC3 and DSPC present a protruded interesting feature or “blebs” (see Figure 5). However, such blebs are not observed for LNP formulated with C12-200. This suggests that different ionizable lipids may lead to different nanostructure packing with an electron dense interior (Figure 6, Figure 7).

[0130] The addition of reference polymer R1 and glycopolymers D1 , D2 and D3 did not affect the particle morphology.

[0131] Effect of glycopolymers on physicochemical profile of LNPs

[0132] Referring now to Figure 8, addition of glycopolymers was shown not to impact the desired physicochemical profile. LNPs prepared with MC3 or C12-200 ionisable lipids exhibited both a mean average diameter of 60 nm, while the addition of the R1 reference polymer led to the highest increase in size for both formulations, reaching around 120 nm in the case of MC3 LNPs (Figure 8A). Addition of the glycopolymers D1 , D2, and D3 resulted in a moderate increase in size, with LNPs having around 70-90 nm (Figures 8A, 8C, 8E). Overall, addition of the glycopolymers was not shown to negatively impact the LNP physicochemical characteristics, which maintained standard size, PDI, and slightly negative zeta potential (Figures 8B, 8D, 8F). The addition of glycopolymers also did not affect the RNA encapsulation efficiency (EE), and all LNPs were observed to have more than 80% EE.

[0133] Effect of glycopolymer on saRNA expression in vitro: As the glycopolymers are known to bind lectin receptors on the surface of immune cells, we transfected four different cell lines with LNPs with and without glycopolymers. HEK 293T / 17 cells are not interferon-competent and thus have little regulation of saRNA expression and hence showed the highest levels of RNA expression (see Figures 9A,10A,11A). HeLa cells are interferon-competent and saRNA expression levels were shown to decrease approximately 10-fold (Figure 9B, 10B). However, C12-200 DOPC seems to be an exception to this rule as it was shown to have similar levels of saRNA expression in both HEK and HeLa cells (Figure 11 B).

[0134] Since these formulations serve as a proxy for a saRNA-LNP vaccine, we investigated expression levels in human skeletal muscle cells (hSkMC) as vaccine’s administration route is usually intramuscular, and in THP-1 cells, which are a monocytic cell line that may indicate the level of RNA expression in immune cells. Additionally, monocytic cells are also known for expressing high levels of lectin receptors.

[0135] All formulations were shown to have good levels of expression in hSkMC, with levels being comparable to HeLa cells (Figure 9C,10C). Again, C12-200 DOPC exhibited the highest levels of expression compared to the other two formulations (Figure 11C). The expression levels of saRNA in the monocytic cell line, THP-1 , were the lowest across all four cell lines, being 10- to 100-fold less compared to the other cell types (Figures 9D,10D,11 D). However, the addition of glycopolymers in the MC3 formulation showed significantly higher levels of saRNA expression compared to the standard LNP, and levels were comparable to lipofectamine (Figure 11 D).

[0136] Addition of glycopolymers on the LNP surface maintains the immunogenicity of LNP formulations in vivo and have similar protective capacity

[0137] As the addition of glycopolymers are shown to provide some increase in the levels of saRNA expression, we proceeded to analyse the immune response in vivo. As the C12- 200 DOPC formulation resulted in the highest levels of expression in vitro, as well as it showed good immune response across C12-200 formulations with different helper lipids, mice were immunised with C12-200 DOPC encapsulating HA-encoding saRNA.

[0138] BALB / c mice were immunised with prime and boost vaccinations of C12-200 DOPC LNPs encapsulating 1 pg of HA saRNA, 21 days apart. Blood samples were collected for both ELISA and antibody neutralisation assays, and at the end of the study, mice were challenged with Cal / 09 influenza virus. The percentage of body weight loss was followed daily, and lungs were collected at the end of the study to determine their viral load.

[0139] The naive group (PBS) lost 14% of body weight by day 5 (see Figure 12A) and were euthanised in accordance with the humane endpoints for challenge studies.

[0140] All vaccinated mice were still protected by day 5, having less than 2% of body weight loss on average (see Figure 12A). The viral load in lungs corroborate these findings, with PBS group showing significantly higher viral load compared to the vaccinated mice.

[0141] The HA IgG antibody titers (see Figure 12B) are in accordance with these results, in which all vaccinated groups exhibited similar levels of antibody, with group D1 having the highest antibody titer in absolute numbers (-940,000 ng / mL) compared to the control (-688,000 ng / mL), while the other groups had equivalent antibody titers (-760,000 ng / mL). These results are reinforced by the levels of neutralising antibodies (see Figure 12D), in which all vaccinated mice showed similar levels of neutralising antibodies, with group D1 showing the highest levels of neutralising antibodies in absolute numbers, compared to the control and R1 groups.

[0142] MC3 was observed to have a better effect when combined with glycopolymer D3 in vitro).

[0143] Induction of cytokines after vaccination differs between mice and human models

[0144] Referring now to Figures 13 to 16, the cytokine profile induced by the different formulations were investigated in both mice sera and human peripheral blood cells (PBMCs). Both LNPs and RNA are known to induce some inflammation and it is important to assess cytokines related to the innate immune response, as an exacerbation of the inflammatory response may dampen the levels of RNA expression. A panel of more than 10 different cytokines and chemokines was analysed by MSD assay.

[0145] Human PBMCs were shown to have a significantly higher production of cytokines after stimulation with LNPs based glycopolymers, unlike the control. Mice sera, conversely, exhibited low levels of cytokine induction after vaccination.

[0146] Accordingly, it has been shown that what is observed in animal models may not directly translate to human responses. Further, the results indicate the importance of adding carbohydrate-based polymers as tools to enhance the inflammatory response from the immune system.

[0147] MIP-ip levels were high in human PBMCs, while, levels of GM-CSF were higher in mice sera. MIP-ip can induce the release of other pro-inflammatory cytokines, while GM-CSF can induce differentiation of precursor cells into macrophages, as well as lead to the activation of T cells.

[0148] In human PBMCs, the addition of glycopolymers was shown to consistently result in higher levels of cytokines compared with the formulations without glycopolymers.

[0149] Cytokines are small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells. Advantageously, understanding and being able to tune the specific release of cytokines can help in regulating the maturation, growth, and responsiveness of particular cell populations in host immune responses to infection and reproduction. Cytokine release has also been associated with the progress of inflammation. It has been observed that cytokine production following immunization was linked to an enhanced antibody immune response and protection against infection, this suggests that vaccine-induced inflammation is necessary to elicit an adaptive immune reaction. Studying the difference between innate sensing of RNA in mice and humans and the consequent cytokine profile is critical in further optimizations of RNA vaccines.

[0150] GM-CSF: The cytokine GM-CSF, or granulocyte-macrophage colony-stimulating factor, is a glycoprotein secreted by different cell types (macrophages, T cells, mast cells, natural killer cells, endothelial cells, fibroblasts). Its function is directly correlated to myeloid cells: firstly, in the stage of differentiation and proliferation of myeloid precursors in the bone marrow, and secondly during the activation and migration of such cells to inflammation sites. An imbalance in its production and signalling may result in a harmful inflammatory process. All LNPs had low levels of this cytokine being secreted upon stimulation of human PBMCs.

[0151] MIP-1a: MIP-1a is a macrophage-inflammatory protein. This is a monocyte-specific cytokine that can also act a chemoattractant. In human PBMCs, cells stimulated with C12- 200 LNPs had high levels of this cytokine when combined with D2 and D3 glycopolymer, while the levels for MC3 LNPs was low. MIP-1 / 3: MIP-ip is also a macrophage-inflammatory protein that is highly related to MIP- 1a but has a diverging signalling pathway. It belongs to CC chemokine subgroup and activates human granulocytes (neutrophils, eosinophils, basophils). It can lead to neutrophilic inflammation. The addition of glycopolymers to both MC3 and C12-200 formulations led to increased levels of this chemokine, especially for C12-200 LNPs.

[0152] MCP-3: MCP-3 is the monocyte chemotactic-protein 3. It belongs to the CC chemokine subgroup, and it is a very pluripotent chemokine, in the sense that it can activate all leukocytes. C12-200 LNPs with glycopolymers D2 and D3, and MC3 LNP with glycopolymer D2 induced significantly higher levels of this chemokine, which may contribute to an increased immune response.

[0153] IP-10: the interferon-induced protein 10 is a chemokine, as the name says, secreted by many cells types in response to IFN-y. IP-10 is produced in response to IFN-y and it recruits immune cells to the site of inflammation. It functions as a chemotactic for T cells, natural killer cells, monocytes, macrophages, and dendritic cells. All LNPs induced high levels of IP-10 secretion, with glycopolymer D1 showing significantly higher levels in the C12-200 DSPC formulation.

[0154] ENA-78: ENA-78 is well known to have chemotactic and activating functions on neutrophil, especially during acute inflammatory responses.

[0155] MCP-1 (or CCL-2): MCP-1 stands for monocyte-chemoattractant protein-1 and it is a chemokine that attracts monocytes, macrophages, and basophils but not neutrophils and eosinophils. It has a role in pathogenesis, in particular related to monocyte infiltration. It is produced by a variety of cell types after oxidative stress, growth factors, or cytokines and it is correlated with antiviral immune response. LNPs induce some level of MCP-1 secretion in PBMCs, however there were no significant differences between the groups.

[0156] IFN- . it is a cytokine involved in downregulating the inflammatory response, and for this reason it is used to treat multiple sclerosis. Human PBMCs stimulated with C12-200 LNPs had very low levels of this cytokine, while stimulation with MC3 LNPs resulted in secretion of IFN-p. IL-1 a: the interleukin 1-a is a cytokine directly correlated with the production of inflammation. This cytokine was present at low levels at stimulation with LNPs, however the combination of C12-200 with glycopolymers induced higher levels of I L-1 a.

[0157] IL-1 / 3’. the interleukin 1-p is a pro-inflammatory cytokine essential to host-defence responses to infection and injuries. It is secreted by a variety of cell types, with many studies reporting that it is produced in response to pathogen-associated molecular patterns (PAMPs).

[0158] MDC: MDC stands for macrophage-derived chemokine. It is constitutively expressed, being regulated by cytokines. It is a pro-inflammatory chemokine that acts on many cells (dendritic cells, natural killer cells, T cell subsets). High levels of MDC were observed after cell stimulation with LNPs, and significantly higher levels with the addition of glycopolymers was observed for both MC3 and C12-200 formulations.

[0159] Eotaxin-2: it is a potent chemoattractant specific to eosinophils, but also to basophils. It is selective for the CCR3 receptor. The addition of glycopolymers to both MC3 and C12-200 LNPs induced higher levels of this chemokine secretion.

[0160] GRO-a: also known as GRO1 oncogene or neutrophil-activating protein 3, GRO-a acts as a chemoattractant for different immune cells, specially by recruiting neutrophils or non- hematopoietic cells to the infection site.

[0161] Referring now to Figure 17, the formulations were analysed in vivo for RNA expression levels in different organs, to assess whether the addition of glycopolymers would have an impact on biodistribution. Mice were immunised with either MC3 DSPC or C12-200 DOPC LNP with and without the Glycopolymer D2. LNPs were formulated with a 10 pg dose of saRNA encoding for firefly luciferase. Bioluminescence imaging was obtained 5 h and 24h post injection. Mice were then culled, and the organs were harvested for bioluminescence imaging as well. Figure 17A shows saRNA expression levels at 5- and 24-hours postinjection. Figure 17B shows the total expression signal and Figure 17C shows the expression in organs normalised by muscle expression, at 24 hours.

[0162] The incorporation of glycopolymer D2 into MC3 and C12-200 LNPs did not result in significant changes in saRNA expression, which was predominantly observed at the muscle injection site (Figure 17A). Formulations with glycopolymer attached (MC3 and D2 or C12-200 and D2) show higher presentation of lipid nanoparticles in organs than the formulations absent of the glycopolymer (MC3 or C12-200 alone). A significantly increased expression of saRNA was observed in the lymph nodes of mice that received the MC3 and D2 formulation (Figure 17B). This enhanced expression is likely due to the presence of mannose receptors, such as DC-SIGN and MR, on antigen-presenting cells (APCs). These receptors can bind mannosylated LNPs, facilitating dendritic cell maturation and migration to secondary lymphoid organs, such as draining lymph nodes, where carbohydrate-binding proteins like L-SIGN are also expressed. In the lymph nodes, antigens are presented to lymphocytes, initiating adaptive immune responses. Therefore, elevated RNA expression in the lymph nodes may lead to stronger immune responses.

[0163] Example 2

[0164] Evaluation of hydrodynamic diameter of glycosylated LNPs in cell culture medium

[0165] Because mannosylated LNPs can interact and aggregate with serum proteins present in the cell culture medium, it was hypothesized that this interaction might modify protein corona formation, thereby influencing downstream processes such as cellular uptake and gene expression. To evaluate this, LNPs were incubated in complete DMEM at 37 °C for 4 hours, a timeframe considered sufficient for LNP uptake and the onset of translation.

[0166] Figure 18 shows the particle hydrodynamic diameter as measured by dynamic light scattering (DLS) before and after incubation in cell culture medium. MC3 and C12-200 LNPs formulated with glycopolymers were incubated in complete DMEM at 37 °C for up to 4 hours. Symbols indicate mean ± SD from three measurements (n = 3). Statistical analysis was performed using Two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0167] The majority of LNP formulations were not significantly affected by the incubation with serum proteins. However, C12-200 LNPs containing glycopolymer D3 showed a notable increase in size, expanding from 97 nm to 139 nm after 4 hours of incubation. This size change suggests that polymer length, rather than ligand density, is more critical for LNP stability. It is plausible that the specific interaction between C12-200 and D3 glycopolymer may present enhanced affinity toward serum proteins of the cell culture medium, resulting in aggregation. Ex vivo and in vivo saRNA expression of mannosylated LNPs

[0168] The next phase of the study focused on evaluating responses in ex vivo and in vivo systems. Glycopolymer D2 was selected as the model glycopolymer to streamline experimental design and adhere to the 3Rs (Replacement, Reduction, Refinement) in animal research.

[0169] With reference to Figures 19A and 19B, Human skin explants were transfected with saRNA LNPs at a dose of 1 pg, and expression levels were assessed using bioluminescence imaging. In Figure 19A, RNA expression levels are presented as photons / second. Figure 19B shows area under the curve (AUG) analysis, which was performed to quantify RNA expression over the entire study period. Bars represent the mean ± SD (n = 3). Statistical significance was determined using Two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0170] A bioluminescent signal was detectable for up to 14 days (Figure 19A). The addition of glycopolymer D2 did not affect the expression of MC3 LNPs but enhanced the expression levels of C12-200 LNPs (Figure 19B).

[0171] These results suggest that the initially lower performance of C12-200 alone in the skin explants, compared to MC3 alone, may account for the increased expression observed when glycosylated. This pattern was consistent with in vitro findings, where C12-200 LNPs showed high expression levels, and the addition of glycopolymers primarily boosted the expression of MC3 LNPs.

[0172] In vivo cytokine response and antibody production of mannosylated LNPs

[0173] To analyze vaccine immunogenicity, mannosylated LNPs were formulated with hemagglutinin (HA) saRNA at a dose of 1 pg. Given the promising response of MC3 LNPs to glycopolymer addition, this formulation was tested at two doses: 1 pg and 5 pg. The experimental regimen involved administering a prime dose followed by a booster dose 21 days later. Blood serum was collected at 2- and 5-weeks post-prime immunization, and animals were challenged with an influenza strain to evaluate vaccine protection against infection. With reference to Figure 20, blood samples were collected at 4- and 24-hours postimmunization with LNPs and analyzed using MSD assays. Significant differences in cytokine levels compared to baseline are shown. (A) Cytokine panel for MC3 LNPs at 24 hours, including the PBS group, and (B) without the PBS group in the analysis. (C) Cytokine panel for C12-200 LNPs at 4 hours, with the PBS group included, and (D) without the PBS group. Data represent n = 2 per group. Statistical significance was determined using Two-way ANOVA compared to baseline levels (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0174] With reference to Figure 21, binding antibody titres against hemagglutinin (HA) were measured by ELISA at 2- and 5-weeks post-immunization with (A) MC3 LNPs and (B) C12-200 LNPs. Neutralizing antibody titres were also assessed using an ELISA protocol for (C) MC3 LNPs and (D) C12-200 LNPs. Statistical significance was determined using Two-way ANOVA, with n = 5 per group (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0175] For C12-200 LNPs, there was no strong activation of the innate immune response 4 hours post-vaccination. The C12-200 + D1 glycopolymer formulation showed a moderate increase in IFN-p, IL-1p, and MCP-1 levels (Figure 21 C, D). In contrast, MC3 LNPs triggered strong activation of all cytokines 24 hours post-immunization compared to the PBS control, with GM-CSF, IFN-a, and IFN-p being significantly elevated only in the higher 5-pg dose group (Figure 21A, B). Among the different formulations, the MC3 + D2 glycopolymer exhibited overall lower cytokine levels but induced a significant increase in IL-ip.

[0176] IL-1 plays a crucial role in combating pathogens and initiating tissue repair. However, excessive or dysregulated production of IL-ip can lead to harmful inflammation, potentially causing tissue damage and contributing to disease progression.

[0177] The evaluation of adaptive immune responses to vaccination with glycosylated LNPs involved analyzing binding and neutralizing antibody titres. At a 1 pg dose of saRNA, MC3 and C12-200 LNPs showed similar levels of binding and neutralizing antibody titres, irrespective of the presence of glycopolymers (Figure 20). However, at the 5 pg dose, the addition of glycopolymer D2 to MC3 LNPs resulted in significantly higher binding and neutralizing antibody titres (Figure 20A-D). Following the completion of the immunization schedule, animals were subjected to a challenge study with the Cal09 influenza strain to assess the protection conferred by the mannosylated LNP vaccines. Animals were monitored daily for body weight loss and were culled if a loss of >25% was observed or anticipated. At the study’s conclusion, lungs were harvested, and viral load was quantified by measuring the flu M gene copy number via qPCR.

[0178] With reference to Figure 22, animals were challenged with the Cal09 influenza strain following the completion of the immunization schedule. (A, B) Body weight was monitored daily, and weight loss was calculated relative to the weight before the challenge. Symbols represent mean ± SD (n = 5 animals). Statistical significance was determined using Two- way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). (C, D) Lung viral load was assessed by quantifying the flu M gene copy number via qPCR. Bars represent mean ± SD (n = 5). Statistical significance was determined using One-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0179] Both C12-200 and MC3 LNPs, at doses of 1 pg and 5 pg, provided complete protection against the influenza challenge. None of the LNP-vaccinated groups experienced significant body weight reduction, whereas the PBS control group showed approximately 15% weight loss by day 5 post-challenge (Figures 22A, B). Additionally, quantification of viral load in the lungs revealed significantly lower flu M gene copy numbers in all vaccinated groups compared to the PBS control (Figures 22C, D). Notably, animals vaccinated with MC3 + glycopolymer D2 at 1 pg or MC3 + reference polymer R1 at 5 pg had virtually no detectable virus in their lungs (Figure 22D).

[0180] In conclusion, mannosylated MC3 LNPs at the higher 5 pg dose demonstrated significantly higher binding and neutralizing antibody titres against influenza HA.

[0181] Conclusion

[0182] Overall, the integration of glycopolymers into LNPs improved their performance both in vitro and in vivo. These findings suggest that glycopolymer addition can enhance LNP characteristics by improving expression at the cellular and organ targeting and uptakelevels. By leveraging sugar-decorated saRNA-based LNPs, these vaccines can achieve effective immunisation with lower doses, potentially reducing the amount of cargo required. It will be appreciated by those skilled in the art that several variations to the aforementioned embodiments are envisaged without departing from the scope of the invention.

[0183] It will also be appreciated by those skilled in the art that any number of combinations of the aforementioned features and / or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein.

[0184] Materials and Methods

[0185] Materials’. A mannose-derived acrylamide (AcManEAA) and a cholesterol-derived RAFT agent were synthesized following literature procedures.1 2N-hydroxylethyl acrylamide (97%, contains 1000 ppm MEHQ as inhibitor), D-(+)-mannose (>99%), sodium methoxide (CH3ONa, 25 wt % in methanol), boron trifluoride diethyl etherate (>46.5% BF3 basis), acetic anhydride (>99.5%), 4,4'-azobis(2-methylpropionitrile) (AIBN), ConcanavalinA (ConA), sodium acetate, sodium chloride were purchased from Sigma-Aldrich. Human lectins DC-SIGN and MBL were purchased from Bio-Techne and used as received. Dialysis tubes (MWCO 500 Da) were purchased from Spectrum Laboratories (California, U.S.A.). DLin-MC3-DMA was purchased from Biorbyt, and C12-200 from Corden Pharma. 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (plant-derived) and 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (DMPE-PEG2000) were purchased from Avanti Polar Lipids, US. Dulbecco's phosphate-buffered saline (DPBS) and trypsin (TrypLE Express) were obtained from Gibco. VivaSpin 6 filter tubes with (MWCO 10kDa) were purchased from (Sartorius, Germany). Quant-iT RiboGreen assay, TE Buffer, Triton X-100, Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), L- glutamine, penicillin / streptomycin, the 96-well Costar plate were purchased from ThermoFisher. Bright-Glo™ luciferin substrate from Promega. The NanoAssemblr was obtained from Precision NanoSystems (Vancouver, BC, Canada).

[0186] NMR Spectroscopy.1H NMR and13C NMR spectra were recorded on a Bruker III HD 400 MHz using deuterated solvents. Gel Permeation Chromatography (GPC): GPC measurements of polymers were carried out at a known concentration on an Agilent 1260 Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual-angle light scattering (LS), and variable wavelength LIV detectors. The system was equipped with 2* PLgel Mixed-D columns (300 x 7.5 mm) and a PLgel 5 pm guard column. The eluent was DMF with 5 mmol of the NH4BF4 additive. Samples were run at 1 mL / min at 50 °C. Poly(methyl methacrylate) standards (Agilent EasiVials) were used for calibration between 955,000 and 550 grnol’1. Analyte samples were filtered through a nylon membrane with 0.22 pm pore size before injection. Experimental molar mass (Mn,GPC) and dispersity (D) values of synthesized polymers were determined by conventional calibration and universal calibration using Agilent GPC / SEC software.

[0187] Surface Plasmon Resonance (SPR): SPR was used for interaction analysis of DC-SIGN and MBL. The extent of interaction between the glycopolymers and lectins was evaluated on a BIAcore T200 system (GE Healthcare). DC-SIGN and MBL (0.005 mg / mL) were immobilized via a standard amino-coupling protocol onto a CM5 sensor chip that was activated by flowing a 1 / 1 mixture of 0.1 M N-hydroxysuccinimide and 0.05 M N-ethyl-N'- (dimethylaminopropyl)- carbodiimide over the chip for 5 min at 25 °C at a flow rate of 5 pL / min. Immobilization of lectins was targeted to 5000 response units (RU-1), in order to ensure a fair comparison between MBL and DCSIGN. Subsequently, all channels were blocked with ethanolamine (1 M pH 8.5) for 10 min at 5 pL / min to remove remaining reactive groups. All experiments were conducted with HEPES-buffered saline (HBS) (0.10 M HEPES, 0.9 M NaCI, 1 mM CaCI2, adjusted to pH 7.4) and filtered using a 0.2 pm regenerated cellulose syringe filter. Sensorgrams for each glycopolymer concentration (1.25-40 pM) were recorded using a 350 s (on period), followed by 200 s of buffer alone (off period). Regeneration of the sensor chip surfaces was performed using 10 mM HEPES (pH 7.4), 150 mM NaCI, 10 mM EDTA, and 0.01% TWEEN 20 surfactant solution. Kinetic data were evaluated using a single set of sites (1 / 1 Langmuir binding) model, by using the BIAevaluation 3.1 software.

[0188] Theoretical Molar Mass Calculation: Calculation of the theoretical number-average molar mass (Mn,th) is shown in the following: where [M]oand [CTA]0are the initial concentrations (in mol dm-3) of the monomer and chain transfer agent, respectively; p is the monomer conversion as determined by1H NMR spectroscopy; and MM and MCTA are the molar masses (g mol-1) of the monomer and chain transfer agent, respectively.

[0189] Acetylation of D-Mannose: D-Mannose (20 g, 111 mmol) and acetic anhydride (105 mL, 1.11 mol) were added to a round-bottom flask and allowed to cool down. Thereafter, a minimum amount of sulfuric acid was added dropwise. The crude was extracted using ice cold water, a NaHCO3solution and distilled water. The extract was dried over MgSO4, and the solvent removed by vacuum. The product (37.0 g) was obtained as a colourless and viscose oil in an 85% yield.

[0190] Synthesis of Acetylated D-Mannose Acrylamide: The acetylated D-mannose (18.5 g, 47.4 mmol) was dissolved in DCM (200 mL) and added to a 500 mL round-bottom flask. Subsequently, hydroxyl acrylamide (8.2 g, 71.1 mmol) was added to the flask whilst it was allowed to cool down in an ice bath. Subsequently, BF3OEt2(29.3 mL, 0.24 mol) was added dropwise into the mixture which was allowed to stir overnight. Finally, the product was extracted using ice-cold water, an aqueous solution of sodium carbonate and distilled water. The extract was dried over MgSO4, the solvent removed and the product purified via column chromatography (EtOAc:Hex, 7:3) to obtain a white solid (9.7 g) in 46% yield.

[0191] Synthesis of cholesterol CTA: PABTC (871 mg, 3.65 mmol), cholesterol (1.7 g, 4.38 mmol) and DMAP (34.8 mg, 0.38 mmol) were dissolved in DCM (36 mL, anhydrous) and added to a 50 mL round-bottom flask. Subsequently, the reaction mixture was cooled to 0°C and a solution of DIC (488 mg, 3.83 mmol) in anhydrous DCM was added dropwise while vigorously stirring. After 2 h the reaction mixture reached room temperature and was stirred overnight. The solution was filtered, and the solvent evaporated under reduced pressure. The residual product was purified via column chromatography (Hex:EtOAc, 7:3) to obtain a yellow solid (2.1 g) in 95 % yield.

[0192] Synthesis of reference polymer R1 via RAFT polymerization: To a nitrogen-purged microwave vial, a solution of Cholesterol CTA 1 (212.9 mg, 0.35 mmol), HEAA (807.5 mg, 7.01 mmol), and AIBN (5.7 mg, 0.035 mmol) in 1 ,4-dioxane was added and degassed with N2for 20 min. The resulting mixture was heated at 70°C in an oil bath until the polymerization reached 95% monomer conversion as determined by1H NMR spectroscopy. Once complete, the polymerization solution was removed from the oil bath, cooled to room temperature, and exposed to oxygen to quench the polymerization. The polymer was purified by dialysis against water for 3 days. The purified polymer was obtained as yellow powder after lyophilization.

[0193] Table 3. RAFT polymerization conditions for R1 , and P1-P3. All polymerizations were conducted in 1 ,4-dioxane as solvent, with Cholesterol CTA as RAFT agent, AIBN as initiator, and at 70°C. determined by 1 H NMR spectroscopy.

[0194] Synthesis of P1-P3 via RAFT polymerization: To a nitrogen-purged microwave vial, a solution of Cholesterol CTA, AcManEAA, and AIBN in 1 ,4-dioxane (see Table 3) was added and degassed with N2for 20 min. The resulting mixture was heated at 70°C in an oil bath until the polymerization reached 99% monomer conversion as determined by1H NMR spectroscopy. Once complete, the polymerization solution was removed from the oil bath, cooled to room temperature, and exposed to oxygen to quench the polymerization.

[0195] Deprotection of Glycopolymers'. Deacetylation of the mannose moieties was performed directly on the crude polymerization. After 1 ,4-dioxane was removed in vacuo, the concentrated solution was diluted with a methanol / DCM 1 :1 mixture to a final polymer concentration of 50 mg / mL. A solution of sodium methoxide in methanol (2 wt%) was added dropwise under vigorous stirring. After six hours, the deprotected polymer precipitated out and, after concentrated the solution in vacuo, was further purified by extensive dialysis against milliQ water (3 days, with frequent exchanges of the dialysis medium). The resulting clear solution was lyophilized and yielded a fluffy white powder that could readily be redispersed in aqueous solutions. Cryo-Transmission Electron Microscopy (Cryo-TEM): A 5 l aliquot of sample was pipetted onto a carbon coated copper grid (HC300Cu, Holey Carbon film on Copper 300 mesh, EM Resolutions, UK). Excess liquid was blotted away with filter paper (Whatman number 1) and the grid was plunge-frozen in a liquid mixture of ethane / propane cooled by liquid nitrogen. The sample was then kept at liquid nitrogen temperatures throughout the analysis. TEM images were taken on a JEOL 2200FS TEM at 200 keV using a Gatan K2 Summit and Gatan 914 cryo-holder.

[0196] UV- Vis Measurements’. UV-vis measurements were performed on an Agilent Cary Series UV-vis spectrophotometer. The LNPs were diluted in HBS buffer to a final concentration of 0.25 mg / mL). Each solution was added into a ConA solution (90 uM) and placed in the UV spectrometer. The absorbance of the mixture was recorded at 420 nm for 10 min every 0.12 s.

[0197] In vitro transcription of self-amplifying mRNA: Self-amplifying mRNA (saRNA) derived from VEEV alphavirus genome and encoding firefly luciferase (fLuc) or hemagglutinin (HA) was prepared by in vitro transcription. pDNA was linearised using Mlul (New England BioLabs, UK) for 2 h at 37 °C, Mlul was added again and incubated for another 1 h at 37 °C. Linearization was confirmed by agarose gel electrophoresis. For transcription into saRNA, 750 ng of linearised fLuc DNA template was synthesized into RNA transcripts via the mMessage mMachine kit (Invitrogen, Thermo Fisher Scientific, UK) according to manufacturer’s instructions. For the synthesis of HA saRNA, 900 ng of linearised DNA templated was transcribed into saRNA via the MEGAScript T7 kit (Invitrogen, Thermo Fisher Scientific, UK), according to manufacturer’s instructions. Transcripts were then purified by lithium chloride (LiCI) precipitation. Transcripts were frozen overnight at -20 °C and precipitated the next morning by centrifugation at 14000 rpm for 20 min at 4 °C. Pellets were resuspended in 70% ethanol and centrifuged at 14000 rpm for 5 min at 4 °C. The ethanol was removed, pellets were allowed to dry for 5 min, and transcripts were resuspended in ultrapure H2O. RNA quantification was done using a NanoDrop One (Thermo Fisher Scientific, UK) and RNA integrity was evaluated by RNA gel electrophoresis using a FlashGel™ System (Lonza, UK).

[0198] LNP formulation: Stock solutions of the following lipids were prepared in absolute ethanol at different solubilizing concentrations: DLin-MC3-DMA (Biorbyt, UK), C12-200 (Gorden Pharma, Switzerland), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti® Polar Lipids, US), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti® Polar Lipids, US), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti® Polar Lipids, US), cholesterol (plant-derived) (Avanti® Polar Lipids, US) and 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DMPE- PEG2000) (Avanti® Polar Lipids, US).

[0199] MC3 LNPs were prepared with MC3, a helper lipid (either DSPC, DOPC, or DOPE), cholesterol, and DMPE-PEG2000 at a molar ratio of 50:10:38.5:1.5. C12-200 LNPs were prepared with C12-200, a helper lipid (either DSPC, DOPC, or DOPE), cholesterol, and DMPE-PEG2000 at three different molar ratios: 50:10:38.5:1.5, 35:16:46.5:2.5, and 56:12:22:10. For LNPs containing the glycopolymers (see Table 1), a weight ratio of 1 :1 between DMPE-PEG2000 and glycopolymer was used. Glycopolymer stock solutions were prepared in RNase-free water. saRNA solutions were prepared in 50 mM sodium acetate (Sigma®, US) and 100 mM sodium chloride buffer (Sigma®, US) at pH = 5.5. MC3 LNPs were prepared at a nitrogen to phosphate (N / P) ratio of 14 and C12-200 LNPs at N / P ratio of 8. LNPs were formulated on a NanoAssemblr (Precision NanoSystems Incorporated, Vancouver, BC, Canada) using a flow rate ratio of RNAJipid of 3:1 and a total flow rate of 8 mL / min. The formulated material was then concentrated in Dulbecco's phosphate-buffered saline (DPBS) 1X (Gibco, UK) in approximately 5 times the volume of total formulation. This diluted sample was spun down using VivaSpin 6 filter tubes with a molecular weight cut-off (MWCO) of 10 kDa (Sartorius, Germany), at 4000 rpm and 18 °C until desired concentration was achieved. Samples were stored at 4 °C until usage.

[0200] RNA encapsulation efficiency by RiboGreen Assay: The quantification of saRNA encapsulated inside lipoplexes was analysed using a Quant-iT RiboGreen assay (ThermoFisher, UK) according to Precision NanoSystems’ protocol. Briefly, samples were diluted to a concentration in the range of 4-7 pg / mL and added 1 :1 in either 1X TE Buffer or Triton X-100, using a black 96-well Costar® plate (ThermoFisher, UK). Samples were incubated at 37 °C for 10 min to allow for the lysis of LNPs and then kept for 5 min at room temperature. RiboGreen reagent was prepared at a 1 :100 dilution in 1X TE buffer and 100 pL was added per well. An RNA standard curve was also prepared as recommended by the protocol. Fluorescence intensity was analysed on a microplate reader (BMG LABTECH, UK) at an excitation of 480 nm and emission of 520 nm. The concentration of total saRNA and saRNA on the outside (non-encapsulated) was calculated based on the standard the curve. The concentration of encapsulated saRNA was calculated by subtraction (total saRNA minus non-encapsulated saRNA). The percentage of encapsulation efficiency (EE) was calculated as: EE (%) = 100 * (RNA encapsulated I RNA added to assay)

[0201] Physicochemical characterization: Z-average diameter and zeta potential were analysed using a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were equilibrated at room temperature and then diluted 1 :100 in DPBS 1X (Gibco, UK), in a total of 850 pL. DTS1070 cuvettes were used, and the parameters as follows: material refractive index of 1.529, absorbance of 0.010, dispersant viscosity of 0.8820 cP, refractive index of 1.330 and dielectric constant of 79.

[0202] Cell line and culture conditions: HEK293T.17 and HeLa cells (ATCC, US) were routinely grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Thermo Fisher, UK) supplemented with 10% (v / v) foetal bovine serum (FBS), 1% (v / v) L-glutamine, and 1% (v / v) penicillin / streptomycin (Thermo Fisher, UK), at 37 °C under 5% CO2. When confluent, cells were washed with DPBS 1X (Gibco, UK) and treated with trypsin (TrypLE Express 1X) (Gibco, UK) for seeding in new culture flasks (Corning, US).

[0203] THP-1 cells (ATCC, US) were routinely grown in RPMI-1640 Medium (Sigma, UK) supplemented with supplemented with 10% (v / v) foetal bovine serum (FBS), 1% (v / v) L- glutamine, and 1% (v / v) penicillin / streptomycin (Thermo Fisher, UK), at 37 °C under 5% CO2. When confluent, the whole cell suspension in culture media was centrifuged at 1750 rpm for 5 min, and the pellet was re-suspended in fresh RPMI-1640 medium for seeding in new culture flasks (Corning, US).

[0204] Finally, an immortalised cell line of human skeletal muscle cells (hSkMC) (PromoCell, UK) was routinely grown in Skeletal Muscle Cell Growth Medium (PromoCell, Germany), supplemented with SupplementMix (PromoCell, Germany). When confluent, cells were washed with DPBS 1X (Gibco, UK) and treated with trypsin. Neutralisation was done with DPBS 1X containing 10% FBS, and cells were centrifuged and re-suspended in the skeletal muscle cell growth medium for seeding in new culture flasks (Corning, US). Cell transfection assay: The cell experiments were carried out via the following procedure: For both HEK293T.17 and HeLa cell lines, a concentration of 5x104cells per well were seeded in a 96-well plate 24 h prior to the experiment. For THP-1 cells, the concentration was 8x104cells per well, and for the immortalised hSkMC, the concentration was 10x104cells per well.

[0205] On the day of the experiment, 100 ng of RNA-LNPs was added to each well. Samples were allowed to transfect for 24 h. After, the transfection efficiency was analysed by removing 50 pL of medium and adding 50 pL of Bright-Glo™ luciferin substrate (Promega, UK) to each well. The total volume was transferred to a white plate (Falcon®, US) and luminescence intensity was analysed on a FLUOstar Omega plate reader (BMG LABTECH, UK). in vivo studies: Female BALB / c mice (Charles River, UK) (n = 5) were injected i.m. with 1 pg hemagglutinin (HA) saRNA complexed with LNPs with and without glycopolymers, and after 21 days received a boost dose. Blood was collected 2 and 5 weeks after the prime dose.

[0206] Antigen-specific IgG ELISA: MaxiSorp high binding ELISA plates (Nunc) were coated with 1 pg / mL HA protein in 100 pL per well. Standard columns were coated with goat antimouse Kappa (Southern Biotech) and Lambda light chains (Southern Biotech) at 1:1000 dilution each. After overnight incubation at 4 °C, plates were washed with PBS-Tween 20 0.05% (v / v) and blocked with 1% BSA (w / v) in PBS-Tween 20 0.05% (v / v), for 1h at 37 °C. After another round of washing, serum samples were diluted and added to the wells at appropriate concentrations, while standards were diluted 1 :5000 and titrated down in a 5- fold dilution series. Plates were incubated for 1h at 37 °C and washed again. Secondary antibody, anti-mouse IgG-HRP (Southern Biotech), was added at 1 :2000 dilution at a volume of 100 pL per well and plates were incubated for 1h at 37 °C. Plates were developed using SureBlue TMB (3,3',5,5'-tetramethylbenzidine) substrate at 50 pL / well and the reaction was stopped after 5 min using 50 pL / well of stop solution (Insight Biotechnologies). Absorbance was read on a spectrophotometer (VersaMax, Molecular Devices) at 450 nm.

[0207] Pseudotype virus neutralization assay: One day before the assay, MDCK cells were seeded at 1x104 cells per well in a 96-well plate using complete DMEM. Sera was mixed with receptor-destroying enzyme (RDE) in a proportion of 3:1 and heat-inactivated for 30 minutes at 56 °C. Next, serum samples were diluted 1 :10 in serum-free DMEM containing 1 pg / ml TPCK-Trypsin. Further serial dilutions at a ratio of 1 :5 serial were performed in serum-free DMEM supplemented with 1 pg / ml TPCK-Trypsin, 1% L-glutamine, 1% streptomycin-penicillin. The same volume of virus was added to the wells at a concentration of 100 TCID50, and samples were incubated for 1h at 37 °C, 5% CO2. MDCK cells were washed with serum-free media and the mixture of serum and virus was transferred to the cells and allowed to incubate for 24h at 37 °C, 5% CO2. Cells were then fixed with cold 80% acetone (v / v) and plates were blocked with 5% (w / v) nonfat milk in PBS + 0.05% (v / v) Tween20 for 1 h. Then, rabbit anti-NP antibody (Thermo) diluted at 1 :1000 was added to the wells and incubated for 1h. Mouse anti-rabbit IgG-HRP (Santa Cruz) diluted at 1 :5000 was added to the wells and incubated for 1h. Plates were developed using TMB solution for 5 min at RT and then by adding stop solution. Measurements were done at CD450 / OD800 and the IC50 was calculated for each sample.

[0208] Viral challenge: Five weeks after the prime dose, mice were challenged with 5 x 104pfu of influenza (Cal / 09) suspended in 100 pL of PBS. Mice were challenged intranasally and weighed each day to determine weight loss. If they reached 20% weight loss, mice were culled following the humane endpoints for the challenge protocol.

[0209] RNA extraction from lungs for viral load determination: RNA was extracted from frozen lungs by adding 500 pL of trizol to eppendorf tubes containing ball bearings. Tissue was homogenized using a tissuelyser at 50 oscillations / sec for 4 min. A volume of 100 pL of chloroform was added to each sample and centrifuged at 12,000g for 15 min at 4 °C. Then, 250 pL of isopropranol was added to each sample, centrifuged, and the aqueous upper layer was transferred to new tubes. Samples were incubated for 10 min at room temperature and centrifuged again. Supernatant was discarded and samples were resuspended in 75% ethanol. Samples were then vortexed and centrifuged at 7,500g for 5 min at 4 °C. Remove the supernatant and air dry the pellet for 10 min. Resuspend samples in RNase-free water and incubate them at 55 °C for 10 min. Determine the RNA concentration using a Nanodrop (Thermo Fisher Scientific, UK).

[0210] Lung viral load by RT-PCR and flu M gene qPCR: RNA samples were diluted to 200 ng / pL in 10 pL and RT-PCR was performed using Promega GoScript (Promega, UK). Master mix was prepared with enzyme mix, reaction buffer / oligo (dT), and nuclease-free water in a proportion of 1 :2:2. A volume of 10 pL of master mix was mixed with 10 pL of RNA sample and the reaction was ran using the following parameters: annealing (25 °C for 5min), extension (42 °C for 60 min), and inactivation (70 °C for 15 min). Next, cDNA samples were added to the qPCR plate at a volume of 2 pL. The qPCR master mix was prepared using the Taqman universal mix (Fisher Scientific, UK) and M IAV gene primers. A proportion of 10:0.4:0.4:0.8:6.4 was used for TaqMan universal mix, forward primer, reverse primer, probe, and nuclease-free water. A volume of 18 pL of the mix was added to each well. The reaction was ran using the following parameters: 1 cycle of denaturation (95 °C for 600 s), and then 40 cycles of denaturation (95 °C for 30 s), annealing (55 °C for 60 s), and extension (72 °C for 30 s).

[0211] MSD assay (human PBMCs): Human peripheral blood mononuclear cells (PBMCs) obtained from a clinical trial donor were seeded at 2,000,000 cells / mL in a 24-well plate using RPMI media (Sigma, UK), supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. PBMCs were transfected with LNPs containing 1 pg of saRNA for both 24h and 48h. Cell media was then collected and spun down for 5 min at 1750 rpm and the supernatant was collected for the MSD assay. The MSD assay was performed according to the manufacturer’s instructions. Briefly, on the day prior to the assay, plates were coated with a multiplex solution (mix of linker with biotinylated antibody), washed 3 times with PBS-T solution (0.05% Tween-20 in DPBS 1X), and stored overnight at 4-8 °C. On the next day, a volume of 25 pL of diluent was added to each well and another 25 pL of either sample or standards. Standard solutions were prepared in a 1:4 dilution factor. Plates were incubated for 1h at RT using a Titramax 1000 platform shaker (Heidolph Instruments, Germany). Plates were then washed 3 times with PBS-T, 50 pL of antibody solution was added to each well, and plates were incubated shaking for 1h at RT again. Finally, plates were washed 3 times with PBS-T and 150 pL of MSD Gold Read buffer was added to each well and plates were read on the MESO QuickPlex SQ 120MM reader (Meso Scale Diagnostics, US).

[0212] MSD assay (mice sera): The same procedure as described above was followed, except that serum samples were added directly to the wells and no previous step of cell transfection was required. The panel of cytokines was specific to the mouse MSD kit and included: GM-CSF, IFN-a, IFN-p, IP-10, IL-ip, IL-6, KC / GRO, MCP-1 , MIP-1a, and MIP- 1 . Biodistribution in vivo: Female BALB / c mice (Charles River, UK) (n = 5) were injected with 10 g of firefly luciferase (fLuc) saRNA complexed with LNPs with and without Glycopolymer D2. After 24h, mice received 100 pL of IVISbrite D-Luciferin Bioluminescent Substrate (PerkinElmer, UK) and were imaged after 10 min for bioluminescence production using the Ami-HT Optical Imaging System (Spectral Instruments Imaging, US). Immediately after, animals were culled following the schedule 1 humane methods and organs were harvested for bioluminescence imaging as well using the same imaging system.

[0213] References

[0214] Monaco, A.; Beyer, V. P.; Napier, R.; Becer, C. R. Multi-Arm Star-Shaped Glycopolymers with Precisely Controlled Core Size and Arm Length. Biomacromolecules 2020, 21 (9), 3736-3744.

[0215] De Vrieze, J.; Louage, B.; Deswarte, K.; Zhong, Z.; De Coen, R.; Van Herck, S.; Nuhn, L.; Kaas Frich, C.; Zelikin, A. N.; Lienenklaus, S.; et al. Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer-Lipid Amphiphile Conjugates of Small-Molecule TLR7 / 8 Agonists. Angew Chem Int Ed Engl 2019, 58 (43), 15390- 15395.

[0216] Hassett, K. J. et al. Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol Ther Nucleic Acids 15, 1-11 (2019).

[0217] Blakney, A. K. et al. Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines. Journal of Controlled Release (2021) doi:10.1016 / j.jconrel.2021.08.029.

[0218] Geijtenbeek, T. B. H., Engering, A. & van Kooyk, Y. DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology. J Leukoc Biol 71 , 921-931 (2002).

[0219] Koppel, E. A., van Gisbergen, K. P. J. M., Geijtenbeek, T. B. H. & van Kooyk, Y. Distinct functions of DC-SIGN and its homologues L-SIGN (DC-SIGNR) and mSIGNRI in pathogen recognition and immune regulation. Cellular Microbiology vol. 7 157-165 (2005). Nahar, II. J., Toth, I. & Skwarczynski, M. Mannose in vaccine delivery. Journal of Controlled Release vol. 351 , 284-300 (2022).

[0220] Slaats, J., ten Oever, J., van de Veerdonk, F. L. & Netea, M. G. IL-1 p / IL-6 / CRP and IL- 18 / ferritin: Distinct Inflammatory Programs in Infections. PLoS Pathogens vol. 12 (2016).

[0221] Tahtinen, S. et al. IL-1 and IL-1 ra are key regulators of the inflammatory response to RNA vaccines. Nat Immunol (2022).

Claims

39CLAIMS1. A lipid nanoparticle functionalised with a glycopolymer, wherein the glycopolymer comprises at least four carbohydrate units, wherein each carbohydrate unit is independently selected from a monosaccharide, a disaccharide and an oligosaccharide.

2. A lipid nanoparticle according to Claim 1 , wherein the lipid nanoparticle comprises a first molecule comprising a lipophilic moiety, wherein the first molecule is functionalised with the glycopolymer.

3. A lipid nanoparticle according to Claim 2, wherein the first molecule is a sterol (e.g. cholesterol), a steroid or a phospholipid.

4. A lipid nanoparticle according to any preceding Claim, comprising an ionizable lipid, a helper lipid and a PEG lipid.

5. A lipid nanoparticle according to Claim 4, wherein the ionizable lipid is selected from 4-(dimethylamino)-butanoic acid, (10Z, 13Z)-1 -(9Z, 12Z)-9, 12-octadecadien-1-yl- 10,13-nonadecadien-1-yl ester (DLin-MC3-DMA), 1,1'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1- piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), dimethyldioctadecylammonium (DDA), dioleoyl-3-trimethylammonium propane (DOTAP), cephalin, and combination thereof.

6. A lipid nanoparticle according to Claim 4 or 5, wherein the helper lipid is selected from 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), hydrogenated soy phosphatidylcholine (HSPC), phosphatidylcholine (POPC), diacylglycerophosphoethanolamine (DAPE), 1 ,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), phosphatidylethanolamine (POPE), and any combination thereof.

7. A lipid nanoparticle according to any of Claims 4 to 6, wherein the PEG lipid has a chain length of from 1000 to 4000, optionally wherein the PEG lipid is selected from401 ,2-dimyristoyl-rac-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), 1 , 2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)-2000 (DSPE-PEG2000), 1 ,2-dimyristoyl-rac-glycero- 3-methoxypolyethylene glycol-2000 (DMG-PEG-2000), and any combination thereof.

8. A lipid nanoparticle according to any of Claims 4 to 7 when dependent on Claim 2, wherein the ionizable lipid, the helper lipid, the first molecule (e.g. sterol), and the PEG lipid are present in the lipid nanoparticle at a molar ratio of: from 25 to 75 ionizable lipid: from 5 to 20 helper lipid: from 10 to 60 first molecule, e.g. sterol: from 0.5 to 20 PEG lipid, for example the ionizable lipid, the helper lipid, the sterol, and the PEG lipid may be present in the lipid nanoparticle at a molar ratio of 50:10:38.5:1.5, 35:16:46.5:2.5 or 56:12:22:10.

9. A lipid nanoparticle according to any preceding Claim, having a particle size in the range of from 2 to 400 nm, for example from 3 to 350 nm, from 4 to 300 nm, from 5 to 250 nm, from 10 to 200 nm, from 15 to 190 nm, from 20 to 180 nm, from 30 to 170 nm, from 40 to 165 nm, from 50 to 160 nm, from 60 to 155 nm or from 70 to 150 nm.

10. A lipid nanoparticle according to any preceding Claim, wherein the glycopolymer comprises a backbone formed from an acrylate, methacrylate, acrylamide, methacrylamide, vinylpyrrolidone, 2-oxazoline or 2-oxazine monomer or mixtures thereof.

11. A lipid nanoparticle according to any preceding Claim, wherein each carbohydrate unit is independently selected from glucose, fucose, mannose, galactose, N-acetyl- galactosamine, N-acetyl-glucosamine and lactose, optionally wherein each carbohydrate unit is a monosaccharide, further optionally wherein each monosaccharide is mannose.

12. A lipid nanoparticle according to any preceding Claim, wherein the glycopolymer comprises from 5 to 1000 carbohydrate units, for example from 6 to 900, from 7 to 800, from 8 to 700, from 9 to 600, from 10 to 500, from 12 to 400, from 15 to 300, from 20 to 200 or from 30 to 100 carbohydrate units, e.g. from 5 to 100, from 10 to 90, from 15 to 80, from 20 to 75, from 25 to 70, from 30 to 60 or from 40 to 50, such41 as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 carbohydrate units.

13. A lipid nanoparticle according to any preceding Claim, wherein the lipid nanoparticle encapsulates one or more molecules independently selected from a nucleic acid, a protein, a polysaccharide, a lipid, a radioactive substance, a therapeutic agent, a prodrug, a nutritional supplement, a biomarker, an enzyme (e.g. a restriction enzyme), a dye or any combination thereof.

14. A lipid nanoparticle according to Claim 13, wherein the nucleic acid(s) is / are selected from deoxyribonucleic acid (DNA), (ribonucleic acid) RNA or a combination thereof, wherein the DNA is selected from plasmid DNA, vector DNA, A-DNA, Z-DNA, C-DNA, D-DNA and E-DNA and / or wherein the RNA is selected from messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), self-amplifying RNA (saRNA), short interfering RNA (siRNA) and guide RNA, optionally wherein the RNA is saRNA.

15. A pharmaceutical composition comprising a lipid nanoparticle of any of Claims 1 to 14 and a pharmaceutically acceptable excipient or carrier.

16. The pharmaceutical composition of Claim 15, wherein the pharmaceutical composition is formulated for administration perorally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intradermally or transdermally.

17. The lipid nanoparticle of any of claims 1 to 14 or the pharmaceutical composition according to claim 15, for use as a medicament.

18. The lipid nanoparticle of any of Claims 1 to 14 or the pharmaceutical preparation according to Claim 15 for use in a method of inducing an immune response in a subject.

19. A method of delivering an encapsulated molecule, e.g. a nucleic acid, to a subject, the method comprising administering to said subject an effective amount of a lipid nanoparticle according to any of Claims 1 to 14 or the pharmaceutical preparation according to Claim 15 or 16.

20. A method of inducing an immune response in a subject, the method comprising administering to the subject an immune stimulatory amount of a lipid nanoparticle composition according to any one of Claims 1 to 14 or the pharmaceutical preparation according to Claim 15 or 16.

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