Lipid nanoparticles for cardiac protection

Abstract

The present invention relates to a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA for use in treating or preventing a cardiac disease or condition in a subject in need thereof, wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the disease or condition. The present invention also provides LNPs comprising an mRNA encoding a cardiotherapeutic polypeptide and an ionisable lipid selected from ALC-0315, SM-102, or DLin-MC3-DMA or analogues thereof, particularly where the mRNA encodes Chrdl1, Fam3b, Fam3c, Mdk, pleiotrophin, HtrA1, Nhlrc3, or Rln-1 polypeptide. The present invention also provides kits for producing said LNPs, and a pharmaceutical composition or unit dose composition comprising said LNPs.

Description

[0001] LIPID NANOPARTICLES FOR CARDIAC PROTECTION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of lipid nanoparticles comprising mRNA for treating or preventing cardiac diseases, or conditions. In particular, the present invention relates to transiently expressing mRNA from said lipid nanoparticles to treat or prevent a cardiac disease, or condition, particularly an acute disease, or condition.

[0004] BACKGROUND

[0005] Heart failure (HF) affects around 1-3% of the global adult population (more than 64.3M people worldwide in 2017), has a 5-year mortality at 50-75%, and absorbs 2-3% of national health expenditures in high-income countries (Savarese et al., 2022, Cardiovascular Research, 118(17): 3272-3287, Roth et al., 2017, J Am Coll Cardiol, 70(1): 1-25), Cook et al., 2014, Int J Cardiol, 171(3): 368-376). An essential component underlying heart failure with reduced ejection fraction is the loss or death of cardiomyocytes (CMs) in adult life. Cardiomyocyte loss can be sudden, for example after acute ST-elevation myocardial infarction (STEMI), or chronic, and it accompanies virtually all the diseases that affect the myocardium (see Secco and Giacca, 2023, J Mol Cell Cardiol, 179: 80-89). Cardiomyocyte death is not offset by new cardiomyocyte generation in adult hearts, as the proliferative capacity of cardiomyocytes stops abruptly after birth.

[0006] In particular, acute myocardial infarction (Ml) can cause the death of up to 25% of the left ventricle, which corresponds to 2 to 4 billion cardiomyocytes. Myocardial infarct size is clearly linked with the development of adverse left ventricular remodelling and heart failure and thus with disease prognosis (Heusch et al., 2014, Lancet, 383(9932): 1933-1943 and Byrne et al., 2023, Eur Heart J, 44(38): 3720-3826). Thus, interventions that serve to limit infarct size are needed.

[0007] Current interventions focus on prompt myocardial reperfusion by percutaneous coronary intervention (PCI) in the first 90 minutes after the acute event, and this reduces the infarcted area but contributes to cardiomyocyte death due to causing sudden oxidative stress (Bulluck et al., 2016, Circ Cardiovasc Imaging, 9(3): e003900). Indeed, even if a subject has prompt reperfusion by PCI, the probability of developing heart failure after an acute STEMI remains high and is estimated by various studies to range between 4% and 28% of patients.

[0008] Whilst a large body of work over the last several years has shown that acute cardiomyocyte loss can be prevented by brief episodes of coronary occlusion / reperfusion preceding or following the myocardial infarction (referred to as ischemic pre- and post- conditioning, respectively) or even by ischemia / reperfusion in distant organs (referred to as remote ischemic conditioning), these studies have yet to be translated into effective clinical applications (Ibáñez et al., 2015, J Am Coll Cardiol, 65(14)L 1454-1471, Skyschally et al., 2008, Herz, 33(2):88-100, Heusch et al., 2015, J Am Coll Cardiol, 65(2): 177-195, and Bulluck et al., 2016). In addition, although these treatments aim to prevent acute cardiomyocyte death, there is a large number of cardiomyocytes that die at later timepoints, e.g., between 48-72 hours after myocardial infarction, in the area-at-risk, which can be as large as 88% of the eventual infarcted area (Rochitte and Azevedo, 2012, Heart, 98(5): 348-350).

[0009] Therefore, a therapeutic strategy which protects against both acute and longer-term cardiomyocyte loss would be of immense therapeutic value to reduce damage after myocardial infarction, particularly in patients who undergo PCI too late or who cannot be revascularized. The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 787971). The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 874764.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention relates to a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA for use in treating or preventing a cardiovascular (e.g., cardiac) disease or condition in a subject in need thereof. Similarly, there is provided a method of treating or preventing a cardiac disease or condition in a subject in need thereof, wherein the method comprises or consists of administering a pharmaceutical composition comprising said lipid nanoparticles (LNPs) comprising an mRNA to a subject in need thereof. In examples, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the cardiovascular (cardiac) disease or condition. Therefore, the LNPs are directed to the liver, and in the liver cells the mRNA is translated into a polypeptide. This has the benefit of avoiding direct protein production in the tissue to be treated, which is particularly important for tissues that are sensitive to protein overload, e.g., cardiomyocytes where proteotoxicity increases the risk of arrhythmias after myocardial infarction. The use of mRNA has the benefit of allowing rapid production of the therapeutic protein in acute conditions. The present data showed that the mRNA is synthesized into a protein which reaches therapeutic levels in the blood within a few hours after administration. The use of mRNA in LNPs also has the benefit that the mRNA and LNPs are degraded with time, and therefore the cardiotherapeutic protein is only present for a transient period of time after administration. This avoids potential side effects from long term expression of therapeutic proteins. The mRNA may encode a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), or relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof, optionally wherein the mRNA encodes a polypeptide with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any of SEQ ID NOs: 1-15, optionally wherein the mRNA comprises or consists of a polynucleotide with a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an mRNA encoded any of SEQ ID NOs: 16-42. These polypeptides have the benefit of being cardiotherapeutic and preserve cardiomyocyte viability, sustain cardiac function, and prevent pathological remodelling (Ruozi et al., 2022. Science translational medicine, 14(660), p.eabo0699 and Giacca et al., 2023, Stem Cells Translational Medicine, 12(8): 527-535).

[0012] The cardiovascular or cardiac disease or condition may be associated with loss of cardiac myocytes, optionally wherein the cardiovascular or cardiac disease or condition is selected from heart failure, myocardial infarction, ischemic or non-ischemic cardiomyopathy, myocarditis or cardiac damage induced by cardiotoxic treatments. The cardiotherapeutic polypeptides described above are particularly beneficial for treating or preventing diseases or conditions associated with a loss of cardiac myocytes, as they protect against cardiomyocyte death after myocardial infarction (Ruozi et al., 2022. Science translational medicine, 14(660), p.eabo0699). In some embodiments, the cardiovascular or cardiac disease or condition is myocardial infarction.

[0013] The cardiovascular or cardiac disease or condition may be acute. The transient treatment described herein may be particularly beneficial when used to treat an acute condition, as the treatment is not present for a prolonged period of time. This avoids potential side effects from long term expression of therapeutic proteins.

[0014] The composition may be administered shortly after the onset of a cardiovascular or cardiac disease or condition (e.g. an acute cardiovascular or cardiac disease or condition). For example, the composition may be administered 120, 96, 72, 60, 48, 36, 24, 12, 9, 6, 3, 2, 1, or 0.5 hours (or less) after the onset of the symptoms of the cardiovascular or cardiac disease or condition. The composition may be administered before cardiomyocyte loss. The composition may be administered after myocardial infarction. For example, the composition may be administered 120, 96, 72, 60, 48, 36, 24, 12, 9, 6, 3, 2, 1, or 0.5 hours (or less) after myocardial infarction. The polypeptide may be present in the blood of the subject for up to (a maximum of) 24, 48, 72, 96 or 120 hours after administration of the composition to the subject. This is beneficial because the therapeutic polypeptide is only present transiently. This avoids potential side effects from long term expression of therapeutic proteins. Similarly, the mRNA and / or LNP may be present in the subject for up to (a maximum of) 24, 48, 72, 96 or 120 hours after administration of the composition to the subject.

[0015] The composition may be administered to the subject intravenously. This has the benefit of allowing easy administration of a cardiotherapeutic agent to the subject without requiring more complex procedures or direct administration to the heart.

[0016] Lipids of the LNPs may comprise or consist of ionisable lipids, structural lipids, phospholipids, and / or PEGylated lipids. This has the benefit of providing the base of a stable nucleic acid lipid particle (once the mRNA cargo is introduced).

[0017] The ionisable lipids may comprise or consist of 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxyoctadeca-9,12-dienyl]amino]butyl]piperazine-2,5-dione (OF-02), ethyl 1-[3-(2-ethylpiperidin-1-yl)propyl]-5,5-bis[(Z)-heptadec-8-enyl]-2H-imidazole-2-carboxylate (A18-Iso5-2DC18), di(oct-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A1), di(non-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A2), di(dec-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A3), di(oct-3-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A4), di(non-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A5), di(dec-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A6), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (C12-200), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]piperazine-2, 5-dione (cKK-E12), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9), 3-[2-[2-[2-[bis[3-(dodecylamino)-3-oxopropyl]amino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]ethylamino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]- / V-dodecylpropanamide (98N12-5), 3-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[2-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[3-[2-(2-hydroxytetradecylamino)ethylamino]-3-oxopropyl]amino]ethyl]amino]-N-[2-(2-hydroxytetradecylamino)ethyl]propanamide (G0-C14), bis[(Z)-non-2-enyl] 9-[4- (dimethylamino)butanoyloxy]heptadecanedioate (L319), 2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-dienoate (OF-Deg-Lin), tridecyl 3-[2-[bis[2-[methyl-(3-oxo-3-tridecoxypropyl)amino]ethyl]amino]ethyl-methylamino]propanoate (304Oi3), 2-(octyldisulfanyl)ethyl 3-[3-[3-[bis[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propyl-methylamino]propyl-[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propanoate (306-O12B), 8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3-oxopropyl]amino]propanoate (306Oi10), octan-3-yl 9-[3-[[3,5-bis[3-[bis(9-octan-3-yloxy-9-oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-3-yloxy-9-oxononyl)amino]nonanoate (FTT5), (1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12-dienoxy]piperidine (YSK05) [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (LP01), [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate (CL1), [7-[4- (dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9-enoate (CL4H6), and / or [7-[4-(dipropylamino)butyl]-13-(2-hexyloctanoyloxy)-7-hydroxytridecyl] 2-hexyloctanoate (CL4F8-6), or analogues thereof. Use of ALC-0315 ionisable lipids in LNPs have the benefit of not activating inflammatory or innate immune responses to the LNP administration, thereby reducing the risk of side effects or immune rejection.

[0018] The structural lipids may comprise or consist of a sterol, optionally cholesterol or analogues thereof.

[0019] The phospholipids may comprise or consist of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Egg Yolk Phosphatidylcholine, or 1.2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM), ceramide or any related phosphatidylcholine or analogues thereof.

[0020] The PEGylated lipids may comprise or consist of 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG 2000), Methoxy Polyethylene Glycol-Distearoylphosphatidylethanolamine (mPEG-DSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (POPE-PEG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DOPE-PEG), 1.2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE- PEG), Distearoyl-rac-glycerol-PEG2000 (PEG-DSG), or 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA) or analogues thereof. The lipids may be present in the LNPs of the composition at a mole percent between 30-60% for the ionisable lipid, 5-40% for the phospholipid, 20-45% for the cholesterol, and 0.5-3.5% for the PEGylated lipid, optionally wherein the lipids are present in the LNPs of the composition at a molar ratio of 50:10:38.5:1.5 or 40:30:27.5:2.5 for the ionisable lipid:phospholipid:cholesterol: PEGylated lipid.

[0021] The mRNA may be present in the LNPs of the composition at a ratio of 10-50 lipids per 1 mRNA.

[0022] The composition may be administered to the subject in a dose which provides between 0.15-2.0, 0.15-1.75, 0.15-1.5, 0.15-1.25, 0.15-1.0, 0.15-0.9, 0.15-0.8, 0.15-0.7, 0.2-2.0, 0.2-1.75, 0.2-1.5, 0.2-1.25, 0.2-1.0, 0.2-0.9, 0.2-0.9, 0.2-0.7, 0.25-2.0, 0.25-1.75, 0.25-1.5, 0.25-1.25, 0.25-1.0, 0.25-0.9, 0.25-0.8, 0.25-0.7, 0.3-2.0, 0.3-1.75, 0.3-1.5, 0.3-1.25, 0.3-1.0, 0.3-0.9, 0.3-0.8, or 0.3-0.7 mg of mRNA / kg body weight of the subject.

[0023] The composition may be administered in a dose which provides between 1-90, 1-80, 1-70, 1-60, 1-50mg, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-45, 5-40, 5-35, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-45, 10-40, and 10-35mg mRNA per dose.

[0024] The polypeptide may be present in the blood of the subject within 2, 4, 6, 8 or 12 hours after administration of the composition. This is beneficial because the production of the cardiotherapeutic polypeptide is rapid, allowing rapid treatment of the disease or condition.

[0025] Over 97, 97.5, 98, 98.5, or 99% of LNPs administered in the composition may be located in spleen or liver of the subject after administration, optionally wherein the ratio of LNPs in liver:spleen is between 3:1 and 12:1, optionally wherein less than 1% of LNPs administered in the composition are located in heart, muscle or lungs of the subject after administration. The lack of targeting of the LNPs to the heart and muscle has the benefit of avoiding direct protein production in the cardiac tissue, which is important as cardiomyocytes sensitive to protein overload. Therefore, lack of targeting to cardiac tissue reduces or prevents proteotoxicity and therefore reduced or prevents the risk of arrhythmias after myocardial infarction.

[0026] The mRNA may comprise one or more modifications, optionally wherein the modification is a 5’-methoxyuridine, N1-methylpseudouridine, or 5’ cap! Such modifications increase the stability, half-life or translation of the mRNA, or reduce immunogenicity of the mRNA, thereby improving the levels of protein in the blood, or reducing side effects of LNP administration, respectively.

[0027] Also provided herein is a lipid nanoparticle (LNP) comprising an mRNA encoding a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), or relaxin-1 (Rln-1) polypeptide or a variant, fragment or isoform thereof.

[0028] Similarly, there is provided herein a lipid nanoparticle (LNP) comprising a cardiotherapeutic mRNA, wherein lipids of the LNP comprise or consist of an ionisable lipid selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof, optionally wherein the mRNA encodes a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeatcontaining protein 3 (Nhlrc3), relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof. ALC-0315 LNPs have the benefit of not activating inflammatory or innate immune responses to the LNP administration, thereby reducing the risk of side effects or immune rejection.

[0029] Lipids of the LNP may comprise or consist of an ionisable lipid, optionally wherein the ionisable lipid is selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxyoctadeca-9,12-dienyl]amino]butyl]piperazine-2, 5-dione (OF-02), ethyl 1-[3-(2-ethylpiperidin-1-yl)propyl]-5,5-bis[(Z)-heptadec-8-enyl]-2H-imidazole-2-carboxylate (A18-Iso5-2DC18), di(oct-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A1), di(non-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A2), di(dec-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A3), di(oct-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A4), di(non-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A5), di(dec-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A6), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1- yl]ethyl]amino]dodecan-2-ol (C12-200), 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]piperazine-2, 5-dione (cKK-E12), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9), 3-[2-[2-[2-[bis[3-(dodecylamino)-3-oxopropyl]amino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]ethylamino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]- / V-dodecylpropanamide (98N12-5), 3-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[2-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[3-[2-(2-hydroxytetradecylamino)ethylamino]-3-oxopropyl]amino]ethyl]amino]-N-[2-(2-hydroxytetradecylamino)ethyl]propanamide (G0-C14), bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate (L319), 2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-dienoate (OF-Deg-Lin), tridecyl 3-[2-[bis[2-[methyl-(3-oxo-3-tridecoxypropyl)amino]ethyl]amino]ethyl-methylamino]propanoate (304Oi3), 2-(octyldisulfanyl)ethyl 3-[3-[3-[bis[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propyl-methylamino]propyl-[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propanoate (306-O12B), 8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3-oxopropyl]amino]propanoate (306Oi10), octan-3-yl 9-[3-[[3,5-bis[3-[bis(9-octan-3-yloxy-9-oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-3-yloxy-9-oxononyl)amino]nonanoate (FTT5), (1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12-dienoxy]piperidine (YSK05) [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (LP01), [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate (CL1), [7-[4- (dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9-enoate (CL4H6), and / or [7-[4-(dipropylamino)butyl]-13-(2-hexyloctanoyloxy)-7-hydroxytridecyl] 2-hexyloctanoate (CL4F8-6), or analogues thereof. ALC-0315 LNPs have the benefit of not activating inflammatory or innate immune responses to the LNP administration, thereby reducing the risk of side effects or immune rejection.

[0030] Lipids of the LNP may further comprise structural lipids, phospholipids, and / or PEGylated lipids.

[0031] Optionally, the structural lipids comprise or consist of a sterol, optionally cholesterol or analogues thereof.

[0032] Optionally, the phospholipids comprise or consist of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Egg Yolk Phosphatidylcholine, or 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM), ceramide or any related phosphatidylcholine or analogues thereof.

[0033] Optionally, the PEGylated lipids comprise or consist of 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG 2000), Methoxy Polyethylene Glycol-Distearoylphosphatidylethanolamine (mPEG-DSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (POPE-PEG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DOPE-PEG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), Distearoyl-rac-glycerol-PEG2000 (PEG-DSG), or 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA) or analogues thereof.

[0034] The lipids in the LNP may be at a mole percent between 30-60% for the ionisable lipid, 5-40% for the phospholipid, 20-45% for the cholesterol, and 0.5-3.5% for the PEGylated lipid, optionally wherein the lipids are in the LNP at molar ratios of 50:10:38.5:1.5 or 40:30:27.5:2.5 for the ionisable lipid:phospholipid:cholesterol: PEGylated lipid.

[0035] The mRNA may be in the LNP at a ratio of 10-50 lipids per 1 mRNA.

[0036] There is also provided a pharmaceutical composition comprising or consisting of one or more LNPs defined herein. Optionally the pharmaceutical composition further comprises or consists of a pharmaceutically acceptable adjuvant, diluent, carrier, or excipient.

[0037] There is also provided a unit dosage composition comprising one or more LNPs defined herein, wherein said unit dosage provides a dose of mRNA between 1mg-100mg.

[0038] There is also provided the LNPs defined herein, the pharmaceutical composition defined herein, or the unit dosage composition defined herein, for use as a medicament.

[0039] There is also provided the LNPs defined herein, the pharmaceutical composition defined herein, or the unit dosage composition defined herein, for use in treating or preventing a cardiac disease or condition. There is also provided use of the LNPs defined herein, the pharmaceutical composition defined herein, or the unit dosage composition defined herein for the manufacture of a medicament.

[0040] There is also provided use of the LNPs defined herein, the pharmaceutical composition defined herein, or the unit dosage composition defined herein for the manufacture of a medicament for treating or preventing a cardiac disease or condition.

[0041] There is also provided a method of treating or preventing a cardiac disease or condition comprising administering the LNPs defined herein, the pharmaceutical composition defined herein, or the unit dosage composition defined herein, to a subject in need thereof.

[0042] The use or method may comprise any use or method described herein. For example, following administration of the LNPs, pharmaceutical composition, or unit dosage to a subject, a polypeptide encoded by the mRNA may be transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide may exert a therapeutic effect on the heart to treat or prevent a disease or condition.

[0043] There is also provided herein a kit comprising an ionisable lipid and an mRNA encoding a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof, optionally wherein the kit further comprises a phospholipid, PEGylated lipid and / or structural lipid, optionally wherein the ionisable lipid is selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof.

[0044] There is also provided herein a kit comprising or consisting a cardiotherapeutic mRNA and an ionisable lipid selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof, optionally wherein the kit further comprises a phospholipid, PEGylated lipid and / or structural lipid. The mRNA of the kit may encode a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), or relaxin-1 (Rin- 1 ) polypeptide, or a variant, fragment or isoform thereof.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1. Generation of lipid nanoparticles (LNPs). (A) Schematic representation of stable nucleic acid lipid particle (SNALP) LNPs. These LNPs comprise an ionisable lipid, helper lipid (e.g., DSPC or DOPE), cholesterol, and a PEGylated lipid (e.g., DMG-PEG200 ‘DMG-PEG’ or PEG200). These LNP formulations were used to encapsulate an in vitro transcribed mRNA (e.g., EGFP or Chrdl1). (B) The size, polydispersity index (PDI) and encapsulation efficiency of three different SNALP LNP formulations was tested. Formulation 1 (‘MC3’): the LNPs were formed of DLin-MC3-DMA, DSPC, Cholesterol, and DMG-PEG 2000 at 50:10:38.5:1.5 molar ratio, respectively. Formulation 2 (‘SM-102’): the LNPs were formed of SM-102, DSPC, Cholesterol, and DMG-PEG at 50:10:38.5:1.5 molar ratio, respectively. Formulation 3 (‘ALC-0315’): the LNPs were formed of ALC-0315, DOPE, Cholesterol, and PEG at 40:30:27.5:2.5 molar ratio, respectively. mRNA (either EGFP or Chrdl1) was loaded into the LNPs of each formulation at a ratio of 40:1 (w / w) lipid: RNA ratio. Formulations are indicated by the ionisable lipid they contained and the mRNA cargo. (C) MC3-EGFP, SM-102-EGFP and ALC-0315-EGFP formulations were injected intravenously (i.v.) into mice providing 10 µg of EGFP mRNA. 24 hours after injection, biodistribution of EGFP was tested by ELISA, and the innate immune response was tested using RT-PCR and ELISA. (D) Biodistribution of EGFP protein in mouse tissues 24 hours after intravenous (i.v.) injection of 10 µg of EGFP mRNA in each of the three formulations measured by ELISA. The most effective transfection was observed in liver and spleen (n=3). (E) Ratio of EGFP protein in liver and spleen as measured by ELISA in (D). The formulation containing the ALC-0315 ionisable lipid was the most effective and specific for liver transduction. Control: animals injected with buffer only. (F) Immunofluorescent detection of EGFP (green) in liver 24 hours after i.v. injection of 10 µg of EGFP mRNA in each of the three formulations. Kupffer cells are identified as F4 / 80-positive (white), and nuclei are stained with DAPI (blue). Scale bar: 100 µm (n=3). (G) Assessment of inflammatory and immune response in liver 24 hours after i.v. injection of 10 µg of EGFP modified mRNA in each of the three formulations. Upper panel: protein levels of Interferon-y-lnduced protein 10 (IP-10, or CXCL10), Interleukin-6 (IL-6) and Tumor Necrosis Factor (TNF)-α by ELISA. Lower panel: detection of antiviral Innate Immune Response Receptor (RIG-I, or DDX58), Interferon Induced With Helicase C Domain 1 (IFIH-1) and Toll-Like Receptor 3 (TLR3) mRNA by qPCR. (n=3).

[0047] Figure 2. Kinetics of protein secretion from the liver after systemic LNP injection. (A) Schematic of the experiment. Mice were administered the ALC-0315 LNP formulation carrying Chrdl1-Flag mRNA by intravenous ( / .v.) injection. At different time points, the levels of circulating Chrdl1 protein were assessed by immunoblotting (IB). (B) Levels of circulating Chrdl1 after LNP administration demonstrated by two representative immunoblots using an anti-Flag antibody in blood microsamples (1 µl blood per sample) from two mice (upper and lower panel) at different times after i.v. LNP mRNA administration. Transferrin was used as a loading control. (C) Quantification of the levels of circulating Chrdl1 (i.e., Chrdl1 in serum) at different times after i.v. administration (n=5-7 per time point).

[0048] Figure 3. Myocardial protection and preservation of cardiac function by systemic administration of LNP-Chrdl1 after myocardial infarction. (A) Schematic of the experiment. Mice were administered the ALC-0315 LNP formulation carrying Chrdl1-Flag mRNA by intravenous (i.v.) injection 20 minutes after ligation of the left anterior descending (LAD) coronary artery. Analysis of cardiac function and morphometric parameters was performed after 1 month. (B) Echocardiography results in mice treated with two different doses (10pg or 20pg) of Chrdl1 mRNA in ALC-0315 LNPs. LVEF: left ventricular ejection fraction; LVEDV: end-diastolic volume LVESV: end-systolic volume. Data are mean±SEM; n=8-10; P values are one-way ANOVA with Dunnett’s post-hoc correction. (C) Representative images of hearts from two control and two treated mice (4 sections / mouse) treated with 10 pg Chrdl1 mRNA in ALC-0315 LNP. Hearts are stained with Masson trichome (fibrotic tissue in blue).

[0049] (D) Quantification of heart scar size at 4 weeks after ligation of the LAD coronary artery and treatment in mice treated with 10 µg Chrdl1 mRNA in ALC-0315 LNPs (n=6 mice).

[0050] DETAILED DESCRIPTION

[0051] Unless otherwise defined below, all technical terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art in the field to which this disclosure belongs.

[0052] Definitions

[0053] Any reference to ‘or’ herein is intended to encompass ‘and / or’ unless otherwise stated.

[0054] As used herein, the singular forms ‘a’, ‘an’, and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

[0055] The terms ‘comprising’, ‘comprises’ and ‘comprised of’ may be synonymous with including, includes or containing, contains, and may be inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses ‘consisting of’. ‘Consisting’ may be understood to mean that only the specified items are present. In other words, this term may be exclusive and closed, and may exclude additional, non-recited members, elements or method steps.

[0056] Whereas the term ‘one or more’, such as one or more members of a group of members, is clear perse, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0057] ‘Around’ and ‘about’ may be used to indicate that the stated value includes values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated value unless otherwise stated or otherwise evident from the context. For example, ‘about’ or ‘around’ may indicate + / - 10% of the stated value.

[0058] ‘Up to’ may be used to indicate a maximum value. ‘Up to’ may include the value indicated. For example, ‘up to 10’ may include all possible values up to and including the number 10.

[0059] ‘At least’ may be used to indicate a minimum value. ‘At least’ may include the value indicated. For example, ‘at least 10’ may include all possible values above 10, and including the number 10.

[0060] ‘Improve’, ‘increase’, or ‘reduce’ may, or grammatical equivalents, may be used to indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or control population or control value) who have not received the treatment.

[0061] ‘Nucleotide’, ‘nucleic acid’ or ‘nucleic acid residue’ may refer to any nucleoside having one or more phosphate groups joined in ester linkages to a sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. In ‘ribonucleic acid’ (RNA), the sugar is ribose. In deoxyribonucleic acid’ (DNA) the sugar is deoxyribose. The term nucleotide is used generally to refer to nucleotides found in DNA and RNA; adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), and any analogues thereof. The term encompasses modified nucleotides, and nucleotides with modified backbone residues or linkages, including modifications which are synthetic, naturally occurring, and non-naturally occurring, and may have similar binding properties as the reference polynucleotide. ‘Polynucleotide’ may refer to a polymer of nucleotides or nucleic acid residues joined together by a phosphodiester or phosphorothioate linkage between 5' and 3' carbon atoms, including DNA and RNA. In examples, ‘polynucleotide’ may refer to a messenger RNA (mRNA). A polynucleotide may be any length, including at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 50, 100, 1000, 10,000 or more nucleotides in length.

[0062] ‘Messenger mRNA’ (mRNA) may be used to refer to a polynucleotide (i.e., polyribonucleotide) which encodes at least one polypeptide or a variant, fragment or isoform thereof. mRNA may contain one or more coding and non-coding regions. ‘mRNA’ may be single stranded RNA molecules. The ‘mRNA’ may be synthetic, or non-naturally occurring, e.g., recombinant mRNA. ‘mRNA’ also includes modified mRNA, particularly mRNA with one or more modifications that increase stability, half-life or translation of the mRNA, or reduce immunogenicity of the mRNA. Examples of modifications include nucleotide analogues or modified backbone residues or linkages, a cap structure, a chain terminating nucleoside, a stem loop, or a poly-A sequence. Therefore, ‘mRNA’ may be used to refer to mature messenger mRNA. ‘mRNA’ sequences may also implicitly include conservative modifications, e.g., where a nucleotide is changed without changing the polypeptide sequence encoded by said mRNA (e.g., degenerate codon substitutions or silent substitution), and / or wherein one or more nucleotides result in a conservative amino acid substitution in the encoded polypeptide (i.e., where one or more amino acids are replaced by one or more amino acids with the same properties as the original amino acids, such that the properties (e.g., chemical or secondary or tertiary structure) of the encoded polypeptide do not change or do not substantially change).

[0063] ‘Amino acid’ may be used to refer to a molecules which has an amine and carboxyl functional group. Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral, nonpolar amino acids. Representative amino acids within these various groups include, but are not limited to, (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

[0064] ‘Polypeptide’ or ‘protein’ may be used to refer to a polymer of amino acids or amino acid residues, e.g. at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 amino acid residues, or a variant, fragment or isoform thereof. One or more amino acid residues in a polypeptide may be an artificial chemical analogue of a corresponding naturally occurring amino acid. The terms are also inclusive of modifications amino acids including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. A polypeptide sequence may include conservative modifications, e.g., where a nucleotide is changed without changing the polypeptide sequence encoded by said mRNA (e.g., degenerate codon substitutions or silent substitution), and / or wherein one or more nucleotides result in a conservative amino acid substitution in the encoded polypeptide (i.e., where one or more amino acids are replaced by one or more amino acids with the same properties as the original amino acids, such that the properties, e.g., chemical or secondary or tertiary structure, of the encoded polypeptide do not change or do not substantially change). In examples, conservative amino acid substitutions include replacing an amino acid with an amino acid in the same group (i.e., acidic, basic, neutral polar or neutral non-polar). Therefore, ‘variant’ may include amino acid modifications of the polypeptide, but still retain the function.

[0065] ‘Isoform’ may refer to variants of a polypeptide which original from the same gene (polynucleotide), but the variants have different amino acid sequences, for example, as a result of splicing, variable promoter use, or other post-transcriptional modifications.

[0066] ‘Fragment’ may refer to a portion or region of a full-length sequence, such as a polynucleotide or polypeptide, optionally a polypeptide fragment which still has the intended function. Accordingly, a fragment may comprise or consist of a sequence that is identical (or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% sequence identity) to a specified portion of the full-length sequence. The portion or region may be between 10-1000, 10-750, 10-500, 10-250, 10-200, 10-150, 10-100, 20-1000, 20-750, 20-500, 20-250, 20-200, 20-150, 20-100, 50-1000, 50-750, 50-500, 50-250, 50-200, 50-150, 50-100, 100-1000, 100-750, 100-500, 100-250, 5-100, 5-75, 5-50, 50-25, 5-20, 5-15, 5-10, 5-1000, 5-750, 5-500, 5-250, 5-200, 5-150, 5-100, 5-75, 5-50, 1-100, 1-75, 1-50, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 nucleic acid residues or amino acid residues from the full length polynucleotide or polypeptide from which the fragment is derived.

[0067] Conservative substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the resulting amino acid residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below.

[0068]

[0069] ‘Sequence’ may refer to any contiguous string of nucleotides or peptides. A ‘sequence’ may be at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 nucleotides or amino acids in length. Unless otherwise specified, all sequences are by convention written from 5’ to 3’ end.

[0070] ‘Encoding’ or ‘coding’ used herein may be used to refer to a polynucleotide which is able is suitable for producing a specified product. For example, mRNA may be referred to as encoding a specific polypeptide or a variant, fragment or isoform thereof to indicate that the mRNA is translated into the specified polypeptide or a variant, fragment or isoform thereof. Therefore ‘encoding’ may be used to indicate that the mRNA, once in a cell, is suitable for expression of a specific polypeptide or a variant, fragment or isoform thereof.

[0071] ‘Homology’, ‘sequence identity’ or ‘sequence similarity’ in the context or two or more polynucleotides may refer to the extent to which the sequence of nucleotides are the same over a specified region. The extent of homology may also be reported as a ‘percentage sequence similarity’, ‘percentage sequence identity’ or ‘percentage homology’, which may be calculated by aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which identical nucleotides occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. The similarity between nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of the nucleotide sequence will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of aligning sequences for comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. Identity may be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman.

[0072] ‘Expression’ may be used to refer to translation of mRNA into a precursor or mature polypeptide, or a variant, fragment or isoform thereof, and therefore may not require the presence of DNA. Therefore, ‘expression’ may also be used to indicate that a specified molecule, e.g., mRNA or polypeptide, or a variant, fragment or isoform thereof, is present in the specified organ, tissue or cells. ‘Expressed in’ and ‘expressed from’ may be used interchangeably.

[0073] ‘Lipid’ may refer to organic compounds that include fatty acids or a derivative of a fatty acid and are generally poorly soluble in water, but soluble in many non-polar solvents. Lipids may be hydrophobic or amphiphilic. Lipids can include waxes, fatty acyls, glycerides, cationic lipids, phospholipids, sphingolipids, steroids (including sterols), prenol lipids, glycolipids, and polyketides.

[0074] ‘Analogue’ when referring to a ‘lipid analogue’ may refer to molecules which are structurally similar to a specific lipid but have further modifications. Examples of modifications that are known in the art include alteration of fatty acid chain lengths (to alter the melting temperature and membrane fluidity of the lipid), altering the degree of saturation (i.e., increasing or decreasing the number of double or triple bonds, to influence membrane fluidity and lipid packing density), replacement of functional groups (to alter chemical reactivity and interaction of the lipid with other molecules), introducing branches or side chains to the fatty acid chains (increased branches decreases lipid packing density), altering the polar head group (can change the lipid’s polarity and interaction with other molecules), adding ring structures within the fatty acid chains or polar head group (increases lipid rigidity and stability), isomerisation (changing the configuration e.g., from cis to trans isomers, affects the spatial arrangement of the lipid and biological activity), replacing an ester bond in the fatty acid to the glycerol backbone with an ether bond (increases lipid resistance to hydrolysis and oxidation), and / or adding halogen atoms (increases lipid stability and interaction with biological membranes).

[0075] ‘Nanoparticle’ may be used to refer to a particle having at least one dimension, optionally diameter, in the order of nanometres (e.g. 1-1000nm).

[0076] ‘Lipid nanoparticle’ (LNP) may refer to a nanoparticle comprising one or more lipids. The LNP may comprise or consist of one or more lipid types. The one or more lipids may contribute to, or provide, the structure of the nanoparticle. The LNP may comprise an outer lipid layer (e.g., lipid monolayer and / or lipid bilayer) and an inner aqueous space. The lipids may form the outer layer of the LNP. The outer lipid layer may be substantially a lipid monolayer (i.e., over 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the outer lipid layer is a monolayer). However, inside the LNP, there may be additional lipids that form a lipid bilayer with regions of the outer lipid layer. ‘Outer lipid layer’ may therefore refer to the exposed surface of an LNP being formed of lipids. This term may refer to the entire lipid e.g., a hydrophilic head which is exposed, and a hydrophobic region which may project towards the centre of the LNP to form the inner aqueous space. The LNP may be substantially spherical. The LNP may be capable or configured to deliver a cardiotherapeutic agent to subject, i.e., the LNP may act as a vehicle for a cardiotherapeutic agent. Therefore, LNP may refer to a LNP comprising or containing a cardiotherapeutic agent or cargo, such as an mRNA. The LNP may be a stable nucleic acid lipid particle (SNALP). The lipids of a SNALP may comprises or consist of an ionisable lipid, a structural lipid, a phospholipid and a PEGylated lipid described herein, optionally only one type of each of these lipids.

[0077] Unless otherwise indicated, reference to LNPs implicitly includes reference to compositions comprising said LNPs.

[0078] ‘lonisable lipid’ may be used to refer to a cationic lipid which is capable of being positively charged, lonisable lipids may have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, optionally at or above physiological pH. In other words, the lipid can exist as a charged or neutral form depending on pH. It is understood in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a cationic or ionisable lipid refers to the nature of the predominant species and does not require that all of the lipids be present in the charged or neutral form.

[0079] ‘Structural lipid’ may be used to refer to a lipid which helps to stabilize the outer lipid layer, particularly, the formation of any lipid bilayer. ‘Sterol’ may refer to a naturally occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A-ring.

[0080] ‘Phospholipid’, ‘helper lipid’, ‘neutral lipid’ or ‘neutral helper lipid’ may be used interchangeably to refer to a lipid which includes a hydrophilic head and a hydrophobic tail. The head region may comprise a phosphate moiety. The tail region may comprise one or more hydrocarbon chains, such as fatty acid chains. The tail may be saturated or unsaturated. Phospholipids may exist either in an uncharged or neutral zwitterionic form at physiological pH.

[0081] ‘Saturation’ or ‘saturated’ may refer to each carbon atom in a hydrocarbon having the maximum possible number of hydrogen atoms, i.e., no double bonds between the carbon atoms.

[0082] ‘Unsaturated’ may refer to a hydrocarbon where more than one bond is present between the carbon atoms, i.e., double or triple bonds.

[0083] ‘PEGylated lipid’, ‘PEG-modified lipid’ or ‘PEG-derived lipid’ may be used interchangeably to refer to a lipid comprising polyethylene glycol (PEG) chain. The PEGylated lipid may be a phospholipid comprising PEG. The PEG may be attached to the lipid through a covalent bond, such as to the head group (i.e., the hydrophilic portion of the lipid).

[0084] ‘Mole percent’, ‘molar percentage’, ‘mole fraction’ or ‘molar fraction’ may be used to refer to the quantity of moles of a specified component, out of the total amount of said component in a composition. For example, ‘mol percent’ may be used to indicate the quantity of moles of lipid out of the total lipid amount in an LNP. The mole percent may be given of the LNP before mRNA cargo is added, or after mRNA cargo is added.

[0085] ‘Aqueous solution’ may refer to a composition comprising water.

[0086] ‘Polydispersity index’ (PDI) may be used to refer to a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution. The PDI values described may refer to a single LNP, all LNPs in the composition, or the mean value of LNPs in a composition.

[0087] ‘Cargo’ or ‘payload’ may be used to define a substance or molecule partially or fully encapsulated by, or contained within, the LNP, and therefore delivered by the LNP. The cargo may be a cardiocardiotherapeutic agent. The cargo may be an mRNA encoding a cardiotherapeutic polypeptide of interest.

[0088] ‘Therapeutic effect’ may be used to may refer to reduced or eliminated symptoms or pathology of a disease, disorder or condition in a subject. ‘Therapeutic effect’ may be measured using self-reports of symptom severity or frequency, or reduced observation of pathological markers associated with a disease. ‘Therapeutic effect’ may be used to indicate that a substance, such as a polypeptide described herein, binds to a receptor or target molecule on an organ to correct or modify the disease, disorder or condition. ‘Binding to a receptor’ may indicate that the protein acts as an agonist, antagonist, partial agonist, inverse agonist, and allosteric modulators.

[0089] A ‘therapeutic effect’ be direct or indirect. For example, a polypeptide may be exerting a therapeutic effect directly or indirectly on an organ. In cases of a direct therapeutic effect, the polypeptide may be acting on a receptor or target molecule which is causative of the disease, disorder or condition, which reduces or eliminates a pathological marker. An indirect therapeutic effect may occur through a secondary or intermediate mechanism or pathway that is not causative of the disease, disorder or condition.

[0090] Therefore, ‘cardiotherapeutic’ may be used to define reduced or eliminated symptoms or pathology of a cardiovascular or cardiac disease, disorder or condition in a subject. A cardiotherapeutic effect may be identified by reduced or eliminated symptoms or pathology of a cardiovascular or cardiac disease, disorder or condition. For example, there may be reduced or eliminates self-reports of symptoms severity or frequency, or reduced observation of markers of the disease, disorder or condition. Examples of measurements or observations of a cardiotherapeutic effect may include increased left ventricular ejection fraction (LVEF), reduced left ventricular end diastolic volume (LVEDV), reduced left ventricular end systolic volume (LVESV), reduced fibrotic tissue area, reduced number of fibrotic cells, increased number of cardiomyocytes (including reduced loss of cardiomyocytes), stroke volume, cardiac output, wall motion score index (WMSI), myocardial strain, exercise tolerance / capacity and brain natriuretic peptide (BNP) levels. A cardiotherapeutic mRNA may therefore encode a polypeptide, or a variant, fragment or isoform thereof, which prevents or treats one or more cardiovascular or cardiac pathologies.

[0091] Therefore, a ‘cardiotherapeutic agent’ may be a substance, composition, compound or molecule which directly or indirectly reduces or eliminates symptoms or pathology of a cardiovascular or cardiac condition, disease or disorder. A cardiotherapeutic agent may be an mRNA encoding a cardiotherapeutic polypeptide of interest, or a variant, fragment or isoform thereof.

[0092] ‘Present’ may define that a substance, compound or molecule is detectable in a specified organ, tissue, cell, or fluid using standard techniques in the art. ‘Subject’ may be used to refer to any organism to which an LNP or composition described herein may be administered e.g., for experimental or therapeutic purposes. The subject may be diagnosed with, or suspected of having, a cardiovascular or cardiac disease, disorder, condition or injury as specified herein. The subject may be at increased risk of a cardiovascular or cardiac disease, disorder, condition or injury as specified herein. The subject may be an animal, including a mammal, non-human primate or human. In embodiments, the subject is a human. The subject may be an infant, child or adult.

[0093] ‘Organ’ may refer to a collection of tissues that form a functional unit. The organ may perform a specific function or group of functions.

[0094] ‘Liver’ may refer to a part or all of a liver organ, liver tissues, or liver cells. ‘Liver cells’ may refer to any cell that forms a liver, and may include hepatocytes, Kupffer cells, hepatic stellate cells, cholangiocytes, and endothelial cells (including liver sinusoidal endothelial cells).

[0095] ‘Cardiovascular’ may refer to components of the cardiovascular system (e.g., heart and blood vessels). Therefore, ‘cardiovascular diseases’ may include ‘cardiac diseases’.

[0096] ‘Cardiac’ may refer to the heart, e.g. the heart organ, tissue, cells or diseases, conditions, injury, disorders or pathology of the heart.

[0097] ‘Secreted’ may refer to release of a substance from a specified organ, tissue or cell. Secretion may be from a specified organ tissue or cell into a different location, fluid, organ, tissue or cell, such as the blood. Secretion may be by active processes.

[0098] ‘Bloodstream’ may be used interchangeably with ‘blood’. Bloodstream may be taken to refer to blood within a circulatory system (e.g., within a body of a subject). ‘Blood’ may include erythrocytes, leukocytes, thrombocytes, and plasma. ‘Blood’ may include ‘serum’.

[0099] ‘Whole blood’ may refer to a blood sample before processing, e.g. a blood sample with all components of blood (e.g., erythrocytes, leukocytes, thrombocytes, and plasma) intact.

[0100] ‘Serum’ may refer to portion of whole blood that remains after coagulated materials have been removed from whole blood. In particular, serum may be the fluid and solvent component of blood which do not play a role in clotting. Serum may define the blood plasma without clotting factors, or blood without blood cells and clotting factors. ‘Disease’, ‘disorder’, ‘condition’ may be used to indicate a pathology, injury or abnormal physiology in a subject, which may be associated with negative symptoms. ‘Disease’ may be used to define a pathology which be generally known to have a clear cause and / or diagnostic criteria, ‘disorder’ may be used to define a pathology where normal function is disrupted, but a cause and / or diagnostic criteria is not defined, and ‘condition’ may be used more broadly to refer to any abnormal physiology, including injuries and acute events such as heart attacks.

[0101] ‘Cardiovascular disease, disorder or condition’ may refer to a disease, disorder or condition of the cardiovascular system. The cardiovascular disease, disorder or condition may be associated with an abnormality in the structure or function of one or more components of the cardiovascular system (heart and blood vessels) compared to healthy subjects, or compared to subjects who do not have the disease, disorder or condition. In other words, the cardiovascular disease, disorder or condition primarily affects the structure or function of one or more components of the cardiovascular system, and / or the pathology is in the cardiovascular system. Therefore, the pathology in the one or more components of the cardiovascular system may be a primary pathology (where the one or more components of the cardiovascular system are directly affected by the disease or condition), or a secondary pathology (complications as a result of a first disease e.g. heart failure as a result of chronic hypertension). As such, a ‘cardiovascular disease, disorder or condition’ may be associated with symptoms or pathology in non-cardiovascular cells, organs or tissues, but where treatment of the pathology in the cardiovascular system is expected to alleviate or reduce the symptoms or pathology in non-cardiovascular cells, organs or tissues.

[0102] ‘Cardiac disease, disorder or condition’ may refer to a disease, disorder or condition of the heart. The cardiac disease, disorder or condition may be associated with an abnormality in the structure or function of one or more components of the heart compared to healthy subjects, or compared to subjects who do not have the disease, disorder or condition. In other words, the cardiac disease, disorder or condition primarily affects the structure or function of one or more components of the heart, and / or the pathology is in the heart. Therefore, the pathology in the one or more components of the heart may be a primary pathology (where the one or more components of the heart are directly affected by the disease or condition), or a secondary pathology (complications as a result of a first disease). As such, a ‘cardiac disease, disorder or condition’ may be associated with symptoms or pathology in non-cardiac cells, organs or tissues, but where treatment of the heart pathology is expected to alleviate or reduce the symptoms or pathology in the non-cardiac cells, organs or tissues. ‘Acute’ may be used to indicate that a cardiovascular or cardiac disease, disorder or condition has rapid onset. Acute cardiovascular or cardiac disease, disorders or conditions may be relatively short in duration (e.g., as a single event, for minutes, hours, days or weeks) in that the cause may be eliminated to alleviate or eliminate symptoms. The acute cardiovascular or cardiac disease, disorder or condition may persist for less than, or up to two or three months. Acute cardiovascular or cardiac diseases, disorders or conditions tend to be associated with severe symptoms and require emergency treatment. Some acute cardiovascular or cardiac conditions may be resolved, but some may lead to long term (chronic) pathology or symptoms in a subject.

[0103] ‘Chronic’ may be used to indicate that a cardiovascular or cardiac disease or condition is long-lasting e.g., at least 3 months, years or lifetime. A chronic cardiovascular or cardiac disease or condition may be stable, progressive or have intermittent episodes of exacerbation.

[0104] ‘Onset of symptoms’ may refer to when a subject first experiences, or displays symptoms of a cardiovascular or cardiac disease, disorder or condition. Therefore, the subject may not note the symptoms, but they may be detected through tests.

[0105] ‘Transient’ may be used to a temporary state, i.e., non-permanent state. ‘Transient’ may refer to a state that exists for a defined period of time, such as hour or days. Therefore, ‘transient’ may refer to a time period up to (e.g., a maximum of) 24, 48, 72, 96 or 120 hours. As such, ‘transiently expressed’ may refer to temporary expression of an mRNA or polypeptide from the liver, liver tissue or liver cells. In this case, an mRNA or polypeptide may be present or increased in the liver, liver tissue, liver cells, blood, serum, heart, heart tissue or heart cells for a defined period of time before reverting to physiological levels (i.e., before administration of the mRNA or polypeptide). In other words, ‘transiently expressed’ may be used to indicate that an mRNA or polypeptide is not present for a prolonged period of time, particularly of weeks, or years.

[0106] ‘Treating’ or ‘treatment’ may refer to reducing or eliminating the severity and / or frequency of symptoms, arresting the development or progression of symptoms and / or pathology, slowing the progression of symptoms and / or pathology, eliminating the symptoms and / or pathology, or improving or ameliorating pathology / damage already caused, reducing the presence of underlying pathology, improving (e.g., reducing) or eliminating pathological markers.

[0107] ‘Preventing’ may refer to may refer to averting, delaying, impeding or hindering the contraction of symptoms and / or pathology. For example, reducing the likelihood of a subject developing or contracting a cardiac disease or condition. In some embodiments, preventing a cardiac disease or condition may reduce the risk of a cardiac disease or condition.

[0108] ‘Pharmaceutically acceptable’ may be used herein to refer to substances, compounds, materials, compositions, and / or dosage forms which are, which are suitable for use in human beings in a clinical environment, e.g., without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0109] ‘Therapeutically effective amount’ may refer to an amount of a cardiotherapeutic agent, e.g. mRNA, which produces the intended cardiotherapeutic result. For example, if a treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a cardiotherapeutic agent for the treatment of that disease or disorder is the amount necessary to affect at least a 25% reduction in that parameter.

[0110] ‘Unit dose’ may be used to define a discrete amount of a composition (optionally a pharmaceutical composition). As such, a unit dose may comprise or consist of a specified amount of LNPs and / or cargo (e.g., cardiotherapeutic agent, such as mRNA). In embodiments, the unit dose may be configured to deliver a single dosage to a subject.

[0111] ‘Dysregulated’ may refer to the increase or decrease of a given substance, compound or molecule as a result of, cause of, or association with a disease, disorder or condition. In other words, the substance, compound or molecule is dysregulated by comparison to a healthy subject or a control subject, who does not have the specified disease, disorder or condition.

[0112] Lipids and LNPs

[0113] The LNPs described herein may be between 50-150, 75-125, 80-125, 90-125, 90-120, 90-110, 95-105 or around 100 nm in diameter. Size of the nanoparticles may be determined using standard techniques in the art, such as dynamic light scattering, for example, using a Zetasizer Nano ZS (Malvern Instruments) with the LNPs.

[0114] The lipids forming the LNP (e.g., the outer layer of the LNP) may comprise or consist of an ionisable lipid. In other words, the LNP, or the lipids of the LNP, may comprise an ionisable lipid. The LNP may further comprise a structural lipid, a phospholipid, and a PEGylated lipid. The lipids of the LNP may comprise or consist of an ionisable lipid, a structural lipid, a phospholipid, and a PEGylated lipid. In embodiments, the lipids of the LNP may comprise or consist of only one type of each of: ionisable lipid, structural lipid, phospholipid, and PEGylated lipid. The LNP may be conjugated to an agent, such as a nucleic acid agent (e.g., targeting moiety) as described in WO2010 / 144740, which is incorporated by reference. The LNP may further comprise other comprise additional lipid types that are known in the art to improve properties of the LNP, such as particle stability, delivery efficacy, tolerability and biodistribution.

[0115] The skilled person understands that any suitable ionisable lipid may be used in a LNP described herein. However, in embodiments, the LNP comprises an ionisable lipid which comprises or consists of DLin-MC3-DMA, SM-102, ALC-0315, OF-02, A18-Iso5-2DC18, A1, A2, A3, A4, A5, A6, C12-200, CKK-E12, 9A1P9, 98N12-5, G0-C14, 7C1, L319, OF-Deg-Lin, 304013, 306-012B, 3060, FTT5 YSK05, YSK13, YSK15, LP01, CL1, CL4H6, and / or CL4F6, or analogues thereof, or combinations thereof. Optionally, the ionisable lipid is DLin-MC3-DMA, SM-102, or ALC-0315 or a combination thereof. Optionally the ionisable lipid is ALC-0315.

[0116] DLin-MC3-DMA (also referred to as DLin-M-C3-DMA, MC3 or M-C3) may comprise or consist of [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (IUPAC name), or analogues thereof. DLin-MC3-DMA may be identified with CAS number 1224606-06-7. DLin-MC3-DMA may include [(6Z,9Z,28Z,31Z)-(18, 19,20-13C3)heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (IUPAC name). DLin-MC3-DMA is commercially available to purchase. DLin-MC3-DMA may comprise or consist of a lipid according to formula I, or analogues thereof.

[0117]

[0118] Formula I

[0119] LNPs comprising DLin-MC3-DMA are described in W02010 / 144740 which is incorporated herein in its entirety.

[0120] SM-102 may comprise or consist of heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (IUPAC name), or analogues thereof. SM-102 may be identified with CAS number 2089251-47-6. SM-102 may include heptadecan-9-yl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate, 8-heptadecan-9-yloxy-A / -(2-hydroxyethyl)-8-oxo-A / -(6-oxo-6-undecoxyhexyl)octan-1 -amine oxide, or 5-[5-(4,4-dioctoxybutanoyloxy)pentyl-(2- hydroxyethyl)amino]pentyl 4,4-dioctoxybutanoate (IUPAC names). SM-102 is commercially available to purchase. SM-102 may comprise or consist of a lipid according to formula II, or analogues thereof.

[0121]

[0122] Formula II

[0123] LNPs comprising SM-102 are described in WO2017 / 049245, which is incorporated herein in its entirety.

[0124] ALC-0315 may comprise or consist of 6-[6-(2-hexyldecanoyloxy)hexyl-(4- hydroxybutyl)amino]hexyl 2-hexyldecanoate (IUPAC name), or analogues thereof. ALC-0315 may be identified with CAS number 2036272-55-4. ALC-0315 may include 6-[6-(2- hexyldecanoyloxy)hexyl-[2-(2-hydroxyethoxy)ethyl]amino]hexyl 2-hexyldecanoate, 8-[8-(2- hexyldecanoyloxy)octyl-(2-hydroxyethyl)amino]octyl 2-hexyldecanoate, 6-[6-(2- hexyldecanoyloxy)hexyl-(3-hydroxypropyl)amino]hexyl 2-hexyldecanoate, 6-[6-(3- hexylundecanoyloxy)hexyl-[2-(2-hydroxyethoxy)ethyl]amino]hexyl 3-hexylundecanoate or 6- [2-(2-hydroxyethoxy)ethyl-[6-(2-octyldodecanoyloxy)hexyl]amino]hexyl 2-octyldodecanoate (IUPAC names). ALC-0315 is commercially available to purchase. ALC-0315 may comprise or consist of a lipid according to formula III, or analogues thereof.

[0125]

[0126] o Formula III

[0127] LNPs comprising ALC-0315 are described in WO 2017 / 075531, which is incorporated herein in its entirety.

[0128] OF-02 may comprise or consist of 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxyoctadeca-9,12- dienyl]amino]butyl]piperazine-2, 5-dione (IUPAC name), or analogues thereof. OF-02 may be identified with CAS number 1883431-67-1. OF-02 may comprise or consist of a lipid according to formula IV, or analogues thereof.

[0129]

[0130] A18-Iso5-2DC18 may comprise or consist of ethyl 1-[3-(2-ethylpiperidin-1 -yl)propyl]-5,5- bis[(Z)-heptadec-8-enyl]-2H-imidazole-2-carboxylate (IIIPAC name), or analogues thereof. A18-Iso5-2DC18 may be identified with CAS number 2412492-09-0. A18-Iso5-2DC18 may comprise or consist of a lipid according to formula V, or analogues thereof.

[0131]

[0132] A1 may comprise or consist of di(oct-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate, or analogues thereof. A2 may comprise or consist of di(non-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, or analogues thereof. A3 may comprise or consist of di(dec-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate, or analogues thereof. A4 may comprise or consist of di(oct-3-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, or analogues thereof. A5 may comprise or consist of di(non-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate. A6 may comprise or consist of di(dec-3-yn- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, or analogues thereof. A1 may comprise or consist of a lipid according to formula Vl-A, or analogues thereof. A2 may comprise or consist of a lipid according to formula Vl-B, or analogues thereof. A3 may comprise or consist of a lipid according to formula Vl-C, or analogues thereof. A4 may comprise or consist of a lipid according to formula Vl-D, or analogues thereof. A5 may comprise or consist of a lipid according to formula Vl-E, or analogues thereof. A6 may comprise or consist of a lipid according to formula Vl-F, or analogues thereof.

[0133]

[0134] C12-200 may comprise or consist of 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (IIIPAC name), or analogues thereof. C12-200 may be identified with CAS number 1220890-25-4. C12-200 may comprise or consist of a lipid according to formula VII, or analogues thereof.

[0135]

[0136] Formula VII

[0137] CKK-E12 may comprise or consist of 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]piperazine-2, 5-dione (IIIPAC name), or analogues thereof. CKK-E12 may be identified with CAS number 1432494-65-9. CKK-E12 may comprise or consist of a lipid according to formula VIII, or analogues thereof.

[0138]

[0139] Formula VIII

[0140] 9A1P9 may comprise or consist of 2-(dioctylamino)ethyl nonyl hydrogen phosphate (IIIPAC name), or analogues thereof. 9A1P9 may be identified with CAS number 2760467-57-8. OF- 02 may comprise or consist of a lipid according to formula IX, or analogues thereof.

[0141]

[0142] Formula IX

[0143] 98N12-5 may comprise or consist of 3-[2-[2-[2-[bis[3-(dodecylamino)-3-oxopropyl]amino]ethyl- [3-(dodecylamino)-3-oxopropyl]amino]ethylamino]ethyl-[3-(dodecylamino)-3- oxopropyl]amino]- / \ / -dodecylpropanamide (IIIPAC name), or analogues thereof. 98N12-5 may be identified with CAS number 917572-74-8. 98N12-5 may comprise or consist of a lipid according to formula X, or analogues thereof.

[0144]

[0145] Formula X

[0146] G0-C14 may comprise or consist of 3-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3- oxopropyl]-[2-[[3-[2-[bis(2-hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[3-[2-(2- hydroxytetradecylamino)ethylamino]-3-oxopropyl]amino]ethyl]amino]- / \ / -[2-(2- hydroxytetradecylamino)ethyl]propanamide (IIIPAC name), or analogues thereof. G0-C14 may be identified with CAS number 1510653-27-6. G0-C14 may comprise or consist of a lipid according to formula XI, or analogues thereof.

[0147]

[0148] Formula XI

[0149] 7C1 may comprise or consist of a lipid according to formula XII, or analogues thereof.

[0150]

[0151] L319 may comprise or consist of bis[(Z)-non-2-enyl] 9-[4- (dimethylamino)butanoyloxy]heptadecanedioate (IIIPAC name), or analogues thereof. L319 may be identified with CAS number 1351586-50-9. L319 may comprise or consist of a lipid

[0152]

[0153] OF-Deg-Lin may comprise or consist of 2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2-[(9Z,12Z)-octadeca-9,12- dienoyl]oxyethyl]amino]ethyl (9Z,12Z)-octadeca-9,12-dienoate (IIIPAC name), or analogues thereof. OF-Deg-Lin may be identified by CAS number 1853202-95-5. OF-Deg-Lin may comprise or consist of a lipid according to formula XIV, or analogues thereof.

[0154]

[0155] Formula XIV

[0156] 304O₁₃ may comprise or consist of tridecyl 3-[2-[bis[2-[methyl-(3-oxo-3- tridecoxypropyl)amino]ethyl]amino]ethyl-methylamino]propanoate (IUPAC name), or analogues thereof. 304O₁₃ may be identified with CAS number 1566559-80-5. 304O₁₃ may comprise or consist of a lipid according to formula XV, or analogues thereof.

[0157]

[0158] Formula XV

[0159] 306-O12B may comprise or consist of 2-(octyldisulfanyl)ethyl 3-[3-[3-[bis[3-[2- (octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propyl-methylamino]propyl-[3-[2- (octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propanoate (IUPAC name), or analogues thereof.

[0160] 306-O12B may be identified with CAS number 2566523-06-4. 306-O12B may comprise or consist of a lipid according to formula XVI, or analogues thereof.

[0161]

[0162] Formula XVI

[0163] 306Oi10may comprise or consist of 8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3- oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3- oxopropyl]amino]propanoate (IUPAC name), or analogues thereof. 306Oi10may be identified with CAS number 2322290-93-5. 306Oi10may comprise or consist of a lipid according to formula XVII, or analogues thereof.

[0164]

[0165] Formula XVII

[0166] FTT5 may comprise or consist of octan-3-yl 9-[3-[[3,5-bis[3-[bis(9-octan-3-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-3-yloxy-9- oxononyl)amino]nonanoate or octan-4-yl 9-[3-[[3,5-bis[3-[bis(9-octan-4-yloxy-9- oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-4-yloxy-9- oxononyl)amino]nonanoate (IIIPAC name), or analogues thereof. FTT5 may be identified with CAS number 2803606-43-9 or 2328129-27-5. FTT5 may comprise or consist of a lipid according to formula XVIII-A or XVIII-B, or analogues thereof.

[0167]

[0168] CL4F8-6 may comprise or consist of [7-[4-(dipropylamino)butyl]-13-(2-hexyloctanoyloxy)-7- hydroxytridecyl] 2-hexyloctanoate (IUPAC name), or analogues thereof. CL4F8-6may be identified with CAS number 2766493-12-1. CL4F8-6 may comprise or consist of a lipid according to formula XIX, or analogues thereof.

[0169]

[0170] Formula XIX

[0171] CL4H6 may comprise or consist of [7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9- enoyl]oxytridecyl] (Z)-octadec-9-enoate (IUPAC name), or analogues thereof. CL4H6 may be identified with CAS number 2256087-35-9. CL4H6 may comprise or consist of a lipid according to formula XX, or analogues thereof.

[0172]

[0173] Formula XX

[0174] CL1 may comprise or consist of [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5- (dimethylamino)pentanoate (IUPAC name), or analogues thereof. CL1 may be identified with CAS number 1450888-71-7. CL1 may comprise or consist of a lipid according to formula XXI, or analogues thereof.

[0175]

[0176] Formula XXI

[0177] LP01 may comprise or consist of [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4- dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (IUPAC name), or analogues thereof. LP01 may be identified with CAS number 1799316-64-5. LP01 may comprise or consist of a lipid according to formula XXII, or analogues thereof.

[0178]

[0179] Formula XXII

[0180] YSK05 may comprise or consist of 1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12- dienoxy]piperidine (IUPAC name), or analogues thereof, such as YSK13 or YSK15. YSK05 may be identified with CAS number 1318793-78-0. YSK05 or analogues may comprise or consist of a lipid according to formula XXIII-A or analogues thereof, YSK13 may comprise or consist of a lipid according to formula XXIII-B, and YSK15 may comprise or consist of a lipid according to formula XXIII-C.

[0181]

[0182] The structural lipid may be a sterol. The structural lipid may comprise or consist of cholesterol fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, and / or brassicasterol, or derivatives thereof.

[0183] The phospholipid may be a 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DU PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 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-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-di linolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glycero-3-phosphoethanolamine, 1.2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), Egg Yolk Phosphatidylcholine, or 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), sphingomyelin (SM), ceramide or any related phosphatidylcholine. Neutral lipids may be synthetic or naturally derived.

[0184] The LNP may comprise a polyethylene glycol (PEG), including PEG2000, PEG3000 or PEG5000, optionally in the form of a PEGylated lipid. A PEG lipid may be selected from the nonlimiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. The PEGylated lipid may be any of the phospholipids described herein and further modified with PEG. In other words, a maximum of 1, 2, 5, 10, 15, 20, 25, 30, 35% of the phospholipids of an LNP may comprise PEG. For example, the PEGylated lipids may comprise or consist of 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG 2000), Methoxy Polyethylene Glycol-Distearoylphosphatidylethanolamine (mPEG-DSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (POPE-PEG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DOPE-PEG), 1.2-didodecanoyl-sn-glycero-3-phosphoethanolamine-peg-succinimidyl ester (PEG-DLPE), 1.2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DMPE) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), Distearoyl-rac-glycerol-PEG2000 (PEG-DSG), or 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA), R-3-[(w-methoxy poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxl-propyl-3-amine (PEG-c-DOMG). Note that the abbreviated lipid name and PEG may be switched, e.g., PEG-DSPE or DSPE-PEG, to refer to the same lipid. The numbers following PEG refer to the molecular weight of PEG. In embodiments, the PEG may be PEG 2000.

[0185] In some embodiments, the LNP comprises DLin-MC3-DMA, DSPC, Cholesterol, and DMG-PEG 2000. In some embodiments, the LNP comprises SM-102, DSPC, Cholesterol, and DMG-PEG.

[0186] In some embodiments, the LNP comprises ALC-0315, DOPE, Cholesterol, and PEG.

[0187] Mole percent used below refers to the quantity of moles of a specified lipid out of the total lipid amount in a LNP before the mRNA is added. In other words, the mole percent refers to the LNP both with and without mRNA.

[0188] The LNP (e.g., the lipids forming the LNP) may comprise an ionisable lipid at a mole percent between 20-60%, 30-60%, 35-55%, 39-51%, 35-45%, 40-50%, 45-55%, 40-60%, 35-45%, 38-42%, 39-41%, 48-52%, 49-51%, or about 40% or 50%, optionally wherein the ionisable lipid is in the LNP at a mole percent between 40-50%, optionally where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA or SM-102, the ionisable lipid may be in the LNP at a mole percent between 20-60%, 30-60%, 35-55%, 39-51%, 40-50%, 45-55%, 40-60%, 48-52%, 49-51%, or about 50%. In embodiments where the ionisable lipid is ALC-0315, the ionisable lipid may be in the LNP at a mole percent between 20-60%, 30-60%, 35-55%, 39-51%, 35-45%, 40-50%, 40-60%, 35-45%, 38-42%, 39-41%, or about 40%.

[0189] LNP may comprise a phospholipid at a mole percent between 5-40%, 5-35%, 7.5-32%, 5-25%, 10-30%, 5-15%, 20-35%, 22-33%, 20-40%, 25-35%, 8-12%, 9-11%, 28-32%, 29-31%, or about 10% or 30%, optionally wherein the phospholipid is in a LNP at a mole percent between 10-30%, optionally where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. The phospholipid may be DSPC in a LNP at a mole percent between 5-15%, or about 10%. The phospholipid may be DOPE in the LNP at a mole percent between 20-40%, 25-25% or about 30%. In embodiments where the ionisable lipid is DLin-MC3-DMA or SM-102, the phospholipid may be in the LNP at a mole percent between 5-40%, 5-35%, 7.5-32%, 5-25%, 10-30%, 5-15%, 8-12%, 9-11%, or about 10%. In embodiments where the ionisable lipid is ALC-0315, the phospholipid may be in the LNP at a mole percent between 5-40%, 5-35%, 7.5-32%, 10-30%, 20-40%, 20-35%, 22-33%, 20-40%, 25-35%, 28-32%, 29-31%, or about 30%.

[0190] The LNP may comprise a sterol at a mole percent between 15-50%, 25-55%, 20-45%, 22-43%, 25-40%, 27-39%, 27.5-38.5%, 27-28%, 38-39%, 25-30%, 35-40%, or about 27.5% or around 38.5%, optionally wherein the cholesterol is in a LNP at a mole percent between 25-40%, optionally where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA or SM-102, the sterol, e.g., cholesterol, may be in the LNP at a mole percent between 15-50%, 25-55%, 20-45%, 22-43%, 25-40%, 27-39%, 27.5-38.5%, 30-45%, 33-42%, 35-42%, 36.5-40%, 38-39%, or about 38.5%. In embodiments where the ionisable lipid is ALC-0315, the sterol, e.g., cholesterol, may be in the LNP at a mole percent between 15-50%, 25-55%, 20-45%, 22-43%, 25-40%, 27-39%, 27.5-38.5%, 27-28%, 20-35%, 22-33%, 25-30%, or about 27.5%.

[0191] The LNP may comprise a PEGylated lipid at a mole percent between 0.5-15%, 0.5-10%, 0.5-5%, 0.5-3.5%, 1.0-3.0%, 1.25-2.75%, 1.5-2.5%, 1.25-1.75%, 1.4-1.6%, 1.0-2.0%, 2.0-3.0%, 2.25-2.75%, 2.4-2.6%, or about 1.5% or 2.5%, optionally wherein the PEGylated lipid is in a LNP at a mole percent between 1.0-3.0%, optionally where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. The PEGylated lipid may be a DMG-PEG in the LNP at a mole percent between 0.5-15%, 0.5-10%, 0.5-5%, 0.5-3.5%, 1.0-3.0%, 1.0-2.0%, 1.25-1.75%, or about 1.5%. In embodiments where the ionisable lipid is DLin-MC3-DMA or SM-102, the PEGylated lipid may be DMG-PEG 2000 or DMG-PEG, respectively, and be in the LNP at a mole percent between 0.5-15%, 0.5-10%, 0.5-5%, 0.5-3.5%, 1.0-3.0%, 0.5-2.5%, 1.0-2.0%, 1.25-1.75%, or 1.4-1.6%, or about 1.5%. In embodiments where the ionisable lipid is ALC-0315, the PEGylated lipid may be PEG and be in the LNP at a mole percent between 0.5-15%, 0.5-10%, 0.5-5%, 0.5-3.5%, 1.0-3.0%, 1.5-3.5%, 2.0-3.0%, 2.25-2.75%, 2.4-2.6% or about 2.5%.

[0192] The LNP may comprise lipids at molar ratios of 50:10:38.5:1.5 or 40:30:27.5:2.5 for the ionisable lipid:phospholipid:sterol: PEGylated lipid, respectively, optionally for embodiments where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG 2000, the ionisable lipid:phospholipid:sterol: PEGylated lipid the lipids may be at molar ratio 50:10:38.5:1.5, respectively. In embodiments where the ionisable lipid is SM-102, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG the lipids may be at molar ratio of 50:10:38.5:1.5, respectively. In embodiments where the ionisable lipid is ALC-0315, the phospholipid is DOPE, the sterol is cholesterol, and the PEGylated lipid is PEG, the lipids may be at molar ratio of 40:30:27.5:2.5, respectively. Alternatively, the amount of a specified lipid in a LNP may be defined by a percentage of the weight of the specified lipid from the total lipid weight in an LNP, i.e., weight (wt) / total lipid weight (wt).

[0193] The LNP may comprise an ionisable lipid at a percentage between 30-75%, 35-70%, 40-65%, 45-60%, 50-60%, 53-57%, 53.9-56.4% or about 53.9%, 55.5% or 56.4% wt / total lipid wt, optionally for embodiments where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA, the ionisable lipid may be in the LNP at a percentage between 50-60%, 52-58%, 53-55%, 53.5-54.5%, or about 53.9% or 53.94% wt / total lipid wt. In embodiments where the ionisable lipid is ALC-0315, the ionisable lipid may be in the LNP at a percentage between 50-60%, 53-59%, 54-58%, 55.5-56.5%, 56-57%, or about 56.4% or 56.37% wt / total lipid wt. In embodiments where the ionisable lipid is SM-102, the ionisable lipid may be in the LNP at a percentage between 50-60%, 52-58%, 53-57%, 54.5-57%, 55-56%, or about 55.5% or 55.56% wt / total lipid wt.

[0194] The LNP may comprise a phospholipid at a percentage between 5-25%, 7-20%, 10-20%, 10-18%, 10-16%, 10-15%, 12-15%, 12-14.5%, 12.2-14.3% or about 12.2%, 13.7% or 14.2% wt / total lipid wt, optionally for embodiments where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA, the phospholipid may be in the LNP at a percentage between 10-16%, 10-15%, 12-15%, 13-14%, or about 13.7% or 13.69% wt / total lipid wt. In embodiments where the ionisable lipid is ALC-0315, the phospholipid may be in the LNP at a percentage between 10-15%, 11-14%, 12-13%, or about 12.2% or 12.22% wt / total lipid wt. In embodiments where the ionisable lipid is SM-102, the phospholipid may be in the LNP at a percentage between 11-17%, 12-16%, 13-15%, 13.5-14.5%, 14-15% or about 14.2% or 14.21% or wt / total lipid wt.

[0195] The LNP may comprise a sterol at a percentage between 10-40%, 15-35%, 20-30%, 23-27%, 24-26%, 24.2-25.8% or about 24.3%, 24.4% or 25.7% wt / total lipid wt, optionally for embodiments where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA, the sterol, e.g., cholesterol, may be in the LNP at a percentage between 10-40%, 15-35%, 20-30%, 23-27%, 24-27%, 25-26%, or about 25.7% or 25.73% wt / total lipid wt. In embodiments where the ionisable lipid is ALC-0315, the sterol, e.g., cholesterol, may be in the LNP at a percentage between 10-40%, 15-35%, 20-30%, 22-27%, 23-26%, 24-25% or about 24.4% or 24.43% wt / total lipid wt. In embodiments where the ionisable lipid is SM-102, the sterol, e.g., cholesterol, may be in the LNP at a percentage between 10-40%, 15-35%, 20-30%, 22-26%, 23.5-24.5%, 24-25%, or about 24.3% or 24.29% wt / total lipid wt.

[0196] The LNP may comprise a PEGylated lipid at a percentage between 2-10%, 3-10%, 4-9%, 5-7%, 5.9-7% or about 5.9%, 6.6% or 7.0% wt / wt i.e., mg / total lipid wt, optionally for embodiments where the ionisable lipid is DLin-MC3-DMA, ALC-0315 or SM-102. In embodiments where the ionisable lipid is DLin-MC3-DMA, the PEGylated lipid may be in the LNP at a percentage between 2-10%, 3-10%, 4-9%, 5-7%, 5.9-7%, 6-7%, 6.4-6.8%, 6.6-6.7% or about 6.6% or 6.64% wt / total lipid wt. In embodiments where the ionisable lipid is ALC- 0315, the PEGylated lipid may be in the LNP at a percentage between 2-10%, 3-10%, 4-9%, 5-7%, 5.9-7%, 6.5-7.5%, 6.8-7.2%, 6.9-7.1%, 6.95-7.05%, or about 6.98% or 7.0% wt / total lipid wt. In embodiments where the ionisable lipid is SM-102, the PEGylated lipid may be in the LNP at a percentage between 2-10%, 3-10%, 4-9%, 5-7%, 5.9-7%, 5.5-6.5%, 5.7-6.2%, 5.9-6.0, or about 5.94 or 6.0% wt / total lipid wt.

[0197] The LNP may comprise lipids at wt / total lipid wt ratio of 5.4: 1.4:2.6:0.6, 5.6: 1.2:2.5:0.7, or 5.6:1.4:2.4:0.6for the ionisable lipid:phospholipid:sterol: PEGylated lipid. Optionally, where the ionisable lipid is DLin-MC3-DMA, the lipids may be in a LNP at wt / total lipid wt ratio of 5.4:1.4:2.6:0.6. Optionally, where the ionisable lipid is ALC-0315, the lipids may be in a LNP at wt / total lipid wt ratio of 5.6: 1.2:2.5:0.7. Optionally, where the ionisable lipid is SM-102, the lipids may be in a LNP at wt / total lipid wt ratio of 5.6: 1.4:2.4:0.6. In embodiments where the ionisable lipid is DLin-MC3-DMA, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG 2000, the lipids are in the LNP at a wt / total lipid wt ratio of ionisable lipid:phospholipid:sterol: PEGylated lipid of 5.4:1.4:2.6:0.6. In embodiments, where the ionisable lipid is SM-102, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG, the lipids are in the LNP at a wt / total lipid wt ratio of ionisable lipid:phospholipid:sterol: PEGylated lipid of 5.6:1.4:2.4:0.6. In embodiments where the ionisable lipid is ALC-0315, the phospholipid is DOPE, the sterol is cholesterol, and the PEGylated lipid is PEG, the lipids are in the LNP at a wt / total lipid wt ratio of ionisable lipid:phospholipid:sterol: PEGylated lipid of 5.6:1.2:2.5:0.7.

[0198] Quantification of lipids forming an LNP can be measured using standard techniques in the art, such as gas chromatography coupled with flame ionization detection, liquid chromatographymass spectrometry (LC / MS), mass spectrometry (MS), high performance liquid chromatography (HPLC) or variations thereof such as ultra-high performance liquid chromatography (UHPLC), UHPLC coupled with an evaporative light scattering detector (ELSD), UHPLC coupled with charged aerosol detector (CAD), reversed phase (RP) HPLC, size exclusion (SE) HPLC, ion pair (IP) HPLC and ion exchange (I EX) HPLC.

[0199] LNPs of the invention may have a PDI of between 0.05-0.20, 0.075-0.20, 0075-0.175, 0.075-0.15, 0.08-0.15, 0.08-0.14, 0.08-0.12, 0.09-0.1, around 0.1. The PDI may be measured using standard techniques in the art, such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) / tuneable resistive pulse sensing (TRPS), size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) with multi-angle static light scattering (MALS) and microscopy. Examples of DLS include using a Zetasizer Nano ZS (Malvern Instruments), following manufacturer instructions. The LNPs described herein may encapsulate or further comprise a cargo or payload. The cargo may be a cardiotherapeutic agent (e.g., mRNA) as described herein. The cargo may be present in (e.g., encapsulated within) the inner aqueous space or in a lipid portion of the LNP. The cargo may be partially or fully encapsulated within the LNP (i.e., the cargo is not exposed to conditions outside the LNP).

[0200] Encapsulation may be measured for LNPs with an mRNA cargo using standard techniques in the art. For example,, a QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of an RNA by the nanoparticle composition. In this example, the samples are diluted to a concentration of approximately 5 pg / mL in a TE buffer solution (10 mM Tris-HCI, 1 mM EDTA, pH 7.5). 50 pL of the diluted samples are transferred to a polystyrene 96 well plate and either 50 pL of TE buffer or 50 pL of a 2% Triton X-100 solution is added to the wells. The plate is incubated at a temperature of 37° C for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 in TE buffer, and 100 pL of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0201] The cargo may encode a cardiotherapeutic polypeptide of interest, or a variant, fragment or isoform thereof. The cargo may comprise or consist of messenger RNA (mRNA, including modified mRNA) encoding a cardiotherapeutic polypeptide of interest, or a variant, fragment or isoform thereof. mRNA may be present in the LNP as a single stranded or double-stranded polynucleotide, or be encoded by a DNA. Translation of the mRNA in vivo (i.e., in the subject to which the LNP comprising the mRNA is administered), may produce the encoded cardiotherapeutic polypeptide, or a variant, fragment or isoform thereof.

[0202] mRNA may be produced by in vitro transcription. In brief, in vitro transcription includes template-directed synthesis of mRNA molecules from a DNA template comprised of an upstream bacteriophage promoter sequence (e.g. including that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Bowman et al., in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed), New York, N. Y. Humana Press, 2012). Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, for example, Brunelle and Green, 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114, incorporated herein by reference). The in vitro transcribed mRNA is then purified from the components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolation of the mRNA transcripts are well known in the art and include phenol / chloroform extraction, precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride, size exclusion chromatography, silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits including, SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). Contaminants, such as short RNAs that result from abortive transcription initiation, as doublestranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3' extension, can be removed using well known techniques including HPLC purification.

[0203] The mRNA may have one or more modifications that increase stability, half-life or translation of the mRNA, or reduce immunogenicity of the mRNA. Therefore ‘mRNA’ includes modified mRNA. These modifications may include modifications to the nucleobases, nucleosides, or nucleotides, including non-naturally occurring nucleobase, nucleoside or nucleotide modification, a cap structure, a chain terminating nucleoside, a stem loop, or a poly-A sequence, optionally wherein the modification is a 5-methoxyuridine, N1-methylpseudouridine or 5’ Cap-1, which are standard in the art. Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co- transcriptionally or post-transcriptionally. Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase. 5' capping and 3 '-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.

[0204] In some embodiments, the mRNA is N1-methyl-pseudouridine-modified and comprises a 5’ Cap-1.

[0205] In embodiments, the mRNA encodes a polypeptide, or a variant, fragment or isoform thereof, which is cardiotherapeutic. The mRNA may encode a chordin-like 1 (Chrdl1, SEQ ID NOs: 1-4), family with sequence similarity 3 member b (Fam3b, SEQ ID NOs: 5-7), family with sequence similarity 3 member C (Fam3c, SEQ ID NO: 8), midkine (Mdk, SEQ ID NO: 9), pleiotrophin (SEQ ID NO: 10), serine protease HtrA1 (Htral, SEQ ID NO: 11), NHL repeatcontaining protein 3 (Nhlrc3, SEQ ID NOs: 12-13), or relaxin-1 (Rln-1, SEQ ID NOs: 14-15) polypeptide. The mRNA may encode a polypeptide having an amino acid sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any of SEQ ID NOs 1-15, or a variant, fragment or isoform thereof. The mRNA may comprise or consist of polynucleotide having a sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an mRNA encoded any of SEQ ID NOs: 16-42. The mRNA may be encoded by a polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any of SEQ ID NOs: 16-42, optionally a coding sequence of any of SEQ ID NOs: 16-42. The mRNA may be encoded by a polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to between 100-4000, 200-3500, 200-3000, 200-2500, 200-2000, 200-1500, 400-4000, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 500-4000, 500-3500, 500-3000, 500-2500, 500-2000, or 500-1500 nucleotides of SEQ ID NOs: 16-42, optionally a coding sequence of any of SEQ ID NOs: 16-42.

[0206] The cardiotherapeutic polypeptide or a variant, fragment or isoform thereof (and thus mRNA encoding said polypeptide) may be identified using standard techniques in the art, such as the FunSel method described in Ruozi et al., 2022, which is incorporated by reference. In brief, pools of barcoded AAVs which include a cDNA encoding a polypeptide of interest and injected into the heart of a mouse at a low multiplicity of infection. Myocardial infarction (Ml) is then induced. Cardiomyocytes die as a consequence of the Ml, but cells gaining selective advantage survive. After 3 weeks, vector DNA is recovered from the surviving myocardial cells, and the frequency of each vector determined using next generation sequences (NGS) to detect a DNA barcode within the viral genome. Barcode read frequencies were compared to those in non-infarcted animals injected with the same AAV9 pool and to those in the viral preparation before injection. When a vector induced CM protection, it was positively selected and, thus, enriched over the others. The opposite holds true for factors favouring CM death. Selected polypeptides can then be verified by individual administration.

[0207] The cargo (e.g., cardiotherapeutic agent) may be present in the LNP at ratios of 5-60 lipids per mRNA, optionally 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 20-50, 20-45, 20-40, 20-35, 20-30 lipids per mRNA. This may be determined using standard techniques in the art. For example, nanodrop or ultraviolet-visible spectroscopy can be used to determine the concentration of a cargo (e.g., cardiotherapeutic agent, such as mRNA) in LNP compositions. In this example, 100 pL of the diluted formulation in 1 xPBS is added to 900 pL of a 4: 1 (v / v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of cargo (e.g., cardiotherapeutic agent such as mRNA) in the LNP composition can be calculated based on the extinction coefficient of the cargo (e.g., cardiotherapeutic agent) used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm. Quantification of cargo (e.g., cardiotherapeutic agent such as mRNA) can also be measured using gas chromatography coupled with flame ionization detection, liquid chromatography-mass spectrometry (LC / MS), mass spectrometry (MS), high performance liquid chromatography (HPLC) or variations thereof such as ultra-high performance liquid chromatography (UHPLC), UHPLC coupled with an evaporative light scattering detector (ELSD), UHPLC coupled with charged aerosol detector (CAD), reversed phase (RP) HPLC, size exclusion (SE) HPLC, ion pair (IP) HPLC and ion exchange (I EX) HPLC. Determining the amount of cardiotherapeutic agent per nanoparticle may enable determination of the LNPs needed for a particular dose.

[0208] Methods of producing LNPs are standard in the art, and are described, for example in WO2010 / 144740, WO2017 / 075531, and WO2017 / 049245, which are incorporated by reference. In summary, a mixture of lipids that are to form the LNP are mixed at the desired amounts (e.g., molar ratios, concentrations or mole percent) in an aqueous solution, and may contain ethanol. This produces pre-formed LNPs. The pre-formed LNPs may then be mixed with a solution containing the cargo (e.g., cardiotherapeutic agent) to form LNPs encapsulating the cargo. Alternatively, a mixture of lipids that are to form the LNP are combined at the desired amounts (e.g., molar ratios, concentrations or mole percent) in the presence of cargo (e.g., cardiotherapeutic agent) in a buffered aqueous solution to directly from LNPs encapsulating the cargo (e.g., cardiotherapeutic agent).

[0209] The alcohol, e.g., ethanol, may be present at 20% (w / w) to 45% (w / w). In addition, The mixture may be mixed, for example, using a microfluidic mixing chip. The mixture may be warmed (e.g., to a temperature of about 25° C to about 50° C) and / or sonicated before mixing. The cargo (e.g., cardiotherapeutic agent, such as mRNA) may be added in either process at a total lipid to cardiotherapeutic agent weight ratio of between 5:1 to 50: 1, 10:1 to 50: 1, 10:1 to 45: 1, 15:1 to 45:1, 20:1-50:1, 20:1-45:1, 20:1-40:1, 30:1-50:1, 35:1-45:1, optionally around 40:1. The composition comprising LNPs can be further processed to improve the composition. For example, the composition may be dialyzed to remove the alcohol. The pH of the solution may then be raised to neutralize at least a portion of the surface charges on the LNPs in order to release some of the unencapsulated cargo (e.g., cardiotherapeutic agent) which may have attached to the LNP surface. Examples of suitable buffers include HEPES, aqueous, citrate, phosphate, acetate, and MES. The released cargo (e.g., cardiotherapeutic agent) can then be removed from the solution via chromatography using standard methods, and then the solution switched into a buffer with a pH above the pKa of the lipids used. The LNPs may be optionally sized using standard methods in the art, including filtration, sonication, homogenization or extrusion. Particle size can be measured by dynamic light scattering (DLS) analysis using conventional techniques in the art such as using a Zetasizer Nano ZS (Malvern, UK), or NanoSight NS300 (Malvern, UK) following manufacturer instructions.

[0210] Cardiotherapeutic polypeptides

[0211] The mRNA may encode a cardiotherapeutic polypeptide. A ‘cardiotherapeutic polypeptide’ may be a polypeptide which directly or indirectly reduces or eliminates symptoms or pathology of a cardiovascular or cardiac condition, disease or disorder.

[0212] In some embodiments, the cardiotherapeutic polypeptide is a cardioprotective polypeptide. A ‘cardioprotective polypeptide’ may be a polypeptide which preserves myocyte viability, to protect cardiomyocytes from ischemic damage, for example due to an acute cardiovascular or cardiac disease.

[0213] Examples of cardiotherapeutic polypeptides include chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), and relaxin-1 (Rln-1) polypeptides, or variants, fragments or isoforms thereof. In some embodiments, the mRNA encodes a Chrdl1, a Fam3b, or a Fam3c polypeptide or variants, fragments or isoforms thereof. In some embodiments, the mRNA encodes a Chrdl1, a Fam3b, or a Fam3c polypeptide. Chrdl1, Fam3b, and Fam3c have been identified as cardioprotective factors against myocardial infarction (see Ruozi, G., et al., 2022. Science translational medicine, 14(660), p.eabo0699).

[0214] Chordin-like 1 (Chrdl1) polypeptides

[0215] In some embodiments, the mRNA encodes a Chrdl1 polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Chrdl1 polypeptide.

[0216] Chordin-like 1 (Chrdl1) is a Bone Morphogenetic Protein (BMP) extracellular inhibitor, mainly expressed in mesenchyme-derived cell types, pericytes in the retina and in neural cells (Sakuta, H., et al. Science 293, 111-115 (2001); Nakayama, N., et al. Dev Biol 232, 372-387 (2001); Chandra, A., etal. Biochem Biophys Res Commun 344, 786-791 (2006); and Coffinier, C., Tran, U., Larrain, J. & De Robertis, E. M. Meeh Dev 100, 119-122 (2001)). The Chrdl1 name derives from its sequence similarity with Chordin, another BMP inhibitor identified as a factor dorsal izi ng Xenopus embryo. Chrdl1 has a spatiotemporal expression pattern distinct from Chordin, but both genes contain cysteine-rich units designed procollagen repeats (CRs), which are also present in a variety of extracellular matrix proteins. CR1 and CR3 are responsible for Chrdl1-BMPs binding. The protein binds with high affinity to BMP4 and with less affinity to BMP5, BMP6 and BMP7.

[0217] Example Chrdl1 polypeptides, variants, fragments and isoforms thereof are provided herein. The Chrdl1 polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Chrdl1. The cardiotherapeutic or cardioprotective activity of the Chrdl1 polypeptide or a variant, fragment or isoform thereof can be readily determined by the skilled person. For example, suitable in vitro assays include: (a) protection from hydrogen peroxide- or doxorubicin-induced cell death, for example using TUN EL assays or Caspase activation assays; (b) induction of autophagy, for example the assaying the formation of LC3-positive autophagy vesicles; and / or (c) reduction of BMP and TGFbeta activities, for example activation of aSMA expression in cardiac fibroblasts upon treatment with recombinant TGFbeta, or reduction of SMAD 1 / 5 / 8 phosphorylation upon treatment with recombinant BMP4.

[0218] The Chrdl1 polypeptide or a variant, fragment or isoform thereof may be a human Chrdl1 polypeptide or a variant, fragment or isoform thereof. Example amino acid sequence of Chrdl1 are provided by SEQ ID NOs: 1-4 and 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 1-4 or 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to any of SEQ ID NOs: 1-4 or 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to any of SEQ ID NOs: 1-4 or 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to any of SEQ ID NOs: 1-4 or 43. In some embodiments, the Chrdl1 polypeptide ora variant, fragment or isoform thereof comprises or consists of any of SEQ ID NOs: 1-4 or 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of any of SEQ ID NOs: 1-4 or 43.

[0219] An example amino acid sequence of Chrdl1 is provided by UniProt ID: Q9BU40-6. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 1. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 1. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 1. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 1.

[0220] A further example amino acid sequence of Chrdl1 is provided by UniProt ID: Q9BU40-3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 2. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 2. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 2. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 2. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 2.

[0221] A further example amino acid sequence of Chrdl1 is provided by UniProt ID: Q9BU40-4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 3. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 3.

[0222] A further example amino acid sequence of Chrdl1 is provided by UniProt ID: Q9BU40-5. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 4. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 4.

[0223] In some embodiments, the Chrdl1 polypeptide is a secreted form of Chrdl1. In some embodiments, the Chrdl1 polypeptide does not comprise a signal peptide. An example amino acid sequence of a Chrdl1 polypeptide lacking the signal peptide is amino acids 28-456 of SEQ ID NO: 1, shown as SEQ ID NO: 43.

[0224] In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 43. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 43.

[0225] Further Chrdl1 amino acid sequences could be generated by a person of skill in the art by suitable amino acid substitutions, deletions, and / or insertions. Known amino acid substitutions compared to SEQ ID NO: 1 include M7V, R195Q, R208H, R208L, K317N, R382Q, andY447H. Other amino acid substitutions compared to SEQ ID NO: 1 which are considered to be tolerated by the SIFT programme include Y12C, Y12H, F14L, S15L, L17F, L20V, L21F, G25D, Q29H, V30I, T35A, Y57H, V60I, S68L, N72S, N72H, R77Q, L86F, H90R, H90P, 191 M, V106A, N108S, T111A, S112R, Y117H, N118S, H124P, L127V, V129I, G132E, T143A, G155S, K157Q, T163A, F166L, S169P, R176Q, V177A, L184P, L184V, L184M, F194L, E201G, H207R, S209F, S209C, S209Y, Y211C, P213L, P213R, P213S, P214R, P215S, S216N, R217Q, Q218P, A219P, G220V, L222V, S223A, R224H, A228V, A228T, R232Q, G233E, G233R, A234V, A234P, A234T, M236I, M236T, Q240E, S242T, I245T, N251D, K253R, N276K, A279V, I282T, N291K, V292I, T293N, K294N, H301Y, N304S, R305Q, Q312K, D315E, P331A, G332D, Q333R, S334N, D336A, N337S, G339A, Y340C, Y340H, E344K, T346M, E351A, V353I, G358R, T360I, T360S, T368I, P372S, V374A, V374I, V378I, R382Q, H390R, H390Y, E392K, P403S, E423A, A424T, S431A, and E440K. Known amino acid insertions compared to SEQ ID NO: 1 include P — > PE at position 100 and K — > KE at position 328. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof comprises a CR1 domain and a CR3 domain. In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof binds to BMP4. The binding to BMP4 may be determined by a co-immunoprecipitation assay (see e.g. Larrain, J., et al., 2000. Development, 127(4), pp.821-830). In some embodiments, the Chrdl1 polypeptide or a variant, fragment or isoform thereof binds to and reduces the activity of BMP4. The activity of BMP4 may be determined by reduction of SMAD 1 / 5 / 8 phosphorylation upon treatment with recombinant BMP4.

[0226] Any mRNA coding sequences for the above Chrdl1 polypeptides or variants, fragments or isoforms thereof is comprised in the present invention. Example mRNA coding sequences are provided by SEQ ID NOs: 16-25. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 16-25. In some embodiments, the mRNA coding sequence comprises or consists of any of SEQ ID NOs: 16-25. In some embodiments, the mRNA coding sequence consists of any of SEQ ID NOs: 16-25.

[0227] An example mRNA coding sequence of Chrdl1 is provided by NCBI reference sequence NM_001143981.2. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 16. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 16.

[0228] Family with sequence similarity 3 member b (Fam3b) polypeptides

[0229] In some embodiments, the mRNA encodes a Fam3b polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Fam3b polypeptide.

[0230] Family with sequence similarity 3 member b (Fam3b), also known as Pancreatic-derived factor (PANDER), is highly expressed in the pancreas, where it participates in regulation of glucose homeostasis and p-cell function (Robert-Cooperman, C. E., Wilson, C. G. & Burkhardt, B. R. FEBS Lett 585, 1345-1349 (2011); Robert-Cooperman, C. E., et al. Diabetes 59, 2209-2218 (2010); Yang, J., etal. Diabetes 54, 3217-3228 (2005)). Fam3b is composed of two antiparallel β sheets lined by three short helices packing to form a conserved water-filled cavity, thereby forming a FAM3 β-β-α Fold (see Johansson, P., et al., 2013. Structure, 21(2), pp.306-313).

[0231] Example Fam3b polypeptides, variants, fragments and isoforms thereof are provided herein. The Fam3b polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Fam3b. The cardiotherapeutic or cardioprotective activity of the Fam3b polypeptide or a variant, fragment or isoform thereof can be readily determined by the skilled person. For example, suitable in vitro assays include: (a) protection from hydrogen peroxide- or doxorubicin-induced cell death, for example using TUN EL assays or Caspase activation assays; and (b) induction of autophagy, for example the assaying the formation of LC3-positive autophagy vesicles.

[0232] The Fam3b polypeptide or a variant, fragment or isoform thereof may be a human Fam3b polypeptide or a variant, fragment or isoform thereof. Example amino acid sequence of Fam3b are provided by SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to any of SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to any of SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to any of SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of any of SEQ ID NOs: 5-7 or 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof consists of any of SEQ ID NOs: 5-7 or 44.

[0233] An example amino acid sequence of Fam3b is provided by UniProt ID: P58499-1. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 5. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 5. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 5. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 5. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 5.

[0234] A further example amino acid sequence of Fam3b is provided by UniProt ID: P58499-2. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 6. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 6. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 6. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 6. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 6.

[0235] A further example amino acid sequence of Fam3b is provided by UniProt ID: P58499-3. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 7. In some embodiments, the Fam3b polypeptide is a secreted form of Fam3b. In some embodiments, the Fam3b polypeptide does not comprise a signal peptide. An example amino acid sequence of a Fam3b polypeptide lacking the signal peptide is amino acids 30-235 of SEQ ID NO: 5, shown as SEQ ID NO: 44.

[0236] In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 44. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 44.

[0237] Further Fam3b amino acid sequences could be generated by a person of skill in the art by suitable amino acid substitutions, deletions, and / or insertions. Known amino acid substitutions compared to SEQ ID NO: 1 include V14M. Other amino acid substitutions compared to SEQ ID NO: 5 which are considered to be tolerated by the SIFT programme include P3T, L4V, L9F, K10R, V12M, F13L, F13V, A17T, A17S, Y23C, S24L, L28V, E30D, L311, D34E, A35S, A35V, P36S, P36L, S39G, S39T, Y42C, I44V, R45H, S46G, S46N, I47L, I47V, G48R, G48R, G48A, E49Q, V52L, V57A, K59R, R60K, D72E, S85G, S85R, K86R, M98V, M98T, M98I, E100G, Q101E, N104S, V105I, V105G, A112V, T122R, A123E, A123V, T124I, R125Q, C126S, C126Y, M129V, M129T, M129I, E131K, G132S, P137S, T139I, K140N, S144N, K148E, K148N, F152L, M153I, V154A, N165S, N165K, A167T, A170V, 1171V, 1179V, R180K, M182I, M182I, 1191V, S200P, E201K, I202M, R204K, A212D, I226V, I230V, and K232R. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises a GG-type lectin domain. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof comprises a FAM3 β-β-α fold. In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof binds to fibroblast growth factor receptors (FGFRs). The binding to FGF may be determined by a co-immunoprecipitation assay (see e.g. Zhang, F., et al., 2021. PNAS, 118(20), p.e2100342118). In some embodiments, the Fam3b polypeptide or a variant, fragment or isoform thereof induces apoptosis in α and β cell lines. The apoptosis in α and β cell lines may be determined by a cell viability assay (see e.g. Cao, X., et al., 2003. Diabetes, 52(9), pp.2296-2303).

[0238] Any mRNA coding sequences for the above Fam3b polypeptides or variants, fragments or isoforms thereof is comprised in the present invention. Example mRNA coding sequences are provided by SEQ ID NOs: 26-27. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 26-27. In some embodiments, the mRNA coding sequence comprises or consists of any of SEQ ID NOs: 26-27. In some embodiments, the mRNA coding sequence consists of any of SEQ ID NOs: 26-27.

[0239] An example mRNA coding sequence of Fam3b is provided by NCBI reference sequence NM_058186.4. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 26. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 26. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 26.

[0240] Family with sequence similarity 3 member c (Fam3c) polypeptides

[0241] In some embodiments, the mRNA encodes a Fam3c polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Fam3c polypeptide.

[0242] Fam3c, also known as Interleukin-like EMT inducer (I LEI), is ubiquitously expressed. It induces inner-ear cell proliferation (Pilipenko, V. V., Reece, A., Choo, D.l. & Greinwald, J. H., Jr. Gene 335, 159-168 (2004)), modulates osteogenic differentiation (Bendre, A., Buki, K. G. & Maatta, J. A. Differentiation 93, 50-57 (2017)) and has a role in epithelial-mesenchymal transition (EMT) during cancer progression (Waerner, T., et al. Cancer Cell 10, 227-239 (2006); Lahsnig, C., et al. Oncogene 28, 638-650 (2009)). Restoration of its levels in the liver of obese diabetic mice improves insulin resistance and reduces fatty liver (Chen, Z., et al. Diabetes 66, 1185-1197 (2017); Chen, Z., etal. Oncotarget8, 106038-106049 (2017)). Fam3c exists as monomers but also as covalent dimers and comprises a FAM3 β-β-α fold (see Johansson, P., et al., 2013. Structure, 21(2), pp.306-313; and Jansson, A. M., et al., 2017. Journal of Biological Chemistry, 292(37), pp.15501-15511). Example Fam3c polypeptides, variants, fragments and isoforms thereof are provided herein. The Fam3c polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Fam3c. The cardiotherapeutic or cardioprotective activity of the Fam3c polypeptide or a variant, fragment or isoform thereof can be readily determined by the skilled person. For example, suitable in vitro assays include: (a) protection from hydrogen peroxide- or doxorubicin-induced cell death, for example using TUN EL assays or Caspase activation assays; and (b) induction of autophagy, for example the assaying the formation of LC3-positive autophagy vesicles.

[0243] The Fam3c polypeptide or a variant, fragment or isoform thereof may be a human Fam3c polypeptide or a variant, fragment or isoform thereof. Example amino acid sequence of Fam3c are provided by SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to any of SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to any of SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to any of SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of any of SEQ ID NOs: 8 and 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof consists of any of SEQ ID NOs: 8 and 45.

[0244] An example amino acid sequence of Fam3c is provided by UniProt ID: Q92520-1. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 8. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 8. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 8. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 8. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 8.

[0245] In some embodiments, the Fam3c polypeptide is a secreted form of Fam3c. In some embodiments, the Fam3c polypeptide does not comprise a signal peptide. An example amino acid sequence of a Fam3c polypeptide lacking the signal peptide is amino acids 25-227 of SEQ ID NO: 8, shown as SEQ ID NO: 45.

[0246] In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 45. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 45.

[0247] Further Fam3c amino acid sequences could be generated by a person of skill in the art by suitable amino acid substitutions, deletions, and / or insertions. Amino acid substitutions compared to SEQ ID NO: 8 which are considered to be tolerated by the SIFT programme include V3A, A4T, V10M, A14V, V15G, V15L, F20S, V22I, M311, M31T, D32G, D32N, A33V, A33S, A33T, S34R, S34N, L35F, N37K, F39I, A43V, A43S, A43T, D45N, T46A, A47V, A48T, R49C, K52E, P54S, A63P, M73I, M73V, I85M, N90D, G95A, N98D, K112T, V116L, T119S, T119A, A130V, A130S, P131L, A138V, I139L, Q140R, Q140K, 1144V, G148A, E160K, A161T, R162Q, R163Q, 1173V, T174I, N175S, N175T, N181S, V183I, 1190V, K191R, T192K, T192A, N203S, N203D, K204E, V215A, V216L, and Q224H. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises a GG-type lectin domain. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof comprises a FAM3 β-β-α fold. In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof binds to leukemia inhibitory factor receptor (LIFR). The binding to FGF may be determined by a co-immunoprecipitation assay (see e.g Woosley, A. N., et al., 2019. Oncogene, 38(20), pp.3794-3811). In some embodiments, the Fam3c polypeptide or a variant, fragment or isoform thereof activates STAT3 activation. STAT3 activation may be determined by a mammosphere assay (see e.g. Woosley, A. N., et al., 2019. Oncogene, 38(20), pp.3794-3811).

[0248] Any mRNA coding sequences for the above Fam3c polypeptides or variants, fragments or isoforms thereof is comprised in the present invention. Example mRNA coding sequences are provided by SEQ ID NOs: 28-29. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 28-29. In some embodiments, the mRNA coding sequence comprises or consists of any of SEQ ID NOs: 28-29. In some embodiments, the mRNA coding sequence consists of any of SEQ ID NOs: 28-29.

[0249] An example mRNA coding sequence of Fam3c is provided by NCBI reference sequence NM_014888.3. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 28. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 28. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 28.

[0250] Midkine (Mdk) polypeptides

[0251] In some embodiments, the mRNA encodes a Mdk polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Mdk polypeptide.

[0252] Midkine (MK or MDK), also known as neurite growth-promoting factor 2 (NEGF2), is a protein that in humans is encoded by the MDK gene. Midkine may play a protective role against cardiac ischemia / reperfusion injury through a reduction of apoptotic reaction (see e.g. Horiba, M., et al., 2006. Circulation, 114(16), pp.1713-1720).

[0253] The Mdk polypeptide or a variant, fragment or isoform thereof may be a human Mdk polypeptide or a variant, fragment or isoform thereof. The Mdk polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Mdk. An example amino acid sequence of Mdk is provided by UniProt ID: P21741-1. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 9. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 9. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 9. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 9. In some embodiments, the Mdk polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 9.

[0254] An example mRNA coding sequence of Mdk is provided by NCBI reference sequence NM_001012334.2. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 30. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 30. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 30.

[0255] Pleiotrophin polypeptides

[0256] In some embodiments, the mRNA encodes a pleiotrophin polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a pleiotrophin polypeptide.

[0257] Pleiotrophin (PTN) also known as heparin-binding brain mitogen (HBBM) or heparin-binding growth factor 8 (HBGF-8) or neurite growth-promoting factor 1 (NEGF1) or heparin affinity regulatory peptide (HARP) or heparin binding growth associated molecule (HB-GAM) is a protein that in humans is encoded by the PTN gene. Pleiotrophin potentiates cardiomyocyte apoptosis through inhibition of endogenous AKT / PKB activity (see e.g. Li, J., et al., 2007. Journal of Biological Chemistry, 282(48), pp.34984-34993).

[0258] The pleiotrophin polypeptide or a variant, fragment or isoform thereof may be a human pleiotrophin polypeptide or a variant, fragment or isoform thereof. The pleiotrophin polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of pleiotrophin. An example amino acid sequence of pleiotrophin is provided by UniProt ID: P21246. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 10. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 10. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 10. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 10. In some embodiments, the pleiotrophin polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 10.

[0259] An example mRNA coding sequence of pleiotrophin is provided by NCBI reference sequence NM_002825.7. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 31. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 31.

[0260] Serine protease HtrA1 (HtrA1) polypeptides

[0261] In some embodiments, the mRNA encodes a HtrA1 polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a HtrA1 polypeptide.

[0262] Serine protease HtrA1 is an enzyme that in humans is encoded by the HTRA1 gene. Htral has been shown to be effective in reducing dilatation and partially improving function post-myocardial infarction (see Ruozi, G., et al., 2022. Science translational medicine, 14(660), p.eabo0699).

[0263] The HtrA1 polypeptide or a variant, fragment or isoform thereof may be a human HtrA1 polypeptide or a variant, fragment or isoform thereof. The HtrA1 polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of HtrA1. An example amino acid sequence of HtrA1 is provided by UniProt ID: Q92743. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 11. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 11. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 11. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 11. In some embodiments, the HtrA1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 11.

[0264] An example mRNA coding sequence of HtrA1 is provided by NCBI reference sequence NM_002775.5. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 32. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 32.

[0265] NHL repeat-containing protein 3 (Nhlrc3) polypeptides

[0266] In some embodiments, the mRNA encodes a Nhlrc3 polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Nhlrc3 polypeptide.

[0267] NHL repeat-containing protein 3 (Nhlrc3) is a protein that in humans is encoded by the NHLRC3 gene. Nhlrc3 has been shown to be effective in reducing dilatation and partially improving function post-myocardial infarction (see Ruozi, G., et al., 2022. Science translational medicine, 14(660), p.eabo0699).

[0268] The Nhlrc3 polypeptide or a variant, fragment or isoform thereof may be a human Nhlrc3 polypeptide or a variant, fragment or isoform thereof. The Nhlrc3 polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Nhlrc3. An example amino acid sequence of Nhlrc3 is provided by UniProt ID: Q5JS37-1 and Q5JS37-2. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12 or 13. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 12 or 13. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 12 or 13. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 12 or 13. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 12 or 13. In some embodiments, the Nhlrc3 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 12 or 13.

[0269] An example mRNA coding sequence of Nhlrc3 is provided by NCBI reference sequence NM_001012754.4. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 33. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 33. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 33.

[0270] Relaxin-1 (Rln-1) polypeptides

[0271] In some embodiments, the mRNA encodes a Rln-1 polypeptide or a variant, fragment or isoform thereof. In some embodiments, the mRNA encodes a Rln-1 polypeptide.

[0272] Relaxin-1 (Rln-1) is a protein that in humans is encoded by the RLN1 gene. Rln-1 belongs to the relaxin family of proteins for which there is evidence on cardiovascular effects (see e.g. Martin, B., et al., 2019. Molecular and Cellular Endocrinology, 487, pp.45-53).

[0273] The Rln-1 polypeptide or a variant, fragment or isoform thereof may be a human Rln-1 polypeptide or a variant, fragment or isoform thereof. The Rln-1 polypeptide or a variant, fragment or isoform thereof may retain the endogenous cardiotherapeutic or cardioprotective activity of Rln-1. An example amino acid sequence of Rln-1 is provided by UniProt ID: P04808-2. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof comprises or consists of an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof has five or fewer, four or fewer, three or fewer, two or fewer, or one amino acid substitution, deletion, and / or insertion compared to SEQ ID NO: 15. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof has one, two, three, four, or five amino acid substitutions compared to SEQ ID NO: 15. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof has one or two amino acid insertions compared to SEQ ID NO: 15. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof comprises or consists of SEQ ID NO: 15. In some embodiments, the Rln-1 polypeptide or a variant, fragment or isoform thereof consists of SEQ ID NO: 15.

[0274] An example mRNA coding sequence of Rln-1 is provided by NCBI reference sequence NM_006911.4. In some embodiments, the mRNA coding sequence comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 34. In some embodiments, the mRNA coding sequence comprises or consists of SEQ ID NO: 34. In some embodiments, the mRNA coding sequence consists of SEQ ID NO: 34.

[0275] Signal peptides

[0276] The mRNA may encode one or more signal peptides. For example, the cardiotherapeutic polypeptide may comprise or be operably linked to one or more signal peptide.

[0277] As used herein, the term “signal peptide” may refer to a short peptide that prompts a cell to translocate the cardiotherapeutic polypeptide so that it may be secreted out of a cell. Any signal peptide that directs translocation and secretion of the cardiotherapeutic polypeptide from the cell may be used. The signal peptide may be cleaved from the cardiotherapeutic polypeptide upon secretion. Optionally, the signal peptide is fused to the N-terminus of the cardiotherapeutic polypeptide. Typically, signal peptides are 16 to 30 amino acid residues in length and comprised of a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain and a C-terminal region with the cleavage site for signal peptidase.

[0278] Suitable signal peptides may include those with are operable in liver cells, e.g. signal peptides that direct translocation and secretion of the cardiotherapeutic polypeptide from a liver cell. In some embodiments, the cardiotherapeutic polypeptide comprises or is operably linked to an endogenous signal peptide. For example, a Chrdl1 polypeptide or variant, fragment or isoforms thereof may comprise a Chrdl1 signal peptide; a Fam3b polypeptide or variant, fragment or isoforms thereof may comprise a Fam3b signal peptide; and a Fam3c polypeptide or variant, fragment or isoforms thereof may comprise a Fam3c signal peptide.

[0279] In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity thereto.

[0280] In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity thereto.

[0281] In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity thereto.

[0282] Kits

[0283] Also provided is a kit comprising or consisting of a phospholipid as described herein, an ionisable lipid as described herein, a structural lipid as described herein, a PEGylated lipid as described herein, and an mRNA selected from Chrdl1, Fam3c or Fam3b as described herein. Alternatively, there is provided a kit comprising or consisting of an ionisable lipid as described herein and a cardiotherapeutic mRNA as described herein. The kit may further comprise a phospholipid as described herein, a PEGylated lipid as described herein and / or a structural lipid as described herein. In embodiments, the lipids in the kit may be in pre-formed LNPs, and the mRNA may be provided in a different container to the LNPs, such that the pre-formed LNPs and mRNA can be mixed. In embodiments, the lipids in the kit may be in individual containers, such that the lipids and mRNA can be mixed to form the LNPs. The kit may further comprise instructions, other lipids for LNPs, and any required buffers or solutions.

[0284] Compositions

[0285] Also provided are compositions comprising the LNPs described herein, including pharmaceutical compositions and unit dose.

[0286] Compositions provided herein may comprise or consist of a one or more LNPs. A composition provided herein (including pharmaceutical composition or unit dose composition) may comprise or consist of between 0.1-100%, 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 0.1-95%, 1-95%, 10-95%, 20-95%, 30-95%, 40-95%, 50-95%, 60-95%, 70-95%, 80-95%, 0.1-90%, 1-90%, 10-90%, 20-90%, 30- 90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 0.1-80%, 1-80%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 0.1-70%, 1-70%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 0.1-60%, 1-60%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%, 0.1-50%, 1-50%, 10-50%, 20-50%, 30-50%, or 40-50% (wt / wt) of LNPs described herein.

[0287] Pharmaceutical compositions or unit doses may optionally a pharmaceutically acceptable diluent, carrier, and / or excipient. Examples of known diluents, carriers and / or excipients are known in the art. Examples of diluents, carriers or excipients include water, saline, physiological saline, Ringer’s solution, fixed oils such as synthetic mono or diglycerides which may serve as a solvent, solubilizing agents and emulsifiers such as ethyl alcohol, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulphate, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and advents for adjusting tonicity such as sodium, chloride or dextrose, and cryoprotectants such as sucrose or trehalose, and / or any mixture thereof. Diluents, carriers or excipients may be sterile.

[0288] Compositions described herein may further comprise or consist of, or be administered with, another cardiotherapeutic agent. The other cardiotherapeutic agent may treat or prevent the same cardiovascular or cardiac disease, disorder or condition that the LNPs are being provided to treat. The other cardiotherapeutic agent may reduce or eliminate symptoms associated with the same cardiovascular or cardiac disease, disorder or condition that the LNPs are being provided to treat. The composition may be administered separately to the other cardiotherapeutic agent, e.g., sequentially, or within 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 36, or 48 hours of each other.

[0289] The composition may have a pH between 5.5-8.5, 5.9-8.1, 6.0-8.0, 6.2-8.0, 6.4-8.0, 6.4-7.5, or 7.0-8.0.

[0290] Relative amounts of LNPs and pharmaceutically acceptable diluents, carriers or excipients, and / or any additional ingredients in a pharmaceutical composition or unit dose may be altered by the skilled person depending upon the identity, size, and / or condition of the subject treated and the route by which the composition is to be administered. The compositions described herein may be sterilized by conventional, well known sterilization techniques. The aqueous solutions can then be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. In other words, the composition may be a liquid formulation or lyophilized formulation.

[0291] The compositions or unit dose may comprise a therapeutically effective amount of cargo (e.g., the cardiotherapeutic agent, optionally mRNA). In embodiments, the cargo of the composition or unit dosage may comprise or consist of 1mg-100mg cargo (e.g., cardiotherapeutic agent, optionally mRNA). In embodiments, the composition or unit dosage may comprise 1-90, 1-80, 1-70, 1-60, 1-50mg, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-45, 5-40, 5-35, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-45, 10-40, and 10-35mg cargo (e.g., cardiotherapeutic agent, optionally mRNA) per dose. In embodiments, the composition or unit dosage may comprise between 0.0001-20, 0.0001-15, 0.0001-10, 0.0001-7, 0.0001-5, 0.0001-2, 0.0001-1, 0.0001-0.1, 0.0001-0.01, 0.001-20, 0.001-15, 0.001-10, 0.001-7, 0.001-5, 0.001-2, 0.001-1, 0.001-0.1, 0.001-0.01, 0.01-20, 0.01-15, 0.01-10, 0.01-7, 0.01-5, 0.01-2, 0.01-1, 0.01-0.1, 0.1-20, 0.1-15, 0.1-10, 0.1-7, 0.1-5, 0.1-2, 0.1-1, 0.15-2.0, 0.15-1.75, 0.15-1.5, 0.15-1.25, 0.15-1.0, 0.15-0.9, 0.15-0.8, 0.15-0.7, 0.2-20, 0.2-15, 0.2-10, 0.2-7, 0.2-5, 0.2-2.0, 0.2-1.75, 0.2-1.5, 0.2-1.25, 0.2-1.0, 0.2-0.9, 0.2-0.9, 0.2-0.7, 0.25-2.0, 0.25-1.75, 0.25-1.5, 0.25-1.25, 0.25-1.0, 0.25-0.9, 0.25-0.8, 0.25-0.7, 0.3-20, 0.3-15, 0.3-10, 0.3-7, 0.3-5, 0.3-2.0, 0.3-1.75, 0.3-1.5, 0.3-1.25, 0.3-1.0, 0.3-0.9, 0.3-0.8, or 0.3-0.7 mg of cargo / kg body weight of the subject, optionally wherein the cargo is a cardiotherapeutic agent, optionally mRNA. The subject may optionally be a human subject, more optionally an adult human subject when calculating dose.

[0292] Administration and uses

[0293] The LNPs, compositions or unit doses described herein may be for use in treating or preventing a cardiovascular or cardiac disease, disorder, condition or injury in a subject in need thereof. The LNPs, compositions or unit doses described herein may be used in a method of treating or preventing a cardiovascular or cardiac disease, disorder, condition or injury in a subject in need thereof.

[0294] The method or use may comprise administering the LNPs, compositions or unit doses described herein to the subject. The subject may be any animal, including mammals, nonhuman primates and humans. The subject may be a child, adolescent or adult. In embodiments the subject is an adult human. According to the invention, the subject has a cardiovascular or cardiac disease, disorder, condition or injury. The disease, disorder, condition or injury may be acute.

[0295] The cardiovascular or cardiac disease, disorder, condition or injury may be selected from: heart failure, consequences of myocardial infarction, reperfusion-injury after percutaneous coronary intervention (coronary angioplasty), cardiac toxic damage (e.g., by cancer chemotherapy), myocardial infarction, ischemic and non-ischemic cardiomyopathy, ventricular dysfunction, and myocarditis. The cardiac disease, disorder, condition or injury may be associated with a loss of cardiac myocytes. The loss of cardiac myocytes may be up to 5, 10, 15, 20 or 25% of cardiac myocytes in the left ventricle. Loss of cardiac myocytes may be detected by non-invasive imaging or measurement of biomarkers. Non-invasive imaging may include cardiac magnetic resonance imaging (MRI, optionally with gadolinium dye), positron emission tomography (PET), or echocardiography. Biomarkers (for example, present in blood samples) for detecting death of cardiomyocytes may include cardiac troponin I (CTn I), cardiac troponin T (CTnT), B-type natriuretic peptide (BNP), and / or N-terminal proBNP. In embodiments where the disease, disorder, condition or injury is cardiovascular, treatment may result in reduced infarct size, reduced ventricular dilation, improved LVEF, improved LVESV, and / or reduced fibrosis in the heart of the subject.

[0296] In some embodiments, the risk of heart failure is reduced in a subject suffering from a cardiovascular or cardiac disease, disorder, condition or injury. In some embodiments, the risk of heart failure is reduced in a subject at risk of a cardiovascular or cardiac disease, disorder, condition or injury. In some embodiments, cardiac muscle cell viability is preserved in a subject suffering from a cardiovascular or cardiac disease, disorder, condition or injury. In some embodiments, cardiac muscle cell viability is preserved in a subject at risk of a cardiovascular or cardiac disease, disorder, condition or injury.

[0297] Treatment may result in a reduction in cardiac remodeling. Cardiac remodeling may refer to a group of molecular, cellular and interstitial changes that manifest clinically as changes in size, mass, geometry and function of the heart after injury and may be determined by any suitable method, for example by changes in the cavity diameter, mass (hypertrophy and atrophy), geometry (heart wall thickness and shape), areas of scar, fibrosis and inflammatory infiltrate (see e.g. Cohn, Ferrari and Sharpe, 2000, J Am Coll Cardiol, 35(3): 569-582).

[0298] Treatment may result in an improved LV ejection fraction may be improved by the method of the invention. Left ventricular ejection fraction (LVEF) may refer to the fraction of chamber volume ejected in systole in relation to the volume of the blood in the ventricle at the end of diastole and may be determined by any suitable method, for example by echocardiography, magnetic resonance imaging (MRI), or computed tomography (CT). LV ejection fraction can be increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with an untreated subject.

[0299] Treatment may result in a reduction in left ventricular volume. LV volume may be determined by any suitable method, for example by echocardiography, magnetic resonance imaging (MRI), or computed tomography (CT). In some embodiments, the LV volume is the end-diastolic volume (EDV) or the end-systolic volume (ESV). LV volume can be decreased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with an untreated subject.

[0300] Treatment may result in reduced myocardial fibrosis. Myocardial fibrosis may be defined as the expansion of the cardiac interstitium due to net accumulation of extracellular matrix proteins, and may arise as a result of cardiac diseases (see e.g. Frangogiannis, N. G., 2021. Cardiac fibrosis. Cardiovascular research, 117(6), pp.1450-1488). The presence and extent of myocardial fibrosis may be determined by any suitable method, for example by endomyocardial biopsy (EMB) histopathology or multiparametric cardiac magnetic resonance (CMR) imaging (see e.g. Abecasis, J., et al., 2023. Cardiovascular Pathology, 65, p.107541 and Grani, C., et al., 2019. Journal of Cardiovascular Magnetic Resonance, 21(1), p.14). Myocardial fibrosis can be decreased by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% when compared with an untreated subject.

[0301] Compositions may be administered to the subject using parenteral techniques. The composition may be administered as an intravenous, intraperitoneal, subcutaneous intramuscularly, intracardiac, or intra-arterial injection or catheterisation and the like. Optionally, the composition is delivered intravenously.

[0302] The composition may be administered to the subject within 1, 2, 3 or 4 weeks, or within 144, 120, 96, 72, 60, 48, 36, 24, 16, 12, 10, 8, 6, 5, 4, 3, 2, 1 or 0.5 hours of onset of one or more symptoms of the cardiovascular or cardiac disease, disorder, condition or injury. The composition may be administered to the subject within 1, 2, 3 or 4 weeks, or within 144, 120, 96, 72, 60, 48, 36, 24, 16, 12, 10, 8, 6, 5, 4, 3, 2, 1 or 0.5 hours of onset of the cardiovascular or cardiac disease, disorder, condition or injury. The composition may be administered to the subject within 1, 2, 3 or 4 weeks, or within 144, 120, 96, 72, 60, 48, 36, 24, 16, 12, 10, 8, 6, 5, 4, 3, 2, 1 or 0.5 hours of an event capable of triggering, or triggering, the cardiovascular or cardiac disease, disorder, condition or injury. Therefore, symptoms may not be experienced by the subject before administration of the composition. In this case, the cardiovascular or cardiac disease, disorder, condition or injury may be detectable upon examination by a medical professional or medical tests.

[0303] A dose may be administered once, for example, to treat or prevent an acute cardiovascular or cardiac disease, disorder or condition. A dose may be provided multiple times, e.g. one or more times per day, in the same or a different amount. For example, multiple doses may be administered, followed by testing of blood sample, to determine if a desired level of polypeptide encoded by the mRNA has been reached. If the level of polypeptide encoded by the mRNA is not sufficient, a further dose may be administered. This may occur within 6, 12, 24, 48, or 72 hours, 4, 5, 6, 7 days, or 2, 3, 4 weeks.

[0304] Upon administration to the subject, particularly after intravenous administration, LNPs may accumulate in, be localised in, be taken up by, or be distributed to, the liver and / or spleen of the subject. At least 90, 91, 92, 93, 94, 95, 96, 97, 97.5, 97, 97.5, 98, 98.5, 99, or 99.5% of administered LNPs may accumulate in, be localised in, be taken up by, or be distributed to, the liver and / or spleen of the subject. The ratio of LNPs in the liver:spleen may be between 1:1 and 30:1, 1:1 and 25:1, 1:1 and 20:1, 1:1 and 15:1, 1:1 and 12:1, 1:1 and 10:1, 1:1 and 9:1, 10:1 and 8:1, 2:1 and 30:1, 2:1 and 25:1, 2:1 and 20:1, 2:1 and 15:1, 2:1 and 12:1, 2:1 and 10:1, 2:1 and 9:1, 2:1 and 8:1, 3:1 and 30:1, 3:1 and 25:1, 3:1 and 20:1, 3:1 and 15:1, 3:1 and 12:1, 3:1 and 10:1, 3:1 and 9:1, or 3:1 and 8:1. Less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25 or 0.1% of LNPs administered may accumulate in, be localised in, be taken up by, or be distributed to, the heart, muscle and / or lungs of the subject.

[0305] The LNPs may enter liver and / or spleen cells through various mechanisms, such as receptor mediated uptake (e.g., apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR)-mediated endogenous uptake to hepatocytes, or scavenger receptor-mediated uptake into hepatic Kupffer cells), or endocytosis. Once in the liver (liver cells) or spleen (spleen cells), the LNPs may release their cargo into the cytosol of the cell, and may then translated to produce the encoded polypeptide, or fragment, variant or isoform thereof. The mRNA may then be degraded within the cell. Therefore, production of the encoded polypeptide, or fragment, variant or isoform thereof, may be limited e.g., in terms of quantity that is able to be produced and time that the polypeptide may be produced for. In other words, expression or production of the polypeptide, or fragment, variant or isoform thereof, may be transient in the liver and / or spleen cells after administration. The polypeptide or fragment, variant or isoform thereof may then be secreted (e.g., released from) the liver and / or spleen cells, optionally into the blood of the subject. The polypeptide or fragment, variant or isoform thereof may be secreted into the serum, erythrocytes, leukocytes, and / or plasma of the subject, optionally into the serum. The secretion from liver and / or spleen cells may be through various mechanisms, such as exocytosis or vesicular transport. The polypeptide or fragment, variant or isoform thereof may enter the blood through mechanisms such as diffusion. There may be intermediate steps in this process that are not recited, such as post-translational modifications of the polypeptide, fragment, variant or isoform thereof.

[0306] The polypeptide, or fragment, variant or isoform thereof, may be transiently increased in the blood of the subject. In examples, the polypeptide, or fragment, variant or isoform thereof may not be present (i.e., are undetectable) in the blood of the subject to be treated before administration of the LNPs, however, after administration of the LNPs to the subject, the polypeptide, or fragment, variant or isoform thereof may be present (i.e., detectable) in the blood for a transient period of time. In other words, levels of the polypeptide, or fragment, variant or isoform thereof in the blood of a treated subject may increase for a specified period of time and then be undetectable after this period of time. Alternatively, the polypeptide, or fragment, variant or isoform thereof may be detectable in the blood of the subject before administration of the LNPs. In this case, after administration of the LNPs to the subject, levels of the polypeptide, or fragment, variant or isoform thereof may be increased in the blood of the patient for a transient period of time. In other words, levels of the polypeptide, or fragment, variant or isoform thereof in the blood may increase for a specified period of time and then return to normal levels in the subject. Reference to ‘before administration’ may refer to the levels in the specific subject to be treated, or to reference values of a population of subjects who have not been treated with the LNPs. Similarly, reference to ‘normal levels’ may refer to the levels in the specific subject before treatment, or to reference values of a population of subjects who have not been treated with the LNPs.

[0307] The polypeptide, or fragment, variant or isoform thereof, may be transiently present in the serum, erythrocytes, leukocytes, and / or plasma of the subject. This may be because the mRNA may be degraded within the cell, and so the production of the encoded polypeptide, or fragment, variant or isoform thereof, may be limited e.g., in terms of quantity that is able to be produced and time that the polypeptide may be produced for. Therefore, polypeptide, or fragment, variant or isoform thereof, may be present in the blood of the subject within 0.5, 1, 2, 3, 4, 5, 6, 8 or 12 hours of (after) administration of the composition. The polypeptide, or fragment, variant or isoform thereof, may be present in the blood of the subject for up to (i.e., a maximum of) 24, 48, 72, 96 or 120 hours after administration of the composition. After this, the polypeptide, fragment, variant or isoform may return to normal (physiological levels) in the blood, or may not be detectable in the blood. The polypeptide, or fragment, variant or isoform thereof, may be present in the blood of the subject between 1-168, 1-160, 1-150, 1-140, 1-130, 1-120, 1-114, 1-108, 1-102, 1-96, 1-90, 1-84, 1-78, 1-72, 1-66, 1-60, 1-54, 1-48, 1-42, 1-36, 1-30, 1-24, 2-168, 2-160, 2-150, 2-140, 2-130, 2-120, 2-114, 2-108, 2-102, 2-96, 2-90, 2-84, 2-78, 2-72, 2-66, 2-60, 2-54, 2-48, 2-42, 2-36, 2-30, 2-24, 3-168, 3-160, 3-150, 3-140, 3-130, 3-120, 3-114, 3-108, 3-102, 3-96, 3-90, 3-84, 3-78, 3-72, 3-66, 3-60, 3-54, 3-48, 3-42, 3-36, 3-30, 3-24, 4-168, 4-160, 4-150, 4-140, 4-130, 4-120, 4-114, 4-108, 4-102, 4-96, 4-90, 4-84, 4-78, 4-72, 4-66, 4-60, 4-54, 4-48, 4-42, 4-36, 4-30, 4-24, 5-168, 5-150, 5-140, 5-130, 5-120, 5-114, 5-108, 5-102, 5-96, 5-90, 5-84, 5-78, 5-72, 5-66, 5-60, 5-54, 5-48, 5-42, 5-36, 5-30, 5-24, 6-168, 6-150, 6-140, 6-130, 6-120, 6-114, 6-108, 6-102, 6-96, 6-90, 6-84, 6-78, 6-72, 6-66, 6-60, 6-54, 6-48, 6-42, 6-36, 6-30, or 6-24 hours after administration.

[0308] Presence of the polypeptide or fragment, variant or isoform thereof in the blood may be detected in a blood sample obtained from the subject after the composition of LNPs has been administered. Optionally, the blood sample is taken at any time point between 1-168, 1-160, 1-150, 1-140, 1-130, 1-120, 1-114, 1-108, 1-102, 1-96, 1-90, 1-84, 1-78, 1-72, 1-66, 1-60, 1-54, 1-48, 1-42, 1-36, 1-30, 1-24, 1-16, 1-12, 1-10, 2-168, 2-160, 2-150, 2-140, 2-130, 2-120, 2-114, 2-108, 2-102, 2-96, 2-90, 2-84, 2-78, 2-72, 2-66, 2-60, 2-54, 2-48, 2-42, 2-36, 2-30, 2-24, 2-16, 2-12, 2-10, 3-168, 3-160, 3-150, 3-140, 3-130, 3-120, 3-114, 3-108, 3-102, 3-96, 3-90, 3-84, 3-78, 3-72, 3-66, 3-60, 3-54, 3-48, 3-42, 3-36, 3-30, 3-24, 4-168, 4-160, 4-150, 4-140, 4-130, 4-120, 4-114, 4-108, 4-102, 4-96, 4-90, 4-84, 4-78, 4-72, 4-66, 4-60, 4-54, 4-48, 4-42, 4-36, 4-30, 4-24, 5-168, 5-150, 5-140, 5-130, 5-120, 5-114, 5-108, 5-102, 5-96, 5-90, 5-84, 5-78, 5-72, 5-66, 5-60, 5-54, 5-48, 5-42, 5-36, 5-30, 5-24, 6-168, 6-150, 6-140, 6-130, 6-120, 6-114, 6-108, 6-102, 6-96, 6-90, 6-84, 6-78, 6-72, 6-66, 6-60, 6-54, 6-48, 6-42, 6-36, 6-30, 6-24, 6-16, 6-12, or 6-10 hours after administration.

[0309] Serum may be separated from the blood sample before any assay to detect the present of the polypeptide or fragment, variant or isoform thereof. Separation techniques are also well known in the art. Briefly, blood may be collected with or without anticoagulants. If collected without anticoagulants, for example, in a serum separator tube, the collected blood may be allowed to clot, optionally for 10-90, 10-60, 10-45, 10-40, 10-30, 15-90, 15-60, 15-45, 15-40, 15-30, 20-90, 20-60, 30-90, 30-60 minutes, optionally at room temperature. Alternatively, if the sample is collected in a tube with anticoagulants present, centrifugation can proceed immediately. The clotted blood may then be centrifuged to separate serum from other components in blood. Centrifugation may occur at around 500-5000, 1000-5000, 500-4000, 1000-4000, 500-3000, 1000-3000 or 1000-2000 xg, optionally for between 5-30, 5-25, 5-20, 5-15, 10-30, 10-25, 10- 20, 10-15 minutes. This separates the blood into three layers. The top layer is serum, the middle layer is white blood cells and platelets, the bottom layer is red blood cells. The middle layer may not be visible by eye. The serum layer is then isolated for testing.

[0310] Presence of a specific polypeptide or fragment, variant or isoform thereof (for example, in the blood) can be detected using standard protein assays in the art, immunoassays and enzyme assays, such as enzyme-linked immunosorbent assay (ELISA), western blotting, mass spectrometry (MS), radioimmunoassay (RIA), Luminex xMAP technology, and protein microarrays. Suitable antibodies are known in the art.

[0311] Once in the blood, the polypeptide, or fragment, variant or isoform thereof, may be delivered via the circulatory system (e.g., circulated) to a heart cell, heart tissue or the heart of the subject. In this embodiment, the polypeptide, or fragment, variant or isoform thereof, may accumulate, be located in, be taken up by, or be distributed to, the heart cells, tissue or the heart where the polypeptide, or fragment, variant or isoform thereof can exert its cardiotherapeutic effect. For example, to treat or prevent a cardiovascular or cardiac disease, disorder, condition or injury. Alternatively, the polypeptide, or fragment, variant or isoform thereof, may exert its cardiotherapeutic effect on the heart via the blood, for example by activating or inhibiting a particular pathway within the blood which in turn results in a therapeutic effect in the heart. In order words, the effect of the polypeptide, or fragment, variant or isoform thereof on a heart cell, tissue or the heart may be direct or indirect. Thus, in some embodiments, the polypeptide, or fragment, variant or isoform thereof, may activate a cardiotherapeutic pathway in the blood, or inhibit a cardiotoxic pathway in the blood, which in turn exerts a cardiotherapeutic effect to treat or prevent a cardiovascular or cardiac disease, disorder, condition or injury.

[0312] Myocardial infarction

[0313] In some embodiments, the cardiovascular or cardiac disease, disorder, condition or injury is myocardial infarction. In some embodiments, the myocardial infarction is acute myocardial infarction.

[0314] The present invention provides a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA for use in treating or preventing myocardial infarction in a subject in need thereof, wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the myocardial infarction. The present invention provides a method of treating or preventing myocardial infarction in a subject, wherein the method comprises or consists of administering a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA to a subject, wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent myocardial infarction.

[0315] In one aspect, the present invention provides a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA encoding a Chrdl1, Fam3b, or Fam3c polypeptide, or a variant, fragment or isoform thereof, for use in treating myocardial infarction in a subject, optionally wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject.

[0316] In another aspect, the present invention provides a method of treating myocardial infarction, the method comprising administering a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA encoding a Chrdl1, Fam3b, or Fam3c polypeptide, or a variant, fragment or isoform thereof, to a subject after myocardial infarction, optionally wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject.

[0317] In some embodiments, the heart is protected from myocardial infarction. In some embodiments, cardiac function is preserved after myocardial infarction. In some embodiments, cardiac function is preserved after percutaneous coronary intervention (coronary angioplasty). In some embodiments, fibrosis after infarction is reduced. In some embodiments, cardiomyocyte loss after infarction is reduced. In some embodiments, the treatment results in heart protection from myocardial infarction and / or other conditions leading to loss of cardiomyocytes, preferably preserving cardiac function and reducing reparative fibrosis.

[0318] The composition may be administered shortly after myocardial infarction. For example, the composition may be administered within 1, 2, 3 or 4 weeks, or within 144, 120, 96, 72, 60, 48, 36, 24, 16, 12, 10, 8, 6, 5, 4, 3, 2, 1 or 0.5 hours after myocardial infarction. In some embodiments, the composition is administered 24 hours or less after myocardial infarction. In some embodiments, the composition is administered 12 hours or less after myocardial infarction. In some embodiments, the composition is administered 6 hours or less after myocardial infarction. In some embodiments, the composition is administered 3 hours or less after myocardial infarction. In some embodiments, the composition is administered 1 hour or less after myocardial infarction.

[0319] The treatment may improve outcomes after myocardial infarction. For example, the treatment may reduce the risk of heart failure, reduce cardiomyocyte loss, and / or reduce myocardial fibrosis after myocardial infarction.

[0320] The treatment may reduce the risk of heart failure after myocardial infarction. In some embodiments, heart failure is prevented and / or the risk of heart failure is reduced compared to an untreated subject.

[0321] The treatment may reduce cardiomyocyte loss after myocardial infarction. In some embodiments, cardiac muscle cell viability is preserved and / or improved compared to an untreated subject. In some embodiments, cardiomyocyte loss is prevented and / or reduced compared to an untreated subject.

[0322] The treatment may result in reduced infarct size, reduced ventricular dilation, improved LVEF, improved LVESV, and / or reduced fibrosis in the heart of the subject. In some embodiments, cardiac remodeling is reduced compared to an untreated subject. In some embodiments, LV ejection fraction is improved compared to an untreated subject. In some embodiments left ventricular volume is reduced compared to an untreated subject. In some embodiments, myocardial fibrosis is prevented and / or reduced compared to an untreated subject.

[0323] EXAMPLES EXAMPLE 1

[0324] Background

[0325] We previously identified Chordin-like 1 (Chrdl1) as the most effective cardioprotective protein once administered immediately after Ml using cardiotropic AAV vectors (Ruozi et al., 2022. Science translational medicine, 14(660), p.eabo0699). However, the therapeutic use of a Chrdl1-expressing AAV vector to reduce myocardial damage after Ml remains problematic, as vectors persist indefinitely in the target tissues. We therefore have tried to identify alternative methods for delivering cardioprotective factors to the heart.

[0326] Methods

[0327] Production of LNPs

[0328] To do this, we produced lipid nanoparticles (LNPs) based on the Stable Nanoparticle Acid Lipid Particle (SNALP) technology, by which LNPs are formulated using four different lipids (cholesterol, phospholipid, PEGylated lipid and an ionisable lipid) to form a nanoparticle of 100 nm or smaller (Figure 1A) (Kulkarni et al., 2018, Nucleic Acid Ther, 28(3): 146-157). We used three different LNP formulations which have entered clinical application for targeting the liver or for COVID-19 vaccine use. In the first formulation (‘MC3’), the LNPs were formed of DLin-MC3-DMA, DSPC, Cholesterol, and DMG-PEG 2000 at 50:10:38.5:1.5 molar ratio, respectively. The ‘MC3’ formulation entered clinical trials with patisiran, an anti-transthyretin siRNA, as the cargo, using the LNPs to deliver patisiran to the liver for the treatment of liver amyloidosis (Adams et al., 2018, N Engl J Med, 379, 11-21). In the second formulation (‘SM-102’), the LNPs were formed of SM-102, DSPC, Cholesterol, and DMG-PEG at 50:10:38.5:1.5 molar ratio, respectively. The ‘SM-102’ formulation entered clinical trials with COVID-19 vaccines from Moderna using mRNA-1273 (Baden et al., 2021, N Engl J Med, 384(5): 403-416). In the third formulation (‘ALC-0315’), the LNPs were formed of ALC-0315, DOPE, Cholesterol, and PEG at 40:30:27.5:2.5 molar ratio, respectively. The ‘ALC-0315’ formulation entered clinical trials with the Pfizer / BioNTech vaccine to administer the SARS-CoV-2 spike mRNA (Polack et al., 2020, N Engl J Med, 383(27):2603-2615).

[0329] mRNA was transcribed in vitro from linearised plasmid DNA (coding for EGFP or Chrdl1 and carrying 3’ and 5’ UTRs for in vitro transcription) using T7 RNA polymerase using N1-methyl-pseudouridine in substitution to uridine to lower immunogenicity. The mRNA transcripts were capped co-transcriptionally using the Cap1 technology, followed by purification.

[0330] For the generation of LNPs, lipids were dissolved in 100% ethanol to form stocks and stored at -20°C until use. ALC-0315 SNALPs were produced using the SORT vortex method (Wang X et al., 2023, Nature Protocols 18:265-291). The required amount of lipids to achieve a 40:1 mass ratio (total lipid: RNA) were combined and 100% ethanol added to make final lipid mix 2.5 mg / mL. The required amount of RNA was then combined with citrate-buffer (pH 4) to a final volume 1,5x that of the lipid mix. The lipid mix was then added dropwise to the RNA tube under vortex at 2000 rpm. After mixing, one volume of pH 7.4 Tris buffer (50 mM Tris-HCL, 150 mM NaC) was added to the SNALP mixture before the buffer was exchanged using 30K ultrafiltration columns by centrifuging at 14,000 xg and replacing buffer with pH 7.4 Tris buffer. Herein, the formulations are referred to by the formulation and mRNA cargo e.g., ‘ALC-0315-EGFP’ is formulation 3 with eGFP mRNA cargo.

[0331] Characterisation of LNPs

[0332] Encapsulation efficiency was determined by the ribogreen assay (Leung, A. K. K. etal., 2012, J. Phys. Chem. C 116, 18440-18450). Particle size and polydispersity were measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument.

[0333] Biodistribution and response upon systemic administration The three LNP-EGFP mRNA formulations in PBS were then administered intravenously into mice at a dose of 0.33 mg mRNA / kg body weight, which corresponded to 10 pg mRNA per mouse per dose (Figure 1C). Control animals were injected with buffer only.

[0334] After 24 hours, mice were culled. eGFP protein biodistribution was assessed by anti-EGFP ELISA of liver, spleen, heart, muscle and lung tissue extracts. Livers were also immunostained against eGFP, F4 / 80 and DAPI to visualise eGFP expression in liver cells. Inflammatory immune response was tested using ELISA for I P-10, IL-6 and TNF-a. Innate immune response was tested using RT-PCR for RIG-I, IFIH1 and TLR3 using GAPDH as a control.

[0335] Circulating Chrdl1

[0336] ‘ALC-0315’ LNPs containing in vitro transcribed Cap1, N1-methylpseudouridine -modified mRNA coding for a Flag epitope (DYKDDDDK)-tagged version of mouse Chrdl1 were produced as described above. The C-terminal tag was introduced to permit more effective monitoring and does not affect protein function (Ruozi et al., 2022. Science translational medicine, 14(660), p.eabo0699). The LNPs (0.33 mg mRNA / kg body weight) were injected intravenously into mice, and circulating protein was detected in serum obtained from blood microsamples (1 µl blood per sample) taken at different times after administration (experimental scheme in Figure 2A). The timings were 1 hour before administration, 4, 6, 9, 12, 16, 24, 48, 72, 96 and 120 hours after administration. Chrdl1 was detected by immunoblotting using an anti-Flag antibody. Transferrin was used as a loading control. Levels of circulating Chrdl1 (i.e., Chrdl1 in serum) were quantified in 10 pl serum by immunoblotting using an anti-Flag antibody.

[0337] Therapeutic efficacy of Chrdl1 LNPs

[0338] Mice underwent ligation of the left anterior descending (LAD) coronary artery (a commonly used model of myocardial infarction), and then received intravenous injection of ‘ALC-0315’ LNPs carrying the Chrdl1 modified mRNA 20 minutes after ligation. Three Chrdl1 mRNA doses were tested; 0.165, 0.33 and 0.66 mg RNA / kg body weight (corresponding to 5, 10 and 20 pg RNA / mouse) (see Figure 3A). 30 days after ligation / LNP administration, mice underwent echocardiography and the left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) was measured. Hearts were then removed and stained with Masson trichome.

[0339] Results

[0340] Characterisation of LNPs, biodistribution upon systemic administration Each of the three formulations showed a size of around 100 nm diameter, average polydispersity index (PDI) around 0.1 and encapsulation efficiency greater than 90% (Figure 1B). Biodistribution and response upon systemic administration

[0341] As shown in Figure 1D, despite intravenous administration, all formulations surprisingly mostly transfected the liver and spleen (>98% total EGFP detected). There was also a small amount of transfection of the lung (<1%). Transfection of heart and skeletal muscle was negligible (<0.1%). The ratio between liver and spleen transfection was the highest for the ALC-0315 formulation (Figure 1E).

[0342] As shown in Figure 1F, immunohistology of the liver revealed that all the three formulations uniformly transfected hepatocytes. There was no noticeable infiltration of the liver by inflammatory cells or increase in F4 / 80-positive macrophage number in treated mice compared to control. Similarly, the vast majority of the F4 / 80-positive cells were negative for EGFP expression, indicating that they were not transfected.

[0343] Activation of inflammatory and innate immune response was then measured. As shown in Figure 1G, none the formulations increased the expression of Interferon Gamma-Induced Protein 10 (IP-10, also referred to as CXCL10), or expression of the NF-kB-stimulated pro-inflammatory cytokines Interleukin 6 (IL-6) and Tumor Necrosis Factor (TNF)-a, which are commonly activated upon innate immunity stimulation (Carpenter, and O’Neill, 2024, Cell, 187(9): 2030-2051).

[0344] However, as shown in Figure 1G, the ‘MC3’ and ‘SM-102’ LNPs significantly upregulated the cellular transcripts for a few intracellular Pattern Recognition Receptors (PRRs), including Antiviral Innate Immune Response Receptor (RIG-I), Interferon Induced With Helicase C Domain 1 (IFIH1, coding for MDA5) and Toll-Like Receptor 3 (TLR3). These genes commonly respond to viral infection by sensing the presence of viral RNA and stimulate type I interferon (IFN) production (Carroll et al., 2024, Immunological Reviews, 323(1 ):276-287). In contrast, the ‘ALC-0315’ formulation was surprisingly inert.

[0345] Circulating Chrdl1

[0346] Based on the immune data, we chose the ALC-0315 LNPs for our subsequent investigations. We obtained LNPs containing in vitro transcribed Cap1, N1-methylpseudouridine-modified mRNA coding for a Flag-tagged version of mouse Chrdl1.

[0347] As can be seen from Figures 2B and 2C, despite the LNPs mostly transfecting the liver and spleen, Chrdl1 was surprisingly detected in the serum of mice. Circulating Chrdl1 was detectable as early as 4 hours after intravenous injection in both example mice, reached maximal concentration between 12 and 16 hours, and continued to be detectable as long as at least 72 hr. After 96 hours, Chrdl1 was not detected by immunoblotting. Therefore, expression of Chrdl1 using LNPs results in transient expression in the serum. Note that immunoblotting is not a sensitive method and therefore the Chrdl1 may be present in the serum earlier than 4 hours, and may be present for between 72-96 hours.

[0348] Therapeutic efficacy of Chrdl1 mRNA administration

[0349] 30 minutes after ligation of the LAD coronary artery, mice were administered ‘ALC-0315’ LNPs carrying the Chrdl1 modified mRNA at low (0.165 mg mRNA / kg body weight), medium (0.33 mg mRNA / kg body weight) and high (0.66 mg mRNA / kg body weight) doses. Figure 3B shows that mice treated with the medium and high doses had significantly (P<0.001 for both doses) improved left ventricular ejection fraction (LVEF, mean LVEF of 37.2±1.2 for the medium dose) compared to controls (mean LVEF of 21.4±1.0) one month after treatment. Similarly, Figure 3B shows that mice treated with the medium and high doses had significantly (P<0.002 and P<0.005, respectively) reduced left ventricular end diastolic volume (LVEDV, mean LVEDV of 147.2±11.9 for the medium dose) compared to controls (mean LVEDV of 201.4±6.4) one month after treatment. Similarly, Figure 3B shows that mice treated with the medium and high doses had a significantly (P<0.001 for both doses) reduced left ventricular end systolic volume (LVESV, mean LVESV of 92.2±.7.3 for the medium dose) compared to controls (mean LVESV of 157.0±5.7) one month after treatment. As shown in Figure 3B, the highest dose (0.66 mg RNA / kg) was as effective as the middle dose (0.33 mg RNA / kg). The lowest dose (0.165 mg RNA / kg) had only marginal efficacy (not shown).

[0350] As shown in Figure 3C, liver-secreted, circulating Chrdl1 also significantly limited left ventricular fibrosis and reduced infarct size (27.7±5.4% vs. 49.9±4.2% of LV in treated vs. control). Figure 3D quantitatively shows that there is a significant reduction in scar size between treated and untreated mice one month after treatment.

[0351] This long-lasting effect is surprising as we have shown that LNPs only transiently express Chrdl1 from the liver, into the circulation for up to 96 hours (based on the immunoblot data).

[0352] Discussion

[0353] This data surprisingly shows that Chrdl1, and other mRNAs, can be administered via intravenous administration of LNPs carrying the respective mRNA, where the LNPs are delivered to the liver, the mRNA is rapidly synthesized into a protein, which reaches therapeutic levels in the blood within a few hours after administration. The protein in the blood is then able to treat diseases in a tissue that is not the liver (in this example, the heart). This is, to our knowledge, the first report of using LNPs targeting the liver to protect a second, distant organ.

[0354] In addition, the fact that LNPs direct protein production in liver hepatocytes is particularly beneficial for treatment of cardiac conditions, as protein overload in the endoplasmic reticulum of cardiomyocytes can activate the unfolded protein response, which can lead to proteotoxicity and, specifically in the heart, increases the risk of arrhythmias after myocardial infarction. Furthermore, the transient expression of mRNA from the LNPs described here is particularly beneficial for treatment of acute conditions because it avoids unwanted side effects from longterm expression, which can occur in other delivery modalities such as AAV vectors.

[0355] In addition, compared to recombinant proteins, mRNA treatment can be quickly adapted to virtually any therapeutic factor, avoiding the complexities of protein-production platforms where protein folding and glycosylation impacts efficacy.

[0356] In addition, the present findings are straightforward for clinical translation, due to the successful use of these LNPs for use in vaccines against SARS-CoV2 (Baden et al., 2021 and Polack et al., 2020). In addition, LNP-mRNA administration has already reached clinical application for two inherited conditions; replacement therapy for propionic acidaemia (Koeberl et al., 2024, Nature, 628: 872-877) and gene editing of transthyretin amyloidosis of the liver (Gillmore et al., 2021, N Engl J Med, 385: 493-502). Thus, the steps required for clinical development can recapitulate those of these previous applications.

[0357] EXAMPLE 2

[0358] In further examples, LNPs as described in Example 1 are delivered hours or days after a cardiac event, such as ligation of the LAD coronary artery, and show increased LVEF, reduced LVEDV and LVESV, reduced fibrosis and reduced infarct size.

[0359] In further examples, LNPs as described in Example 1 are used to deliver other cardioprotective mRNAs, such as Fam3b and Fam3c, and show similar cardioprotective effects.

[0360] SEQUENCE LISTING SEQ ID NO: 1 - Chrdl1 protein (human) Isoform 1 - canonical sequence UniProt ID: Q9BU40-6 MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICS ENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQN RQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARH SYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGES WHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGY FCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLV TRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC

[0361] SEQ ID NO: 2 - Chrdl1 protein (human) Isoform 2 UniProt ID: Q9BU40-3 MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICS ENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPGDGELSWEHSDGDIFRQPANREARHSYHRSHYDPPP SRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIV ECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKEELPGQSFDNKGYFCGEETMPVY ESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKI FTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC

[0362] SEQ ID NO: 3 - Chrdl1 protein (human) Isoform 3 UniProt ID: Q9BU40-4 MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICS ENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPEDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQ NRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDI FRQ PANREAR HSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGE SWHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKEELPGQSFDNK GYFCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFK LVTRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC

[0363] SEQ ID NO: 4 - Chrdl1 protein (human) Isoform 4 UniProt ID: Q9BU40-5 MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICS ENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQN RQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARH SYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGES WHPNLRAFGIVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKEELPGQSFDNKG YFCGEETMPVYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKL VTRTTLSQWKIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC SEQ ID NO: 5 - Fam3b protein (human) Isoform B - canonical sequence UniProt ID: P58499-1 MRPLAGGLLKVVFVVFASLCAWYSGYLLAELIPDAPLSSAAYSIRSIGERPVLKAPVPKRQKCDHWTP CPSDTYAYRLLSGGGRSKYAKICFEDNLLMGEQLGNVARGINIAIVNYVTGNVTATRCFDMYEGDNSG PMTKFIQSAAPKSLLFMVTYDDGSTRLNNDAKNAIEALGSKEIRNMKFRSSWVFIAAKGLELPSEIQR EKINHSDAKNNRYSGWPAEIQIEGCI PEERS

[0364] SEQ ID NO: 6- Fam3b protein (human) Isoform A UniProt ID: P58499-2 MRPLAGGSRWACWLTRCLISCFDINVQGRLLVKLRPKPTANTTCPGLLKVVFVVFASLCAWYSGYLLA ELIPDAPLSSAAYSIRSIGERPVLKAPVPKRQKCDHWTPCPSDTYAYRLLSGGGRSKYAKICFEDNLL MGEQLGNVARGINIAIVNYVTGNVTATRCFDMYEGDNSGPMTKFIQSAAPKSLLFMVTYDDGSTRLNN DAKNAIEALGSKEIRNMKFRSSWVFIAAKGLELPSEIQREKINHSDAKNNRYSGWPAEIQIEGCIPKE RS

[0365] SEQ ID NO: 7 - Fam3b protein (human) Isoform C UniProt ID: P58499-3 MRPLAGAPVPKRQKCDHWTPCPSDTYAYRLLSGGGRSKYAKICFEDNLLMGEQLGNVARGINIAIVNY VTGNVTATRCFDMYEGDNSGPMTKFIQSAAPKSLLFMVTYDDGSTRLNNDAKNAIEALGSKEIRNMKF RSSWVFIAAKGLELPSEIQREKINHSDAKNNRYSGWPAEIQIEGCI PEERS SEQ ID NO: 8 - Fam3c protein (human) UniProt ID: Q92520-1 MRVAGAAKLVVAVAVFLLTFYVISQVFEIKMDASLGNLFARSALDTAARSTKPPRYKCGISKACPEKH FAFKMASGAANVVGPKICLEDNVLMSGVKNNVGRGINVALANGKTGEVLDTKYFDMWGGDVAPFIEFL KAIQDGTIVLMGTYDDGATKLNDEARRLIADLGSTSITNLGFRDNWVFCGGKGIKTKSPFEQHIKNNK DTNKYEGWPEVVEMEGCIPQKQD

[0366] SEQ ID NO: 9 - Mdk protein (human) UniProt ID: P21741-1 MQHRGFLLLTLLALLALTSAVAKKKDKVKKGGPGSECAEWAWGPCTPSSKDCGVGFREGTCGAQTQRI RCRVPCNWKKEFGADCKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETIRVTKPCTPKTKAKAKA KKGKGKD

[0367] SEQ ID NO: 10 - pleiotrophin protein (human) UniProt ID: P21246 MQAQQYQQQRRKFAAAFLAFIFILAAVDTAEAGKKEKPEKKVKKSDCGEWQWSVCVPTSGDCGLGTRE GTRTGAECKQTMKTQRCKIPCNWKKQFGAECKYQFQAWGECDLNTALKTRTGSLKRALHNAECQKTVT ISKPCGKLTKPKPQAESKKKKKEGKKQEKMLD

[0368] SEQ ID NO: 11 - HtrA1 protein (human) UniProt ID: Q92743 MQIPRAALLPLLLLLLAAPASAQLSRAGRSAPLAAGCPDRCEPARCPPQPEHCEGGRARDACGCCEVC GAPEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDANTYANLCQLRAAS RRSERLHRPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELFRKLPFSKREVPVASG SGFIVSEDGLIVTNAHVVTNKHRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSE LRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVI GINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAKELKDRHRDFPDV ISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITVIPE EIDP

[0369] SEQ ID NO: 12 - Nhlrc3 protein (human) Isoform 1 - canonical sequence UniProt ID: Q5JS37-1 MARFWVCVAGAGFFLAFLVLHSRFCGSPVLRNFTFAVSWRTEKILYRLDVGWPKHPEYFTGTTFCVAV DSLNGLVYIGQRGDNIPKILVFTEDGYFLRAWNYTVDTPHGIFAASTLYEQSVWITDVGSGFFGHTVK KYSSFGDLVQVLGTPGKKGTSLNPLQFDNPAELYVEDTGDIYIVDGDGGLNNRLIKLSQDFMILWLHG ENGTGPAKFNIPHSVTLDSAGRVWVADRGNKRIQVFDKDTGEWLGAWNNCFTEEGPSSVRFTPDGKYL IVAQLNLSRLSVVAAPPVGSIGECSVISTIQLADQVLPHLLEVDRKTGAVYVAEIGAKQVQKYVPLNS YVPSFGS

[0370] SEQ ID NO: 13 - Nhlrc3 protein (human) Isoform 2 UniProt ID: Q5JS37-2 MARFWVCVAGAGFFLAFLVLHSRFCGSPVLRNFTFAVSWRTEKILYRLDVGWPKHPEYFTGTTFCVAV DSLNGLVYIGQRGDNIPKILVFTEDGYFLRAWNYTVDTPHGIFAASTLYEQSVWITDVGSDFMILWLH GENGTGPAKFNIPHSVTLDSAGRVWVADRGNKRIQVFDKDTGEWLGAWNNCFTEEGPSSVRFTPDGKY LIVAQLNLSRLSVVAAPPVGSIGECSVISTIQLADQVLPHLLEVDRKTGAVYVAEIGAKQVQKYVPLN SYVPSFGS

[0371] SEQ ID NO: 14 - Rln-1 protein (human) Isoform 1 - canonical sequence UniProt: ID: P04808-1 MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRP VAEIVPSFINKDTETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNR QSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLAKYC

[0372] SEQ ID NO: 15 - Rln-1 protein (human) Isoform 2 -UniProt: ID: P04808-2 MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRP VAGDFIQTVSLGISPDGGKALRTGSCFTREFLGALSKLYHPSSTKIQKL

[0373] SEQ ID NO: 16 - Chrdl1 mRNA coding sequence transcript variant 1 NCBI reference sequence NM_001143981.2 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataaggtgaccagca agtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcag aatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagac ttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggag atggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaaga cattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcc tggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattg tcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgag tcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaa gcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaa aatgctgcaaggtgtgtccaggtaaaaaagcaaaagaagaacttccaggccaaagctttgacaataaa ggctacttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaac cagaaaaatagcactggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagg gcattctccagcacttccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaag ctggtgaccagaacaaccctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagat gtgttcaagtcgtgtatgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctg aaaagggccactgttag

[0374] SEQ ID NO: 17 - Chrdl1 mRNA coding sequence transcript variant 2 NCBI reference sequence NM_001143982.2 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagactccttacccccagtgaacaataaggtgaccagcaagt cttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcagaat cggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagacttg ccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggagatg gagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaagacat tcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcctgg ggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattgtca tcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgagtcc tggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaagca agagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaaaat gctgcaaggtgtgtccaggtaaaaaagcaaaagaagaacttccaggccaaagctttgacaataaaggc tacttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccag aaaaatagcactggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggca ttctccagcacttccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctg gtgaccagaacaaccctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtg ttcaagtcgtgtatgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaa agggccactgttag SEQ ID NO: 18 - Chrdl1 mRNA coding sequence transcript variant 3 NCBI reference sequence NM_145234.4 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagactccttacccccagtgaacaataaggtgaccagcaagt cttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcagaat cggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagacttg ccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggagatg gagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaagacat tcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcctgg ggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattgtca tcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgagtcc tggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaagca agagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaaaat gctgcaaggtgtgtccaggtaaaaaagcaaaagaacttccaggccaaagctttgacaataaaggctac ttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccagaaa aatagcactggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggcattc tccagcacttccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctggtg accagaacaaccctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtgttc aagtcgtgtatgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaaagg gccactgttag

[0375] SEQ ID NO: 19 - Chrdl1 mRNA coding sequence transcript variant 4 NCBI reference sequence NM_001143983.3 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccaggagatggagaactgtcatgggaacattctgatggtgata tcttccggcaacctgccaacagagaagcaagacattcttaccaccgctctcactatgatcctccacca agccgacaggctggaggtctgtcccgctttcctggggccagaagtcaccggggagctcttatggattc ccagcaagcatcaggaaccattgtgcaaattgtcatcaataacaaacacaagcatggacaagtgtgtg tttccaatggaaagacctattctcatggcgagtcctggcacccaaacctccgggcatttggcattgtg gagtgtgtgctatgtacttgtaatgtcaccaagcaagagtgtaagaaaatccactgccccaatcgata cccctgcaagtatcctcaaaaaatagacggaaaatgctgcaaggtgtgtccaggtaaaaaagcaaaag aagaacttccaggccaaagctttgacaataaaggctacttctgcggggaagaaacgatgcctgtgtat gagtctgtattcatggaggatggggagacaaccagaaaaatagcactggagactgagagaccacctca ggtagaggtccacgtttggactattcgaaagggcattctccagcacttccatattgagaagatctcca agaggatgtttgaggagcttcctcacttcaagctggtgaccagaacaaccctgagccagtggaagatc ttcaccgaaggagaagctcagatcagccagatgtgttcaagtcgtgtatgcagaacagagcttgaaga tttagtcaaggttttgtacctggagagatctgaaaagggccactgttag

[0376] SEQ ID NO: 20 - Chrdl1 mRNA coding sequence transcript variant 5 NCBI reference sequence NM_001367204.1 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataaggtgaccagca agtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcag aatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagac ttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggag atggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaaga cattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcc tggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattg tcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgag tcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaa gcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaa aatgctgcaaggtgtgtccaggtaaaaaagcaaaagaagaacttccaggccaaagctttgacaataaa ggctacttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaac cagaaaaatagcactggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagg gcattctccagcacttccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaag ctggtgaccagaacaaccctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagat gtgttcaagtcgtgtatgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctg aaaagggccactgttag

[0377] SEQ ID NO: 21 - Chrdl1 mRNA coding sequence transcript variant 6 NCBI reference sequence NM_001367205.1 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataaggtgaccagca agtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcag aatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagac ttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggag atggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaaga cattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcc tggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattg tcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgag tcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaa gcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaa aatgctgcaaggtgtgtccagaagaacttccaggccaaagctttgacaataaaggctacttctgcggg gaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccagaaaaatagcact ggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggcattctccagcact tccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctggtgaccagaaca accctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtgttcaagtcgtgt atgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaaagggccactgtt ag

[0378] SEQ ID NO: 22 - Chrdl1 mRNA coding sequence transcript variant 7 NCBI reference sequence NM_001367206.1 agtggtccaaatgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttc ttttgctagaaggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaag aagtacagagtgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactg catctgctcagagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctc ctgtgcatattcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataag gtgaccagcaagtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagg gctctttcagaatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtg gtctcaagacttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggta tgcagaggagatggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacag agaagcaagacattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgt cccgctttcctggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccatt gtgcaaattgtcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattc tcatggcgagtcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgta atgtcaccaagcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaa atagacggaaaatgctgcaaggtgtgtccagaagaacttccaggccaaagctttgacaataaaggcta cttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccagaa aaatagcactggagactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggcatt ctccagcacttccatattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctggtgaccagaacaaccctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtgtt caagtcgtgtatgcagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaaag ggccactgttag

[0379] SEQ ID NO: 23 - Chrdl1 mRNA coding sequence transcript variant 8 NCBI reference sequence NM_001367207.1 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagactccttacccccagtgaacaataaggtgaccagcaagt cttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcagaat cggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagacttg ccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggagatg gagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaagacat tcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcctgg ggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattgtca tcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgagtcc tggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaagca agagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaaaat gctgcaaggtgtgtccagaagaacttccaggccaaagctttgacaataaaggctacttctgcggggaa gaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccagaaaaatagcactgga gactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggcattctccagcacttcc atattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctggtgaccagaacaacc ctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtgttcaagtcgtgtatg cagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaaagggccactgttag SEQ ID NO: 24 - Chrdl1 mRNA coding sequence transcript variant 9 NCBI reference sequence NM_001367208.1 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataaggtgaccagca agtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcag aatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagac ttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggag atggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaaga cattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcc tggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattg tcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgag tcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaa gcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaa aatgctgcaaggtgtgtccagaacttccaggccaaagctttgacaataaaggctacttctgcggggaa gaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaaccagaaaaatagcactgga gactgagagaccacctcaggtagaggtccacgtttggactattcgaaagggcattctccagcacttcc atattgagaagatctccaagaggatgtttgaggagcttcctcacttcaagctggtgaccagaacaacc ctgagccagtggaagatcttcaccgaaggagaagctcagatcagccagatgtgttcaagtcgtgtatg cagaacagagcttgaagatttagtcaaggttttgtacctggagagatctgaaaagggccactgttag SEQ ID NO: 25 - Chrdl1 mRNA coding sequence transcript variant 10 NCBI reference sequence NM_001367209.1 atgagaaaaaagtggaaaatgggaggcatgaaatacatcttttcgttgttgttctttcttttgctaga aggaggcaaaacagagcaagtaaaacattcagagacatattgcatgtttcaagacaagaagtacagag tgggtgagagatggcatccttacctggaaccttatgggttggtttactgcgtgaactgcatctgctca gagaatgggaatgtgctttgcagccgagtcagatgtccaaatgttcattgcctttctcctgtgcatat tcctcatctgtgctgccctcgctgcccagaagactccttacccccagtgaacaataaggtgaccagca agtcttgcgagtacaatgggacaacttaccaacatggagagctgttcgtagctgaagggctctttcag aatcggcaacccaatcaatgcacccagtgcagctgttcggagggaaacgtgtattgtggtctcaagac ttgccccaaattaacctgtgccttcccagtctctgttccagattcctgctgccgggtatgcagaggag atggagaactgtcatgggaacattctgatggtgatatcttccggcaacctgccaacagagaagcaaga cattcttaccaccgctctcactatgatcctccaccaagccgacaggctggaggtctgtcccgctttcc tggggccagaagtcaccggggagctcttatggattcccagcaagcatcaggaaccattgtgcaaattg tcatcaataacaaacacaagcatggacaagtgtgtgtttccaatggaaagacctattctcatggcgag tcctggcacccaaacctccgggcatttggcattgtggagtgtgtgctatgtacttgtaatgtcaccaa gcaagagtgtaagaaaatccactgccccaatcgatacccctgcaagtatcctcaaaaaatagacggaa aatgctgcaaggtgtgtccaggtaaaaaagcaaaagaagaacttccaggccaaagctttgacaataaa ggctacttctgcggggaagaaacgatgcctgtgtatgagtctgtattcatggaggatggggagacaac cagaaaaatagcactggagactgagagaccacctcaggcattctccagcacttccatattgagaagat ctccaagaggatgtttgaggagcttcctcacttcaagctggtga

[0380] SEQ ID NO: 26 – Fam3b coding sequence transcript variant 1 NCBI reference sequence NM_058186.4 atgcgcccattggctggtggcctgctcaaggtggtgttcgtggtcttcgcctccttgtgtgcctggta ttcggggtacctgctcgcagagctcattccagatgcacccctgtccagtgctgcctatagcatccgca gcatcggggagaggcctgtcctcaaagctccagtccccaaaaggcaaaaatgtgaccactggactccc tgcccatctgacacctatgcctacaggttactcagcggaggtggcagaagcaagtacgccaaaatctg ctttgaggataacctacttatgggagaacagctgggaaatgttgccagaggaataaacattgccattg tcaactatgtaactgggaatgtgacagcaacacgatgttttgatatgtatgaaggtgataactctgga ccgatgacaaagtttattcagagtgctgctccaaaatccctgctcttcatggtgacctatgacgacgg aagcacaagactgaataacgatgccaagaatgccatagaagcacttggaagtaaagaaatcaggaaca tgaaattcaggtctagctgggtatttattgcagcaaaaggcttggaactcccttccgaaattcagaga gaaaagatcaaccactctgatgctaagaacaacagatattctggctggcctgcagagatccagataga aggctgcatacccaaagaacgaagctga

[0381] SEQ ID NO: 27 – Fam3b coding sequence transcript variant 2 NCBI reference sequence NM_206964.2 atgcgcccattggctggtgctccagtccccaaaaggcaaaaatgtgaccactggactccctgcccatc tgacacctatgcctacaggttactcagcggaggtggcagaagcaagtacgccaaaatctgctttgagg ataacctacttatgggagaacagctgggaaatgttgccagaggaataaacattgccattgtcaactat gtaactgggaatgtgacagcaacacgatgttttgatatgtatgaaggtgataactctggaccgatgac aaagtttattcagagtgctgctccaaaatccctgctcttcatggtgacctatgacgacggaagcacaa gactgaataacgatgccaagaatgccatagaagcacttggaagtaaagaaatcaggaacatgaaattc aggtctagctgggtatttattgcagcaaaaggcttggaactcccttccgaaattcagagagaaaagat caaccactctgatgctaagaacaacagatattctggctggcctgcagagatccagatagaaggctgca tacccaaagaacgaagctga

[0382] SEQ ID NO: 28 – Fam3c coding sequence transcript variant 1 NCBI reference sequence NM_014888.3 atgagggtagcaggtgctgcaaagttggtggtagctgtggcagtgtttttactgacattttatgttat ttctcaagtatttgaaataaaaatggatgcaagtttaggaaatctatttgcaagatcagcattggaca cagctgcacgttctacaaagcctcccagatataagtgtgggatctcaaaagcttgccctgagaagcat tttgcttttaaaatggcaagtggagcagccaacgtggtgggacccaaaatctgcctggaagataatgt tttaatgagtggtgttaagaataatgttggaagagggatcaatgttgccttggcaaatggaaaaacag gagaagtattagacactaaatattttgacatgtggggaggagatgtggcaccatttattgagtttctg aaggccatacaagatggaacaatagttttaatgggaacatacgatgatggagcaaccaaactcaatga tgaggcacggcggctcattgctgatttggggagcacatctattactaatcttggttttagagacaact gggtcttctgtggtgggaagggcattaagacaaaaagcccttttgaacagcacataaagaacaataag gatacaaacaaatatgaaggatggcctgaagttgtagaaatggaaggatgcatcccccagaagcaaga ctaa SEQ ID NO: 29 – Fam3c coding sequence transcript variant 2 NCBI reference sequence NM_001040020.2 atgagggtagcaggtgctgcaaagttggtggtagctgtggcagtgtttttactgacattttatgttat ttctcaagtatttgaaataaaaatggatgcaagtttaggaaatctatttgcaagatcagcattggaca cagctgcacgttctacaaagcctcccagatataagtgtgggatctcaaaagcttgccctgagaagcat tttgcttttaaaatggcaagtggagcagccaacgtggtgggacccaaaatctgcctggaagataatgt tttaatgagtggtgttaagaataatgttggaagagggatcaatgttgccttggcaaatggaaaaacag gagaagtattagacactaaatattttgacatgtggggaggagatgtggcaccatttattgagtttctg aaggccatacaagatggaacaatagttttaatgggaacatacgatgatggagcaaccaaactcaatga tgaggcacggcggctcattgctgatttggggagcacatctattactaatcttggttttagagacaact gggtcttctgtggtgggaagggcattaagacaaaaagcccttttgaacagcacataaagaacaataag gatacaaacaaatatgaaggatggcctgaagttgtagaaatggaaggatgcatcccccagaagcaaga ctaa

[0383] SEQ ID NO: 30 – Mdk coding sequence transcript variant 1 NCBI reference sequence NM_001012334.2 atgcagcaccgaggcttcctcctcctcaccctcctcgccctgctggcgctcacctccgcggtcgccaa aaagaaagataaggtgaagaagggcggcccggggagcgagtgcgctgagtgggcctgggggccctgca cccccagcagcaaggattgcggcgtgggtttccgcgagggcacctgcggggcccagacccagcgcatc cggtgcagggtgccctgcaactggaagaaggagtttggagccgactgcaagtacaagtttgagaactg gggtgcgtgtgatgggggcacaggcaccaaagtccgccaaggcaccctgaagaaggcgcgctacaatg ctcagtgccaggagaccatccgcgtcaccaagccctgcacccccaagaccaaagcaaaggccaaagcc aagaaagggaagggaaaggactag

[0384] SEQ ID NO: 31 – pleiotrophin coding sequence transcript variant 1 NCBI reference sequence NM_002825.7 atgcaggctcaacagtaccagcagcagcgtcgaaaatttgcagctgccttcttggcattcattttcatactggcagctgtggatactgctgaagcagggaagaaagagaaaccagaaaaaaaagtgaagaagtctg actgtggagaatggcagtggagtgtgtgtgtgcccaccagtggagactgtgggctgggcacacgggag ggcactcggactggagctgagtgcaagcaaaccatgaagacccagagatgtaagatcccctgcaactg gaagaagcaatttggcgcggagtgcaaataccagttccaggcctggggagaatgtgacctgaacacag ccctgaagaccagaactggaagtctgaagcgagccctgcacaatgccgaatgccagaagactgtcacc atctccaagccctgtggcaaactgaccaagcccaaacctcaagcagaatctaagaagaagaaaaagga aggcaagaaacaggagaagatgctggattaa SEQ ID NO: 32 – HtrA1 coding sequence mRNA transcript NCBI reference sequence NM_002775.5 atgcagatcccgcgcgccgctcttctcccgctgctgctgctgctgctggcggcgcccgcctcggcgca gctgtcccgggccggccgctcggcgcctttggccgccgggtgcccagaccgctgcgagccggcgcgct gcccgccgcagccggagcactgcgagggcggccgggcccgggacgcgtgcggctgctgcgaggtgtgc ggcgcgcccgagggcgccgcgtgcggcctgcaggagggcccgtgcggcgaggggctgcagtgcgtggt gcccttcggggtgccagcctcggccacggtgcggcggcgcgcgcaggccggcctctgtgtgtgcgcca gcagcgagccggtgtgcggcagcgacgccaacacctacgccaacctgtgccagctgcgcgccgccagc cgccgctccgagaggctgcaccggccgccggtcatcgtcctgcagcgcggagcctgcggccaagggca ggaagatcccaacagtttgcgccataaatataactttatcgcggacgtggtggagaagatcgcccctg ccgtggttcatatcgaattgtttcgcaagcttccgttttctaaacgagaggtgccggtggctagtggg tctgggtttattgtgtcggaagatggactgatcgtgacaaatgcccacgtggtgaccaacaagcaccg ggtcaaagttgagctgaagaacggtgccacttacgaagccaaaatcaaggatgtggatgagaaagcag acatcgcactcatcaaaattgaccaccagggcaagctgcctgtcctgctgcttggccgctcctcagag ctgcggccgggagagttcgtggtcgccatcggaagcccgttttcccttcaaaacacagtcaccaccgg gatcgtgagcaccacccagcgaggcggcaaagagctggggctccgcaactcagacatggactacatcc agaccgacgccatcatcaactatggaaactcgggaggcccgttagtaaacctggacggtgaagtgatt ggaattaacactttgaaagtgacagctggaatctcctttgcaatcccatctgataagattaaaaagtt cctcacggagtcccatgaccgacaggccaaaggaaaagccatcaccaagaagaagtatattggtatcc gaatgatgtcactcacgtccagcaaagccaaagagctgaaggaccggcaccgggacttcccagacgtg atctcaggagcgtatataattgaagtaattcctgataccccagcagaagctggtggtctcaaggaaaa cgacgtcataatcagcatcaatggacagtccgtggtctccgccaatgatgtcagcgacgtcattaaaa gggaaagcaccctgaacatggtggtccgcaggggtaatgaagatatcatgatcacagtgattcccgaa gaaattgacccatag

[0385] SEQ ID NO: 33 – Nhlrc3 coding sequence transcript variant 1 NCBI reference sequence NM_001012754.4 atggcgagattctgggtctgcgtagccggtgctggcttctttcttgcatttttggttttgcattcgcg tttttgtggctctccagttttgaggaactttacttttgcagtttcctggagaactgagaaaattcttt accggctggatgtgggttggcctaagcacccagaatattttaccggaacaacattttgtgttgcagtt gactccctcaatggattggtttacataggtcaaagaggggataacatcccaaagatattagtgttcac agaggatggatatttcctacgagcctggaattatacagttgacacacctcatggtatatttgcagcca gtactctatatgaacaatccgtctggatcacggatgtaggaagtggattctttggtcatactgttaaa aaatacagttcttttggtgatcttgttcaagtcttgggtactccaggcaaaaaaggcactagtttgaa tcctttgcagtttgataacccagcagaattatatgtagaggacacaggagatatttacattgtggatg gagatggaggattgaataacagattgatcaaactgtcccaagatttcatgatcctttggctgcatgga gaaaatgggacagggcctgctaagttcaacatacctcacagtgttacacttgattcagctggtcgggt gtgggttgctgaccgaggaaataaaagaatccaagtatttgataaagacactggggagtggt taggag catggaataattgtttcacagaagagggaccttcttcagtcaggtttactcctgatgggaagtacttg attgtggcccagctgaatcttagcaggctctcagtcgtagcagcacccccagtgggaagcattgggga gtgttctgtgatcagcacaatccaactagcagatcaagttttgccacatctcctagaagtcgacagaa agactggagcagtctatgtagcagaaattggagcaaaacaagtacaaaaatatgtccctttgaatagc tatgttccttcatttggttcataa

[0386] SEQ ID NO: 34 – Rln-1 coding sequence mRNA transcript NCBI reference sequence NM_006911.4 atgcctcgcctgttcttgttccacctgctagaattctgtttactactgaaccaattttccagagcagt cgcggccaaatggaaggacgatgttattaaattatgcggccgcgaattagttcgcgcgcagattgcca tttgcggcatgagcacctggagcaaaaggtctctgagccaggaagatgctcctcagacacctagacca gtggcagaaattgtaccatccttcatcaacaaagatacagaaactataattatcatgttggaattcat tgctaatttgccaccggagctgaaggcagccctatctgagaggcaaccatcattaccagagctacagc agtatgtacctgcattaaaggattccaatcttagctttgaagaatttaagaaacttattcgcaatagg caaagtgaagccgcagacagcaatccttcagaattaaaatacttaggcttggatactcattctcaaaa aaagagacgaccctacgtggcactgtttgagaaatgttgcctaattggttgtaccaaaaggtctcttg ctaaatattgctga

[0387] SEQ ID NO: 35 - Chrdl1 gene, NCBI reference NG_012816.1

[0388] SEQ ID NO: 36 – Fam3b gene, NCBI reference NC_000021.9, nucleotides 41304242 to 41357727

[0389] SEQ ID NO: 37 – Fam3c gene, NCBI reference NC_000007.14, nucleotides complement c121396396 to 121348878

[0390] SEQ ID NO: 38 – Mdk gene, NCBI reference NC_000011.10, nucleotides 46380784-46383837

[0391] SEQ ID NO: 39 – pleiotrophin gene, NCBI reference NC_000007.14, nucleotides complement c137343733-13722734

[0392] SEQ ID NO: 40 – HtrA1 gene, NCBI reference NG_011554.1

[0393] SEQ ID NO: 41 – Nhlrc3 gene, NCBI reference NC_000013.11, nucleotides 39038311-39050109

[0394] SEQ ID NO: 42 – Rln-1 gene, NCBI reference NC_000009.12, nucleotides complement C5340916-5334930

[0395] SEQ ID NO: 43 - Chrdl1 protein (human) Isoform 1 lacking the signal peptide EQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVLCSRVRCPNVHCLSPVHIPHLC CPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLT CAFPVSVPDSCCRVCRGDGELSWEHSDGDI FRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSH RGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKK IHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFMEDGETTRKIALE TERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKI FTEGEAQISQMCSSRVC RTELEDLVKVLYLERSEKGHC

[0396] SEQ ID NO: 44 - Fam3b protein (human) canonical sequence lacking the signal peptide MRPLAGGLLKVVFVVFASLCAWYSGYLLAELIPDAPLSSAAYSIRSIGERPVLKAPVPKRQKCDHWTP CPSDTYAYRLLSGGGRSKYAKICFEDNLLMGEQLGNVARGINIAIVNYVTGNVTATRCFDMYEGDNSG PMTKFIQSAAPKSLLFMVTYDDGSTRLNNDAKNAIEALGSKEIRNMKFRSSWVFIAAKGLELPSEIQR EKINHSDAKNNRYSGWPAEIQIEGCI PEERS

[0397] SEQ ID NO: 45 - Fam3c protein (human) sequence lacking the signal peptide QVFEIKMDASLGNLFARSALDTAARSTKPPRYKCGISKACPEKHFAFKMASGAANVVGPKICLEDNVL MSGVKNNVGRGINVALANGKTGEVLDTKYFDMWGGDVAPFIEFLKAIQDGTIVLMGTYDDGATKLNDE ARRLIADLGSTSITNLGFRDNWVFCGGKGIKTKSPFEQHIKNNKDTNKYEGWPEVVEMEGCIPQKQD

[0398] SEQ ID NO: 46 - example Chrdl1 signal peptide MRKKWKMGGMKYIFSLLFFLLLEGGKT

[0399] SEQ ID NO: 47 - example Fam3b signal peptide MRPLAGGLLKVVFVVFASLCAWYSGYLLA

[0400] SEQ ID NO: 48 - example Fam3c signal peptide

[0401] MRVAGAAKLVVAVAVFLLTFYVIS

Claims

CLAIMS1. A pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA for use in treating or preventing a cardiac disease or condition in a subject in need thereof, wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the disease or condition.

2. A method of treating or preventing a cardiac disease or condition in a subject, wherein the method comprises or consists of administering a pharmaceutical composition comprising lipid nanoparticles (LNPs) comprising an mRNA to a subject, wherein, following administration to the subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the disease or condition.

3. The pharmaceutical composition for use according to claim 1 or the method according to claim 2, wherein the mRNA encodes a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), or relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof, optionally wherein the mRNA encodes a polypeptide with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any of SEQ ID NOs: 1- 15, optionally wherein the mRNA comprises or consists of a polynucleotide with a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an mRNA encoded by any of SEQ ID NOs: 16-42.

4. The pharmaceutical composition for use according to claim 1 or 3 or the method according to claim 2 or 3, wherein the cardiac disease or condition is associated with loss of cardiac myocytes, optionally wherein the cardiac disease or condition is selected from heart failure, myocardial infarction, ischemic or non-ischemic cardiomyopathy, myocarditis or cardiac damage induced by cardiotoxic treatments.

5. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 4 or the method according to any of claims 2 to 4, wherein the cardiac disease or condition is acute.

6. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 5 or the method according to any of claims 2 to 5, wherein the polypeptide is present in the blood of the subject for up to 24, 48, 72, 96 or 120 hours after administration of the composition to the subject.

7. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 6 or the method according to any of claims 2 to 6, wherein the composition is administered to the subject intravenously.

8. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 7 or the method according to any of claims 2 to 7, wherein lipids of the LNPs comprise or consist of ionisable lipids, structural lipids, phospholipids, and / or PEGylated lipids, optionallywherein the ionisable lipids comprise or consist of 6-[6-(2-hexyldecanoyloxy)hexyl-(4- hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2- hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), [(6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3- DMA), 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxyoctadeca-9,12-dienyl]amino]butyl]piperazine- 2, 5-dione (OF-02), ethyl 1-[3-(2-ethylpiperidin-1-yl)propyl]-5,5-bis[(Z)-heptadec-8- enyl]-2H-imidazole-2-carboxylate (A18-Iso5-2DC18), di(oct-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A1), di(non-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A2), di(dec-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A3), di(oct-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A4), di(non-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A5), di(dec-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A6), 1-[2-[bis(2- hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1- yl]ethyl]amino]dodecan-2-ol (C12-200), 3,6-bis[4-[bis(2- hydroxydodecyl)amino]butyl]piperazine-2, 5-dione (cKK-E12), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9), 3-[2-[2-[2-[bis[3-(dodecylamino)-3- oxopropyl]amino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]ethylamino]ethyl-[3- (dodecylamino)-3-oxopropyl]amino]- / \ / -dodecylpropanamide (98N12-5), 3-[[3-[2-[bis(2- hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[2-[[3-[2-[bis(2- hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[3-[2-(2- hydroxytetradecylamino)ethylamino]-3-oxopropyl]amino]ethyl]amino]- / \ / -[2-(2- hydroxytetradecylamino)ethyl]propanamide (G0-C14), bis[(Z)-non-2-enyl] 9-[4-(dimethylamino)butanoyloxy]heptadecanedioate (L319), 2-[4-[5-[4-[bis[2-[(9Z,12Z)-octadeca-9,12-dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2- [(9Z, 12Z)-octadeca-9, 12-dienoyl]oxyethyl]ami no]ethyl (9Z, 12Z)-octadeca-9, 12-dienoate (OF-Deg-Lin), tridecyl 3-[2-[bis[2-[methyl-(3-oxo-3-tridecoxypropyl)amino]ethyl]amino]ethyl-methylamino]propanoate (304Oi3), 2- (octyldisulfanyl)ethyl 3-[3-[3-[bis[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propyl-methylamino]propyl-[3-[2-(octyldisulfanyl)ethoxy]-3-oxopropyl]amino]propanoate (306-O12B), 8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3-oxopropyl]amino]propanoate (306Oi10), octan-3-yl 9-[3-[[3,5-bis[3-[bis(9-octan-3-yloxy-9-oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-3-yloxy-9-oxononyl)amino]nonanoate (FTT5), (1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12-dienoxy]piperidine (YSK05) [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4-dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (LP01), [(6Z,16Z)-12-[(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate (CL1), [7-[4-(dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9-enoate (CL4H6), and / or [7-[4-(dipropylamino)butyl]-13-(2-hexyloctanoyloxy)-7-hydroxytridecyl] 2-hexyloctanoate (CL4F8-6), or analogues thereof, and / orwherein the structural lipids comprise or consist of a sterol, optionally cholesterol or analogues thereof; and / orwherein the phospholipids comprise or consist of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Egg Yolk Phosphatidylcholine, or 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DM PC), sphingomyelin (SM), ceramide or any related phosphatidylcholine or analogues thereof; and / orwherein the PEGylated lipids comprise or consist of 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG 2000), Methoxy Polyethylene Glycol-Distearoylphosphatidylethanolamine (mPEG-DSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (POPE-PEG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DOPE-PEG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), Distearoyl-rac- glycerol-PEG2000 (PEG-DSG), or 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA) or analogues thereof.

9. The pharmaceutical composition for use or method according to claim 8, wherein the lipids are present in the LNPs of the composition at a mole percent between 30-60% for the ionisable lipid, 5-40% for the phospholipid, 20-45% for the cholesterol, and 0.5- 3.5% for the PEGylated lipid, optionally wherein the lipids are present in the LNPs of the composition at a molar ratio of 50:10:38.5:1.5 or 40:30:27.5:2.5 for the ionisable lipid:phospholipid:cholesterol: PEGylated lipid.

10. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 9 or the method according to any of claims 2 to 9, wherein the composition is administered to the subject in a dose which provides between 0.15-2.0, 0.15-1.75, 0.15-1.5, 0.15- 1.25, 0.15-1.0, 0.15-0.9, 0.15-0.8, 0.15-0.7, 0.2-2.0, 0.2-1.75, 0.2-1.5, 0.2-1.25, 0.2- 1.0, 0.2-0.9, 0.2-0.9, 0.2-0.7, 0.25-2.0, 0.25-1.75, 0.25-1.5, 0.25-1.25, 0.25-1.0, 0.25- 0.9, 0.25-0.8, 0.25-0.7, 0.3-2.0, 0.3-1.75, 0.3-1.5, 0.3-1.25, 0.3-1.0, 0.3-0.9, 0.3-0.8, or 0.3-0.7 mg of mRNA / kg body weight of the subject, and / orwherein the composition is administered in a dose which provides between 1-90, 1-80, 1-70, 1-60, 1-50mg, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-45, 5-40, 5-35, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-45, 10-40, and 10-35mg mRNA per dose, and / orwherein the mRNA is present in the LNPs of the composition at a ratio of 10-50 lipids per 1 mRNA.

11. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 10 or the method according to any of claims 2 to 10, wherein the composition is first administered within 72, 60, 48, 36, 24, 12, 6, 3, 2, 1 or 0.5 hours of onset of symptoms of the disease or condition, and / orwherein the polypeptide is present in the blood of the subject within 2, 4, 6, 8 or 12 hours after administration of the composition.

12. The pharmaceutical composition for use according to claim 1 or any of claims 3 to 11 or the method according to any of claims 2 to 11, wherein over 97, 97.5, 98, 98.5, or99% of LNPs administered in the composition are located in spleen or liver of the subject after administration, optionally wherein the ratio of LNPs in liver:spleen is between 3:1 and 12:1, optionally wherein less than 1% of LNPs administered in the composition are located in heart, muscle or lungs of the subject after administration, and / orwherein the mRNA comprises one or more modifications, optionally wherein the modification is a 5-methoxyuridine, N1-methylpseudouridine, or 5’ cap1.

13. A lipid nanoparticle (LNP) comprising an mRNA encoding a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), or relaxin-1 (Rln-1) polypeptide or a variant, fragment or isoform thereof.

14. A lipid nanoparticle (LNP) comprising a cardiotherapeutic mRNA, wherein lipids of the LNP comprise or consist of an ionisable lipid selected from 6-[6-(2- hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM- 102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4- (dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof, optionally wherein the mRNA encodes a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof.

15. The LNP according to claim 13, wherein lipids of the LNP comprise or consist of an ionisable lipid, optionally wherein the ionisable lipid is selected from 6-[6-(2- hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM- 102), or [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4- (dimethylamino)butanoate (DLin-MC3-DMA), 3,6-bis[4-[bis[(9Z,12Z)-2- hydroxyoctadeca-9,12-dienyl]amino]butyl]piperazine-2, 5-dione (OF-02), ethyl 1-[3-(2- ethylpiperidin-1-yl)propyl]-5,5-bis[(Z)-heptadec-8-enyl]-2H-imidazole-2-carboxylate (A18-Iso5-2DC18), di(oct-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A1), di(non-2-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A2), di(dec-2-yn-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (A3), di(oct-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A4), di(non-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A5), di(dec-3-yn-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (A6), 1-[2-[bis(2- hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1- yl]ethyl]amino]dodecan-2-ol (C12-200), 3,6-bis[4-[bis(2- hydroxydodecyl)amino]butyl]piperazine-2, 5-dione (cKK-E12), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9), 3-[2-[2-[2-[bis[3-(dodecylamino)-3- oxopropyl]amino]ethyl-[3-(dodecylamino)-3-oxopropyl]amino]ethylamino]ethyl-[3- (dodecylamino)-3-oxopropyl]amino]- / \ / -dodecylpropanamide (98N12-5), 3-[[3-[2-[bis(2- hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[2-[[3-[2-[bis(2- hydroxytetradecyl)amino]ethylamino]-3-oxopropyl]-[3-[2-(2- hydroxytetradecylamino)ethylamino]-3-oxopropyl]amino]ethyl]amino]- / \ / -[2-(2- hydroxytetradecylamino)ethyl]propanamide (G0-C14), bis[(Z)-non-2-enyl] 9-[4- (dimethylamino)butanoyloxy]heptadecanedioate (L319), 2-[4-[5-[4-[bis[2-[(9Z,12Z)- octadeca-9,12-dienoyl]oxyethyl]amino]butyl]-3,6-dioxopiperazin-2-yl]butyl-[2- [(9Z, 12Z)-octadeca-9, 12-dienoyl]oxyethyl]ami no]ethyl (9Z, 12Z)-octadeca-9, 12- dienoate (OF-Deg-Lin), tridecyl 3-[2-[bis[2-[methyl-(3-oxo-3- tridecoxypropyl)amino]ethyl]amino]ethyl-methylamino]propanoate (304Oia), 2- (octyldisulfanyl)ethyl 3-[3-[3-[bis[3-[2-(octyldisulfanyl)ethoxy]-3- oxopropyl]amino]propyl-methylamino]propyl-[3-[2-(octyldisulfanyl)ethoxy]-3- oxopropyl]amino]propanoate (306-O12B), 8-methylnonyl 3-[3-[3-[bis[3-(8- methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3- oxopropyl]amino]propanoate (306Oi10), octan-3-yl 9-[3-[[3,5-bis[3-[bis(9-octan-3- yloxy-9-oxononyl)amino]propylcarbamoyl]benzoyl]amino]propyl-(9-octan-3-yloxy-9- oxononyl)amino]nonanoate (FTT5), (1-methyl-4,4-bis[(9Z,12Z)-octadeca-9,12- dienoxy]piperidine (YSK05) [2-[3-(diethylamino)propoxycarbonyloxymethyl]-3-(4,4- dioctoxybutanoyloxy)propyl] (9Z,12Z)-octadeca-9,12-dienoate (LP01), [(6Z,16Z)-12- [(Z)-dec-4-enyl]docosa-6,16-dien-11-yl] 5-(dimethylamino)pentanoate (CL1), [7-[4- (dipropylamino)butyl]-7-hydroxy-13-[(Z)-octadec-9-enoyl]oxytridecyl] (Z)-octadec-9- enoate (CL4H6), and / or [7-[4-(dipropylamino)butyl]-13-(2-hexyloctanoyloxy)-7- hydroxytridecyl] 2-hexyloctanoate (CL4F8-6), or analogues thereof.

16. The LNP according to claim 14 or 15, wherein the mRNA in the LNP is at a ratio of 10-50 lipids per 1 mRNA, and / orwherein lipids of the LNP further comprise structural lipids, phospholipids, and / or PEGylated lipids, optionallywherein the structural lipids comprise or consist of a sterol, optionally cholesterol or analogues thereof; and / orwherein the phospholipids comprise or consist of 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Egg Yolk Phosphatidylcholine, or 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DM PC), sphingomyelin (SM), ceramide or any related phosphatidylcholine or analogues thereof; and / orwherein the PEGylated lipids comprise or consist of 1,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol (DMG-PEG 2000), Methoxy Polyethylene Glycol- Distearoylphosphatidylethanolamine (mPEG-DSPE), 1-Palmitoyl-2-oleoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (POPE-PEG), 1,2- Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DPPE-PEG), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DOPE-PEG), 1,2-Distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), Distearoyl-rac- glycerol-PEG2000 (PEG-DSG), or 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine (PEG-cDMA) or analogues thereof.

17. The LNP according to claim 16, wherein the lipids in the LNP are at a mole percent between 30-60% for the ionisable lipid, 5-40% for the phospholipid, 20-45% for the cholesterol, and 0.5-3.5% for the PEGylated lipid, optionally wherein the lipids are in the LNP at molar ratios of 50:10:38.5:1.5 or 40:30:27.5:2.5 for the ionisable lipid:phospholipid:cholesterol: PEGylated lipid.

18. A pharmaceutical composition comprising one or more LNPs defined in any of claims 13 to 17, and optionally a pharmaceutically acceptable adjuvant, diluent, carrier, or excipient.

19. A unit dosage composition comprising one or more LNPs defined in any of claims 13 to 17, wherein said unit dosage provides a dose of mRNA between 1mg-100mg.

20. The LNPs according to any of claims 13 to 17, pharmaceutical composition according to claim 18, or unit dosage according to claim 19, for use as a medicament.

21. The LNPs according to any of claims 13 to 17, pharmaceutical composition according to claim 18, or unit dosage according to claim 19, for use in treating or preventing a cardiac disease or condition.

22. The LNPs, pharmaceutical composition, or unit dosage according to claim 21, wherein following administration of the LNPs, pharmaceutical composition, or unit dosage to a subject, a polypeptide encoded by the mRNA is transiently expressed by liver cells and secreted into the blood of the subject, wherein the polypeptide exerts a therapeutic effect on the heart to treat or prevent the disease or condition.

23. A kit comprising an ionisable lipid and an mRNA encoding a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeat-containing protein 3 (Nhlrc3), relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof, optionally wherein the kit further comprises a phospholipid, PEGylated lipid and / or structural lipid, optionally wherein the ionisable lipid is selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2- hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6- undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof.

24. A kit comprising or consisting a cardiotherapeutic mRNA and an ionisable lipid selected from 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6- undecoxyhexyl)amino]octanoate (SM-102), or [(6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (DLin-MC3-DMA) or analogues thereof, optionally wherein the kit further comprises a phospholipid, PEGylated lipid and / or structural lipid.

25. The kit of claim 24, wherein the mRNA encodes a chordin-like 1 (Chrdl1), family with sequence similarity 3 member b (Fam3b), family with sequence similarity 3 member c (Fam3c), midkine (Mdk), pleiotrophin, serine protease HtrA1 (HtrA1), NHL repeatcontaining protein 3 (Nhlrc3), or relaxin-1 (Rln-1) polypeptide, or a variant, fragment or isoform thereof.

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