Novel treatment for diabetes and obesity

Abstract

The present invention relates to compositions for the prevention or treatment of a metabolic disorder. In one aspect, the invention relates to the regulation of blood glucose levels and / or lipid metabolism with a composition of the invention.

Description

[0001] NOVEL TREATMENT FOR DIABETES AND OBESITY

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of Australian Provisional Application No. 2024902183 filed on 15 July 2024, the entire contents of which is incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to compositions for the prevention or treatment of a metabolic disorder. In one aspect, the invention relates to the regulation of blood glucose levels and / or lipid metabolism with a composition of the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Diabetes and obesity are interconnected metabolic disorders posing significant health challenges globally. Type 2 diabetes is the most common of form of diabetes with approximately 1.2 million (or 4.6%) of Australians diagnosed as having type 2 diabetes, whereas approximately only 10% of diabetic patients are diagnosed as having type 1 diabetes.

[0008] Diabetes impacts over 1.3 million Australians, with an estimated 500 million people globally afflicted with the disease. By 2050, it is expected that about 3.1 million of Australians will be living with diabetes. Obesity rates have also significantly increased, with the Australian Bureau of Statistics (ABS) reporting that around 36% of Australians are overweight and 31% are obese, while global statistics indicate that 1.9 billion adults are overweight and 650 million adults are obese. Individually, both diseases have major impacts on health and healthcare costs, but significantly they are also major risk factors for complications including heart disease, stroke, kidney disease, eye disease, sleep apnea and several types of cancers.

[0009] Treatment and management of diabetes is typically approached through a combination of lifestyle modifications (diet and exercise), education programs, medications including insulin therapy and in some cases surgery, particularly when diabetes is present as a comorbidity with obesity. Treatment of diabetes includes oral medication such as metformin, which is considered a first line of therapy, sulphonylureas, thiazolidinediones (TZD), sodium-glucose transporter 2 inhibitors (SGLT2 inhibitors), dipeptidyl peptidase-4 (DPP4) inhibitors); and injectables such as insulin and incretins such as glucagon like 1 peptides. Despite the availability of these treatments, the consistent increase in the prevalence of diabetes and obesity globally indicates that these medications are not sufficiently reducing or impacting the diabetes and obesity pandemic. Furthermore, the longer a person has type 2 diabetes, the greater the need for multiple combination therapies to control glucose levels. Moreover, the response to therapies between and within drug classes can differ at an individual level.

[0010] In view of the above-described limitations and relative importance and urgency thereof of developing new strategies for the treatment of metabolic disorders, there is a need for improved therapeutics that overcome one or more of the above-described limitations.

[0011] Reference to any prior art in the specification is not an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art.

[0012] SUMMARY OF THE INVENTION

[0013] The inventors have developed a novel therapeutic modality for the treatment of metabolic disorders, specifically those associated with elevated blood glucose or metabolic disorders such as obesity. In particular, the proposed therapy comprising compositions that include one or more synthetic nucleic acids, preferably mRNA including but not limited to a self-amplifying mRNA, encoding one or more components of the incretin axis have been developed that stimulate the production of specific hormones that regulate glucose metabolism, energy expenditure and satiety. The expression of components of the incretin axis including preproglucagon and pre-pro-gastric inhibitory peptide (preproGIP) in tissues that naturally produce these hormones is utilised herein to regulate blood glucose in a manner that can effectively regulate blood glucose levels to control hyperglycemia in diabetes and / or to reduce body weight. Moreover, the encapsulation of the synthetic nucleic acid, preferably mRNA, within an LNP engineered to be stable at low pH protects the cargo from degradation in the digestive system.

[0014] Importantly, whilst GLP-1 and GIP agonists are currently indicated for the treatment of diabetes, for example, Tirzepatide which is a combination therapeutic comprising GIP as a coagonist with GLP-1, these drugs are a synthetic or modified version of the endogenous hormone(s). The work described herein is unique in the targeting of mRNA encoding pre-pro hormone genes in tissues that naturally produce these hormones. This is associated with advantages including reduced off-target effects and the potential for personalised treatment given that the pre-pro hormone is utilized which can lead to expressions of differing types of incretins in various organs. The technology described herein thus provides for the targeting of genes critical for maintaining glucose metabolism and body weight to treat associated metabolic disorders, thus providing an effective treatment regime for such, as well as reducing incidence of multiple co-morbidities, complications arising therefrom. In an aspect of the invention, there is therefore provided a composition comprising a nucleic acid encoding a component of the incretin axis, preferably wherein the nucleic acid encodes a pre-pro hormone of the incretin axis, and wherein the nucleic acid is capable of regulating glucose metabolism and / or lipid metabolism.

[0015] In an embodiment, the nucleic acid encodes one or both of preproglucagon and pre-pro- gastric inhibitory peptide (preproGIP). In another embodiment, the nucleic acid encodes one or more of the components selected from the group consisting of IP1, IP2, GRPP, glucagon, gastric inhibitory peptide (GIP; also known as also known as glucose-dependent insulinotropic polypeptide), glucagon-like peptide-I (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM). In one embodiment, the nucleic acid encodes a preproglucagon and a pre- pro-gastric inhibitory peptide (preproGIP).

[0016] In one embodiment, there is provided a composition comprising a nucleic acid encoding pre-pro-gastric inhibitory peptide (preproGIP) and a nucleic acid encoding preproglucagon. The composition is used for regulating glucose metabolism and / or lipid metabolism.

[0017] In one embodiment, there is provided a composition for regulating glucose metabolism and / or lipid metabolism, the composition comprising a self-amplifying mRNA encoding a pre- pro-gastric inhibitory peptide (preproGIP) and a self-amplifying mRNA encoding a preproglucagon. The preproGIP encoded by the self-amplifying mRNA may comprise an amino acid modification selected from A45V, A45T, and A45G. In one embodiment, the amino acid modification is A45G. The preproglucagon encoded by the self-amplifying mRNA may comprise an amino acid modification selected from A99V, A99T, and A99G. In one embodiment, the amino acid modification is A99G.

[0018] In some embodiments, nucleic acids that encode a combination of components of the incretin axis, for example, preproGIP and preproglucagon, act synergistically to produce a desired effect such as reduced blood glucose level, reduced body weight, and / or increased lipid metabolism, beyond the level observed with either of the individual components (e.g., a nucleic acid that encodes preproGIP only or a nucleic acid that encodes preproglucagon only).

[0019] In some embodiments, a composition comprising a nucleic acid that encodes a preproGIP and a nucleic acid that encodes a preproglucagon, produces a desired synergistic effect such as reduced blood glucose level, reduced body weight, and / or increased lipid metabolism, beyond the level observed with either of the individual components comprised in a composition (e.g., a composition comprising a nucleic acid that encodes preproGIP only or a composition comprising a nucleic acid that encodes preproglucagon only). In some embodiments, a composition comprising a mRNA that encodes a preproGIP and a mRNA that encodes a preproglucagon produces a desired synergistic effect, beyond the level observed with either of the individual components comprised in a composition, where the preproglucagon comprises an A99G modification and the preproGIP comprises an A45G modification. The desired synergistic effect may be reduced blood glucose level, reduced body weight, and / or increased lipid metabolism.

[0020] In another embodiment, the nucleic acid encodes one or more of the components selected from the group consisting of peptide YY (PYY); chromogranin A (CgA), synaptophysin (Syn), serotonin (5-HT), tryptophan 5-hydroxylase (TPH), Oxyntomodulin, Glicentin, Somatostatin, Ghrelin, Gastrin, Serotonin, Neurotensin, Growth differential factor 15, Fibroblast growth factor 19, Guanylin, Uroguanylin, Insulin-Like Peptide 5 (INSL5), Cholecystokinin (CKK), Secretin, Motilin, Nesfatin-1, Leptin, Zenin, Histamine, Proglucagon, Glucagon, Fibroblast growth factor 21.

[0021] In an embodiment, the nucleic acid comprises one or more or all of a furin cleavage site, a leader sequence or a secretion signal.

[0022] In an embodiment, the nucleic acid is mRNA and encodes preproglucagon preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 1. In some embodiments, the preproglucagon comprises an amino acid modification selected from A99V, A99T, and A99G. In one embodiment, the preproglucagon comprises an A99G modification.

[0023] In an embodiment, the nucleic acid is mRNA and encodes preproGIP preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 2. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 2. In some embodiments, the preproGIP comprises an amino acid modification selected from A45V, A45T, and A45G. In one embodiment, the preproGIP comprises an A45G modification.

[0024] In an embodiment, the nucleic acid is mRNA and encodes GLP-1 preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 3, 4, 5 or 10. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 3, 4, 5 or 10.

[0025] In an embodiment, the nucleic acid is mRNA and encodes glucagon preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 6 or 11. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 6 or 11.

[0026] In an embodiment, the nucleic acid is mRNA and encodes GLP-2 preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 7 or 12. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 7 or 12.

[0027] In an embodiment, the nucleic acid is mRNA and encodes GIP preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 8 or 13. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 8 or 13.

[0028] In another aspect of the invention, there is provided a composition for regulating glucose metabolism and / or lipid metabolism, the composition comprising a nucleic acid encoding pre-pro- gastric inhibitory peptide (preproGIP).

[0029] In an embodiment, the composition further comprises a nucleic acid encoding preproglucagon.

[0030] In an embodiment, the composition regulates glucose metabolism and lipid metabolism. In an embodiment, the composition regulates glucose metabolism. In an embodiment, the composition regulates lipid metabolism.

[0031] In an embodiment, the nucleic acid is selected from the group consisting of a messenger RNA (mRNA), deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Preferably, the nucleic acid is a mRNA such as a self-amplifying mRNA. In an embodiment, the mRNA comprises a cap, 5'UTR, coding sequence, a 3'UTR and a poly A tail.

[0032] In an embodiment, the 5'UTR comprises or consists of a sequence set forth in SEQ ID NO: 20. In an embodiment, the 3'UTR comprises or consists of a sequence set forth in SEQ ID NO: 21. In an embodiment, the poly A tail comprises or consists of a sequence set forth in SEQ ID NO: 22.

[0033] In an embodiment, the mRNA comprises an optimised codon and / or a chemical modification. In one embodiment, the chemical modification is a uridine-5’ -triphosphate nucleoside modification, optionally modified to Nl-methylpseudouridine-5’ -triphosphate (m^TP) or 5-methoxyuridine-5’-triphosphate (5moUTP). In an embodiment, the optimised codon comprises substituting adenine (A) or uracil (U) containing codons with codons enriched in guanine (G) or cytosine (C). In an embodiment, the chemical modification increases mRNA stability and / or mRNA translation when compared to a mRNA without the chemical modification.

[0034] In an embodiment, the uridine modification is selected from the group consisting of pseudouridine (y), pyridin-4-one ribonucleoside, 5 -aza-uridine, 6-aza-uridine, 2-thio-5-zauridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thiopseudouridine, 2-thio-pseudouridine, 5-hydroxy- uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromouridine), 3- methyluridine (m3U), 5 -methoxy -uridine (5moU), uridine 5-oxyacetic acid (cmo5U), uridine 5- oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-20 methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5 -carbarn oylmethyl-uri dine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnnfU), 5-carboxymethylaminomethyl-2 -thio-uridine (cmnmVU), 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (tm5U), 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (tm5s2U), l-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methylpseudouridine (m1!] / ), 5-methyl-2-thiouridine (m5s2U), l-methyl-4-thio-pseudouridine 4-thio-l-methyl-pseudouridine, 3 -methyl -pseudouridine 2-thio-l -methylpseudouridine, 1-m ethyl- 1-deaza-pseudouri dine, 2-3 0 thio- 1 -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5 ,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2- thio-dihydrouridine, 2-thiodihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy -pseudouridine, 4-methoxy -2-thio-pseudouridine, Nl-methyl-pseudouridine (also known as 1 -methylpseudouridine (m1!] / ), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl- 3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl- uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ym), 2-thio-2'-O- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-Omethyl-uridine (ncm5Um), 5-carboxymethylaminom ethyl -2'-0- methyluridine (cmnm5Um), 3,2'O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O- methyl-uridine (inm5Um), l-thio-5 uridine, deoxythymidine, 2'-F-arauridine, 2'-F-uridine, 2'-0H- ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)]uridine.

[0035] In an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least or 100% of uridines are modified.

[0036] In another embodiment, the nucleoside modification is a modified cytosine. In this embodiment, suitable cytidine modifications may be selected from the group consisting of 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (PC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-methy-lcytidine 5' -triphosphate (5mCTP), 5-halo-cytidine (e.g., 5 -iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4- thio-l-methyl-l-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5- aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thiozebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, lysidine (k2C), 2-thiocytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl- cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl- 2'-O-methyl-cytidine (CCm), N4,N4,2'-O-trimethyl-cytidine (m Cm), 1 -thiocytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0037] In an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least or 100% of cytosines are modified.

[0038] In an embodiment, the optimised codon increases mRNA stability and / or mRNA translation when compared to a mRNA without the optimised codon. In another embodiment, the optimised codon increases guanine (G) and / or cytosine (C) codon content. Preferably, the optimised codon comprises substituting adenine (A) or uracil (U) containing codons with codons enriched in guanine (G) or cytosine (C). In an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least or 100% of codons are modified.

[0039] In an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least or 100% of codons are modified to increase guanine (G) and / or cytosine (C) codon content.

[0040] In an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least or 100% of adenine (A) or uracil (U) containing codons are substituted with codons enriched in guanine (G) or cytosine (C).

[0041] In an embodiment, the nucleic acid encoding preproglucagon is mRNA and encodes an amino acid sequence having 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% or at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 1.

[0042] In an embodiment, the nucleic acid encoding preproGIP is mRNA, and encodes an amino acid sequence having 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% or at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 2. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 2.

[0043] In an embodiment, the composition comprises an additional nucleic acid selected from the group consisting of IP1, IP2, GRPP, glucagon, glucagon-like peptide-I (GLP-1), gastric inhibitory peptide (GIP), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM).

[0044] In another embodiment, the nucleic acid encodes one or more of the components selected from the group consisting of peptide YY (PYY); chromogranin A (CgA), synaptophysin (Syn), serotonin (5-HT), tryptophan 5-hydroxylase (TPH), Oxyntomodulin, Glicentin, Somatostatin, Ghrelin, Gastrin, Serotonin, Neurotensin, Growth differential factor 15, Fibroblast growth factor 19, Guanylin, Uroguanylin, Insulin-Like Peptide 5 (INSL5), Cholecystokinin (CKK), Secretin, Motilin, Nesfatin-1, Leptin, Zenin, Histamine, Proglucagon, Glucagon, Fibroblast growth factor 21 (FGF21). In an embodiment, the nucleic acid encodes Oxyntomodulin comprising a sequence set forth in SEQ ID NO: 26. In an embodiment, the nucleic acid encodes FGF21 comprising a sequence set forth in SEQ ID NO: 27.

[0045] In an embodiment, the additional nucleic acid is mRNA and encodes GLP-1 preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 3, 4, 5 or 10. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 3, 4, 5 or 10.

[0046] In an embodiment, the additional nucleic acid is mRNA and encodes glucagon preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 6 or 11. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 6 or 11.

[0047] In an embodiment, the additional nucleic acid is mRNA and encodes GLP-2 preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID NOs: 7 or 12. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID NOs: 7 or 12.

[0048] In an embodiment, the additional nucleic acid is mRNA and encodes GIP preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 8 or 13. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 8 or 13.

[0049] In an embodiment, a nucleic acid described herein encodes a chimeric polypeptide. In an embodiment, a suitable chimeric polypeptide comprises a sequence encoding a GLP-1 and a preproglucagon leader sequence set forth in SEQ ID NO: 4.

[0050] In an embodiment, the nucleic acid, preferably mRNA, comprises a modification and / or an additional sequence for one or more or all of tissue specificity, increased solubility and / or halflife, increased affinity, increased activity, increased stability and resistance to protease cleavage. In an embodiment, the additional sequence is a heterologous moiety that encodes a protein, a peptide, a protein domain, a linker, an albumin, a human serum albumin (HSA), a HSA FcRn binding portion, an antibody, an antibody fragment, a single chain antibody, a domain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, an epitope tag, a recombinant polypeptide polymer, a cytokine, or any combination of two or more of such moi eties.

[0051] In an embodiment, the heterologous moiety comprises a sequence encoding a linker. In another embodiment, the mRNA is bound to the heterologous moiety by a sequence encoding a linker.

[0052] In one example, the sequence encodes a flexible linker. A “flexible” linker is an amino acid sequence which does not have a fixed structure (secondary or tertiary structure) in solution. Such a flexible linker is therefore free to adopt a variety of conformations. Flexible linkers suitable for use in the present disclosure are known in the art. An example of a flexible linker for use in the present disclosure is the linker sequence SGGGGS / GGGGS / GGGGS or (Gly4Ser)3. Another example of a flexible linker is an alanine linker.

[0053] The linker may comprise any amino acid sequence that does not substantially hinder interaction of the component of the incretin axis with its cognate receptor. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine proline, lysine, arginine, glutamine and glutamic acid.

[0054] In some examples, the linker sequences comprise five or more amino acid residues. In one example, the flexible linker consists of 5 or more residues, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more residues. In another example, the flexible linker sequences consist of 5, 7, 10 or 16 residues.

[0055] In one example, the linker is a rigid linker. A “rigid linker” (including a “semi-rigid linker”) refers to a linker having limited flexibility. For example, the relatively rigid linker comprises the sequence (EAAAK)n, where n is between 1 and 3. The value of n can be between 1 and about 10 or between about 1 and 100. For example, n is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10.

[0056] In one example, n is less than 100. For example, n is less than 90, or less than about 80, or less than about 70, or less than about 60, or less than about 50, or less than about 40, or less than about 30, or less than about 20, or less than about 10. A rigid linker need not completely lack flexibility.

[0057] In an embodiment, the heterologous moiety for tissue specificity comprises an antibody, a domain of an antibody, an antibody fragment, a single chain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, an epitope tag, a recombinant polypeptide polymer, a cytokine, or any combination of two or more of such moieties.

[0058] In an embodiment, the heterologous moiety increases solubility and / or prolongs half-life and comprises a hydrophilic moiety.

[0059] In another embodiment, solubility and / or half-life is increased by the addition of a heterologous moiety that encodes charged amino acids, for example with charged amino acids selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid, or by the addition of charged amino acids to the amino or carboxy terminus of the peptide. For example, the peptide encoded by a mRNA of the invention may comprise one, two, three, four or more charged amino acids selected from the group consisting of lysine, arginine, histidine, aspartic acid and glutamic acid.

[0060] In another embodiment, solubility and / or half-life is increased by the addition of a heterologous moiety that encodes albumin, a human serum albumin (HSA) or a HSA FcRn binding portion.

[0061] In an embodiment, solubility and / or half-life is increased by at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least, 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold or at least 100 fold, when compared to the solubility and / or half-life of a polypeptide without the additional sequence or modification.

[0062] In an embodiment, the modification or heterologous moiety increases affinity of GIP and / or GLP-1 for GIP-R and / or GLP-1R respectively.

[0063] In an embodiment, affinity is at least 10 fold, at least 25 fold, at least, 50 fold, at least 100 fold, at least 200 fold, at least 400 fold, at least 600 fold, at least 800 fold, at least 1000 fold, at least 1200 fold, at least 1400 fold, at least 1600 fold, at least 1800 fold, or at least 2000 fold higher when compared to the affinity without the heterologous moiety or modification.

[0064] In an embodiment, the heterologous moiety or modification increases activity. In an embodiment, activity is increased by the addition of a heterologous moiety that encodes negatively charged amino acids. In another embodiment, activity is increased by a modification that comprises substituting the naturally occurring serine with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with glutamine.

[0065] In an embodiment, the heterologous moiety or modification increases activity by at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least, 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold or at least 100 fold, when compared to the activity of a polypeptide encoded by a mRNA without the heterologous moiety or modification.

[0066] In another example, the heterologous moiety increases stability.

[0067] In an embodiment, stability of a mRNA of the disclosure may be increased by at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least, 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold or at least 100 fold, when compared to the stability of a mRNA without the heterologous moiety or modification.

[0068] In another example, resistance to proteolytic cleavage, for example by dipeptidyl peptidase 4 (DPP -4), is increased, preferably by a modification comprising substituting amino acids intended for cleavage with the amino acid glycine. In an embodiment, a suitable GLP-1 sequence for inhibiting cleavage of the peptide by DPP-4 is that encoding an amino acid sequence set forth in SEQ ID NO: 10.

[0069] In an embodiment, a composition described herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0070] In another aspect of the invention, the composition is suitable, or formulated for delivery in a lipid nanoparticle (LNP).

[0071] Thus, in one aspect of the invention, there is provided a lipid nanoparticle (LNP) comprising a composition described herein.

[0072] In an embodiment, the LNP encapsulates the nucleic acid. In another embodiment, the nucleic acid is bound to the LNP. In another embodiment, the nucleic acid is adsorbed on to the LNP.

[0073] In an embodiment, the LNP comprises an ionizable lipid, a phospholipid, a sterol, and a PEG-lipid.

[0074] In an embodiment, the molar ratio of ionizable lipid, phospholipid, sterol, and PEG-lipid is 50: 10:38.5: 1.5. In an embodiment, the molar ratio of ionizable lipid, phospholipid, sterol, and PEG-lipid is selected from the group consisting of 60:5: 10:25, 55:30:45:0.2, 52:8:38.5: 1.5, 52:8:37:3, 50:20:23.5:6.5 50: 10.5:38:1.5, 50:12.5:35:2.5, 45: 13:39.5:2.5, 35: 16:46.5:2.5, 35:40:22.5:2.5, 26.5:20:52: 1.5, 25:30:30:1, 40:10:38.5: 1.5, 30: 10:38.5:1.5, 40: 10:38.5: 1.5, 60: 10:38.5: 1.5, 70: 10:38.5: 1.5, 50:5:38.5: 1.5, 50: 15:38.5: 1.5, 50:20:38.5: 1.5, 50:25:38.5: 1.5, 50: 10: 18.5: 1.5, 50: 10:28.5: 1.5, 50: 10:48.5: 1.5, 50: 10:58.5: 1.5, 50: 10:38.5:0.5, 50: 10:38.5: 1.0, 50: 10:38.5:2.0, 50: 10:38.5:2.5 or any combination thereof. In an embodiment, the lipid nanoparticle comprises 20-60 mol % (e.g., 20-30 mol %, 20-

[0075] 40 mol %, 20-50 mol %, 20-60 mol %, 30-40 mol %, 30-50 mol %, 30-60 mol %, 40-50 mol %,

[0076] 40-60 mol %, 45-55 mol %, or 45-50 mol % or 50-60 mol%) ionizable lipid; 5-25 mol % (e.g., 5-

[0077] 10 mol %, 5-15 mol %, 5-20 mol %, 5-25 mol %, or 10-15 mol %, 10-20 mol %, 10-25 mol %,

[0078] 15-20 mol %, 15-25 mol %) phospholipid; 25-55 mol % (e.g., 25-35 mol %, 25-45 mol %, 25-55 mol %, 35-45 mol %, 35-55 mol % or 45-55 mol %) sterol; and 0.5-15 mol % (e.g., 0.5-5 mol %,

[0079] 0.5-10 mol %, or 0.5-15 mol %, 2.5-5 mol %, 2.5-10 mol %, 2.5-15 mol %, 5-10 mol %, 5-15 mol

[0080] %) PEG-lipid.

[0081] In an embodiment, the ionizable lipid comprises a pKa between 5 and 8, between 6 and 8 or between 6 and 7 and may include any ionizable lipid known in the art. In another embodiment, the ionizable lipid comprises a pKa between 6.1 and 7, between 6.2 and 7, between 6.3 and 7, between 6.4 and 7, between 6.5 and 7, between 6.6 and 7, between 6.7 and 7, between 6.8 and 7 or between 6.9 and 7. In another embodiment, the ionizable lipid comprises a pKa between 6 and 6.9, between 6 and 6.8, between 6 and 6.7, between 6 and 6.6, between 6 and 6.5, between 6 and 6.4, between 6 and 6.3, between 6 and 6.2 or between 6 and 6.1.

[0082] In an embodiment, the ionizable lipid is selected from the group consisting of 9- Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), DLin-MC3-DMA (MC3), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2- dioleyloxy-N,Ndimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethyl-3 -aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-l-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750), 4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate (ALC0315), C12-200, 306- O12B, 4A3-SC8, cKK-E12, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Preferably, the ionizable lipid is DLin-MC3-DMA.

[0083] In an embodiment, the ionizable lipid is a cationic lipid. In this embodiment, the cationic lipid is selected from the group consisting of l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-dioleoyl-s-glycero-3 -ethylphosphocholine (EPC), dimethyldioctadecylammonium bromide (DDAB), and l,2-di-O-octadecenyl-3 -trimethylammonium -propane (DOTMA).

[0084] In another embodiment, the phospholipid is selected from the group consisting of 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine,

[0085] 1.2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine(DSPE),

[0086] 1.2-dilinoleoyl-sn-glycero-3 - phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 - phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof. Preferably, the phospholipid is DSPC.

[0087] In an embodiment, the sterol is selected from the group consisting of B-sitosterol, cholesterol, 24(S)-hydroxycholesterol, 20a-hydroxycholesterol, cholesterol oleate, other cholesterol esters, fecosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol and combinations thereof. Preferably, the sterol is cholesterol.

[0088] In another embodiment, the PEG-lipid is selected from the group consisting of PEG2000- c-DMG, PEG2000-DMG, PEG2000-DLPE, PEG2000-DMPE, PEG2000-DPPC, a PEG2000- DSPE lipid and combinations thereof. Preferably, the PEG2000-lipid is PEG2000-DSPE or PEG2000-DMG. In another embodiment, the PEG-lipid may be a different molecular weight as whose suitability is determined by a skilled person in the art. For example, the PEG-lipid may have a molecular weight of 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600 or 4000. Preferably, the PEG-lipid is PEG2000-DMG.

[0089] In a preferred embodiment, the ionizable lipid is DLin-MC3-DMA; the phospholipid is a DSPC; the sterol is cholesterol and the PEG-lipid is PEG2000-DMG. In this embodiment, the molar ratio of ionizable lipid, phospholipid, sterol, and PEG-lipid is preferably 50: 10:38.5: 1.5.

[0090] In an embodiment, the LNP further comprises a fluorescent label, chromophoric label, electron-dense label, chemiluminescent label or radioactive label.

[0091] In an embodiment, the composition or LNP is suitable, or formulated for delivery to one or more or all of the liver, stomach, pancreas, duodenumjejunum, ileum, colon and adipose tissue.

[0092] In an embodiment, the composition further comprises a polymeric microparticle. In an embodiment, the mRNA is encapsulated in a polymeric microparticle. In an embodiment, the mRNA is bound to a polymeric microparticle. In another embodiment, the mRNA is adsorbed on to a polymeric microparticle.

[0093] In an embodiment, the composition further comprises an oil-in-water emulsion. For example, the mRNA is encapsulated in an oil-in-water emulsion. In an embodiment, the mRNA is bound to an oil-in-water emulsion. In another embodiment, the mRNA is adsorbed on to an oil-in- water emulsion. In another embodiment, the mRNA is resuspended in an oil-in-water emulsion.

[0094] In another aspect, there is provided a host cell, preferably a host cell of the liver, stomach, pancreas, duodenum, jejunum, ileum, colon or adipose tissue, comprising a nucleic acid of the invention. In an embodiment, the host cell is a beta cell, alpha cell, adipocyte, hepatocyte, epithelial cell, enterocyte, parietal cell, goblet cell or enteroendocrine cell. In an embodiment, the host cell is a human or mammalian cell.

[0095] In another aspect, there is provided a method of synthesis of a LNP described herein, the method comprising producing a nucleic acid, preferably mRNA, from a DNA polynucleotide, and encapsulating the nucleic acid within the LNP.

[0096] In an embodiment, the method further comprises labelling the LNP with a lipid fluorescent label.

[0097] Preferably, the method of producing the nucleic acid is carried out using a cell free assay, preferably in vitro transcription. In an embodiment, the method further comprises introducing a chemical modification to the mRNA.

[0098] In an embodiment, the DNA polynucleotide is provided in a plasmid or vector.

[0099] In an aspect, the present invention provides a LNP obtained from a method described herein.

[0100] In another aspect of the invention, there is provided a method of producing glucagon-like peptide-I (GLP-1) or gastric inhibitory peptide (GIP) comprising expressing a nucleic acid encoding pro-pro-glucagon or pre-pro-gastric inhibitory peptide (preproGIP) respectively, under conditions sufficient for the production of GLP-1 or GIP. Preferably the nucleic acid is mRNA. Preferably the mRNA is formulated in a LNP. Optionally, the mRNA is provided in a plasmid or vector.

[0101] In an aspect, the present invention provides a kit comprising one or more of the following:

[0102] (i) a nucleic acid of the invention;

[0103] (ii) a composition of the invention; (iii) a lipid nanoparticle (LNP) of the invention.

[0104] In the case of a kit for therapeutic use, the kit preferably comprises a pharmaceutical composition of the invention and additionally comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0105] Optionally, a kit of the invention is packaged with instructions for use in a method described herein according to any example.

[0106] In another aspect, there is provided a method of treating or preventing a metabolic disorder associated with elevated blood glucose levels in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject, thereby treating or preventing the metabolic disorder in the subject.

[0107] In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for treating or preventing a metabolic disorder associated with elevated blood glucose levels in a subject.

[0108] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in treating or preventing a metabolic disorder associated with elevated blood glucose levels in a subject.

[0109] In an embodiment, the metabolic disorder is selected from the group consisting of type 2 diabetes, type 1 diabetes, insulin resistance, prediabetes, fatty liver disease, metabolic associated fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH), and obesity.

[0110] In another aspect, there is provided a method of regulating blood glucose levels in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) described herein to the subject, thereby regulating blood glucose levels in the subject.

[0111] In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for regulating blood glucose levels in a subject.

[0112] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in regulating blood glucose levels in a subject.

[0113] In an embodiment, the subject is overweight or obese. In an embodiment, the subject is pre-diabetic and has elevated blood glucose levels. In an embodiment, the subject is insulin resistant. In an embodiment, the subject is hyperglycemic. In an embodiment, the subject has been diagnosed with type 2 diabetes. In another embodiment, the subject has type 1 diabetes. In an embodiment, the subject has a baseline HbAlc percentage of greater than 5.7% or 6.5%. In an embodiment, a treatment described herein decreases the subject’s fasting plasma glucose concentration by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% or more, when compared to a subject not having received the treatment.

[0114] In an embodiment, the treatment decreases plasma glucose concentration within at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes at least 110 minutes, at least 120 minutes, at least 130 minutes or at least 140 minutes of an oral glucose tolerance test in a subject. Preferably, the treatment decreases plasma glucose concentration within at least 30 minutes of an oral glucose tolerance test.

[0115] In an embodiment, the method further comprises identifying a subject having, or at risk of having a metabolic disease.

[0116] In an embodiment, the subject exhibits one or more of the following symptoms: elevated blood glucose levels, baseline HbAlc percentage of greater than 5.7% or 6.5%, frequent urination, increased thirst, loss of body weight, presence of ketones in urine, weakness and tiredness, irritability, poor wound healing and susceptibility to infection.

[0117] In an embodiment, the subject is also obese or overweight. In this embodiment, the obese subject has a body mass index of at least 30. Alternatively, the overweight subject has a body mass index of between about 25 to 29.9. In an embodiment, the subject is a female having a waist circumference of at least 80cm. In an embodiment, the subject is a male having a waist circumference of at least 94cm.

[0118] In an embodiment, the treatment improves one or more of the above symptoms by at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more when compared to a subject not receiving the treatment.

[0119] In an embodiment, where prevention of a metabolic disease or disorder is contemplated, the subject preferably has no clinical symptoms of the metabolic disease.

[0120] In another aspect, there is provided a method of treating or preventing obesity or weight gain in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject, thereby treating or preventing obesity or weight gain in the subject. In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for treating or preventing obesity or weight gain in a subject.

[0121] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in treating or preventing obesity or weight gain in a subject.

[0122] In another aspect, there is provided a method of regulating lipid metabolism in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject, thereby regulating lipid metabolism in the subject.

[0123] In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for regulating lipid metabolism in a subject.

[0124] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in regulating lipid metabolism in a subject.

[0125] In another aspect, there is provided a method of promoting weight loss in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject, thereby promoting weight loss in the subject.

[0126] In another aspect, there is provided a method for regulating cardiovascular risk in a subject comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject, thereby regulating cardiovascular risk in the subject.

[0127] In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for regulating cardiovascular risk in a subject.

[0128] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in regulating cardiovascular risk in a subject.

[0129] In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for promoting weight loss in a subject.

[0130] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in promoting weight loss in a subject.

[0131] In another aspect, there is provided a method of increasing expression of one or both of pre-pro-gastric inhibitory peptide (preproGIP) and preproglucagon in a subject in need thereof, comprising administering a composition or lipid nanoparticle (LNP) of the invention to the subject. In another aspect, there is provided use of a composition or lipid nanoparticle (LNP) of the invention in the manufacture of a medicament for increasing expression of one or both of pre-pro- gastric inhibitory peptide (preproGIP) and preproglucagon in a subject.

[0132] In another aspect, there is provided a composition or lipid nanoparticle (LNP) of the invention for use in increasing expression of one or both of pre-pro-gastric inhibitory peptide (preproGIP) and preproglucagon in a subject.

[0133] In an embodiment, the treatment further comprises administering an additional therapeutic agent. In an embodiment, the additional therapeutic agent is bound to a LNP in the composition. In an embodiment, the LNP encapsulates the additional therapeutic agent. In an embodiment, the additional therapeutic agent is adsorbed on to the LNP.

[0134] In an embodiment, the additional therapeutic agent is an anti-obesity agent selected from Bupropion-naltrexone (Contrave), Liraglutide (Saxenda), Orlistat (Xenical, Alli), Phenterminetopiramate (Qsymia), Semaglutide (Wegovy), Retatrutide (LY-3437943), Setmelanotide (Imcivree), Survotutide, Mazdutide, Efinopegdutide, Cagrisema, Dulaglutide (Trulicity), Exenatide (Byetta, Bydureon, and Bydureon BCise), Efpeglenatide, Lixisenatide, Cotadutide, Danuglipron and Orgorglipton.

[0135] In an embodiment, the additional therapeutic agent is an anti-diabetic and / or obesity agent selected from the group consisting of native insulin, native glucagon and functional analogs thereof, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose / Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SOLT (sodium-dependent glucose transporter 1) inhibitors; FBPase (fructose 1,6-bisphosphatase) inhibitors, Tirzepatide (Mounjaro), SAR441255 and semaglutide (Ozempic) or analogues thereof.

[0136] In this embodiment, administration of the additional therapeutic agent may be at the same time or a different time to the administration of a composition of the invention.

[0137] In an embodiment, the additional therapeutic is mRNA and encodes Exenatide preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in any one of SEQ ID Nos: 9, 14, 18, 19, or 23. In another embodiment, the sequence comprises or consists of the sequence set forth in any one of SEQ ID Nos: 9, 14, 18, 19, or 23. In this embodiment, the Exenatide sequence is fused to albumin or an albumin binding domain, for example, according to those sequences set forth in SEQ ID NO: 18 and 19 respectively.

[0138] In an embodiment, the additional therapeutic is mRNA and encodes an analogue of Tirzepatide preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 15. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 15.

[0139] In an embodiment, the additional therapeutic is mRNA and encodes an analogue of Retatrutide preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 16. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 16.

[0140] In an embodiment, the additional therapeutic is mRNA and encodes an analogue of SAR441255 preferably comprising or consisting of a sequence having 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% or at least 99% or 100% sequence identity to the sequences set forth in SEQ ID NO: 17. In another embodiment, the sequence comprises or consists of the sequence set forth in SEQ ID NO: 17.

[0141] In another aspect, there is provided a method of expressing a nucleic acid in a cell of the liver, stomach, pancreas, duodenum, jejunum, ileum, colon and adipose tissue of a subject, comprising administering a LNP or composition of the invention to the subject, thus expressing the nucleic acid in the cell of the subject.

[0142] In another aspect, there is provided use of a LNP or composition of the invention in the manufacture of a medicament for expressing a nucleic acid in a cell of the liver, stomach, pancreas, duodenum jejunum, ileum, colon and adipose tissue of a subject.

[0143] In another aspect, there is provided a LNP or composition of the invention for use in expressing a nucleic acid in a cell of the liver, stomach, pancreas, duodenum, jejunum, ileum, colon and adipose tissue of a subject. In an embodiment, the nucleic acid is expressed in cells of the liver, stomach, pancreas, duodenumjejunum, ileum, colon and adipose tissue.

[0144] Preferably, the nucleic acid is a mRNA and the cell is selected from the group consisting of a beta cell, alpha cell, adipocyte, hepatocyte, epithelial cell, enterocyte, parietal cell, goblet cell and an enteroendocrine cell.

[0145] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0146] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0147] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0148] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0149] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0150] DESCRIPTION OF THE DRAWINGS

[0151] Figure 1 Blood glucose levels following an oral glucose tolerance test (2g / kg) following administration of mRNA compounds (preproglucagon, preproGIP, lipid nanoparticle (LNP) control and TRIS buffer control. Compounds were administered intraperitoneally 4 hours prior and mice fasted for 4 hours. A. Glucose excursion over 120 minutes post oral glucose. B. Total area under curve (AUC) of blood glucose levels following an oral glucose tolerance test (2g / kg) following administration of mRNA compounds (preproglucagon, preproGIP, lipid nanoparticle (LNP) control and TRIS buffer control. Compounds were administered intraperitoneally 4 hours prior and mice fasted for 4 hours. C. Glucose excursion (OGTT) following exenatide administration IP, 1 hour prior to OGTT (2g / kg). Figure 2 Nanoluciferase protein expression in tissues and plasma of B6 control mice administered LNPs containing mScarlet-I-nLuc mRNA. Compounds were administered IP 4h prior to tissue collection. Data presented as mean ± SD, n=5.

[0152] Figure 3 A. Change in body weight (g) in B6 control mice and DIO mice over 7 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon / preproGIP (native) DIO mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs TRIS Buffer Control; b (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs Tirzepatide; c (selfamplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs LNP B6 mice; d (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs LNP DIO mice. B. Change in body weight (g) in B6 control mice only over 7 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs TRIS Buffer Control; b (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) vs Tirzepatide; c (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs LNP B6 mice. C. Change in body weight (g) in DIO mice over 7 days following a single IP injection of mRNA compounds self-amplifying preproglucagon / preproGIP (native), selfamplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) DIO mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs TRIS Buffer Control; b (self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs Tirzepatide. D. Average change in body weight (g) in B6 control mice and DIO mice over 7 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP B6 mice, d vs preproGIP native mRNA - B6 mice; e vs selfamplifying preproGIP mRNA (native) - B6 mice; f vs self-amplifying preproglucagon; g vs LNP DIO mice. E. % change in body weight (g) in B6 control mice and DIO mice over 7 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP B6 mice, d vs preproGIP native mRNA - B6 mice; e vs self-amplifying preproglucagon mRNA (native) - B6 mice; f vs self-amplifying preproGIP mRNA (native) - B6 mice; g vs LNP DIO mice.

[0153] Figure 4 A. Change in body weight (g) in B6 control mice and DIO mice over 14 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon / preproGIP (native) DIO mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs TRIS Buffer Control; b (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) vs Tirzepatide; c (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) DIO mice) vs LNP B6 mice; d self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, vs self-amplifying preproGIP (native) - B6 mice; e self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, mice vs self-amplifying preproglucagon (native) - B6 mice; f self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, mice vs LNP - DIO mice; g self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, mice vs self-amplifying preproglucagon / preproGIP (native) - DIO mice. B. Average change in body weight (g) in B6 control mice and DIO mice over 14 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP B6 mice, d self-amplifying preproGIP mRNA (native) - B6 mice; e vs self-amplifying preproglucagon (native) - B6 mice; f vs LNP - DIO mice; g vs self-amplifying preproglucagon / preproGIP mRNA (native)- DIO mice. C. % change in body weight (g) in B6 control mice and DIO mice over 14 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), selfamplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), selfamplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP B6 mice, d self-amplifying preproGIP mRNA (native) - B6 mice; e vs selfamplifying preproglucagon (native) - B6 mice; f vs LNP - DIO mice; g vs self-amplifying preproglucagon / preproGIP mRNA (native)- DIO mice.

[0154] Figure 5 A. Change in blood glucose levels (mmol / L) in B6 control mice and DIO mice over 7 days following a single IP injection of mRNA compounds preproGIP (native), selfamplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs TRIS Buffer Control; b (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon / preproGIP mRNA (native)- DIO mice, self-amplifying preproglucagon A99G / preproGIP A45GmRNA (modified) DIO mice) vs Tirzepatide; c (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs LNP DIO mice; d (selfamplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs self-amplifying preproglucagon (native) - B6 mice. B. Average change in blood glucose (mmol / L) in B6 control mice and DIO mice over 7 days following a single IP injection of mRNA compounds preproGIP (native), selfamplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified)and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP DIO mice, d vs selfamplifying preproglucagon mRNA (native) - B6 mice. C. Change in blood glucose levels (mmol / L) in B6 control mice and DIO mice over 14 days following a single IP injection of mRNA compounds preproGIP (native), self-amplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05, where significant results are indicated by the letter codes: a (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice vs TRIS Buffer Control; b (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice, self-amplifying preproglucagon / preproGIP mRNA (native)- DIO mice, self-amplifying preproglucagon A99G / preproGIP A45GmRNA (modified) DIO mice) vs Tirzepatide; c (self-amplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs LNP DIO mice; d (selfamplifying preproglucagon / preproGIP (native) B6 mice, self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) B6 mice) vs self-amplifying preproglucagon (native) - B6 mice. D. Average change in blood glucose (mmol / L) in B6 control mice and DIO mice over 14 days following a single IP injection of mRNA compounds preproGIP (native), selfamplifying preproGIP (native), self-amplifying preproglucagon (native), self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control, Tirzepatide peptide (30nmol / kg, subcutaneous) and LNP. Data presented as mean ± SEM (n=4-5 per group). Two-way ANOVA, p<0.05: a vs TRIS Buffer Control; b vs Tirzepatide peptide, c vs LNP DIO mice, d vs selfamplifying preproglucagon mRNA (native) - B6 mice.

[0155] Figure 6 A. Blood glucose (mmol / L) excursion following an OGTT (2g / kg) 1 week (7 days) post administration in B6 and DIO mice of mRNA compounds self-amplifying preproglucagon / preproGIP (native), self-amplifying preproglucagonA99G / preproGIP A45G mRNA (modified) and comparators TRIS Buffer control and LNP. B. Total AUC of the glucose excursion (120 minutes). Data presented as mean ± SEM (n=4-5 per group).

[0156] Figure 7 Circulating hormone levels 3 hours after a single IP injection of preproGIP mRNA (native) in B6 mice compared to TRIS Buffer Control administered mice: A) GIP B) Glucagon C) GLP-1. Data as mean ± SEM (n=4-5 per group). Students 2 tail t-test: *** p<0.01 vs TRIS Buffer control; * p<0.05 vs TRIS Buffer control. KEY TO SEQUENCE LISTING

[0157] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0158] General Techniques and Definitions

[0159] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, molecular biology, protein chemistry and biochemistry).

[0160] Unless otherwise indicated, the recombinant polynucleotide, polypeptide, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as Perbal (1984), Sambrook (1989), Brown (1991), Glover and Hames (1995 and 1996), Ausubel et al. (1988) and Coligan et al. (including all updates until present). The term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0161] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0162] The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the invention. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10%, more preferably 5%, more preferably 1%, of the particular term.

[0163] Incretin axis

[0164] Glucose metabolism is tightly regulated by numerous pathways. The incretin pathway (also referred to as the incretin axis) is one such key regulator of glucose metabolism and includes gastric inhibitory peptide (GIP) and a number of proglucagon derived peptides including glucagon, glucagon-like peptide-I (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM). In particular, preproglucagon and preproGIP, which are large prohormones that undergo cleavage intracellularly, produce individual hormones including GLP-1, GLP-2, glucagon, intervening peptide 1 (IP1), intervening peptide 2 (IP2), glicentin-related pancreatic polypeptide (GRPP; from preproglucagon) and GIP (from preproGIP). These are then secreted in their cleaved form the cell to exert their effects. Release of GLP-1 and GIP from specific cells within the small intestine in response to nutrients stimulates the release of insulin from pancreatic beta-cells to lower blood glucose levels and suppress glucagon release (which acts to increase glucose from stored glycogen breakdown). These incretins also have an appetite suppressing effect by slowing down gastric emptying to make an individual feel fuller for longer.

[0165] GLP-1 and GIP are pre-pro hormones produce specific prohormones that are cleaved further by prohormone convertases (Pcsks) to become functional hormones. GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of preproglucagon. The precursor gene for GLP-1 production, known as preproglucagon, is specifically expressed in the L-cells of the distal and proximal gut, colon, brain, and pancreatic a-cells. Within L-cells and the brain, preproglucagon encodes for proglucagon which is further processed by Pcsk 1 / 3 to form GLP-1, GLP-2, oxyntomodulin, glicentin, and IP2. Conversely, in a-cells, Pcsk 2 cleavage of proglucagon is responsible for producing glucagon, glicentin-related pancreatic polypeptide, intervening peptide 1, and a significant proglucagon fragment (GLP-1, IP2 and GLP-2 connected).

[0166] GIP, also known as a glucose-dependent insulinotropic peptide, is generated from the 153 amino acid precursor molecule pro-GIP in the K-cells of the duodenum, upper jejunum and lower gut (itself produced from preproGIP after removal of a signal peptide), through the actions of Pcskl / 3, which liberates the full-length GIP that circulates as a biologically active 42 amino acid peptide. After release, it is cleaved further by DPP-4 to generate GIP.

[0167] It is envisaged herein that the delivery of compositions comprising nucleic acids encoding at least one of pre-pro-gastric inhibitory peptide (preproGIP) and at least one of preproglucagon leads to the generation of active forms of GIP and GLP-1 as well as other incretins expressed in other organs or tissues which in turn provides for the beneficial effects described herein including regulation of blood glucose levels, weight lowering effects and / or lipid metabolism. The invention described herein differs from the use of GLP-1 or GIP peptides which are the basis for the design of peptide drugs. By using mRNA, the preprohormones (such as preproGIP and preproglucagon) are expressed intracellularly and can give rise to multiple hormones such as GLP-1, GLP-2, glucagon, and GIP. Without being bound by theory, the effects of the compositions and nucleic acids described herein can be due to other breakdown products, not just GLP-1 (e.g., from preproglucagon) or GIP (e.g., from preproGIP). It is envisaged that the breakdown products are cell-type dependent and therefore there are different hormones being produced in the gut, pancreas or liver.

[0168] Most biologically active peptide hormones are made as larger precursor forms called prohormones. Prohormones act as an inactive storage form of peptide hormones. The conversion of prohormones to active peptide hormones is a mechanism which helps regulate the levels of the hormone. Prohormones are broken down by the prohormone convertases (PCs) to release the hormones. Processing by several post-translational modifications enables the full potential biological activity of the prohormone. These modifications include cleavage by enzymes and modifications to their structure. Multiple forms of biologically active peptides are produced by the actions of these enzymes.

[0169] Without being bound by theory, by administering a preprohormone, the body can act to finely tune the efficient production of active hormones and counter potential dysregulation of the incretin pathway. This will likely amplify increased formation of appropriate and correct sequence of the active hormones the body needs at the time of a meal. The administration of preprohormones is therefore associated with advantages including making the therapy more personalised i.e., providing the precursor of the natural peptides, leading to amplified production of the natural peptides that would be produced by that person at the time of the meal. It is likely that the cleavage of the preprohormone is slightly different for each individual, leading to slightly different amounts of the active hormones that occur at the time of a meal for a person. Providing the preprohormone could also reduce potential side effects because it will allow a more amplified natural response, as opposed to providing simply a single protein which is only one of the end products. Providing preprohormones therefore has the potential to lead to a more personalized approach to incretin therapy for the treatment of a metabolic disorder and / or obesity described herein.

[0170] Without being bound by theory, it is expected that using mRNA technologies to target pre- pro hormones could offer a multifaceted approach by regulating glucose metabolism, reducing body weight, and having other metabolic effects, and exerting diverse effects on various organs throughout the body including modulation of gastric emptying and gastrointestinal motility, stimulation of lipolysis, improving metabolic dysfunction associated fatty liver disease, lung protection, promotion of satiety, renal protection, cerebroprotection, prevention of fat accumulation, promotion of bone formation, improving cardiovascular disease, and evident cardioprotective and vascular effects.

[0171] In some embodiments, a mRNA described herein can further comprise a heterologous moiety or modification. As used herein, the term heterologous moiety refers to a sequence that may be directly or indirectly linked to a nucleic acid or mRNA described herein for the purpose of increasing or improving a functional parameter of the nucleic acid or mRNA. In some embodiments, the heterologous moiety acts to provide one or more or all of tissue specificity, increased solubility and / or half-life, increased affinity, increased activity, increased stability or resistance to protease cleavage. Examples of heterologous moieties are described herein. The term modification is known in the art and encompasses mutations such as substitutions, additions and deletions. For example, activity can be increased by engineering nucleic acids such that naturally occurring serine is substituted with glutamic acid or with another negatively charged amino acid having a side chain with a length of 4 atoms, or alternatively with glutamine.

[0172] In one example, as GIP and GLP-1, respectively produced from pre-pro-gastric inhibitory peptide and preproglucagon, bind to their cognate receptors GIP-R and GLP-1R, which are predominantly located on beta cells in the pancreas, mRNA of the invention may be targeted to the beta cells in the pancreas or intestines. In one example, mRNA of the invention may be targeted to the beta cells in the pancreas or to cells of the intestine or liver by conjugating the LNP with nanobodies, or antibody fragments that bind to cell-specific target molecules. Such mechanisms may act to advantageously prevent off target effects in other tissues or cells.

[0173] In one embodiment, solubility and / or half-life is increased by a mechanism described herein. In addition to improving solubility and / or half-life, it is envisaged that the addition of a hydrophilic moiety may also act to prolong half-life in the circulation. As used herein, the term "hydrophilic moiety" refers to any compound that is readily water-soluble or readily absorbs water, and which are tolerated in vivo by mammalian species without toxic effects (i.e. are biocompatible). Examples are described herein. The determination of increased solubility and / or half-life can be determined by methods known in the art including ELISA assay to detect expression of a protein encoded from a mRNA described herein.

[0174] In some embodiments, a polypeptide encoded by a modified mRNA or comprising a heterologous moiety of the invention binds to a GLP-1 receptor or GIP receptor with higher affinity than the affinity of a polypeptide encoded by an unmodified mRNA or not having a heterologous moiety. In another embodiment, a GLP-1 polypeptide encoded by a modified mRNA of the invention binds to a GLP-1 receptor (GLP-1R) with higher affinity than the affinity of a GLP-1 polypeptide encoded by an unmodified mRNA that encodes an amino acid sequence such as that set forth in SEQ ID NO: 3. In another embodiment, a GIP polypeptide encoded by a modified mRNA of the invention binds to a GIP receptor (GIP-R) with higher affinity than the affinity of a GIP polypeptide encoded by an unmodified mRNA that encodes an amino acid sequence such as that set forth in SEQ ID NO: 8. The determination of increased affinity of the encoded polypeptide can be determined by methods known in the art including surface plasmon resonance.

[0175] Where it is envisaged that peptides encoded by the mRNA of the invention provide for increased activity, this may be achieved through amino acid modification with various negatively charged amino acids. Activity of peptides encoded by the mRNA of the disclosure may be assessed in vivo, through assessment by oral glucose test for example, or in vitro, by ability to stimulate cAMP synthesis in a validated in vitro model assay. As used herein, activity is understood to include activity for a cognate receptor. For example, a GIP polypeptide encoded by a modified mRNA of the disclosure may have increased activity once bound to its cognate GIP-R, by for example, by increasing the initiation of G protein signalling, including the stimulation of adenylate cyclase (AC), the second messenger cAMP and downstream protein kinase A (PKA).

[0176] Where it is envisaged that a heterologous moiety or modification provide for enhanced stability, it is envisaged that the term stability may encompass increased stability in vivo or in vitro, for example reduced precipitation or aggregation and / or degradation. For example, to improve stability in vivo, the mRNA sequence may be codon optimized as described herein. Modifying the mRNA structural elements particularly the 5' cap, 5 '-and 3 '-untranslated regions (UTRs), the coding region, and polyadenylation tail help reduce the excessive mRNA immunogenicity and consistently improve its intracellular stability and translational efficiency. Examples of such modifications are described herein. Enhanced stability in vivo may be determined by analysis of polypeptide translation as a function of time, typically by analyzing the incretin concentration in the blood circulation, by ELISA, after administration of the formulated mRNA product.

[0177] Where it is envisaged that peptides encoded by the mRNA of the invention provide for increased resistance to proteolytic cleavage, for example by dipeptidyl peptidase 4 (DPP -4), it is envisaged that amino acids intended for cleavage can be substituted with an alternative amino acid selected from the group of natural amino acids. For example, the preproGIP described herein may carry an amino acid modification selected from A45V, A45T, and A45G, which prolongs half-life of GIP due to reduced DPP -4 degradation. Increased resistance to cleavage may be determined by techniques known in the art including surface plasmon resonance.

[0178] In some embodiments, the preproGIP encoded by a self-amplifying mRNA may comprise an amino acid modification selected from A45V, A45T, and A45G. In one embodiment, the amino acid modification is A45G. In some embodiments, the preproglucagon encoded by a selfamplifying mRNA may comprise an amino acid modification selected from A99V, A99T, and A99G. In one embodiment, the amino acid modification is A99G.

[0179] In one embodiment, there is provided a composition comprising a self-amplifying mRNA encoding preproGIP and a self-amplifying mRNA encoding a preproglucagon, where the preproGIP encoded by the self-amplifying mRNA comprises an A45G modification, and the preproglucagon encoded by the self-amplifying mRNA comprises an A99G modification.

[0180] In one example, a suitable mRNA of the invention may encode a chimeric polypeptide. Suitable chimeric polypeptides may comprise various domains from components of the incretin axis as understood by a skilled person in the art. For example, a suitable chimeric polypeptide comprises a sequence encoding a GLP-1 and a preproglucagon leader sequence set forth in SEQ ID NO: 4.

[0181] Lipid nanoparticles

[0182] In an embodiment of the invention, a nucleic acid, preferably a mRNA of the invention may be formulated in a liposome, lipoplex or a lipid nanoparticle (LNP), so as to increase stability and transfection of the mRNA. Liposomes are artificially-prepared vesicles which are primarily composed of a lipid bilayer and may be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposomes, lipoplexes or lipid nanoparticles may include, but are not limited to, opsonins or ligands in order to improve the attachment of liposomes, lipoplex or lipid nanoparticles to different tissues or to activate events such as, but not limited to, endocytosis and transcytosis. Liposomes, lipoplexes or lipid nanoparticles may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. The formation of liposomes, lipoplexes or lipid nanoparticles may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposome, lipoplex or lipid nanoparticle products.

[0183] Preferably, an mRNA of the invention is formulated in a LNP. LNPs are composed primarily of ionizable along with other lipid ingredients. These typically include “helper lipids” such as neutral phospholipid molecules belonging to the phosphatidylcholine (PC) or phosphoethanolamine (PE) class, or charged lipids such as DOTAP or DOTMA, and sterols, such as cholesterol. As used herein, the term “helper lipid” is a term understood in the art to be a lipid that increases one or more or all of particle stability, fluidity, tissue targeting specificity, and / or transfection efficiency. Another common lipid ingredient is known as a PEGylated phospholipid - a polyethylene glycol (PEG) polymer covalently attached to the head-group of a phospholipid.

[0184] As used herein, the term lipid nanoparticle (LNP) shall therefore be understood to refer to lipid-based particles having at least one dimension in the order of nanometers (e.g., 1- 1,000 nm) and which typically comprise a nucleic acid as described herein. In embodiments, LNPs are formulated in a composition for delivery of a nucleic acid to a desired target. For example, the LNP may be any lipid composition, including, but not limited to, liposomes or vesicles, where an aqueous volume is encapsulated by amphipathic lipid bilayers (e.g., single; unilamellar or multiple; multilamellar), micelle-like lipid nanoparticles having a non-aqueous core and solid lipid nanoparticles, wherein solid lipid nanoparticles lack lipid bilayers.

[0185] Whilst liposomes include one or more rings of lipid bilayer surrounding an aqueous pocket, not all LNPs have a contiguous bilayer like liposomes. Instead, it is understood that some LNPs assume a micelle-like structure, encapsulating drug molecules in a non-aqueous core. Suitable cationic lipids may include those described in the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. W02012040184, WO201 1153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373 and WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836. Other suitable cationic lipids, non-cationic lipids, PEG lipids and structural lipids, and suitable ratios thereof include those disclosed in WO 2015164674 and WO 2013090648.

[0186] Exemplary LNP compositions and methods of making same are described, for example, in Semple et al., Nature Biotechnology, 28: 172-176 (2010); and Jayarama et al., Angew Chem Int Ed Engl 51(34): 8529-33 (2012). Alternatively, the LNP formulation may be formulated by the methods described in WO201 1 127255 or W02008103276.

[0187] Further, the particle size of the lipid nanoparticle may be increased and / or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the nucleic acid delivered to a given subject.

[0188] LNPs suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein. The lipids can have an anionic, cationic or zwitterionic hydrophilic head group. In one embodiment, the lipid nanoparticle comprises a PEG-lipid, a sterol structural lipid and / or a neutral lipid.

[0189] In an embodiment, the LNP encapsulates the nucleic acid. In another embodiment, the nucleic acid is bound to the LNP. In another embodiment, the nucleic acid is adsorbed on to the LNP.

[0190] In an embodiment, the LNP comprises an ionizable lipid, a phospholipid, sterol, and PEG- lipid. In an embodiment, the molar ratio of ionizable lipid, a phospholipid, sterol, and PEG-lipid is 50: 10:38.5: 1.5. In an embodiment, the molar ratio of ionizable lipid, a phospholipid, sterol, and PEG-lipid is selected from the group consisting of 60:5: 10:25, 55:30:45:0.2, 52:8:38.5: 1.5, 52:8:37:3, 50:20:23.5:6.5 50: 10.5:38: 1.5, 50: 12.5:35:2.5, 45: 13:39.5:2.5, 35: 16:46.5:2.5, 35:40:22.5:2.5, 26.5:20:52: 1.5, 25:30:30: 1, 40:10:38.5: 1.5, 30: 10:38.5:1.5, 40: 10:38.5: 1.5, 60: 10:38.5: 1.5, 70: 10:38.5: 1.5, 50:5:38.5: 1.5, 50: 15:38.5: 1.5, 50:20:38.5: 1.5, 50:25:38.5: 1.5, 50: 10: 18.5: 1.5, 50: 10:28.5: 1.5, 50: 10:48.5: 1.5, 50: 10:58.5: 1.5, 50: 10:38.5:0.5, 50: 10:38.5: 1.0, 50: 10:38.5:2.0, 50: 10:38.5:2.5 or any combination thereof.

[0191] In an embodiment, the lipid nanoparticle comprises 20-60 mol % (e.g., 20-30 mol %, 20- 40 mol %, 20-50 mol %, 20-60 mol %, 30-40 mol %, 30-50 mol %, 30-60 mol %, 40-50 mol %, 40-60 mol %, 45-55 mol %, or 45-50 mol % or 50-60 mol%) ionizable lipid; 5-25 mol % (e.g., 5- 10 mol %, 5-15 mol %, 5-20 mol %, 5-25 mol %, or 10-15 mol %, 10-20 mol %, 10-25 mol %, 15-20 mol %, 15-25 mol %) phospholipid; 25-55 mol % (e.g., 25-35 mol %, 25-45 mol %, 25-55 mol %, 35-45 mol %, 35-55 mol % or 45-55 mol %) sterol; and 0.5-15 mol % (e.g., 0.5-5 mol %, 0.5-10 mol %, or 0.5-15 mol %, 2.5-5 mol %, 2.5-10 mol %, 2.5-15 mol %, 5-10 mol %, 5-15 mol %) PEG-lipid.

[0192] In an embodiment, the ionizable lipid is selected from the group consisting of 9- Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), DLin-MC3-DMA (MC3), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2- di oleyloxy- N,Ndimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethyl-3 -aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-l-yloxyl)benzyl-4-(dimethylamino)butnoate (LKY750), 4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate (ALC0315), C12-200, 306- O12B, 4A3-SC8, cKK-E12, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Preferably the ionizable lipid is SM-102 or MC3.

[0193] In an embodiment, the ionizable lipid is a cationic lipid. In this embodiment, the cationic lipid is selected from the group consisting of l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-dioleoyl-s-glycero-3 -ethylphosphocholine (EPC), dimethyldioctadecylammonium bromide (DDAB), and l,2-di-O-octadecenyl-3 -trimethylammonium -propane (DOTMA).

[0194] In an embodiment, the phospholipid is selected from the group consisting of 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-3 -trimethylammonium -propane (DOTAP),

[0195] 1.2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl- sn- glycero-3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine,

[0196] 1.2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin and combinations thereof. Preferably, the phospholipid is DSPC.

[0197] In an embodiment, the sterol is selected from the group consisting of B-sitosterol, cholesterol, 24(S)-hydroxycholesterol, 20a-hydroxycholesterol, cholesterol oleate, other cholesterol esters, fecosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol and combinations thereof. Preferably, the sterol is cholesterol.

[0198] In another embodiment, the PEG-lipid is selected from the group consisting of PEG2000- c-DMG, PEG2000-DMG, PEG2000-DLPE, PEG2000-DMPE, PEG2000-DPPC, a PEG2000- DSPE lipid and combinations thereof. Preferably, the PEG2000-lipid is PEG2000-DMG. In another embodiment, the PEG-lipid may be a different molecular weight as whose suitability is determined by a skilled person in the art. For example, the PEG-lipid may have a molecular weight of 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600 or 4000.

[0199] In an embodiment, the LNP further comprises one or more additional helper lipids. For example, the LNP may comprise an additional permanently cationic or anionic lipid described herein or known in the art, or another lipid component.

[0200] In one example, the pharmaceutical composition of the present disclosure further comprises a polymeric microparticle.

[0201] The skilled person will be aware that various polymers can form microparticles to encapsulate or adsorb the nucleic acid of the present disclosure. It will be apparent that use of a substantially non-toxic polymer means that particles are safe, and the use of a biodegradable polymer means that the particles can be utilised after delivery to avoid long-term persistence. Useful polymers are also utilised to assist in the preparation of pharmaceutical grade formulations.

[0202] Exemplary non-toxic and biodegradable polymers include, but are not limited to, polyphydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl- pyrrolidinones or polyesteramides, and combinations thereof.

[0203] In an embodiment, the pharmaceutical composition of the present disclosure further comprises an oil-in-water cationic emulsion. Suitable oils for use in an oil-in-water emulsion will be apparent to the skilled person and / or are described herein. For example, the emulsion comprises one or more oils derived, for example, from an animal (e.g., fish) or a vegetable source (e.g., nuts, seeds, grains). The skilled person will understand that biocompatible and biodegradable oils are preferentially used. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oils, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, com oil. In addition to the oil, the oil-in-water emulsion also comprises a cationic lipid to facilitate formation and stabilisation of the emulsion. Suitable cationic lipids will be apparent to the skilled person and / or are described herein. Exemplary cationic lipids include, but are not limited to, limited to: l,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3’-[N-(N’,N’- Dimethylaminoethane)-carbamoyl] Cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA), l,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane (DMTAP), dipalmitoyl [C16:0]trimethyl ammonium propane (DPTAP) and distearoyltrimethylammonium propane (DSTAP).

[0204] In some embodiments, the oil-in-water emulsion also comprises a non-ionic surfactant and / or a zwitterionic surfactant. The skilled person will be aware of surfactants suitable for use in the present disclosure. Exemplary surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (e.g., polysorbate 20 and polysorbate 80) and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0205] As delivery to particular tissues including the pancreas, intestine and fat tissue is contemplated, the formulation may further comprise one or more components to aid delivery. In an embodiment, there is therefore provided LNP formulations that additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064.

[0206] Nucleic acid cargos

[0207] In an aspect of the invention, there is provided a nucleic acid that encodes pre-pro-gastric inhibitory peptide (preproGIP) and optionally preproglucagon. In a preferred embodiment the nucleic acid is messenger ribonucleic acid (mRNA) but may otherwise be selected from the group consisting of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0208] In another aspect of the invention, there is provided a composition comprising a nucleic acid that encodes a preproGIP and a nucleic acid that encodes a preproglucagon. In a preferred embodiment, the nucleic acid is mRNA, more preferably a self-amplifying mRNA.

[0209] In some embodiments, nucleic acids that encode a combination of components of the incretin axis, for example, preproGIP and preproglucagon, act synergistically to produce a desired effect.

[0210] In some embodiments, a composition comprising a nucleic acid that encodes a preproGIP and a nucleic acid that encodes a preproglucagon, produces a desired synergistic effect such as reduced blood glucose level, reduced body weight, and / or increased lipid metabolism, beyond the level observed with either of the individual components comprised in a composition (e.g., a composition comprising a nucleic acid that encodes preproGIP only or a composition comprising a nucleic acid that encodes preproglucagon only). In some embodiments, the preproGIP encoded by the nucleic acid, preferably a self-amplifying mRNA, comprises an amino acid modification selected from A45V, A45T, and A45G. In one embodiment, the amino acid modification is A45G. In some embodiments, the preproglucagon encoded by the nucleic acid, preferably a selfamplifying mRNA, comprises an amino acid modification selected from A99V, A99T, and A99G. In one embodiment, the amino acid modification is A99G.

[0211] The term “synergistically” or “synergistic” as used herein refers to but is not limited to a synergistic effect encountered when two or more components of the incretin axis such as preproGIP and preproglucagon are combined in a composition, where the combined effect of the two or more components exceeds the sum of the effects achieved from the individual components. The presence of a synergistic effect is determined using methods that will be apparent to those skilled in the art. mRNA is a single-stranded molecule of ribonucleic acid (RNA) that corresponds to the genetic sequence of a gene. mRNA is created during the process of transcription, where an enzyme (RNA polymerase) converts the gene into primary transcript mRNA (also known as pre- mRNA). In accordance with the invention, the term messenger RNA (mRNA) refers to a polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce an encoded polypeptide in vitro, in vivo, in situ or ex vivo, preferably in vivo. The term “mRNA” may be used interchangeably with “mRNA molecule” herein.

[0212] As used herein, the term “mRNA” includes but is not limited to a self-amplifying mRNA, also known as a “self-replicating RNA”, “replicon RNA”, or “SAM”. When introduced into a cell, the self-amplifying mRNA replicates itself, leading to an amplification of the amount of RNA encoding the desired gene product. The self-amplifying mRNA may enhance efficiency of RNA delivery and expression of the encoded gene products. Examples of SAMs include but is not limited to an alphavirus-based SAMs such as Venezuelan equine encephalitis virus, Semliki Forest virus, and Chikungunya virus, Flavivirus based vectors, West Nile virus-based vectors, and synthetic non-viral SAMs.

[0213] In some embodiments, the self-amplifying mRNA may comprise a 5’ m7G(5')ppp(5')(2'OMeA)pU cap, sequence encoding for the non- structural proteins 1-4 (nsPl-4) from the Venezuelan equine encephalitis virus (VEEV) strain TC-83, a subgenomic promoter, followed by the sequence encoding for native preproglucagon, native preproGIP, preproglucagon with an A99G mutation, or preproGIP with an A45G mutation, and a polyA-tail downstream of the gene of interest. The translation of the four nsPs form a replicase protein machinery, which facilitates the amplification of the RNA sequence encoding the gene of interest, such as the preproglucagon and preproGIP. This enables the proteins to be expressed at a higher amount for a longer duration compared to a conventional mRNA.

[0214] In producing a mRNA of the invention, a skilled person would understand that mRNA sequences can be synthesised according to the methods described herein or known in the art including those described in the Example. It is envisaged that the mRNA sequence may be optimised by in vitro transcription from a DNA template. In an embodiment, the mRNA sequence may then be chemically modified to increase mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification. For example, the chemical modification may be a nucleoside modification whereby a base is modified to a uracil (U) or a cytosine but the modification may be to any mRNA base including adenine (A), and guanine (G) if the modification increases mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification.

[0215] In an embodiment, the “corresponding mRNA” may be a mRNA without the chemical and / or nucleoside modification which may also be referred to as a wild-type mRNA. A “wildtype mRNA” refers to any mRNA wild-type gene that is capable of having normal (level of function absent disease or disorder) biological activity when expressed as a protein in vivo.

[0216] In an embodiment, the coding region of the mRNA molecule or the region that encodes a polypeptide may comprise from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 1000 to about 1200, from about 1200 to about 1400, from about 1400 to about

[0217] 1600, from about 1600 to about 1800, from about 1800 to about 2000, from about 2000 to about

[0218] 2200, from about 2200 to about 2400, from about 2400 to about 2600, from about 2600 to about

[0219] 2800, from about 2800 to about 3000, from about 3000 to about 3200, from about 3200 to about

[0220] 3400, from about 3400 to about 3600, from about 3600 to about 3800, from about 3800 to about

[0221] 4000, from about 4000 to about 4200, from about 4200 to about 4400 or more nucleotides or bases.

[0222] It is understood that the basic components of an mRNA molecule include at least a 5' cap, a 5’UTR, a coding region, a 3’UTR and a poly -A tail (in 5’ to 3’ direction). The mRNA may also comprise an optimised codon and / or a chemical modification. A skilled person will understand that similar optimisation can be made to other mRNA sequences or molecules for the purpose of increasing or restoring protein expression, enhancing mRNA stability, thermal stability or functionality. Such methods may include those described herein or known in the art.

[0223] Untranslated regions 5’UTRs are regions of a gene that are transcribed but not translated. In mRNA, the 5’UTR starts at the transcription start site and continues to the start codon but does not include the start 5 codon; whereas the 3’UTR starts immediately following the stop codon and continues until the end of the transcript. 5’UTRs play a role in stability and translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(NG)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5’UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0224] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of mRNA of the invention. For example, introduction of 5’ UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein NB / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a mRNA, in hepatocytes or other liver cells. Likewise, use of 5’ UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-I, CD36), for myeloid cells (C / EBP, AMLI, G-CSF, GM-CSF, CDllb, MSR, Fr-1, i- NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-NB / CID). Other 5’UTRs are well known in the art. An exemplary 5’UTR useful in the present invention is the sequence according to SEQ ID NO: 20.

[0225] Other non-UTR sequences may be incorporated into the 5’ (or 3’ UTR) UTRs. For example, introns or portions of intron sequences, or RNA aptamers may be incorporated into the flanking regions of the polynucleotides, primary constructs or mRNA of the invention. Incorporation of intronic sequences may increase protein production as well as mRNA levels.

[0226] 3’ UTRs are known to have stretches of uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-p. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. C-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Therefore, it is envisaged that HuR specific binding sites may be engineered into the 3' UTR of nucleic acid molecules which will lead to HuR binding and thus, stabilization of the message in vivo.

[0227] Introduction, removal or modification of 3’ UTR AU rich elements (AREs) can be used to modulate the stability of mRNA of the invention. Although less preferable, when engineering specific mRNA, one or more copies of an ARE can be introduced to make mRNA of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using mRNA of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection. Suitable 3’UTRs are well known in the art. An exemplary 3 ’UTR useful in the present invention is the sequence according to SEQ ID NO: 21.

[0228] 5 ’Capping

[0229] The 5’ cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5’ proximal intrans removal during mRNA splicing.

[0230] Endogenous mRNA molecules may be 5’-end capped generating a 5 ’-ppp-5 ’-triphosphate linkage between a terminal guanosine cap residue and the 5 ’-terminal transcribed sense nucleotide of the mRNA molecule. This 5 ’-guanylate cap may then be methylated to generate an NY-methyl- guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5’ end of the mRNA may optionally also be 2 ’-O-m ethylated. 5’-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0231] Modifications to the mRNA of the present invention may generate a non-hydrolysable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 ’-ppp-5’ phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with p-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5 ’-ppp-5’ cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0232] Additional modifications include, but are not limited to, 2’-O-methylation of the ribose sugars of 5’-terminal and / or 5’-anteterminal nucleotides of the mRNA on the 2’-hydroxyl group of the sugar ring. Multiple distinct 5’-cap structures can be used to generate the 5’-cap of a nucleic acid molecule, such as an mRNA molecule.

[0233] Cap analogs, or synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5 ’-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non- enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule. For example, the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5’-5’- triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3’-O-methyl group (i.e., N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G-3’mppp-G; which may equivalently be designated 3’ O-Me-m7G(5’)ppp(5’)G). The 3’-0 atom of the other, unmodified, guanine becomes linked to the 5 ’-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA). The N7- and 3’-O-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA). Another cap is mCAP, which is similar to ARCA but has a 2’-O-methyl group on guanosine (i.e., N7,2’-O- dimethyl-guanosine-5’triphosphate-5 guanosine, m7Gm-ppp-G).

[0234] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 ’-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability. mRNA of the invention may also be capped post-transcriptionally, using enzymes, in order to generate a cap representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 ’-cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’ endonucleases and / or reduced 5 ’decapping, as compared to synthetic 5 ’cap structures known in the art (or to a wild-type, natural or physiological 5 ’cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2’-O-methyltransferase enzyme can create a canonical 5’-5’triphosphate linkage between the 5’-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5’-terminal nucleotide of the mRNA contains a 2’-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 ’cap analog structures known in the art. Cap structures include, but are not limited to 7mG(5’)ppp(5’)N,pN2p (cap 0), 7mG(5’)ppp(5’)NlmpNp (cap 1), and 7mG(5’)ppp(5’)NlmpN2mp (cap 2).

[0235] Because the mRNA may be capped post-transcriptionally, and because this process is more efficient, it is preferred that the mRNA be capped. 5’ terminal caps may include endogenous caps or cap analogs. A 5’ terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2’fluoro-O guanosine, 7-deaza-guanosine, 8- oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine.

[0236] A suitable non-limiting example of a cap structure that is useful in the invention is a Capl structure, preferably the sequence as set forth in the sequence, AG. A skilled person will be able to determine other suitable cap structures useful in the invention.

[0237] IRES Sequences

[0238] Further provided are mRNA which may contain an internal ribosome entry site (IRES). IRES plays an important role in initiating protein synthesis in absence of the 5’ cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When mRNA are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picomaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0239] Poly- A tails

[0240] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3’ end of the transcript may be cleaved to free a 3’ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 100 and 250 residues long. Generally, the length of a poly-A tail of the present invention is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300,350,400,450,500,600,700,800,900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polynucleotide or mRNA includes a poly-A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0241] In another embodiment, the poly A tail comprises between 25 to 200 nucleotides, between 50 to 150 nucleotides, between 80 to 120 nucleotides, preferably about 100 nucleotides. An exemplary poly A sequence useful in the present invention is the sequence according to SEQ ID NO: 22.

[0242] In one embodiment, the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions) or based on the length of the ultimate product expressed from the mRNA. In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotides or mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of mRNA for Poly-A binding protein may enhance expression.

[0243] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3 ’-end using modified nucleotides at the 3 ’-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr and day 7 post-transfection.

[0244] In one embodiment, a mRNA of the present invention may be designed to include a polyA- G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant mRNA construct is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.

[0245] Codon optimisation

[0246] A nucleotide sequence of a mRNA useful for the invention may be codon optimised. In performing codon optimisation, codon frequencies in target and host organisms are generally matched to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post translation modification sites in encoded protein (e.g., glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the mRNA.

[0247] Codon composition is known to affect translation efficiency. Replacing rare codons with synonymous frequent codons improves translational yield because reuse of the same tRNA accelerates translation owing to amino-acylation of tRNAs in the vicinity of the ribosomes. Codon context (that is, neighboring nucleotides and codons) also affects the translational elongation rate and translational efficiency. Similar to recombinant DNA-based approaches, codon-optimised in vitro transcribed (IVT) mRNAs have been successfully used. However, in some cases, there may be valid reasons to refrain from using optimised codons as understood by a skilled person. Some proteins require slow translation, which is ensured by rare codons, for their proper folding. It may also be beneficial for some IVT mRNA-encoded to maintain the original ORF.

[0248] Codon optimisation methods are useful to increase expression, structural stability, thermal stability or increased function of the encoded protein. Codon optimisation tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) or other methods known in the art. However, specific strategies for codon optimisation vary considerably based on underlying assumptions about which codon features are important to translation. One approach involves substituting in the most frequently used codon for all instances of a given amino acid. Another approach involves only replacing rare codons with more abundant synonymous codons. Still other approaches involve adjusting the codon usage frequency to match the natural frequencies in a host organism, or choosing codons based on cognate transfer RNA (tRNA) abundance. A skilled person will generally understand that codon optimisation involves the replacement of a codon with an optimised codon that is synonymous with the replaced codon. In one embodiment, the ORF sequence is optimised using optimisation algorithms. In a preferred embodiment, codon optimisation is conducted by enriching a DNA template that will encode a mRNA of the invention for guanine (G) and cytosine content. Even more preferably, Gand C content is enriched by substituting codons with adenine (A) or uracil (U) at the third base with codons enriched in guanine (G) or cytosine (C). A skilled person will understand methods for determining suitable nucleotide substitutions based on codon options for each amino acid as they are known in the art.

[0249] Whilst preferable, codon optimised mRNA need not be uniformly codon optimised along the entire length of the mRNA molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the modification(s) may be located at any position(s) of a mRNA such that the polypeptide function, polypeptide expression, mRNA thermal stability or structure is preferably increased or improved. A modification may also be a 5’ or 3’ terminal modification. Further, the mRNA may contain at a minimum one and at maximum 100% optimised codons, or any intervening percentage, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% optimised codons.

[0250] The % identity of a nucleic acid or mRNA is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 900 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 900 nucleotides. Preferably, the query sequence is at least 975 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 975 nucleotides. Even more preferably, the query sequence is at least 1,050 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 1,050 nucleotides. Even more preferably, the GAP analysis aligns two sequences over their entire length.

[0251] With regard to the defined nucleic acid or mRNA, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the mRNA comprises a sequence which is at least 50%, at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9 identical to the relevant sequence.

[0252] In a further embodiment, the present invention relates to nucleic acids or mRNA which are substantially identical or identical to those specifically described herein. As used herein, with reference to a mRNA or polynucleotide the term “substantially identical” means the substitution of one or a few (for example 2, 3, or 4) nucleotides whilst maintaining activity of the native protein encoded by the polynucleotide. In addition, this term includes the addition or deletion of nucleotides which results in the increase or decrease in size of the encoded native protein by one or a few (for example 2, 3, or 4) amino acids whilst maintaining activity of the native protein encoded by the polynucleotide.

[0253] Chemical modifications

[0254] The terms “modification” or “modified” refer to modification of a mRNA of the invention with respect to A, G, U or C ribonucleotides. Generally, the modification refers to the coding region of the mRNA. However, the modification may also be introduced into the flanking regions and / or the terminal regions if the modification increases protein expression, function, thermal stability or structure.

[0255] Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

[0256] The mRNA or nucleic acids can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage to the phosphodiester backbone). For example, the major groove of a polynucleotide, or the major groove face of a nucleobase may comprise one or more modifications. One or more atoms of a pyrimidine nucleobase (e.g. on the major groove face) may he replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain cases (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., the substitution of the 2’ OH of the ribofuranysyl ring to 2’H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are known in the art. In some embodiments, the chemical modification increases mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification. Preferably the modification is to a uracil (U) or a cytosine (C) but may be to any mRNA base including adenine (A), and guanine (G) if the modification increases mRNA stability and / or mRNA translation when compared to a mRNA without chemical modification.

[0257] Suitable uridine modifications may include but are not limited to pseudouridine (y), 5-aza- uridine, 6-aza-uridine, 2-thio-5-zauridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4- thiopseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo- uridine (e.g., 5 -iodo-uridine or 5 -bromouridine), 3 -methyluridine (m3U), 5-methoxyuridine (5moU), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5- carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-20 methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio- uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno- uridine (mnm5se2U), 5 -carbamoyl methyl -uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyluridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (tm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine (tm5s2U), l-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1 -methylpseudouridine (nfy), 5-methyl-2- thiouridine (m5s2U), l-methyl-4-thio-pseudouridine (m4s4y), 4-thio-l-methyl-pseudouridine, 3- methyl-pseudouridine (m3y), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2- 3 0 thio- 1 -methyl- 1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5 ,6- dihydrouridine, 5-methyldihydrouridine (m5D), 2-thio-dihydrouridine, 2- thiodihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine (also known as 1- methylpseudouridine (nfy), 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino- 3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2’-O-methyl-uridine (Um), 5,2’-O-dimethyl-uridine (m5Um), 2’-O-methyl-pseudouridine (ym), 2-thio-2’-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2’-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2’- Omethyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2’-O-methyluridine (cmnm5Um), 3,2’O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2’-O-methyl-uridine (inm5Um), 1- thio-5 uridine, deoxythymidine, 2’-F-arauridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)]uridine. Suitable cytidine modifications may include but are not limited to 5-aza- cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl- cytidine (PC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-methylcytidine (5mC), 5- halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l- deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5- aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thiozebularine, 2-methoxy- cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine (k2C), 2-thiocytidine, 2’-O-methyl-cytidine (Cm), 5,2’-O-dimethyl- cytidine (m5Cm), N4-acetyl- 2’-O-methyl-cytidine (ac4Cm), N4,2’-O-dimethyl-cytidine (m4Cm), 5-formyl- 2’-O-methyl- cytidine (CCm), N4,N4,2’-O-trimethyl-cytidine (m Cm), 1 -thiocytidine, 2’-F-ara-cytidine, 2’-F- cytidine, and 2’-OH-ara-cytidine.

[0258] Modified nucleic acids need not be uniformly chemically modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the modification(s) may be located at any position(s) of a mRNA such that the polypeptide function, polypeptide expression, mRNA thermal stability or structure is preferably increased or improved. A modification may also be a 5' or 3' terminal modification. Further, the mRNA may contain at a minimum one and at maximum 100% chemical modifications, preferably nucleoside modifications, or any intervening percentage, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% modified nucleotides.

[0259] Methods of synthesis of mRNA mRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT), enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art.

[0260] In a preferred embodiment, a mRNA of the invention is prepared by IVT using methods known in the art. The machinery of the transfected cell is utilized for in vivo translation of the message to the corresponding protein. IVT mRNA is engineered to structurally resemble naturally occurring mature and processed mRNA in the cytoplasm of eukaryotic cells. Hence, the IVT mRNA is single-stranded, has a 5’ cap and a 3’ poly(A) tail. The open reading frame (ORF) encoding the protein of interest is marked by start and stop codons and is flanked by untranslated region (UTRs). The mRNA is generally synthesized in a cell-free system by IVT from a DNA template, such as a linearized plasmid or a PCR product. With the exception of the 5’ cap, this DNA template encodes all the structural elements of a functional mRNA. IVT is performed with T7 or SP6 RNA polymerase in the presence of nucleotides and thereafter the mRNA is capped enzymatically. The template DNA is then digested by DNAses and the mRNA is purified by conventionally used methods for isolating nucleic acids.

[0261] Nucleic acids and vectors

[0262] The present invention relates to various nucleic acids, preferably encoding mRNA, particularly those used in IVT for the generation of a mRNA of the invention. As used herein, a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means a polymer of nucleotides, which may be DNA or RNA or a combination thereof, and includes genomic DNA, mRNA, cRNA, and cDNA. A given polynucleotide may be of cellular, genomic or synthetic origin, for example made on an automated synthesizer, and may be combined with carbohydrate, lipids, protein or other materials, labelled with fluorescent or other groups, or attached to a solid support to perform a particular activity defined herein, or comprise one or more modified nucleotides not found in nature, well known to those skilled in the art. The polymer may be single -stranded, essentially double-stranded or partly double-stranded. Basepairing as used herein refers to standard basepairing between nucleotides, including G:U basepairs. “Complementary” means two polynucleotides are capable of basepairing (hybridizing) along part of their lengths, or along the full length of one or both. The term “polynucleotide” is used interchangeably herein with the term “nucleic acid”.

[0263] Self-amplifying mRNAs may harbor the RDRP genes and mimic the characteristic replicative features of positive-strand RNA viruses. SAMs can be produced by IVT from cDNA templates. The structural genes of the RNA virus are replaced by the heterologous genes of interest, which are controlled by a sub-genomic promoter.

[0264] The polynucleotides or nucleic acid sequences of the present application may be deoxyribonucleic acid (DNA) sequences or ribonucleic acid (RNA) sequences and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences.

[0265] The term "nucleic acid molecule" or its derivatives, as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA For example, it may be useful for the nucleic acid molecules of the disclosure to be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double stranded regions, hybrid molecules comprising DNA and RNA that may be single stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, it may be useful for the nucleic acid molecules to be composed of triple stranded regions comprising RNA or DNA or both RNA and DNA The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” encompasses chemically, enzymatically, or metabolically modified forms. The term “polynucleotide” shall have a corresponding meaning.

[0266] The term "isolated polynucleotide" or “isolated nucleic acid” means a polynucleotide or nucleic acid which has generally been separated from the polynucleotide sequences with which it is associated or linked in its native state if the polynucleotide is found in nature. Preferably, the isolated polynucleotide is at least 90% free from other components with which it is naturally associated, if it is found in nature. Preferably, the polynucleotide is not naturally occurring, for example by covalently joining two shorter polynucleotide sequences in a manner not found in nature (chimeric polynucleotide).

[0267] A genomic form or clone of a gene containing the transcribed region may be interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences”, which may be either homologous or heterologous with respect to the “exons” of the gene. An “intron” as used herein is a segment of a gene which is transcribed as part of a primary RNA transcript but is not present in the mature mRNA molecule. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA). Introns may contain regulatory elements such as enhancers. “Exons” as used herein refer to the DNA regions corresponding to the RNA sequences which are present in the mature mRNA or the mature RNA molecule in cases where the RNA molecule is not translated. An mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term “gene” includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.

[0268] As used herein, a “chimeric gene” refers to any gene that comprises covalently joined sequences that are not found joined in nature. Typically, a chimeric gene comprises regulatory and transcribed or protein coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. The term “endogenous” is used herein to refer to a mRNA that is normally present in a mammalian cell and refers to a native gene in its natural location in the genome of an organism. It may also be referred to as wild-type. As used herein, “recombinant nucleic acid molecule”, “recombinant polynucleotide” or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA / RNA technology. The terms “foreign polynucleotide” or “exogenous polynucleotide” or “heterologous polynucleotide” and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulations.

[0269] Foreign or exogenous genes may be genes that are inserted into a non-native organism or cell, native genes introduced into a new location within the native host, or chimeric genes. Alternatively, foreign or exogenous genes may be the result of editing the genome of the organism or cell, or progeny derived therefrom. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0270] The term "genetically engineered", "genetically modified”, “genetic modification” or variants thereof refers to any genetic manipulation by man and includes introducing genes into cells by transformation or transduction, gene editing, cisgenesis, mutating genes in cells and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny and so on.

[0271] Furthermore, the term “exogenous” in the context of a polynucleotide (nucleic acid) refers to the polynucleotide when present in a cell that does not naturally comprise the polynucleotide. The cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide, for example an exogenous polynucleotide which increases the expression of an endogenous polypeptide, or a cell which in its native state does not produce the polypeptide. Increased production of a polypeptide is also referred to as “overexpression”. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and / or synthetic) joined to form a single polynucleotide. Typically, such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable for driving transcription of the open reading frame in a cell of interest. The present invention also relates to the use of oligonucleotides, for instance in methods of detecting or screening a mRNA molecule of the invention. As used herein, “oligonucleotides” are polynucleotides up to 50 nucleotides in length. The minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. They can be RNA, DNA, or combinations or derivatives of either. Oligonucleotides are typically relatively short single stranded molecules of 10 to 30 nucleotides, commonly 15-25 nucleotides in length. When used as a guide for genome editing, probe or as a primer in an amplification reaction, the minimum size of such an oligonucleotide is the size required for the formation of a stable hybrid between the oligonucleotide and a complementary sequence on a target nucleic acid molecule. Preferably, the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 22 nucleotides, even more preferably at least 25 nucleotides in length. Oligonucleotides of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

[0272] As those skilled in the art would be aware, the sequence of the oligonucleotide primers described herein can be varied to some degree without affecting their usefulness for the methods of the invention. A “variant” of an oligonucleotide disclosed herein (also referred to herein as a “primer” or “probe” depending on its use) useful for the methods of the invention includes molecules of varying sizes of, and / or are capable of hybridising to the genome close to that of, the specific oligonucleotide molecules defined herein. For example, variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region. Furthermore, a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise the target region. In addition, variants may readily be designed which hybridise close (for example, but not limited to, within 50 nucleotides or within 100 nucleotides) to the region of the genome where the specific oligonucleotides defined herein hybridise.

[0273] The present invention includes oligonucleotides that can be used as, for example, guides for RNA-guided endonucleases, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Probes and / or primers can be used to clone homologues of the polynucleotides of the invention from other species. Furthermore, hybridization techniques known in the art can also be used to screen genomic or cDNA libraries for such homologues.

[0274] Nucleic acids and oligonucleotides of the present invention include those which hybridize under stringent conditions to one or more of the sequences disclosed herein. As used herein, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 MNaCl / 0.0015 M sodium citrate / 0.1% NaDodSQ4 at 50°C; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol / vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt’ s solution, sonicated salmon sperm DNA (50 g / ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS.

[0275] Nucleic acids of the present invention may possess, when compared to naturally occurring molecules, one or more modifications which may be a mutation such as a deletion, insertion, or substitution of one or more nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid). A variant of a polynucleotide of the invention includes molecules of varying sizes when compared to the reference polynucleotides defined herein. For example, variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they encode a functional protein. Furthermore, a few nucleotides may be substituted without influencing the integrity of the encoded protein. In addition, variants may include polynucleotides which encode the same polypeptide or amino acid sequence but which vary in nucleotide sequence by redundancy of the genetic code. The terms “polynucleotide variant” and “variant” also include naturally occurring allelic variants.

[0276] Cells

[0277] In an aspect of the invention, there is provided a host cell comprising a nucleic acid of the invention. In an embodiment, the host cell is a mammalian cell. In an embodiment, the host cell is a non-human cell. In an embodiment, the host cell is preferably a human cell but may be any mammalian cell including those from pets (i.e., cats, rabbits, guinea pigs, ferrets, dogs) or livestock animals (cattle, pigs, sheep, goats, chickens, camelids, deer, bison, buffalo and related species including wild and zoo animals). In an embodiment, the human cell is a cell of the liver, stomach, pancreas, duodenumjejunum, ileum, colon or adipose tissue.

[0278] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cells and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Pharmaceutical compositions

[0279] One method of treating a metabolic disease in accordance with the present disclosure comprises the step of administering a composition described herein to a patient using any standard route of administration, including parenterally, such as intravenously, intraperitoneally, subcutaneously or intramuscularly, intrathecally, transdermally, rectally, orally, nasally or by inhalation. In one embodiment the composition is administered subcutaneously or intramuscularly. In one embodiment, the composition is administered parenterally and the incretin-insulin conjugate is prepackaged in a syringe.

[0280] In an aspect of the invention, the compositions is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient. In some examples, a pharmaceutical composition described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically- acceptable carriers, or by any other convenient dosage form. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques. It is envisaged that the pharmaceutical compositions are most suitable for intravenous administration, oral administration or inhalation.

[0281] Methods for preparing a LNP into a suitable form for administration to a subject (e.g. a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington and Gennaro (1990) and U.S. Pharmacopeia: National Formulary (1984).

[0282] The pharmaceutical compositions of this invention are useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ or joint. The compositions for administration will commonly comprise a suspension of LNPs suspended in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., PBS, Tris buffer, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of a nucleic acid of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient’s needs. Exemplary carriers include water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0283] Pharmaceutically acceptable acidic / anionic salts for use in the invention include and are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate and triethiodide. Organic or inorganic acids also include, and are not limited to, hydriodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2- naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharinic or trifluoroacetic acid. Pharmaceutically acceptable basic / cationic salts include, and are not limited to aluminum, 2- amino-2-hydroxymethyl-propane- 1,3 -diol (also known as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”), ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, lysine, magnesium, meglumine, N- methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0284] Upon formulation, a pharmaceutical composition of the present invention will be administered in a manner compatible with the dosage formulation and in such an amount as is therapeutically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, LNP forms and the like. Pharmaceutical “slow release” capsules or compositions may also be used. Slow-release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver a LNP of the present invention.

[0285] Suitable dosages of a pharmaceutical composition of the present invention will vary depending on the specific nucleic acid used and / or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED50 of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0286] In some examples, a method of the present invention comprises administering a therapeutically effective amount of a pharmaceutical composition described herein. The term “therapeutically effective amount” is the quantity which, when administered to a subject in need of treatment, improves the prognosis and / or state of the subject and / or that reduces or inhibits one or more symptoms of a clinical condition described herein to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that condition. The amount to be administered to a subject will depend on the particular characteristics of the condition to be treated, the type and stage of condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of protein(s), rather the present invention encompasses any amount of the nucleic acid sufficient to achieve the stated result in a subject.

[0287] Methods of treatment

[0288] The disclosed compositions are suitable for any use that has previously been described for members of the incretin axis including GLP-1 and GIP, including for the purpose of reducing weight, preventing weight gain, treating or preventing hyperglycemia, or treating other metabolic disorders such as those that are associated with high blood glucose levels. Accordingly, the present invention encompasses pharmaceutical compositions comprising a mRNA disclosed herein and a pharmaceutically acceptable carrier for treating a patient suffering from a metabolic disorder. As used herein, the term “metabolic disorder” is a term understood in the art to refer to a disorder characterized by abnormal chemical reactions in the body and includes disorders such as type 2 diabetes, type 1 diabetes, gestational diabetes, insulin resistance, prediabetes, fatty liver disease (hepatosteatosis) such as metabolic associated fatty liver disease (MAFLD) and non-alcoholic steatohepatitis (NASH), and obesity.

[0289] “MAFLD” is also known in the art as “non-alcoholic fatty liver disease” (NAFLD) and “metabolic (dysfunction)-associated fatty liver disease” (MASLD). In some embodiments, the present invention encompasses pharmaceutical compositions comprising a mRNA disclosed herein and a pharmaceutically acceptable carrier for treating a patient suffering from kidney disease related to diabetes and / or obesity. For example, the kidney disease may be diabetic kidney disease (DKD, also known as diabetic nephropathy), chronic kidney disease (CKD) or end-stage renal disease (ESRD).

[0290] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a metabolic disorder described herein via delivery of a pharmaceutical composition described herein.

[0291] The term “prophylactically effective amount” shall be taken to mean a sufficient quantity of a pharmaceutical composition that prevents or inhibits or delays the onset of one or more detectable symptoms of a clinical condition. The skilled person will be aware that such an amount will vary depending on, for example, the specific nucleic acid formulated in a LNP administered and / or the particular subject and / or the type or severity or level of condition and / or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of nucleic acid or a pharmaceutical composition thereof, rather the present invention encompasses any amount of a pharmaceutical composition thereof sufficient to achieve the stated result in a subject.

[0292] As used herein, a subject “at risk” of developing a metabolic disorder described herein may have or may not have detectable symptoms of the metabolic disorder, and may have or may not have displayed detectable symptoms of a metabolic disorder described herein prior to a treatment according to the present disclosure. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of a metabolic disorder described herein, as known in the art and / or described herein.

[0293] As used herein, “preventing” or “prevention” is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

[0294] Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians. In relation to the present disclosure, prevention of neurological disease or disorder may be achieved where a subject does not have any clinical symptoms of the disease or disorder and may be considered asymptomatic.

[0295] A subject in need of treatment may present a number of symptoms depending on the type of disorder that is present. In an aspect, a subject in need of treatment may exhibit well characterised symptoms associated with a disease or condition described herein. Preferably the subject is a human but may be a mammal described herein or known in the art.

[0296] The term “treatment” or "treating" refers to any indication of success in the treatment or amelioration of a metabolic disorder or other condition described herein, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury. In particular, treating refers to a reduction in a symptoms associated with the disease or condition. Any improvement may be determined directly from the subject, or a sample or biopsy therefrom. The sample or biopsy may be of the diseased tissue.

[0297] In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent such as an anti-diabetic agent. Such additional treatments which may be administered together or separately.

[0298] In an embodiment, the additional therapeutic agent is an anti-obesity agent selected from Bupropion-naltrexone (Contrave), Liraglutide (Saxenda), Orlistat (Xenical, Alli), Phenterminetopiramate (Qsymia), Semaglutide (Wegovy), Retatrutide (LY-3437943), Setmelanotide (Imcivree), Survotutide, Mazdutide, Efinopegdutide, Cagrisema, Dulaglutide, Exenatide, Efpeglenatide, Lixisenatide, Cotadutide, Danuglipron and Orgorglipton.

[0299] In an embodiment, the additional therapeutic agent is an anti-diabetic and / or obesity agent selected from the group consisting of native insulin, native glucagon and functional analogs thereof, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose / Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SOLT (sodium-dependent glucose transporter 1) inhibitors; FBPase (fructose 1,6-bisphosphatase) inhibitors, Tirzepatide (Mounjaro), SAR441255 and semaglutide (Ozempic) or analogues thereof.

[0300] In certain embodiments, an additional therapeutic agent is a therapeutic agent effective to treat the same or a different condition as the composition described herein is being employed to treat. In certain embodiments, the at least one additional therapeutic agent is selected for its ability to mitigate one or more side effects of the composition described herein. The additional therapeutic agent can be administered in the same or separate formulations and administered together or separately with the composition described herein. The composition described herein can be administered prior to, simultaneously with, and / or following, the administration of the additional therapeutic agent and / or adjuvant.

[0301] In an embodiment, the additional therapeutic is mRNA is one or more or all of an analogue of Exenatide, an analogue of Tirzepatide, an analogue of Retatrutide or an analogue of SAR441255 as described herein.

[0302] Where analogues are contemplated, the mRNA may contain substitutions compared to the synthetic peptide product. For example, SEQ ID NO: 15 is an exemplary amino acid sequence of an analogue of Tirzepatide with a secretion signal from SEAP containing the following substitutions:

[0303] 1. Aib2G - Replace Aib with natural amino acid glycine;

[0304] 2. Aibl3G - Replace Aib with natural amino acid glycine; and

[0305] 3. K(C20)21K - Fatty acid chain removed.

[0306] For example, SEQ ID NO: 16 is an exemplary amino acid sequence of an analogue of Retatrutide with a secretion signal from SEAP containing the following substitutions:

[0307] 1. Aib2G - Replace Aib with natural amino acid glycine;

[0308] 2. a-Me-L13L - Replace a-methyl-leucine with natural amino acid leucine;

[0309] 3. K(C20)17I - Fatty acid chain replaced with isoleucine residue from GIP; and

[0310] 4. Aib20Q - Replace Aib with natural amino acid glutamine from glucagon and GIP.

[0311] For example, SEQ ID NO: 17 is an exemplary amino acid sequence of an analogue of SAR441255 with a secretion signal from SEAP containing the following substitutions:

[0312] 1. Aib2G - Replace Aib with natural amino acid glycine.

[0313] 2. K(Glu-Glu-C16)14L - Replace unnatural fatty acid chain with natural amino acid leucine from GLP-1 and glucagon.

[0314] 3. Aib20K - Replace Aib with natural amino acid lysine from GLP-1.

[0315] 4. dAla29G - Replace D-alanine with glycine.

[0316] 5. Aib34G - Replace Aib with natural amino acid glycine. Kits

[0317] In an aspect, the present invention provides a kit comprising one or more of the following:

[0318] (iv) a nucleic acid of the invention;

[0319] (ii) a pharmaceutical composition of the invention.

[0320] In the case of a kit for therapeutic use, the kit can additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.

[0321] Optionally, a kit of the invention is packaged with instructions for use in a method described herein according to any example.

[0322] In another aspect, the application relates to an article of manufacture (such as a kit) containing materials useful for the treatment or prevention a metabolic disorder or other condition described herein. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating or preventing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a nucleic acid. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can include (a) a first container with a composition described herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent. The article of manufacture in this embodiment of the invention can further include a package insert indicating that the compositions can be used to treat a particular condition. Optionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer’s solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. EXAMPLES

[0323] Example 1

[0324] This Example was conducted to determine whether administration of pre-pro Glucagon mRNA (which produces GLP-1) and preproGIP (which produces GIP) can lower glucose levels during an oral glucose tolerance test (OGTT) in wildtype C57B16 / J (B6) mice.

[0325] The studies described herein utilise the body’s natural cellular processes to produce specific hormones, harnessing the body’s own mechanisms to regulate physiological processes like glucose metabolism. In the case of GLP-1 and GIP, the pre-pro hormones produce specific prohormones that are cleaved further by prohormone convertases (Pcsks) to become functional hormones.

[0326] Materials and methods

[0327] 8-week-old male C57BL6 / J (B6) mice (n=5 per treatment group) were weight matched into groups to receive a single dose of one of the following treatments: preproglucagon mRNA (1 Opg) within a LNP, preproGIP mRNA (1 Opg) within a LNP, LNP control (10pg; containing an empty vector) or TRIS buffer control. Mice were intraperitoneally injected with their allocated treatment 4 hours prior to the OGTT with concurrent 4 hour fasting. A baseline blood sample was taken for a glucose measurement (0 minutes) after the 4 hour fast and gavaged with a single bolus of glucose (2g / kg). Glucose levels were measured via a tail snip, with a drop of blood tested on a handheld glucometer (Accu-Chek Instant) at 2, 5, 10, 30, 45, 60 and 120 minutes thereafter.

[0328] Results

[0329] Mice responded favourably in lowering glucose following treatment with the compounds as shown by the glucose excursion graph. Two-Way ANOVA demonstrated a significant effect of treatment on glucose levels (p<0.005) and significant interaction between treatment and time (p<0.001). Post-hoc analysis using Fishers LSD showed statistically significant changes from as early as 5 minutes post oral glucose (Figure 1A), with reductions in glucose seen in the preproglucagon group compared to the pre-pro GIP group (p=0.01) and compared to the LNP control group (p=0.002). The data demonstrates that the greatest changes happened in the latter phase of the glucose excursion curve. The data shows marked reductions in glucose following both the preproglucagon and preproGIP treatment when compared to the TRIS buffer control at 30, 45 and 60 minutes (p<0.05 in all treatment groups (a and b on Figure 1 A)). By 120 minutes, the levels were below baseline, with no significant differences between the treatment groups.

[0330] When the data was calculated as a total AUC (from 0 to 120 minutes), preproGIP maintains a sustained reduction in glucose compared to the TRIS buffer control (p=0.04), with the preproglucagon showing a strong pattern towards significance, yet not statistically significant (p=0.09) (Figure IB). Whilst there are specific timepoint differences with these compounds, the overall effect as shown by the total AUC, would suggest the preproGIP has the more profound effect across the 120-minute test.

[0331] These data demonstrate that the use of mRNA compounds can alter glucose levels in an acute setting (4 hours after administration) and that mice tolerated the compound well. The data suggest that under conditions of high glucose (2g / kg of glucose given to the mice), the compounds are presumably increasing hormone production of both GLP-1 and GIP to reduce glucose in the expectant manner. The data not only shows that there is biological activity following administration of the mRNA compounds in wildtype mice, but that the compounds respond to the high glucose environment and reduce blood glucose levels over the course of the glucose tolerance test.

[0332] On comparison with a short-acting GLP-1 receptor agonist (Exenatide), our historical data (20mg / kg, 1 hour prior to the OGTT), demonstrated a similar glucose lowering (and expectant) profile, but was lower when compared to the profiles seen with the mRNA compounds (at the 10- minute peak) (Figure 1C). This is likely due to Exenatide being a full protein acting at a faster rate compared to the mRNA compounds, which requires more time to produce an active hormone.

[0333] Tissue distribution of nanoluciferase protein expression encoded by mScarlet-I-nLuc mRNA encapsulated in LNPs: In a group of male B6 mice, a single injection of LNP encoding the mScarlet-I-nLuc mRNA was administered IP. At 4 hours post administration, mice were sacrificed for blood and tissue collection (with tissues snap frozen in liquid nitrogen) and assayed for luciferase protein expression. Figure 2 shows that following a single injection, expression was detected across multiple tissues including the liver and pancreas, but importantly in the ileum and colon which are the predominant sites for incretin production. This data confers that the delivery method of the LNPs and the LNPs itself are able to enter tissues of interest.

[0334] Our data shows promising results that targeting and modulating hormone production via mRNA therapies is a valid approach. The increased glucose environment in the wildtype animal suggest that the mRNA therapy can be modulated by excess nutrients and could likely be extended to an animal model with chronically high levels of glucose as found in type 2 diabetes, such as the genetic db / db mouse model that shows all characteristics of type 2 diabetes.

[0335] Example 2 - Acute animal studies: Dose response and optimal route of administration To determine what dose is the most efficacious, an optimisation study will be performed looking at varying doses of mRNA. All routes of administration: IP; subcutaneous; intravenous; and oral delivery will be assessed utilising 5, 10 and 30pg of each of the mRNAs and controls (LNP and TRIS Buffer saline). A 10-fold increase in mRNAs (50, 100 and 300pg) will be delivered orally using Tirzepatide as a benchmark. Human equivalent doses will be calculated as a physiological equivalent. Power calculations based on the total AUC data in example 1 between the pre-pro GIP and TRIS buffer saline, mean ± SD, pre-pro GIP mice: 83.8±134.6 mmol / L vs TRIS buffer saline: 455.6±152 mmol / L). A sample size n=10 mice / group will be used to detect a difference (p<0.05, power=85%). A two-arm glucose analysis study in male wildtype and db / db mouse models will be conducted. The db / db mouse model is a standard model of type 2 diabetes that is used in pre-clinical evaluation of potential drug studies as well as discovery studies and is the most appropriate for this work to be developed.

[0336] ARM 1 will be a timed glucose tracking experiment where the various doses of mRNA and controls will be given via the different routes and blood glucose levels measured at time 0 mins (prior to dosing), 1, 2, 3, 6, 24, and 48 h after dosing. Blood will be collected at each of these timepoints (along with body weight and food intake data) and at the conclusion, a terminal cardiac blood collection taken along with tissue collection (gastrointestinal tract [stomach, duodenum, jejunum, ileum, colon], heart, pancreas, liver, kidney, muscle, white fat [gonadal], brown fat and brain [hypothalamus isolated]). These samples will then be used for pharmacokinetic, and circulating hormone profiling and for expression. Change in blood glucose levels (delta change) and AUC will be determined from the data.

[0337] ARM 2 will measure glucose tolerance in response to administration of these mRNA compounds (all doses and all routes) and follow the same protocol as was performed in the recent Tirzepatide preclinical studies. A 4-hour duration between administration and testing will be used, where mice will also be fasted for this time. Glucose (2g / kg) will be delivered via gavage following a baseline blood glucose sample and then taken at 2, 5, 10, 30, 45, 60 and 120 minutes thereafter. Blood samples will be collected from the tail at intervals across the tolerance test (due to limitations on total volume taken from a mouse at any one time, blood at 0, 5 and 30 minutes) will be taken and glucose measured. An aliquot at these timepoints will also be taken for plasma insulin levels to determine the effects of these mRNAs on insulin secretion. At conclusion, a terminal cardiac blood sample and tissues will be taken for future analysis. Data will be analysed as a delta change in glucose levels and insulin levels, total AUC (0-120 mins), first phase (0-10 mins) and second phase (10-120 mins) AUC.

[0338] Timing of mRNA administration study: Timed experiments will be performed in both wildtype and the db / db mouse model, where the mRNA compounds and the controls will be administered and fasting blood glucose levels (at baseline) and glucose tolerance with the OGTT assessed 2, 6, 10 and 18 hours (overnight fast) after the drug administration, with mice fasted for the same duration.

[0339] Example 3 - Acute animal studies: Pharmacokinetic and Circulating Hormone Studies

[0340] Using blood samples collected from wildtype mice in AIM 1, the following assessments will be performed:

[0341] Aim 2.1. Pharmacokinetic studies: The concentration of incretins in the blood circulation will be assessed, to give an indication of the pharmacokinetics profile of incretins resulting from mRNA administration. Assay will be subject to development of suitable ELISA assays using beadbased multiplex assays (Luminex Magpix reader). This assay procedure will be established using commercial antibodies as available to detect incretins.

[0342] Aim 2.2. Circulating hormone profile analysis: Levels of circulating hormones will be measured using specific multiplex ELISA panels measuring diabetes and obesity biomarkers (leptin; the products of pro-glucagon processing GLP-1, GLP-2, oxyntomodulin; products of pro- GIP processing GIP; peptide YY; ghrelin; Insl5; insulin; glucagon).

[0343] Example 4 - Chronic Animal Studies: Once Weekly Dosing in Wildtype, Type 2 Diabetic and Diet-Induced Obese Mouse Models. db / db mice at 10-12 weeks of age will be utilised, when their fasting blood glucose levels reach >10mmol / L. A model of diet-induced obesity as a standalone obese model independent of diabetes will also be used. The wildtype mice will be fed a 60% high-fat diet from the age of 8 weeks for a period of 8 weeks (mice will be 16 weeks of age) to establish obesity with chow-fed wildtype mice used as the obesity comparator. All mice will be weight matched into their respective treatment groups. This will provide evidence for an anti-diabetic and anti-obesity effect of these mRNAs.

[0344] A once weekly injection of the mRNA compounds, controls and Tirzepatide, over 4 weeks using the optimal dose and the optimal route of administration will be conducted. During the 4 weeks, body weight, food intake and blood glucose levels every 2-3 days will be monitored. After 4 weeks, OGTT will be assessed with blood collection taken at each of the timepoints for measurement of circulating levels of key gut hormones. Tissues will be taken (fat [white and brown], muscle, heart, liver, kidney, gastrointestinal tract sectioned into stomach, duodenum, jejunum, ileum and colon) for gene and protein expression analysis. Pancreatic islets will be isolated to determine the effect on beta-cell mass and number and determine the effect of chronic administration on glucose-mediated insulin secretion in isolated islets (2mM and 20mM glucose). Mice will be observed for tolerability and safety parameters and monitored for any signs of distress or physiological abnormalities in response.

[0345] Example 5 - Chronic Animal Studies: Gene and Protein Expression Profiling mRNA and protein expression of genes and proteins relevant to the target pathway will be assessed including assessment of Gcg, GIP, GLP-1R, GIP-R genes and Pcskl / 3 and Pcsk 2 proteins. Gene expressions of central satiety peptides specifically within the hypothalamic arcuate nucleus will be measured including neuropeptide Y [NPY], pro-opiomelanocortin [POMC], cocaine-and amphetamine-regulated transcript [CART] and agouti related peptide [AgRP]) and as a control, the paraventricular nucleus.

[0346] It is expected that gene expressions of Gcg (proglucagon) and GIP, and their respective receptors will be increased in the ileum and colon, particularly in chronic studies. It is also expected that an increase in the protein levels of Pcsk 1 / 3 and 2 indicating an enhancement in this pathway and subsequent reductions in orexigenic neuropeptides, NPY, AgRP and increases in anorexigenic neuropeptides.

[0347] Example 6 - Clinical trials

[0348] Phase 1 Study design-. A single dose escalating study, 3 doses of the selected pre-pro mRNA and optimal route will be administered to 15 healthy volunteers (n=5 per group). Safety signals, including fever, headaches, abdominal discomfort, increased heart rate, and blood pressure, will be monitored every 2 hours for 48 hours. Anticipated outcomes aim to demonstrate safety and minimal side effects of the pre-pro mRNA compound within 48 hours, facilitating the phase 2A clinical trial initiation.

[0349] Phase 2A Study Design'. A randomised, double-blind, placebo-controlled trial with 6 arms will assign participants (with and without diabetes) to receive formulations of optimal pre-pro mRNA, LNP, or placebo alongside standard care for 12 weeks. Endpoints include glucose tolerance, HbAlc, glucose, GIP, GLP-1 levels, body weight, hunger, and cardiovascular output. Anticipated outcomes involve improved glucose tolerance, comparable HBAlc and fasting blood glucose (as well as time in range) for those without diabetes, and no safety concerns with dosing or administration route.

[0350] Example 7 - Self-Amplifying Constructs with Preproglucagon and PreproGIP in Wildtype and Diet-Induced Obese Mouse Models. Materials and methods

[0351] Male B6 mice (8-10 weeks of age) and male diet-induced obese mice fed a 60% high-fat dietfor 10 weeks (DIO, 16-18w of age; SF02-00660% Fat Modified Rodent Diet, Specialty Feeds, Western Australia) were randomised to receive one of following combination mRNA compounds encased in lipid nanoparticles (LNP) intraperitoneally (IP): (1) self-amplifying preproglucagon / preproGIP mRNA (native sequences) denoted sa-preproglucagon / preproGIP mRNA (native) on figures or (2) self-amplifying preproglucagon A99G / preproGIP A45G mRNA (mutated sequences): denoted sa-preproglucagon A99G / preproGIP A45G mRNA (modified) on figures. Each mouse received a 10pg total dose of the combination mRNA compounds (5 pg of self-amplifying preproglucagon (native) + 5 pg self-amplifying preproGIP (native) or 5 pg of selfamplifying preproglucagon A99G (modified) + 5 pg self-amplifying preproGIP A45G (modified). The two compounds were combined into an eppendorf tube and gently inverted to ensure mixing of the two compounds. Following IP administration, body weights and fed blood glucoses were measured at day 1, 2, 3, 7 and 14. On the seventh day, an oral glucose tolerance test (OGTT, 2g / kg oral glucose bolus) was completed to assess the combination mRNAs ability to lower blood glucose levels over 120 minutes. Blood samples for glucose were taken at 0, 2, 5, 10, 30, 45, 60, 90 and 120 minutes thereafter and the excursion was calculated as a change from baseline. Total area under the curve (AUC) was also calculated over the 120-minute duration. Comparators included: TRIS Buffer control, Tirzepatide peptide (30nmol / kg delivered subcutaneously as previously published (Coskun T et al. LY3298176, a novel dual GIP and GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus: From discovery to clinical proof of concept. Mol Metab. 2018;18:3-14.), LNPs containing mScarlet-I-nLuc mRNA (lOpg), and mRNA compounds (preproGIP (native sequences), self-amplifying (sa)- preproGIP (native sequences), selfamplifying (sa)- preproglucagon (native sequences), (10pg)).

[0352] Results

[0353] Change in body weight in response to a single IP injection of combination mRNA: Body weight was measured after 1, 2, 3, 7 and 14 days following a single IP injection of the mRNA compounds and comparator compounds.

[0354] For the 7 day data, two-way ANOVA demonstrated a statistically significant effect of treatment on body weight change over 7 days. Specifically, within the B6 cohort of mice (Figure 3A and 3B), there was a significant effect of the self-amplifying preproglucagon / preproGIP mRNAs (both native and modified sequences) on body weight when compared to TRIS, Tirzepatide peptide, and the LNP - B6 mice (p<0.05). Within the DIO group (Figure 3 A and 3C), there were significant effects of the self-amplifying preproglucagon / preproGIP combination (native and modified sequences) compared to the TRIS Buffer control B6 group. Compared to Tirzepatide peptide treated B6 mice and both the LNP administered B6 and DIO mice, the selfamplifying preproglucagon / preproGIP combination (modified) group was the only group that was significantly different.

[0355] Calculating the data as an average body weight change over the 7 days (in grams, Figure 3D) and presented as a % change in body weight over the 7 days (Figure 3E), it can be seen that the self-amplifying preproglucagon / preproGIP mRNA (native) in the B6 mice lowered body weight by 8.2% compared to the TRIS Buffer control, Tirzepatide peptide, LNP-B6 mice, preproGIP mRNA (native), the self-amplifying preproGIP mRNA (native) and the self-amplifying preproglucagon mRNA (native). The self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) treated B6 mice reduced their body weight by 6.2% compared to TRIS Buffer control, and the single compounds, but not Tirzepatide peptide (p=0.06). In the DIO group, both self-amplifying preproglucagon / preproGIP mRNA (native and modified) treated DIO mice reduced their body weight by 2.7% and 5.8% respectively, compared to TRIS buffer, but the native compound was not statistically significant when compared to the Tirzepatide peptide. Further, body weight in the self-amplifying preproglucagon / preproGIP mRNA (modified) treated DIO mice was significantly reduced compared to the LNP treated B6 and DIO mice, the preproGIP mRNA (native) - B6 mice, and the self-amplifying preproglucagon mRNA (native) and the selfamplifying preproGIP mRNA (native) treated B6 mice.

[0356] The data after 7 days demonstrates a strong body weight reducing effect of the selfamplifying preproglucagon / preproGIP mRNA (native) in the B6 mice compared to the standard care of Tirzepatide peptide and to single preproGIP (native) and self-amplifying preproglucagon and self-amplifying preproGIP (native) compounds. The self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) treatment reduced body weight in the B6 mice compared to TRIS Buffer control, LNP and the single mRNA compounds (preproGIP (native), self-amplifying preproGIP (native) and self-amplifying preproglucagon (native) groups), and there is a very close significant effect when compared to Tirzepatide peptide (when calculated as the % change). In the DIO mouse model, both self-amplifying preproglucagon / preproGIP mRNA compounds (native and modifed) reduced body weight compared to TRIS Buffer control, yet the self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) significantly reduced body weight compared to the LNP control (and the single compounds in the B6 mice). Importantly, the LNPs in both mouse models do not have any toxic effect on body weight.

[0357] The mice were followed for a further 7 days which took the effects out to 14 days (Figure 4). The data in the B6 mice showed a sustained reduction in body weight when the average over 14 days is calculated. In the model, the Tirzepatide peptide treated B6 mice gained weight above baseline (Figure 4A), while the mRNA compounds, in particular the self-amplifying combinations, remained at their lower levels. Figure 4B and 4C illustrate the average body weight change over the 14 days and as a % change respectively. It can be clearly seen that in the B6 mice both the selfamplifying preproglucagon / preproGIP mRNA (native and modified) treatment maintained a significant 7.3% and 5.8% body weight reduction respectively compared to TRIS Buffer control, Tirzepatide peptide, LNP-B6 and the single mRNA compounds (self-amplifying preproGIP (native), self-amplifying preproglucagon (native); preproGIP (native) data not available for 14 days). More profound was the significant 6.3% reduction in body weight in the DIO mice treated with self-amplifying preproglucagon A99G / preproGIP A45G mRNA (modified) compared to the TRIS Buffer control, Tirzepatide peptide, LNPs (B6 and DIO) and the two single self-amplifying mRNA compounds (preproGIP and preproglucagon). Further, there was a highly significant reduction in body weight (p<0.0001), when compared against the self-amplifying preproglucagon / preproGIP mRNA (native) treated DIO mice, indicating that the two modifications (A99G on the preproglucagon and A45G on the preproGIP) keeps a sustained body weight effect in the current model of obesity.

[0358] The overall body weight data clearly showed the impact that the self-amplifying combination mRNAs have on maintaining sustained reductions over 14 days after a single injection. The combination of the native sequence is the product of the two individual selfamplifying preprohormones (native); self-amplifying preproGIP and self-amplifying preproglucagon, where there is an enhanced effect when the two are combined in the B6 mice (self-amplifying preproglucagon / preproGIP mRNA (native)). The DIO model showed that the two mutations (A45V / A99G) on the preprohormones, when combined, provides an even greater maintenance of body weight reduction compared to the native sequences in the same model. Change in blood glucose in response to a single IP injection of combination mRNA: Blood glucose levels were also measured at the same time as the body weight was taken at 1, 2, 3, 7 and 14 days following the single IP injection of the mRNA compounds.

[0359] Figure 5 illustrates the change in blood glucose levels over the first 7 days in the B6 and DIO mice and includes comparators tested in the B6 mice: TRIS Buffer control, Tirzepatide peptide, LNP, pre-pro GIP mRNA (native), self-amplifying pre-pro GIP (native) and selfamplifying preproglucagon (native); and DIO mice: LNP. The data shows the Tirzepatide peptide can maintain a reduction in blood glucose levels over 7 days (Figure 5A). When the average blood glucose over the 7 days was calculated (Figure 5B), the self-amplifying preproglucagon / preproGIP mRNA (native and modified) in the B6 mice, could lower blood glucose compared to TRIS Buffer control, and self-amplifying preproglucagon mRNA (native), whilst in the DIO mice, there was no significant effect of lowering in blood glucose levels. When the data was extended to 14 days, the same pattern as seen at 7 days was maintained in both the B6 and DIO mice (Figure 5C and 5D).

[0360] Glucose Tolerance in response to a single injection of combination mRNA: Glucose tolerance was measured in both the B6 and DIO mice via an oral glucose tolerance test (OGTT), 1 week following a single injection of the self-amplifying combination mRNAs (Figure 6). The data shows that both the glucose excursion as a change from baseline and the total AUC of the glucose excursion were not significantly affected by the treatment in either the B6 mice or the DIO mouse model, when compared to the LNP controls.

[0361] Example 8 - Circulating hormone levels following a single IP injection of preproGIP mRNA

[0362] To determine whether the mRNA encoding one of the incretins could increase circulating levels of its final product and other hormones along its and associated pathways, a single injection of preproGIP mRNA (native) was administered to B6 mice and TRIS buffer for the control, and cardiac blood samples 3 hours post administration were collected. Blood was collected in tubes containing protease inhibitors and spun down to collect plasma. Plasma was analysed for circulating GIP, GLP-1, GLP-2, Oxyntomodulin and Glucagon levels using a milliplex ELISA kit (Figure 7). The data showed that 3 hours after a single injection of preproGIP mRNA, there was a significant increase in circulating GIP levels (Figure 7A) compared to TRIS treated mice as well as a significantly elevated Glucagon levels (Figure 7B). GLP-1 levels (Figure 7C) appeared to be elevated, but this did not reach statistical significance (p=0.113). No differences were observed in circulating GLP-2 or Oxyntomodulin levels (data not shown). Example 7 indicates that preproGIP mRNA works to produce the specific targeted incretin and it also increases the level of other associated hormones that are critical in the incretin pathway. The data further illustrates that LNP delivery of the target mRNA is working as expected.

[0363] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0364] Sequences of the Disclosure

Claims

CLAIMS1. A composition for regulating glucose metabolism and / or lipid metabolism, the composition comprising a nucleic acid encoding pre-pro-gastric inhibitory peptide (preproGIP) and a nucleic acid encoding preproglucagon.

2. The composition of claim 1, wherein the nucleic acid is a mRNA.

3. The composition of claim 2, wherein the mRNA is a self-amplifying mRNA.

4. The composition of claim 2 or 3, wherein the mRNA comprises a cap, 5'UTR, coding sequence, a 3'UTR and a poly A tail.

5. The composition of claim 4, wherein the 5'UTR comprises or consists of a sequence set forth in SEQ ID NO: 20.

6. The composition of claim 4, wherein the 3'UTR comprises or consists of a sequence set forth in SEQ ID NO: 21.

7. The composition of claim 4, wherein the poly A tail comprises or consists of a sequence set forth in SEQ ID NO: 22.

8. The composition of claim 4, wherein the mRNA comprises an optimised codon and / or a chemical modification, optionally comprising replacement of uridine with pseudouridine or Nl-methyl-pseudouridine.

9. The composition of any one of claims 1 to 8, wherein the nucleic acid encoding preproGIP encodes an amino acid sequence having 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% sequence identity to the sequence set forth in SEQ ID NO: 2.

10. The composition of any one of claims 1 to 9, wherein the nucleic acid encoding preproglucagon encodes an amino acid sequence having 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 least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 1.

11. The composition of any one of claims 1 to 10, wherein the preproGIP comprises an amino acid modification selected from A45V, A45T, and A45G.

12. The composition of any one of claims 1 to 11, wherein the preproglucagon comprises an amino acid modification selected from A99V, A99T, and A99G.

13. The composition of any one of claims 1 to 12, wherein the preproGIP comprises an A45 V modification, and the preproglucagon comprises an A99G modification.

14. The composition of any one of claims 1 to 13, wherein the composition comprises an additional nucleic acid encoding one or more of the components selected from the group consisting of IP1, IP2, GRPP, glucagon, glucagon-like peptide-I (GLP-1), gastric inhibitory peptide (GIP), glucagon-like peptide-2 (GLP-2), oxyntomodulin (OXM), peptide YY (PYY); chromogranin A (CgA), synaptophysin (Syn), serotonin (5-HT), tryptophan 5-hydroxylase (TPH), Oxyntomodulin, Glicentin, Somatostatin, Ghrelin, Gastrin, Serotonin, Neurotensin, Growth differential factor 15, Fibroblast growth factor 19, Guanylin, Uroguanylin, Insulin-Like Peptide 5 (INSL5), Cholecystokinin (CKK), Secretin, Motilin, Nesfatin-1, Leptin, Zenin, Histamine, Proglucagon, Glucagon, Fibroblast growth factor 21.

15. The composition of claim 14, wherein the additional nucleic acid is a mRNA and wherein:(a) the GLP-1 encodes an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 5 or 10;(b) the glucagon encodes an amino acid sequence set forth in any one of SEQ ID NOs: 6 or 11;(c) the GLP-2 encodes an amino acid sequence set forth in any one of SEQ ID NOs: 7 or 12;(d) the GIP encodes an amino acid sequence set forth in SEQ ID NO: 13.

16. The composition of any one of claims 1 to 15, wherein the nucleic acid further comprises a modification or heterologous moiety for one or more or all of tissue specificity,increased solubility and / or half-life, increased affinity, increased activity, increased stability and resistance to protease cleavage.

17. The composition of claim 16, wherein the heterologous moiety for increasing solubility and / or half-life is selected from the group consisting of albumin, human serum albumin (HSA) or HSA FcRn binding portion or wherein the modification for increasing resistance to protease cleavage comprises substituting amino acids for cleavage, preferably with glycine.

18. The composition of any one of claims 1 to 17, further comprising an additional therapeutic selected from the group consisting of an analogue of Exenatide, an analogue of Tirzepatide, an analogue of Retatrutide and an analogue of SAR441255, optionally wherein the additional therapeutic is:(a) an analogue of Exenatide encoding an amino acid sequence according to those set forth in any one of SEQ ID Nos: 9, 14, 18, 19, or 23;(b) an analogue of Tirzepatide encoding an amino acid sequence according to that set forth in SEQ ID No: 15;(c) an analogue of Retatrutide encoding an amino acid sequence according to that set forth in SEQ ID No: 16; or(d) an analogue of SAR441255 encoding an amino acid sequence according to that set forth in SEQ ID No: 17.

19. The composition of any one of claims 1 to 18, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

20. A lipid nanoparticle (LNP) comprising the composition of any one of claims 1 to 19.

21. The LNP of claim 20, wherein the LNP encapsulates the nucleic acid.

22. The LNP of claim 20 or 21, wherein the LNP comprises an ionizable lipid, a phospholipid, a sterol, and PEG-lipid, optionally wherein the molar ratio of ionizable lipid, phospholipid, sterol, and PEG-lipid is 50: 10:38.5: 1.5.

23. The LNP of claim 22, wherein the ionizable lipid is DLin-MC3-DMA; the phospholipid is a DSPC; the sterol is cholesterol, and the PEG-lipid is PEG2000-DMG.

24. The LNP of claim 23, wherein the composition or LNP is suitable, or formulated for delivery to one or more or all of the following locations: liver, stomach, pancreas, duodenumjejunum, ileum, colon and adipose tissue.

25. A host cell comprising the composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24.

26. The host cell of claim 25, wherein the cell is a cell of the liver, stomach, pancreas, duodenum ejunum, ileum, colon or adipose tissue.

27. A method of synthesis of the lipid nanoparticle (LNP) of any one of claims 20 to 24, the method comprising producing a nucleic acid, preferably mRNA, from a DNA polynucleotide optionally provided in a vector, and encapsulating the nucleic acid within the LNP.

28. The method of claim 27, wherein the mRNA is generated by in vitro transcription.

29. A lipid nanoparticle obtained from the method of claim 27.

30. A kit comprising a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24.

31. A method of treating or preventing a metabolic disorder associated with elevated blood glucose levels in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 to the subject, thereby treating or preventing the metabolic disorder in the subject.

32. A method of regulating blood glucose levels in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 to the subject, thereby regulating blood glucose levels in the subject.

33. A method of treating or preventing obesity or weight gain in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of anyone of claims 20 to 24 to the subject, thereby treating or preventing obesity or weight gain in the subject.

34. A method of regulating lipid metabolism in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 to the subject, thereby regulating lipid metabolism in the subject.

35. A method of promoting weight loss in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 to the subject, thereby promoting weight loss in the subject.

36. A method of increasing expression of both pre-pro-gastric inhibitory peptide (preproGIP) and preproglucagon in a subject in need thereof, comprising administering a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 to the subject.

37. The method of claim 31, wherein the metabolic disorder is type 2 diabetes, type 1 diabetes, insulin resistance, prediabetes, metabolic associated fatty liver disease (MAFLD), nonalcoholic steatohepatitis (NASH), or obesity.

38. The method of any one of claims 31 to 37, wherein the subject is pre-diabetic and / or insulin resistant.

39. The method of claim 31 or 32, wherein the treatment decreases plasma glucose concentration within about 30 minutes.

40. The method of claim 31 or 32, wherein the subject exhibits one or more of the following symptoms: elevated blood glucose levels, baseline HbAlc percentage of greater than 5.7% or 6.5%, frequent urination, increased thirst, loss of body weight, presence of ketones in urine, weakness and tiredness, irritability, poor wound healing and susceptibility to infection.

41. The method of claim 31 or 32, wherein the subject is obese or overweight, optionally wherein the obese subject has a body mass index of at least 30 or wherein the overweight subject has a body mass index of between about 25 to 29.9.

42. The method of claim 31 or 32, wherein the treatment further comprises administering an additional therapeutic agent.

43. The method of claim 42, wherein the additional therapeutic agent is an anti-diabetic agent selected from the group consisting of native insulin, native glucagon and functional analogs thereof, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose / Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP -4) inhibitors such as vildagliptin or sitagliptin; SOLT (sodium-dependent glucose transporter 1) inhibitors; FBPase (fructose 1,6-bisphosphatase) inhibitors, Tirzepatide (Mounjaro), SAR441255 and semaglutide (Ozempic) or analogues thereof.

44. The method of any one of claims 33 to 35, wherein the treatment further comprises administering an additional therapeutic agent.

45. The method of claim 44, wherein the additional therapeutic agent is an anti-obesity agent selected from Bupropion-naltrexone (Contrave), Liraglutide (Saxenda), Orlistat (Xenical, Alli), Phentermine-topiramate (Qsymia), Semaglutide (Wegovy), Retatrutide (LY- 3437943), Setmelanotide (Imcivree), Survotutide, Mazdutide, Efinopegdutide, Cagrisema, Dulaglutide, Exenatide, Efpeglenatide, Lixisenatide, Cotadutide, Danuglipron and Orgorglipton.

46. The method of any one of claims 42 to 45, wherein the additional therapeutic may be administered at the same time or a different time to the administration of a composition of any one of claims 1 to 19.

47. The method of claim 43 or 45, wherein the additional therapeutic is selected from the group consisting of an analogue of Exenatide, an analogue of Tirzepatide, an analogue of Retatrutide and an analogue of SAR441255.

48. The method of claim 47, wherein the additional therapeutic is an analogue of Exenatide encoding an amino acid sequence according to those set forth in any one of SEQ ID Nos: 9, 14, 18, 19, or 23.

49. The method of claim 47, wherein the additional therapeutic is an analogue of Tirzepatide encoding an amino acid sequence according to that set forth in SEQ ID No: 15.

50. The method of claim 47, wherein the additional therapeutic is an analogue of Retatrutide encoding an amino acid sequence according to that set forth in SEQ ID No: 16.

51. The method of claim 47, wherein the additional therapeutic is an analogue of SAR441255 encoding an amino acid sequence according to that set forth in SEQ ID No: 17.

52. A method of producing glucagon-like peptide-I (GLP-1) or gastric inhibitory peptide (GIP) comprising expressing a nucleic acid, preferably mRNA, encoding pro-pro-glucagon or pre-pro-gastric inhibitory peptide (preproGIP) respectively, under conditions sufficient for the production of GLP-1 or GIP.

53. The method of claim 52, wherein the mRNA is formulated in a LNP.

54. A composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 for use in the treatment or prevention of a metabolic disorder associated with elevated blood glucose levels.

55. A composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 for use in the regulation of blood glucose levels.

56. A composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 for use in the treatment or prevention of obesity or weight gain.

57. A composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 for use in the regulation of lipid metabolism.

58. A composition of any one of claims 1 to 19 or the LNP of any one of claims 19 to 24 for use in the promotion of weight loss.

59. A composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 for use in increasing expression of both pre-pro-gastric inhibitory peptide (preproGIP) and preproglucagon in a subject in need thereof.

60. Use of a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 in the manufacture of a medicament for treating or preventing a metabolic disorder associated with elevated blood glucose levels.

61. Use of a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 in the manufacture of a medicament for regulating blood glucose levels.

62. Use of a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 in the manufacture of a medicament for treating or preventing obesity or weight gain.

63. Use of a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 in the manufacture of a medicament for regulating lipid metabolism.

64. Use of a composition of any one of claims 1 to 19 or the LNP of any one of claims 20 to 24 in the manufacture of a medicament for promoting weight loss.

65. The use of claim 54 or 60, wherein the metabolic disorder is type 2 diabetes, type 1 diabetes, insulin resistance, prediabetes, metabolic associated fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH), or obesity.

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