WO2024121160A1 - Regulator(s) of energy homeostasis-encoding rna molecule(s) with increased translation efficiency - Google Patents

Regulator(s) of energy homeostasis-encoding rna molecule(s) with increased translation efficiency Download PDF

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WO2024121160A1
WO2024121160A1 PCT/EP2023/084367 EP2023084367W WO2024121160A1 WO 2024121160 A1 WO2024121160 A1 WO 2024121160A1 EP 2023084367 W EP2023084367 W EP 2023084367W WO 2024121160 A1 WO2024121160 A1 WO 2024121160A1
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utr
rna molecule
seq
rna
sequence
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Carsten Rudolph
Manish ANEJA
Rebekka Kubisch-Dohmen
Günther HASENPUSCH
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Ethris Gmbh
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • RNA molecules and their applications in medical and therapeutic contexts. Specifically, the invention relates to an RNA molecule that includes a coding region for a regulator of energy homeostasis (like GLP-1 and/or GDF-15) and untranslated regions (UTRs) with specified sequences. These UTRs are designed to enhance the translation efficiency of the RNA molecule.
  • a regulator of energy homeostasis like GLP-1 and/or GDF-15
  • UTRs untranslated regions
  • the invention also encompasses a set of RNA molecules, each coding for different regulators of energy homeostasis, and nucleic acid molecules encoding these RNA molecules. Furthermore, it includes vectors containing these nucleic acid molecules and host cells comprising these vectors or nucleic acid molecules. Additionally, the invention pertains to pharmaceutical compositions containing these RNA molecules, nucleic acid molecules, vectors, or host cells, and kits comprising these components. These are intended for use in RNA-based therapies, particularly for regulating energy homeostasis, controlling body weight, and treating or preventing metabolic disorders. The invention also includes methods for decreasing food intake, controlling body weight, and treating or preventing metabolic disorders.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • DNA-based gene therapeutics mRNA does not need to be transported into the nucleus but is directly translated into protein in the cytoplasm (1,2). This makes mRNA safer in avoiding potential insertional mutagenesis, an unlikely but existent risk of DNA gene medicines.
  • mRNA therapeutics are emerging as promising alternatives for gene and protein replacement therapies in a broad variety of medical indications (1-4).
  • the strong immunogenicity as well as the limited stability of conventional mRNA has to be overcome to further establish its clinical applicability.
  • mRNA stability and in particular the translation rate of the mRNA is an essential parameter for envisaged medical applications because it determines, for example, dosing and the dosing intervals of mRNA drugs.
  • UTRs untranslated regions
  • mRNA stability and mRNA translation have been reported to play a pivotal role in regulating both mRNA stability and mRNA translation. UTRs are known to influence translational initiation, elongation, and termination, as well as mRNA stabilization and intracellular localization through their interaction with RNA binding proteins (6,7). Depending on the specific motives within the UTR, it can either enhance or decrease mRNA turnover (8-11). Recently, data on mRNA half- lives and the corresponding UTR sequences have been published (12, 43).
  • the translation efficiency is an essential parameter for envisaged medical applications because it determines, for example, dosing and the dosing intervals of mRNA drugs and, ultimately, determines the bioavailability of the final product, i.e., the encoded peptide or protein.
  • the increased occurrence of nutrition- dependent diseases and metabolic disorders, respectively, e.g. obesity/adipositas, hypercholesterolemia, diabetes, hyperglycaemia, hypertension and the like
  • obesity/adipositas e.g. obesity/adipositas, hypercholesterolemia, diabetes, hyperglycaemia, hypertension and the like
  • diseases/disorders are secondary diseases and pathological consequences caused, for example by obesity, as a consequence of overnutrition.
  • pathological consequences of increased glucose concentrations in the blood due to diabetes are retinopathia and renal failures.
  • overweight and diabetes are risk factors for diseases such as hypertension, heart attack, other cardiovascular diseases, stroke, biliary stones or other bile disorders, gout, and the like.
  • Especially obesity has risen to alarming levels world-wide (McLellan (2002), Lancet 359, 1412). For example, the average weight of German conscripts increases by almost 400g/year. Similar data were obtained in Austria, Norway and the UK.
  • GLP-1 Glucagon-like Peptide 1
  • GDF15 growth differentiation factor 15
  • DPP-4 dipeptidyl-peptidase-4
  • GLP-1 is known to be a neuropeptide and an incretin, secreted in the periphery from the intestinal L cell as a gut hormone.
  • the major source of GLP-1 in the brain is the nucleus of the solitary tract.
  • GLP-1 increases the secretion of insulin, slows down the emptying process in the stomach and increases the feeling of saturation. Low levels of GLP-1 are known to result in increased food intake and, as a consequence, in obesity and related metabolic disorders.
  • exemplary reference is made to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1760073/pdf/v044p00081.pdf, Gutzwiller (Gut 44, 1999, 81-86) and Schick (Glucagon-like peptide 1: a novel brain peptide involved in feeding regulation. In: Obesity in Europe 1993, Ditschuneit et al. (Eds), 1994, 363–367).
  • GLP-1 and its agonists have therefore been used in the treatment of metabolic disorders like obesity.
  • Examples of respective pharmaceuticals are: Exenatide (Byetta ®® , AstraZeneca; acts as a GLP-1 mimic), Exenatide (Byduroen ®® , Amylin, AstraZeneca), Liraglutide (Victoza ®® , 97% aa-similarity to GLP- 1; NovoNordisk).
  • GDF15 is a member of the TGFbeta superfamily, also known as placental bone morphogenetic factor (PLAB) (Hromas R 1997, Biochim Biophys Acta.1354:40-4), prostate derived factor (PDF) (Paralkar V M 1998, J Biol Chem.
  • GDF15 macrophage inhibitory cytokine 1 (MIC1) (Bootcov M R, 1997, Proc Natl Acad Sci 94:11514-9), placental transforming growth factor beta (PTGFB) (Lawton L N 1997, Gene.203:17-26), and NSAID activated gene-1 / nonsteroidal anti- inflammatory drug-activated gene 1 (NAG-1) (Baek S J 2001, J Biol Chem.276: 33384-92). It is a cellular stress-responsive factor mediating cytoprotection. GDF15 is expressed almost ubiquitous; it is (on mRNA level) most abundant in the placenta and prostate but also present in heart, liver, kidney, pancreas and colon.
  • GDF15 is highly expressed in macrophages, choroid plexus, prostate, lung, kidney proximal tubules, placenta and intestinal mucosa and is overexpressed by a variety of cancers, which may relate to its antitumorigenic and proapoptotic properties. Although it has been described as a prognostic marker in acute myocardial infarction it is only poorly expressed in the heart. In this context, exemplary reference is made to Strelau (J Neural Transm Suppl.65, 2003, 197-203).
  • GDF15 has been discussed to be involved in body weight control and its levels have been associated with obesity, risk for insulin resistance and glucose control in patients (Cartsensen, European J of Endocrinology 162 (5), 2010, 913-917; Dostalova, European J. of Endocrinology 161, 2009, 397-404; Vila, Clinical Chemistry 57 (2), 2011, 309-316). Moreover, GDF15 has been considered as a protective in heart and kidney disease and as a biomarker for cancer and cardiovascular diseases.
  • exemplary reference is made to Xu (Circ Res 98(3), 2006, 342-50), Bauskin (Cancer Res 66(10), 2006, 4983-6), Macia (PLoS ONE 7(4), 2012, ArticleID e34868) and Kempf (Nature Medicine 17(5), 2011,581–288).
  • GDF15 levels are elevated before the manifestation of Type 2 diabetes mellitus (Cartsensen, European J of Endocrinology 162 (5), 2010, 913-917; Dostalova; European J. of Endocrinology 161, 2009, 397-404).
  • GDF15 decreases food intake and improves glucose tolerance in mice (Macia, PLoS ONE 7 (4), 2012, Article e34 (6)). Consequently, GDF15 and its agonists has been suggested to treat obesity and other metabolic disorders (Chrysovergis, International Journal of Obesity (38), 2014, 1555–1564; WO2012138919 (Amgen)). For example, Lim et al. showed a weight-lowering effect in obese mice models of YH34160, an Fc fusion GDF15 variant (Meeting abstract: OBESITY—ANIMAL, JUNE 012021, Diabetes 2021; 70(Supplement_1); 217-LB).
  • GDF15 has also been described as a diagnostic marker, e.g. for diabetes I (WO2009141357; Roche) and myocardial infarction (US 8771961; Roche). GDF15 has also been discussed to be involved in tumor-induced anorexia and weight loss (Johnen, Nat Med.13 (11): 2007, 1333-40). It is well established that gut-derived hormones, such as GLP-1, play an important role in the control of energy homeostasis (Burcelin, J. Nutr. 137 (11 Suppl.), 2007, 2534S-2538S).
  • TGF- ⁇ superfamily cytokine GDF15 The action of the TGF- ⁇ superfamily cytokine GDF15 is mainly mediated by direct action on the feeding centers in the hypothalamus (Tsai, 2014 PLoS ONE 2014, 9(6): e100370. doi:10.1371/journal.pone.0100370). Although the lack of a rapid and substantial increase of GDF15 serum levels after taking a meal provides some evidence that GDF15 does not act as a satiety factor it may, similarly to GLP-1, act as a physiological long-term regulator of energy homeostasis (Tsai, PLoS One 2015, 10 (7): e0133362. doi: 10.1371/journal.pone.0133362).
  • exemplary reference is made to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238422/pdf/ijcp0068-0557.pdf and Brunton (Int J Clin Pract 68(5), 2014, 557–567).
  • DPP-4 inhibitors are Sitagliptin, Vidagliptin, Saxagliptin, Linagliptin, Alogliptin, Dutogliptin and Gemigliptin.
  • WO 2017/001554 describes UTRs derived from an mRNA of the human cytochrome b-245 alpha polypeptide (CYBA) gene which increase the translation efficiency of RNA molecules.
  • CYBA cytochrome b-245 alpha polypeptide
  • RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis like GLP-1 and/or GDF-15 (MIC-1); and (b) upstream of said coding region one (or more) untranslated region(s) (UTR(s)) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40; or (B) the 5’-UTR of alpha globin (Ag) or a functional derivative of said 5’-UTR; and/or (c) downstream of said coding region one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions
  • the present invention further relates to a set of 2 or more RNA molecules, wherein the coding region of one RNA molecule of said set codes for one regulator of energy homeostasis (like GLP- 1) and wherein the coding region of another RNA molecule of said set codes for another regulator of energy homeostasis (like GDF-15).
  • the present invention relates to a nucleic acid molecule encoding the RNA molecule or the set of RNA molecules according to the present invention and to a set of 2 or more nucleic acid molecules encoding the 2 or more RNA molecules, respectively, of the set of the 2 or more RNA molecules.
  • the present invention relates to a vector comprising the nucleic acid molecule or the set of nucleic acid molecules according to the present invention and to a set of 2 or more vectors comprising the 2 or more nucleic acid molecules, respectively, of the set of 2 or more nucleic acid molecules.
  • the present invention further relates to a host cell comprising the nucleic acid molecule or set of nucleic acid molecules according to the present invention or the vector or the set of vectors according to the present invention and to a set of 2 or more host cells comprising the 2 or more nucleic acid molecules, respectively, of the set of 2 or more nucleic acid molecules or the 2 or more vectors, respectively, of the set of 2 or more vectors.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set, and optionally a pharmaceutically acceptable carrier.
  • the present invention relates to a kit comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set.
  • the present invention further relates to the pharmaceutical composition for use in an RNA-based therapy and as a regulator of energy homeostasis; in body weight control, in particular in decreasing (aberrant) body weight; and/or in the treatment or prevention of a metabolic disorder.
  • the present invention further relates to a method for decreasing food intake; restraining appetite; controlling body weight, in particular decreasing (aberrant) body weight; and/or treating or preventing a metabolic disorder.
  • the present invention relates to the use of one or more UTR(s) as defined in (b) and/or one or more UTR(s) as defined in (c) for increasing the efficiency of translating a coding region of an RNA molecule into a regulator of energy homeostasis like GLP-1 and/or GDF-15 (MIC-1) encoded by said coding region.
  • GLP-1 and/or GDF-15 MIC-1
  • particular UTRs confer an increased translational efficiency when fused to a given (foreign) mRNA. More particularly, it was found that particular UTRs confer an increased translational efficiency (and/or expression) when fused to (foreign) mRNA encoding regulators of energy homeostasis, especially GLP-1 (or an agonist thereof) or GDF15 (or an agonist thereof).
  • regulators of energy homeostasis especially GLP-1 (or an agonist thereof) or GDF15 (or an agonist thereof).
  • One of these particular UTRs is derived from an mRNA of the cytochrome b-245 alpha polypeptide (CYBA) gene, preferably from an RNA of the human CYBA gene (see also WO 2017/001554).
  • CYBA cytochrome b-245 alpha polypeptide
  • the CYBA gene comprises specific 5’ and 3’ UTRs (also referred to herein as (5’ and 3’, respectively) “eth”, “ETH” or “ethris” (this covers also the variants used)).
  • 5’ UTR motives such as upstream open reading frames (uORFs) or internal ribosomal entry sites (IRES) are known to be involved in gene regulation, particularly in translational initiation (13).
  • the 3’ UTRs can comprise even more regulatory functions than the 5’UTRs, some of them even hindering mRNA translation (14).
  • the CYBA’s 3’ UTR is known to contain two regulatory motives, the finding of the present invention that the CYBA UTRs confer an increased translational efficiency when fused to a given mRNA encoding a regulator of energy homeostasis, especially GLP-1 (or an agonist thereof) or GDF15 (or an agonist thereof) is nevertheless surprising since these two motives are described in the context of the mRNA’s stability but not in the increase of the translational efficiency.
  • the 3’ UTR of CYBA is known to harbour a polyadenylation signal (PAS) which is known to interact with the cytoplasmic polyadenylation element binding protein (CPEB), as well as with the cleavage and polyadenylation signaling factor (CPSF) (11).
  • CPEB cytoplasmic polyadenylation element binding protein
  • CPSF cleavage and polyadenylation signaling factor
  • a second regulatory motif contained in the CYBA 3’ UTR is the insulin 3’ UTR stability element (INS_SCE) (15).
  • the INS_SCE sequence has been shown to bind to the polypyrimidine tract binding protein (PTB) under reducing conditions, increasing the mRNA half-life of insulin (15).
  • PTB polypyrimidine tract binding protein
  • both regulatory motives of the CYBA’s 3’ UTR are predominantly linked with the mRNA stability.
  • Another of the particular UTRs that confers an increased translational efficiency (and/or expression) in accordance with the invention is derived from the alpha globin (Ag) gene, preferably from the human alpha globin (hAg) gene.
  • the hAg gene comprises a specific 5’-UTR.
  • the following polyribonucleotide sequence is an example of the 5’-UTR derived from the Ag gene, in particular the hAg gene: CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC (SEQ ID NO: 3).
  • the DNA sequences displaying the nucleotide sequence of the human CYBA gene’s 5’- and 3’- UTRs present on the coding strand of the human CYBA gene are shown in the following Table 1.
  • Table 1 Genetic code of the human CYBA gene UTRs
  • Table 1 shows the exact genetic code of the human CYBA gene UTRs. DNA sequences are shown from the 5’ to the 3’ end.
  • the polyadenylation signal (PAS) of the 3’ UTR is shown in bold letters and the insulin 3’UTR stability element (INS_SCE) is underlined.
  • the 5’ UTR consists of 71 base pairs, whereas the 3’ UTR contains 64 base pairs. Both UTRs are shorter than average human UTRs, which consist of around 200 nucleotides in the case of 5’UTRs and approximately 1000 nucleotides in the case of 3’UTRs.
  • the DNA sequences displaying the human CYBA gene 5’- and 3’ UTRs are shown as SEQ ID NO:25 and SEQ ID NO:26, respectively.
  • RNA sequence In view of the fact that the present invention predominantly relates to an RNA molecule, reference is made in the following to the corresponding RNA sequences.
  • UTR sequence on the RNA level can be derived (preferred CYBA 5‘UTR; a 5 ⁇ C residue has been added; a (further) Kozak sequence has been added (GCCACC)): 5’-CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC-3‘ (SEQ ID NO:1).
  • This 5’UTR sequence (which may comprise a (partial) Kozak sequence (e.g.
  • GUCGCC or GCCGCC, or, preferably in the context of the invention, GCCACC at its 3 ⁇ end) immediately precedes the start codon of the human CYBA gene.
  • the following UTR sequence on the RNA level depicts a preferred CYBA 5‘UTR (without the (partial) Kozak sequence and without the 5 ⁇ C residue: 5’-CGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC-3‘ (SEQ ID NO:40) From the above DNA sequence SEQ ID NO:26, the following UTR sequence on the RNA level can be derived (preferred CYBA 3‘UTR): 5’-CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCC ACCUGCAAUAAAUGCAGCGAAGCCGGGA-3‘ (SEQ ID NO:2)).
  • the DNA sequence displaying the nucleotide sequence of the human Ag gene’s 5’-UTR present on the coding strand of the human Ag gene is shown in the following Table 2.
  • Table 2 Genetic code of the human Ag gene UTR Table 2 shows the exact genetic code of the hAg gene UTR. The DNA sequence is shown from the 5’ to the 3’ end. The 5’ UTR consists of 36 base pairs. The UTR is shorter than average human 5 ⁇ UTRs, which consist of around 200 nucleotides.
  • the DNA sequence displaying the hAg gene 5’UTR is shown as SEQ ID NO:28.
  • the following UTR sequence on the RNA level can be derived (preferred hAg 5‘UTR; a G residue has been added to create a full Kozak sequence (GCCACC)): 5’-(CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC-3‘ (SEQ ID NO:3).
  • This 5’UTR sequence (which may comprise a (partial) Kozak sequence (e.g. (C)CCACC or, preferred in the context of the invention, GCCACC) at its 3 ⁇ end) immediately precedes the start codon of the hAg gene.
  • Another important feature influencing mRNA translation efficiency is the poly-A tail, which is located on the 3’ end.
  • the present application surprisingly found that an increase of the translational efficiency (and/or expression) of a natural CYBA or Ag (e.g. hAg) mRNA can be conferred to a foreign mRNA, namely by virtue of flanking its coding sequence with combinations of (shortened) CYBA 5’-UTRs or (shortened) Ag 5’-UTR and/or (shortened) CYBA 3’-UTRs. It is of note in this respect that both, the 5’UTR and the 3’UTR of the present invention (e.g.
  • SEQ ID NO:1/40/3 and SEQ ID NO:2, respectively may be shorter than the DNA sequences displaying the human CYBA gene 5’- and 3’ UTRs and hAg gene 5’-UTR shown as SEQ ID NO:25/31/26/29 and SEQ ID NO:27/28/30, respectively.
  • This can be shown by a single-cell analysis of mRNA transfection time-lapse movies which has recently been shown to be capable of assessing individual expression time courses (26) while it has been reported that it is possible to use regular micropatterns to position cells on a regular grid of adhesion sites (27).
  • This technology offers the resolution to rapidly screen and compare different UTR combinations on a foreign mRNA.
  • the coding sequence of destabilized enhanced green fluorescence protein has been chosen to artificially shorten the life cycle of the reporter protein inside the cell (28).
  • the combinations included insertion of, for example, the respective CYBA UTRs at 5’ or 3’ ends, respectively, at both 5’ and 3’ ends, at the 5’ end combined with two repeats of the 3’ UTR at the 3’ end, or two repeats of 3’ UTR without 5’ UTR. All of these were compared to a control construct without UTRs. Protein and functional mRNA life times and the expression rate from each of the compared transcripts were assessed.
  • regulators of energy homeostasis for example GLP-1 (or an GLP-1 analogue) or GDF15 (or an GLP-1 analogue)
  • UTR(s) CYBA 5’- or Ag 5-’UTRs and/or 3’- UTR(s)
  • GLP-1 may, for example, be a native GLP-1 or an analogue thereof (i.e.
  • GLP-1 may be selected from the following group: Native GLP-1: The native (human) GLP-1 molecule has two biologically active forms, GLP-1(7-36)amide and GLP-1(7-37). The sequence for GLP-1(7-37) is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO:57).
  • Exenatide (Byetta ®® /Bydureon®, AstraZeneca, Amylin, AstraZeneca; derived from the saliva of the Gila monster; acts as a GLP-1 mimic, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:58), Liraglutide (Victoza ®® , 97% aa-similarity to GLP-1 with a modification that includes a fatty acid moiety which extends its half-life; NovoNordisk.
  • the sequence of Liraglutide is: HAEGTFTSDVSSYLEEQAAKEFIAWLVKGRG (SEQ ID NO:59), Semaglutide (Ozempic®): Similar to Liraglutide, Semaglutide has modifications for longer action.
  • the sequence is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 60), Dulaglutide (Trulicity®): This is a GLP-1 analogue designed for once-weekly administration. Its sequence is a fusion of two GLP- 1 sequences to an Fc fragment of human IgG4, Albiglutide (Tanzeum): This GLP-1 analogue is a fusion protein consisting of two GLP-1 analogues linked to human albumin.
  • GDF-15 may, for example, be a native GDF-15 or an analogue thereof (i.e. a therapeutic analogue).
  • Therapeutic analogues of GDF-15 are known that aim to enhance the stability, potency, or specificity of GDF-15 for clinical applications. Examples include: a) Fc-fusion proteins where GDF-15 is fused with the Fc fragment of an antibody to increase its stability and half-life, b) modified peptides or small molecules that mimic or modulate the activity of GDF-15.
  • Fc-fusion proteins where GDF-15 is fused with the Fc fragment of an antibody to increase its stability and half-life
  • modified peptides or small molecules that mimic or modulate the activity of GDF-15.
  • GDF15 in particular GDF15 WT, in combination with the CYBA 5‘- and 3‘-UTRs, shows physiological activity (reduction of food intake and weight loss); in particular at a dosage of about 0.5 mg/kg body weight (BW).
  • the GDF-15 and GLP-1 constructs in combination with the CYBA 5‘- and 3‘-UTRs and the hAg 5‘-UTR, respectively) are superior against corresponding non- translated control RNAs; in particular at a dosage of about 1 mg/kg BW.
  • GDF-15 levels are significantly elevated up to 24 hours post injection.
  • RNA molecules comprising (a) a coding region coding for a regulator of energy homeostasis (e.g.
  • UTR(s) comprising (A) a 5’-UTR of cytochrome b-245 alpha polypeptide (CYBA), or a functional derivative of said 5’-UTR; or (B) a 5’-UTR of alpha globin (Ag), or a functional derivative of said 5’-
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis (e.g. GLP-1 or GDF-15, or an analogue thereof), or for two such regulators (or even for more); and (b) upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40, or a functional derivative of said sequence; or (B) the sequence as shown in SEQ ID NO:3, or a functional derivative of said sequence; and/or (c) downstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2, or a functional derivative of said sequence.
  • a regulator of energy homeostasis e.g. GLP-1 or GDF-15, or an analogue thereof
  • upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis (e.g. GLP-1 or GDF-15, or an analogue thereof), or for two such regulators (or even for more); and (b) upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40; or (B) the sequence as shown in SEQ ID NO:3 or a sequence which shows 1 to 6 substitutions in comparison to SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID
  • the coding region to be comprised in the RNA molecule of the invention is selected from the group consisting of: (i) a coding region coding for GLP-1 (or GLP-1 analogue), preferably human GLP-1 (hGLP- 1); (ii) a coding region coding for GDF15 (or GDF15 analogue), preferably human GDF15 (hGDF15) (H6D mutant or, preferably, WT); and (iii) a coding region coding for both, GLP-1 (or GLP-1 analogue), preferably hGLP-1, and GDF15 (or GLP-1 analogue), preferably hGDF15 (H6D mutant or, preferably, WT).
  • the UTR(s) as defined in any of (b), supra is/are located at the 5’ end of the coding region as defined in any of (a), supra.
  • the UTR(s) as defined in any of (c), supra is/are located at the 3’ end of the coding region as defined in any of (a), supra.
  • the UTR(s) as defined in any of (b), supra, in particular as defined in any of (b)(A), supra is/are located at the 5’ end of the coding region as defined in any of (a), supra, and the UTR(s) as defined in any of (c), supra, is/are located at the 3’ end of the coding region as defined in any of (a), supra.
  • the GLP-1 (or GLP-1 analogue) may, for example, be encoded by SEQ ID NO:4, 5 or 6 or by SEQ ID NO: 52 or 53, or have the amino acid sequence as depicted in SEQ ID NO:7 or 8 or as depicted in SEQ ID NO: 57, 58, 59 or 60.
  • the GDF15 WT may, for example, be encoded by SEQ ID NOs:9, 10 or 11 or have an amino acid sequence as depicted/comprised in SEQ ID NO:12 or 13 or in SEQ ID NO:61.
  • the GDF15 H6D may, for example, be encoded by SEQ ID NO:14, 15 or 16 or have the amino acid sequence as depicted in SEQ ID NO:17.
  • the GDF15 may, for example, be encoded by SEQ ID NO: 54, 55 or 56.
  • a GLP-1 or GLP-1 analogue as encoded by SEQ ID NO: 52 or 53 is preferred.
  • a GDF15 (or GDF15 analogue) as encoded by SEQ ID NO: 54, 55 or 56 is preferred.
  • RNA molecule according to the invention are selected from the group consisting of (I) an RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (CYBA-GDF15 WT); (II) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as defined herein elsewhere (hAg-GLP-1); (III) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (hAg-GDF15 WT); (IV) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 H6D as defined herein
  • RNA molecule according to the invention are selected from the group consisting of (I) an RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; (II) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as encoded by SEQ ID NO: 52 or 53; (III) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; (IV) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55
  • RNA molecule according to the invention may comprise between its upstream UTR(s) ((b)) and its coding region a (partial) Kozak sequence (like the one defined herein elsewhere (e.g. (C)CCACC or, preferably, GCCGCC or, more preferably, GCCACC), and/or may comprise upstream of its upstream UTR(s) ((b)) a promoter (like the one defined herein elsewhere (e.g. a T7 promoter)). Between the promoter and the upstream UTR(s) ((b)), (an) additional ribonulceotide(s) may be comprised (e.g.
  • RNA ribonucleic acid
  • G, A, U, and C a polymeric molecule which is assembled as a chain of the nucleotides termed G, A, U, and C.
  • Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'.
  • a nitrogenous base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U).
  • A adenine
  • C cytosine
  • G guanine
  • U uracil
  • a phosphate group is attached to the 3' position of one ribose and the 5' position of the next.
  • the nucleotides in a polymeric RNA molecule are covalently linked to each other wherein the phosphate group from one nucleotide binds to the 3' carbon on the subsequent nucleotide, thereby forming a phosphodiester bond.
  • an RNA strand has a 5' end and a 3' end, so named for the carbons on the ribose ring.
  • upstream and downstream relate to the 5' to 3' direction in which RNA transcription takes place.
  • the RNA molecule is a messenger RNA (mRNA) molecule.
  • mRNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression.
  • pre-mRNA primary transcript mRNA
  • RNA polymerase RNA polymerase
  • mature mRNA is translated into a polymer of amino acids: a protein, as summarized in the central dogma of molecular biology.
  • RNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis.
  • a ribonucleic acid (RNA) molecule of present invention comprises two or even three main modules, i.e., (a) a coding region coding for a polypeptide, in particular for a regulator of energy homeostasis like GLP-1 or GDF-15, or for two such regulators (or even more), (b) upstream of said coding region one (or more) UTR(s), and/or (c) downstream of said coding region one (or more) UTR(s) which are different than the UTR(s) of module (b).
  • RNA molecule of the present invention resembles with respect to its structure a “normal” mRNA molecule which occurs in nature, harboring a coding region as well as (an) (5’ and/or 3’) UTR(s) as well as, optionally, a poly-A tail.
  • coding region as used in accordance with the present invention relates to a polymeric RNA molecule which is composed of codons, which are decoded and translated into proteins by the ribosome in accordance with the information provided by the “genetic code”. Coding regions commonly begin with a start codon and end with a stop codon. In general, the start codon is an AUG triplet and the stop codon is UAA, UAG, or UGA.
  • portions of coding regions may serve as regulatory sequences in the pre-mRNA as exonic splicing enhancers or exonic splicing silencers.
  • the coding region of a gene coding for a polypeptide or a protein as used in accordance with the present invention is also known as the coding sequence or CDS (from coding DNA sequence) and is that portion of a gene's DNA or RNA, composed of exons, that codes for a polypeptide or protein. As mentioned, the region is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon.
  • the coding region in mRNA is usually flanked by the five prime untranslated region (5’-UTR) and the three prime untranslated region (3’-UTR) which are also parts of the exons.
  • the coding region or CDS is that portion of the mRNA transcript, i.e., of the coding region coding for a polypeptide as used in accordance with the present invention, that is translated by a ribosome into a polypeptide or a protein.
  • untranslated region or “UTR” as used in accordance with the present invention relates sections of the mRNA upstream the start codon and downstream the stop codon that are not translated, and are, therefore, termed the five prime untranslated region (5'-UTR) and three prime untranslated region (3'-UTR), respectively. These regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA.
  • the 3’ untranslated region (3'-UTR) relates to the section of messenger RNA (mRNA) that immediately follows the translation termination codon. An mRNA molecule is transcribed from the DNA sequence and is later translated into protein.
  • the 5' untranslated region (also known as a Leader Sequence or Leader RNA) is the region of an mRNA that is directly upstream from the start codon.
  • the 5′-UTR begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region.
  • the length of the 5′-UTR tends to be 3- 10 nucleotides long while in eukaryotes it tends to be, longer, generally from 100 to several thousand nucleotides long, but sometimes also shorter UTRs occur in eukaryotes.
  • the 3’-UTR may comprise regulatory regions within the 3'- untranslated region which are known to influence polyadenylation and stability of the mRNA. Many 3'-UTRs also contain AU-rich elements (AREs). Furthermore, the 3'-UTR contains the sequence AAUAAA that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript.
  • an RNA molecule as used in accordance with the present invention may also contain a poly-A tail.
  • a poly-A tail is a long sequence of adenine nucleotides (often several hundred) added to the 3' end of the pre-mRNA by a process called polyadenylation. This tail promotes export from the nucleus and translation, and protects the mRNA from degradation.
  • Polyadenylation is the addition of a poly(A) tail to a messenger RNA.
  • the poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases.
  • the RNA molecule of the present invention preferably comprises two or three main modules, i.e., (a) a coding region coding for a polypeptide, in particular for a regulator of energy homeostasis like GLP-1 or GDF-15 (or analog thereof), or for two such regulators (or even more); and (b) upstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40, or a functional derivative thereof (for example a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40, respectively); or the sequence as shown in SEQ ID NO:3, or a
  • the RNA molecule of the present invention comprises two main modules, i.e., the module (a) (as described herein elsewhere) and at least one of module (b) (as described herein elsewhere) and module (c) (as described herein elsewhere).
  • the RNA molecule of the present invention comprises three main modules, i.e., the module (a) (as described herein elsewhere) and module (b) (as described herein elsewhere) and module (c) (as described herein elsewhere).
  • module (a) is mandatory, it is also envisaged that the RNA molecule may also lack one of the modules (b) or (c).
  • a coding region... is not particularly limited and may be any desired coding region which is to be expressed in a given cell, as long as it codes for a regulator of energy homeostasis like GLP-1 or GDF15 (or analog thereof), or for two such regulators (or even more).
  • this module may be a coding region coding for the desired regulator of energy homeostasispolypeptide, i.e., the desired final product.
  • a coding region in accordance with the invention may also be a nucleotide sequence which differs from a known natural sequence and contains mutations (i.e. point mutations, insertion mutation, deletions and combinations thereof).
  • a coding region in accordance with the invention may partly or to the full extent be a codon optimized sequence derived from the natural sequence to be used as module (a). Codon optimization is a technique to maximize the protein expression by increasing the translational efficiency of a gene of interest. It is known that natural genes do not use the available codons randomly, but show a certain preference for particular codons for the same amino acid.
  • the codon optimized sequence may, for example, be codon optimized for (the expression in) mice or, preferably, for (the expression in) humans.
  • Particular examples of nucleotide sequence encoding such a codon optimized sequence are depicted in SEQ ID NOs:5, 6, 10, 11, 15 and 16 and in SEQ ID NOs:52, 53, 54, 55 and 56.
  • Examples of polypeptides encoded by such a codon optimized sequence are depicted in SEQ ID NOs: 7, 8, 12 and 17.
  • a coding region i.e. GLP-1 and GDF15 (or the respective analogue).
  • the coding region may comprise (or consist of) the respective codon optimized sequence(s) (see above for particular examples).
  • “coding region” should be understood to mean any polyribonucleotide molecule which, if introduced into a cell, is translatable to a polypeptide/protein or fragment thereof in accordance with the invention.
  • polypeptide and “protein” here encompass any kind of amino acid sequence, i.e., chains of two or more amino acids which are each linked via peptide bonds and also includes peptides and fusion proteins. It is particularly envisaged in the context of the invention that the “coding region coding for a polypeptide” contains a ribonucleotide sequence which encodes a polypeptide/protein or fragment thereof whose function in the cell or in the vicinity of the cell is needed or beneficial, e.g., a protein the lack or defective form of which is a trigger for a disease or an illness, the provision of which can moderate or prevent a disease or an illness, or a protein which can promote a process which is beneficial for the body, in a cell or its vicinity.
  • the coding region may contain the sequence for the complete protein or a functional variant thereof.
  • the ribonucleotide sequence of the coding region encodes a polypeptide/protein (or 2 or more polypeptides/proteins) which act(s) as (a), regulator(s) of energy homeostasis, or a functional fragment thereof.
  • a polypeptide/protein or 2 or more polypeptides/proteins
  • a particular example of such a regulator is an anorectic.
  • the particular polypeptide/protein, regulator of energy homeostasis and/or anorectic, to be encoded by the “coding region...” (module (a)) is GLP-1 and GDF15 (or a functional variant/derivative or fragment thereof).
  • Each of these proteins is one whose function is necessary in order to remedy a related disorder (for example a metabolic disorder like obesity, diabetes mellitus, insulin resistance or metabolic syndrome) and/or in order to initiate related processes in vivo (such as decreasing (aberrant) body weight, decreasing food intake, restraining appetite, etc.
  • a related disorder for example a metabolic disorder like obesity, diabetes mellitus, insulin resistance or metabolic syndrome
  • functional variant/derivative is understood to mean a fragment or other variant which in the cell can undertake the function of the protein whose function in the cell is needed or the lack or defective form whereof is pathogenic.
  • the “coding region... ” (module (a)) encodes a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect on the conditions, diseases or disorders as defined herein elsewhere.
  • RNA molecule of the present invention comprising said “coding region...” may be used in nucleic acid therapy and related applications.
  • an increased efficiency of translating a coding region of an RNA molecule into the polypeptide or a protein encoded by said coding region of an introduced exogenous RNA molecule is envisaged to compensate or complement endogenous gene expression, in particular in cases where an endogenous gene is defective or silent, leading to no, insufficient or a defective or a dysfunctional product of gene expression such as is the case with many metabolic diseases like obesity, diabetes mellitus, insulin resistance, metabolic syndrome, etc. to name a few.
  • RNA molecule of the present invention comprising a “coding region...” can appropriately be used in any case where a polypeptide or a protein as defined herein elsewhere, which would naturally be present in the body but is not present or is present in deficient form or in too small quantity because of gene defects or diseases, is to be provided to the body.
  • Respective proteins and the genes encoding them, the deficiency or defect whereof are linked with a disease as defined herein elsewhere, are known and likewise defined herein elsewhere.
  • the respective intact version of the coding region coding for the intact polypeptide or protein can be used in accordance with the present invention.
  • Numerous genetic disorders which may be treated/prevented in accordance with the invention, and which may be caused by the mutation of a single gene, are known and are candidates for the respective mRNA therapeutic approaches. Disorders caused by single-gene mutations, can be dominant or recessive with respect to the likelihood that a certain trait will appear in the offspring.
  • polygenic disorders are caused by two or more genes and the manifestation of the respective disease is often fluent and associated to environmental factors. Examples for polygenic disorders are hypertension, elevated cholesterol level and others.
  • therapeutic mRNA representing one or more of these genes may be beneficial to those patients.
  • a genetic disorder must not have been passed down from the parents' genes, but can also be caused by new mutations. Also in these cases therapeutic mRNA representing the correct gene sequence may be beneficial to the patients.
  • compositions of the invention may comprise an mRNA encoding a therapeutic fusion protein, wherein the encoded therapeutic protein or a homolog thereof is one as described herein elsewhere and the second protein is a signal peptide that allows the secretion of the therapeutic protein.
  • a signal peptide is a short, typically 5-30 amino acids long, amino acids sequence present at the N-terminus of said therapeutic protein and that leads the fusion protein towards the cell’s secretory pathway via certain organelles (i.e. the endoplasmic reticulum, the golgi-apparatus or the endosomes).
  • fusion protein is secreted from the cell or from a cellular organelle or inserted into a cellular membrane (e.g. multi-spanning trans- membrane proteins) at a cellular compartment or at the cell’s surface.
  • the “coding region coding for a polypeptide” may encode one (or two or more) product(s) of genes that cause, predispose or protect from conditions, disorders or diseases as defined herein elsewhere. Examples of such conditions, disorders or diseases that may be treated (or prevented) include those outlined in the following Table 3.
  • the “coding region...” (module (a)) may be translated into a partial or full length protein comprising cellular activity at a level equal to or greater than that of the native protein.
  • the “coding region... (module (a))” encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or preventive effect with respect to the conditions, diseases or disorders selected from the group consisting of the ones as outlined herein elsewhere and, in particular, in the following Table 3.
  • the “coding region... “(module (a)) may be used to express a partial or full length protein with cellular activity at a level equal to or less than that of the native protein. This may allow the treatment of diseases for which the administration of an RNA molecule can be indicated.
  • Table 3 Examples of particular conditions, diseases and disorders to be treated or prevented in accordance with the invention (aberrant) body weight
  • the above Table 3 shows examples of conditions, disorders or diseases which can be treated with the RNA molecule of the present invention wherein the RNA molecule comprises a “coding region...” which encodes an intact or even improved version of a protein/polypeptide or a functional fragment or variant thereof, (the deficient expression of) which is involved in the respective condition, disorder or disease and to which the respective condition, disorder or disease relates, respectively.
  • the protein/polypeptide is GLP-1 and/or GDF-15.
  • Obesity e.g. abdomal obesity
  • diabetes mellitus e.g.
  • the metabolic syndrome to be treated in accordance with the invention may be a set of syndromes.
  • the metabolic syndrome and set of symptoms, respectively, may include obesity (e.g. abdominal obesity), hypertension, cardiovascular disease, elevated fasting plasmid glycose, dyslipidemia, and/or an enhanced inflammatory state.
  • a protein e.g. an enzyme, may be defective (or at least undesirably low expressed), which can be medically/addressedby administering the RNA according to the invention.
  • nucleotide and amino acid sequences can, for example, be obtained from the pertinent gene/protein databases like, for example, UniProt KB.
  • the nucleotide and amino acid sequence of hGLP-1 can, for example, be obtained via the database entry P01275 (e.g. via UniProt KB).
  • the amino acid sequence of hGLP-1 is also depicted in SEQ ID NO:7.
  • An example of a corresponding coding nucleotide sequence is depicted in SEQ ID NO:4 (the nucleotide sequence of EX4GLP1Gly8 as disclosed in Parsons Gene Therapy 14, 2007, 38-48).
  • An example for a corresponding nucleotide sequence which is codon optimized for humans is depicted in SEQ ID NO:5.
  • the nucleotide and amino acid sequence of mGLP-1 can, for example, be obtained via the database entry P55095 (e.g. via UniProt KB).
  • the amino acid sequence of mGLP-1 is also depicted in SEQ ID NO:8.
  • An example of the corresponding codon optimized nucleotide sequence for mouse is depicted in SEQ ID NO:6.
  • the GLP-1 (Glucagon) UTRs may also be obtained from reference sequence in NCBI (NM_002054.4).
  • the 5’-UTR nt position in the reference sequence is 1-256.
  • the 3’-UTR nt position in the reference sequence is 800-1294.
  • the nucleotide and amino acid sequence of hGDF15 can, for example, be obtained via the pertinent database entries (e.g. http://www.ebi.ac.uk/ena/data/view/BC000529 and http://www.uniprot.org/uniprot/Q99988, respectively).
  • An amino acid sequence of hGDF15 WT is also depicted/comprised in SEQ ID NO:12.
  • the amino acid sequence of hGDF15 WT is also depicted in SEQ ID NO:61.
  • An example of a corresponding encoding nucleotide sequence is depicted in SEQ ID NO:9.
  • SEQ ID NO:10 An example for a corresponding coding nucleotide sequence which is codon optimized for humans is depicted in SEQ ID NO:10.
  • the nucleotide and amino acid sequence of mGDF15 can, for example, be obtained by the database entry Q9Z0J7 (e.g. via UniProt KB).
  • the amino acid sequence of is also depicted in SEQ ID NO:13.
  • An example for a corresponding coding nucleotide sequence which is codon optimized for mouse is depicted in SEQ ID NO:11.
  • a particular mutant form of hGDF15 is known in the art, namely hGDF15 H6D.
  • the histidin (H) has been replaced by an aspertate (D) at amino acid position 6 of the hGDF15 WT. It is known for this mutant form that T1/2 is increased.
  • the amino acid sequence of hGDF15 H6D is depicted in SEQ ID NO:17.
  • An example of a corresponding coding nucleotide sequence is depicted in SEQ ID NO:14.
  • An example for a respective coding nucleotide sequence being codon optimized for humans is depicted in SEQ ID NO:15 and an example for a respective coding nucleotide sequence being codon optimized for mouse is depicted in SEQ ID NO:16.
  • the coding region to be employed in the context of the invention may comprise multimers of sequences coding for a regulator of energy homeostasis, like GLP-1 and/or GDF-15 (MIC-1).
  • the multimers may be heteromultimers or homomultimers.
  • Heteromultimers may comprise sequences coding for different regulators of energy homeostasis or sequences coding one regulator of energy homeostasis and a mutant form of the same regulator of energy homeostasis.
  • a multimer may comprise 2, 3, 4, 5, or even more coding sequences for a regulator of energy homeostasis. Dimers, in particular heterodimers are preferred.
  • a non-limiting example of a heterodimer comprises the coding sequences for hGDF15 WT and hGDF15 H6D.
  • the “coding region...” may also encode a variant/derivative of the protein/polypeptide, a variant/derivative of GLP-1 and/or a variant/derivative of GDF-15, respectively.
  • the terms “GLP-1” and “GDF-15” encompass GLP-1 itself and GDF-15 itself, respectively, and also the variants/derivatives of GLP-1 and the variants/derivatives of GDF-15, respectively.
  • the GLP-1 analogues and the GDF-15 analogues are also encompassed by the meaning of variant/derivative of GLP-1 and variant/derivative of GDF-15, respectively. What is said with respect to variant/derivative of GLP-1 and variant/derivative of GDF-15 herein elsewhere also applies to the GLP-1 analogue and the GDF-15 analogue, respectively.
  • a “variant” or “derivative” of a protein/polypeptide is envisaged to exhibit the same function(s), in particular the same biological function(s), more particular the same biological function(s) within a cell, tissue and/or body/patient as the respective protein/polypeptide itself.
  • a GLP-1 variant/derivative is envisaged to exhibit the same (biological) function(s) as GLP-1 itself and a GDF-15 variant/derivative is envisaged to exhibit the same (biological) function(s) as GDF-15 itself, respectively.
  • “The same (biological) function(s)”, for example means, at least one of the biologically relevant functions (see below for examples).
  • GLP-1 and GDF15 may act as (physiological) regulators of energy homeostasis, in particular, as (physiological) long-term regulators of energy homeostasis.
  • Examples of (biological) functions of GLP-1 are (i) incretin function; (ii) increasing the secretion of insulin; (iii) slowing down the emptying process in the stomach; (iv) increasing the feeling of saturation; and/or (v) decreasing of food intake.
  • GLP-1 peripheral functions, like: increasing insulin secretion from the pancreas in a glucose-dependent manner; increasing insulin-sensitivity in both alpha cells and beta cells; increasing beta cells mass and insulin expression, PTM, and secretion; inhibiting acid secretion and gastric emptying in the stomach; promoting insulin sensitivity; and/or decreasing glucagon secretion from the pancreas via GPCR binding; and/or CNS functions, like: increasing hippocampus-related function; increasing acquisition/strength of conditioned taste aversions; increasing anxiety; increasing nausea or visceral malaise (illness); decreasing the hedonic value (pleasure) of food; decreasing the motivation (reward) to eat; decreasing quantity and frequency of food consumption; and/or decreasing general levels of motor activity.
  • GLP-1 biologically active protein-1
  • GDF-15 mediating site protection; (ii) decreasing food intake (in particular in mice); (iii) improving glucose tolerance (in particular in mice); and/or (iv) regulating appetite in a patient.
  • the GLP-1 and GDF-15 as employed in the context of the invention may exhibit at least one, several or even all of the above mentioned (biological) functions.
  • the GLP-1 and GDF-15 as employed in the context of the invention may exhibit those (biological) functions which, upon administration of the GLP-1 or GDF-15 in accordance with the invention, results in the prevention, amelioration and/or cure of the (medical) indications and/or symptoms disclosed herein elsewhere.
  • a prominent example of a GDF-15 variant is the herein elsewhere described GDF-15 H6D variant (e.g. SEQ ID NOs:15, 16, 17)
  • the “coding region...” (module (a)) may also encode fragments of the herein defined proteins/polypeptides, GLP-1 and GDF-15, or fragments of the respective variants/derivatives.
  • fragments can also be considered as “variants”/“derivatives” in accordance with the invention. Consequently, also the GLP-1 fragments and the GDF-15 fragments to be employed in accordance with the invention are envisaged to exhibit the respective (biological) functions (see above). “Variant”/“derivative” in accordance with the invention particularly means that the respective protein/polypeptide has the (biological) activity in accordance with the invention (see above) and the respective “variant”/“derivative” polynucleotide encodes for such a (biologically) active biomolecule, respectively.
  • a variant/derivative protein/polypeptide and polynucleotide, respectively, in accordance with the invention is envisaged to share an identity (in particular sequence identity) of at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98% and even more preferably at least 99% with a reference protein/polypeptide (e.g. the GLP-1 or the GDF15 amino acid sequence as depicted in SEQ ID NOs: 7 and 8 and 12 and 13 and 17, respectively) and polynucleotide (e.g.
  • a reference protein/polypeptide e.g. the GLP-1 or the GDF15 amino acid sequence as depicted in SEQ ID NOs: 7 and 8 and 12 and 13 and 17, respectively
  • polynucleotide e.g.
  • the GLP-1 or the GDF15 encoding nucleic acid molecule as depicted in SEQ ID NOs: 4, 5 and 6 and 9, 10 and 11 and 13, 14 and 15, respectively; or the UTRs as depicted in any of SEQ ID NOs: 1, 2, 3 and 31), respectively (for example based on the number of nucleotides and amino acids comprised in the reference sequence, respectively).
  • the reference protein/polypeptide and polynucleotide, respectively is envisaged to be GLP-1 itself or GDF15 itself, and the respective amino acid and nucleotide sequences, respectively.
  • variant/derivative polynucleotide in accordance with the invention is a polynucleotide that comprises or consists of a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule (e.g. the GLP-1 or the GDF15 encoding nucleic acid molecule as depicted in SEQ ID NOs: 4, 5 and 6 and 9, 10 and 11 and 13, 14 and 15, respectively) encoding a protein/polypeptide in accordance with the invention (e.g. the GLP-1 or the GDF15 amino acid sequence as depicted in SEQ ID NOs: 7 and 8 and 12 and 13 and 17, respectively).
  • a nucleic acid molecule e.g. the GLP-1 or the GDF15 encoding nucleic acid molecule as depicted in SEQ ID NOs: 4, 5 and 6 and 9, 10 and 11 and 13, 14 and 15, respectively
  • a protein/polypeptide in accordance with the invention e.g. the GLP-1 or the
  • hybridizing means that hybridization can occur between one nucleic acid molecule and another (complementary) nucleic acid molecule. Hybridization of two nucleic acid molecules usually occurs under conventional hybridization conditions. In the context of the invention, stringent hybridization conditions are preferred. Hybridization conditions are, for instance, described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA.
  • a “variant”/“derivative” being a polypeptide/protein it is, for example, envisaged that it is or comprises the amino acid sequence of the reference polypeptide/protein (e.g. GLP-1 itself and/or GDF15 itself) having (about) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 40, 50, 60 (or even more) amino acid residue(s) deleted or substituted by (a) different amino acid residue(s). Preferred is/are (a) conservative substitution(s). Any of the herein defined particular polypeptides/proteins (e.g. SEQ ID NO:7, 8, 12, 13 and 17) may be a reference polypeptide/protein.
  • polynucleotides that are codon optimized (in order to ensure proper expression of a corresponding peptide) and/or that are different due to the degeneracy of the genetic code are considered “variant”/ “derivative” in accordance with the invention.
  • any of the variants/derivatives described herein is envisaged to have the (biological) activity and (biological) function, respectively, in accordance with the invention (e.g. decreasing food intake and the like; see herein elsewhere), or to encode the respective proteins/polypeptides.
  • a “variant”/“derivative” in accordance with the invention also encompasses a (biologically active) fragment of the coding region and comprised polypeptide, respectively, as defined herein (e.g. as depicted in SEQ ID NO:4, 5, 6, 9, 10, 11, 13, 14 or 15) or of the encoded protein/polynucleotide (e.g. as depicted in SEQ ID NO:7, 8, 12, 13 and 17).
  • a fragment may be a nucleic acid sequence stretch of at least 30, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140 or at least 150 nucleotides.
  • a fragment may be an amino acid stretch of at least 10, at least 20, at least 30, at least 40, at least 45, at least 46, at least 47, at least 48 or at least 49 amino acid residues.
  • the fragment of the polynucleotide encodes for an amino acid sequence which exhibits (biological) activity (e.g. GLP-1 or GDF-15 activity) in accordance with the invention (see above for examples) and the fragment of the polypeptide/protein exhibits (biological) activity (e.g. GLP-1 or GDF-15 activity) in accordance with the invention (see above for examples), respectively.
  • proglucagon in the intestinal L cells may be cleaved into GLP- 1 (AA positions 1-36).
  • GLP-1 Prior to secretion into the circulation, GLP-1 (AA positions 1-36) is further processed into amidated GLP-1 (AA positions 7-36) and small amounts of glycine-extended GLP- 1 (AA positions 7-37). Both, GLP-1 (AA positions 7-36) and GLP-1 (AA positions 7-37), cause glucose dependent release of insulin by pancreatic beta-cells, stimulate gastric emptying, suppress glucose production and may promote satiety and stimulate glucose disposal in peripheral tissues independent of the actions of insulin.
  • Such fragments are preferred fragments of GLP-1 to be employed in the context of the invention.
  • a GLP-1 fragment to be employed in the context of the invention may be an amino acid stretch of about 25 to 40, preferably about 30 to 37, preferably about 31 or 36 amino acid residues; most preferably such fragments are from the N-terminal part of GLP-1.
  • the premature GDF15/MIC-1 protein consists of 308 amino acids that contain a 29 amino acid signal peptide, a 167 amino acid propeptide, and a 112 amino acid mature protein. Respective fragments are preferred fragments of GDF15/MIC-1 to be employed in the context of the invention.
  • a GDF15/MIC-1 fragment to be employed in the context of the invention may be an amino acid stretch of about 100 to 120, preferably about 105 to 116, preferably about 110 or 114, preferably about 112 amino acid residues; most preferably such fragments comprise the mature GDF15/MIC-1 protein.
  • a polypeptide/protein which is encoded by a “variant”/“derivative” polynucleotide described herein is envisaged to be a “variant”/“derivative” in accordance with the invention.
  • RNA Ribonucleotide
  • module (a) the nucleotide sequence encoding it
  • DNA DNA
  • BspQI motiv This motive is used for plasmid template linearization during in vitro transcription (IVT).
  • IVT in vitro transcription
  • Such a component are a 5’-UTR (e.g. a 5’-UTR as depicted in or encoded by any of SEQ ID NOs: 1, 3, 18, 19, 20, 21, 25, 27, 28, 31 and 32), or a 3’-UTR (e.g. a 3’-UTR as depicted in or encoded by any of SEQ ID NOs: 2, 19, 21 and 26).
  • “Variants”/“derivatives” or “fragments” of these components may also be employed in accordance with the invention. Particular examples of such “variants”/“derivatives” are described in more detail herein elsewhere.
  • the relevant (biological) function of the component itself needs to be maintained.
  • the “variant”/“fragment” of an UTR needs to exhibit the respective UTR function.
  • the “variant”/“derivative”/“fragment” of an UTR needs to result in an RNA molecule having the same or a higher translation efficiency as an RNA molecule which comprises the reference UTR itself.
  • the second module (b) is (are) the one (or more) 5’-UTR(s) as defined herein, in particular the one (or more) CYBA or hAg 5’-UTR(s) as described herein elsewhere (e.g. in Tables 1 and 2, supra, and in the explanations to these tables) and as depicted in SEQ ID NOs:1 and 3, respectively, or the herein defined “variants”, “derivatives” or “fragments” thereof.
  • the second module (b) is (are) the one (or more) 5’-UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40, respectively.
  • second molecule (b) is (are) the one (or more) 5’-UTR(s) comprising the sequence as shown in SEQ ID NO:3 or a sequence which shows 1-6 substitutions in SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:3.
  • “One or more” in this context means that module (b) of the RNA molecule may harbor at least one of the defined 5’-UTRs.
  • the RNA molecule may also harbor two, three or four of these UTRs of the present invention.
  • the RNA molecule may also harbor five or even more of these UTRs of the present invention.
  • the second module (b) is (or comprises) the one (or more) CYBA or hAg 5’-UTR(s) as described herein elsewhere.
  • other (functional) 5’-UTR(s) may, in principle, be employed in accordance with the invention, e.g. in lieu of the one (or more) CYBA or hAg 5’-UTR(s). This may apply especially in cases were the third module (c) is (or comprises) the one (or more) CYBA 3’-UTR(s) as described herein elsewhere.
  • 5’-UTR(s) which may be employed in accordance with the invention may, for example, be (a) minimal UTR(s), e.g. as disclosed in WO 2017/167910.
  • Alternative 5’-UTR(s), which are preferably located directly upstream of said coding region (module (a); optionally without an additional promoter sequence) may, for example, be defined as follows: (A) UTR(s) selected from the group consisting of: (b1) a UTR of the sequence R 2 -CGCCACC (SEQ ID NO:41), or a sequence wherein in said UTR sequence the C at position 6 of SEQ ID NO:41 is substituted by an A and the C at position 7 of SEQ ID NO:41 is substituted by a G; and/or the A at position 5 of SEQ ID NO:41 is substituted by a G; and (b2) a UTR of the sequence R 2 -CNGCCACC (SEQ ID NO:42), wherein the nucleotide N at position 2 of SEQ ID NO
  • the third module (c) is (are) the one (or more) 3’-UTR(s) as defined herein, in particular the one (or more) CYBA 3’-UTR(s) as defined herein elsewhere (e.g. in Table 1, supra, and in the explanations to this table) and as depicted in SEQ ID NO:2, or the herein defined “variants”, “derivatives” or “fragments” thereof.
  • the third module (c) is (are) the one (or more) 3’-UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 (i.e., the above module (c)).
  • “One or more” in this context means that module (c) of the RNA molecule may harbor at least one of the defined 3’-UTRs. The RNA molecule, however, may also harbor two, three or four of these UTRs of the present invention.
  • the RNA molecule may also harbor five or even more of these UTRs of the present invention. However, harboring one of these UTRs is preferred.
  • the full-length sequence of the native human cytochrome b-245 alpha polypeptide (CYBA) mRNA is known in the art and has the sequence as shown in SEQ ID NO:29.
  • the sequence from nucleotides 36 to 71 of the native human CYBA mRNA (SEQ ID NO:25) has been used as the 5’ UTR fragment of the CYBA mRNA (resulting in, for example, the nucleotide sequence 5’- CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC-3‘ (SEQ ID NO:1); a C has been added at the 5 ⁇ end; a full Kozak sequence has been added at the 3 ⁇ end) and the sequence from nucleotides 657 to 723 of the native human CYBA mRNA (SEQ ID NO:26) has been used as the 3’ UTR of the CYBA mRNA (resulting in, for example, the nucleotide sequence 5’-CCUC GCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGAAGCCGG GA-3‘ (SEQ ID NO:2)).
  • the full-length sequence of the native hAg mRNA is also known in the art and has the sequence as shown in SEQ ID NO:30.
  • sequence from nucleotides 31 to 66 of the native hAg mRNA (SEQ ID NO:28) has been used as the 5’-UTR fragment of the hAg mRNA (resulting in, for example, the nucleotide sequence 5’- CTCTTCTGGTCCCCACAGACT CAGAGAGAACGCCACC-3’ (SEQ ID NO:3); a G has been added to create a full Kozak sequence at the 3 ⁇ end).
  • the 5’-UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) but may also be a “variant”/“derivative” 5’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40. In principle, the lower numbers of substitutions are preferred.
  • the above nucleotide substitution in comparison to SEQ ID NO:1 may be performed at position 33 in the sequence of SEQ ID NO:1 or at position 32 in the sequence of SEQ ID NO:40.
  • the nucleotide “U” at this position may be substituted by a “C”. This substitution brings a sequence element of CYBA which is present in SEQ ID NO:1 or SEQ ID NO:40 (GUCGCC) closer to the Kozak consensus sequence of vertebrates.
  • GCCGCC (SEQ ID NO:23).
  • the Kozak consensus sequence of vertebrates has the sequence of GCCRCCAUG (SEQ ID NO:24; the start codon is underlined while “R” indicates any purine) while the mentioned element of CYBA has the sequence of GuCGCCAUG, comprising the motive CuCGCC (the start codon is underlined while the deviation from the vertebrate consensus sequence is indicated by the lower case letter “u”).
  • the UTR sequence(s) which have one or more of the above substitutions may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g. SEQ ID NO:1 or SEQ ID NO:40, respectively), preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:1 or SEQ ID NO:40).
  • the property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples.
  • the translation efficiency is the rate of mRNA translation into polypeptides or proteins within cells.
  • the translation efficiency of a given mRNA is measured as the number of proteins or polypeptides which are translated per mRNA per time unit.
  • Translation is the process in which cellular ribosomes create proteins and is well-known to the skilled person. Briefly, in translation, messenger RNA (mRNA) which is produced by transcription from DNA is decoded by a ribosome to produce a specific amino acid chain or a polypeptide or a protein.
  • mRNA messenger RNA
  • the translation efficiency of a given RNA molecule harboring a modified UTR sequence is preferably higher in comparison to a translation efficiency of the same given RNA but harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)).
  • the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring a modified UTR sequence which are translated per RNA per time unit is higher than the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)) which are translated per RNA per time unit.
  • the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring a modified UTR sequence which are translated per RNA per time unit is similar to or the same as the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)) which are translated per RNA per time unit.
  • translation efficiency can, e.g., be determined by methods described in the appended examples and as outlined in the following.
  • Translation efficiency in the context of the present invention, is the rate of mRNA translated into protein within a cell at a certain time point in relation to the amount of mRNA encoding the respective protein in said cell at the same time point.
  • the translation efficiency is the quotient of the mRNA translated into protein within a cell at a certain time point and the amount of mRNA encoding the respective protein. Both parameters, i.e., the mRNA translated into a protein as well as the amount of mRNA encoding the respective protein, can be determined by methods known in the art.
  • the amount of mRNA translated into protein within a cell can, e.g., be determined by as determined by flow cytometry (FC) while the amount of mRNA encoding the respective protein can, e.g., be measured by qPCR.
  • the UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) or a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising unmodified UTR (e.g.
  • SEQ ID NO:25 (or SEQ ID NO:1 or SEQ ID NO:40)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s), e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40.
  • the addition of (a) nucleotide(s) can be flanking.
  • the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention.
  • the additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides.
  • the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:1 or SEQ ID NO:40, respectively, preferably higher translation efficiency as SEQ ID NO:1 or SEQ ID NO:40, respectively, as defined above.
  • nucleotide(s) can be interspersed.
  • the additional nucleotide(s) may be added/inserted within the nucleotide sequence of the UTR(s) of the present invention.
  • These nucleotide(s) insertions comprise 1, 2, or 3 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:1 or SEQ ID NO:40, preferably higher translation efficiency as SEQ ID NO:1 or SEQ ID NO:40 as defined above.
  • the UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) and modifications thereof. Rather, the specific sequence of SEQ ID NO:1 or SEQ ID NO:40 and modifications thereof merely define the CYBA 5’ core region.
  • the UTR as shown in SEQ ID NO:1 or SEQ ID NO:40 is extended on the 5’ end (i.e., upstream) by at least 1 nucleotide.
  • the UTR as shown in SEQ ID NO:1 or SEQ ID NO:40, respectively is extended on the 5’ end (i.e., upstream) by 1 to 20 nucleotides.
  • the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 20 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40).
  • the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 18, 15, 13, 10, 7 or 5 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40).
  • sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 4, 5 or 2 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40). In other preferred embodiment, the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 1 nucleotide on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40).
  • UTR sequences which are extended on the 5’ end may also be modified as defined herein above, e.g. for SEQ ID NO:1 or SEQ ID NO:40. Accordingly, the same applies, mutatis mutandis, to the UTRs which are extended on the 5’ end as defined above as has been set forth above in the context of the UTR of SEQ ID NO:1 or SEQ ID NO:40.
  • the 5’-UTRs as used in the present invention are also not particularly limited to the above specific sequence of SEQ ID NO:3 (or SEQ ID NO:28) but may also be a “variant”/“derivative” 5’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:3.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 5, 1 to 4 or 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:3.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g. in comparison to SEQ ID NO:3.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:3.
  • the lower numbers of substitutions are preferred.
  • the position of the above nucleotide substitution is performed at position 32 and between positions 31 and 32, respectively, in the sequence of SEQ ID NO:28.
  • a nucleotide “G” at position 32 and between positions 31 and 32, respectively, is inserted into SEQ ID NO:28. This insertion is preferred since it brings the Kozak element of hAG which is (partially) present in SEQ ID NO:28 (CCCACC) closer to the Kozak consensus sequence of vertebrates.
  • the UTR sequence(s) which have one or more of the above substitutions may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g. SEQ ID NO:3), preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:3).
  • the property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples. What has been said herein above with respect to the “translation efficiency” also applies here, mutatis mutandis.
  • the UTR(s) comprising the sequence as shown in SEQ ID NO:3 (or SEQ ID NO:28) or a sequence which shows 1 to 6 substitutions, e.g. in comparison to SEQ ID NO:3, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an unmodified UTR (e.g.
  • SEQ ID NO:28 (or SEQ ID NO:3)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s) in comparison to SEQ ID NO:3.
  • the addition of (a) nucleotide(s) can be flanking.
  • the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention.
  • the additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides.
  • the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:3, preferably higher translation efficiency as SEQ ID NO:3 as defined above.
  • the addition of (a) nucleotide(s) can be interspersed.
  • the additional nucleotide(s) may be added/inserted within the nucleotide sequence of the UTR(s) of the present invention.
  • These nucleotide(s) insertions comprise 1, 2, or 3 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:3, preferably higher translation efficiency as SEQ ID NO:3 as defined above.
  • the UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO: 3 ((or SEQ ID NO:28)) and modifications thereof. Rather, in one embodiment, the UTR as shown in SEQ ID NO:3 is extended at the 5’ end (i.e., upstream) by at least 1 nucleotide.
  • the UTR as shown in SEQ ID NO:3 is extended on the 5’ end (i.e., upstream) by 1 to 20 nucleotides.
  • the sequence of SEQ ID NO:3 extends by 18, 15, 13, 10, 7 or 5 nucleotides on the 5’ end (i.e., upstream).
  • the sequence of SEQ ID NO:3 extends by 4, 5 or 2 nucleotides on the 5’ end (i.e., upstream).
  • the sequence of SEQ ID NO:3 extends by 1 nucleotide on the 5’ end (i.e., upstream).
  • UTR sequences which are extended on the 5’ end may also be modified as defined herein above, e.g. for SEQ ID NO:3. Accordingly, the same applies, mutatis mutandis, to the UTRs which are extended on the 5’ end as defined above as has been set forth above in the context of the UTR of SEQ ID NO:3.
  • the 3’-UTRs as used in the present invention are also not particularly limited to the above specific sequence of SEQ ID NO:2 (or SEQ ID NO:26) but may also be a “variant”/“derivative” 3’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 7 substitutions, e.g.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 6 substitutions, e.g. in comparison to SEQ ID NO:2.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 5 substitutions, e.g. in comparison to SEQ ID NO:2.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:2.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:2.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g.
  • the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:2. Most preferably, the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:2. In principle, the lower numbers of substitutions are preferred.
  • the UTR sequence(s) which have one or more of the above substitutions may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g.
  • SEQ ID NO:2 preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:2).
  • the property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples. What has been said herein above with respect to the “translation efficiency” also applies here, mutatis mutandis.
  • the UTR(s) comprising the sequence as shown in SEQ ID NO:2 (or SEQ ID NO:26) or a sequence which shows 1 to 7 substitutions, e.g.
  • RNA molecule in comparison to SEQ ID NO:2, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an unmodified UTR (e.g. SEQ ID NO:26 (or SEQ ID NO:2)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s) in comparison to SEQ ID NO:2.
  • the addition of nucleotide(s) can be flanking or interspersed.
  • the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention.
  • the additional nucleotide(s) may also be within the nucleotide sequence of the UTR(s) of the present invention.
  • the additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides.
  • the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:2, preferably higher translation efficiency as SEQ ID NO:2 as defined above.
  • the UTR(s) of the present invention as well as RNA molecules containing such UTR(s) may be recombinantly (e.g., in an in vivo or an in vitro system) or synthetically generated/synthesized by methods known to the person skilled in the art. More specifically, the UTRs of the present invention and RNA molecules containing such UTR(s) may be produced either recombinantly in in vivo systems by methods known to the person skilled in the art. Alternatively, the UTRs of the present invention and RNA molecules containing such UTR(s) may be produced in an in vitro system using, for example, an in vitro transcription system.
  • In vitro transcription systems are commonly known and usually require a purified linear DNA template containing a DNA sequence “encoding” module (b) and/or module (c) as outlined in detail further below wherein said DNA sequence is under the control of an appropriate promoter.
  • an in vitro transcription system also commonly requires ribonucleoside triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase which provides the enzymatic activity for the in vitro transcription of the DNA sequence “encoding” the modules (b) and/or (c) into the UTR(s) of the present invention.
  • the UTRs of the present invention and RNA molecules containing such UTR(s) may be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques or by chemical synthesis of the respective DNA-sequences and subsequent in vitro or in vivo transcription of the same.
  • the RNA molecules/polyribonucleic acid molecules of the invention may be modified RNA molecules/polyribonucleic acid molecules.
  • RNA molecule, ribonucleic acid and polyribonucleotide are used interchangeably and, in certain embodiments, include any compound and/or substance that comprises a polymer of nucleotides wherein greater than 50% of the nucleotides are ribonucleotides.
  • polyribonucleotides comprise a polymer of nucleotides wherein greater than 60%, 70%, 75%, 80%, 90%, greater than 95%, greater than 99% or 100% of the nucleotides are ribonucleotides.
  • Polyribonucleotides wherein one or more nucleotides are modified nucleotides may be referred to as modified polyribonucleotides.
  • polyribonucleotides may include modified polyribonucleotides.
  • the sequence of the RNA molecules/polyribonucleotides can be derived from, for example, any suitable nucleic acid that comprises the genetic information of a gene of interest (i.e. of at least one gene of GLP-1 and GDF-15).
  • nucleic acids include genomic DNA, RNA, or cDNA comprising the gene(s) of interest.
  • the polynucleotides can be derived from nucleic acids carrying mutated genes and polymorphisms.
  • RNA molecule/polyribonucleotide of the present invention comprises a sequence which is not particularly limited but comprises, as module (a), any desired coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF- 15.
  • said sequence may be a coding region coding for a desired GLP- 1 and/or GDF-15 polypeptide/protein as outlined herein elsewhere.
  • the RNA molecule/polyribonucleotide further comprises an untranslated sequence positioned upstream (5’) of the module (a)’s start codon or an untranslated sequence positioned downstream (3’) of module (a)’s stop codon, or both, an untranslated sequence positioned upstream (5’) of module (a)’s start codon and an untranslated sequence positioned downstream (3’) of module (a)’s stop codon.
  • an RNA molecule/polyribonucleotide of the present invention may be a modified RNA molecule/polyribonucleotide.
  • module (a) the coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF-15, is modified.
  • This aspect is also encompassed by the meaning of modified RNA molecule/polyribonucleotide in accordance with the invention.
  • ribonucleotides namely, adenosine, guanosine, cytidine and uridine, there exist numerous analogs of each of these nucleobases (modified nucleobases).
  • RNA molecules/polyribonucleotides that include one or more of these analogs are referred to as modified (e.g., modified nucleotides or modified ribonucleotides). Some analogs differ from the above canonical nucleobases, but yet can exist in nature. Other analogs are non-naturally occurring. Either type of analog is contemplated, in principle.
  • RNA molecules/polyribonucleotides of the present invention comprise nucleotide analogs (e.g., the polyribonucleotide comprises a modified polyribonucleotide).
  • RNA molecule/polyribonucleotide or other nucleic acid of the disclosure may also comprise (in addition to or alternatively) modifications in the phosphodiester backbone or in the linkage between nucleobases.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GAAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • LNAs including LNA having a beta- D -ribo configuration, alpha -LNA having an alpha - L -ribo configuration (a diastereomer of LNA), 2 ⁇ -amino-LNA having a 2 ⁇ -amino functionalization, and 2 ⁇ -amino-alpha- LNA having a 2 ⁇ -amino functionalization
  • LNAs locked nucleic acids
  • a modification may be on one or more nucleoside(s) or the backbone of the nucleic acid/polynucleotide molecule. In certain embodiments, a modification may be on both a nucleoside and a backbone linkage. In certain embodiments, a modification may be engineered into a polynucleotide in vitro. In certain embodiments, a modified ribonucleotide/nucleotide may also be synthesized post-transcriptionally by covalent modification of the classical/natural nucleotides/ribonucleotides.
  • RNA molecule/polyribonucleotide of the present invention can be a modified RNA molecule/polyribonucleotide and, in certain embodiments, can comprise analogs of purines and/or analogs of pyrimidines.
  • a modified RNA molecule/polyribonucleotide of the present invention comprises a pyrimidine analog, such as an analog of uridine and/or an analog of cytidine.
  • a modified RNA molecule/polyribonucleotide of the present invention comprises an analog of uridine and an analog of cytidine.
  • the modified RNA molecule/polyribonucleotide does not comprise analogs of adenosine and/or analogs of guanosine.
  • the RNA molecule/polyribonucleotide comprises a single type of analog of uridine and a single type of analog of cytidine (e.g., one type of analog, not a single molecule of analog – the single analog may be present at any of several percentages described herein).
  • the RNA molecule/polyribonucleotide comprises more than one type of analog of uridine and/or cytidine and, optionally and if present, one or more analogs of adenosine and/or guanosine (or none of either or both).
  • a modified uridine (e.g., analog of uridine) is selected from 2-thiouridine, 5 ⁇ - methyluridine, pseudouridine, 5-iodouridine (I5U), 4-thiouridine (S4U), 5-bromouridine (Br5U), 2 ⁇ - methyl-2 ⁇ -deoxyuridine (U2 ⁇ m), 2 ⁇ -amino-2 ⁇ -deoxyuridine (U2 ⁇ NH2), 2 ⁇ -azido-2 ⁇ -deoxyuridine (U2 ⁇ N3), and 2 ⁇ -fluoro-2 ⁇ -deoxyuridine (U2 ⁇ F).
  • 2-thiouridine 5 ⁇ - methyluridine, pseudouridine, 5-iodouridine (I5U), 4-thiouridine (S4U), 5-bromouridine (Br5U), 2 ⁇ - methyl-2 ⁇ -deoxyuridine (U2 ⁇ m), 2 ⁇ -amino-2 ⁇ -deoxyuridine (U2 ⁇ NH2)
  • a modified cytidine (e.g., analog of cytidine) is selected from 5-methylcytidine, 3-methylcytidine, 2-thio-cytidine, 2 ⁇ -methyl-2 ⁇ - deoxycytidine (C2 ⁇ m), 2 ⁇ -amino-2 ⁇ -deoxycytidine (C2 ⁇ NH2), 2 ⁇ -fluoro-2 ⁇ -deoxycytidine (C2 ⁇ F), 5- iodocytidine (I5C), 5-bromocytidine (Br5C) and 2 ⁇ -azido-2 ⁇ -deoxycytidine (C2 ⁇ N3).
  • the foregoing also refers to analogs in their 5’ triphosphate form.
  • the cytidine analog is 5-iodocytidine and the uridine analog is 5-iodouridine.
  • the modified RNA molecule/polyribonucleotide is at least 25% more stable as compared to a non-modified (or unmodified) RNA molecule/polyribonucleotide.
  • the modified RNA molecule/polyribonucleotide can be at least 30% more stable, at least 35% more stable, at least 40% more stable, at least 45% more stable, at least 50% more stable, at least 55% more stable, at least 60% more stable, at least 65% more stable, at least 70% more stable, at least 75% more stable, at least 80% more stable, at least 85% more stable, at least 90% more stable, or at least 95% more stable as compared to a non-modified RNA molecule/polyribonucleotide.
  • stability is measured in vivo.
  • stability is measured in vitro.
  • stability is quantified by measuring the half-life of the polyribonucleotide.
  • a RNA molecule/polyribonucleotide of the present invention can have nucleotides that have been modified in the same form or else a mixture of different modified nucleotides.
  • the modified nucleotides can have modifications that are naturally or not naturally occurring in messenger RNA.
  • a mixture of various modified nucleotides can be used.
  • one or more modified nucleotides within an RNA molecule/polyribonucleotide can have natural modifications, while another part has modifications that are not naturally found in mRNA.
  • some modified nucleotides can have a base modification, while other modified nucleotides have a sugar modification. In the same way, it is possible that all modifications are base modifications or all modifications are sugar modifications or any suitable mixture thereof.
  • an analog e.g., a modified nucleotide
  • an analog can be selected from the group comprising pyridin-4-one ribonucleoside, 5-iodouridine, 5-iodocytidine, 5-aza-uridine, 2’-amino- 2’-deoxycytidine, 2’-fluor-2’-deoxycytidine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-p
  • a modified RNA molecule/polyribonucleotide of the present invention does not include pseudouridine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include 5-methyl cytidine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include 5-methyl uridine.
  • a modified RNA molecule/polyribonucleotide of the present invention comprises analogs of U and analogs of C, wherein such analogs of U may all be the same analog or may be different analogs (e.g., more than one type of analog), and wherein such analogs of C may all be the same analog or may be different analogs (e.g., more than one type of analog).
  • a modified RNA molecule/polyribonucleotide of the present invention does not include analogs of adenosine and analogs of guanosine.
  • RNA molecule/polyribonucleotide comprises a modified polyribonucleotide
  • analogs may be present as a certain proportion of the nucleotides in the compound (e.g., a given percentage of a given nucleobase may be analog, as described herein).
  • An RNA molecule/polyribonucleotide that comprises at least one modified nucleotide is a modified RNA molecule/polyribonucleotide.
  • the modified RNA molecule/polyribonucleotide includes modified or non-naturally occurring (e.g., analogs of or modified) adenosine, cytidine, guanosine, or uridine, such as the analog nucleotides described herein. In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% of the modified RNA molecule/polyribonucleotide includes modified or non- naturally occurring (e.g., analogs of or modified) adenosine, cytidine, guanosine, or uridine.
  • the modified RNA molecule/polyribonucleotide includes modified or non-naturally occurring adenosine, cytidine, guanosine, or uridine.
  • the RNA molecule of the present invention contains a combination of modified and unmodified nucleotides.
  • the RNA molecule of the present invention contains a combination of modified and unmodified nucleotides as described in WO 2011/012316 or WO 2018/127382.
  • modified RNA molecules are also known and commercialized as “SNIM ® RNA”.
  • RNA molecule described in, for example, WO 2011/012316 is reported to show an increased stability and diminished immunogenicity.
  • 5 to 50% of the cytidine nucleotides and 5 to 50% of the uridine nucleotides are modified.
  • the adenosine- and guanosine- containing nucleotides can be unmodified.
  • the adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form.
  • the cytidine and uridine nucleotides are modified and particularly preferably the content of the modified cytidine nucleotides lies in a range from 7.5 to 25% and the content of the modified uridine nucleotides lies in a range from 7.5 to 25%. It has been found that in fact a relatively low content, e.g. only 10% each, of modified cytidine and uridine nucleotides can achieve the desired properties. It is particularly preferred that the modified cytidine nucleotides are methylcytidine residues (in particular 5-methylcytidine residues) and that the modified uridine nucleotides are thiouridine residues (in particular 2-thiouridine residues).
  • the content of modified cytidine nucleotides e.g. (5-)methylcytidines
  • the content of the modified uridine nucleotides is (about) 25% (e.g. ⁇ 2, 3, 4 or 5 %), respectively.
  • the modified RNA molecule of the invention may be prepared by using a particular combination of unmodified nucleotides, adenosine-triphosphate (ATP), guanosine-triphosphate (GTP), uridine- triphosphate (UTP) and cytosine-triphosphate (CTP), as well as chemically modified nucleotides like, for example, (5-)methyl-CTP and (2-)thio-UTP.
  • a certain combination/ratio of unmodified and modified nucleotides may be used (e.g. in an in vitro transcription (IVT), and in an in vitro transcription mix (IVT mix), respectively).
  • the unmodified and modified nucleotides may be present (for an IVT, and in an IVT mix, respectively) at a concentration ratio of ATP : CTP : UTP : (5-)methyl-CTP : (2-)thio-UTP : GTP of 7.57mM : 5.68mM : 5.68mM : 1.89mM : 1.89mM : 1.21mM, respectively, or at a concentration ratio of ATP : GTP : UTP : CTP: (5-)methyl-CTP : (2-)thio-UTP of 7.13mM : 1.14mM : 5.36mM : 5.36mM :0.536mM : 0.536mM, respectively.
  • RNA molecule/polyribonucleotide molecule 5 to 50% of the cytidines are analogs of C and 5 to 50% of the uridines are analogs of U.
  • 5 to 40% of the cytidines are analogs of C and 5 to 40% of the uridines are analogs of U.
  • 5 to 30% of the cytidines are analogs of C and 5 to 30% of the uridines are analogs of U.
  • RNA molecule/polyribonucleotide molecule 10 to 30% of the cytidines are analogs of C and 10 to 30% of the uridines are analogs of U.
  • uridines in such a modified polyribonucleotide molecule 5 to 20% of the cytidines are analogs of C and 5 to 20% of the uridines are analogs of U.
  • in such a modified RNA molecule/polyribonucleotide molecule 5 to 10% of the cytidine nucleotides and 5 to 10% of the uridine nucleotides are modified.
  • RNA molecule/polyribonucleotide molecule 25% of the cytidine nucleotides and 25% of the uridine nucleotides are modified.
  • the adenosine- and guanosine-containing nucleotides can be unmodified.
  • the adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form.
  • analogs of U refers to a single type of analog of U. In certain embodiments, analogs of U refers to two or more types of analogs of U.
  • analogs of C refers to a single type of analog of C. In certain embodiments, analogs of C refers to two or more types of analogs of C.
  • the percentage of cytidines in an RNA molecule/polyribonucleotide that are analogs of cytidine is not the same as the percentage of uridines in the RNA molecule/polyribonucleotide that are analogs of uridine. In certain embodiments, the percentage of analogs of cytidine is lower than the percentage of analogs of uridine.
  • polyribonucleotides of the disclosure comprises less than 15%, less than 10%, less than 5% or less than 2% analogs of adenosine, analogs of guanosine or both.
  • an RNA molecule/polyribonucleotide of the present inention comprises analogs of cytidine and analogs of uridine, and 5 to 20% of the cytidines are analogs of cytidine and 25 to 45% of the uridines are analogs of uridine.
  • the RNA molecule/polyribonucleotide comprises modified and unmodified cytidines and modified and unmodified uridines, and 5 to 20% of the cytidines comprise analogs of cytidine while 25 to 45% of the uridines comprise analogs of uridine.
  • the RNA molecule/polyribonucleotide comprises 5 to 10% analogs of cytidine and 30 to 40% analogs of uridine, such as 7-9% analogs of cytidine, such as about 7, 7.5 or 8% and, such as 32-38% analogs of uridine, such as about 33, 34, 35, 36%.
  • analogs of uridine and analogs of cytidine described herein may be used, optionally excluding pseudouridine.
  • the analog of cytidine comprises or consists of (e.g., in the case of consists of, it is the single analog type used) 5- iodocytidine and the analog of uridine comprises or consists of (e.g., in the case of consists of, it is the single analog type used) 5-iodouridine.
  • the percentage of analogs of a given nucleotide refers to input percentage (e.g., the percentage of analogs in a starting reaction, such as a starting in vitro transcription reaction).
  • the percentage of analogs of a given nucleotide refers to output (e.g., the percentage in a synthesized or transcribed compound).
  • the RNA molecules/polyribonucleotide molecules of the present invention may be produced recombinantly in in vivo systems by methods known to a person skilled in the art which are described in more detail furher below.
  • the modified polyribonucleotide molecules of the present invention may be produced in an in vitro system using, for example, an in vitro transcription system which is described in more detail further below.
  • An in vitro transcription system capable of producing RNA molecules/polyribonucleotides requires an input mixture of modified and unmodified nucleoside triphosphates to produce modified RNA molecules/polyribonucleotides with the desired properties of the present invention.
  • 5 to 50% of the cytidines are analogs of cytidine in such an input mixture and 5 to 50% of the uridines are analogs of uridine in such an input mixture.
  • 5 to 40% of the cytidines are analogs of cytidine in such an input mixture and 5 to 40% of the uridines are analogs of uridine in such an input mixture.
  • 5 to 30% of the cytidines are analogs of cytidine in such a mixture and 5 to 30% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 5 to 30% of the cytidines are analogs of cytidine in such mixture and 10 to 30% of the uridines are analogs of uridine in such mixture. In certain embodiments, 5 to 20% of the cytidines are analogs of cytidine in such an input mixture and 5 to 20% of the uridines are analogs of uridine in such an input mixture.
  • the input mixture does not comprise analogs of adenosine and/or guanosine. In other embodiments, optionally, the input mixture comprises one or more analogs of adenosine and/or guanosine (or none of either or both).
  • the percentage of cytidines in an input mixture that are analogs of cytidine is not the same as the percentage of uridines in an input mixture that are analogs of uridine. In certain embodiments, the percentage of analogs of cytidine in an input mixture is lower than the percentage of analogs of uridine in an input mixture. As noted above, this may be in the presence or the absence of analogs of adenosine and guanosine in the input mixture but, in certain embodiments, is in the absence of analogs of adenosine and analogs of guanosine in the input mixture.
  • an input mixture of nucleotides for an in vitro transcription system that produces a RNA molecule/polyribonucleotide of the present invention comprises analogs of cytidine and analogs of uridine, and 5 to 20% of the cytidines of the input mixture are analogs of cytidine and and 25 to 45% of the uridines of the input mixture are analogs of uridine.
  • the input mixture comprises modified and unmodified cytidines and modified and unmodified uridines, and 5 to 20% of the cytidines of the input mixture comprise analogs of cytidine while 25 to 45% of the uridines of the input mixture comprise analogs of uridine.
  • the input mixture comprises 5 to 10% analogs of cytidine and 30 to 40% analogs of uridine, such as 7-9% analogs of cytidine, such as 7, 7.5 or 8% and, such as 32-38% analogs of uridine, such as 33, 34, 35, 36%.
  • any of the analogs of uridine and analogs of cytidine described herein may be used, optionally excluding pseudouridine.
  • the analog of cytidine comprises or consists of (e.g., it is the single C analog type used) 5-iodocytidine and the analog of uridine comprises or consists of (e.g., it is the single U analog type used) 5-iodouridine.
  • the RNA molecule of the invention may comprise one or more modified nucleosides.
  • the or each modified nucleoside may be selected from the following group of modified nucleosides: Pseudouridine ( ⁇ ), N1-methylpseudouridine (m1 ⁇ ), N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl- pseudouridine, 4-thio-
  • the one or more modified nucleosides may comprise at least one N1-methylpseudouridine (m1 ⁇ ) modification (preferred modification).
  • m1 ⁇ N1-methylpseudouridine
  • at least 50% of the uridines have been modified, in particular in the RNA molecule of/deriving from the coding region (module (a).
  • at least 50% of the uridines have been modified to m1 ⁇ , in particular in the RNA molecule of/deriving from the coding region (module (a)).
  • the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine. Exemplary analogs are also described in the tables above.
  • modified polyribonucleotides encoding the desired polypeptide (module (a)) the analogs and level of modification is, unless indicated otherwise, considered across the entire polyribonucleotide encoding the desired polypeptide (module (a)), including 5’ and(/or) 3’ untranslated regions (e.g., the level of modification is based on input ratios of analogs in an in vitro transcription reaction such that analogs may be incorporated at positions that are transcribed).
  • modified RNA molecules/polyribonucleotide molecules may be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques or by chemical synthesis of the respective DNA sequences and subsequent in vitro or in vivo transcription of the same.
  • upstream and downstream both refer to a relative position in an RNA molecule. In the context of the present invention, upstream is toward the 5' end of the RNA molecule and downstream is toward the 3' end of the molecule.
  • the UTR module (b) e.g., the one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which has 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, as defined hereinabove
  • the UTR module (a) is located upstream of the coding region of module (a).
  • the UTR module (c) e.g., the one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 as defined hereinabove
  • the UTR module (a) is located downstream of the coding region of module (a).
  • the coding region coding for a polypeptide is located between the UTR module (b) and the UTR module (c) and, accordingly, the RNA molecule preferably has the arrangement of 5’-(b)-(a)-(c)-3’.
  • the RNA molecule only harbors one UTR module (i.e., either module (b) (e.g., the one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, as defined hereinabove) or module (c) (e.g., the one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 as defined hereinabove)) the RNA molecule preferably has the arrangement of 5’-(b)-(a)
  • the RNA molecule may be present in the form of fused RNA sequences of modules (a), (b) and/or (c), i.e., a (fusion) RNA molecule which is formed by the expression of a hybrid gene made by combining at least two nucleotide sequences encoding said modules.
  • a cDNA a hybrid gene made by combining at least two nucleotide sequences encoding said modules.
  • this can be accomplished by cloning a cDNA into an expression vector which allows for the translation of the RNA molecule.
  • the DNA molecule encoding the RNA molecule of the present invention may be a fused DNA sequence, i.e., a chimeric molecule which is formed by joining two or more polynucleotides via the phosphate group from one nucleotide bound to the 3' carbon on another nucleotide, forming a phosphodiester bond between the respective ends of one module and the end of another molecule.
  • a fused DNA sequence i.e., a chimeric molecule which is formed by joining two or more polynucleotides via the phosphate group from one nucleotide bound to the 3' carbon on another nucleotide, forming a phosphodiester bond between the respective ends of one module and the end of another molecule.
  • RNA nucleic acid sequence encoding said Protein, polypeptide or enzyme molecule.
  • the at least two modules, preferably all three modules may also be covalently coupled by a chemical conjugate.
  • the modules of the RNA molecule may be chemically synthesized individually and subsequently coupled in a covalent linkage by a phosphodiester bond as outlined above.
  • the UTR module (b) and/or (c) of the present invention in relation to the coding region (a) are described wherein the UTR module (b) (corresponding to the above-defined 5’-UTR fragment of the CYBA or hAg mRNA) is located upstream of the coding region (i.e., at the 5’ end of the coding region) and/or the UTR module (c) (corresponding to the above-defined 3’ UTR of the CYBA or hAg mRNA) is located downstream of the coding region (i.e., at the 3’ end of the coding region).
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said UTR(s) as defined in (b) is/are located at the 5’ end of the coding region as defined in (a).
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (c) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is not a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said UTR(s) as defined in (c) is/are located at the 3’ end of the coding region as defined in (a).
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively; and (c) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is a coding region
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one UTR comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively; and (c) two UTRs comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2; wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis G
  • the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (c) two UTRs comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said RNA molecule comprises said two UTRs as defined in (c) at the 3’ end of the coding region as defined in (a).
  • the herein employed 5’-UTR(s) may contain at its 5 ⁇ -end sequences which correspond to (residual 3’) parts of a promoter and/or at its 3 ⁇ -end a so-called Kozak sequence.
  • a Kozak sequence may be required for ribosome recognition and translation of many genes.
  • Kozak sequences may have a consensus comprising CCR(A/G)CC (see also above), where R is a purine (adenine or guanine) that is located three bases upstream of the start codon (AUG).
  • Non limiting examples of a Kozak sequence to be employed in the context of the invention are GUGGCC, CCCACC, GCCGCC (preferred, especially for the 5 ⁇ -CYBA-UTR), GCCACC (preferred, especially for the 5 ⁇ -hAg-UTR).
  • the RNA molecule of the present invention may also harbor a poly-A tail.
  • a poly- A tail relates to a sequence of adenine nucleotides located at the 3’ end of the RNA.
  • a poly-A tail is commonly added to the 3' end of the RNA by a process called polyadenylation.
  • the present invention relates to any of the above-described RNA, wherein the RNA molecule comprises a poly-A tail at the 3’ end.
  • the length of the poly-A tail is not particularly limited.
  • the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly-A tail has a length of at least 50, 60, 70, 80, 90, 100 or 110 nucleotides.
  • the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly- A tail has a length of at least 120 nucleotides.
  • the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly-A tail has a length of at least 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 nucleotides.
  • a poly-A tail of an optimal length can be established and can be added RNA molecule.
  • the poly-A tail can either be added directly from an encoding DNA template, by using poly(A) polymerase (see, e.g. Pardi, Nature Reviews Drug Discovery 17, 2018, 261–79) or ligation after in-vitro transcription.
  • poly-A tail to be added to the RNA molecule of the invention may have a length of 90 A nucleotides (A90) or more, 100 A nucleotides (A100) or more, 110 A nucleotides (A110) or more, 120 A nucleotides (A120) or more, 130 A nucleotides (A130) or more, 150 A nucleotides (A 150 ) or more, 180 A nucleotides (A 180 ) or more, 190 A nucleotides (A 190 ) or more.
  • An example of a particularly suitable length of poly-A tail is poly( ⁇ A 120 ).
  • the poly-A tail may be a segmented poly-A tail, e.g.
  • the segmented poly-A tail may have the structure A 55-65 -S-A 55-65 wherein S is a single nucleotide selected from C, G, T or U.
  • the poly-A tail may have the structure: A 55-65 -N-S 4 -N-A 55-65 , wherein N is a nucleotide that is not adenine, and wherein S 4 are four nucleotides selected from A, C, G, T or U. .
  • RNA molecule of the present invention is produced by an in vitro transcription method, e.g.
  • the poly-A tail is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct while the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail.
  • the construct according to the present invention may not only comprise the above three main modules (a), (b) and/or (c). Rather, it may be desirable that between the individual modules (a) linker moiety/moieties and/or (a) multiple cloning site(s) is/are placed which may, e.g., facilitate the construction of the construct.
  • Suitable linker moieties and multiple cloning sites are known to the skilled person.
  • the construct of the present invention harbors a multiple cloning site, which is, for example, derived from the plasmid pVAX1 (Invitrogen). All the constructs as outlined in the Example section originate from the construct pVAX A120 which has previously been described in WO2013/182683 A1.
  • a UTR to be employed in accordance with the invention, in particular the 3’ UTR may comprise a sequence for generation of a restriction site, e.g. when comprised in form of a nucleotide sequence in a vector. Suitable restriction sites are known in the art. One non-limiting example of a restriction site is GAAUU.
  • the position of the UTR modules (b) and/or (c) within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is not particularly limited and, accordingly, between the individual modules of the RNA molecule of the present invention there may be a spacing or a gap filled with one or more nucleotides G, A, U and/or C which are not part of the main modules (a), (b) and/or (c).
  • “One or more nucleotides G, A, U and/or C” in this context means that the spacing or gap between the individual modules of the RNA molecule of the present invention is/are filled with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides G, A, U and/or C.
  • the spacing or gap between the individual modules of the RNA molecule of the present invention are filled with 20, 30, 40, 50, 60, 70, 80, 90, 100 or 110 or more nucleotides G, A, U and/or C.
  • the UTR module (b) or (c), within the RNA molecule of the present invention in relation to module (a) i.e., the coding region
  • the UTR module (b) or (c), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the termination codon (i.e., the stop codon) of the coding region of module (a) without any spacing or gap in between, i.e., directly downstream of the termination codon/stop codon of the coding region of module (a).
  • the UTR module (b), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the start codon of the coding region of module (a) without any spacing or gap in between, i.e., directly upstream of the start codon of the coding region of module (a) and the UTR module (c), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the termination codon (i.e., the stop codon) of the coding region of module (a) without any spacing or gap in between, i.e., directly downstream of the termination codon/stop codon of the coding region of module (a).
  • the present invention further relates to a set of 2 (or more) RNA molecules of the invention and as defined herein elsewhere, respectively.
  • the set comprises at least 2 different RNA molecules of the invention, in particular, 2 RNA molecules the “coding regions...” (module (a)) of which encode 2 different regulators of energy homeostasis, respectively.
  • these 2 different regulators are GLP-1 and GDF15 (or “variants”, “derivatives” or “fragments” thereof), e.g. as defined herein, and that the 2 RNA molecules encode GLP-1 and GDF15 (or “variants”, “derivatives” or “fragments” thereof), e.g. as defined herein, respectively.
  • a particular but non-limiting example of a set of 2 (or more) RNA molecules comprises one RNA molecule as defined in one of item (I) (or (III), (IV) or (V)), infra and herein elsewhere, and another RNA molecule as defined in one of item (II) (or (VI)), infra and herein elsewhere.
  • RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (CYBA-GDF15 WT);
  • CYBA-GDF15 WT an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as defined herein elsewhere
  • hAg-GLP-1 an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (hAg-GDF15 WT)
  • IV an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 H6D as defined herein elsewhere (CYBA-GDF15 H6D);
  • V an RNA
  • RNA molecule typically, this is accomplished by cloning a cDNA into an expression vector which allows for the transcription of the entire RNA molecule.
  • a variety of methods are known for making fusion constructs, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid molecule “encoding” the RNA molecule of the present invention.
  • Such a double-stranded nucleic acid molecule i.e., DNA molecule harbors on one strand (i.e., on the coding strand) the DNA sequence corresponding to the RNA molecule of the present invention and, accordingly, “encodes” the RNA molecule of the present invention.
  • such a double-stranded nucleic acid/DNA molecule comprises on a strand the genetic information, when transcribed, the RNA molecule of the present invention as defined herein above.
  • the term “coding” or “encoding” in the context of the present invention is not only used in its conventional sense, i.e., to relate to a gene's DNA that codes for a protein (and, accordingly, the genetic information which may be translated into a polypeptide or a protein amino acid sequence).
  • the construct in a construct wherein the individual DNA sequences encoding the modules (a), (b) and/or (c) are “fused” or linked into a single (chimeric) DNA molecule, the construct also comprises components (i.e., module (b) and/or module (c)) which are not translated into a protein.
  • the DNA sequence corresponding to module (b) and/or module (c) provide the information, i.e., the “code”, for the UTRs’ structure of the present invention and, accordingly, the term “encoding” in the present invention also relates to the genetic information for the UTRs which may be expressed, i.e., transcribed, if, e.g., present in a double-stranded nucleic acid molecule which harbors on one strand the RNA molecule of the present invention.
  • the term “encoding” in the context of the present invention is to be understood in a way that the nucleic acid molecule can be transcribed into the RNA molecule of the present invention which harbours parts encoding a protein or a polypeptide (i.e., module (a)) and parts “encoding” the UTRs (i.e., modules (b) and/or (b)) wherein the latter represent the final product when expressed since UTRs are not translated into proteins or polypeptides.
  • modules (b) and/or (b) i.e., modules (b) and/or (b)
  • Such a double-stranded nucleic acid may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g.
  • vector such as “expression vector” or “cloning vector” in the sense of the present invention is understood as a circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA and which is used as a vehicle to carry genetic material into a cell, where it can be replicated and/or expressed (i.e., transcribed into RNA and translated into a amino acid sequence).
  • a vector containing foreign DNA is termed recombinant DNA.
  • the vector itself is generally a DNA sequence that typically consists of an insert (i.e., module (b) and/or module (c) which are not translated into a protein and module (a) the coding region) and a larger sequence that serves as the "backbone" of the vector. Plasmids in the sense of the present invention are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells and are as such a subpopulation of “vectors” as used in the sense of the present invention. Particular, non-limiting, examples of the RNA molecule of the invention are depicted in SEQ ID NOs:18 and 19.
  • the present invention also relates to a nucleic acid molecule encoding the RNA molecule of the present invention or the set of 2 (or more) RNA molecules of the invention.
  • the present invention further relates to a set of 2 (or more) nucleic acid molecules, at least 2 nucleic acids of which encode 2 different RNA molecules of the invention, respectively.
  • the nucleic acid is, for example a DNA, encoding two of the three main modules (i.e., module (a) and module (b) or module (c)) of the RNA molecule of the present invention.
  • the nucleic acid preferably a DNA, encodes all three main modules (i.e., module (a) and module (b) and module (c)).
  • the above nucleic acid molecule of the present invention preferably is a recombinant nucleic acid molecule but may also comprise naturally occurring nucleic acid molecules.
  • the nucleic acid molecule of the invention may, therefore, be of natural origin, synthetic or semi-synthetic. It may comprise DNA, RNA, locked nucleic acid as well as PNA and it may be a hybrid thereof. It is evident to the person skilled in the art that (a)regulatory sequence(s) may be added to the nucleic acid molecule of the invention encoding the RNA molecule.
  • promoters for example, promoters, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, enhancer or activator sequences, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide, i.e., the RNA molecule, of the invention may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen, Trends Biotech. 12 (1994), 58-62, or a dexamethasone-inducible gene expression system as described, e.g.
  • the nucleic acid molecule encoding the RNA molecule of the invention may comprise one or more promoter sequence(s), and, optionally, any associated regulatory sequences. It may comprise either a whole promoter, and, optionally, associated regulatory sequences, or a fragment thereof. Suitable promoters may be constitutive or inducible promoters as known in the art; these are also contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. Preferred but non-limiting examples of promoters to be employed in the context of the invention are disclosed herein elsewhere, e.g in Table 10.
  • MRNA is transcribed from a gene by a DNA-dependent RNA polymerase, which begins transcribing at the transcription start site (TSS).
  • TSS transcription start site
  • the position of the TSS is determined by the specific promoter sequence (and any other regulatory sequences) upstream of the start codon of the gene.
  • the TSS may be within the promoter sequence.
  • the 5’ UTR to be employed in accordance with the invention may comprise a portion of a promoter sequence.
  • the promoter sequence (and any associated regulatory sequence) or portion thereof can be positioned at the 5’-end of the 5’-UTR.
  • a promoter sequence and/or an associated regulatory sequence can comprise any number of modified or unmodified nucleotides.
  • Promoter sequences and/or any associated regulatory sequences can comprise, for example, at least 150 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, or at least 10000 bases or base pairs.
  • a promoter sequence and/or an associated regulatory sequence can comprise any number of modified or unmodified nucleotides, for example, at most 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or base pairs, 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, or 100 bases or base pairs.
  • DNA sequences of promoters of the disclosure include, but are not limited to, DNA sequences corresponding to the RNA sequences listed in Table 10.
  • modified RNA sequences versions of the promoters listed in Table 10 may be employed.
  • said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
  • Said nucleic acid molecules may be transcribed from an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. In the context of the present invention said nucleic acid molecules may also be labeled.
  • nucleic acid molecule(s) of the invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.
  • the nucleic acid molecule of the invention is part of a vector.
  • nucleic acid molecule which encodes the RNA molecule of the invention comprises the following (1) to (8), most preferably in the depicted order (5’ to 3’): (1) a 5’-cloning site (optional); (2) a promoter (optional but preferred), for example a T7 promoter; (3) a 5’-UTR encoding site as defined herein elsewhere; (4) a (partial) Kozak element (optional but preferred), for example as defined herein elsewhere; (5) a coding region as defined herein elsewhere; (6) a 3’-UTR encoding site as defined herein elsewhere; (7) a poly-A addition site (optional but preferred; e.g.
  • RNA molecule may comprise the following (3) to (7), most preferably in the depicted order (5’ to 3’): (3) a 5’-UTR as defined herein elsewhere; (4) a Kozak element (optional but preferred), for example as defined herein elsewhere; (5) a coding region as defined herein elsewhere; (6) a 3’-UTR as defined herein elsewhere; and (7) a poly-A tail (optional), for example as defined herein elsewhere.
  • a preferred promoter to be employed in the context of the invention may be a T7 promoter, like the T7 promoter as comprised or depicted in any of SEQ ID NOs:18, 19, 20, 21 and 22.
  • the present invention also relates to a vector comprising the nucleic acid molecule of the present invention.
  • the vector is an expression vector.
  • the present invention further relates to a set of 2 (or more) vectors, at least 2 vectors of which comprise 2 different nucleic acid molecules of the invention, respectively.
  • the vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g.
  • the vector of the present invention may, in addition to the sequences of the nucleic acid molecule encoding the RNA molecule of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts.
  • control elements are known to the skilled person and may include a promoter, a splice cassette, translation start codon, translation and insertion site for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.
  • the present invention relates to a vector comprising the nucleic acid molecule of the present invention, wherein the nucleic acid molecule is operably linked to control sequences that are recognized by a host cell when the eukaryotic and/or prokaryotic (host) cell is transfected with the vector.
  • Control elements ensuring expression in eukaryotic and prokaryotic (host) cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Yet, in accordance of the present invention, it is not crucial that the vector itself harbors a sequence for a poly-A tail.
  • the above poly-A tail is part of the construct of the present invention (and not necessarily originally located on the cloning vector) and is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct.
  • the RNA molecule of the present invention is produced by an in vitro transcription method the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail.
  • Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions.
  • Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous Sarcoma Virus), human elongation factor 1 ⁇ - promoter, the glucocorticoid-inducible MMTV-promoter Mouse Mammary Tumor Virus), metallothionein- or tetracyclin-inducible promoters, or enhancers, like CMV enhancer or SV40- enhancer.
  • neurofilament-, PGDF-, NSE-, PrP- , or thy-1-promoters can be employed.
  • Said promoters are known in the art and, inter alia, described in Charron, J. Biol. Chem.270 (1995), 25739-25745.
  • promoters including, for example, the tac-lac-promoter or the trp promoter, has been described.
  • transcription termination signals such as SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), pX (Pagano, Science 255 (1992), 1144-1147), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris, Cell 75 (1995), 791-803), or prokaryotic expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia).
  • the vector of the present invention may also be an expression vector.
  • the nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • the present invention also relates to a host cell comprising the vector of the set of vectors of the present invention.
  • the present invention further relates to a set of 2 (or more) host cells, at least 2 host cells of which comprise 2 different nucleic acid molecules of the invention, respectively, or 2 different vectors of the invention, respectively.
  • the present invention relates to a host (or set of host cells) transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i.e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule.
  • a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule.
  • the term "genetically modified" means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents.
  • the nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.
  • the transformation of the host cell with a vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
  • the host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.
  • the host cell of the present invention may be any prokaryotic or eukaryotic cell.
  • Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis.
  • eukaryotic cells comprise, for example, fungal or animal cells. Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae.
  • suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g.
  • Further suitable cell lines known in the art are obtainable from cell line depositories, like, e.g., the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) or the American Type Culture Collection (ATCC).
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • ATCC American Type Culture Collection
  • primary cells/cell cultures may function as host cells.
  • Said cells are in particular derived from insects (like insects of the species Drosophila or Blatta) or mammals (like human, swine, mouse or rat).
  • Said host cells may also comprise cells from and/or derived from cell lines like neuroblastoma cell lines.
  • the above mentioned primary cells are well known in the art and comprise, inter alia, primary astrocytes, (mixed) spinal cultures or hippocampal cultures.
  • primary astrocytes primary astrocytes
  • spinal cultures or hippocampal cultures.
  • RNA molecule, nucleic acid, vector or host cell also applies to the respective sets thereof, mutatis mutandis.
  • the present invention also relates to methods of producing the RNA molecule (or set of RNA molecule) of the present invention by culturing a host cell (or set of host cells) harbouring an expression vector (or set of expression vector(s) encoding the individual modules of the present invention or the entire RNA molecule (or set thereof) of the invention in culture medium, and recovering the RNA molecule (or set thereof) from the host cell (or set thereof) or culture medium.
  • the present invention may also relate to a method for producing an RNA molecule of the present invention comprising the cultivation of the host cell of the present invention and optionally recovering the RNA molecule from the culture. Methods of recovering and/or subsequently purifying the RNA molecule of the present invention are known to the person skilled in the art.
  • the present invention also relates to methods of producing in an in vitro reaction the RNA molecule of the present invention by methods known to the person skilled in the art. More specifically, the RNA molecule of the present invention may be produced in vitro using an in vitro transcription system. In vitro transcription systems are commonly known and usually require a purified linear DNA template containing a DNA sequence “encoding” module (b) and/or module (c) as outlined above wherein said DNA sequence is under the control of an appropriate promoter.
  • an in vitro transcription system also commonly requires ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase which provides the enzymatic activity for the in vitro transcription of the DNA sequence into the RNA molecule of the present invention.
  • Methods which are commonly used to produce RNA molecules using in vitro transcription are well-known to the person skilled in the art and are, e.g., described in Methods Mol. Biol. 703 (2011):29-41.
  • the above poly-A tail may be part of the construct of the present invention (and not necessarily originally located on the cloning vector) and is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct.
  • the RNA molecule of the present invention is produced by an in vitro transcription method the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail.
  • the RNA molecule of the present invention may also be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques.
  • the present invention also relates to methods of producing in an in vitro reaction the RNA molecule of the present invention by methods known to the person skilled in the art and as outlined above and recovering the RNA molecule from the reaction. Methods of recovering and/or subsequently purifying the RNA molecule of the present invention are known to the person skilled in the art.
  • the RNA molecules as defined above are particularly useful in medical settings and in the treatment or prevention of a certain disease (e.g. as defined herein elsewhere) and, in particular, in RNA-based therapies.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the RNA molecule (or set of RNA molecules) of the present invention, the nucleic acid molecule (or set of nucleic acid molecules) of the present invention, the vector (or the set of vectors) of the present invention or the host cell (or the set of host cells) of the present invention and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the invention is for use in an RNA-based therapy.
  • the pharmaceutical composition according to the invention is for use (i) as an anorectic; (ii) in body weight control, in particular in decreasing (aberrant) body weight; and/or (iii) in the treatment or prevention of a metabolic disorder.
  • the present invention further relates to a method for (i) decreasing food intake; (ii) restraining appetite; (iii) controlling body weight, in particular decreasing (aberrant) body weight; and/or (iv) treating or preventing a metabolic disorder.
  • said method is envisaged to comprise the step of administering a pharmaceutically active amount of the pharmaceutical composition of the invention to a patient in need thereof.
  • the metabolic disorder to be medically addressed in accordance with the invention (treated or prevented) may be selected from the group consisting of (i) obesity, in particular abdominal obesity; (ii) diabetes mellitus, in particular type II diabetes mellitus; (iii) insulin resistance; and/or (iv) metabolic syndrome.
  • the metabolic syndrome may (be a set of symptoms which) include(s) obesity (in particular abdominal obesity), hypertension, cardiovascular disease, elevated fasting plasma glucose, dyslipidemia, and/or an enhanced inflammatory state.
  • the term “treatment” (or “prevention”) and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect.
  • the treatment of the present invention may relate to the treatment of (acute) states of a certain disease but may also relate to the prophylactic treatment in terms of completely or partially preventing a disease or symptom thereof.
  • the term “treatment” is to be understood as being therapeutic in terms of partially or completely curing a disease and/or adverse effect and/or symptoms attributed to the disease.
  • Treatment in this respect means that the subject shows symptoms of the disease.
  • the subject to be treated is in actual need of a treatment and the term “acute treatment” in the context of the present invention relates to the measures taken to actually treat the disease after the onset of the disease or the breakout of the disease.
  • the treatment may also be prophylactic or preventive treatment, i.e., measures taken for disease prevention, e.g., in order to prevent the infection and/or the onset of the disease.
  • the pharmaceutical composition of the present invention may be administered via a large range of classes of forms of administration known to the skilled person.
  • the present invention relates to a pharmaceutical composition, comprising an effective amount of the RNA molecule (or the nucleic acid molecule, the vector or the host cell, or the respective sets) of the present invention in accordance with the above and at least one pharmaceutically acceptable excipient or carrier.
  • An excipient or carrier is an inactive substance formulated alongside the active ingredient, i.e., construct of the present invention in accordance with the above, for the purpose of bulking-up formulations that contain potent active ingredients.
  • Excipients are often referred to as “bulking agents,” “fillers,” or “diluents”. Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. They also can serve various therapeutic-enhancing purposes, such as facilitating drug absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients also depends upon the route of administration and the dosage form, as well as the active ingredient and other factors.
  • the pharmaceutical composition comprising an effective amount of the RNA molecule (or the nucleic acid, vector or host cell, or the respective set) of the present invention may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). It is preferred that said pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier and/or diluent.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose, i.e., in “an effective amount” which can easily be determined by the skilled person by methods known in the art. The dosage regimen will be determined by the attending physician and clinical factors.
  • the construct of the present invention is included in an effective amount.
  • the term "effective amount” refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered.
  • the content of the construct of the present invention in the pharmaceutical composition is not limited as far as it is useful for treatment as described above, but preferably contains 0.0000001-10% by weight per total composition.
  • the construct described herein is preferably employed in a carrier.
  • an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic.
  • the carrier include but are not limited to saline, Ringer's solution and dextrose solution.
  • acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter-ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g.
  • polyvinylpyrrolidone amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine
  • carbohydrates including glucose, mannose, or dextrins
  • monosaccharides e.g. sucrose, mannitol, trehalose or sorbitol
  • chelating agents e.g. EDTA
  • non-ionic surfactants e.g. Tween, Pluronics or polyethylene glycol
  • antioxidants including methionine, ascorbic acid and tocopherol
  • preservatives e.g.
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol).
  • Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. Therapeutic progress can be monitored by periodic assessment.
  • RNA molecule of the present invention or the pharmaceutical composition of the invention may be in sterile aqueous or non-aqueous solutions, suspensions, and emulsions as well as creams and suppositories.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition.
  • Said agents may be, e.g., polyoxyethylene sorbitan monolaurate, available on the market with the commercial name Tween, propylene glycol, EDTA, Citrate, Sucrose as well as other agents being suitable for the intended use of the pharmaceutical composition that are well-known to the person skilled in the art.
  • the term “pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient.
  • the pharmaceutical composition of the present invention may be for use in RNA-based therapies.
  • RNA molecule of the present invention comprising a “coding region...” can be used in RNA-based therapies wherein the “coding region...” encodes a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect.
  • the pharmaceutical composition of the present invention may be for use in RNA-based therapies in the treatment or prevention of a condition, disorder or disease as recited in the above Table 3, or herein elsewhere.
  • RNA-based therapies in accordance with the present invention may be for use in the treatment or prevention of a condition, disorder or disease as recited in the above Table 3, or herein elsewhere.
  • the pharmaceutical composition of the present invention may be for use in RNA-based therapies in cases where gene defects lead to a disease which can then be treated or prevented by a transcript replacement therapy/enzyme replacement therapy with the RNA molecule of the present invention, wherein the RNA molecule comprises a “coding region...” which encodes an intact version of the protein or a functional variant or functional fragment thereof compensating the defective gene.
  • the pharmaceutical composition of the present invention may be for use in RNA-based therapies in the treatment or prevention of a metabolic disorder (e.g. as defined herein elsewhere), in particular, a metabolic disorder related to GLP-1 and/or GDF15.
  • “Related” in this context particularly means that the respective metabolic disorder is, partially or totally, due to a situation, where functional GLP-1 and/or GDF15 is not present (in the body of a subject) or present in deficient form or in too small quantity (for example because of gene defects or diseases); and/or that the respective metabolic disorder is treatable (or preventable) by enhancing GLP-1 and/or GDF15 function.
  • Transcript replacement therapies/enzyme replacement therapies beneficially do not affect the underlying genetic defect, but increase the concentration of the enzyme in which the patient is deficient.
  • the transcript replacement therapy/enzyme replacement therapy may replace the deficient GLP-1 and/or GDF15.
  • the pharmaceutical composition of the present invention may be for use in RNA-based therapies in accordance with the present invention wherein the “coding region...” encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or preventive effect, wherein said polypeptide, protein or peptide is GLP-1 and/or GDF15, or an analogue thereof, respectively.
  • RNA-based therapies in accordance with the present invention may be for use in treating a disease or disorder that is, partially or totally, due to a situation, where functional GLP-1 and/or GDF15 is not present (in the body of a subject) or present in deficient form or in too small quantity (for example because of gene defects or diseases); or any disease or disorder where GLP-1 and/or GDF15 produced in a cell may have a beneficial effect for the patient.
  • cardiovascular diseases include atherosclerosis, coronary heart disease, pulmonary heart disease and cardiomyopathy.
  • Examples of (inherited) metabolic disorders include, but are not limited to, Gaucher’s disease and Phenylketonuria.
  • the invention also relates to a method of an RNA-based therapy.
  • the present invention relates to a method for the treatment of a disease, such a cardiovascular disease, a (inherited) metabolic disorders or a genetic disorder by an RNA-based therapy.
  • a disease such as a cardiovascular disease, a (inherited) metabolic disorders or a genetic disorder by an RNA-based therapy.
  • the preferred embodiments of the method for treatment the same applies, mutatis mutandis, as has been set forth above in the context of the RNA molecule or the pharmaceutical composition for use in RNA- based therapy as defined above.
  • the subject is, in a preferred embodiment, a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee, and human.
  • the subject is a human.
  • the present invention also relates to a kit comprising the RNA molecule of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention or the host cell of the present invention (or the respective sets of the invention).
  • the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of the above and below uses and methods.
  • kits of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
  • the kit of the present invention may be advantageously used, inter alia, for carrying out the methods of the invention, the preparation of the RNA molecule of the invention and could be employed in a variety of applications referred herein, e.g., in the uses as outlined above and below.
  • Another component that can be included in the kit is instructions to a person using a kit for its use. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
  • the present invention also relates to the use of one (or more) 5’-UTR(s) as defined herein elsewhere (e.g.
  • RNA molecule comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively); and/or of one (or more) 3’-UTR(s) as defined herein elsewhere (e.g.
  • RNA molecule comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2) for increasing the efficiency of translating a coding region of an RNA molecule into a polypeptide or a protein encoded by said coding region as being defined above.
  • the present invention relates to the use of one or more UTR(s) as defined herein elsewhere, in particular as defined in any of (b), more particular as defined in any of 1(b)(A), and/or one or more UTR(s) as defined in any of (c), for increasing the efficiency of translating a coding region of an RNA molecule into at least one of the regulators of energy homeostasis GLP- 1 and GDF15 as defined herein elsewhere encoded by said coding region.
  • RNA molecule, the set of RNA molecules, the nucleic acid molecule, the vector, the pharmaceutical composition or the kit of the present invention may be combined with one or more oligomer(s), polymer(s) or lipidoid(s) (for example one or more oligomer(s), polymer(s) or lipidoid(s) as disclosed in WO 2014/207231 and WO 2015/128030) and/or with one or more liposomal transfection reagent(s) (for example one or more liposomal transfection reagent(s) as disclosed in WO 2016/075154).
  • RNA molecule, the set of RNA molecules, the nucleic acid molecule or the vector may be administered together with one or more oligomer(s), polymer(s) or lipidoid(s) and/or one or more liposomal transfection reagent(s).
  • the RNA molecule, the set of RNA molecules, the nucleic acid molecule or the vector may be complexed with one or more oligomer(s), polymers or lipidoid(s) and/or one or more liposomal transfection reagent(s).
  • RNA molecule or set of RNA molecules (or the nucleic acid molecule or the vector) may be formulated with liposomes, to generate lipoplexes, or with subsequent generations of lipid nanocarriers, such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LiNPs), nanostructured lipid carriers, and/or cationic lipid-nucleic acid complexes.
  • LNPs lipid nanoparticles
  • LiNPs lipidoid nanoparticles
  • nanostructured lipid carriers and/or cationic lipid-nucleic acid complexes.
  • cationic lipid-nucleic acid complexes such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LiNPs), nanostructured lipid carriers, and/or cationic lipid-nucleic acid complexes.
  • the LNPs or LiNPs may be comprised in the pharmaceutical composition or the kit of the invention.
  • the pharmaceutical composition or the kit may comprise one or more oligomer(s), polymer(s) or lipidoid(s) and/or one or more liposomal transfection reagent(s).
  • Oligomers, polymers or lipidoids and liposomal transfection reagents are known in the art and are, for example, distributed by OzBiosciences, Marseille, France and Invitrogene, CA, USA.
  • Oligomers, polymers or lipidoids and/or liposomal transfection reagents to be employed according to the invention may be oligomers, polymers or lipidoids as disclosed in WO 2014/207231, WO 2015/128030, WO 2016/075154, Zhang (loc.
  • lipids as, e.g., disclosed in EP2285772 (e.g. DOGTOR) and lipopolyamines as, e.g., disclosed in EP1003711 (e. g. DreamFect TM and DreamFect Gold TM ).
  • Oligomers, polymers, lipidoids and (liposomal) transfection reagents to be employed in accordance with the invention may also comprise one or more (e.g. two, three or four) (further) lipid(s) (e.g.
  • helper lipids like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g- PEG2k-lipids) (e.g. DMPE-PEG (e.g. DMPE-PEG2k), DMG-PEG (e.g. DMG-PEG2k)).
  • PEG-lipids e.g. DMPE-PEG (e.g. DMPE-PEG2k), DMG-PEG (e.g. DMG-PEG2k)
  • DMPE-PEG e.g. DMPE-PEG2k
  • DMG-PEG e.g. DMG-PEG2k
  • Non-limiting examples of (an) oligomer(s), polymer(s) or lipidoid(s) and/or (a) liposomal transfection reagent(s) to be employed in the context of the invention are DOGTOR (preferred) and Lipofectamine TM 2000 (more preferred).
  • Non-limiting examples of (an) oligomer(s), polymer(s) or lipidoid(s) to be employed in the context of the invention are also oligomers, polymers or lipidoids which contain, as one main characteristic, the following common structural entity of formula (I):
  • Such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from: a) an oligomer or polymer comprising a plurality of groups of formula (II) as a side chain and/or as a terminal group: wherein the variables a, b, p, m, n and R 2 to R 6 are defined as follows, independently for each group of formula (II) in a plurality of such groups: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ⁇ 2; and
  • such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from a) and b), wherein a) is an oligomer or polymer comprising a plurality of groups of formula (IIa) as a side chain and/or as a terminal group: -NR 2 ⁇ CH2-(CH2)a-NR 3 -CH2-(CH2)b-NR 4 ⁇ m-[CH2-(CH2)a-NR 5 ]n-R 6 (IIa), wherein a, b, m, n, and R 2 to R 6 are defined as described above, and wherein one or more of the nitrogen atoms indicated in formula (IIa) may be protonated to provide a cationic oligomer or polymer structure; and b) is an oligomer or polymer comprising a plurality of groups of formula (IIIa) as repeating units: -NR 2 ⁇ CH2-(CH2)a-NR 3
  • such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from a lipidoid having the structure of formula (IVa): R 1 -NR 2 ⁇ CH2-(CH2)a-NR 3 -CH2-(CH2)b-NR 4 ⁇ m-[CH2-(CH2)a-NR 5 ]n-R 6 (IVa), wherein a, b, m, n, and R 1 to R 6 are defined as described above, and wherein one or more of the nitrogen atoms indicated in formula (IVa) may be protonated to provide a cationic lipidoid.
  • formula (IVa) R 1 -NR 2 ⁇ CH2-(CH2)a-NR 3 -CH2-(CH2)b-NR 4 ⁇ m-[CH2-(CH2)a-NR 5 ]n-R 6 (IVa), wherein a, b, m, n, and R 1 to R 6 are defined as described above, and wherein one
  • polymers or lipidoids, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa) n may be 1; or m may be 1 and n may be 1.
  • polymers or lipidoids, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa) a may be 1 and b may be 2; or a may be 2 and b may be 1.
  • the oligomer, polymer or lipidoid may be a cationic (e.g. protonated) oligomer, polymer or lipidoid.
  • a cationic lipid which, for example, was prepared by mixing (e.g.100mg) N,N′-Bis(2-aminoethyl)-1,3-propanediamine (e.g. 0.623mmol) with (e.g. 575.07mg) 1,2- Epoxydodecane (e.g.3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)); mixed, for example, for 96h at, for example, 80°C, preferably under constant shaking.
  • oligomer, polymer or lipidoid is also referred to as lipidoid “C12-(2-3-2)”.
  • Other non-limiting examples of oligomers, polymers or lipidoids that may be employed in the context of the invention is a (cationic) oligomer, polymer or lipidoid as disclosed in, for example, Zhang (Tissue Eng Part A 25(1-2), 2019, 131-144; doi: 10.1089/ten.TEA.2018.0112) and/or Jarzebinska (Angew Chem Int Ed Engl 8;55(33), 2016, 9591-5; doi: 10.1002/anie.201603648; and in the supporting information).
  • Such an oligomer, polymer or lipidoid may also be referred to as lipidoid “C12-(2-3-2)”.
  • the above oligomer, polymer or lipidoid (or any other (of the herein described) oligomer, polymer or lipidoid) may be produced as described in Zhang (loc. cit.) and/or Jarzebinska (loc. cit.)
  • An oligomer, polymer or lipidoid, in particular a polymer, to be employed in accordance with the invention may be a copolymer, in particular a statistical copolymer.
  • Such a copolymer may be a copolymer which contains a statistical/random arrangement of alkylene amine repeating units of alternating length (e.g. in contrast to a less preferred polymer which contains analogous arrangements of alkylene amine repeating units of non-alternating length).
  • the copolymer may be a cationic (e.g. protonated) copolymer.
  • Copolymers to be employed in accordance with the invention are known in the art and are, for example, described in EP 14199439.2, WO 01/00708, EP-A11198489 and CA-A12,377,207.
  • the copolymer may be a statistical copolymer comprising a plurality of repeating units (a) independently selected from repeating units of the following formulae (a1) and (a2): C H2 CH2 NH (a1) a plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) to (b4): wherein the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, and wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer.
  • the copolymer may be a statistical copolymer, wherein any repeating units (a) and any repeating units (b) are statistically distributed in the copolymer macromolecule. It is typically obtained from the copolymerization of a mixture of monomers yielding, during the polymerization reaction, the repeating units (a) with monomers yielding, during the polymerization reaction, the repeating units (b).
  • the copolymer is a random copolymer wherein any repeating units (a) and any repeating units (b) are randomly distributed in the polymer macromolecule.
  • the copolymer in accordance with the invention can be a linear, branched or dendritic copolymer.
  • a repeating unit of the formula (a1), (b1) or (b3) with two valencies leads to a propagation of the copolymer structure in a linear manner.
  • a linear copolymer of the invention comprises repeating units of formula (a1) and one or more types of the repeating units of formulae (b1) and (b3), but no repeating units of formula (a2), (b2) or (b4).
  • the presence of a repeating unit of formula (a2), (b2) or (b4) with three valencies provides a branching point in the copolymer structure.
  • a branched copolymer comprises one or more types of the repeating units of formulae (a2), (b2) and (b4), and may further comprise one or more types of the repeating units of formulae (a1), (b1) and (b3).
  • the copolymer in accordance with the invention comprises a plurality of repeating units (a) independently selected from repeating units of formulae (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulae (b1) to (b4) defined above.
  • copolymers comprising a plurality of repeating units (a) independently selected from repeating units of formulae (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulae (b1) and (b2) defined above.
  • the copolymer in accordance with the invention is a branched copolymer comprising one or more types of repeating units selected from repeating units (a2), (b2) and (b4), and which optionally further comprises one or more types of the repeating units of formulae (a1), (b1) and (b3), and in particular a copolymer which comprises repeating units of the formula (a2) and one or more type of the repeating units of formulae (b2) and (b4), and which optionally further comprises one or more types of the repeating units of formulae (a1), (b1) and (b3).
  • a more preferred copolymer is thus a branched copolymer which comprises repeating units of the formula (a2) and repeating units of formula (b2), and which optionally further comprises one or more types of the repeating units of formulae (a1) and (b1).
  • the total number of the repeating units (a) and repeating units (b) is typically 20 or more, preferably 50 or more and more preferably 100 or more.
  • the total number of the repeating units (a) and repeating units (b) is 10,000 or less, preferably 5,000 or less, more preferably 1,000 or less.
  • repeating units (a) and (b) account for 80 mol% or more, more preferably 90 mol% or more of all repeating units in the copolymer. Further preferred are copolymers wherein repeating units (a) selected from (a1) and (a2) and repeating units (b) selected from (b1) and (b2) account for 80 mol% or more, more preferably 90 mol% or more of all repeating units in the copolymer.
  • all of the repeating units in the copolymer are repeating units (a) or (b), in particular that all of the repeating units in the copolymer are repeating units (a) selected from (a1) and (a2) or repeating units (b) selected from (b1) and (b2).
  • the weight average molecular weight of the copolymer in accordance with the present invention generally ranges from 1,000 to 500,000 Da, preferably from 2,500 to 250,000 Da and more preferably 5,000-50,000 less.
  • the terminal groups of the copolymer in accordance with the invention typically comprise one or more types of groups (c) independently selected from groups of the formulae (c1) to (c3) below, preferably from groups of the formulae (c1) and (c2) below: C H2 CH2 NH2 (c1) CH2 CH2 CH2 NH2 (c2) C H2 CH2 CH2 CH2 NH2
  • the terminal groups in the copolymer consist of one or more types of groups (c) independently selected from groups of the formulae (c1) to (c3) below, preferably from groups of the formulae (c1) and (c2).
  • the number of terminal groups depends on the structure of the copolymer in accordance with the invention.
  • the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, and preferably within the range of 0.8/1.0 to 1.0/0.8. This molar ratio can be determined, e.g., via NMR.
  • the ratio is usually determined for a plurality of macromolecules of the copolymer in accordance with the invention, and typically indicates the overall ratio of the sum of repeating units (a) to the sum of repeating units (b) in the plurality of macromolecules.
  • one or more of the nitrogen atoms of the copolymer in accordance with the invention may be protonated to result in a copolymer in a cationic form, typically an oligocationic or polycationic form.
  • the primary, secondary, or tertiary amino groups in the repeating units (a) or (b) or in the terminal groups (c) can act as proton acceptors, especially in water and aqueous solutions, including physiological fluids.
  • the copolymers of the present invention typically have an overall positive charge in an aqueous solution at a pH of below 7.5.
  • An aqueous solution is a solution wherein the solvent comprises 50 % (vol./vol.) or more, preferably 80 or 90 % or more, and most preferably 100 % of water.
  • the compositions in accordance with the invention are in contact with a physiological fluid having a pH of below 7.5, including e.g. blood and lung fluid, they typically contain repeating units (a) and (b) wherein the nitrogen atoms are protonated.
  • the pKa values of the copolymers used in the compositions in accordance with the invention can be determined by acid-base titration using an automated pKa titrator. The net charge at a given pH value can then be calculated e.g. from the Henderson–Hasselbach equation. Any charge may be shared across several of the basic centres and cannot necessarily be attributed to a single point.
  • the copolymers used in the compositions in accordance with the invention comprise repeating units with amino groups in protonated state and repeating units with amino groups in unprotonated state.
  • the copolymers in accordance with the invention as well as the compositions in accordance with the invention may also be provided as a dry salt form which contains the copolymer in a cationic form.
  • counterions (anions) for the positive charges of protonated amino groups in compositions according to the invention comprising the copolymer and nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA are typically provided by anionic moieties contained in the nucleic acid.
  • a preferred copolymer in accordance with the invention is a random copolymer, wherein 80 mol% or more of all repeating units, more preferably all repeating units, are formed by a plurality of repeating units (a) independently selected from repeating units of the following formulae (a1) and (a2): C H2 CH2 NH (a1) a plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) and (b2): wherein the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, more preferably within the range of 0.8/1.0 to 1.0/0.8; wherein the terminal groups of the copolymer are formed by groups (a) independently selected from repeating units of the following formulae (a1) and (a2): C H2 CH2 NH (a1) a plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) and (b2)
  • the copolymer is a branched copolymer, comprising units (a2) and (b2), optionally together with units (a1) and/or (b1).
  • the copolymers in accordance with the invention can be conveniently prepared with procedures analogous to those known for the preparation of polyalkyleneimines, such as branched or linear polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the monomers used for the production of the copolymers will have to be adjusted accordingly.
  • the monomers can be conveniently reacted in a quantitative manner, such that the ratio of the units (a) and (b) in the copolymer can be adjusted by adjusting the monomer ratio accordingly in the monomer mixture subjected to polymerization.
  • polyethyleneimine can be prepared e.g. via ring-opening polymerization of aziridine
  • the copolymers in accordance with the invention can be prepared via ring opening polymerization of a monomer mixture comprising or consisting of aziridine, azetidine and, where applicable pyrrolidine, or, in preferred embodiments, of aziridine and azetidine.
  • the expression “where applicable” refers to the presence or absence of repeating units (b3) and (b4) or terminal groups (c3) which would be formed by the pyrrolidine.
  • the ring opening polymerization of the non-substituted cyclic amines usually leads to branched copolymers.
  • Linear copolymers in accordance with the invention can be prepared, e.g., via polymerization of suitable N-substituted aziridines, N-substituted azetidines and N-substituted pyrrolidines, or N-substituted aziridines and N-substituted azetidines, which may be followed e.g. by a hydrolytic cleavage of N-substituents attached to the resulting polyalkyleneimine chain, e.g. in analogy to the procedure published in Katrien F. Weyts, Eric J. Goethals, New synthesis of linear polyethyleneimine, Polymer Bulletin, January 1988, Volume 19, Issue 1, pp 13-19.
  • dendrimer for the preparation of a dendrimer (or dendritic copolymer), synthetic strategies can be analogously applied which are known for the production of polyethyleneimine or polypropyleneamine dendrimers.
  • Polypropylenimine dendrimers can be synthesized from acrylonitrile building blocks using a repetitive sequence of a Michael addition to a primary amine, followed by a heterogeneously catalyzed hydrogenation (Newkome and Shreiner Poly(amidoamine), polypropylenimine, and related dendrimers and dendrons possessing different 1 ⁇ 2 branching motifs: An overview of the divergent procedures. Polymer 49 (2008) 1- 173; De Brabander-Van Den Berg et al.
  • Polyethylenimine dendrimers can be produced using a repetitive sequence of a Michael addition of a vinyl bromide building block to a primary amine followed by a conversion of alkylbromide to amine using a Gabriel amine synthesis method (Yemul & Imae, Synthesis and characterization of poly(ethyleneimine) dendrimers, Colloid Polym Sci (2008) 286:747–752).
  • a Gabriel amine synthesis method Yemul & Imae, Synthesis and characterization of poly(ethyleneimine) dendrimers, Colloid Polym Sci (2008) 286:747–752
  • dendrimer generations with layers comprising or consisting of random compositions of repeating units of formula (a2), (b2) and (b4) and preferably repeating units (a2) and (b2) can be generated.
  • the ring opening polymerization of aziridine and azetidine, or of aziridine, azetidine and pyrrolidine, can be carried out in solution, e.g. in water.
  • the total monomer concentration is not particularly limited, typical concentrations range from 10% wt/wt to 80% wt/wt, preferably 30% wt/wt to 60% wt/wt.
  • the polymerization is initiated by protons, such that it is preferred to add a Br ⁇ nsted acid, in particular a mineral acid such as sulphuric acid to the reaction system.
  • a Br ⁇ nsted acid in particular a mineral acid such as sulphuric acid
  • Small amounts of acid are generally sufficient, such as 0.001 to 0.01 equivalents, based on the total concentration of monomers.
  • the reaction proceeds at convenient rates e.g. in the temperature range of 50 to 150°C, in particular 90 to 140°C. In these ranges, higher molecular weight copolymers are usually at higher temperatures, and lower molecular weight copolymers at lower temperatures.
  • a lipidoid is preferred among the oligomers, polymers or lipidoids to be employed in accordance with the invention, in particular as compared to an oligomer and, more particular particular, to a polymer
  • Said cationic lipid may be prepared by mixing, e.g., 100mg N,N′-Bis(2-aminoethyl)-1,3-propanediamine (0.623mmol) with, e.g., 575.07mg 1,2- Epoxydodecane (3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)).
  • Said mixing may be, for example, for 96h at, for example, 80°C, preferably under constant shaking.
  • a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R 1 to R 6 are defined as follows: a is 1 and b is 2, p is 1, m is 1, n is 1; and R 1 to R 6 are independently of each other selected from hydrogen, a group -CH 2 -CH(OH)-R 7 , a group -CH(R 7 )-CH 2 -OH, wherein R 7 is C10 alkyl; provided that at least two residues among R 1 to R 6 are selected from a group -CH 2 -CH(OH)-R 7 , a group -CH(R 7 )-CH 2 -OH, wherein R 7 is C10 alkyl; wherein it may be provided that at least one of R 1 , R 2 , R 5 and(/or) and R 6 is hydrogen; and wherein one
  • a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R 1 to R 6 are defined as follows: a is 1 and b is 2; p is 1; m is 1; and n is 1; and R 1 to R 6 are independently of each other selected from hydrogen; a group -CH 2 -CH(OH)-R 7 ; a group -CH(R 7 )-CH2-OH, wherein R 7 is C10 alkyl; wherein it may be provided that at least one of R 1 , R 2 , R 5 and(/or) and R 6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV)
  • a cationic lipidoid that may be employed in the context of the invention is
  • Said cationic lipid may be prepared by mixing N,N′- Bis(2-aminoethyl)-1,3-propanediamine with 1,2-Epoxydodecane. More specifically, said cationic lipid may be prepared by mixing, e.g., 100mg N,N′-Bis(2-aminoethyl)-1,3-propanediamine (0.623mmol) with, e.g., 575.07mg 1,2-Epoxydodecane (3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)).
  • Non-limiting examples of (lipofection) complexes or combinations to be employed in accordance with the invention are Lipofectamine TM 2000/RNA lipoplexes, DOGTOR/RNA lipoplexes, C12-(2- 3-2)/RNA lipoplexes and “helper lipid(s)”/RNA lipoplexes.
  • the RNA (or (lipofection) complexes or combinations) to be employed in accordance with the invention may also be prepared for magnetofection, prepared to be transfected by magnetofection, delivered/introduced via magnetofection and/or administered via magnetofection.
  • a cationic lipidoid of the following formula (V) and/or formula (VI) may be employed according to the invention (e.g. comprised in the pharmaceutical composition, the kit, or LiNPs): and/or
  • the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector) of the invention can be delivered to target cells and/or target tissues in vivo, ex-vivo and/or in vitro using LNPs or LiNPs.
  • LNPs and LiNPs can be distinguished from other carriers due to their small size, their homogenous size distribution and their structure; and are especially suited for administration of a subject.
  • the skilled person knows method for the production of LNPs and LiNPs.
  • the production of LNPs or LiNPs involves a combination of lipids and/or lipidoids, such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol. This mixture is then subjected to a process called nanoprecipitation, which involves rapidly mixing the lipid solution with a non-solvent, such as a nucleic acid dissolved in water, under controlled conditions of temperature, pressure, and stirring rate.
  • a non-solvent such as a nucleic acid dissolved in water
  • the lipids self-assemble into complex nanoscale structures, which trap and protect the therapeutic nucleic acids of the invention inside.
  • the nanoparticles may also be further modified with various surface coatings, such as polyethylene glycol (PEG), to improve their stability and reduce their tendency to be cleared by the immune system.
  • the LiNPs may comprise as component (a) the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector) of the invention, as component (b) a (ionizable) lipid or an (ionizable) lipidoid, and optionally as component (c) helper lipids, e.h. as defined herein elewhere.
  • the pharmaceutical composition, kit or LiNP may (further) comprise helper lipids as described in the following or herein elsewhere.
  • the herein described agents and reagents for administering, delivering and/or introducing the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector), e.g. into a target cell or a target tissue, and the herein described lipids and lipidoids may be combined with one or more (e.g., two, three or four) further lipid(s) (like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g. DMPE- PEG, DMG-PEG2000)).
  • helper lipids may support the desired function of the therapeutic agents and the lipidoids (support and/or increase the delivery and/or introduction of RNA into the cell or tissue and improve transfection efficiency, respectively); and function as respective “helper lipids”.
  • helper lipids are cholesterol, DPPC, DOPE and/or PEG- lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG-PEG2000).
  • the further lipids e.g., “helper lipids” may also be part(s) of the herein disclosed complexes/particles. The skilled person is readily in the position to prepare complexes/particles in accordance with the invention.
  • helper lipids examples include lipids, “helper lipids”) and ratios of the cationic lipidoid(s) and the further lipids (e.g. “helper lipids”).
  • Such ratios may be molar ratios of [1-4 : 1-5], [3-4 : 4-6], [about 4 : about 5], [about 4 : about 5.3] of cationic lipidoid(s) : further lipid(s), (the more narrow ranges are preferred).
  • the cationic lipidoid may be combined with three further lipids, like DPPC, cholesterol, and DMG-PEG2000, preferably at a molar ratio of ⁇ 8.0 : ⁇ 5.3 : ⁇ 4.4 : ⁇ 0.9, respectively, or, more particularly, 8.00 : 5.29 : 4.41 : 0.88, respectively.
  • the lipidoids according to formula (b-1), (b-1b), (b-V), (b-VI) and (b-VII) are as described above and used with helper lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00:5.29:4.41:0.88 for formulating lipidoid nanoparticles.
  • the pharmaceutical composition, kit or LiNPs may comprises a LiNP comprising the following components: a) an RNA molecule, or set of RNA molecules (or a nucleic acid molecule or a vector) according to the invention, b) a cationic lipidoid as defined herein elsewhere, and c) optionally one or more helper lipid(s), e.g.
  • the LiNP/NLP comprises a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks.
  • a composition in which the R-isomer of formula (V) may be formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000, e.g.
  • composition in which the lipidoid of formula (VI) may be formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000, e.g. at the molar ratios 8.00 : 5.29 : 4.41 : 0.88; also referred herein as “Formulation II”.
  • the LiNPs may comprise Formulation I and/or Formulation II.
  • the cationic lipidoid to mRNA ratios in the LiNP can readily be controlled in terms of the mole ratio of nitrogen atoms of the cationic lipidoid (N) to phosphate groups in the mRNA (P) (N/P ratio).
  • the other lipid components can be calculated according the target molar lipid proportions relative to the cationic lipidoid as discussed above, and may be for example 8.00 : 5.29 : 4.41 : 0.88 for cationic lipidoid, DPPC, cholesterol and PEG-lipid DMG-PEG2000, respectively.
  • the final N/P ratio of a cationic lipidoid e.g.
  • lipid or lipidoid nanoparticles e.g. as contained in the (suspension or aerosol) formulation in accordance with the invention, preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm.
  • the indicated particle diameter is the hydrodynamic diameter of the particles, as determined by dynamic light scattering (DLS).
  • the polydispersity index of the nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2.
  • the polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C.
  • LDS dynamic light scattering
  • Figure 1 Fluorescence microscopy and flow cytometry data of A549 cells.
  • FIG. 1 Schematic illustration of therapeutic mRNA, consisting of a 5’ CAP, a 5’ UTR, an encoding region, a 3’ UTR and a poly-A tail.
  • B Fluorescence microscopy pictures taken with 4x magnification (JULYTM) at 24 h post-transfection. All constructs showed improved protein expression levels as compared to the control.
  • C The percentage of d2EGFP positive cells as determined by FC is similar for all constructs. Propidium iodide was used to detect dead cells. The applied gates ensured exclusion of dead cells and untransfected cells.
  • D At 48 h post transfection, sustained protein expression was higher for the stabilized constructs as compared to the control.
  • FIG. 2 Time courses of protein expression as determined by FC for A549 cells (A) and Huh7 cells (B). Mean fluorescence intensities normalized to the control are plotted versus time in a log- linear plot. With increasing time post transfection, the elevated protein expression levels of the stabilized constructs become more and more evident. The bars corresponding to the control, 5’UTR and 3’UTR constructs, respectively, as well as to the constructs 5’+3’, 5’+2x3’ and 2x3’ are differently shaded as shown on the right hand side of the figure.
  • Figure 3 Microstructured multi-channel slides for parallel single-cell assays to test differently stabilized mRNA constructs.
  • FIG. 1 Cell-adhesive, microstructured protein patterns with cell-repellent PEG areas in between allow ordered cell arrangement. Fluorescently labeled fibronectin was used to visualize the micropattern.
  • B Fluorescent A549 cells adhering to fibronectin patterns inside a microchannel (three hours after seeding).
  • C Schematic drawing of mRNA lipofection (on the left) and reaction scheme underlying our analytical solution (on the right).
  • D Exemplary time courses of mRNA- mediated d2EGFP expression in A549 cells. Bold black lines are representative fits to the theoretical translation model.
  • Figure 4 Distributions of expression rates K, mRNA life times, and d2EGFP life times and corresponding mean values with schematic representations of the constructs.
  • A Distributions of expression rate K, which is the product of the initial number of mRNA molecules and the translation rates. The fact that the distributions are similarly shaped indicates that the transfection kinetics and the translation rates are very similar.
  • B The distributions of the mRNA half-lives show great variations in their broadness. As a guide to the eye, dotted lines indicate the mean half-life of the control.
  • C Distributions of d2EGFP half-lives. As expected, the distributions of the different constructs are similarly shaped and show comparable mean values. As a guide to the eye, the overall mean half-life of d2EGFP based on all measured half-lives is shown as a dotted line.
  • FIG. 6 AUC and mRNA life time prolongation factors of the different constructs.
  • A Schematic representation of the AUC to illustrate the interplay between mRNA translation and degradation of mRNA and protein.
  • B and (C) AUC of the different constructs as analysed for ⁇ . Crosses show relative AUCs of different experiments, the bars correspond to the mean of all single-cell AUCs.
  • D and (E) mRNA life time prolongation factors. All modifications result in prolonged mRNA life times as compared to the control.
  • FIG. 7 Fluorescence microscopy and flow cytometry data of Huh7 cells.
  • A Fluorescence microscopy pictures taken with 4x magnification (JULYTM) at 24 h post- transfection. All constructs showed improved protein expression levels as compared to the control.
  • B The percentage of d2EGFP positive cells as determined by FC is similar for all constructs. Propidium iodide was used to detect dead cells. The applied gates ensured exclusion of dead cells and untransfected cells.
  • C At 48 h post transfection, sustained protein expression was higher for the stabilized constructs as compared to the control.
  • Figure 8 Determination of mRNA half-life by qRT-PCR in A549 and Huh7 cells.
  • the cells were transfected according to the protocol as described in Materials & Methods part. Absolute mRNA quantification at 4, 8, 24, 36, 48, 60, 72 hours for all mRNA constructs was determined in A549 (see Figure 8 A) and in Huh7 (see Figure 8 B). Out of this data the mRNA half-life was calculated. The physical half-life was normalized to the control.
  • Figure 9 Transfection efficiencies on microstructured substrates. Percentage of transfected cells and corresponding standard deviations for A549 cells and Huh7 cells transfected with modified RNA/SNIM ® RNA with help of LipofectamineTM2000 or DOGTOR.
  • FIG. 10 Distributions of directly measured d2EGFP half-lives.
  • A Exemplary time courses of cycloheximide-induced d2EGFP degradation in Huh7 cells. Bold black lines are simple exponential fits for protein degradation.
  • B Distribution of d2EGFP half- lives measured in A549 cells, yielding a mean half-life of 2.46 h (std 0.71 h).
  • C Distribution of d2EGFP half-lives measured in Huh7 cells, yielding a mean half-life of 4.04 h (std 1.82 h).
  • Figure 11 Distribution of the single-cell AUCs. AUCs were calculated according to equation 3 below.
  • FIG. 12 A549 data are shown in the left column, Huh7 data are shown in the right column.
  • Figure 12 Comparison of the constructs #2 o #5 having UTRs of different genes as indicated in Table 4 with the CYBA-UTR #1 construct.
  • Figure 13 GLP1 expression in HEK293 cells quantified at 24 h post transfection.
  • Figure 14 Kinetic of GLP1 expression in HEK293 cells (63 ng/well).
  • Figure 15 GLP1 expression in HEK293 cells (24 h).
  • Figure 16 GDF15 expression in HEK293 cells quantified at 72 h* post transfection. The six groups of bars correspond to ETH, hAg, nat, ETH, hAg, nat, from left to right, respectively (cf.
  • Figure 17 Kinetics of GDF15 expression in HEK293 cells (250 ng/well).
  • Figure 18 modified RNA/SNIM ® RNA containing WT sequence of GDF-15 resulted in highest plasma protein levels in vivo after 6 hours.
  • Figure 19 modified RNA/SNIM ® RNA GDF-15 reduces food consumption and facilitates weight loss at already low doses.
  • A Food consumed within the last 24 hours after injection of test item.
  • B Weight lost within 24 hours after injection of test item.
  • Figure 20 1 mg/kg BW modified RNA/SNIM ® RNA GDF-15 results in elevated GDF-15 plasma levels up to 24 hours post injection.
  • A GDF-15 plasma levels measured 24 hours after modified RNA/SNIM ® RNA injection modified RNA/SNIM ® RNA.
  • B Plasma glucose, determined using a Bayer ContourXT 24 hours after the application of test item.
  • Figure 21 modified RNA/SNIM ® RNA GLP-1 reduces food consumption (36% vs Succrose, 19% vs STOP) and body weight.
  • A Food consumed within the last 24 hours after modified RNA/SNIM ® RNA injection.
  • B Weight lost within 24 hours after injection of modified RNA/SNIM ® RNA.
  • Figure 22 GLP-1 plasma levels are elevated against Succrose control, but not against STOP control.
  • FIG. 24 Active GLP-1 in plasma 24 hours after injection of modified RNA/SNIM ® RNA.
  • Figure 23 Both, GDF-15 and GLP-1 coding modified RNA/SNIM ® RNA facilitate statistically significant reduction of food consumption.
  • Figure 24 For hGDF15, normalizing food consumption to 100 g/mouse does not result in significant changes of results.
  • A Food consumed within the last 24 hours after modified RNA/SNIM ® RNA injection.
  • B Decrease of food intake within 24 hours upon application of hGDF15 coding modified RNA/SNIM ® RNA and controls.
  • Figure 25 For GLP-1, normalizing food consumption to 100 g/mouse does not result in significant changes of results.
  • A Food consumed within the last 24 hours after modified RNA/SNIM ® RNA injection.
  • B Decrease of food intake within 24 hours upon application of GLP-1 coding modified RNA/SNIM ® RNA and controls.
  • Figure 26 Application of 2% Sucrose solution does not alter food intake of test animals.
  • Figure 27 Inflammatory cytokines are within physiological ranges 24 hours after application of Test items.
  • Figure 28 Application GLP-1 coding modified RNA/SNIM ® RNA results in significantly increased IGF-1 levels after 24 hours.
  • RNA/SNIM ® mRNAs for the production of modified RNA/SNIM ® mRNAs, chemically modified nucleotides namely methyl-CTP and thio-UTP (Jena Bioscience, Germany) were added to a final concentration of ATP:CTP:UTP:methyl-CTP:thio- UTP:GTP of 7.57mM:5.68mM:5.68mM:1.89mM:1.89mM:1.21mM.
  • Figure 1A A schematic representation of an exemplary mRNA construct can be seen in Figure 1A. The exact sequences of the UTRs are given herein elsewhere (e.g. in the above Tables 1 and 2.
  • Table 5 Secondary structures (mfold) In Table 5, features of the mRNA constructs such as free minimum energy ( ⁇ G) and secondary structures found at both ends and within the UTRs are listed. The folding platform mfold was used to predict mRNA secondary structures (40). For each construct, we compared the eight secondary structures that have the highest free energy. The highest free energy values are predicted for the 2x3’ UTR and the 3’ UTR constructs. The 5’ end of each mRNA construct partially binds with the 3’UTR or the 5’UTR, except for the control construct, which binds to the coding sequence (cds). Interestingly, the 5’ end of the 2x3’ mRNA construct forms a stabilizing hairpin with itself.
  • Lipofectamine TM 2000 Complexes were prepared at a ratio of 2.5 ⁇ l Lipofectamine TM 2000 per 1 ⁇ g mRNA.
  • Lipofectamine TM 2000 and mRNA were diluted separately in OptiMEM transfection medium in a total volume of 50 ⁇ l, each. These mixtures were incubated at room temperature for 5 minutes. The mRNA solution was then mixed with the Lipofectamine TM 2000 solution, followed by another 20 minutes of incubation at room temperature. After incubation, 900 ⁇ l of OptiMEM were added to the lipoplex solution.
  • the reaction was neutralized by adding 80 ⁇ l 1xPBS, supplemented with 2% FBS. Cells were mixed by pipetting and were transferred into a 96 well plate appropriate for flow cytometric measurements. Finally, 5 ⁇ l of Propidium iodide (final concentration 1 ⁇ g/ml) were added per well and measured with Attune Auto Sampler (Applied Biosystems). Fluorescence images were taken prior to FC analysis with a JULY TM microscope. Quantitative real-time PCR A qRT-PCR analysis was used to determine the d2EGFP mRNA amount at time intervals of 4, 8, 24, 36, 48, 60 and 72 hours in A549 and Huh7 cells.
  • RNA isolation was performed according to the manufacturer’s protocol using NucleoSpin RNA (Macherey Nagel). The isolated total RNA was examined in RNA concentration and quality by spectrophotometric measurements and gel analysis. Further, 0,5 ⁇ g of the total RNA of each UTR constructs and the control were used for cDNA synthesis using Oligo(dT)s from First Strand cDNA Synthesis Kit (Thermo Scientific).
  • Microstructured surfaces were produced by selective oxygen plasma treatment (Femto Diener, 40 W for 3 min) on a top as substrate (ibidi GmbH) with subsequent passivation. Selectivity was achieved using a polydimethylsiloxane (PDMS) stamp (cast from a master produced by photolithography) as a mask. The parts exposed to plasma were passivated by incubation for 30 min with PLL(20k)–g(3.5)-PEG(2k) at a concentration of 1 mg/ml in aqueous buffer (10 mM HEPES pH 7.4 and 150 mM NaCl). Thereafter, the samples were rinsed with PBS and the PDMS stamps were removed.
  • aqueous buffer 10 mM HEPES pH 7.4 and 150 mM NaCl
  • the foils were then fixed to adhesive six-channel slides (sticky ⁇ -slide VI). Each channel was filled with a solution of 50 ⁇ g/ml fibronectin in PBS for one hour to render the remaining sectors cell-adhesive. Probes were thoroughly rinsed with PBS three times. The samples were stored in cell medium at room temperature before cell seeding. For this study, square adhesion sites of 30 ⁇ m x 30 ⁇ m were used because this size turned out to be reasonable for single-cell adhesion of A549 as well as Huh7 cells. Cells were seeded at a density of 10,000 cells per channel so that roughly one cell could adhere on each cell-adhesive island.
  • a mixture of 20 ⁇ g/ml fibronectin and 30 ⁇ g/ml fibrinogen conjugated with Alexa Fluor 488 was used.
  • Materials FBS, Leibovitz’s L-15 Medium (Gibco), Lipofectamine TM 2000, and OptiMEM (Gibco) were purchased from Invitrogen, Germany. Sterile PBS was prepared in-house.
  • Ham’s F-12K, DMEM, and Trypsin-EDTA were purchased from c.c.pro GmbH, Germany. Channel slides were purchased from ibidi, Germany.
  • Fibronectin was purchased from Yo Proteins, Sweden.
  • PLL-g- PEG was purchased from SuSoS AG, Switzerland.
  • Alexa Fluor 488 was purchased from Life Technologies, Germany.
  • the plasmid pd2EGFP-N1 was purchased from BD Biosciences Clontech, Germany.
  • Cell Culture A human alveolar adenocarcinoma cell line (A549, ATCC CCL-185) was grown in Ham’s F12K medium supplemented with 10% FBS.
  • a human hepatoma epithelial cell line (Huh7, JCRB0403, JCRB Cell Bank, Japan) was cultured in DMEM medium, supplemented with 10% fetal bovine serum. All cell lines were grown in a humidified atmosphere at 5% CO 2 level.
  • In vitro Transfection Three hours prior to transfection, 10.000 cells per channel were seeded in a 6-channel slide.
  • Cells were transfected at a dose of 5pg mRNA/cell using the commercial transfection reagent Lipofectamine TM 2000 at a ratio of 2.5 ⁇ l Lipofectamine TM 2000per 1 ⁇ g mRNA.
  • the complex formation was prepared as follows: Lipofectamine TM 2000 and mRNA were separately diluted in OptiMEM transfection medium to add up to a total volume of 45 ⁇ l, each. These mixtures were incubated at room temperature for 5 minutes. The Lipofectamine TM 2000 solution was then mixed with the mRNA solution, followed by another 20 minutes of incubation at room temperature. Please note that the microchannels were never empty during all subsequent rinsing steps: Immediately before transfection, the cells were washed with PBS.
  • the lipoplex solutions containing different mRNAs constructs were filled into the six channels. All five different mRNA constructs plus the reference construct could thus be measured under the same experimental conditions.
  • the cells were incubated in a total transfection volume of 90 ⁇ l at 37°C (5% CO 2 level) for one hour. The transfection medium was thereafter removed and the cells were washed with PBS. Subsequently, the cells were re-incubated with Leibovitz’s L-15 Medium containing 10% FBS. A drop of anti-evaporation oil (ibidi GmbH, Germany) was added on top of each medium reservoir before microscopic monitoring of d2EGFP expression.
  • Live-cell imaging was performed on a motorized inverted microscope (Nikon, Eclipse Ti-E) equipped with an objective lens (CFI PlanFluor DL-10 ⁇ , Phase1, N.A. 0.30; Nikon) and with a temperature-controlled mounting frame for the microscope stage.
  • a motorized inverted microscope Nakon, Eclipse Ti-E
  • CFI PlanFluor DL-10 ⁇ , Phase1, N.A. 0.30; Nikon a temperature-controlled mounting frame for the microscope stage.
  • ibidi heating system Ibidi GmbH, Germany
  • a temperature controller to stabilize the temperature of the samples at 37°C ( ⁇ 2°C) throughout the measurements.
  • CCD camera CCD camera
  • a mercury light source (C-HGFIE Intensilight, Nikon) was used for illumination and a filter cube with the filter set 41024 (Chroma Technology Corp., BP450-490, FT510, LP510-565) was used for d2EGFP detection.
  • An illumination shutter control was used to prevent bleaching. Images were taken at 10 fold magnification with a constant exposure time of 600 ms at 10 minute-intervals for at least 25 hours post-transfection. Fluorescence images were consolidated into single-image sequence files. Quantitative analysis of characteristic parameters of single-cell expression kinetics allows the comparison of various vector performances in terms of expression efficiency and stability. Image analysis consisted of several steps and was done using in-house-developed software based on ImageJ.
  • a rectangular grid was overlaid with the original time-lapse movie and adjusted to the size and orientation of the underlying cell- pattern.
  • the software automatically detected d2EGFP-expressing cells by reading out the fluorescence intensities of all squares. Unoccupied squares were used for background correction.
  • the software calculates the cells’ fluorescence over the entire sequence and connects corresponding intensities to time courses of the fluorescence per cell. Finally, single-cell fluorescence intensities per square were extracted.
  • Example 1 Fluorescence microscopy and analysis via flow cytometry (FC) To evaluate the effect of different UTR combinations on transgene expression kinetics, two different cells lines were transfected using LipofectamineTM2000 with different d2EGFP mRNA constructs containing a 5’ UTR alone, a 3’ UTR, 5’+3’ UTR, two copies of 3’UTR and 5’+2x3’ UTR. A schematic representation of the building blocks of all constructs can be seen in Figure 1A. At different time points through three days post-transfection, d2EGFP expression was quantified using FC.
  • Figure 1B and D and Figure 7A and C Comparable transfection efficiencies for all mRNA constructs were confirmed 24 hours post transfection ( Figure 1B and Figure 8A). Thereby, differential transfer efficiencies to be a causal factor for the observed differences in expression kinetics can be ruled out.
  • a drastic reduction of d2EGFP expression for all constructs at 48 h post- transfection was detected (see Figures 1B and D, Figure 7A and C).
  • Example 2 Quantitative real-time PCR qRT-PCR measurement as an additional approach was conducted to determine the “physical” mRNA half-life of the different constructs. Binding of our selected primers to d2EGFP occurred 600nt downstream of the start codon. Hence, measurements of physical mRNA half-life compromise both intact mRNAs and those which have either been decapped but not yet degraded or both decapped and degraded up to base 599. It also includes mRNA that has been removed from the translational pool and stored in P-bodies (29-32).
  • Example 3 Single-cell expression arrays Microstructured, cell-adhesive substrates as shown in Figure 3A and B were fabricated as a platform for single-cell time-lapse microscopy.
  • the rectangular squares are functionalized with the extracellular matrix protein fibronectin, while the surrounding dark area is passivated with cell repellent PLL-g-PEG.
  • Cells were seeded at an appropriately dilute cell density such that after about three hours, cells adhered to the rectangular squares. This cellular self-organization process has been studied in detail before (27).
  • the size of the squares was 30 ⁇ m for optimal filling with single cells. The distance between the squares was just big enough (60 ⁇ m) to minimize bridging effects of cells adhering to more than one square at the same time.
  • a typical set of background corrected raw data is shown in Figure 3D.
  • the black lines represent exemplary fits to the mathematical expression for mRNA translation (see also Materials and Methods section).
  • Data were analyzed as described recently (26) by fitting each time-course with the analytical solution for mRNA-induced protein expression, (Equation 1) using IgorPro software.
  • G denotes the amount of protein
  • K is the expression rate
  • ⁇ is the mRNA degradation rate
  • ⁇ is the degradation rate of the reporter protein d2EGFP.
  • the expression rate K m 0 ⁇ k TL is the product of the initial amount of mRNA molecules inside the cell (m0) and the translation rate kTL.
  • the time-course that is described by Equation 1 will be discussed in detail in below section “mastercurves of protein expression”.
  • V. Example 4 In vitro transfection on cell arrays In a typical experiment, cells were allowed to adhere to the micropatterns for three hours before transfection. Each of the six microchannels was filled with a different lipoplex solution, containing one of the constructs of interest. In initial experiments, we compared two different, commercially available transfection reagents (namely LipofectamineTM 2000 and DOGTOR). Higher transfection efficiencies were found for LipofectamineTM 2000 than for DOGTOR (see Figure 9).
  • Example 5 Expression rates All results for the two cell types are based on four independent measurements under the same experimental conditions. Time-lapse data of about thousand A549 cells and thousand Huh7 cells have been analyzed. The distributions of the obtained expression rates K are shown in Figure 4A and the corresponding mean values can be seen in Figure 4D. Both the mean expression rates and the shape of their distributions were found to be rather similar for the different constructs. VII.
  • Example 6 mRNA half-lives
  • ⁇ ln2 ⁇ .
  • Figure 4B shows the half-life distributions of differently stabilized mRNA constructs in A549 and Huh7 cells, respectively.
  • ⁇ ln2 ⁇ .
  • Figure 4D An overview of all determined half-lives is given in Figure 4D.
  • the coefficients of variation are about 0.29 (A549) and 0.45 (Huh7) and hence is significantly smaller than the coefficient of variation of up to 0.6 that we found for the distribution on mRNA life-times.
  • the half-lives in an alternative approach were also measured, where translation was inhibited by addition of cycloheximide at a given time point, t0, after transfection (see Figure 10). In this case, protein expression is induced for a while and then stopped. The exponential decay in fluorescence after inhibition yields protein life times.
  • Example 8 Mastercurves of protein expression The features of mRNA induced protein expression become evident in the so-called mastercurve of protein expression as depicted in Figure 5A (A549) and B (Huh7).
  • the mastercurve is the population average of the onset-time corrected single cell traces, i.e. all onset-times were shifted to time point zero. Fluorescence intensities were converted into actual numbers of d2EGFP as described before in reference (26).
  • the superior properties of the 3’ and the 5’+3’-stabilized mRNA constructs are illustrated in the mastercurve plot. These constructs showed the shallowest decrease in protein expression with time and hence the longest half-lives in addition with higher protein expression values as compared to the other constructs. X.
  • Example 9 Area under the curve (AUC) In pharmacokinetics, the total exposure of a drug is known as the “area under the curve”.
  • the analogous expression in gene therapy is the integral of the amount of artificially expressed protein over time, i.e. the area under the (expression-vs.-time) curve (AUC).
  • AUC is a means to simultaneously quantify the translational efficiency and the stability of an mRNA construct. It can be interpreted as the cumulative time-dose of the protein that is encoded on the mRNA and hence describes the efficacy of a chosen mRNA construct.
  • AUC 0.48 ⁇ m 0 ⁇ k TL ⁇ ⁇ mRNA ⁇ ⁇ d2EGFP (Equation 3)
  • an optimal therapeutic mRNA construct should desirably have both long mRNA, ⁇ mRNA, as well as protein half-life, ⁇ d2EGFP, and high translational efficiency, kTL.
  • the transfer efficiency which determines the initial amount of therapeutic mRNA, m0 is directly proportional to the AUC.
  • Example 11 Comparison of constructs having UTRs of different genes compared to the CYBA-UTR construct The constructs #2 to #5 having UTRs of different genes as indicated in the below Table 4 have been compared to the CYBA-UTR construct #1 in order to optimize the mRNA structure in terms of stability and productivity. Five different cellular UTRs of a gene were selected based on publication data (Hoen et al., 2010) featuring long mRNA half-lives. These cellular UTRs are CYBA, DECR1, GMFG, MAPBPIP and MYL6B.
  • the sequences of 5’and 3’ untranslated regions of each cellular gene were obtained from the UTR database (http://utrdb.ba.itb.cnr.it/search) and were cloned into five different combinations, which were 5’UTR alone, 3’UTR alone, 5’+3’UTR, 5’+2x3’UTR and 2x3’UTR.
  • the untranslated region sequences were cloned into the backbone pVAX1-A120.
  • cloning occurred via HindIII restriction site on the 5’end and BamHI restriction site on the 3’end and was inserted upstream of the reporter gene coding for Metridia luciferase (MetLuc).
  • the restriction sites for 3’UTRs were EcoRI (5’end) and PstI (3’end) and were cloned downstream of MetLuc.
  • the plasmids containing 5’ UTR alone and 5’+3’UTR for each cellular UTR were produced by Eurofins MWG Operon. These plasmids were transformed into E. coli bacteria (DH10B) via electroporation. The other combinations, including 3’UTR alone, 5’+2x3’UTR and 2x3’ UTR were cloned in-house. Cloning of plasmids with 3’UTR was performed by simply cutting out the 5’UTR of the backbone via HindIII (blunt) and BamHI (blunt) digestion.
  • Constructs containing 5’UTR+2x3’UTR were cloned by inserting MetLuc containing 3’UTR (BamHI/PstI blunt) into the backbone of pVAX1-A120 MetLuc comprising 5’+3’UTR, thereby replacing MetLuc and inserting a second 3’UTR in front of the respective 3’UTR of the backbone. Finally, the constructs containing 2x3’UTR were generated by removing the 5’UTR (HindIII and BamHI, both blunt) from the plasmid containing 5’+2x3’UTR. After cloning, all plasmids were amplified in E. coli bacteria (DH10B) after electroporation.
  • DH10B E. coli bacteria
  • mRNA was produced by in vitro transcription.
  • the plasmids were linearized with XbaI digestion and were purified with chloroform/ethanol precipitation.
  • the in vitro transcription kit included the required T7 polymerase enzyme mix as well as the suitable buffers.
  • the transcription mix also contained the unmodified nucleotides adenosine-triphosphate (ATP), guanosine-triphosphate (GTP), uridine-triphosphate (UTP) and cytosine-triphosphate (CTP) as well as the chemically modified nucleotides methyl- CTP and thio-UTP (Jena Bioscience, GmbH, Jena, Germany) with a final concentration of ATP:GTP:UTP:CTP:methyl-CTP:thio-UTP of 7.13 mM:1.14 mM:5.36 mM:5.36 mM:0.536 mM:0.536 mM.
  • ATP adenosine-triphosphate
  • GTP guanosine-triphosphate
  • UDP uridine-triphosphate
  • CTP cytosine-triphosphate
  • the cap structure analog ARCA anti-reverse cap analog
  • the linearized DNA was added into the reaction mix.
  • the IVT mix was incubated at 37°C for 2 h. Digestion of the remaining DNA was enabled by the addition of DNase I and further incubation at 37°C for another 20 min.
  • RNA precipitation was performed by the addition of pre-cooled ammonium- acetate to a final concentration 2.5 M. The RNA pellet was washed with 70% ethanol. The washing step was performed twice. At last, the RNA was re-suspended in RNase-free water. The RNA concentration was determined with a spectrophotometric device and purity was tested on an agarose gel.
  • the different mRNAs were tested in two different cell lines, i.e., in NIH3T3 and A549.
  • a non-viral nucleic acid delivery system like lipofection, was used.
  • different transfection agents were tested to compare protein expression and cell viability (data not shown).
  • dose titration were conducted to evaluate dose dependent effects.
  • the experimental set-up is as follows: 5000 cells (NIH3T3) in 150 ⁇ l DMEM complete medium were seeded per well in 96-well plates and transfected 24 hours post-seeding.
  • the dilution series were prepared. In the remaining seven wells subjacent of the complex mix, 180 ⁇ l DMEM without supplements per well was added. After incubation time 180 ⁇ l of the complex solution was removed and added into the first well of dilution series. This procedure was conducted until the last dilution step. Finally, 50 ⁇ l of the complex solution were added to the cells and incubated for 4 hour. For every mRNA construct, biological triplicates were prepared. After 4 hours, the complete supernatant was removed from the cell culture plate for measurement and fresh 150 ⁇ l medium was added to each well. Bioluminescence was measured after 4, 24, 48, 72, 96, 120 and 144 hours using a multilabel plate reader.
  • the AUC of the d2EGFP time course is also evaluated, because the total protein expression is relevant for a sustained therapeutic effect.
  • microstructured single-cell arrays for parallel, quantitative measurements of mRNA stability and translational efficiency were used.
  • the regular arrangement of cells guaranteed reproducible microenvironments and enabled fast and automated image-analysis, which are prerequisites for comparative, high-throughput single-cell studies.
  • the approach allows the determination of distribution functions for (i) protein half-life, (ii) expression rates, and (iii) functional mRNA half- life.
  • mean protein half-lives of d2EGFP were narrowly distributed and independent of the UTR sequence.
  • the mean half-life determined for A549 cells with the mean half-life determined for Huh7 cells is compared, the same ratio of roughly 1.64 for both measurement methods is found. Also, even a possible systematic over-estimation of mRNA and protein half-lives does not change the qualitative order of the mRNA performance.
  • the expression rate depends on the initial number of mRNA molecules, m0, as well as on the translation rate KTL. It is to be noted that the number of successfully delivered mRNA molecules varies due to the intrinsic stochasticity of the delivery process. The mean number of mRNA molecules, however, is expected to be the same, since the transfection protocol has scrupulously been kept up in all experiments. In contrast, the translational activity (KTL) of the various UTR constructs might vary.
  • the physical mRNA half-life determined by qRT-PCR showed a decrease in mRNA stability for 5’, 3’, 5’+2x3’ and 2x3’ UTR in both cell lines (see Figure 8A and B).
  • One major difference is the translational capacity for every measured mRNA in both methods.
  • the mRNA is involved in active translation, whereas the physical mRNA half-life is monitored regardless of the translational status of the detected mRNA. Similar findings have been reported by Gallie et al. (35). It is believed that the physical mRNA half-life is not an appropriate indicator of the translational capacity of the mRNA.
  • mRNA half-lives for the constructs with UTRs ranged from 13.0 h to 23.0 h as compared to 5.8 h for the control.
  • Huh7 cells half-lives from 9.9 h to 13.6 h were measured for UTR-containing constructs, as opposed to a half-life of 7.8 h for the control mRNA.
  • the half-life of the 3’UTR-stabilized mRNA in A549 cells is in good agreement with mRNA life times of similarly stabilized mRNAs that were reported previously (16,26).
  • single-cell analysis of mRNA-induced protein expression is a means to characterize and improve pharmacokinetic properties of mRNA constructs.
  • Messenger RNA constructs with persistent protein expression over a period of time (AUC) is desirable and allows proper reduced dosing into a patient with a final therapeutic outcome.
  • GDF15 UTRs Reference Sequence in NCBI (NM_004864.2) 5’UTR: Position in the reference sequence: 1-32 3’UTR: Position in the reference sequence: 960-1200 ⁇ Glucagon UTRs: Reference Sequence in NCBI (NM_002054.4) 5’UTR: Position in the reference sequence: 1-256 3’UTR: Position in the reference sequence: 800-1294 ⁇ Nucleic acid sequences for GDF15 and GDF15H6D were derived from amino acid sequence via reverse translation (Expasy). ⁇ Obtained nucleic acid sequence was codon optimized for expression in humans. ⁇ Final sequence to be synthesized was assembled in the following order: 1. 5‘cloning site 2. T7 promoter 3.
  • 5‘UTR natural from the gene (GDF15 or Glucagon), CYBA, human alpha globin 4. Kozak element 5.
  • SOI sequence of interest: Codon optimized GDF15 or GDF15H6D 6.
  • 3‘UTR natural from the gene, CYBA 7.
  • RNA/SNIM ® RNAs passed the Quality control criteria of: ⁇ Size – agarose gel electrophoresis ⁇ Integrity – lack of smearing on gel ⁇ Purity – ratios of 260/280 and 230/280 ⁇ 1.8 ⁇ Concentration of each modified RNA/SNIM ® RNA was adjusted to 1 mg/mL in water for injection. ⁇ modified RNA/SNIM ® RNAs were stored at -80 ° C till further use. modified RNA/SNIM ® RNA tested in the present study: GLP1 ELISA For GLP1 quantification Glucagon-Like Peptide-1 (Active) ELISA Kit (Millipore) was used.
  • ELISA was performed as described in the manufactures manual. Samples were diluted 1:5 or 1:2. Measurement was performed on a Tecan infinite 200Pro device (Excitation Wavelength: 355 nm; Emission Wavelength: 460 nm; Excitation Bandwidth: 9 nm; Emission Bandwidth: 20 nm; Gain: 62 Calculated From: B2 (100%); Number of Flashes: 25; Integration Time: 20 ⁇ s)
  • GDF15 ELISA For GDF15 quantification Human GDF-15 Quantikine ELISA Kit (R&D Systems) was used. GDF15 ELISA was performed as described in the manufactures manual. Samples were diluted 1:100.
  • IVC individually ventilated cages
  • RNA/SNIM ® RNAs were taken, transferred into 1,5 mL Eppendorf Tubes ® and subsequently snap frozen and stored at -80 °C until further analysis. Statistical significances between groups were calculated using Mann-Whitney’s U-Test. P-values ⁇ 0,05 were considered as statistically significant.
  • RNA/SNIM ® RNA transfection efficiency using firefly luciferase reporter modified RNA/SNIM ® RNA.
  • HEK293 an appropriate cellular model for each of the target proteins
  • RNA/SNIM ® RNA transfection efficiency using firefly luciferase reporter modified RNA/SNIM ® RNA.
  • ⁇ 2 Expression and quantification of secreted proteins in each of the cellular systems (supernatants and lysates) via ELISA
  • 3) Analysis of expression kinetics dose-dependent expression time-course over one week) Transfection into HEK293 cells ⁇
  • the produced modified RNA/SNIM ® RNAs were tested for their functionality in HEK293 cells and the resulting protein expression was quantified in cell culture supernatants via ELISA.
  • ⁇ HEK293 cells were transfected in 96-well plates using Lipofectamine2000 (Reagent to modified RNA/SNIM ® RNA ratio 2 ⁇ L / 1 ⁇ g modified RNA/SNIM ® RNA).
  • Lipofectamine2000 Reagent to modified RNA/SNIM ® RNA ratio 2 ⁇ L / 1 ⁇ g modified RNA/SNIM ® RNA.
  • 2x104 HEK293 cells were seeded / well in 96-well plates.
  • Lipofectamine2000 Lipofectamine2000/mRNA ratio 2/1).
  • mRNA was diluted in dH2O.
  • Lipofectamine2000 was diluted in medium w/o serum and P/S and mixed by pipetting.
  • mRNA solution was added to each Lipofectamine2000 solution and incubated for 5 min at RT.
  • ⁇ ELISA Kits used in the present study ⁇ EGLP-35K Glucagon-Like Peptide-1 (Active) ELISA Kit: Merck Millipore ⁇ Human GDF-15 Quantikine ELISA Kit: R&D Systems
  • Dose response - GLP1 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.24 h after transfection supernatants were taken for analysis. Values of PBS treated control and modified eGFP-RNA/eGFP-SNIM ® RNA (500 ng/well) treated samples were below the lower limit of quantification.
  • hAg resulted in highest GLP1 expression for all doses compared (see Fig.13).
  • Kinetics of protein expression - GLP1 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.50 ⁇ L of supernatant were taken for analysis at time points 24, 48 and 72 h. Mean +/- SEM of 3 experimental replicates (3 separately transfected wells) is shown.
  • GLP1“hAg“ resulted in highest GLP1 expression at all the time points compared (see Fig.14).
  • late time point (72h) was chosen to analyze potential differences between the stabilized (H6D) and not stabilized version of GDF-15 No significant differences at time point 72 h (see Fig.16).
  • Kinetics of protein expression - GDF15 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.50 ⁇ L of supernatant were taken for analysis at time points 24, 48 and 72 h. Mean +/- SEM of 3 experimental replicates (3 separately transfected wells) is shown. At 24 h and 48 h, higher values for H6D mutants were observed compared to their corresponding wild type GDF-15 sequences, no significant differences at time point 72 h (see Fig.17).
  • GLP1“hAg“ with human-alpha-globin as a 5‘UTR could be identified as a lead candidate with respect to expression of GLP1.
  • H6D mutant sequences lead to higher GDF15 expression.
  • GDF15H6D“eth“/CYBA appears to be the lead candidate among the modified GDF15- RNA/GDF15-SNIM ® RNAs compared in the present study.
  • XVI. In vivo testing of modified RNA/SNIM ® RNAs concerning GLP-1 and GDF15 Primary read-out of in vivo study: Reduction of food intake within 24 hours.
  • modified RNA/SNIM ® RNA coding for GDF-15 and GLP-1 was tested against two control groups (see below for a schematic overview). If not indicated otherwise, hGDF15 WT (with 5 ⁇ - and 3 ⁇ - CYBA-UTRs) and hGLP-1 (with 5 ⁇ -hAg- UTR) were used in the in vivo testings. modified RNA/SNIM ® RNA containing WT sequence of GDF-15 (with 5 ⁇ - and 3 ⁇ - CYBA UTRs) resulted in highest plasma protein levels in vivo after 6 hours.
  • a lipid carrier such as C12-(2-3-2)
  • RNA/SNIM ® RNA GLP-1 (with 5 ⁇ -hAg-UTR) reduces food consumption (36% vs Succrose, 19% vs STOP) and body weight. Data are shown in Fig.21. GLP-1 plasma levels are elevated against sucrose control, but not against STOP control. Data are shown in Fig.22. Both, GDF-15 and GLP-1 coding modified RNA/SNIM ® RNA facilitate statistically significant reduction of food consumption. Dosis: 1 mg/kg BW, Reduction of food consumption within 24 hours Data are shown in Fig.23.
  • ⁇ GDF-15 shows physiological activity (reduction of food intake and weight loss) at a dosage of 0.5 mg/kg BW ⁇ GDF-15 and GLP-1 are superior against corresponding non-translated control modified RNA/SNIM ® RNAs at a dosage of 1 mg/kg BW ⁇ GDF-15 levels are significantly elevated up to 24 hours post injection
  • Normalizing food consumption to 100 g/mouse does not result in significant changes of results.
  • Data are shown in Fig.24. Normalizing food consumption to 100 g/mouse does not result in significant changes of results.
  • Data are shown in Fig.25.
  • Application of 2% Sucrose solution does not alter food intake of test animals
  • Data are shown in Fig.26.
  • Inflammatory cytokines are within physiological ranges 24 hours after application of Test items. Data are shown in Fig.27. Application GLP-1 coding modified RNA/SNIM ® RNA results in significantly increased IGF- 1 levels after 24 hours. Data are shown in Fig.28.
  • SEQ ID NO: 1 Nucleotide sequence of the used 5’cyba-UTR (RNA; on DNA level, “U” will be “T”; Kozak sequence is included (underlined); additional 5 ⁇ C is included (underlined)) CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC
  • SEQ ID NO: 2 Nucleotide sequence of the used 3’cyba-UTR (RNA; on DNA level, “U” will be “T”) CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGAAGCCGGGA
  • SEQ ID NO: 3 Nucleotide sequence of the used 5’hAg-UTR (RNA; on DNA level, “U” will be “T”; additional G is included (bold)) CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC

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Abstract

Described is an RNA molecule comprising (a) a region coding for a regulator of energy homeostasis like GLP-1 and/or GDF-15; and (b) upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a functional derivative thereof; or (B) the 5'-UTR of alpha globin (Ag) or a functional derivative thereof; and/or (c) downstream of said coding region one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a functional derivative thereof. Further described is a set of 2 or more RNA molecules, wherein the coding region of one RNA molecule codes for one regulator of energy homeostasis (like GLP-1) and wherein the coding region of another RNA molecule codes for another such regulator (like GDF-15). Moreover, described is a nucleic acid molecule encoding the RNA molecule or the set of RNA molecules and a set of 2 or more nucleic acid molecules encoding the 2 or more RNA molecules, respectively, of the set of the 2 or more RNA molecules. Further described are respective vectors and host cells and respective sets of vectors and host cells. Further described is a pharmaceutical composition comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set. Also described is a kit comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set. Further described is the pharmaceutical composition of the invention for use in an RNA-based therapy and/or as an anorectic; in body weight control, in particular in decreasing (aberrant) body weight; and/or in the treatment or prevention of a metabolic disorder/metabolic syndrome. Also described are respective methods for (medical) intervention. Finally, described is the use of one or more UTR(s) as defined in (b) and/or one or more UTR(s) as defined in (c) in increasing the efficiency of translating a coding region of an RNA molecule into a regulator of energy homeostasis like GLP-1 and/or GDF-15 (MIC-1) encoded by said coding region.

Description

New PCT Patent Application Based on EP 22211336.7 Ethris GmbH Our Ref.: AF3725 PCT S3 Regulator(s) of energy homeostasis-encoding RNA molecule(s) with increased translation efficiency Field of the Invention The present invention relates to RNA molecules and their applications in medical and therapeutic contexts. Specifically, the invention relates to an RNA molecule that includes a coding region for a regulator of energy homeostasis (like GLP-1 and/or GDF-15) and untranslated regions (UTRs) with specified sequences. These UTRs are designed to enhance the translation efficiency of the RNA molecule. The invention also encompasses a set of RNA molecules, each coding for different regulators of energy homeostasis, and nucleic acid molecules encoding these RNA molecules. Furthermore, it includes vectors containing these nucleic acid molecules and host cells comprising these vectors or nucleic acid molecules. Additionally, the invention pertains to pharmaceutical compositions containing these RNA molecules, nucleic acid molecules, vectors, or host cells, and kits comprising these components. These are intended for use in RNA-based therapies, particularly for regulating energy homeostasis, controlling body weight, and treating or preventing metabolic disorders. The invention also includes methods for decreasing food intake, controlling body weight, and treating or preventing metabolic disorders. Lastly, it relates to the use of certain UTRs to increase the efficiency of translating coding regions of RNA molecules into regulators of energy homeostasis. Introduction In recent years, messenger RNA (mRNA) has become increasingly relevant as a new drug entity. As opposed to DNA-based gene therapeutics, mRNA does not need to be transported into the nucleus but is directly translated into protein in the cytoplasm (1,2). This makes mRNA safer in avoiding potential insertional mutagenesis, an unlikely but existent risk of DNA gene medicines. As a consequence, mRNA therapeutics are emerging as promising alternatives for gene and protein replacement therapies in a broad variety of medical indications (1-4). However, the strong immunogenicity as well as the limited stability of conventional mRNA has to be overcome to further establish its clinical applicability. With respect to this, mRNA stability and in particular the translation rate of the mRNA is an essential parameter for envisaged medical applications because it determines, for example, dosing and the dosing intervals of mRNA drugs. Also, untranslated regions (UTRs) in mRNAs have been reported to play a pivotal role in regulating both mRNA stability and mRNA translation. UTRs are known to influence translational initiation, elongation, and termination, as well as mRNA stabilization and intracellular localization through their interaction with RNA binding proteins (6,7). Depending on the specific motives within the UTR, it can either enhance or decrease mRNA turnover (8-11). Recently, data on mRNA half- lives and the corresponding UTR sequences have been published (12, 43). Accordingly, there is still a need for improvements in reducing the immunogenic response triggered by mRNA administered to cells or organisms and increasing the translation efficiency, in particular as regards further or alternate means to increase the translation efficiency since the translation efficiency is an essential parameter for envisaged medical applications because it determines, for example, dosing and the dosing intervals of mRNA drugs and, ultimately, determines the bioavailability of the final product, i.e., the encoded peptide or protein. Further, especially in the affluent industrial nations, the increased occurrence of nutrition- dependent diseases and metabolic disorders, respectively, (e.g. obesity/adipositas, hypercholesterolemia, diabetes, hyperglycaemia, hypertension and the like) is a serious problem. In many cases, such diseases/disorders are secondary diseases and pathological consequences caused, for example by obesity, as a consequence of overnutrition. For instance, pathological consequences of increased glucose concentrations in the blood due to diabetes are retinopathia and renal failures. Further, overweight and diabetes are risk factors for diseases such as hypertension, heart attack, other cardiovascular diseases, stroke, biliary stones or other bile disorders, gout, and the like. Especially obesity has risen to alarming levels world-wide (McLellan (2002), Lancet 359, 1412). For example, the average weight of German conscripts increases by almost 400g/year. Similar data were obtained in Austria, Norway and the UK. In the treatment of obesity and other nutrition-dependent diseases and metabolic disorders, respectively, regulators of energy homeostasis and/or appetite suppressants, also named anorectics, have been established and are used widely. These comprise Glucagon-like Peptide 1 (GLP-1) and growth differentiation factor 15 (GDF15; also known as MIC-1) and respective agonists as well as inhibitors of dipeptidyl-peptidase-4 (DPP-4) which is known to rapidly inactivate GLP-1. GLP-1 is known to be a neuropeptide and an incretin, secreted in the periphery from the intestinal L cell as a gut hormone. The major source of GLP-1 in the brain is the nucleus of the solitary tract. GLP-1 increases the secretion of insulin, slows down the emptying process in the stomach and increases the feeling of saturation. Low levels of GLP-1 are known to result in increased food intake and, as a consequence, in obesity and related metabolic disorders. In this context, exemplary reference is made to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1760073/pdf/v044p00081.pdf, Gutzwiller (Gut 44, 1999, 81-86) and Schick (Glucagon-like peptide 1: a novel brain peptide involved in feeding regulation. In: Obesity in Europe 1993, Ditschuneit et al. (Eds), 1994, 363–367). GLP-1 and its agonists have therefore been used in the treatment of metabolic disorders like obesity. Examples of respective pharmaceuticals are: Exenatide (Byetta®®, AstraZeneca; acts as a GLP-1 mimic), Exenatide (Byduroen®®, Amylin, AstraZeneca), Liraglutide (Victoza®®, 97% aa-similarity to GLP- 1; NovoNordisk). GDF15 is a member of the TGFbeta superfamily, also known as placental bone morphogenetic factor (PLAB) (Hromas R 1997, Biochim Biophys Acta.1354:40-4), prostate derived factor (PDF) (Paralkar V M 1998, J Biol Chem. 273:13760-7), macrophage inhibitory cytokine 1 (MIC1) (Bootcov M R, 1997, Proc Natl Acad Sci 94:11514-9), placental transforming growth factor beta (PTGFB) (Lawton L N 1997, Gene.203:17-26), and NSAID activated gene-1 / nonsteroidal anti- inflammatory drug-activated gene 1 (NAG-1) (Baek S J 2001, J Biol Chem.276: 33384-92). It is a cellular stress-responsive factor mediating cytoprotection. GDF15 is expressed almost ubiquitous; it is (on mRNA level) most abundant in the placenta and prostate but also present in heart, liver, kidney, pancreas and colon. GDF15 is highly expressed in macrophages, choroid plexus, prostate, lung, kidney proximal tubules, placenta and intestinal mucosa and is overexpressed by a variety of cancers, which may relate to its antitumorigenic and proapoptotic properties. Although it has been described as a prognostic marker in acute myocardial infarction it is only poorly expressed in the heart. In this context, exemplary reference is made to Strelau (J Neural Transm Suppl.65, 2003, 197-203). GDF15 has been discussed to be involved in body weight control and its levels have been associated with obesity, risk for insulin resistance and glucose control in patients (Cartsensen, European J of Endocrinology 162 (5), 2010, 913-917; Dostalova, European J. of Endocrinology 161, 2009, 397-404; Vila, Clinical Chemistry 57 (2), 2011, 309-316). Moreover, GDF15 has been considered as a protective in heart and kidney disease and as a biomarker for cancer and cardiovascular diseases. In this context, exemplary reference is made to Xu (Circ Res 98(3), 2006, 342-50), Bauskin (Cancer Res 66(10), 2006, 4983-6), Macia (PLoS ONE 7(4), 2012, ArticleID e34868) and Kempf (Nature Medicine 17(5), 2011,581–288). In particular, it is known that GDF15 levels are elevated before the manifestation of Type 2 diabetes mellitus (Cartsensen, European J of Endocrinology 162 (5), 2010, 913-917; Dostalova; European J. of Endocrinology 161, 2009, 397-404). Furthermore, GDF15 decreases food intake and improves glucose tolerance in mice (Macia, PLoS ONE 7 (4), 2012, Article e34 (6)). Consequently, GDF15 and its agonists has been suggested to treat obesity and other metabolic disorders (Chrysovergis, International Journal of Obesity (38), 2014, 1555–1564; WO2012138919 (Amgen)). For example, Lim et al. showed a weight-lowering effect in obese mice models of YH34160, an Fc fusion GDF15 variant (Meeting abstract: OBESITY—ANIMAL, JUNE 012021, Diabetes 2021; 70(Supplement_1); 217-LB). YH34160 was held to be a promising therapeutic candidate for the treatment of human obesity (Lim, loc. cit.). GDF15 has also been described as a diagnostic marker, e.g. for diabetes I (WO2009141357; Roche) and myocardial infarction (US 8771961; Roche). GDF15 has also been discussed to be involved in tumor-induced anorexia and weight loss (Johnen, Nat Med.13 (11): 2007, 1333-40). It is well established that gut-derived hormones, such as GLP-1, play an important role in the control of energy homeostasis (Burcelin, J. Nutr. 137 (11 Suppl.), 2007, 2534S-2538S). The action of the TGF-β superfamily cytokine GDF15 is mainly mediated by direct action on the feeding centers in the hypothalamus (Tsai, 2014 PLoS ONE 2014, 9(6): e100370. doi:10.1371/journal.pone.0100370). Although the lack of a rapid and substantial increase of GDF15 serum levels after taking a meal provides some evidence that GDF15 does not act as a satiety factor it may, similarly to GLP-1, act as a physiological long-term regulator of energy homeostasis (Tsai, PLoS One 2015, 10 (7): e0133362. doi: 10.1371/journal.pone.0133362). It is known, however, that there are also anorectic actions of GDF15. These, however, require an intact brainstem area postrema and nucleus of the solitary tract (Tsai, loc.cit.). Also GLP-1 was discussed in the context of appetite regulation (Dasiley, Trends Endocrinol Metab.24 (2): 2013, 85–91). Dipeptidyl-peptidase-4 (DPP-4) is known to rapidly inactivate GLP-1. In particular, DPP-4 has been discussed to be responsible for the extremely short T1/2 of GLP-1. Consequently, DPP-4 inhibitors in the treatment of obesity and other metabolic disorders have been proposed. GLP-1 agonists act in a similar manner as DPP-4 inhibitors. In this context, exemplary reference is made to https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238422/pdf/ijcp0068-0557.pdf and Brunton (Int J Clin Pract 68(5), 2014, 557–567).Well known examples of DPP-4 inhibitors are Sitagliptin, Vidagliptin, Saxagliptin, Linagliptin, Alogliptin, Dutogliptin and Gemigliptin. WO 2017/001554 describes UTRs derived from an mRNA of the human cytochrome b-245 alpha polypeptide (CYBA) gene which increase the translation efficiency of RNA molecules. Although means and methods for treating nutrition-dependent diseases and metabolic disorders, respectively, (like obesity) have already been described in the prior art, there is still a need for improvements, in particular as regards further or alternative means to increase the translation efficiency in respective mRNA-based approaches. This especially pertains to means and methods which rely on pharmaceuticals based on regulators of energy homeostasis like GLP-1 and its agonists and GDF15 and its agonists. Brief Summary of the invention The present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis like GLP-1 and/or GDF-15 (MIC-1); and (b) upstream of said coding region one (or more) untranslated region(s) (UTR(s)) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40; or (B) the 5’-UTR of alpha globin (Ag) or a functional derivative of said 5’-UTR; and/or (c) downstream of said coding region one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2. The present invention further relates to a set of 2 or more RNA molecules, wherein the coding region of one RNA molecule of said set codes for one regulator of energy homeostasis (like GLP- 1) and wherein the coding region of another RNA molecule of said set codes for another regulator of energy homeostasis (like GDF-15). Moreover, the present invention relates to a nucleic acid molecule encoding the RNA molecule or the set of RNA molecules according to the present invention and to a set of 2 or more nucleic acid molecules encoding the 2 or more RNA molecules, respectively, of the set of the 2 or more RNA molecules. Further, the present invention relates to a vector comprising the nucleic acid molecule or the set of nucleic acid molecules according to the present invention and to a set of 2 or more vectors comprising the 2 or more nucleic acid molecules, respectively, of the set of 2 or more nucleic acid molecules. The present invention further relates to a host cell comprising the nucleic acid molecule or set of nucleic acid molecules according to the present invention or the vector or the set of vectors according to the present invention and to a set of 2 or more host cells comprising the 2 or more nucleic acid molecules, respectively, of the set of 2 or more nucleic acid molecules or the 2 or more vectors, respectively, of the set of 2 or more vectors. Further, the present invention relates to a pharmaceutical composition comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set, and optionally a pharmaceutically acceptable carrier. Moreover, the present invention relates to a kit comprising the RNA molecule, nucleic acid molecule, vector or host cell according to the present invention, or the respective set. The present invention further relates to the pharmaceutical composition for use in an RNA-based therapy and as a regulator of energy homeostasis; in body weight control, in particular in decreasing (aberrant) body weight; and/or in the treatment or prevention of a metabolic disorder. The present invention further relates to a method for decreasing food intake; restraining appetite; controlling body weight, in particular decreasing (aberrant) body weight; and/or treating or preventing a metabolic disorder. Finally, the present invention relates to the use of one or more UTR(s) as defined in (b) and/or one or more UTR(s) as defined in (c) for increasing the efficiency of translating a coding region of an RNA molecule into a regulator of energy homeostasis like GLP-1 and/or GDF-15 (MIC-1) encoded by said coding region. The present application addresses this need by providing the embodiments as defined in the claims. Detailed description The present application surprisingly found that particular UTRs confer an increased translational efficiency when fused to a given (foreign) mRNA. More particularly, it was found that particular UTRs confer an increased translational efficiency (and/or expression) when fused to (foreign) mRNA encoding regulators of energy homeostasis, especially GLP-1 (or an agonist thereof) or GDF15 (or an agonist thereof). One of these particular UTRs is derived from an mRNA of the cytochrome b-245 alpha polypeptide (CYBA) gene, preferably from an RNA of the human CYBA gene (see also WO 2017/001554). The CYBA gene comprises specific 5’ and 3’ UTRs (also referred to herein as (5’ and 3’, respectively) “eth”, “ETH” or “ethris” (this covers also the variants used)). In general, 5’ UTR motives such as upstream open reading frames (uORFs) or internal ribosomal entry sites (IRES) are known to be involved in gene regulation, particularly in translational initiation (13). The 3’ UTRs can comprise even more regulatory functions than the 5’UTRs, some of them even hindering mRNA translation (14). Although the CYBA’s 3’ UTR is known to contain two regulatory motives, the finding of the present invention that the CYBA UTRs confer an increased translational efficiency when fused to a given mRNA encoding a regulator of energy homeostasis, especially GLP-1 (or an agonist thereof) or GDF15 (or an agonist thereof) is nevertheless surprising since these two motives are described in the context of the mRNA’s stability but not in the increase of the translational efficiency. More specifically, the 3’ UTR of CYBA is known to harbour a polyadenylation signal (PAS) which is known to interact with the cytoplasmic polyadenylation element binding protein (CPEB), as well as with the cleavage and polyadenylation signaling factor (CPSF) (11). CPEB is known to be responsible for the prolongation of the poly-A tail in the cytoplasm, whereas CPSF primes the pre-mRNA through cleavage at a specific site for the upcoming addition of poly-A (11, 14). A second regulatory motif contained in the CYBA 3’ UTR is the insulin 3’ UTR stability element (INS_SCE) (15). The INS_SCE sequence has been shown to bind to the polypyrimidine tract binding protein (PTB) under reducing conditions, increasing the mRNA half-life of insulin (15). Thus, both regulatory motives of the CYBA’s 3’ UTR are predominantly linked with the mRNA stability. Another of the particular UTRs that confers an increased translational efficiency (and/or expression) in accordance with the invention is derived from the alpha globin (Ag) gene, preferably from the human alpha globin (hAg) gene. The hAg gene comprises a specific 5’-UTR. The following polyribonucleotide sequence is an example of the 5’-UTR derived from the Ag gene, in particular the hAg gene: CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC (SEQ ID NO: 3). The DNA sequences displaying the nucleotide sequence of the human CYBA gene’s 5’- and 3’- UTRs present on the coding strand of the human CYBA gene are shown in the following Table 1. Table 1: Genetic code of the human CYBA gene UTRs
Figure imgf000008_0001
Table 1 shows the exact genetic code of the human CYBA gene UTRs. DNA sequences are shown from the 5’ to the 3’ end. The polyadenylation signal (PAS) of the 3’ UTR is shown in bold letters and the insulin 3’UTR stability element (INS_SCE) is underlined. The 5’ UTR consists of 71 base pairs, whereas the 3’ UTR contains 64 base pairs. Both UTRs are shorter than average human UTRs, which consist of around 200 nucleotides in the case of 5’UTRs and approximately 1000 nucleotides in the case of 3’UTRs. In the above Table 1, the DNA sequences displaying the human CYBA gene 5’- and 3’ UTRs are shown as SEQ ID NO:25 and SEQ ID NO:26, respectively. In view of the fact that the present invention predominantly relates to an RNA molecule, reference is made in the following to the corresponding RNA sequences. From the above DNA sequence SEQ ID NO:25, the following UTR sequence on the RNA level can be derived (preferred CYBA 5‘UTR; a 5´ C residue has been added; a (further) Kozak sequence has been added (GCCACC)): 5’-CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC-3‘ (SEQ ID NO:1). This 5’UTR sequence (which may comprise a (partial) Kozak sequence (e.g. GUCGCC or GCCGCC, or, preferably in the context of the invention, GCCACC) at its 3´end) immediately precedes the start codon of the human CYBA gene. The following UTR sequence on the RNA level depicts a preferred CYBA 5‘UTR (without the (partial) Kozak sequence and without the 5´ C residue: 5’-CGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC-3‘ (SEQ ID NO:40) From the above DNA sequence SEQ ID NO:26, the following UTR sequence on the RNA level can be derived (preferred CYBA 3‘UTR): 5’-CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCC ACCUGCAAUAAAUGCAGCGAAGCCGGGA-3‘ (SEQ ID NO:2)). The DNA sequence displaying the nucleotide sequence of the human Ag gene’s 5’-UTR present on the coding strand of the human Ag gene is shown in the following Table 2. Table 2: Genetic code of the human Ag gene UTR
Figure imgf000009_0001
Table 2 shows the exact genetic code of the hAg gene UTR. The DNA sequence is shown from the 5’ to the 3’ end. The 5’ UTR consists of 36 base pairs. The UTR is shorter than average human 5´UTRs, which consist of around 200 nucleotides. In the above Table 2, the DNA sequence displaying the hAg gene 5’UTR is shown as SEQ ID NO:28. In view of the fact that the present invention predominantly relates to an RNA molecule, reference is made in the following to the corresponding RNA sequences. From the above DNA sequence SEQ ID NO:28, the following UTR sequence on the RNA level can be derived (preferred hAg 5‘UTR; a G residue has been added to create a full Kozak sequence (GCCACC)): 5’-(CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC-3‘ (SEQ ID NO:3). This 5’UTR sequence (which may comprise a (partial) Kozak sequence (e.g. (C)CCACC or, preferred in the context of the invention, GCCACC) at its 3´end) immediately precedes the start codon of the hAg gene. Another important feature influencing mRNA translation efficiency is the poly-A tail, which is located on the 3’ end. It has been shown that a prolongation of the poly-A tail to 120 nucleotides has beneficial effects on protein expression, presumably because of the protective effect of longer poly-A tails against mRNA degradation (16). In contrast to long poly-A tails, mRNAs with poly-A tails shorter than 50 nucleotides are claimed not to be translated at all (11, 17). Hence, in mRNA therapy, recombinant mRNA constructs are advantageously to be furnished with a poly-A tail of 120 nucleotides or more. Degradation of most mRNA transcripts in eukaryotic cells begins with 3’ to 5’ exonucleolytic deadenylation, resulting in removal of most of the poly A-tail. Subsequently, two major pathways that are responsible for the degradation of the rest of the mRNA body are known to come into play. On the one hand, the 5’ end is decapped by the Dcp1/Dcp2 complex, followed by 5’-3’ exonucleolytic degradation that is catalyzed by Xrn1p. On the other hand, the exosome enables 3’-5’ exoribonucleolytic degradation with the 5’ cap being retained (18). Moreover, it is known that the 5’ cap interaction with the 3’ poly-A tail results in circular forms of the mRNA. It is assumed that the circular shape of the mRNA increases the initiation rate of ribosomes after translating the first stop codon and also protects mRNA against degradation (19). The present application, inter alia, surprisingly found that an increase of the translational efficiency (and/or expression) of a natural CYBA or Ag (e.g. hAg) mRNA can be conferred to a foreign mRNA, namely by virtue of flanking its coding sequence with combinations of (shortened) CYBA 5’-UTRs or (shortened) Ag 5’-UTR and/or (shortened) CYBA 3’-UTRs. It is of note in this respect that both, the 5’UTR and the 3’UTR of the present invention (e.g. as shown in SEQ ID NO:1/40/3 and SEQ ID NO:2, respectively), may be shorter than the DNA sequences displaying the human CYBA gene 5’- and 3’ UTRs and hAg gene 5’-UTR shown as SEQ ID NO:25/31/26/29 and SEQ ID NO:27/28/30, respectively. This can be shown by a single-cell analysis of mRNA transfection time-lapse movies which has recently been shown to be capable of assessing individual expression time courses (26) while it has been reported that it is possible to use regular micropatterns to position cells on a regular grid of adhesion sites (27). The present application has further demonstrated that this technology offers the resolution to rapidly screen and compare different UTR combinations on a foreign mRNA. To address this, the coding sequence of destabilized enhanced green fluorescence protein (d2EGFP) has been chosen to artificially shorten the life cycle of the reporter protein inside the cell (28). The combinations included insertion of, for example, the respective CYBA UTRs at 5’ or 3’ ends, respectively, at both 5’ and 3’ ends, at the 5’ end combined with two repeats of the 3’ UTR at the 3’ end, or two repeats of 3’ UTR without 5’ UTR. All of these were compared to a control construct without UTRs. Protein and functional mRNA life times and the expression rate from each of the compared transcripts were assessed. Single-cell analysis of the dynamics of gene expression after mRNA transfection was compared to population based methods (flow cytometry, fluorescence microscopy imaging, and the bioluminescence measurement of luciferase activity). It has surprisingly been shown that the total protein expression over a period of three days for all UTR combinations compared to the control is improved. Even more surprisingly, it was found in the context of the present invention that particular combinations of regulators of energy homeostasis (for example GLP-1 (or an GLP-1 analogue) or GDF15 (or an GLP-1 analogue)) and (a) particular UTR(s) (CYBA 5’- or Ag 5-’UTRs and/or 3’- UTR(s)) provide for extraordinary good results, in vitro and/or in vivo (as shown in the appended examples), in particular with respect to an increase of the translation efficiency and/or of expression, respectively, of said regulators of energy homeostasis. For example, the excretion of said regulators (e.g. into the supernatant of, e.g., HEK293 cells) is increased (in vitro and/or in vivo). Assays which can be employed to test such an increase are enclosed herein, e.g. in the appended examples. Outstanding in this respect were the particular combinations of GLP-1 with the hAg 5’-UTR (in vitro and in vivo testings), hGDF15 H6D with the CYBA 5’- and 3’-UTRs (in vitro testing) and (h)GDF15 WT and the CYBA 5’- and 3’-UTRs (in vivo testing). In the context of the present invention GLP-1 may, for example, be a native GLP-1 or an analogue thereof (i.e. a therapeutic analogue). GLP-1 may be selected from the following group: Native GLP-1: The native (human) GLP-1 molecule has two biologically active forms, GLP-1(7-36)amide and GLP-1(7-37). The sequence for GLP-1(7-37) is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO:57). Exenatide (Byetta®®/Bydureon®, AstraZeneca, Amylin, AstraZeneca; derived from the saliva of the Gila monster; acts as a GLP-1 mimic, HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:58), Liraglutide (Victoza®®, 97% aa-similarity to GLP-1 with a modification that includes a fatty acid moiety which extends its half-life; NovoNordisk. The sequence of Liraglutide is: HAEGTFTSDVSSYLEEQAAKEFIAWLVKGRG (SEQ ID NO:59), Semaglutide (Ozempic®): Similar to Liraglutide, Semaglutide has modifications for longer action. The sequence is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 60), Dulaglutide (Trulicity®): This is a GLP-1 analogue designed for once-weekly administration. Its sequence is a fusion of two GLP- 1 sequences to an Fc fragment of human IgG4, Albiglutide (Tanzeum): This GLP-1 analogue is a fusion protein consisting of two GLP-1 analogues linked to human albumin. In the context of the present invention GDF-15 may, for example, be a native GDF-15 or an analogue thereof (i.e. a therapeutic analogue). Therapeutic analogues of GDF-15 are known that aim to enhance the stability, potency, or specificity of GDF-15 for clinical applications. Examples include: a) Fc-fusion proteins where GDF-15 is fused with the Fc fragment of an antibody to increase its stability and half-life, b) modified peptides or small molecules that mimic or modulate the activity of GDF-15. In particular, the following was demonstrated in the context of the present invention and is shown in the appended examples. In the in vitro testings made in the context of the present invention, the following was found: The particular combination of GLP1 with the hAg 5‘-UTR could be identified as a lead candidate with respect to expression of GLP1. For GDF15, the H6D mutant sequences lead to higher GDF15 expression. The particular combination of GDF15 H6D with the CYBA 5‘- and 3`-UTRs was shown as lead candidate among the tested GDF15 RNA constructs. In the in vivo testings made in the context of the present invention, the following was found (in particular as regards a reduced food consumption): GDF15, in particular GDF15 WT, in combination with the CYBA 5‘- and 3‘-UTRs, shows physiological activity (reduction of food intake and weight loss); in particular at a dosage of about 0.5 mg/kg body weight (BW). The GDF-15 and GLP-1 constructs (in combination with the CYBA 5‘- and 3‘-UTRs and the hAg 5‘-UTR, respectively) are superior against corresponding non- translated control RNAs; in particular at a dosage of about 1 mg/kg BW. For the GDF-15 construct, GDF-15 levels are significantly elevated up to 24 hours post injection. Chemically modified RNA versions (modified RNA/SNIM® RNA; see below) and/or codon optimized versions provided quite good results in all testings. These findings lead to the provision of the embodiments as characterized in the claims. Thus, the present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis (e.g. GLP-1 or GDF-15, or an analogue thereof), or for two such regulators (or even for more); and (b) upstream of said coding region one (or more) UTR(s) comprising (A) a 5’-UTR of cytochrome b-245 alpha polypeptide (CYBA), or a functional derivative of said 5’-UTR; or (B) a 5’-UTR of alpha globin (Ag), or a functional derivative of said 5’-UTR; and/or (c) downstream of said coding region one (or more) UTR(s) comprising a 3’-UTR of CYBA, or a functional derivative of said 3’-UTR. More particular, the present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis (e.g. GLP-1 or GDF-15, or an analogue thereof), or for two such regulators (or even for more); and (b) upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40, or a functional derivative of said sequence; or (B) the sequence as shown in SEQ ID NO:3, or a functional derivative of said sequence; and/or (c) downstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2, or a functional derivative of said sequence. Even more particular, the present invention relates to an RNA molecule comprising (a) a coding region coding for a regulator of energy homeostasis (e.g. GLP-1 or GDF-15, or an analogue thereof), or for two such regulators (or even for more); and (b) upstream of said coding region one (or more) UTR(s) comprising (A) the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40; or (B) the sequence as shown in SEQ ID NO:3 or a sequence which shows 1 to 6 substitutions in comparison to SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:3; and/or (c) downstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2. Most preferably, the coding region to be comprised in the RNA molecule of the invention is selected from the group consisting of: (i) a coding region coding for GLP-1 (or GLP-1 analogue), preferably human GLP-1 (hGLP- 1); (ii) a coding region coding for GDF15 (or GDF15 analogue), preferably human GDF15 (hGDF15) (H6D mutant or, preferably, WT); and (iii) a coding region coding for both, GLP-1 (or GLP-1 analogue), preferably hGLP-1, and GDF15 (or GLP-1 analogue), preferably hGDF15 (H6D mutant or, preferably, WT). Preferably, the UTR(s) as defined in any of (b), supra, is/are located at the 5’ end of the coding region as defined in any of (a), supra. Preferably, the UTR(s) as defined in any of (c), supra, is/are located at the 3’ end of the coding region as defined in any of (a), supra. More preferably, the UTR(s) as defined in any of (b), supra, in particular as defined in any of (b)(A), supra, is/are located at the 5’ end of the coding region as defined in any of (a), supra, and the UTR(s) as defined in any of (c), supra, is/are located at the 3’ end of the coding region as defined in any of (a), supra. In the context of the invention, the GLP-1 (or GLP-1 analogue) may, for example, be encoded by SEQ ID NO:4, 5 or 6 or by SEQ ID NO: 52 or 53, or have the amino acid sequence as depicted in SEQ ID NO:7 or 8 or as depicted in SEQ ID NO: 57, 58, 59 or 60. The GDF15 WT may, for example, be encoded by SEQ ID NOs:9, 10 or 11 or have an amino acid sequence as depicted/comprised in SEQ ID NO:12 or 13 or in SEQ ID NO:61. The GDF15 H6D may, for example, be encoded by SEQ ID NO:14, 15 or 16 or have the amino acid sequence as depicted in SEQ ID NO:17. Alternatively, the GDF15 (or GDF15 analogue) may, for example, be encoded by SEQ ID NO: 54, 55 or 56. A GLP-1 (or GLP-1 analogue) as encoded by SEQ ID NO: 52 or 53 is preferred. A GDF15 (or GDF15 analogue) as encoded by SEQ ID NO: 54, 55 or 56 is preferred. Particular embodiments of the RNA molecule according to the invention are selected from the group consisting of (I) an RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (CYBA-GDF15 WT); (II) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as defined herein elsewhere (hAg-GLP-1); (III) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (hAg-GDF15 WT); (IV) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 H6D as defined herein elsewhere (CYBA-GDF15 H6D); (V) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(B), supra, and a coding region encoding GDF15 H6D as defined herein elsewhere (hAg-GDF15 H6D); and (W) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GLP-1 as defined herein elsewhere (CYBA-GLP-1). Preferred embodiments of the RNA molecule according to the invention are selected from the group consisting of (I) an RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; (II) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as encoded by SEQ ID NO: 52 or 53; (III) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; (IV) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; (V) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(B), supra, and a coding region encoding GDF15 as encoded by SEQ ID NO: 54, 55 or 56; and (W) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GLP-1 as encoded by SEQ ID NO: 52 or 53. The RNA molecule according to the invention, in particular any of the above described particular RNA molecules, may comprise between its upstream UTR(s) ((b)) and its coding region a (partial) Kozak sequence (like the one defined herein elsewhere (e.g. (C)CCACC or, preferably, GCCGCC or, more preferably, GCCACC), and/or may comprise upstream of its upstream UTR(s) ((b)) a promoter (like the one defined herein elsewhere (e.g. a T7 promoter)). Between the promoter and the upstream UTR(s) ((b)), (an) additional ribonulceotide(s) may be comprised (e.g. at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 additional ribonulceotide(s); e.g. a “C” or “CT”)) Non-limiting examples of Kozak sequences, promoters and 5‘-UTR and 3‘-UTR sequences suitable to be employed in accordance with the invention are described herein elsewhere and, for example, also in WO 2017/001554. A ribonucleic acid (RNA) molecule in accordance with the present invention relates to a polymeric molecule which is assembled as a chain of the nucleotides termed G, A, U, and C. Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A nitrogenous base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). In a polymeric RNA molecule a phosphate group is attached to the 3' position of one ribose and the 5' position of the next. Thus, the nucleotides in a polymeric RNA molecule are covalently linked to each other wherein the phosphate group from one nucleotide binds to the 3' carbon on the subsequent nucleotide, thereby forming a phosphodiester bond. Accordingly, an RNA strand has a 5' end and a 3' end, so named for the carbons on the ribose ring. By convention, upstream and downstream relate to the 5' to 3' direction in which RNA transcription takes place. Preferably, the RNA molecule is a messenger RNA (mRNA) molecule. mRNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids: a protein, as summarized in the central dogma of molecular biology. As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis. As will be outlined in more detail below, a ribonucleic acid (RNA) molecule of present invention comprises two or even three main modules, i.e., (a) a coding region coding for a polypeptide, in particular for a regulator of energy homeostasis like GLP-1 or GDF-15, or for two such regulators (or even more), (b) upstream of said coding region one (or more) UTR(s), and/or (c) downstream of said coding region one (or more) UTR(s) which are different than the UTR(s) of module (b). Thus, the RNA molecule of the present invention resembles with respect to its structure a “normal” mRNA molecule which occurs in nature, harboring a coding region as well as (an) (5’ and/or 3’) UTR(s) as well as, optionally, a poly-A tail. The term “coding region” as used in accordance with the present invention relates to a polymeric RNA molecule which is composed of codons, which are decoded and translated into proteins by the ribosome in accordance with the information provided by the “genetic code”. Coding regions commonly begin with a start codon and end with a stop codon. In general, the start codon is an AUG triplet and the stop codon is UAA, UAG, or UGA. In addition to being protein-coding, portions of coding regions may serve as regulatory sequences in the pre-mRNA as exonic splicing enhancers or exonic splicing silencers. The coding region of a gene coding for a polypeptide or a protein as used in accordance with the present invention is also known as the coding sequence or CDS (from coding DNA sequence) and is that portion of a gene's DNA or RNA, composed of exons, that codes for a polypeptide or protein. As mentioned, the region is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon. The coding region in mRNA is usually flanked by the five prime untranslated region (5’-UTR) and the three prime untranslated region (3’-UTR) which are also parts of the exons. The coding region or CDS is that portion of the mRNA transcript, i.e., of the coding region coding for a polypeptide as used in accordance with the present invention, that is translated by a ribosome into a polypeptide or a protein. The term “untranslated region” or “UTR” as used in accordance with the present invention relates sections of the mRNA upstream the start codon and downstream the stop codon that are not translated, and are, therefore, termed the five prime untranslated region (5'-UTR) and three prime untranslated region (3'-UTR), respectively. These regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA. As used in the present invention, the 3’ untranslated region (3'-UTR) relates to the section of messenger RNA (mRNA) that immediately follows the translation termination codon. An mRNA molecule is transcribed from the DNA sequence and is later translated into protein. Several regions of the mRNA molecule are not translated into protein including the 5'-cap, 5'-UTR, 3'- UTR, and the poly-A tail. As used in the present invention, the 5' untranslated region (5′-UTR) (also known as a Leader Sequence or Leader RNA) is the region of an mRNA that is directly upstream from the start codon. The 5′-UTR begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region. In prokaryotes, the length of the 5′-UTR tends to be 3- 10 nucleotides long while in eukaryotes it tends to be, longer, generally from 100 to several thousand nucleotides long, but sometimes also shorter UTRs occur in eukaryotes. As used in the present invention, the 3’-UTR may comprise regulatory regions within the 3'- untranslated region which are known to influence polyadenylation and stability of the mRNA. Many 3'-UTRs also contain AU-rich elements (AREs). Furthermore, the 3'-UTR contains the sequence AAUAAA that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript. As will be outlined in more detail further below, an RNA molecule as used in accordance with the present invention may also contain a poly-A tail. A poly-A tail is a long sequence of adenine nucleotides (often several hundred) added to the 3' end of the pre-mRNA by a process called polyadenylation. This tail promotes export from the nucleus and translation, and protects the mRNA from degradation. Polyadenylation is the addition of a poly(A) tail to a messenger RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. As mentioned above, the RNA molecule of the present invention preferably comprises two or three main modules, i.e., (a) a coding region coding for a polypeptide, in particular for a regulator of energy homeostasis like GLP-1 or GDF-15 (or analog thereof), or for two such regulators (or even more); and (b) upstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40, or a functional derivative thereof (for example a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40, respectively); or the sequence as shown in SEQ ID NO:3, or a functional derivative thereof (for example a sequence which shows 1 to 6 substitutions in comparison to SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:3); and/or (c) downstream of said coding region one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a functional derivative thereof (for example a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2). Thus, it is mandatory that the RNA molecule of the present invention comprises two main modules, i.e., the module (a) (as described herein elsewhere) and at least one of module (b) (as described herein elsewhere) and module (c) (as described herein elsewhere). In another preferred embodiment, the RNA molecule of the present invention comprises three main modules, i.e., the module (a) (as described herein elsewhere) and module (b) (as described herein elsewhere) and module (c) (as described herein elsewhere). Yet, while module (a) is mandatory, it is also envisaged that the RNA molecule may also lack one of the modules (b) or (c). One module of the RNA molecule, i.e., “a coding region… ” (module (a)) is not particularly limited and may be any desired coding region which is to be expressed in a given cell, as long as it codes for a regulator of energy homeostasis like GLP-1 or GDF15 (or analog thereof), or for two such regulators (or even more). Thus, this module may be a coding region coding for the desired regulator of energy homeostasispolypeptide, i.e., the desired final product. A coding region in accordance with the invention may also be a nucleotide sequence which differs from a known natural sequence and contains mutations (i.e. point mutations, insertion mutation, deletions and combinations thereof). Particular examples of such a coding region are encoded by the nucleotide sequences as depicted in SEQ ID NOs:14, 15 and 16. An Example of a polypeptide encoded by such a nucleotide sequence is depicted in SEQ ID NO:17. A coding region in accordance with the invention may partly or to the full extent be a codon optimized sequence derived from the natural sequence to be used as module (a). Codon optimization is a technique to maximize the protein expression by increasing the translational efficiency of a gene of interest. It is known that natural genes do not use the available codons randomly, but show a certain preference for particular codons for the same amino acid. Thus, because of the degeneracy of the genetic code - one amino acid can be encoded by several codons - transforming the nucleotide sequence of a gene of interest into a set of preferred codons of the same or another species. In the context of the invention, the codon optimized sequence may, for example, be codon optimized for (the expression in) mice or, preferably, for (the expression in) humans. Particular examples of nucleotide sequence encoding such a codon optimized sequence are depicted in SEQ ID NOs:5, 6, 10, 11, 15 and 16 and in SEQ ID NOs:52, 53, 54, 55 and 56. Examples of polypeptides encoded by such a codon optimized sequence are depicted in SEQ ID NOs: 7, 8, 12 and 17. In particular, it is envisaged in the context of the present invention that “a coding region…” (module (a)) codes for GLP-1, GDF15 or both, i.e. GLP-1 and GDF15 (or the respective analogue). Also in this respect, the coding region may comprise (or consist of) the respective codon optimized sequence(s) (see above for particular examples). In the context of the present invention, “coding region” should be understood to mean any polyribonucleotide molecule which, if introduced into a cell, is translatable to a polypeptide/protein or fragment thereof in accordance with the invention. The terms “polypeptide” and “protein” here encompass any kind of amino acid sequence, i.e., chains of two or more amino acids which are each linked via peptide bonds and also includes peptides and fusion proteins. It is particularly envisaged in the context of the invention that the “coding region coding for a polypeptide” contains a ribonucleotide sequence which encodes a polypeptide/protein or fragment thereof whose function in the cell or in the vicinity of the cell is needed or beneficial, e.g., a protein the lack or defective form of which is a trigger for a disease or an illness, the provision of which can moderate or prevent a disease or an illness, or a protein which can promote a process which is beneficial for the body, in a cell or its vicinity. The coding region may contain the sequence for the complete protein or a functional variant thereof. In the context of the invention, the ribonucleotide sequence of the coding region encodes a polypeptide/protein (or 2 or more polypeptides/proteins) which act(s) as (a), regulator(s) of energy homeostasis, or a functional fragment thereof. A particular example of such a regulator is an anorectic. The particular polypeptide/protein, regulator of energy homeostasis and/or anorectic, to be encoded by the “coding region…” (module (a)) is GLP-1 and GDF15 (or a functional variant/derivative or fragment thereof). Each of these proteins is one whose function is necessary in order to remedy a related disorder (for example a metabolic disorder like obesity, diabetes mellitus, insulin resistance or metabolic syndrome) and/or in order to initiate related processes in vivo (such as decreasing (aberrant) body weight, decreasing food intake, restraining appetite, etc. Here, functional variant/derivative is understood to mean a fragment or other variant which in the cell can undertake the function of the protein whose function in the cell is needed or the lack or defective form whereof is pathogenic. As mentioned, it is particularly preferred that the “coding region… ” (module (a)) encodes a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect on the conditions, diseases or disorders as defined herein elsewhere. As such, the RNA molecule of the present invention comprising said “coding region…” may be used in nucleic acid therapy and related applications. In this context, in accordance with the invention, an increased efficiency of translating a coding region of an RNA molecule into the polypeptide or a protein encoded by said coding region of an introduced exogenous RNA molecule is envisaged to compensate or complement endogenous gene expression, in particular in cases where an endogenous gene is defective or silent, leading to no, insufficient or a defective or a dysfunctional product of gene expression such as is the case with many metabolic diseases like obesity, diabetes mellitus, insulin resistance, metabolic syndrome, etc. to name a few. An increased efficiency of translating a coding region of an RNA molecule into a polypeptide of introduced exogenous RNA molecules of the present invention may also be intended to have the product of the expression interact or interfere with any endogenous cellular process such as the regulation of gene expression, signal transduction and other cellular processes. As mentioned, the RNA molecule of the present invention comprising a “coding region…” can appropriately be used in any case where a polypeptide or a protein as defined herein elsewhere, which would naturally be present in the body but is not present or is present in deficient form or in too small quantity because of gene defects or diseases, is to be provided to the body. Respective proteins and the genes encoding them, the deficiency or defect whereof are linked with a disease as defined herein elsewhere, are known and likewise defined herein elsewhere. The respective intact version of the coding region coding for the intact polypeptide or protein can be used in accordance with the present invention. Numerous genetic disorders which may be treated/prevented in accordance with the invention, and which may be caused by the mutation of a single gene, are known and are candidates for the respective mRNA therapeutic approaches. Disorders caused by single-gene mutations, can be dominant or recessive with respect to the likelihood that a certain trait will appear in the offspring. While a dominant allele manifests a phenotype in individuals who have only one copy of the allele, for a recessive allele the individual must have two copies, one from each parent to become manifest. In contrast, polygenic disorders are caused by two or more genes and the manifestation of the respective disease is often fluent and associated to environmental factors. Examples for polygenic disorders are hypertension, elevated cholesterol level and others. Also in these cases therapeutic mRNA representing one or more of these genes may be beneficial to those patients. Furthermore, a genetic disorder must not have been passed down from the parents' genes, but can also be caused by new mutations. Also in these cases therapeutic mRNA representing the correct gene sequence may be beneficial to the patients. An online catalog with presently 22,993 entries of Human Genes and Genetic Disorders (including those related to GLP-1 and/or GDF15) together with their respective genes (including GLP-1 and GDF15 genes) and a description of their phenotypes are available at the ONIM (Online Mendelian Inheritance in Man) webpage (http://www.onim.org); sequences of each are available from the Uniprot database (http://www.uniprot.org). The following Table 3 lists some conditions, diseases and disorders which are intended to be addressed/intervened (e.g. treated or prevented) in accordance with the present invention, i.e. which relate in any manner to the regulator(s) of energy homeostasis like GLP-1 and/or GDF15. In this context, it is noted that due to the high degree of interaction of cellular signaling pathways, the mutation of a certain gene usually causes a multiply of pathogenic symptoms. In principle, compositions of the invention may comprise an mRNA encoding a therapeutic fusion protein, wherein the encoded therapeutic protein or a homolog thereof is one as described herein elsewhere and the second protein is a signal peptide that allows the secretion of the therapeutic protein. A signal peptide is a short, typically 5-30 amino acids long, amino acids sequence present at the N-terminus of said therapeutic protein and that leads the fusion protein towards the cell’s secretory pathway via certain organelles (i.e. the endoplasmic reticulum, the golgi-apparatus or the endosomes). Thus, such fusion protein is secreted from the cell or from a cellular organelle or inserted into a cellular membrane (e.g. multi-spanning trans- membrane proteins) at a cellular compartment or at the cell’s surface. Thus, in preferred embodiments of the present invention the “coding region coding for a polypeptide” (module (a)) may encode one (or two or more) product(s) of genes that cause, predispose or protect from conditions, disorders or diseases as defined herein elsewhere. Examples of such conditions, disorders or diseases that may be treated (or prevented) include those outlined in the following Table 3. In some embodiments, the “coding region…” (module (a)) may be translated into a partial or full length protein comprising cellular activity at a level equal to or greater than that of the native protein. In some embodiments, the “coding region… (module (a))” encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or preventive effect with respect to the conditions, diseases or disorders selected from the group consisting of the ones as outlined herein elsewhere and, in particular, in the following Table 3. The “coding region… “(module (a)) may be used to express a partial or full length protein with cellular activity at a level equal to or less than that of the native protein. This may allow the treatment of diseases for which the administration of an RNA molecule can be indicated. Table 3: Examples of particular conditions, diseases and disorders to be treated or prevented in accordance with the invention (aberrant) body weight
Figure imgf000022_0001
The above Table 3 shows examples of conditions, disorders or diseases which can be treated with the RNA molecule of the present invention wherein the RNA molecule comprises a “coding region…” which encodes an intact or even improved version of a protein/polypeptide or a functional fragment or variant thereof, (the deficient expression of) which is involved in the respective condition, disorder or disease and to which the respective condition, disorder or disease relates, respectively. As mentioned, it is particularly envisaged that the protein/polypeptide is GLP-1 and/or GDF-15. Obesity (e.g. abdomal obesity), diabetes mellitus (e.g. type II diabetes mellitus), insulin resistance and metabolic syndrome are non-limiting examples of a metabolic disorder to be treated/prevented in accordance with the invention. The metabolic syndrome to be treated in accordance with the invention may be a set of syndromes. The metabolic syndrome and set of symptoms, respectively, may include obesity (e.g. abdominal obesity), hypertension, cardiovascular disease, elevated fasting plasmid glycose, dyslipidemia, and/or an enhanced inflammatory state. In the conditions, disorders or diseases to be addressed in accordance with the invention, a protein, e.g. an enzyme, may be defective (or at least undesirably low expressed), which can be medically/addressedby administering the RNA according to the invention. This makes the protein encoded by the defective/low expressed gene or a functional fragment or variant thereof available. Transcript replacement approaches/enzyme replacement approaches do not affect an underlying genetic defect, but increase the concentration of the enzyme in which the patient is deficient or which is undesirably low. As an example, in obesity, the transcript replacement therapy/enzyme replacement therapy replaces the deficient GLP-1 and/or GDF-15. Thus, as mentioned, particular proteins which can be encoded by the “coding region… (module (a)) according to the invention are GLP-1 and GDF-15. GLP-1 and GDF-15, their functions and nucleotide and amino acid sequences are well known in the art (see the background section above and the respective references cited therein). Their nucleotide and amino acid sequences can, for example, be obtained from the pertinent gene/protein databases like, for example, UniProt KB. The nucleotide and amino acid sequence of hGLP-1 can, for example, be obtained via the database entry P01275 (e.g. via UniProt KB). The amino acid sequence of hGLP-1 is also depicted in SEQ ID NO:7. An example of a corresponding coding nucleotide sequence is depicted in SEQ ID NO:4 (the nucleotide sequence of EX4GLP1Gly8 as disclosed in Parsons Gene Therapy 14, 2007, 38-48). An example for a corresponding nucleotide sequence which is codon optimized for humans is depicted in SEQ ID NO:5. The nucleotide and amino acid sequence of mGLP-1 can, for example, be obtained via the database entry P55095 (e.g. via UniProt KB). The amino acid sequence of mGLP-1 is also depicted in SEQ ID NO:8. An example of the corresponding codon optimized nucleotide sequence for mouse is depicted in SEQ ID NO:6. The GLP-1 (Glucagon) UTRs may also be obtained from reference sequence in NCBI (NM_002054.4). The 5’-UTR nt position in the reference sequence is 1-256. The 3’-UTR nt position in the reference sequence is 800-1294. The nucleotide and amino acid sequence of hGDF15 (wildtype; hGDF15 WT) can, for example, be obtained via the pertinent database entries (e.g. http://www.ebi.ac.uk/ena/data/view/BC000529 and http://www.uniprot.org/uniprot/Q99988, respectively). An amino acid sequence of hGDF15 WT is also depicted/comprised in SEQ ID NO:12. The amino acid sequence of hGDF15 WT is also depicted in SEQ ID NO:61. An example of a corresponding encoding nucleotide sequence is depicted in SEQ ID NO:9. An example for a corresponding coding nucleotide sequence which is codon optimized for humans is depicted in SEQ ID NO:10. The nucleotide and amino acid sequence of mGDF15 can, for example, be obtained by the database entry Q9Z0J7 (e.g. via UniProt KB). The amino acid sequence of is also depicted in SEQ ID NO:13. An example for a corresponding coding nucleotide sequence which is codon optimized for mouse is depicted in SEQ ID NO:11. A particular mutant form of hGDF15 is known in the art, namely hGDF15 H6D. In this mutant form, the histidin (H) has been replaced by an aspertate (D) at amino acid position 6 of the hGDF15 WT. It is known for this mutant form that T1/2 is increased. The amino acid sequence of hGDF15 H6D is depicted in SEQ ID NO:17. An example of a corresponding coding nucleotide sequence is depicted in SEQ ID NO:14. An example for a respective coding nucleotide sequence being codon optimized for humans is depicted in SEQ ID NO:15 and an example for a respective coding nucleotide sequence being codon optimized for mouse is depicted in SEQ ID NO:16. The coding region to be employed in the context of the invention may comprise multimers of sequences coding for a regulator of energy homeostasis, like GLP-1 and/or GDF-15 (MIC-1). The multimers may be heteromultimers or homomultimers. Heteromultimers may comprise sequences coding for different regulators of energy homeostasis or sequences coding one regulator of energy homeostasis and a mutant form of the same regulator of energy homeostasis. A multimer may comprise 2, 3, 4, 5, or even more coding sequences for a regulator of energy homeostasis. Dimers, in particular heterodimers are preferred. A non-limiting example of a heterodimer comprises the coding sequences for hGDF15 WT and hGDF15 H6D. In accordance with the invention, beside the protein/polypeptide itself, GLP1 and/or GDF15, the “coding region…” (module (a)) may also encode a variant/derivative of the protein/polypeptide, a variant/derivative of GLP-1 and/or a variant/derivative of GDF-15, respectively. If not otherwise specified herein, the terms “GLP-1” and “GDF-15” encompass GLP-1 itself and GDF-15 itself, respectively, and also the variants/derivatives of GLP-1 and the variants/derivatives of GDF-15, respectively. The GLP-1 analogues and the GDF-15 analogues are also encompassed by the meaning of variant/derivative of GLP-1 and variant/derivative of GDF-15, respectively. What is said with respect to variant/derivative of GLP-1 and variant/derivative of GDF-15 herein elsewhere also applies to the GLP-1 analogue and the GDF-15 analogue, respectively. In context of the present invention, a “variant” or “derivative” of a protein/polypeptide is envisaged to exhibit the same function(s), in particular the same biological function(s), more particular the same biological function(s) within a cell, tissue and/or body/patient as the respective protein/polypeptide itself. Accordingly, a GLP-1 variant/derivative is envisaged to exhibit the same (biological) function(s) as GLP-1 itself and a GDF-15 variant/derivative is envisaged to exhibit the same (biological) function(s) as GDF-15 itself, respectively. “The same (biological) function(s)”, for example means, at least one of the biologically relevant functions (see below for examples). As mentioned, GLP-1 and GDF15 may act as (physiological) regulators of energy homeostasis, in particular, as (physiological) long-term regulators of energy homeostasis. Examples of (biological) functions of GLP-1 are (i) incretin function; (ii) increasing the secretion of insulin; (iii) slowing down the emptying process in the stomach; (iv) increasing the feeling of saturation; and/or (v) decreasing of food intake. More particular examples of (biological) functions of GLP-1 are peripheral functions, like: increasing insulin secretion from the pancreas in a glucose-dependent manner; increasing insulin-sensitivity in both alpha cells and beta cells; increasing beta cells mass and insulin expression, PTM, and secretion; inhibiting acid secretion and gastric emptying in the stomach; promoting insulin sensitivity; and/or decreasing glucagon secretion from the pancreas via GPCR binding; and/or CNS functions, like: increasing hippocampus-related function; increasing acquisition/strength of conditioned taste aversions; increasing anxiety; increasing nausea or visceral malaise (illness); decreasing the hedonic value (pleasure) of food; decreasing the motivation (reward) to eat; decreasing quantity and frequency of food consumption; and/or decreasing general levels of motor activity. Such examples of (biological) functions of GLP-1 are also described in https://en.wikipedia.org/wiki/Glucagon-like_peptide-1. Examples of (biological) functions of GDF-15 are (i) mediating site protection; (ii) decreasing food intake (in particular in mice); (iii) improving glucose tolerance (in particular in mice); and/or (iv) regulating appetite in a patient. The GLP-1 and GDF-15 as employed in the context of the invention may exhibit at least one, several or even all of the above mentioned (biological) functions. In particular, the GLP-1 and GDF-15 as employed in the context of the invention may exhibit those (biological) functions which, upon administration of the GLP-1 or GDF-15 in accordance with the invention, results in the prevention, amelioration and/or cure of the (medical) indications and/or symptoms disclosed herein elsewhere. A prominent example of a GDF-15 variant is the herein elsewhere described GDF-15 H6D variant (e.g. SEQ ID NOs:15, 16, 17) In accordance with the invention, the “coding region…” (module (a)) may also encode fragments of the herein defined proteins/polypeptides, GLP-1 and GDF-15, or fragments of the respective variants/derivatives. In principle, such “fragments” can also be considered as “variants”/“derivatives” in accordance with the invention. Consequently, also the GLP-1 fragments and the GDF-15 fragments to be employed in accordance with the invention are envisaged to exhibit the respective (biological) functions (see above). “Variant”/“derivative” in accordance with the invention particularly means that the respective protein/polypeptide has the (biological) activity in accordance with the invention (see above) and the respective “variant”/“derivative” polynucleotide encodes for such a (biologically) active biomolecule, respectively. For example, a variant/derivative protein/polypeptide and polynucleotide, respectively, in accordance with the invention is envisaged to share an identity (in particular sequence identity) of at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98% and even more preferably at least 99% with a reference protein/polypeptide (e.g. the GLP-1 or the GDF15 amino acid sequence as depicted in SEQ ID NOs: 7 and 8 and 12 and 13 and 17, respectively) and polynucleotide (e.g. the GLP-1 or the GDF15 encoding nucleic acid molecule as depicted in SEQ ID NOs: 4, 5 and 6 and 9, 10 and 11 and 13, 14 and 15, respectively; or the UTRs as depicted in any of SEQ ID NOs: 1, 2, 3 and 31), respectively (for example based on the number of nucleotides and amino acids comprised in the reference sequence, respectively). In particular, the reference protein/polypeptide and polynucleotide, respectively, is envisaged to be GLP-1 itself or GDF15 itself, and the respective amino acid and nucleotide sequences, respectively. Another example of a variant/derivative polynucleotide in accordance with the invention is a polynucleotide that comprises or consists of a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule (e.g. the GLP-1 or the GDF15 encoding nucleic acid molecule as depicted in SEQ ID NOs: 4, 5 and 6 and 9, 10 and 11 and 13, 14 and 15, respectively) encoding a protein/polypeptide in accordance with the invention (e.g. the GLP-1 or the GDF15 amino acid sequence as depicted in SEQ ID NOs: 7 and 8 and 12 and 13 and 17, respectively). In the context of the present invention, "hybridizing" means that hybridization can occur between one nucleic acid molecule and another (complementary) nucleic acid molecule. Hybridization of two nucleic acid molecules usually occurs under conventional hybridization conditions. In the context of the invention, stringent hybridization conditions are preferred. Hybridization conditions are, for instance, described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA. In a particular embodiment, “hybridizing” means that hybridization occurs under the following conditions: Hybridization buffer: 2 x SSC, preferably 1 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG + BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 ^g/ml of herring sperm DNA; 50 ^g/ml of tRNA; or 0.25 M of sodium phosphate buffer, pH 7.2; 1 mM EDTA 7% SDS Hybridization temperature T = 60°C, preferably 65°C Washing buffer: 2 x SSC, preferably 1 x SSC, more preferably 0,1 x SSC; 0.1% SDS Washing temperature T = 60°C, preferably 65°C. As to a “variant”/“derivative” being a polypeptide/protein, it is, for example, envisaged that it is or comprises the amino acid sequence of the reference polypeptide/protein (e.g. GLP-1 itself and/or GDF15 itself) having (about) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 40, 50, 60 (or even more) amino acid residue(s) deleted or substituted by (a) different amino acid residue(s). Preferred is/are (a) conservative substitution(s). Any of the herein defined particular polypeptides/proteins (e.g. SEQ ID NO:7, 8, 12, 13 and 17) may be a reference polypeptide/protein. In particular, polynucleotides that are codon optimized (in order to ensure proper expression of a corresponding peptide) and/or that are different due to the degeneracy of the genetic code are considered “variant”/ “derivative” in accordance with the invention. As mentioned, any of the variants/derivatives described herein is envisaged to have the (biological) activity and (biological) function, respectively, in accordance with the invention (e.g. decreasing food intake and the like; see herein elsewhere), or to encode the respective proteins/polypeptides. As mentioned, a “variant”/“derivative” in accordance with the invention also encompasses a (biologically active) fragment of the coding region and comprised polypeptide, respectively, as defined herein (e.g. as depicted in SEQ ID NO:4, 5, 6, 9, 10, 11, 13, 14 or 15) or of the encoded protein/polynucleotide (e.g. as depicted in SEQ ID NO:7, 8, 12, 13 and 17). As to a polynucleotide, a fragment may be a nucleic acid sequence stretch of at least 30, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140 or at least 150 nucleotides. As to a polypeptide/protein, a fragment may be an amino acid stretch of at least 10, at least 20, at least 30, at least 40, at least 45, at least 46, at least 47, at least 48 or at least 49 amino acid residues. Most preferably, the fragment of the polynucleotide encodes for an amino acid sequence which exhibits (biological) activity (e.g. GLP-1 or GDF-15 activity) in accordance with the invention (see above for examples) and the fragment of the polypeptide/protein exhibits (biological) activity (e.g. GLP-1 or GDF-15 activity) in accordance with the invention (see above for examples), respectively. In response to Glucose ingestion, proglucagon in the intestinal L cells may be cleaved into GLP- 1 (AA positions 1-36). Prior to secretion into the circulation, GLP-1 (AA positions 1-36) is further processed into amidated GLP-1 (AA positions 7-36) and small amounts of glycine-extended GLP- 1 (AA positions 7-37). Both, GLP-1 (AA positions 7-36) and GLP-1 (AA positions 7-37), cause glucose dependent release of insulin by pancreatic beta-cells, stimulate gastric emptying, suppress glucose production and may promote satiety and stimulate glucose disposal in peripheral tissues independent of the actions of insulin. Such fragments are preferred fragments of GLP-1 to be employed in the context of the invention. More general, a GLP-1 fragment to be employed in the context of the invention may be an amino acid stretch of about 25 to 40, preferably about 30 to 37, preferably about 31 or 36 amino acid residues; most preferably such fragments are from the N-terminal part of GLP-1. What has been said herein above with respect to the (biological) functions applies here, mutatis mutandis. The premature GDF15/MIC-1 protein consists of 308 amino acids that contain a 29 amino acid signal peptide, a 167 amino acid propeptide, and a 112 amino acid mature protein. Respective fragments are preferred fragments of GDF15/MIC-1 to be employed in the context of the invention. More general, a GDF15/MIC-1 fragment to be employed in the context of the invention may be an amino acid stretch of about 100 to 120, preferably about 105 to 116, preferably about 110 or 114, preferably about 112 amino acid residues; most preferably such fragments comprise the mature GDF15/MIC-1 protein. What has been said herein above with respect to the (biological) functions applies here, mutatis mutandis. Also a polypeptide/protein which is encoded by a “variant”/“derivative” polynucleotide described herein is envisaged to be a “variant”/“derivative” in accordance with the invention. Also a polynucleotide which encodes a “variant”/“derivative” protein/polypeptide described herein is envisaged to be a “variant”/“derivative” in accordance with the invention. The coding region (RNA; module (a)), or the nucleotide sequence encoding it (DNA), may comprise the BspQI motiv. This motive is used for plasmid template linearization during in vitro transcription (IVT). In principle, what has been said herein above with respect to “variants”/“derivatives” and “fragments” also applies to the other modules (b) and (c) of the RNA molecules of the present invention and to each component of these other modules and of the RNA molecules. Particular examples of such a component are a 5’-UTR (e.g. a 5’-UTR as depicted in or encoded by any of SEQ ID NOs: 1, 3, 18, 19, 20, 21, 25, 27, 28, 31 and 32), or a 3’-UTR (e.g. a 3’-UTR as depicted in or encoded by any of SEQ ID NOs: 2, 19, 21 and 26). “Variants”/“derivatives” or “fragments” of these components may also be employed in accordance with the invention. Particular examples of such “variants”/“derivatives” are described in more detail herein elsewhere. Also in the context of the “variants”/“derivatives”/“fragments” of the components of the other modules of the RNA molecule of the invention, the relevant (biological) function of the component itself needs to be maintained. For example, the “variant”/“fragment” of an UTR needs to exhibit the respective UTR function. For example, the “variant”/“derivative”/“fragment” of an UTR needs to result in an RNA molecule having the same or a higher translation efficiency as an RNA molecule which comprises the reference UTR itself. The second module (b) is (are) the one (or more) 5’-UTR(s) as defined herein, in particular the one (or more) CYBA or hAg 5’-UTR(s) as described herein elsewhere (e.g. in Tables 1 and 2, supra, and in the explanations to these tables) and as depicted in SEQ ID NOs:1 and 3, respectively, or the herein defined “variants”, “derivatives” or “fragments” thereof. In a non-limiting example, the second module (b) is (are) the one (or more) 5’-UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40, respectively. In a further non- limiting example, second molecule (b) is (are) the one (or more) 5’-UTR(s) comprising the sequence as shown in SEQ ID NO:3 or a sequence which shows 1-6 substitutions in SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:3. “One or more” in this context means that module (b) of the RNA molecule may harbor at least one of the defined 5’-UTRs. The RNA molecule, however, may also harbor two, three or four of these UTRs of the present invention. Alternatively, the RNA molecule may also harbor five or even more of these UTRs of the present invention. However, harboring one of these UTRs is preferred. It is, in principle, preferred in the context of the invention that the second module (b) is (or comprises) the one (or more) CYBA or hAg 5’-UTR(s) as described herein elsewhere. However, also other (functional) 5’-UTR(s) may, in principle, be employed in accordance with the invention, e.g. in lieu of the one (or more) CYBA or hAg 5’-UTR(s). This may apply especially in cases were the third module (c) is (or comprises) the one (or more) CYBA 3’-UTR(s) as described herein elsewhere. Other 5’-UTR(s) which may be employed in accordance with the invention may, for example, be (a) minimal UTR(s), e.g. as disclosed in WO 2017/167910. Alternative 5’-UTR(s), which are preferably located directly upstream of said coding region (module (a); optionally without an additional promoter sequence), may, for example, be defined as follows: (A) UTR(s) selected from the group consisting of: (b1) a UTR of the sequence R2-CGCCACC (SEQ ID NO:41), or a sequence wherein in said UTR sequence the C at position 6 of SEQ ID NO:41 is substituted by an A and the C at position 7 of SEQ ID NO:41 is substituted by a G; and/or the A at position 5 of SEQ ID NO:41 is substituted by a G; and (b2) a UTR of the sequence R2-CNGCCACC (SEQ ID NO:42), wherein the nucleotide N at position 2 of SEQ ID NO:42 is a nucleotide selected from the group consisting of U, G, C or A, or a sequence wherein in said UTR sequence the C at position 7 of SEQ ID NO:42 is substituted by an A and the C at position 8 of SEQ ID NO:2 is substituted by a G; and/or the A at position 6 of SEQ ID NO:42 is substituted by a G, wherein R2 is an RNA sequence corresponding to the part of a promoter region starting with the nucleotide where a DNA-dependent RNA-polymerase initiates RNA synthesis, wherein R2 is selected from the group consisting of: (i) GGGAGA (SEQ ID NO: 43); (iii) GAAG (SEQ ID NO: 44); and (iv) GGGA (SEQ ID NO: 45); and Non-limiting examples of such alternative 5’-UTR(s), which are preferably located directly upstream of said coding region (module (a); optionally without an additional promoter sequence), may, for example, be defined as follows: (A) UTR(s) of the sequence R2-CNGCCACC (SEQ ID NO:2), wherein the nucleotide N at position 2 of SEQ ID NO:2 is U, wherein R2 is an RNA sequence corresponding to the part of a promoter region starting with the nucleotide where a DNA-dependent RNA-polymerase initiates RNA synthesis, wherein R2 is selected from the group consisting of: (i) GGGAGA (SEQ ID NO: 43); (ii) GAAG (SEQ ID NO: 44); and (iii) GGGA (SEQ ID NO: 45); and wherein the UTR has a maximal length of 14 nucleotides when R2 is (i); or wherein the UTR has a maximal length of 12 nucleotides when R2 is (ii) or (iii). The third module (c) is (are) the one (or more) 3’-UTR(s) as defined herein, in particular the one (or more) CYBA 3’-UTR(s) as defined herein elsewhere (e.g. in Table 1, supra, and in the explanations to this table) and as depicted in SEQ ID NO:2, or the herein defined “variants”, “derivatives” or “fragments” thereof. In a non-limiting example, the third module (c) is (are) the one (or more) 3’-UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 (i.e., the above module (c)). “One or more” in this context means that module (c) of the RNA molecule may harbor at least one of the defined 3’-UTRs. The RNA molecule, however, may also harbor two, three or four of these UTRs of the present invention. Alternatively, the RNA molecule may also harbor five or even more of these UTRs of the present invention. However, harboring one of these UTRs is preferred. The full-length sequence of the native human cytochrome b-245 alpha polypeptide (CYBA) mRNA is known in the art and has the sequence as shown in SEQ ID NO:29. Herein (examplary) and in the appended examples, the sequence from nucleotides 36 to 71 of the native human CYBA mRNA (SEQ ID NO:25) has been used as the 5’ UTR fragment of the CYBA mRNA (resulting in, for example, the nucleotide sequence 5’- CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC-3‘ (SEQ ID NO:1); a C has been added at the 5´end; a full Kozak sequence has been added at the 3´end) and the sequence from nucleotides 657 to 723 of the native human CYBA mRNA (SEQ ID NO:26) has been used as the 3’ UTR of the CYBA mRNA (resulting in, for example, the nucleotide sequence 5’-CCUC GCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGAAGCCGG GA-3‘ (SEQ ID NO:2)). The full-length sequence of the native hAg mRNA is also known in the art and has the sequence as shown in SEQ ID NO:30. Herein (examplary) and in the appended examples, the sequence from nucleotides 31 to 66 of the native hAg mRNA (SEQ ID NO:28) has been used as the 5’-UTR fragment of the hAg mRNA (resulting in, for example, the nucleotide sequence 5’- CTCTTCTGGTCCCCACAGACT CAGAGAGAACGCCACC-3’ (SEQ ID NO:3); a G has been added to create a full Kozak sequence at the 3´end). However, as mentioned, the 5’-UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) but may also be a “variant”/“derivative” 5’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40, respectively. Alternatively, the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40. Most preferably, the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40. In principle, the lower numbers of substitutions are preferred. The above nucleotide substitution in comparison to SEQ ID NO:1 may be performed at position 33 in the sequence of SEQ ID NO:1 or at position 32 in the sequence of SEQ ID NO:40. The nucleotide “U” at this position may be substituted by a “C”. This substitution brings a sequence element of CYBA which is present in SEQ ID NO:1 or SEQ ID NO:40 (GUCGCC) closer to the Kozak consensus sequence of vertebrates. For example, it results in the Kozak sequence GCCGCC (SEQ ID NO:23). The Kozak consensus sequence of vertebrates has the sequence of GCCRCCAUG (SEQ ID NO:24; the start codon is underlined while “R” indicates any purine) while the mentioned element of CYBA has the sequence of GuCGCCAUG, comprising the motive CuCGCC (the start codon is underlined while the deviation from the vertebrate consensus sequence is indicated by the lower case letter “u”). The UTR sequence(s) which have one or more of the above substitutions (in comparison to SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25)) may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g. SEQ ID NO:1 or SEQ ID NO:40, respectively), preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:1 or SEQ ID NO:40). The property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples. The translation efficiency is the rate of mRNA translation into polypeptides or proteins within cells. The translation efficiency of a given mRNA is measured as the number of proteins or polypeptides which are translated per mRNA per time unit. Translation is the process in which cellular ribosomes create proteins and is well-known to the skilled person. Briefly, in translation, messenger RNA (mRNA) which is produced by transcription from DNA is decoded by a ribosome to produce a specific amino acid chain or a polypeptide or a protein. Thus, the translation efficiency of a given RNA molecule harboring a modified UTR sequence is preferably higher in comparison to a translation efficiency of the same given RNA but harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)). Accordingly, the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring a modified UTR sequence which are translated per RNA per time unit is higher than the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)) which are translated per RNA per time unit. In case the translation efficiency of a given RNA molecule harboring a modified UTR sequence is similar or the same in comparison to a translation efficiency of the same given RNA but harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)), the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring a modified UTR sequence which are translated per RNA per time unit is similar to or the same as the number of proteins or polypeptides encoded by the coding region of the RNA molecule harboring an unmodified UTR (e.g. SEQ ID NOs:25/28/26 (or SEQ ID NO:1/40/3/2)) which are translated per RNA per time unit. The “translation efficiency” can, e.g., be determined by methods described in the appended examples and as outlined in the following. Translation efficiency, in the context of the present invention, is the rate of mRNA translated into protein within a cell at a certain time point in relation to the amount of mRNA encoding the respective protein in said cell at the same time point. Thus, the translation efficiency is the quotient of the mRNA translated into protein within a cell at a certain time point and the amount of mRNA encoding the respective protein. Both parameters, i.e., the mRNA translated into a protein as well as the amount of mRNA encoding the respective protein, can be determined by methods known in the art. As it has been done in the appended examples, as non-limiting examples, the amount of mRNA translated into protein within a cell can, e.g., be determined by as determined by flow cytometry (FC) while the amount of mRNA encoding the respective protein can, e.g., be measured by qPCR. The UTR(s) comprising the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) or a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising unmodified UTR (e.g. SEQ ID NO:25 (or SEQ ID NO:1 or SEQ ID NO:40)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s), e.g. in comparison to SEQ ID NO:1 or SEQ ID NO:40. The addition of (a) nucleotide(s) can be flanking. Thus, the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention. The additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides. In light of the rationale that the addition of nucleotides is likely not to change the above functional properties of the UTR(s) of the invention the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:1 or SEQ ID NO:40, respectively, preferably higher translation efficiency as SEQ ID NO:1 or SEQ ID NO:40, respectively, as defined above. Alternatively, or in addition to these flanking additions of (a) nucleotide(s) the addition of (a) nucleotide(s) can be interspersed. Thus, the additional nucleotide(s) may be added/inserted within the nucleotide sequence of the UTR(s) of the present invention. These nucleotide(s) insertions comprise 1, 2, or 3 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:1 or SEQ ID NO:40, preferably higher translation efficiency as SEQ ID NO:1 or SEQ ID NO:40 as defined above. The UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO:1 or SEQ ID NO:40 (or SEQ ID NO:25) and modifications thereof. Rather, the specific sequence of SEQ ID NO:1 or SEQ ID NO:40 and modifications thereof merely define the CYBA 5’ core region. Thus, in a preferred embodiment, the UTR as shown in SEQ ID NO:1 or SEQ ID NO:40 is extended on the 5’ end (i.e., upstream) by at least 1 nucleotide. In another preferred embodiment, the UTR as shown in SEQ ID NO:1 or SEQ ID NO:40, respectively is extended on the 5’ end (i.e., upstream) by 1 to 20 nucleotides. Hence, in a preferred embodiment, the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 20 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40). In other preferred embodiments, the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 18, 15, 13, 10, 7 or 5 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40). In other preferred embodiments, the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 4, 5 or 2 nucleotides on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40). In other preferred embodiment, the sequence of SEQ ID NO:1 or SEQ ID NO:40 extends by 1 nucleotide on the 5’ end (i.e., upstream) as shown in the nucleotide sequence of SEQ ID NO:31 vis-à-vis SEQ ID NO:1 (or SEQ ID NO:40). These UTR sequences which are extended on the 5’ end (i.e., upstream) may also be modified as defined herein above, e.g. for SEQ ID NO:1 or SEQ ID NO:40. Accordingly, the same applies, mutatis mutandis, to the UTRs which are extended on the 5’ end as defined above as has been set forth above in the context of the UTR of SEQ ID NO:1 or SEQ ID NO:40. Moreover, as mentioned, the 5’-UTRs as used in the present invention are also not particularly limited to the above specific sequence of SEQ ID NO:3 (or SEQ ID NO:28) but may also be a “variant”/“derivative” 5’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:3. Alternatively, the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 5, 1 to 4 or 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:3. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g. in comparison to SEQ ID NO:3. Most preferably, the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:3. In principle, the lower numbers of substitutions are preferred. Preferably, the position of the above nucleotide substitution is performed at position 32 and between positions 31 and 32, respectively, in the sequence of SEQ ID NO:28. Preferably, a nucleotide “G” at position 32 and between positions 31 and 32, respectively, is inserted into SEQ ID NO:28. This insertion is preferred since it brings the Kozak element of hAG which is (partially) present in SEQ ID NO:28 (CCCACC) closer to the Kozak consensus sequence of vertebrates. It results in the Kozak sequence GCCACC (SEQ ID NO:23). The Kozak consensus sequence of vertebrates has the sequence of GCCRCCAUG (SEQ ID NO:24; the start codon is underlined while “R” indicates any purine) while the Kozak element of hAG has the sequence CCCACCAUG, comprising the motive CCCACC. An example of the resulting “variant”/“derivative” 5’-UTR sequence is depicted in SEQ ID NOs: 3 & 32. The UTR sequence(s) which have one or more of the above substitutions (in comparison to SEQ ID NO:3 (or SEQ ID NO:28)) may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g. SEQ ID NO:3), preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:3). The property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples. What has been said herein above with respect to the “translation efficiency” also applies here, mutatis mutandis. The UTR(s) comprising the sequence as shown in SEQ ID NO:3 (or SEQ ID NO:28) or a sequence which shows 1 to 6 substitutions, e.g. in comparison to SEQ ID NO:3, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an unmodified UTR (e.g. SEQ ID NO:28 (or SEQ ID NO:3)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s) in comparison to SEQ ID NO:3. The addition of (a) nucleotide(s) can be flanking. Thus, the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention. The additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides. In light of the rationale that the addition of nucleotides is likely not to change the above functional properties of the UTR(s) of the invention the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:3, preferably higher translation efficiency as SEQ ID NO:3 as defined above. Alternatively, or in addition to these flanking additions of (a) nucleotide(s) the addition of (a) nucleotide(s) can be interspersed. Thus, the additional nucleotide(s) may be added/inserted within the nucleotide sequence of the UTR(s) of the present invention. These nucleotide(s) insertions comprise 1, 2, or 3 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:3, preferably higher translation efficiency as SEQ ID NO:3 as defined above. The UTRs as used in the present invention are not particularly limited to the above specific sequence of SEQ ID NO: 3 ((or SEQ ID NO:28)) and modifications thereof. Rather, in one embodiment, the UTR as shown in SEQ ID NO:3 is extended at the 5’ end (i.e., upstream) by at least 1 nucleotide. In another embodiment, the UTR as shown in SEQ ID NO:3 is extended on the 5’ end (i.e., upstream) by 1 to 20 nucleotides. In other embodiments, the sequence of SEQ ID NO:3 extends by 18, 15, 13, 10, 7 or 5 nucleotides on the 5’ end (i.e., upstream). In other embodiments, the sequence of SEQ ID NO:3 extends by 4, 5 or 2 nucleotides on the 5’ end (i.e., upstream). In another embodiment, the sequence of SEQ ID NO:3 extends by 1 nucleotide on the 5’ end (i.e., upstream). These UTR sequences which are extended on the 5’ end (i.e., upstream) may also be modified as defined herein above, e.g. for SEQ ID NO:3. Accordingly, the same applies, mutatis mutandis, to the UTRs which are extended on the 5’ end as defined above as has been set forth above in the context of the UTR of SEQ ID NO:3. Moreover, as mentioned, also the 3’-UTRs as used in the present invention are also not particularly limited to the above specific sequence of SEQ ID NO:2 (or SEQ ID NO:26) but may also be a “variant”/“derivative” 3’-UTR sequence, like the UTR sequence which comprises a sequence which shows 1 to 7 substitutions, e.g. in comparison to SEQ ID NO:2. Alternatively, the UTR sequence may also be a sequence which comprises a sequence which shows 1 to 6 substitutions, e.g. in comparison to SEQ ID NO:2. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 5 substitutions, e.g. in comparison to SEQ ID NO:2. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 4 substitutions, e.g. in comparison to SEQ ID NO:2. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:2. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 2 substitutions, e.g. in comparison to SEQ ID NO:2. The UTR sequence may also be a sequence which comprises a sequence which shows 1 to 3 substitutions, e.g. in comparison to SEQ ID NO:2. Most preferably, the UTR sequence may also be a sequence which comprises a sequence which shows 1 substitution, e.g. in comparison to SEQ ID NO:2. In principle, the lower numbers of substitutions are preferred. The UTR sequence(s) which have one or more of the above substitutions (in comparison to SEQ ID NO:2 (or SEQ ID NO:26)) may result in an RNA molecule with the same or similar capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising an UTR comprising the unmodified UTR sequence (e.g. SEQ ID NO:2), preferably a higher capability in terms of the translation efficiency (and/or expression) as an RNA molecule comprising the unmodified UTR sequence (e.g. SEQ ID NO:2). The property/capability of a given modified UTR sequence in terms of the translation efficiency (and/or expression) in comparison to the unmodified UTR sequence can be determined by the skilled person by methods known in the art and as outlined in the appended examples. What has been said herein above with respect to the “translation efficiency” also applies here, mutatis mutandis. The UTR(s) comprising the sequence as shown in SEQ ID NO:2 (or SEQ ID NO:26) or a sequence which shows 1 to 7 substitutions, e.g. in comparison to SEQ ID NO:2, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an unmodified UTR (e.g. SEQ ID NO:26 (or SEQ ID NO:2)) as used in the present invention is/are not particularly limited to the above specific sequences and the above described substitutions but may also relate to (an) UTR sequence(s) which comprise(s) a sequence which shows (a) nucleotide(s) addition(s) in comparison to SEQ ID NO:2. The addition of nucleotide(s) can be flanking or interspersed. Thus, the additional nucleotide(s) may be added at the 3’-end or 5’-end of the UTR(s) of the present invention. Alternatively, or in addition to these flanking additional nucleotide(s), the additional nucleotide(s) may also be within the nucleotide sequence of the UTR(s) of the present invention. The additional nucleotide(s) comprise polynucleotide chains of up to 0 (no changes), 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, preferably of up to 20 nucleotides or even more preferably of up to 30 nucleotides. In light of the rationale that the addition of nucleotides is likely not to change the above functional properties of the UTR(s) of the invention the addition of the nucleotides may also have a length of up to 40, 50, 60, 70, 80, 90, or even 100 nucleotides or even more, up to 200, 300, 400 or 500 nucleotides as long as these sequences have a similar capability (in terms of the above-described translation efficiency) as SEQ ID NO:2, preferably higher translation efficiency as SEQ ID NO:2 as defined above. The UTR(s) of the present invention as well as RNA molecules containing such UTR(s) may be recombinantly (e.g., in an in vivo or an in vitro system) or synthetically generated/synthesized by methods known to the person skilled in the art. More specifically, the UTRs of the present invention and RNA molecules containing such UTR(s) may be produced either recombinantly in in vivo systems by methods known to the person skilled in the art. Alternatively, the UTRs of the present invention and RNA molecules containing such UTR(s) may be produced in an in vitro system using, for example, an in vitro transcription system. In vitro transcription systems are commonly known and usually require a purified linear DNA template containing a DNA sequence “encoding” module (b) and/or module (c) as outlined in detail further below wherein said DNA sequence is under the control of an appropriate promoter. Moreover, an in vitro transcription system also commonly requires ribonucleoside triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase which provides the enzymatic activity for the in vitro transcription of the DNA sequence “encoding” the modules (b) and/or (c) into the UTR(s) of the present invention. Furthermore, the UTRs of the present invention and RNA molecules containing such UTR(s) may be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques or by chemical synthesis of the respective DNA-sequences and subsequent in vitro or in vivo transcription of the same. The RNA molecules/polyribonucleic acid molecules of the invention may be modified RNA molecules/polyribonucleic acid molecules. The terms RNA molecule, ribonucleic acid and polyribonucleotide are used interchangeably and, in certain embodiments, include any compound and/or substance that comprises a polymer of nucleotides wherein greater than 50% of the nucleotides are ribonucleotides. In certain embodiments, polyribonucleotides comprise a polymer of nucleotides wherein greater than 60%, 70%, 75%, 80%, 90%, greater than 95%, greater than 99% or 100% of the nucleotides are ribonucleotides. Polyribonucleotides wherein one or more nucleotides are modified nucleotides may be referred to as modified polyribonucleotides. However, the term polyribonucleotides may include modified polyribonucleotides. The sequence of the RNA molecules/polyribonucleotides can be derived from, for example, any suitable nucleic acid that comprises the genetic information of a gene of interest (i.e. of at least one gene of GLP-1 and GDF-15). Examples of nucleic acids include genomic DNA, RNA, or cDNA comprising the gene(s) of interest. The polynucleotides can be derived from nucleic acids carrying mutated genes and polymorphisms. An RNA molecule/polyribonucleotide of the present invention comprises a sequence which is not particularly limited but comprises, as module (a), any desired coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF- 15. In a preferred embodiment, said sequence may be a coding region coding for a desired GLP- 1 and/or GDF-15 polypeptide/protein as outlined herein elsewhere. Preferably, in line with the above, the RNA molecule/polyribonucleotide further comprises an untranslated sequence positioned upstream (5’) of the module (a)’s start codon or an untranslated sequence positioned downstream (3’) of module (a)’s stop codon, or both, an untranslated sequence positioned upstream (5’) of module (a)’s start codon and an untranslated sequence positioned downstream (3’) of module (a)’s stop codon. As mentioned, in one embodiment, an RNA molecule/polyribonucleotide of the present invention may be a modified RNA molecule/polyribonucleotide. In one aspect, it is envisaged that only module (a), the coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF-15, is modified. This aspect is also encompassed by the meaning of modified RNA molecule/polyribonucleotide in accordance with the invention. In addition to the four classical ribonucleotides, namely, adenosine, guanosine, cytidine and uridine, there exist numerous analogs of each of these nucleobases (modified nucleobases). Sometimes, throughout the present description and in the literature, these analogs, or RNA molecules/polyribonucleotides that include one or more of these analogs, are referred to as modified (e.g., modified nucleotides or modified ribonucleotides). Some analogs differ from the above canonical nucleobases, but yet can exist in nature. Other analogs are non-naturally occurring. Either type of analog is contemplated, in principle. In certain embodiments, RNA molecules/polyribonucleotides of the present invention comprise nucleotide analogs (e.g., the polyribonucleotide comprises a modified polyribonucleotide). Exemplary nucleotide analogs are provided below (e.g., analogs of U; analogs of C; analogs of A; analogs of G). In addition, in certain embodiments, an RNA molecule/polyribonucleotide or other nucleic acid of the disclosure may also comprise (in addition to or alternatively) modifications in the phosphodiester backbone or in the linkage between nucleobases. Exemplary nucleic acids that can form part or all of an RNA molecule/polyribonucleotide of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a beta-D-ribo configuration, alpha -LNA having an alpha -L-ribo configuration (a diastereomer of LNA), 2´-amino-LNA having a 2´-amino functionalization, and 2´-amino-alpha- LNA having a 2´-amino functionalization) or hybrids thereof. In certain embodiments, a modification may be on one or more nucleoside(s) or the backbone of the nucleic acid/polynucleotide molecule. In certain embodiments, a modification may be on both a nucleoside and a backbone linkage. In certain embodiments, a modification may be engineered into a polynucleotide in vitro. In certain embodiments, a modified ribonucleotide/nucleotide may also be synthesized post-transcriptionally by covalent modification of the classical/natural nucleotides/ribonucleotides. An RNA molecule/polyribonucleotide of the present invention can be a modified RNA molecule/polyribonucleotide and, in certain embodiments, can comprise analogs of purines and/or analogs of pyrimidines. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention comprises a pyrimidine analog, such as an analog of uridine and/or an analog of cytidine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention comprises an analog of uridine and an analog of cytidine. In certain embodiments, the modified RNA molecule/polyribonucleotide does not comprise analogs of adenosine and/or analogs of guanosine. In certain embodiments, the RNA molecule/polyribonucleotide comprises a single type of analog of uridine and a single type of analog of cytidine (e.g., one type of analog, not a single molecule of analog – the single analog may be present at any of several percentages described herein). In other embodiments, the RNA molecule/polyribonucleotide comprises more than one type of analog of uridine and/or cytidine and, optionally and if present, one or more analogs of adenosine and/or guanosine (or none of either or both). In some cases a modified uridine (e.g., analog of uridine) is selected from 2-thiouridine, 5´- methyluridine, pseudouridine, 5-iodouridine (I5U), 4-thiouridine (S4U), 5-bromouridine (Br5U), 2´- methyl-2´-deoxyuridine (U2´m), 2´-amino-2´-deoxyuridine (U2´NH2), 2´-azido-2´-deoxyuridine (U2´N3), and 2´-fluoro-2´-deoxyuridine (U2´F). In some cases, a modified cytidine (e.g., analog of cytidine) is selected from 5-methylcytidine, 3-methylcytidine, 2-thio-cytidine, 2´-methyl-2´- deoxycytidine (C2´m), 2´-amino-2´-deoxycytidine (C2´NH2), 2´-fluoro-2´-deoxycytidine (C2´F), 5- iodocytidine (I5C), 5-bromocytidine (Br5C) and 2´-azido-2´-deoxycytidine (C2´N3). Note that when referring to analogs, the foregoing also refers to analogs in their 5’ triphosphate form. In certain embodiments, the cytidine analog is 5-iodocytidine and the uridine analog is 5-iodouridine. In some cases, the modified RNA molecule/polyribonucleotide is at least 25% more stable as compared to a non-modified (or unmodified) RNA molecule/polyribonucleotide. In some cases, the modified RNA molecule/polyribonucleotide can be at least 30% more stable, at least 35% more stable, at least 40% more stable, at least 45% more stable, at least 50% more stable, at least 55% more stable, at least 60% more stable, at least 65% more stable, at least 70% more stable, at least 75% more stable, at least 80% more stable, at least 85% more stable, at least 90% more stable, or at least 95% more stable as compared to a non-modified RNA molecule/polyribonucleotide. In certain embodiments, stability is measured in vivo. In certain embodiments, stability is measured in vitro. In certain embodiments, stability is quantified by measuring the half-life of the polyribonucleotide. A RNA molecule/polyribonucleotide of the present invention can have nucleotides that have been modified in the same form or else a mixture of different modified nucleotides. The modified nucleotides can have modifications that are naturally or not naturally occurring in messenger RNA. A mixture of various modified nucleotides can be used. For example one or more modified nucleotides within an RNA molecule/polyribonucleotide can have natural modifications, while another part has modifications that are not naturally found in mRNA. Additionally, some modified nucleotides can have a base modification, while other modified nucleotides have a sugar modification. In the same way, it is possible that all modifications are base modifications or all modifications are sugar modifications or any suitable mixture thereof. In some cases, the stability of the modified RNA molecule/polyribonucleotide can be selectively optimized by changing the nature of modified bases within the modified polyribonucleotide. Table 6: Non-limiting examples of analogs of U
Figure imgf000042_0001
Table 7: Non-limiting examples of analogs of C
Figure imgf000042_0002
Table 8: Non-limiting examples of analogs of A
Figure imgf000043_0001
Table 9: Non-limiting examples of analogs of G
Figure imgf000043_0002
In certain embodiments, an analog (e.g., a modified nucleotide) can be selected from the group comprising pyridin-4-one ribonucleoside, 5-iodouridine, 5-iodocytidine, 5-aza-uridine, 2’-amino- 2’-deoxycytidine, 2’-fluor-2’-deoxycytidine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, N1-methyl-pseudouridine (m1ψ; sometimes also termed just “1-methyl-pseudouridine”), 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl- pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-1-methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, 5-methylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2- methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl- 8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl- 6-thio-guanosine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include pseudouridine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include 5-methyl cytidine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include 5-methyl uridine. In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention comprises analogs of U and analogs of C, wherein such analogs of U may all be the same analog or may be different analogs (e.g., more than one type of analog), and wherein such analogs of C may all be the same analog or may be different analogs (e.g., more than one type of analog). In certain embodiments, a modified RNA molecule/polyribonucleotide of the present invention does not include analogs of adenosine and analogs of guanosine. As described in detail herein, when an RNA molecule/polyribonucleotide comprises a modified polyribonucleotide, analogs may be present as a certain proportion of the nucleotides in the compound (e.g., a given percentage of a given nucleobase may be analog, as described herein). An RNA molecule/polyribonucleotide that comprises at least one modified nucleotide is a modified RNA molecule/polyribonucleotide. In certain embodiments, at least about 5% of the modified RNA molecule/polyribonucleotide includes modified or non-naturally occurring (e.g., analogs of or modified) adenosine, cytidine, guanosine, or uridine, such as the analog nucleotides described herein. In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% of the modified RNA molecule/polyribonucleotide includes modified or non- naturally occurring (e.g., analogs of or modified) adenosine, cytidine, guanosine, or uridine. In some cases, at most about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, of the modified RNA molecule/polyribonucleotide includes modified or non-naturally occurring adenosine, cytidine, guanosine, or uridine. In a preferred embodiment the RNA molecule of the present invention contains a combination of modified and unmodified nucleotides. Preferably, the RNA molecule of the present invention contains a combination of modified and unmodified nucleotides as described in WO 2011/012316 or WO 2018/127382. Such modified RNA molecules are also known and commercialized as “SNIM®RNA”. The RNA molecule described in, for example, WO 2011/012316 is reported to show an increased stability and diminished immunogenicity. In a preferred embodiment, in a modified RNA molecule of the invention, 5 to 50% of the cytidine nucleotides and 5 to 50% of the uridine nucleotides are modified. The adenosine- and guanosine- containing nucleotides can be unmodified. The adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form. Preferably 10 to 35% of the cytidine and uridine nucleotides are modified and particularly preferably the content of the modified cytidine nucleotides lies in a range from 7.5 to 25% and the content of the modified uridine nucleotides lies in a range from 7.5 to 25%. It has been found that in fact a relatively low content, e.g. only 10% each, of modified cytidine and uridine nucleotides can achieve the desired properties. It is particularly preferred that the modified cytidine nucleotides are methylcytidine residues (in particular 5-methylcytidine residues) and that the modified uridine nucleotides are thiouridine residues (in particular 2-thiouridine residues). Most preferably, the content of modified cytidine nucleotides (e.g. (5-)methylcytidines) and the content of the modified uridine nucleotides (e.g. (2-)thiouridines) is (about) 25% (e.g. ± 2, 3, 4 or 5 %), respectively. The modified RNA molecule of the invention may be prepared by using a particular combination of unmodified nucleotides, adenosine-triphosphate (ATP), guanosine-triphosphate (GTP), uridine- triphosphate (UTP) and cytosine-triphosphate (CTP), as well as chemically modified nucleotides like, for example, (5-)methyl-CTP and (2-)thio-UTP. In this context, a certain combination/ratio of unmodified and modified nucleotides may be used (e.g. in an in vitro transcription (IVT), and in an in vitro transcription mix (IVT mix), respectively). For example, the unmodified and modified nucleotides may be present (for an IVT, and in an IVT mix, respectively) at a concentration ratio of ATP : CTP : UTP : (5-)methyl-CTP : (2-)thio-UTP : GTP of 7.57mM : 5.68mM : 5.68mM : 1.89mM : 1.89mM : 1.21mM, respectively, or at a concentration ratio of ATP : GTP : UTP : CTP: (5-)methyl-CTP : (2-)thio-UTP of 7.13mM : 1.14mM : 5.36mM : 5.36mM :0.536mM : 0.536mM, respectively. In certain other embodiments, in such a modified RNA molecule/polyribonucleotide molecule, 5 to 50% of the cytidines are analogs of C and 5 to 50% of the uridines are analogs of U. In certain embodiments, in such a modified polyribonucleotide molecule 5 to 40% of the cytidines are analogs of C and 5 to 40% of the uridines are analogs of U. In certain embodiments, in such a modified RNA molecule/polyribonucleotide molecule 5 to 30% of the cytidines are analogs of C and 5 to 30% of the uridines are analogs of U. In certain embodiments, in such a modified RNA molecule/polyribonucleotide molecule 10 to 30% of the cytidines are analogs of C and 10 to 30% of the uridines are analogs of U. In certain embodiments, in such a modified polyribonucleotide molecule 5 to 20% of the cytidines are analogs of C and 5 to 20% of the uridines are analogs of U. In certain embodiments, in such a modified RNA molecule/polyribonucleotide molecule 5 to 10% of the cytidine nucleotides and 5 to 10% of the uridine nucleotides are modified. In certain embodiments, in such a modified RNA molecule/polyribonucleotide molecule 25% of the cytidine nucleotides and 25% of the uridine nucleotides are modified. In certain embodiments, the adenosine- and guanosine-containing nucleotides can be unmodified. In certain embodiments, the adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form. As noted above, in certain embodiments, analogs of U refers to a single type of analog of U. In certain embodiments, analogs of U refers to two or more types of analogs of U. In certain embodiments, analogs of C refers to a single type of analog of C. In certain embodiments, analogs of C refers to two or more types of analogs of C. In certain embodiments, the percentage of cytidines in an RNA molecule/polyribonucleotide that are analogs of cytidine is not the same as the percentage of uridines in the RNA molecule/polyribonucleotide that are analogs of uridine. In certain embodiments, the percentage of analogs of cytidine is lower than the percentage of analogs of uridine. As noted above, this may be in the presence or the absence of analogs of adenosine and guanosine but, in certain embodiments, is in the absence of analogs of adenosine and analogs of guanosine. In certain embodiments, polyribonucleotides of the disclosure comprises less than 15%, less than 10%, less than 5% or less than 2% analogs of adenosine, analogs of guanosine or both. In certain embodiments, an RNA molecule/polyribonucleotide of the present inention comprises analogs of cytidine and analogs of uridine, and 5 to 20% of the cytidines are analogs of cytidine and 25 to 45% of the uridines are analogs of uridine. In other words, the RNA molecule/polyribonucleotide comprises modified and unmodified cytidines and modified and unmodified uridines, and 5 to 20% of the cytidines comprise analogs of cytidine while 25 to 45% of the uridines comprise analogs of uridine. In other embodiments, the RNA molecule/polyribonucleotide comprises 5 to 10% analogs of cytidine and 30 to 40% analogs of uridine, such as 7-9% analogs of cytidine, such as about 7, 7.5 or 8% and, such as 32-38% analogs of uridine, such as about 33, 34, 35, 36%. In certain embodiments, any of the analogs of uridine and analogs of cytidine described herein may be used, optionally excluding pseudouridine. In certain embodiments, the analog of cytidine comprises or consists of (e.g., in the case of consists of, it is the single analog type used) 5- iodocytidine and the analog of uridine comprises or consists of (e.g., in the case of consists of, it is the single analog type used) 5-iodouridine. In certain embodiments of any of the foregoing, the percentage of analogs of a given nucleotide refers to input percentage (e.g., the percentage of analogs in a starting reaction, such as a starting in vitro transcription reaction). In certain embodiments of any of the foregoing, the percentage of analogs of a given nucleotide refers to output (e.g., the percentage in a synthesized or transcribed compound). The RNA molecules/polyribonucleotide molecules of the present invention may be produced recombinantly in in vivo systems by methods known to a person skilled in the art which are described in more detail furher below. Alternatively, the modified polyribonucleotide molecules of the present invention may be produced in an in vitro system using, for example, an in vitro transcription system which is described in more detail further below. An in vitro transcription system capable of producing RNA molecules/polyribonucleotides requires an input mixture of modified and unmodified nucleoside triphosphates to produce modified RNA molecules/polyribonucleotides with the desired properties of the present invention. In certain embodiments, 5 to 50% of the cytidines are analogs of cytidine in such an input mixture and 5 to 50% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 5 to 40% of the cytidines are analogs of cytidine in such an input mixture and 5 to 40% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 5 to 30% of the cytidines are analogs of cytidine in such a mixture and 5 to 30% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 5 to 30% of the cytidines are analogs of cytidine in such mixture and 10 to 30% of the uridines are analogs of uridine in such mixture. In certain embodiments, 5 to 20% of the cytidines are analogs of cytidine in such an input mixture and 5 to 20% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 5 to 10% of the cytidines are analogs of cytidine in such an input mixture and 5 to 10% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, 25% of the cytidines are analogs of cytidine in such an input mixture and 25% of the uridines are analogs of uridine in such an input mixture. In certain embodiments, the input mixture does not comprise analogs of adenosine and/or guanosine. In other embodiments, optionally, the input mixture comprises one or more analogs of adenosine and/or guanosine (or none of either or both). In certain embodiments, the percentage of cytidines in an input mixture that are analogs of cytidine is not the same as the percentage of uridines in an input mixture that are analogs of uridine. In certain embodiments, the percentage of analogs of cytidine in an input mixture is lower than the percentage of analogs of uridine in an input mixture. As noted above, this may be in the presence or the absence of analogs of adenosine and guanosine in the input mixture but, in certain embodiments, is in the absence of analogs of adenosine and analogs of guanosine in the input mixture. In certain embodiments, an input mixture of nucleotides for an in vitro transcription system that produces a RNA molecule/polyribonucleotide of the present invention comprises analogs of cytidine and analogs of uridine, and 5 to 20% of the cytidines of the input mixture are analogs of cytidine and and 25 to 45% of the uridines of the input mixture are analogs of uridine. In other words, the input mixture comprises modified and unmodified cytidines and modified and unmodified uridines, and 5 to 20% of the cytidines of the input mixture comprise analogs of cytidine while 25 to 45% of the uridines of the input mixture comprise analogs of uridine. In other embodiments, the input mixture comprises 5 to 10% analogs of cytidine and 30 to 40% analogs of uridine, such as 7-9% analogs of cytidine, such as 7, 7.5 or 8% and, such as 32-38% analogs of uridine, such as 33, 34, 35, 36%. In certain embodiments, any of the analogs of uridine and analogs of cytidine described herein may be used, optionally excluding pseudouridine. In certain embodiments, the analog of cytidine comprises or consists of (e.g., it is the single C analog type used) 5-iodocytidine and the analog of uridine comprises or consists of (e.g., it is the single U analog type used) 5-iodouridine. As mentioned, the RNA molecule of the invention (in particular the RNA molecule of/deriving from the coding region (module (a)) may comprise one or more modified nucleosides. In certain embodiments of this aspect, the or each modified nucleoside may be selected from the following group of modified nucleosides: Pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5- iodo-uridine, 2′-O-methyl uridine, 5-methylcytidine, 5-iodo-cytidine, N1-methyladenosine, N6- methyladenosine. In particular embodiments of this aspect, the one or more modified nucleosides may comprise at least one N1-methylpseudouridine (m1ψ) modification (preferred modification). In particular embodiments of this aspect, at least 50% of the uridines have been modified, in particular in the RNA molecule of/deriving from the coding region (module (a). For example, at least 50% of the uridines have been modified to m1ψ, in particular in the RNA molecule of/deriving from the coding region (module (a)). In particular embodiments of this aspect, the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine. In particular embodiments of this aspect, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. In particular embodiments of this aspect, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine. Exemplary analogs are also described in the tables above. It should be understood that for modified polyribonucleotides encoding the desired polypeptide (module (a)), the analogs and level of modification is, unless indicated otherwise, considered across the entire polyribonucleotide encoding the desired polypeptide (module (a)), including 5’ and(/or) 3’ untranslated regions (e.g., the level of modification is based on input ratios of analogs in an in vitro transcription reaction such that analogs may be incorporated at positions that are transcribed). Furthermore, the modified RNA molecules/polyribonucleotide molecules may be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques or by chemical synthesis of the respective DNA sequences and subsequent in vitro or in vivo transcription of the same. In molecular biology and genetics, upstream and downstream both refer to a relative position in an RNA molecule. In the context of the present invention, upstream is toward the 5' end of the RNA molecule and downstream is toward the 3' end of the molecule. Accordingly, in one embodiment, the UTR module (b) (e.g., the one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which has 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, as defined hereinabove) is located upstream of the coding region of module (a). Moreover, in one embodiment, the UTR module (c) (e.g., the one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 as defined hereinabove) is located downstream of the coding region of module (a). Yet, preferably, the coding region coding for a polypeptide (i.e., module (a)) is located between the UTR module (b) and the UTR module (c) and, accordingly, the RNA molecule preferably has the arrangement of 5’-(b)-(a)-(c)-3’. In case the RNA molecule only harbors one UTR module (i.e., either module (b) (e.g., the one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, as defined hereinabove) or module (c) (e.g., the one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2 as defined hereinabove)) the RNA molecule preferably has the arrangement of 5’-(b)-(a)-3’ or 5’-(a)-(c)-3’. The RNA molecule may be present in the form of fused RNA sequences of modules (a), (b) and/or (c), i.e., a (fusion) RNA molecule which is formed by the expression of a hybrid gene made by combining at least two nucleotide sequences encoding said modules. Typically, as will be explained in more detail further below, this can be accomplished by cloning a cDNA into an expression vector which allows for the translation of the RNA molecule. Accordingly, the DNA molecule encoding the RNA molecule of the present invention may be a fused DNA sequence, i.e., a chimeric molecule which is formed by joining two or more polynucleotides via the phosphate group from one nucleotide bound to the 3' carbon on another nucleotide, forming a phosphodiester bond between the respective ends of one module and the end of another molecule. In this way, the above DNA molecules encoding said at least two modules, preferably all three modules are joined together in the form of a DNA molecule in terms of the present invention. Once cloned in frame, such a recombinant DNA molecule is then transcribed into its corresponding RNA nucleic acid sequence encoding said Protein, polypeptide or enzyme molecule. Alternatively, the at least two modules, preferably all three modules may also be covalently coupled by a chemical conjugate. Thus, as will be outlined in more detail further below, the modules of the RNA molecule may be chemically synthesized individually and subsequently coupled in a covalent linkage by a phosphodiester bond as outlined above. In the following, preferred arrangements of the UTR modules (b) and/or (c) of the present invention in relation to the coding region (a) are described wherein the UTR module (b) (corresponding to the above-defined 5’-UTR fragment of the CYBA or hAg mRNA) is located upstream of the coding region (i.e., at the 5’ end of the coding region) and/or the UTR module (c) (corresponding to the above-defined 3’ UTR of the CYBA or hAg mRNA) is located downstream of the coding region (i.e., at the 3’ end of the coding region). Thus, in a preferred embodiment, and in accordance with the foregoing, the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively, wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said UTR(s) as defined in (b) is/are located at the 5’ end of the coding region as defined in (a). In a preferred embodiment, and in accordance with the foregoing, the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (c) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is not a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said UTR(s) as defined in (c) is/are located at the 3’ end of the coding region as defined in (a). In a preferred embodiment, and in accordance with the foregoing, the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively; and (c) one (or more) UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said UTR(s) as defined in (b) is/are located at the 5’ end of the coding region as defined in (a) and wherein said UTR(s) as defined in (c) is/are located at the 3’ end of the coding region as defined in (a). In one embodiment, and in accordance with the foregoing, the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) one UTR comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively; and (c) two UTRs comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2; wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said RNA molecule comprises said one UTR as defined in (b) at the 5’ end of the coding region as defined in (a) and which comprises said two UTRs as defined in (c) at the 3’ end of the coding region as defined in (a). In one embodiment, and in accordance with the foregoing, the present invention relates to an RNA molecule comprising (a) a coding region coding for a polypeptide; and (c) two UTRs comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2, wherein said coding region coding for a polypeptide in (a) is a coding region coding for at least one of the regulators of energy homeostasis GLP-1 and GDF15 as defined herein elsewhere and wherein said RNA molecule comprises said two UTRs as defined in (c) at the 3’ end of the coding region as defined in (a). As indicated above, the herein employed 5’-UTR(s) may contain at its 5´-end sequences which correspond to (residual 3’) parts of a promoter and/or at its 3´-end a so-called Kozak sequence. A Kozak sequence may be required for ribosome recognition and translation of many genes. Kozak sequences may have a consensus comprising CCR(A/G)CC (see also above), where R is a purine (adenine or guanine) that is located three bases upstream of the start codon (AUG). Non limiting examples of a Kozak sequence to be employed in the context of the invention are GUGGCC, CCCACC, GCCGCC (preferred, especially for the 5´-CYBA-UTR), GCCACC (preferred, especially for the 5´-hAg-UTR). The RNA molecule of the present invention may also harbor a poly-A tail. As used herein, a poly- A tail relates to a sequence of adenine nucleotides located at the 3’ end of the RNA. A poly-A tail is commonly added to the 3' end of the RNA by a process called polyadenylation. Thus, the present invention relates to any of the above-described RNA, wherein the RNA molecule comprises a poly-A tail at the 3’ end. The length of the poly-A tail is not particularly limited. Yet, in preferred embodiments, the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly-A tail has a length of at least 50, 60, 70, 80, 90, 100 or 110 nucleotides. In a more preferred embodiment, the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly- A tail has a length of at least 120 nucleotides. In other preferred embodiments, the RNA molecule of the present invention comprises a poly-A tail at the 3’ end wherein the poly-A tail has a length of at least 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 nucleotides. The skilled person is readily in the position to add a suitable poly-A tail to the RNA molecule of the invention. In particular, a poly-A tail of an optimal length can be established and can be added RNA molecule. The poly-A tail can either be added directly from an encoding DNA template, by using poly(A) polymerase (see, e.g. Pardi, Nature Reviews Drug Discovery 17, 2018, 261–79) or ligation after in-vitro transcription. Particular examples of the poly-A tail to be added to the RNA molecule of the invention may have a length of 90 A nucleotides (A90) or more, 100 A nucleotides (A100) or more, 110 A nucleotides (A110) or more, 120 A nucleotides (A120) or more, 130 A nucleotides (A130) or more, 150 A nucleotides (A150) or more, 180 A nucleotides (A180) or more, 190 A nucleotides (A190) or more. An example of a particularly suitable length of poly-A tail is poly(~A120). The poly-A tail may be a segmented poly-A tail, e.g. as disclosed in WO 2020074642 A1 (herein incorporated by reference). Optionally the segmented poly-A tail may have the structure A55-65-S-A55-65 wherein S is a single nucleotide selected from C, G, T or U. Optionally the poly-A tail may have the structure: A55-65-N-S4-N-A55-65, wherein N is a nucleotide that is not adenine, and wherein S4 are four nucleotides selected from A, C, G, T or U..Particular, non-limiting examples of the (segmented) poly-A tail are poly-A tails consisting of or comprising of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51). In case the RNA molecule of the present invention is produced by an in vitro transcription method, e.g. as described herein further below, the poly-A tail is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct while the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail. The construct according to the present invention may not only comprise the above three main modules (a), (b) and/or (c). Rather, it may be desirable that between the individual modules (a) linker moiety/moieties and/or (a) multiple cloning site(s) is/are placed which may, e.g., facilitate the construction of the construct. Suitable linker moieties and multiple cloning sites are known to the skilled person. Preferably, the construct of the present invention harbors a multiple cloning site, which is, for example, derived from the plasmid pVAX1 (Invitrogen). All the constructs as outlined in the Example section originate from the construct pVAX A120 which has previously been described in WO2013/182683 A1. A UTR to be employed in accordance with the invention, in particular the 3’ UTR, may comprise a sequence for generation of a restriction site, e.g. when comprised in form of a nucleotide sequence in a vector. Suitable restriction sites are known in the art. One non-limiting example of a restriction site is GAAUU. The position of the UTR modules (b) and/or (c) within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is not particularly limited and, accordingly, between the individual modules of the RNA molecule of the present invention there may be a spacing or a gap filled with one or more nucleotides G, A, U and/or C which are not part of the main modules (a), (b) and/or (c). “One or more nucleotides G, A, U and/or C” in this context means that the spacing or gap between the individual modules of the RNA molecule of the present invention is/are filled with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides G, A, U and/or C. In other preferred embodiments, the spacing or gap between the individual modules of the RNA molecule of the present invention are filled with 20, 30, 40, 50, 60, 70, 80, 90, 100 or 110 or more nucleotides G, A, U and/or C. Yet, in a preferred embodiment, the UTR module (b) or (c), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the start codon of the coding region of module (a) without any spacing or gap in between, i.e., directly upstream of the start codon of the coding region of module (a). In another preferred embodiment, the UTR module (b) or (c), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the termination codon (i.e., the stop codon) of the coding region of module (a) without any spacing or gap in between, i.e., directly downstream of the termination codon/stop codon of the coding region of module (a). In a preferred embodiment, the UTR module (b), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the start codon of the coding region of module (a) without any spacing or gap in between, i.e., directly upstream of the start codon of the coding region of module (a) and the UTR module (c), within the RNA molecule of the present invention in relation to module (a) (i.e., the coding region), is directly placed adjacent to the termination codon (i.e., the stop codon) of the coding region of module (a) without any spacing or gap in between, i.e., directly downstream of the termination codon/stop codon of the coding region of module (a). The present invention further relates to a set of 2 (or more) RNA molecules of the invention and as defined herein elsewhere, respectively. It is particularly envisaged that the set comprises at least 2 different RNA molecules of the invention, in particular, 2 RNA molecules the “coding regions…” (module (a)) of which encode 2 different regulators of energy homeostasis, respectively. It is particularly envisaged that these 2 different regulators are GLP-1 and GDF15 (or “variants”, “derivatives” or “fragments” thereof), e.g. as defined herein, and that the 2 RNA molecules encode GLP-1 and GDF15 (or “variants”, “derivatives” or “fragments” thereof), e.g. as defined herein, respectively. A particular but non-limiting example of a set of 2 (or more) RNA molecules comprises one RNA molecule as defined in one of item (I) (or (III), (IV) or (V)), infra and herein elsewhere, and another RNA molecule as defined in one of item (II) (or (VI)), infra and herein elsewhere. (I) an RNA molecule comprising the UTR(s) as defined in any one of (b)(A) and (c), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (CYBA-GDF15 WT); (II) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GLP-1 as defined herein elsewhere (hAg-GLP-1); (III) an RNA molecule comprising the UTR(s) as defined in any one of (b)(B), supra, and a coding region encoding GDF15 WT as defined herein elsewhere (hAg-GDF15 WT); (IV) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(A) and (c), supra, and a coding region encoding GDF15 H6D as defined herein elsewhere (CYBA-GDF15 H6D); (V) an RNA molecule comprising the UTR(s) as defined in any one of 1(b)(B), supra, and a coding region encoding GDF15 H6D as defined herein elsewhere (hAg-GDF15 H6D); and As mentioned above, the RNA molecule may be present in the form of fused RNA sequences of modules (a), (b) and/or (c), i.e., a (fusion) RNA molecule which is formed by the transcription of a hybrid gene made by combining at least two nucleotide sequences encoding said modules. Typically, this is accomplished by cloning a cDNA into an expression vector which allows for the transcription of the entire RNA molecule. A variety of methods are known for making fusion constructs, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid molecule “encoding” the RNA molecule of the present invention. Such a double-stranded nucleic acid molecule (i.e., DNA molecule) harbors on one strand (i.e., on the coding strand) the DNA sequence corresponding to the RNA molecule of the present invention and, accordingly, “encodes” the RNA molecule of the present invention. In other words, such a double-stranded nucleic acid/DNA molecule comprises on a strand the genetic information, when transcribed, the RNA molecule of the present invention as defined herein above. The term “coding” or “encoding” in the context of the present invention is not only used in its conventional sense, i.e., to relate to a gene's DNA that codes for a protein (and, accordingly, the genetic information which may be translated into a polypeptide or a protein amino acid sequence). Rather, in terms of the present invention, in a construct wherein the individual DNA sequences encoding the modules (a), (b) and/or (c) are “fused” or linked into a single (chimeric) DNA molecule, the construct also comprises components (i.e., module (b) and/or module (c)) which are not translated into a protein. Nevertheless, the DNA sequence corresponding to module (b) and/or module (c) provide the information, i.e., the “code”, for the UTRs’ structure of the present invention and, accordingly, the term “encoding” in the present invention also relates to the genetic information for the UTRs which may be expressed, i.e., transcribed, if, e.g., present in a double-stranded nucleic acid molecule which harbors on one strand the RNA molecule of the present invention. Thus, the term “encoding” in the context of the present invention, although it is commonly only used to relate to the coding/expression of a protein, is to be understood in a way that the nucleic acid molecule can be transcribed into the RNA molecule of the present invention which harbours parts encoding a protein or a polypeptide (i.e., module (a)) and parts “encoding” the UTRs (i.e., modules (b) and/or (b)) wherein the latter represent the final product when expressed since UTRs are not translated into proteins or polypeptides. Such a double-stranded nucleic acid may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed, 1989). The term “vector” such as “expression vector” or “cloning vector” in the sense of the present invention is understood as a circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA and which is used as a vehicle to carry genetic material into a cell, where it can be replicated and/or expressed (i.e., transcribed into RNA and translated into a amino acid sequence). A vector containing foreign DNA is termed recombinant DNA. The vector itself is generally a DNA sequence that typically consists of an insert (i.e., module (b) and/or module (c) which are not translated into a protein and module (a) the coding region) and a larger sequence that serves as the "backbone" of the vector. Plasmids in the sense of the present invention are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells and are as such a subpopulation of “vectors” as used in the sense of the present invention. Particular, non-limiting, examples of the RNA molecule of the invention are depicted in SEQ ID NOs:18 and 19. The present invention also relates to a nucleic acid molecule encoding the RNA molecule of the present invention or the set of 2 (or more) RNA molecules of the invention. The present invention further relates to a set of 2 (or more) nucleic acid molecules, at least 2 nucleic acids of which encode 2 different RNA molecules of the invention, respectively. The nucleic acid is, for example a DNA, encoding two of the three main modules (i.e., module (a) and module (b) or module (c)) of the RNA molecule of the present invention. Alternatively, the nucleic acid, preferably a DNA, encodes all three main modules (i.e., module (a) and module (b) and module (c)). The above nucleic acid molecule of the present invention preferably is a recombinant nucleic acid molecule but may also comprise naturally occurring nucleic acid molecules. The nucleic acid molecule of the invention may, therefore, be of natural origin, synthetic or semi-synthetic. It may comprise DNA, RNA, locked nucleic acid as well as PNA and it may be a hybrid thereof. It is evident to the person skilled in the art that (a)regulatory sequence(s) may be added to the nucleic acid molecule of the invention encoding the RNA molecule. For example, promoters, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, enhancer or activator sequences, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide, i.e., the RNA molecule, of the invention may be employed. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen, Trends Biotech. 12 (1994), 58-62, or a dexamethasone-inducible gene expression system as described, e.g. by Crook, EMBO J.8 (1989), 513-519. In one aspect, the nucleic acid molecule encoding the RNA molecule of the invention may comprise one or more promoter sequence(s), and, optionally, any associated regulatory sequences. It may comprise either a whole promoter, and, optionally, associated regulatory sequences, or a fragment thereof. Suitable promoters may be constitutive or inducible promoters as known in the art; these are also contemplated by the disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. Preferred but non-limiting examples of promoters to be employed in the context of the invention are disclosed herein elsewhere, e.g in Table 10. MRNA is transcribed from a gene by a DNA-dependent RNA polymerase, which begins transcribing at the transcription start site (TSS). The position of the TSS is determined by the specific promoter sequence (and any other regulatory sequences) upstream of the start codon of the gene. The TSS may be within the promoter sequence. Thus the 5’ UTR to be employed in accordance with the invention may comprise a portion of a promoter sequence. The promoter sequence (and any associated regulatory sequence) or portion thereof can be positioned at the 5’-end of the 5’-UTR. A promoter sequence and/or an associated regulatory sequence can comprise any number of modified or unmodified nucleotides. Promoter sequences and/or any associated regulatory sequences can comprise, for example, at least 150 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, or at least 10000 bases or base pairs. A promoter sequence and/or an associated regulatory sequence can comprise any number of modified or unmodified nucleotides, for example, at most 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or base pairs, 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, or 100 bases or base pairs. DNA sequences of promoters of the disclosure include, but are not limited to, DNA sequences corresponding to the RNA sequences listed in Table 10. As the present disclosure also concerns modified polyribonucleotides, modified RNA sequences versions of the promoters listed in Table 10 may be employed.
Figure imgf000058_0001
Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed from an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. In the context of the present invention said nucleic acid molecules may also be labeled. Methods for the detection of nucleic acids are well known in the art, e.g., Southern and Northern blotting, PCR or primer extension. The nucleic acid molecule(s) of the invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. Preferably, the nucleic acid molecule of the invention is part of a vector. An exemplary, however non-limiting, nucleic acid molecule which encodes the RNA molecule of the invention comprises the following (1) to (8), most preferably in the depicted order (5’ to 3’): (1) a 5’-cloning site (optional); (2) a promoter (optional but preferred), for example a T7 promoter; (3) a 5’-UTR encoding site as defined herein elsewhere; (4) a (partial) Kozak element (optional but preferred), for example as defined herein elsewhere; (5) a coding region as defined herein elsewhere; (6) a 3’-UTR encoding site as defined herein elsewhere; (7) a poly-A addition site (optional but preferred; e.g. G/AATAn (n may be 3)), for example as defined herein elsewhere; and (8) a 3’-cloning site (optional; e.g. including or consisting of a BamHI restriction site (see SEQIDNO:33); may also be item (7)). The resulting RNA molecule may comprise the following (3) to (7), most preferably in the depicted order (5’ to 3’): (3) a 5’-UTR as defined herein elsewhere; (4) a Kozak element (optional but preferred), for example as defined herein elsewhere; (5) a coding region as defined herein elsewhere; (6) a 3’-UTR as defined herein elsewhere; and (7) a poly-A tail (optional), for example as defined herein elsewhere. A preferred promoter to be employed in the context of the invention may be a T7 promoter, like the T7 promoter as comprised or depicted in any of SEQ ID NOs:18, 19, 20, 21 and 22. The present invention also relates to a vector comprising the nucleic acid molecule of the present invention. Preferably, the vector is an expression vector. The present invention further relates to a set of 2 (or more) vectors, at least 2 vectors of which comprise 2 different nucleic acid molecules of the invention, respectively. The vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Furthermore, the vector of the present invention may, in addition to the sequences of the nucleic acid molecule encoding the RNA molecule of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the skilled person and may include a promoter, a splice cassette, translation start codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Accordingly, the present invention relates to a vector comprising the nucleic acid molecule of the present invention, wherein the nucleic acid molecule is operably linked to control sequences that are recognized by a host cell when the eukaryotic and/or prokaryotic (host) cell is transfected with the vector. Control elements ensuring expression in eukaryotic and prokaryotic (host) cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Yet, in accordance of the present invention, it is not crucial that the vector itself harbors a sequence for a poly-A tail. As mentioned above, in case the RNA molecule of the present invention is produced by an in vitro transcription method as described herein further below the above poly-A tail is part of the construct of the present invention (and not necessarily originally located on the cloning vector) and is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct. In case the RNA molecule of the present invention is produced by an in vitro transcription method the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous Sarcoma Virus), human elongation factor 1 ^- promoter, the glucocorticoid-inducible MMTV-promoter Mouse Mammary Tumor Virus), metallothionein- or tetracyclin-inducible promoters, or enhancers, like CMV enhancer or SV40- enhancer. For expression in neural cells, it is envisaged that neurofilament-, PGDF-, NSE-, PrP- , or thy-1-promoters can be employed. Said promoters are known in the art and, inter alia, described in Charron, J. Biol. Chem.270 (1995), 25739-25745. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Besides elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), pX (Pagano, Science 255 (1992), 1144-1147), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris, Cell 75 (1995), 791-803), or prokaryotic expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). Furthermore, the vector of the present invention may also be an expression vector. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. The present invention also relates to a host cell comprising the vector of the set of vectors of the present invention. The present invention further relates to a set of 2 (or more) host cells, at least 2 host cells of which comprise 2 different nucleic acid molecules of the invention, respectively, or 2 different vectors of the invention, respectively. Thus, the present invention relates to a host (or set of host cells) transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i.e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule. The term "genetically modified" means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents. The nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host. The transformation of the host cell with a vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. The host cell of the present invention may be any prokaryotic or eukaryotic cell. Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise, for example, fungal or animal cells. Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae. Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO,COS-7, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A. Further suitable cell lines known in the art are obtainable from cell line depositories, like, e.g., the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) or the American Type Culture Collection (ATCC). In accordance with the present invention, it is furthermore envisaged that primary cells/cell cultures may function as host cells. Said cells are in particular derived from insects (like insects of the species Drosophila or Blatta) or mammals (like human, swine, mouse or rat). Said host cells may also comprise cells from and/or derived from cell lines like neuroblastoma cell lines. The above mentioned primary cells are well known in the art and comprise, inter alia, primary astrocytes, (mixed) spinal cultures or hippocampal cultures. In principle, and if not explicitly stated otherwise, what is said herein elsewhere with respect of the RNA molecule, nucleic acid, vector or host cell also applies to the respective sets thereof, mutatis mutandis. The present invention also relates to methods of producing the RNA molecule (or set of RNA molecule) of the present invention by culturing a host cell (or set of host cells) harbouring an expression vector (or set of expression vector(s) encoding the individual modules of the present invention or the entire RNA molecule (or set thereof) of the invention in culture medium, and recovering the RNA molecule (or set thereof) from the host cell (or set thereof) or culture medium. The present invention may also relate to a method for producing an RNA molecule of the present invention comprising the cultivation of the host cell of the present invention and optionally recovering the RNA molecule from the culture. Methods of recovering and/or subsequently purifying the RNA molecule of the present invention are known to the person skilled in the art. The present invention also relates to methods of producing in an in vitro reaction the RNA molecule of the present invention by methods known to the person skilled in the art. More specifically, the RNA molecule of the present invention may be produced in vitro using an in vitro transcription system. In vitro transcription systems are commonly known and usually require a purified linear DNA template containing a DNA sequence “encoding” module (b) and/or module (c) as outlined above wherein said DNA sequence is under the control of an appropriate promoter. Moreover, an in vitro transcription system also commonly requires ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate RNA polymerase which provides the enzymatic activity for the in vitro transcription of the DNA sequence into the RNA molecule of the present invention. Methods which are commonly used to produce RNA molecules using in vitro transcription are well-known to the person skilled in the art and are, e.g., described in Methods Mol. Biol. 703 (2011):29-41. As mentioned above, in case the RNA molecule of the present invention is produced by an in vitro transcription method as described herein further below the above poly-A tail may be part of the construct of the present invention (and not necessarily originally located on the cloning vector) and is located at the 3’ end of the RNA adjacent to the UTR at the 3’ end of the RNA construct. In case the RNA molecule of the present invention is produced by an in vitro transcription method the plasmid harboring the RNA molecule of the present invention is linearized prior to the in vitro transcription downstream of the poly-A tail in order to assure that the in vitro transcribed RNA molecule contains said poly-A tail. Alternatively, the RNA molecule of the present invention may also be chemically synthesized, e.g., by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid-phase support and standard techniques. The present invention also relates to methods of producing in an in vitro reaction the RNA molecule of the present invention by methods known to the person skilled in the art and as outlined above and recovering the RNA molecule from the reaction. Methods of recovering and/or subsequently purifying the RNA molecule of the present invention are known to the person skilled in the art. The RNA molecules as defined above are particularly useful in medical settings and in the treatment or prevention of a certain disease (e.g. as defined herein elsewhere) and, in particular, in RNA-based therapies. Thus, the present invention also relates to a pharmaceutical composition comprising the RNA molecule (or set of RNA molecules) of the present invention, the nucleic acid molecule (or set of nucleic acid molecules) of the present invention, the vector (or the set of vectors) of the present invention or the host cell (or the set of host cells) of the present invention and optionally a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition of the invention is for use in an RNA-based therapy. In one embodiment, the pharmaceutical composition according to the invention is for use (i) as an anorectic; (ii) in body weight control, in particular in decreasing (aberrant) body weight; and/or (iii) in the treatment or prevention of a metabolic disorder. The present invention further relates to a method for (i) decreasing food intake; (ii) restraining appetite; (iii) controlling body weight, in particular decreasing (aberrant) body weight; and/or (iv) treating or preventing a metabolic disorder. In particular, said method is envisaged to comprise the step of administering a pharmaceutically active amount of the pharmaceutical composition of the invention to a patient in need thereof. The metabolic disorder to be medically addressed in accordance with the invention (treated or prevented) may be selected from the group consisting of (i) obesity, in particular abdominal obesity; (ii) diabetes mellitus, in particular type II diabetes mellitus; (iii) insulin resistance; and/or (iv) metabolic syndrome. The metabolic syndrome may (be a set of symptoms which) include(s) obesity (in particular abdominal obesity), hypertension, cardiovascular disease, elevated fasting plasma glucose, dyslipidemia, and/or an enhanced inflammatory state. In principle, the term “treatment” (or “prevention”) and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. Accordingly, the treatment of the present invention may relate to the treatment of (acute) states of a certain disease but may also relate to the prophylactic treatment in terms of completely or partially preventing a disease or symptom thereof. Preferably, the term “treatment” is to be understood as being therapeutic in terms of partially or completely curing a disease and/or adverse effect and/or symptoms attributed to the disease. “Acute” in this respect means that the subject shows symptoms of the disease. In other words, the subject to be treated is in actual need of a treatment and the term “acute treatment” in the context of the present invention relates to the measures taken to actually treat the disease after the onset of the disease or the breakout of the disease. The treatment may also be prophylactic or preventive treatment, i.e., measures taken for disease prevention, e.g., in order to prevent the infection and/or the onset of the disease. The pharmaceutical composition of the present invention may be administered via a large range of classes of forms of administration known to the skilled person. Administration may be systemically, locally, orally, through aerosols, including but not limited to tablets, needle injection, the use of inhalators, creams, foams, gels, lotions and ointments. As mentioned, the present invention relates to a pharmaceutical composition, comprising an effective amount of the RNA molecule (or the nucleic acid molecule, the vector or the host cell, or the respective sets) of the present invention in accordance with the above and at least one pharmaceutically acceptable excipient or carrier. An excipient or carrier is an inactive substance formulated alongside the active ingredient, i.e., construct of the present invention in accordance with the above, for the purpose of bulking-up formulations that contain potent active ingredients. Excipients are often referred to as "bulking agents," "fillers," or "diluents". Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. They also can serve various therapeutic-enhancing purposes, such as facilitating drug absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients also depends upon the route of administration and the dosage form, as well as the active ingredient and other factors. Thus, in line with the above, the pharmaceutical composition comprising an effective amount of the RNA molecule (or the nucleic acid, vector or host cell, or the respective set) of the present invention may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). It is preferred that said pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose, i.e., in “an effective amount” which can easily be determined by the skilled person by methods known in the art. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's or subject’s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Thus, preferably, the construct of the present invention is included in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered. In accordance with the above, the content of the construct of the present invention in the pharmaceutical composition is not limited as far as it is useful for treatment as described above, but preferably contains 0.0000001-10% by weight per total composition. Further, the construct described herein is preferably employed in a carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter-ions, e.g. sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g. serum albumin, or gelatine; hydrophilic polymers, e.g. polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g. sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g. EDTA; non-ionic surfactants, e.g. Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and/or preservatives, e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g. methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. Therapeutic progress can be monitored by periodic assessment. The RNA molecule of the present invention or the pharmaceutical composition of the invention may be in sterile aqueous or non-aqueous solutions, suspensions, and emulsions as well as creams and suppositories. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition. Said agents may be, e.g., polyoxyethylene sorbitan monolaurate, available on the market with the commercial name Tween, propylene glycol, EDTA, Citrate, Sucrose as well as other agents being suitable for the intended use of the pharmaceutical composition that are well-known to the person skilled in the art. In accordance with this invention, the term “pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention may be for use in RNA-based therapies. As mentioned above, the RNA molecule of the present invention comprising a “coding region…” can be used in RNA-based therapies wherein the “coding region…” encodes a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect. Thus, in preferred embodiments, the pharmaceutical composition of the present invention may be for use in RNA-based therapies in the treatment or prevention of a condition, disorder or disease as recited in the above Table 3, or herein elsewhere. Accordingly, RNA-based therapies in accordance with the present invention may be for use in the treatment or prevention of a condition, disorder or disease as recited in the above Table 3, or herein elsewhere. Thus, the pharmaceutical composition of the present invention may be for use in RNA-based therapies in cases where gene defects lead to a disease which can then be treated or prevented by a transcript replacement therapy/enzyme replacement therapy with the RNA molecule of the present invention, wherein the RNA molecule comprises a “coding region…” which encodes an intact version of the protein or a functional variant or functional fragment thereof compensating the defective gene. In particularly preferred embodiments, the pharmaceutical composition of the present invention may be for use in RNA-based therapies in the treatment or prevention of a metabolic disorder (e.g. as defined herein elsewhere), in particular, a metabolic disorder related to GLP-1 and/or GDF15. “Related” in this context particularly means that the respective metabolic disorder is, partially or totally, due to a situation, where functional GLP-1 and/or GDF15 is not present (in the body of a subject) or present in deficient form or in too small quantity (for example because of gene defects or diseases); and/or that the respective metabolic disorder is treatable (or preventable) by enhancing GLP-1 and/or GDF15 function. Transcript replacement therapies/enzyme replacement therapies beneficially do not affect the underlying genetic defect, but increase the concentration of the enzyme in which the patient is deficient. As an example, in metabolic disorders, the transcript replacement therapy/enzyme replacement therapy may replace the deficient GLP-1 and/or GDF15. In other preferred embodiments, the pharmaceutical composition of the present invention may be for use in RNA-based therapies in accordance with the present invention wherein the “coding region…” encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or preventive effect, wherein said polypeptide, protein or peptide is GLP-1 and/or GDF15, or an analogue thereof, respectively. RNA-based therapies in accordance with the present invention may be for use in treating a disease or disorder that is, partially or totally, due to a situation, where functional GLP-1 and/or GDF15 is not present (in the body of a subject) or present in deficient form or in too small quantity (for example because of gene defects or diseases); or any disease or disorder where GLP-1 and/or GDF15 produced in a cell may have a beneficial effect for the patient. Examples of cardiovascular diseases include atherosclerosis, coronary heart disease, pulmonary heart disease and cardiomyopathy. Examples of (inherited) metabolic disorders include, but are not limited to, Gaucher’s disease and Phenylketonuria. The invention also relates to a method of an RNA-based therapy. Thus, the present invention relates to a method for the treatment of a disease, such a cardiovascular disease, a (inherited) metabolic disorders or a genetic disorder by an RNA-based therapy. As regards the preferred embodiments of the method for treatment the same applies, mutatis mutandis, as has been set forth above in the context of the RNA molecule or the pharmaceutical composition for use in RNA- based therapy as defined above. In the present invention, the subject is, in a preferred embodiment, a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee, and human. In a most preferable embodiment, the subject is a human. The present invention also relates to a kit comprising the RNA molecule of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention or the host cell of the present invention (or the respective sets of the invention). As regards the preferred embodiments, the same applies, mutatis mutandis, as has been set forth above in the context of the RNA molecule, nucleic acid molecule, vector or the host cell according to the present invention (or the respective sets). Advantageously, the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of the above and below uses and methods. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. The kit of the present invention may be advantageously used, inter alia, for carrying out the methods of the invention, the preparation of the RNA molecule of the invention and could be employed in a variety of applications referred herein, e.g., in the uses as outlined above and below. Another component that can be included in the kit is instructions to a person using a kit for its use. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art. Finally, the present invention also relates to the use of one (or more) 5’-UTR(s) as defined herein elsewhere (e.g. comprising the sequence as shown in SEQ ID NO:1, 40 or 3 or a sequence which shows 1 to 4 or 1 to 6 substitutions in comparison to SEQ ID NO:1, 40 or 3, respectively, and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1, 40 or 3, respectively); and/or of one (or more) 3’-UTR(s) as defined herein elsewhere (e.g. comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2) for increasing the efficiency of translating a coding region of an RNA molecule into a polypeptide or a protein encoded by said coding region as being defined above. In one aspect, the present invention relates to the use of one or more UTR(s) as defined herein elsewhere, in particular as defined in any of (b), more particular as defined in any of 1(b)(A), and/or one or more UTR(s) as defined in any of (c), for increasing the efficiency of translating a coding region of an RNA molecule into at least one of the regulators of energy homeostasis GLP- 1 and GDF15 as defined herein elsewhere encoded by said coding region. The RNA molecule, the set of RNA molecules, the nucleic acid molecule, the vector, the pharmaceutical composition or the kit of the present invention may be combined with one or more oligomer(s), polymer(s) or lipidoid(s) (for example one or more oligomer(s), polymer(s) or lipidoid(s) as disclosed in WO 2014/207231 and WO 2015/128030) and/or with one or more liposomal transfection reagent(s) (for example one or more liposomal transfection reagent(s) as disclosed in WO 2016/075154). The RNA molecule, the set of RNA molecules, the nucleic acid molecule or the vector may be administered together with one or more oligomer(s), polymer(s) or lipidoid(s) and/or one or more liposomal transfection reagent(s). The RNA molecule, the set of RNA molecules, the nucleic acid molecule or the vector may be complexed with one or more oligomer(s), polymers or lipidoid(s) and/or one or more liposomal transfection reagent(s). In this context, the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector), may be formulated with liposomes, to generate lipoplexes, or with subsequent generations of lipid nanocarriers, such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LiNPs), nanostructured lipid carriers, and/or cationic lipid-nucleic acid complexes. The LNPs or LiNPs may be comprised in the pharmaceutical composition or the kit of the invention. The pharmaceutical composition or the kit (and LNPs or LiNPs) may comprise one or more oligomer(s), polymer(s) or lipidoid(s) and/or one or more liposomal transfection reagent(s). Oligomers, polymers or lipidoids and liposomal transfection reagents are known in the art and are, for example, distributed by OzBiosciences, Marseille, France and Invitrogene, CA, USA. Oligomers, polymers or lipidoids and/or liposomal transfection reagents to be employed according to the invention may be oligomers, polymers or lipidoids as disclosed in WO 2014/207231, WO 2015/128030, WO 2016/075154, Zhang (loc. cit.) and/or Jarzebinska (loc. cit.) (e.g. cationic lipidoids termed “C12-(2-3-2)”, lipids as, e.g., disclosed in EP2285772 (e.g. DOGTOR) and lipopolyamines as, e.g., disclosed in EP1003711 (e. g. DreamFectTM and DreamFect GoldTM). Oligomers, polymers, lipidoids and (liposomal) transfection reagents to be employed in accordance with the invention may also comprise one or more (e.g. two, three or four) (further) lipid(s) (e.g. “helper lipids”), like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g- PEG2k-lipids) (e.g. DMPE-PEG (e.g. DMPE-PEG2k), DMG-PEG (e.g. DMG-PEG2k)). Such lipids and their use in lipofection/transfection are, for example, described in WO 2016/075154 and Zhang (loc. cit.). Non-limiting examples of (an) oligomer(s), polymer(s) or lipidoid(s) and/or (a) liposomal transfection reagent(s) to be employed in the context of the invention are DOGTOR (preferred) and LipofectamineTM2000 (more preferred). Non-limiting examples of (an) oligomer(s), polymer(s) or lipidoid(s) to be employed in the context of the invention are also oligomers, polymers or lipidoids which contain, as one main characteristic, the following common structural entity of formula (I):
Figure imgf000070_0001
Such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from: a) an oligomer or polymer comprising a plurality of groups of formula (II) as a side chain and/or as a terminal group:
Figure imgf000070_0002
wherein the variables a, b, p, m, n and R2 to R6 are defined as follows, independently for each group of formula (II) in a plurality of such groups: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and R2 to R5 are, independently of each other, selected from hydrogen; a group -CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O-R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; and a poly(ethylene glycol) chain; R6 is selected from hydrogen; a group -CH2-CH(OH)-R7, -CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O- R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; –C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand, and wherein one or more of the nitrogen atoms indicated in formula (II) may be protonated to provide a cationic group of formula (II); b) an oligomer or polymer comprising a plurality of groups of formula (III) as repeating units:
Figure imgf000071_0001
wherein the variables a, b, p, m, n and R2 to R5 are defined as follows, independently for each group of formula (III) in a plurality of such groups: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and R2 to R5 are, independently of each other, selected from hydrogen; a group –CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O-R7 or -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; –C(NH)-NH2; and a poly(ethylene glycol) chain; and wherein one or more of the nitrogen atoms indicated in formula (III) may be protonated to provide a cationic group of formula (III); and c) a lipidoid having the structure of formula (IV): R2 R4 R1 N {CH2 (CH2)a N [CH2 (CH2)b N]p}m [CH2 (CH2)a N]n R6 R3 R5 (IV) wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and R1 to R6 are independently of each other selected from hydrogen; a group -CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O-R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; –C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand; provided that at least two residues among R1 to R6 are a group -CH2-CH(OH)-R7, -CH(R7)-CH2-OH, -CH2- CH2-(C=O)-O-R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV). In a more specific aspect, such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from a) and b), wherein a) is an oligomer or polymer comprising a plurality of groups of formula (IIa) as a side chain and/or as a terminal group: -NR2{CH2-(CH2)a-NR3-CH2-(CH2)b-NR4}m-[CH2-(CH2)a-NR5]n-R6 (IIa), wherein a, b, m, n, and R2 to R6 are defined as described above, and wherein one or more of the nitrogen atoms indicated in formula (IIa) may be protonated to provide a cationic oligomer or polymer structure; and b) is an oligomer or polymer comprising a plurality of groups of formula (IIIa) as repeating units: -NR2{CH2-(CH2)a-NR3-CH2-(CH2)b-NR4}m-[CH2-(CH2)a-NR5]n- (IIIa), wherein a, b, m, n, and R2 to R5 are defined as described above, and wherein one or more of the nitrogen atoms indicated in formula (IIIa) may be protonated to provide a cationic oligomer or polymer structure. In a another more specific aspect, such oligomers, polymers or lipidoids may be (a component comprising) an oligo(alkylene amine) selected from a lipidoid having the structure of formula (IVa): R1-NR2{CH2-(CH2)a-NR3-CH2-(CH2)b-NR4}m-[CH2-(CH2)a-NR5]n-R6 (IVa), wherein a, b, m, n, and R1 to R6 are defined as described above, and wherein one or more of the nitrogen atoms indicated in formula (IVa) may be protonated to provide a cationic lipidoid. As to such oligomers, polymers or lipidoids, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa) n may be 1; or m may be 1 and n may be 1. Further, as to such oligomers, polymers or lipidoids, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa) a may be 1 and b may be 2; or a may be 2 and b may be 1. In one particular aspect, the oligomer, polymer or lipidoid may be a cationic (e.g. protonated) oligomer, polymer or lipidoid. One non-limiting example of such an oligomer, polymer or lipidoid that may be employed in the context of the invention is a cationic lipid which, for example, was prepared by mixing (e.g.100mg) N,N′-Bis(2-aminoethyl)-1,3-propanediamine (e.g. 0.623mmol) with (e.g. 575.07mg) 1,2- Epoxydodecane (e.g.3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)); mixed, for example, for 96h at, for example, 80°C, preferably under constant shaking. Such an oligomer, polymer or lipidoid is also referred to as lipidoid “C12-(2-3-2)”. Other non-limiting examples of oligomers, polymers or lipidoids that may be employed in the context of the invention is a (cationic) oligomer, polymer or lipidoid as disclosed in, for example, Zhang (Tissue Eng Part A 25(1-2), 2019, 131-144; doi: 10.1089/ten.TEA.2018.0112) and/or Jarzebinska (Angew Chem Int Ed Engl 8;55(33), 2016, 9591-5; doi: 10.1002/anie.201603648; and in the supporting information). Such an oligomer, polymer or lipidoid may also be referred to as lipidoid “C12-(2-3-2)”. The above oligomer, polymer or lipidoid (or any other (of the herein described) oligomer, polymer or lipidoid) may be produced as described in Zhang (loc. cit.) and/or Jarzebinska (loc. cit.) An oligomer, polymer or lipidoid, in particular a polymer, to be employed in accordance with the invention may be a copolymer, in particular a statistical copolymer. Such a copolymer may be a copolymer which contains a statistical/random arrangement of alkylene amine repeating units of alternating length (e.g. in contrast to a less preferred polymer which contains analogous arrangements of alkylene amine repeating units of non-alternating length). The copolymer may be a cationic (e.g. protonated) copolymer. Copolymers to be employed in accordance with the invention are known in the art and are, for example, described in EP 14199439.2, WO 01/00708, EP-A11198489 and CA-A12,377,207. In particular, the copolymer may be a statistical copolymer comprising a plurality of repeating units (a) independently selected from repeating units of the following formulae (a1) and (a2): CH2 CH2 NH (a1)
Figure imgf000073_0001
a plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) to (b4):
Figure imgf000073_0002
wherein the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, and wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer. The copolymer may be a statistical copolymer, wherein any repeating units (a) and any repeating units (b) are statistically distributed in the copolymer macromolecule. It is typically obtained from the copolymerization of a mixture of monomers yielding, during the polymerization reaction, the repeating units (a) with monomers yielding, during the polymerization reaction, the repeating units (b). Preferably, the copolymer is a random copolymer wherein any repeating units (a) and any repeating units (b) are randomly distributed in the polymer macromolecule. The copolymer in accordance with the invention can be a linear, branched or dendritic copolymer. As will be understood by the skilled reader, a repeating unit of the formula (a1), (b1) or (b3) with two valencies (i.e. open bonds to neighboring units) leads to a propagation of the copolymer structure in a linear manner. Thus, a linear copolymer of the invention comprises repeating units of formula (a1) and one or more types of the repeating units of formulae (b1) and (b3), but no repeating units of formula (a2), (b2) or (b4). As will be further understood, the presence of a repeating unit of formula (a2), (b2) or (b4) with three valencies provides a branching point in the copolymer structure. Thus, a branched copolymer comprises one or more types of the repeating units of formulae (a2), (b2) and (b4), and may further comprise one or more types of the repeating units of formulae (a1), (b1) and (b3). The copolymer in accordance with the invention comprises a plurality of repeating units (a) independently selected from repeating units of formulae (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulae (b1) to (b4) defined above. Preferred are copolymers comprising a plurality of repeating units (a) independently selected from repeating units of formulae (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from repeating units of formulae (b1) and (b2) defined above. It is also preferred that the copolymer in accordance with the invention is a branched copolymer comprising one or more types of repeating units selected from repeating units (a2), (b2) and (b4), and which optionally further comprises one or more types of the repeating units of formulae (a1), (b1) and (b3), and in particular a copolymer which comprises repeating units of the formula (a2) and one or more type of the repeating units of formulae (b2) and (b4), and which optionally further comprises one or more types of the repeating units of formulae (a1), (b1) and (b3). In line with the above, a more preferred copolymer is thus a branched copolymer which comprises repeating units of the formula (a2) and repeating units of formula (b2), and which optionally further comprises one or more types of the repeating units of formulae (a1) and (b1). In the copolymers in accordance with the invention, the total number of the repeating units (a) and repeating units (b) is typically 20 or more, preferably 50 or more and more preferably 100 or more. Typically, the total number of the repeating units (a) and repeating units (b) is 10,000 or less, preferably 5,000 or less, more preferably 1,000 or less. Furthermore, it is preferred for the copolymers in accordance with the invention that the repeating units (a) and (b) account for 80 mol% or more, more preferably 90 mol% or more of all repeating units in the copolymer. Further preferred are copolymers wherein repeating units (a) selected from (a1) and (a2) and repeating units (b) selected from (b1) and (b2) account for 80 mol% or more, more preferably 90 mol% or more of all repeating units in the copolymer. It is most preferred that all of the repeating units in the copolymer are repeating units (a) or (b), in particular that all of the repeating units in the copolymer are repeating units (a) selected from (a1) and (a2) or repeating units (b) selected from (b1) and (b2). The weight average molecular weight of the copolymer in accordance with the present invention, as measured e.g. via size exclusion chromatography relative to linear poly(ethylene oxide) standards, generally ranges from 1,000 to 500,000 Da, preferably from 2,500 to 250,000 Da and more preferably 5,000-50,000 less. The terminal groups of the copolymer in accordance with the invention typically comprise one or more types of groups (c) independently selected from groups of the formulae (c1) to (c3) below, preferably from groups of the formulae (c1) and (c2) below: CH2 CH2 NH2 (c1) CH2 CH2 CH2 NH2 (c2) CH2 CH2 CH2 CH2 NH2
Figure imgf000076_0001
Preferably, the terminal groups in the copolymer consist of one or more types of groups (c) independently selected from groups of the formulae (c1) to (c3) below, preferably from groups of the formulae (c1) and (c2). As will be understood by the skilled person, the number of terminal groups depends on the structure of the copolymer in accordance with the invention. While a linear copolymer has only two terminals, larger numbers of terminal groups are contained in a branched, in particular in a dendritic copolymer. As will be further understood, also one or more of the nitrogen atoms of the terminal groups (c) contained in the copolymer may be protonated to provide a cationic copolymer. In the copolymer in accordance with the invention, the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, and preferably within the range of 0.8/1.0 to 1.0/0.8. This molar ratio can be determined, e.g., via NMR. It will thus be understood that the ratio is usually determined for a plurality of macromolecules of the copolymer in accordance with the invention, and typically indicates the overall ratio of the sum of repeating units (a) to the sum of repeating units (b) in the plurality of macromolecules. As indicated above, one or more of the nitrogen atoms of the copolymer in accordance with the invention may be protonated to result in a copolymer in a cationic form, typically an oligocationic or polycationic form. It will be understood that the primary, secondary, or tertiary amino groups in the repeating units (a) or (b) or in the terminal groups (c) can act as proton acceptors, especially in water and aqueous solutions, including physiological fluids. Thus, the copolymers of the present invention typically have an overall positive charge in an aqueous solution at a pH of below 7.5. An aqueous solution, as referred to herein, is a solution wherein the solvent comprises 50 % (vol./vol.) or more, preferably 80 or 90 % or more, and most preferably 100 % of water. Also, if the compositions in accordance with the invention are in contact with a physiological fluid having a pH of below 7.5, including e.g. blood and lung fluid, they typically contain repeating units (a) and (b) wherein the nitrogen atoms are protonated. The pKa values of the copolymers used in the compositions in accordance with the invention can be determined by acid-base titration using an automated pKa titrator. The net charge at a given pH value can then be calculated e.g. from the Henderson–Hasselbach equation. Any charge may be shared across several of the basic centres and cannot necessarily be attributed to a single point. Typically, in solutions at physiological pH, the copolymers used in the compositions in accordance with the invention comprise repeating units with amino groups in protonated state and repeating units with amino groups in unprotonated state. However, as will be understood by the skilled reader, the copolymers in accordance with the invention as well as the compositions in accordance with the invention may also be provided as a dry salt form which contains the copolymer in a cationic form. As will be further understood, counterions (anions) for the positive charges of protonated amino groups in compositions according to the invention comprising the copolymer and nucleic acid, in particular RNA, preferably single-stranded RNA such as mRNA, are typically provided by anionic moieties contained in the nucleic acid. If the positively charged groups are present in excess compared to the anionic moieties in the nucleic acid, positive charges may be balanced by other anions, in particular those typically encountered in physiological fluids, such as Cl- or HCO3-. In line with the above, a preferred copolymer in accordance with the invention is a random copolymer, wherein 80 mol% or more of all repeating units, more preferably all repeating units, are formed by a plurality of repeating units (a) independently selected from repeating units of the following formulae (a1) and (a2): CH2 CH2 NH (a1)
Figure imgf000077_0001
a plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) and (b2):
Figure imgf000077_0002
wherein the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) lies within the range of 0.7/1.0 to 1.0/0.7, more preferably within the range of 0.8/1.0 to 1.0/0.8; wherein the terminal groups of the copolymer are formed by groups (c) independently selected from groups of the formulae (c1) and (c2): CH2 CH2 NH2 (c1) CH2 CH2 CH2 NH2 (c2); and wherein one or more of the nitrogen atoms of the repeating units (a) and/or (b) and/or of the terminal groups (c) contained in the copolymer may be protonated to provide a cationic copolymer. It is further preferred that the copolymer is a branched copolymer, comprising units (a2) and (b2), optionally together with units (a1) and/or (b1). The copolymers in accordance with the invention can be conveniently prepared with procedures analogous to those known for the preparation of polyalkyleneimines, such as branched or linear polyethyleneimine (PEI). It will be understood that the monomers used for the production of the copolymers will have to be adjusted accordingly. In the context of the present invention, it has been found that the monomers can be conveniently reacted in a quantitative manner, such that the ratio of the units (a) and (b) in the copolymer can be adjusted by adjusting the monomer ratio accordingly in the monomer mixture subjected to polymerization. While polyethyleneimine can be prepared e.g. via ring-opening polymerization of aziridine, the copolymers in accordance with the invention can be prepared via ring opening polymerization of a monomer mixture comprising or consisting of aziridine, azetidine and, where applicable pyrrolidine, or, in preferred embodiments, of aziridine and azetidine. It will be understood that the expression “where applicable” refers to the presence or absence of repeating units (b3) and (b4) or terminal groups (c3) which would be formed by the pyrrolidine. The ring opening polymerization of the non-substituted cyclic amines usually leads to branched copolymers. Linear copolymers in accordance with the invention can be prepared, e.g., via polymerization of suitable N-substituted aziridines, N-substituted azetidines and N-substituted pyrrolidines, or N-substituted aziridines and N-substituted azetidines, which may be followed e.g. by a hydrolytic cleavage of N-substituents attached to the resulting polyalkyleneimine chain, e.g. in analogy to the procedure published in Katrien F. Weyts, Eric J. Goethals, New synthesis of linear polyethyleneimine, Polymer Bulletin, January 1988, Volume 19, Issue 1, pp 13-19. For the preparation of a dendrimer (or dendritic copolymer), synthetic strategies can be analogously applied which are known for the production of polyethyleneimine or polypropyleneamine dendrimers. Polypropylenimine dendrimers can be synthesized from acrylonitrile building blocks using a repetitive sequence of a Michael addition to a primary amine, followed by a heterogeneously catalyzed hydrogenation (Newkome and Shreiner Poly(amidoamine), polypropylenimine, and related dendrimers and dendrons possessing different 1→2 branching motifs: An overview of the divergent procedures. Polymer 49 (2008) 1- 173; De Brabander-Van Den Berg et al. Large-scale production of polypropylenimine dendrimers, Macromolecular Symposia (1994) 77 (1) 51–62). Polyethylenimine dendrimers can be produced using a repetitive sequence of a Michael addition of a vinyl bromide building block to a primary amine followed by a conversion of alkylbromide to amine using a Gabriel amine synthesis method (Yemul & Imae, Synthesis and characterization of poly(ethyleneimine) dendrimers, Colloid Polym Sci (2008) 286:747–752). Hence the person skilled in the art will be able to produce not only dendrimers with strictly alternating layers of e.g. propylenimine and ethylenimine can be produced. Similarly dendrimer generations with layers comprising or consisting of random compositions of repeating units of formula (a2), (b2) and (b4) and preferably repeating units (a2) and (b2) can be generated. The ring opening polymerization of aziridine and azetidine, or of aziridine, azetidine and pyrrolidine, can be carried out in solution, e.g. in water. The total monomer concentration is not particularly limited, typical concentrations range from 10% wt/wt to 80% wt/wt, preferably 30% wt/wt to 60% wt/wt. Typically, the polymerization is initiated by protons, such that it is preferred to add a Brønsted acid, in particular a mineral acid such as sulphuric acid to the reaction system. Small amounts of acid are generally sufficient, such as 0.001 to 0.01 equivalents, based on the total concentration of monomers. The reaction proceeds at convenient rates e.g. in the temperature range of 50 to 150°C, in particular 90 to 140°C. In these ranges, higher molecular weight copolymers are usually at higher temperatures, and lower molecular weight copolymers at lower temperatures. A lipidoid is preferred among the oligomers, polymers or lipidoids to be employed in accordance with the invention, in particular as compared to an oligomer and, more particular particular, to a polymer One particular example of a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is 2; p is 1, m is 1; and n is 1; and R1 to R6 are independently of each other selected from hydrogen; a group -CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O-R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is C3- C18 alkyl; provided that at least two residues among R1 to R6 are a group -CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-O-R7, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; wherein it may be provided that at least one of R1, R2, R5 and(/or) and R6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV), wherein said lipidoid is a cationic lipid which is prepared by mixing N,N′-Bis(2-aminoethyl)-1,3- propanediamine with 1,2-Epoxydodecane. Said cationic lipid may be prepared by mixing, e.g., 100mg N,N′-Bis(2-aminoethyl)-1,3-propanediamine (0.623mmol) with, e.g., 575.07mg 1,2- Epoxydodecane (3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)). Said mixing may be, for example, for 96h at, for example, 80°C, preferably under constant shaking. One particular example of a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is 2; p is 1, m is 1 and n is 1; and R1 to R6 are independently of each other selected from hydrogen; a group -CH2-CH(OH)-R7, - CH(R7)-CH2-OH, -CH2-CH2-(C=O)-NH-R7 or -CH2-R7 wherein R7 is C3-C18 alkyl; provided that at least two residues among R1 to R6 are a group -CH2-CH(OH)-R7, -CH(R7)-CH2-OH, -CH2-CH2- (C=O)-NH-R7 or -CH2-R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; wherein it may be provided that at least one of R1, R2, R5 and(/or) and R6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV). One particular example of a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is 2, p is 1, m is 1, n is 1; and R1 to R6 are independently of each other selected from hydrogen, a group -CH2-CH(OH)-R7, a group -CH(R7)-CH2-OH, wherein R7 is C10 alkyl; provided that at least two residues among R1 to R6 are selected from a group -CH2-CH(OH)-R7, a group -CH(R7)-CH2-OH, wherein R7 is C10 alkyl; wherein it may be provided that at least one of R1, R2, R5 and(/or) and R6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV). One particular example of a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is 2; p is 1; m is 1; and n is 1; and R1 to R6 are independently of each other selected from hydrogen; a group -CH2-CH(OH)-R7; a group -CH(R7)-CH2-OH, wherein R7 is C10 alkyl; wherein it may be provided that at least one of R1, R2, R5 and(/or) and R6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) may be protonated to provide a cationic lipidoid of formula (IV) One particular example of a cationic lipidoid that may be employed in the context of the invention is a cationic lipidoid having the structure of formula (IV), wherein the variables a, b, p, m, n and R1 to R6 are defined as follows: a is 1 and b is 2; p is 1; m is 1; n is 1; and R1 to R6 are independently of each other hydrogen, a group -CH2-CH(OH)-R7, or a group -CH(R7)- CH2-OH, wherein R7 is C10 alkyl; provided that at least two residues among R1 to R6 are a group -CH2-CH(OH)-R7, or a group -CH(R7)-CH2-OH, wherein R7 is C10 alkyl; wherein it may be provided that at least one of R1, R2, R5 and(/or) and R6 is hydrogen; and wherein one or more of the nitrogen atoms indicated in formula (IV) is optionally protonated to provide a cationic lipidoid of formula (IV). Said cationic lipid may be prepared by mixing N,N′- Bis(2-aminoethyl)-1,3-propanediamine with 1,2-Epoxydodecane. More specifically, said cationic lipid may be prepared by mixing, e.g., 100mg N,N′-Bis(2-aminoethyl)-1,3-propanediamine (0.623mmol) with, e.g., 575.07mg 1,2-Epoxydodecane (3.12mmol; (N-1) eq. where N is 2x amount of primary amine plus 1x amount secondary amine per oligo(alkylene amine)). Said mixing may be, for example, for 96h at, for example, 80°C, preferably under constant shaking. Non-limiting examples of (lipofection) complexes or combinations to be employed in accordance with the invention are LipofectamineTM2000/RNA lipoplexes, DOGTOR/RNA lipoplexes, C12-(2- 3-2)/RNA lipoplexes and “helper lipid(s)”/RNA lipoplexes. The RNA (or (lipofection) complexes or combinations) to be employed in accordance with the invention may also be prepared for magnetofection, prepared to be transfected by magnetofection, delivered/introduced via magnetofection and/or administered via magnetofection. The principles of magnetofection are known in the art and are, for example, described in WO 02/00870. In certain embodiments, a cationic lipidoid of the following formula (V) and/or formula (VI) may be employed according to the invention (e.g. comprised in the pharmaceutical composition, the kit, or LiNPs): and/or
Figure imgf000082_0001
In some embodiments, the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector) of the invention can be delivered to target cells and/or target tissues in vivo, ex-vivo and/or in vitro using LNPs or LiNPs. LNPs and LiNPs can be distinguished from other carriers due to their small size, their homogenous size distribution and their structure; and are especially suited for administration of a subject. The skilled person knows method for the production of LNPs and LiNPs. The production of LNPs or LiNPs, for example, involves a combination of lipids and/or lipidoids, such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol. This mixture is then subjected to a process called nanoprecipitation, which involves rapidly mixing the lipid solution with a non-solvent, such as a nucleic acid dissolved in water, under controlled conditions of temperature, pressure, and stirring rate. During this process, the lipids self-assemble into complex nanoscale structures, which trap and protect the therapeutic nucleic acids of the invention inside. The nanoparticles may also be further modified with various surface coatings, such as polyethylene glycol (PEG), to improve their stability and reduce their tendency to be cleared by the immune system. The LiNPs may comprise as component (a) the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector) of the invention, as component (b) a (ionizable) lipid or an (ionizable) lipidoid, and optionally as component (c) helper lipids, e.h. as defined herein elewhere. The pharmaceutical composition, kit or LiNP (as component (c)) may (further) comprise helper lipids as described in the following or herein elsewhere. In particular, the herein described agents and reagents for administering, delivering and/or introducing the RNA molecule, or set of RNA molecules (or the nucleic acid molecule or the vector), e.g. into a target cell or a target tissue, and the herein described lipids and lipidoids may be combined with one or more (e.g., two, three or four) further lipid(s) (like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g. DMPE- PEG, DMG-PEG2000)). These further lipids may support the desired function of the therapeutic agents and the lipidoids (support and/or increase the delivery and/or introduction of RNA into the cell or tissue and improve transfection efficiency, respectively); and function as respective “helper lipids”. Particular examples of such “helper lipids” are cholesterol, DPPC, DOPE and/or PEG- lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG-PEG2000). The further lipids (e.g., “helper lipids”) may also be part(s) of the herein disclosed complexes/particles. The skilled person is readily in the position to prepare complexes/particles in accordance with the invention. Examples of further lipids (e.g., “helper lipids”) are also known in the art. The skilled person is readily in the position to choose suitable further lipids (e.g., “helper lipids”) and ratios of the cationic lipidoid(s) and the further lipids (e.g. “helper lipids”). Such ratios may be molar ratios of [1-4 : 1-5], [3-4 : 4-6], [about 4 : about 5], [about 4 : about 5.3] of cationic lipidoid(s) : further lipid(s), (the more narrow ranges are preferred). For example, the cationic lipidoid may be combined with three further lipids, like DPPC, cholesterol, and DMG-PEG2000, preferably at a molar ratio of ~8.0 : ~5.3 : ~4.4 : ~0.9, respectively, or, more particularly, 8.00 : 5.29 : 4.41 : 0.88, respectively. Preferably, the lipidoids according to formula (b-1), (b-1b), (b-V), (b-VI) and (b-VII) are as described above and used with helper lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00:5.29:4.41:0.88 for formulating lipidoid nanoparticles. In some embodiments, the pharmaceutical composition, kit or LiNPs may comprises a LiNP comprising the following components: a) an RNA molecule, or set of RNA molecules (or a nucleic acid molecule or a vector) according to the invention, b) a cationic lipidoid as defined herein elsewhere, and c) optionally one or more helper lipid(s), e.g. as defined herein elsewhere, optionally selected from: c1) DPPC, and/or c2) cholesterol, and/or c3) PEG-lipid, like DMG-PEG2000, optionally, components b), and c1-c3), are present, optionally component b) and c1)-c3) are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively. Optionally, the LiNP/NLP comprises a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks. For example, a composition in which the R-isomer of formula (V) may be formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000, e.g. at the molar ratios 8.00 : 5.29 : 4.41 :0.88. This is also referred herein as “Formulation I”. A composition in which the lipidoid of formula (VI) may be formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000, e.g. at the molar ratios 8.00 : 5.29 : 4.41 : 0.88; also referred herein as “Formulation II”. In some embodiments, the LiNPs may comprise Formulation I and/or Formulation II. The cationic lipidoid to mRNA ratios in the LiNP can readily be controlled in terms of the mole ratio of nitrogen atoms of the cationic lipidoid (N) to phosphate groups in the mRNA (P) (N/P ratio). The other lipid components can be calculated according the target molar lipid proportions relative to the cationic lipidoid as discussed above, and may be for example 8.00 : 5.29 : 4.41 : 0.88 for cationic lipidoid, DPPC, cholesterol and PEG-lipid DMG-PEG2000, respectively. In some embodiments, the final N/P ratio of a cationic lipidoid (e.g. as defined herein elewhere) to one phosphate group of mRNA molecule, is preferably 4 to 44, preferably 4 to 16, more preferably 8 nitrogen atoms of a cationic lipidoid (e.g. as defined herein elewhere), per one phosphate group of the mRNA molecule. The lipid or lipidoid nanoparticles, e.g. as contained in the (suspension or aerosol) formulation in accordance with the invention, preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm. The indicated particle diameter is the hydrodynamic diameter of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C. The polydispersity index of the nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C. As regards the (preferred) embodiments of the use of the present invention, the same applies, mutatis mutandis, as has been set forth above in the context of the RNA molecule of the present invention. Figure 1: Fluorescence microscopy and flow cytometry data of A549 cells. (A) Schematic illustration of therapeutic mRNA, consisting of a 5’ CAP, a 5’ UTR, an encoding region, a 3’ UTR and a poly-A tail. (B) Fluorescence microscopy pictures taken with 4x magnification (JULYTM) at 24 h post-transfection. All constructs showed improved protein expression levels as compared to the control. (C) The percentage of d2EGFP positive cells as determined by FC is similar for all constructs. Propidium iodide was used to detect dead cells. The applied gates ensured exclusion of dead cells and untransfected cells. (D) At 48 h post transfection, sustained protein expression was higher for the stabilized constructs as compared to the control. Figure 2: Time courses of protein expression as determined by FC for A549 cells (A) and Huh7 cells (B). Mean fluorescence intensities normalized to the control are plotted versus time in a log- linear plot. With increasing time post transfection, the elevated protein expression levels of the stabilized constructs become more and more evident. The bars corresponding to the control, 5’UTR and 3’UTR constructs, respectively, as well as to the constructs 5’+3’, 5’+2x3’ and 2x3’ are differently shaded as shown on the right hand side of the figure. Figure 3:Microstructured multi-channel slides for parallel single-cell assays to test differently stabilized mRNA constructs. (A) Cell-adhesive, microstructured protein patterns with cell-repellent PEG areas in between allow ordered cell arrangement. Fluorescently labeled fibronectin was used to visualize the micropattern. (B) Fluorescent A549 cells adhering to fibronectin patterns inside a microchannel (three hours after seeding). (C) Schematic drawing of mRNA lipofection (on the left) and reaction scheme underlying our analytical solution (on the right). (D) Exemplary time courses of mRNA- mediated d2EGFP expression in A549 cells. Bold black lines are representative fits to the theoretical translation model. Figure 4: Distributions of expression rates K, mRNA life times, and d2EGFP life times and corresponding mean values with schematic representations of the constructs. (A) Distributions of expression rate K, which is the product of the initial number of mRNA molecules and the translation rates. The fact that the distributions are similarly shaped indicates that the transfection kinetics and the translation rates are very similar. (B) The distributions of the mRNA half-lives show great variations in their broadness. As a guide to the eye, dotted lines indicate the mean half-life of the control. (C) Distributions of d2EGFP half-lives. As expected, the distributions of the different constructs are similarly shaped and show comparable mean values. As a guide to the eye, the overall mean half-life of d2EGFP based on all measured half-lives is shown as a dotted line. (D) Mean values and the corresponding standard deviations (std) of the fitted rates. Although the control construct yields high mean K values in both cell types, the short mRNA half-life of this construct leads to small AUC values as compared to the stabilized constructs. This can be seen in Figure 6. Schematic representations of the constructs can be seen on the right hand side. All constructs have the same 5’cap and a poly-A tail. Data from 895 single A549 and 1355 Huh7 cells were analysed. Figure 5: Mastercurves of the different constructs. Population averages of A549 (A) and Huh7 (B) cells with the onset time shifted to zero. The dark grey, medium grey and light grey curves correspond to the control/5’UTR/3’UTR constructs, respectively. The curves correspond to the constructs as correspondingly indicated on the right hand side. Figure 6: AUC and mRNA life time prolongation factors of the different constructs. (A) Schematic representation of the AUC to illustrate the interplay between mRNA translation and degradation of mRNA and protein. (B) and (C) AUC of the different constructs as analysed for ^ . Crosses show relative AUCs of different experiments, the bars correspond to the mean of all single-cell AUCs. (D) and (E) mRNA life time prolongation factors. All modifications result in prolonged mRNA life times as compared to the control. Similar trends are observed in A549 (D) and Huh7 (E) cells. Error bars in (D) and (E) indicate standard deviation. Figure 7: Fluorescence microscopy and flow cytometry data of Huh7 cells. (A) Fluorescence microscopy pictures taken with 4x magnification (JULYTM) at 24 h post- transfection. All constructs showed improved protein expression levels as compared to the control. (B) The percentage of d2EGFP positive cells as determined by FC is similar for all constructs. Propidium iodide was used to detect dead cells. The applied gates ensured exclusion of dead cells and untransfected cells. (C) At 48 h post transfection, sustained protein expression was higher for the stabilized constructs as compared to the control. Figure 8: Determination of mRNA half-life by qRT-PCR in A549 and Huh7 cells. The cells were transfected according to the protocol as described in Materials & Methods part. Absolute mRNA quantification at 4, 8, 24, 36, 48, 60, 72 hours for all mRNA constructs was determined in A549 (see Figure 8 A) and in Huh7 (see Figure 8 B). Out of this data the mRNA half-life was calculated. The physical half-life was normalized to the control. Figure 9: Transfection efficiencies on microstructured substrates. Percentage of transfected cells and corresponding standard deviations for A549 cells and Huh7 cells transfected with modified RNA/SNIM®RNA with help of LipofectamineTM2000 or DOGTOR. Higher transfection efficiencies were found for cells transfected with LipofectamineTM2000. Figure 10: Distributions of directly measured d2EGFP half-lives. (A) Exemplary time courses of cycloheximide-induced d2EGFP degradation in Huh7 cells. Bold black lines are simple exponential fits for protein degradation. (B) Distribution of d2EGFP half- lives measured in A549 cells, yielding a mean half-life of 2.46 h (std 0.71 h). (C) Distribution of d2EGFP half-lives measured in Huh7 cells, yielding a mean half-life of 4.04 h (std 1.82 h). Figure 11: Distribution of the single-cell AUCs. AUCs were calculated according to equation 3 below. A549 data are shown in the left column, Huh7 data are shown in the right column. Figure 12: Comparison of the constructs #2 o #5 having UTRs of different genes as indicated in Table 4 with the CYBA-UTR #1 construct. Figure 13: GLP1 expression in HEK293 cells quantified at 24 h post transfection. Figure 14: Kinetic of GLP1 expression in HEK293 cells (63 ng/well). Figure 15: GLP1 expression in HEK293 cells (24 h). Figure 16: GDF15 expression in HEK293 cells quantified at 72 h* post transfection. The six groups of bars correspond to ETH, hAg, nat, ETH, hAg, nat, from left to right, respectively (cf. the explanation on the right top down) Figure 17: Kinetics of GDF15 expression in HEK293 cells (250 ng/well). Figure 18: modified RNA/SNIM®RNA containing WT sequence of GDF-15 resulted in highest plasma protein levels in vivo after 6 hours. Figure 19: modified RNA/SNIM®RNAGDF-15 reduces food consumption and facilitates weight loss at already low doses. A: Food consumed within the last 24 hours after injection of test item. B: Weight lost within 24 hours after injection of test item. Figure 20: 1 mg/kg BW modified RNA/SNIM®RNAGDF-15 results in elevated GDF-15 plasma levels up to 24 hours post injection. A: GDF-15 plasma levels measured 24 hours after modified RNA/SNIM®RNA injection modified RNA/SNIM®RNA. B: Plasma glucose, determined using a Bayer ContourXT 24 hours after the application of test item. Figure 21: modified RNA/SNIM®RNAGLP-1 reduces food consumption (36% vs Succrose, 19% vs STOP) and body weight. A: Food consumed within the last 24 hours after modified RNA/SNIM®RNA injection. B: Weight lost within 24 hours after injection of modified RNA/SNIM®RNA. Figure 22: GLP-1 plasma levels are elevated against Succrose control, but not against STOP control. A: total GLP-1 in plasma 24 hours after injection of modified RNA/SNIM®RNA. B: Active GLP-1 in plasma 24 hours after injection of modified RNA/SNIM®RNA. Figure 23: Both, GDF-15 and GLP-1 coding modified RNA/SNIM®RNA facilitate statistically significant reduction of food consumption. Figure 24: For hGDF15, normalizing food consumption to 100 g/mouse does not result in significant changes of results. A: Food consumed within the last 24 hours after modified RNA/SNIM®RNA injection. B: Decrease of food intake within 24 hours upon application of hGDF15 coding modified RNA/SNIM®RNA and controls. Figure 25: For GLP-1, normalizing food consumption to 100 g/mouse does not result in significant changes of results. A: Food consumed within the last 24 hours after modified RNA/SNIM®RNA injection. B: Decrease of food intake within 24 hours upon application of GLP-1 coding modified RNA/SNIM®RNA and controls. Figure 26: Application of 2% Sucrose solution does not alter food intake of test animals. Figure 27: Inflammatory cytokines are within physiological ranges 24 hours after application of Test items. Figure 28: Application GLP-1 coding modified RNA/SNIM®RNA results in significantly increased IGF-1 levels after 24 hours. In the foregoing detailed description of the invention, a number of individual elements, characterizing features, techniques and/or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter. All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety. Examples I. Materials and Methods Plasmid Vectors Destabilized Enhanced Green Fluorescent Protein (d2EGFP) was excised from pd2EGFP-N1 (Clonetech) and cloned in pVAXA120 (3) to generate pVAXA120-d2EGFP. Based on previously published data with respect to mRNA stability, preselected 5’ and 3’ UTR sequences of CYBA gene were synthesized by Eurofins MWG (Germany) and cloned upstream (5’UTR) and/or downstream (3’UTR or 2x3’UTR) of d2EGFP in pVAXA120-d2EGFP, thereby generating the constructs with respective UTR combinations. mRNA production To generate in vitro transcribed mRNA (IVT mRNA), plasmids were linearized downstream of the poly-A tail by NotI digestion and purified by chloroform extraction and ethanol precipitation. Purified linear plasmids were used as template for in vitro transcription using RiboMax Large Scale RNA production System-T7 (Promega, Germany). Anti-Reverse Cap Analog (ARCA) was added to the reaction mix to generate 5’ capped mRNA. Additionally for the production of modified RNA/SNIM® mRNAs, chemically modified nucleotides namely methyl-CTP and thio-UTP (Jena Bioscience, Germany) were added to a final concentration of ATP:CTP:UTP:methyl-CTP:thio- UTP:GTP of 7.57mM:5.68mM:5.68mM:1.89mM:1.89mM:1.21mM. The complete IVT mix was incubated at 37°C for 2 hours followed by a DNA disgestion with DNaseI for 20 minutes at 37°C. RNA was precipitated with ammonium acetate (final concentration 2.5M) and washed with 70% EtOH. The washing step was performed twice. Finally, the RNA pellet was re-suspended in RNAse-free water. All mRNAs were verified on 1% agarose gels. A schematic representation of an exemplary mRNA construct can be seen in Figure 1A. The exact sequences of the UTRs are given herein elsewhere (e.g. in the above Tables 1 and 2.
Figure imgf000090_0001
Table 5: Secondary structures (mfold) In Table 5, features of the mRNA constructs such as free minimum energy (∆G) and secondary structures found at both ends and within the UTRs are listed. The folding platform mfold was used to predict mRNA secondary structures (40). For each construct, we compared the eight secondary structures that have the highest free energy. The highest free energy values are predicted for the 2x3’ UTR and the 3’ UTR constructs. The 5’ end of each mRNA construct partially binds with the 3’UTR or the 5’UTR, except for the control construct, which binds to the coding sequence (cds). Interestingly, the 5’ end of the 2x3’ mRNA construct forms a stabilizing hairpin with itself. However, hairpin loops near the 5’ end can also hinder protein translation (41). Another feature was found in the 3’ end of the 3’ UTR and 5’+3’ UTR mRNA constructs: There, the 3’ end binds with the 5’ end, minimizing the distance from each other and thus enabling faster initiation of translation. Unlike the 5’UTRs, the 3’ UTR of each mRNA construct forms at least one hairpin with itself. Flow cytometry (FC) The experimental set-up looks like as follows: 20.000 cells in 150µl medium were seeded per well in 96-well plates and transfected 24 hours post-seeding. Cells were transfected at a dose of 5pg mRNA/cell using the commercial transfection reagent LipofectamineTM2000. Complexes were prepared at a ratio of 2.5µl LipofectamineTM2000 per 1 µg mRNA. For the formation of lipoplexes, LipofectamineTM2000 and mRNA were diluted separately in OptiMEM transfection medium in a total volume of 50µl, each. These mixtures were incubated at room temperature for 5 minutes. The mRNA solution was then mixed with the LipofectamineTM2000 solution, followed by another 20 minutes of incubation at room temperature. After incubation, 900µl of OptiMEM were added to the lipoplex solution. Finally, 50µl of the complex solution were added to the cells and incubated for 1 hour. For every mRNA construct, biological triplicates were prepared. After incubation, the lipoplex-solution was discarded and fresh 150µl medium was added to each well. d2EGFP expression was measured after 8, 24, 36, 48, 60 and 72 hours using FC. Fluorescence microscopy images were taken at each of these time points. For FC measurements, the cell culture medium was discarded and the cells were washed with 1xDPBS (Gibco Life Technology). Subsequently, 20µl of TrypLE Express (Gibco Life Technology) were added per well and incubated for 5 min at 37°C. The reaction was neutralized by adding 80µl 1xPBS, supplemented with 2% FBS. Cells were mixed by pipetting and were transferred into a 96 well plate appropriate for flow cytometric measurements. Finally, 5µl of Propidium iodide (final concentration 1µg/ml) were added per well and measured with Attune Auto Sampler (Applied Biosystems). Fluorescence images were taken prior to FC analysis with a JULYTM microscope. Quantitative real-time PCR A qRT-PCR analysis was used to determine the d2EGFP mRNA amount at time intervals of 4, 8, 24, 36, 48, 60 and 72 hours in A549 and Huh7 cells. Additionally, the mRNA expression kinetic itself was used to calculate the mRNA half-life of each UTR. Here, the cells were transfected similarly to the protocol described above (see FC). A cell density of 200.000 cells/well was found to be sufficient for RNA isolation. RNA isolation was performed according to the manufacturer’s protocol using NucleoSpin RNA (Macherey Nagel). The isolated total RNA was examined in RNA concentration and quality by spectrophotometric measurements and gel analysis. Further, 0,5 µg of the total RNA of each UTR constructs and the control were used for cDNA synthesis using Oligo(dT)s from First Strand cDNA Synthesis Kit (Thermo Scientific). Equivalent amounts of cDNA (diluted 1:50) were tested with 125nM of each d2EGFP-Primer (forward Primer: 5’-CAA CCA CTA CCT GAG CAC CC-3’ (SEQ ID NO:34); reverse Primer:5’-GTC CAT GCC GAG AGT GAT CC-3’ (SEQ ID NO:35)) using SsoAdvanced™ Universal SYBR® Green Supermix (BioRad). As a standard for the absolute quantification, pure d2EGFP mRNA produced by IVT was used for synthesis of cDNA. Absolute mRNA quantification was performed on a Lightcycler 96 device (Roche). Surface patterning and sample preparation Microstructured surfaces were produced by selective oxygen plasma treatment (Femto Diener, 40 W for 3 min) on a top as substrate (ibidi GmbH) with subsequent passivation. Selectivity was achieved using a polydimethylsiloxane (PDMS) stamp (cast from a master produced by photolithography) as a mask. The parts exposed to plasma were passivated by incubation for 30 min with PLL(20k)–g(3.5)-PEG(2k) at a concentration of 1 mg/ml in aqueous buffer (10 mM HEPES pH 7.4 and 150 mM NaCl). Thereafter, the samples were rinsed with PBS and the PDMS stamps were removed. The foils were then fixed to adhesive six-channel slides (sticky µ-slide VI). Each channel was filled with a solution of 50 µg/ml fibronectin in PBS for one hour to render the remaining sectors cell-adhesive. Probes were thoroughly rinsed with PBS three times. The samples were stored in cell medium at room temperature before cell seeding. For this study, square adhesion sites of 30 µm x 30 µm were used because this size turned out to be reasonable for single-cell adhesion of A549 as well as Huh7 cells. Cells were seeded at a density of 10,000 cells per channel so that roughly one cell could adhere on each cell-adhesive island. To obtain fluorescent micropatterns as shown in Figure 3A, a mixture of 20 µg/ml fibronectin and 30 µg/ml fibrinogen conjugated with Alexa Fluor 488 was used. Materials FBS, Leibovitz’s L-15 Medium (Gibco), LipofectamineTM2000, and OptiMEM (Gibco) were purchased from Invitrogen, Germany. Sterile PBS was prepared in-house. Ham’s F-12K, DMEM, and Trypsin-EDTA were purchased from c.c.pro GmbH, Germany. Channel slides were purchased from ibidi, Germany. Fibronectin was purchased from Yo Proteins, Sweden. PLL-g- PEG was purchased from SuSoS AG, Switzerland. Alexa Fluor 488 was purchased from Life Technologies, Germany. The plasmid pd2EGFP-N1 was purchased from BD Biosciences Clontech, Germany. Cell Culture A human alveolar adenocarcinoma cell line (A549, ATCC CCL-185) was grown in Ham’s F12K medium supplemented with 10% FBS. A human hepatoma epithelial cell line (Huh7, JCRB0403, JCRB Cell Bank, Japan) was cultured in DMEM medium, supplemented with 10% fetal bovine serum. All cell lines were grown in a humidified atmosphere at 5% CO2 level. In vitro Transfection Three hours prior to transfection, 10.000 cells per channel were seeded in a 6-channel slide. Cells were transfected at a dose of 5pg mRNA/cell using the commercial transfection reagent LipofectamineTM2000 at a ratio of 2.5µl LipofectamineTM2000per 1 µg mRNA. The complex formation was prepared as follows: LipofectamineTM2000 and mRNA were separately diluted in OptiMEM transfection medium to add up to a total volume of 45µl, each. These mixtures were incubated at room temperature for 5 minutes. The LipofectamineTM2000 solution was then mixed with the mRNA solution, followed by another 20 minutes of incubation at room temperature. Please note that the microchannels were never empty during all subsequent rinsing steps: Immediately before transfection, the cells were washed with PBS. Finally, the lipoplex solutions containing different mRNAs constructs were filled into the six channels. All five different mRNA constructs plus the reference construct could thus be measured under the same experimental conditions. The cells were incubated in a total transfection volume of 90µl at 37°C (5% CO2 level) for one hour. The transfection medium was thereafter removed and the cells were washed with PBS. Subsequently, the cells were re-incubated with Leibovitz’s L-15 Medium containing 10% FBS. A drop of anti-evaporation oil (ibidi GmbH, Germany) was added on top of each medium reservoir before microscopic monitoring of d2EGFP expression. Data Acquisition and Quantitative Image Analysis Live-cell imaging was performed on a motorized inverted microscope (Nikon, Eclipse Ti-E) equipped with an objective lens (CFI PlanFluor DL-10×, Phase1, N.A. 0.30; Nikon) and with a temperature-controlled mounting frame for the microscope stage. We used an ibidi heating system (Ibidi GmbH, Germany) with a temperature controller to stabilize the temperature of the samples at 37°C (± 2°C) throughout the measurements. To acquire cell images, we used a cooled CCD camera (CLARA-E, Andor). A mercury light source (C-HGFIE Intensilight, Nikon) was used for illumination and a filter cube with the filter set 41024 (Chroma Technology Corp., BP450-490, FT510, LP510-565) was used for d2EGFP detection. An illumination shutter control was used to prevent bleaching. Images were taken at 10 fold magnification with a constant exposure time of 600 ms at 10 minute-intervals for at least 25 hours post-transfection. Fluorescence images were consolidated into single-image sequence files. Quantitative analysis of characteristic parameters of single-cell expression kinetics allows the comparison of various vector performances in terms of expression efficiency and stability. Image analysis consisted of several steps and was done using in-house-developed software based on ImageJ. First, a rectangular grid was overlaid with the original time-lapse movie and adjusted to the size and orientation of the underlying cell- pattern. Next, the software automatically detected d2EGFP-expressing cells by reading out the fluorescence intensities of all squares. Unoccupied squares were used for background correction. The software calculates the cells’ fluorescence over the entire sequence and connects corresponding intensities to time courses of the fluorescence per cell. Finally, single-cell fluorescence intensities per square were extracted. Data were then analyzed as described recently by fitting each time-course with the analytical solution for mRNA-induced protein expression (see equation 1) using IgorPro software, which is the solution to the differential equations for mRNA and d2EGFP, (Equation 4)
Figure imgf000093_0001
(Equation 5) A schematic representation of the underlying simplistic model assumed for mRNA-induced protein expression is depicted in Figure 3C. II. Example 1: Fluorescence microscopy and analysis via flow cytometry (FC) To evaluate the effect of different UTR combinations on transgene expression kinetics, two different cells lines were transfected using LipofectamineTM2000 with different d2EGFP mRNA constructs containing a 5’ UTR alone, a 3’ UTR, 5’+3’ UTR, two copies of 3’UTR and 5’+2x3’ UTR. A schematic representation of the building blocks of all constructs can be seen in Figure 1A. At different time points through three days post-transfection, d2EGFP expression was quantified using FC. An exemplary dot plot for t=24h, illustrating d2EGFP expression levels of live A549 cells, is shown in Figure 1C (see Figure 7B for corresponding Huh7 data). In addition, we imaged the cells using fluorescence microscopy (see Figure 1B and D and Figure 7A and C). Comparable transfection efficiencies for all mRNA constructs were confirmed 24 hours post transfection (Figure 1B and Figure 8A). Thereby, differential transfer efficiencies to be a causal factor for the observed differences in expression kinetics can be ruled out. Based on fluorescence microscopy images, a drastic reduction of d2EGFP expression for all constructs at 48 h post- transfection was detected (see Figures 1B and D, Figure 7A and C). However, higher EGFP expression levels with respect to the control were found for all UTR-stabilized mRNAs. More specifically, mRNA constructs containing 3’ UTRs seemed to enhance expression more than constructs without 3’ UTRs. This was observed for A549 and Huh7 cells (see Figure 1 and Figure 7 , respectively). At time points later than 48h, this effect was pronounced even more (data not shown). In Figure 2 A and B, the time courses of the mean fluorescence intensities (MFI) as determined by FC are shown for all constructs in both cell types. Also here, all UTR-containing mRNA constructs showed higher MFI values than the control construct in both cell lines at all points in time. Taken together, the fluorescence microscopy and FC data suggest that mRNA molecules furnished with CYBA UTRs show persistent d2EGFP expression for more than 24 hours. III. Example 2: Quantitative real-time PCR qRT-PCR measurement as an additional approach was conducted to determine the “physical” mRNA half-life of the different constructs. Binding of our selected primers to d2EGFP occurred 600nt downstream of the start codon. Hence, measurements of physical mRNA half-life compromise both intact mRNAs and those which have either been decapped but not yet degraded or both decapped and degraded up to base 599. It also includes mRNA that has been removed from the translational pool and stored in P-bodies (29-32). Though intact mRNAs contribute to d2EGFP expression, the latter group of decapped and/or partially degraded transcripts, and those in P-bodies do not lead to any expression. Determination of physical mRNA half-life did not reveal any significant life time prolongation of the UTRs compared to the control in the A549 and Huh7cells (see Figure 8A and B, respectively). Interestingly, instead a decrease in mRNA physical half-life for 5’, 3’, 5’+2x3’ and 2x3’ UTR constructs was observed in both cell lines. Determination of mRNA half-life by qRT-PCR in A549 and Huh7 cells In an additional experiment, the mRNA half-life of the different mRNA constructs with qRT-PCR was investigated which is a conventional approach (see Figure 8A and B). Therefore, the mRNA constructs were transfected as described in herein. At the end, the absolute mRNA amount at each specific time point was obtained and calculated the mRNA half-life for each mRNA furnished with UTRs. No significant mRNA stabilization effects for any of the selected mRNA constructs as compared to the control were observed. IV. Example 3: Single-cell expression arrays Microstructured, cell-adhesive substrates as shown in Figure 3A and B were fabricated as a platform for single-cell time-lapse microscopy. The rectangular squares are functionalized with the extracellular matrix protein fibronectin, while the surrounding dark area is passivated with cell repellent PLL-g-PEG. Cells were seeded at an appropriately dilute cell density such that after about three hours, cells adhered to the rectangular squares. This cellular self-organization process has been studied in detail before (27). The size of the squares was 30µm for optimal filling with single cells. The distance between the squares was just big enough (60µm) to minimize bridging effects of cells adhering to more than one square at the same time. Time-lapse fluorescence microscopy and automated image analysis of the fluorescence signal per square yields hundreds of individual time courses. A typical set of background corrected raw data is shown in Figure 3D. The black lines represent exemplary fits to the mathematical expression for mRNA translation (see also Materials and Methods section). Data were analyzed as described recently (26) by fitting each time-course with the analytical solution for mRNA-induced protein expression,
Figure imgf000095_0001
(Equation 1) using IgorPro software. Here, G denotes the amount of protein, K is the expression rate, δ is the mRNA degradation rate, and β is the degradation rate of the reporter protein d2EGFP. The expression rate K = m0 ∙ kTL is the product of the initial amount of mRNA molecules inside the cell (m0) and the translation rate kTL. The time-course that is described by Equation 1 will be discussed in detail in below section “mastercurves of protein expression”. V. Example 4: In vitro transfection on cell arrays In a typical experiment, cells were allowed to adhere to the micropatterns for three hours before transfection. Each of the six microchannels was filled with a different lipoplex solution, containing one of the constructs of interest. In initial experiments, we compared two different, commercially available transfection reagents (namely LipofectamineTM 2000 and DOGTOR). Higher transfection efficiencies were found for LipofectamineTM 2000 than for DOGTOR (see Figure 9). Because additionally obtained high cell viability rates of above 80% were obtained with LipofectamineTM2000 (data not shown), all further transfection experiments were conducted using LipofectamineTM2000. As mRNA-mediated protein expression starts shortly after transfection, incubation time was kept to a minimum. Accordingly, the ratio between mRNA dosage and incubation time was adjusted to achieve high transfection efficiencies (see also Figure 9) and negligible toxic effects caused by over-expression of the reporter protein. At an mRNA dose of 5pg/cell, an incubation time of one hour was found to be optimal. Transfection efficiencies on microstructured substrates The percentage of successfully transfected cells was assessed to compare two different transfection agents and to ensure that transfection efficiencies were not hampered by microstructured cell growth (see Figure 9). Here, all cells grew on microstructured protein arrays. We obtained higher transfection efficiencies for LipofectamineTM2000 as compared to DOGTOR. Using a commercial Live/Dead cell viability assay (Molecular Probes, Germany), we found high cell viability rates above 80% (data not shown). VI. Example 5: Expression rates All results for the two cell types are based on four independent measurements under the same experimental conditions. Time-lapse data of about thousand A549 cells and thousand Huh7 cells have been analyzed. The distributions of the obtained expression rates K are shown in Figure 4A and the corresponding mean values can be seen in Figure 4D. Both the mean expression rates and the shape of their distributions were found to be rather similar for the different constructs. VII. Example 6: mRNA half-lives We converted the fitted mRNA-degradation rates δ into mRNA half-lives according to τ = ln2 δ . (Equation 2) Figure 4B shows the half-life distributions of differently stabilized mRNA constructs in A549 and Huh7 cells, respectively. Here, it becomes evident that for stabilized constructs, both mean half- life and broadness of the underlying distribution increase as compared to the reference construct. An overview of all determined half-lives is given in Figure 4D. Both for A549 and for Huh7 cells, we found longer half-lives for mRNAs stabilized by UTR elements compared to the control construct (5.8 hours for A549 cells and to 7.8 hours for Huh7 cells) that does not contain any stabilizing UTR. The life time prolonging effect was more pronounced in A549 cells. VIII. Example 7: Protein half-lives The distributions of protein (d2EGFP) degradation life times are presented in Figure 4C. As expected the half-lives of the expressed protein do not vary for the different mRNA constructs. The determined mean life times range from 4.2 to 4.9 hours for A549 cells and from 5.6 to 8.5 hours for Huh7 cells as shown in Figure 4D. The coefficients of variation are about 0.29 (A549) and 0.45 (Huh7) and hence is significantly smaller than the coefficient of variation of up to 0.6 that we found for the distribution on mRNA life-times. As a control, the half-lives in an alternative approach were also measured, where translation was inhibited by addition of cycloheximide at a given time point, t0, after transfection (see Figure 10). In this case, protein expression is induced for a while and then stopped. The exponential decay in fluorescence after inhibition yields protein life times. These half-lives were found to be smaller by a factor of about two, compared to the above experiments without inhibition. In both experiments, however, the relative ratios of the protein life times in Huh7 cells as compared to those in A549 cells is the same. Degradation rate of the reporter protein To check the fitted d2EGFP degradation rates, the degradation rate of d2EGFP inside A549 and Huh7 cells were independently measured in microstructured six-channel slides. Protein synthesis was blocked by the antibiotic cycloheximide, which interferes with peptidyl transferase activity (42). Single-cell fluorescence intensity time courses were monitored for approximately 20h (see Figure 10). Control experiments ensured that the decrease in fluorescence intensity was not due to photobleaching of the chromophore. Single-cell time courses were fitted by a single exponential fit, yielding distributions of protein degradation rates. The mean degradation rates were found to be 0.28/h (std 0.08/h) in A549 cells and 0.17/h (std 0.08/h) in Huh7 cells, corresponding to protein life times of 2.46 h and 4.04 h, respectively. Although these life times are significantly shorter than the life times as determined by single-cell time course analysis of mRNA mediated protein expression, the ratio between the mean life times of d2EGFP inside Huh7 and A549 cells is the same (4.04 h/2.46 h=1.64 as measured by translational blocking compared to 7.4 h/4.5 h=1.64 as determined by fitting the analytical solution for mRNA expression). IX. Example 8: Mastercurves of protein expression The features of mRNA induced protein expression become evident in the so-called mastercurve of protein expression as depicted in Figure 5A (A549) and B (Huh7). The mastercurve is the population average of the onset-time corrected single cell traces, i.e. all onset-times were shifted to time point zero. Fluorescence intensities were converted into actual numbers of d2EGFP as described before in reference (26). The superior properties of the 3’ and the 5’+3’-stabilized mRNA constructs are illustrated in the mastercurve plot. These constructs showed the shallowest decrease in protein expression with time and hence the longest half-lives in addition with higher protein expression values as compared to the other constructs. X. Example 9: Area under the curve (AUC) In pharmacokinetics, the total exposure of a drug is known as the “area under the curve”. The analogous expression in gene therapy is the integral of the amount of artificially expressed protein over time, i.e. the area under the (expression-vs.-time) curve (AUC). The AUC is a means to simultaneously quantify the translational efficiency and the stability of an mRNA construct. It can be interpreted as the cumulative time-dose of the protein that is encoded on the mRNA and hence describes the efficacy of a chosen mRNA construct. Given the biochemical rate model (see Figure 3A) the AUC can be explicitly calculated: AUC = 0.48 ∙m0 ∙ kTL ∙ τmRNA ∙ τd2EGFP (Equation 3) Hence an optimal therapeutic mRNA construct should desirably have both long mRNA, τmRNA, as well as protein half-life, τd2EGFP, and high translational efficiency, kTL. In addition, the transfer efficiency which determines the initial amount of therapeutic mRNA, m0, is directly proportional to the AUC. An illustrative explanation for the theoretical time course of protein expression and calculated AUC can be seen in Figure 6A. If there was no protein degradation (β=0), the amount of protein inside a cell would run into a steady state level as a consequence of a balanced flux of mRNA translation and mRNA degradation. In this case the expression dynamics follows −δt
Figure imgf000098_0001
(1 − e ). The same would be true in an analogous manner for the case where δ was equal to zero. The superposition of this with the permanent, exponential decay of the d2EGFP protein (following e−βt) results in the characteristic shape of the AUC as shown in Figure 6A. Figures 6B and C show the overall mean relative AUCs as well as the “per-experiment” relative AUCs normalized to the mean AUC of the control, the latter being the AUC of protein expression after transfection with the control construct. In both cell types, the highest relative AUCs was found for the 3’UTR- and the 5’+3’UTR-stabilized construct. This is consistent with the observed long half-lives for these constructs, because they contribute to the AUC as seen in equation 3. The detailed, single-cell AUC distributions can be found in Figure 11. More specifically, assuming biochemical rate equations (4) and (5) for translation and degradation according to Figure 3C, the amount of expressed protein after mRNA transfection is given by (Equation 1).
Figure imgf000099_0001
The area under the curve (AUC) is calculated by integrating the expression level Gd2EGFP(t) from t0, when expression sets in to long times ( ^^ → ∞ ):
Figure imgf000099_0002
with ^^ = ^^ − ^^0. Using ^^ ^^ ^^ ^^ ^^ = ^^ ^^2⁄ ^^ , ^^ ^^2 ^^ ^^ ^^ ^^ = ^^ ^^2⁄ ^^ , and ^^ = ^^0 ∙ ^^ ^^ ^^ equation 3 is obtained: ^^ ^^ ^^ = 0.48 ∙ ^^0 ∙ ^^ ^^ ^^ ∙ ^^ ^^ ^^ ^^ ^^ ∙ ^^ ^^2 ^^ ^^ ^^ ^^ The time course of Gd2EGFP(t) and the AUC is schematically depicted in Figure 6A. The experimental single-cell AUC distributions can be seen in Figure 11. Because the AUC depends linearly from the mRNA and protein life times, the single-cell AUC distributions are closely related to the mRNA and protein half-life distributions that are shown in Figure 4B and 4C of the main text. XI. Example 10: Life time-prolongation factor The life time-prolongation factors for A549 and Huh7 cells are shown in Figure 6D and E, respectively. As expected, all stabilized constructs yield life time-prolongation factors higher than one, meaning that the insertion of UTRs at either end causes mRNA stabilization. However, the 3’UTR mRNA construct shows longer mRNA life times than the 2x3’UTR construct. Similarly, the 5’+3’UTR construct is more stable than the 5’+ 2x3’ construct. These results hold true for both cell types. Interestingly, the stabilizing effects are significantly more pronounced in A549 cells than in Huh7 cells in all cases. XI. Example 11: Comparison of constructs having UTRs of different genes compared to the CYBA-UTR construct The constructs #2 to #5 having UTRs of different genes as indicated in the below Table 4 have been compared to the CYBA-UTR construct #1 in order to optimize the mRNA structure in terms of stability and productivity. Five different cellular UTRs of a gene were selected based on publication data (Hoen et al., 2010) featuring long mRNA half-lives. These cellular UTRs are CYBA, DECR1, GMFG, MAPBPIP and MYL6B. The sequences of 5’and 3’ untranslated regions of each cellular gene were obtained from the UTR database (http://utrdb.ba.itb.cnr.it/search) and were cloned into five different combinations, which were 5’UTR alone, 3’UTR alone, 5’+3’UTR, 5’+2x3’UTR and 2x3’UTR. Firstly, the untranslated region sequences were cloned into the backbone pVAX1-A120. In case of the 5’UTRs, cloning occurred via HindIII restriction site on the 5’end and BamHI restriction site on the 3’end and was inserted upstream of the reporter gene coding for Metridia luciferase (MetLuc). The restriction sites for 3’UTRs were EcoRI (5’end) and PstI (3’end) and were cloned downstream of MetLuc. The plasmids containing 5’ UTR alone and 5’+3’UTR for each cellular UTR were produced by Eurofins MWG Operon. These plasmids were transformed into E. coli bacteria (DH10B) via electroporation. The other combinations, including 3’UTR alone, 5’+2x3’UTR and 2x3’ UTR were cloned in-house. Cloning of plasmids with 3’UTR was performed by simply cutting out the 5’UTR of the backbone via HindIII (blunt) and BamHI (blunt) digestion. Constructs containing 5’UTR+2x3’UTR were cloned by inserting MetLuc containing 3’UTR (BamHI/PstI blunt) into the backbone of pVAX1-A120 MetLuc comprising 5’+3’UTR, thereby replacing MetLuc and inserting a second 3’UTR in front of the respective 3’UTR of the backbone. Finally, the constructs containing 2x3’UTR were generated by removing the 5’UTR (HindIII and BamHI, both blunt) from the plasmid containing 5’+2x3’UTR. After cloning, all plasmids were amplified in E. coli bacteria (DH10B) after electroporation. Secondly, chemically modified mRNA was produced by in vitro transcription. For that purpose, the plasmids were linearized with XbaI digestion and were purified with chloroform/ethanol precipitation. The in vitro transcription kit (Promega) included the required T7 polymerase enzyme mix as well as the suitable buffers. The transcription mix also contained the unmodified nucleotides adenosine-triphosphate (ATP), guanosine-triphosphate (GTP), uridine-triphosphate (UTP) and cytosine-triphosphate (CTP) as well as the chemically modified nucleotides methyl- CTP and thio-UTP (Jena Bioscience, GmbH, Jena, Germany) with a final concentration of ATP:GTP:UTP:CTP:methyl-CTP:thio-UTP of 7.13 mM:1.14 mM:5.36 mM:5.36 mM:0.536 mM:0.536 mM. Additionally, the cap structure analog ARCA (anti-reverse cap analog) was added to the mix to ensure the incorporation of the 5’-cap in the right direction. Finally, the linearized DNA was added into the reaction mix. The IVT mix was incubated at 37°C for 2 h. Digestion of the remaining DNA was enabled by the addition of DNase I and further incubation at 37°C for another 20 min. RNA precipitation was performed by the addition of pre-cooled ammonium- acetate to a final concentration 2.5 M. The RNA pellet was washed with 70% ethanol. The washing step was performed twice. At last, the RNA was re-suspended in RNase-free water. The RNA concentration was determined with a spectrophotometric device and purity was tested on an agarose gel. After IVT, the different mRNAs were tested in two different cell lines, i.e., in NIH3T3 and A549. For the screening experiments a non-viral nucleic acid delivery system, like lipofection, was used. In a first transfection experiment, different transfection agents were tested to compare protein expression and cell viability (data not shown). Next, the screening experiments including dose titration were conducted to evaluate dose dependent effects. The experimental set-up is as follows: 5000 cells (NIH3T3) in 150µl DMEM complete medium were seeded per well in 96-well plates and transfected 24 hours post-seeding. Cells were transfected at a starting dose of 500ng/well (100pg mRNA/cell) using the commercial transfection reagent Dreamfect Gold (DFG). Complexes were prepared at a ratio of 4 µl Dreamfect Gold per 1µg mRNA. For the formation of lipoplexes, mRNA (3.6µg) was diluted separately in DMEM without supplements in a total volume of 340µl for each mRNA. In a 96well plate 14.4µl DFG was mixed with 5.6µl water in one well prepared for each mRNA dilution. Complex formation took place when the mRNA dilution was added to the DFG and mixed by up and down pipetting. The mixtures were incubated at room temperature for 20 minutes. In the meantime, the dilution series were prepared. In the remaining seven wells subjacent of the complex mix, 180µl DMEM without supplements per well was added. After incubation time 180µl of the complex solution was removed and added into the first well of dilution series. This procedure was conducted until the last dilution step. Finally, 50µl of the complex solution were added to the cells and incubated for 4 hour. For every mRNA construct, biological triplicates were prepared. After 4 hours, the complete supernatant was removed from the cell culture plate for measurement and fresh 150µl medium was added to each well. Bioluminescence was measured after 4, 24, 48, 72, 96, 120 and 144 hours using a multilabel plate reader. To this 50µl of supernatant was mixed with 20µl coelenterazin and the generated light was measured. Finally the protein amount over time was observed and is depicted as area under the curve (AUC). The results are shown in Figure 12.
Figure imgf000101_0001
Figure imgf000102_0001
Table 4: summary of the constructs #1 to #5. XIII. Discussion Determination of mRNA stability and its expression are two major factors to be considered when it comes to developing new mRNA therapeutics. Here, different combinations of UTRs, a 5’ UTR, 3’UTR, a 5’+3’ UTR, 5’+2x3’ UTR, and two copies 3’ UTR were used to improve mRNA in terms of stability and its expression. The AUC of the d2EGFP time course is also evaluated, because the total protein expression is relevant for a sustained therapeutic effect. In order to get detailed time-resolved data and monitor protein expression dynamics at the single-cell level, microstructured single-cell arrays for parallel, quantitative measurements of mRNA stability and translational efficiency were used. The regular arrangement of cells guaranteed reproducible microenvironments and enabled fast and automated image-analysis, which are prerequisites for comparative, high-throughput single-cell studies. The approach allows the determination of distribution functions for (i) protein half-life, (ii) expression rates, and (iii) functional mRNA half- life. In both A549 and Huh7 cells, mean protein half-lives of d2EGFP were narrowly distributed and independent of the UTR sequence. The calculated half-life values of 4.5 hours for A549 cells and 7.4 hours for Huh7 cells could be attributed to cell type specific differences between the compared cell lines. Such cell specific differences in d2EGFP half-life have been published previously. A study in NIH3T3 cells using a similar imaging cytometry approach, recorded a half-life of 2.8 h within a measurement window of 12 hours (33). An even shorter half-life of less than two hours has been reported for CHO cells by Li et al. (34). Here, protein degradation was measured by Western blotting and flow cytometry for three hours only. To validate our findings from single-cell data analysis, d2EGFP life times in direct measurements using cycloheximide were additionally determined (see Figure 10). Shorter life times as compared to the values observed from single-cell data analysis were found. This might be due to the fact that in single-cell data analysis, a constant initial number of mRNA molecules was assumed as part of the combined expression rate K = m0 ∙ kTL (see Equation 1). However, regardless of the fact that cells have been washed after one hour incubation time, it is still likely that the number of mRNA molecules is not constant from the start of observation. As a consequence, mRNA molecules that are available for translation later on, leading to protein expression, might result in longer half-life values obtained from single-cell expression time course fitting. When the mean half-life determined for A549 cells with the mean half-life determined for Huh7 cells is compared, the same ratio of roughly 1.64 for both measurement methods is found. Also, even a possible systematic over-estimation of mRNA and protein half-lives does not change the qualitative order of the mRNA performance. The expression rate depends on the initial number of mRNA molecules, m0, as well as on the translation rate KTL. It is to be noted that the number of successfully delivered mRNA molecules varies due to the intrinsic stochasticity of the delivery process. The mean number of mRNA molecules, however, is expected to be the same, since the transfection protocol has scrupulously been kept up in all experiments. In contrast, the translational activity (KTL) of the various UTR constructs might vary. Still, the fact that the distributions as well as the mean values of the expression rate K are rather similar for all constructs (see Figure 3A and D) indicates that the translation rate is merely influenced by the inserted UTRs. The parameter of highest interest is the mRNA half-life. Here functional mRNA half-life was compared to physical mRNA half-life. The results with single cell transfection studies suggest that any insertion of 5’ or 3’ UTRs into the mRNA sequence increases its functional mRNA half-life. All modifications tested in this study led to prolonged mRNA half-lives (see Figures 2 and 3), thereby resulting in prolonged expression as measured by fluorescence microscopic imaging and FC (see Figure 1). In contrast to the functional mRNA half-life, the physical mRNA half-life determined by qRT-PCR showed a decrease in mRNA stability for 5’, 3’, 5’+2x3’ and 2x3’ UTR in both cell lines (see Figure 8A and B). One major difference is the translational capacity for every measured mRNA in both methods. In the case of measuring functional mRNA half-lives, the mRNA is involved in active translation, whereas the physical mRNA half-life is monitored regardless of the translational status of the detected mRNA. Similar findings have been reported by Gallie et al. (35). It is believed that the physical mRNA half-life is not an appropriate indicator of the translational capacity of the mRNA. Translational capacity for a mRNA could be judged from it’s functional half life (longevity of expression) and the amount of total protein produced (Area Under the Curve). For a therapeutic mRNA, it is imperative that the molecule is functional for as long as possible and produces maximum possible protein. This leads to the conclusion that both functional mRNA half-life and total amounts of produced protein are better measures for identifying, comparing and testing mRNA therapeutics. Furthermore, the heterogenic distribution of the half-lives points out the importance of single-cell measurement techniques, because these effects are obscured in ensemble measurements (see Figures 2, 4, and 8A and B). Interestingly, a positive effect on protein expression was observed for 5’ UTR alone, although so far, no known motif in the CYBA 5’ UTR has been discovered. For the first time, it has been shown that CYBA UTRs at either end suffice to increase both peak and persistence of protein expression in both cell lines. These findings are consistent with publications claiming individual or synergistic behavior of 5’ UTRs and 3’ UTRs (14). In contrast to Holtkamp et al. (16), no additional increase in protein expression or mRNA stability could be observed with two sequential copies of the 3’UTR as compared to one single 3’ UTR (see Figure 4). Conversely, it even resulted in shorter life times both for 5’+3’ versus 5’+2x3’ UTR insertion and for 3’ versus 2x3’ UTR insertion. This might be due to the fact that a different type of cells (namely dendritic cells) was used in the study by Holtkamp et al. (16). Similar cell type specific effects have been reported for hepatocytes, too (39). Another contributing factor affecting both mRNA stability and its translation efficiency might be the secondary structure of the different mRNAs. Such effects of mRNA secondary structure in regulating gene expression have been reported before (36,37). Important structural characteristics together with their minimum free energy for the mRNA constructs used in the current study are summarized in Table 5. The persistent protein expression of the 5’ + 3’UTR stabilized construct could be due to binding of the 5’ to the 3’end, which facilitates circularization of the mRNA (19). Because no stable secondary structures within the 5’ UTR could be found, it is assumed that this feature enables an early expression onset (38). In contrast, secondary structures within the 3’ UTRs were identified. These might protect the mRNA from the 3’-5’ degradation pathway. Two 3’ UTRs showed even more secondary structures (two hairpins) with the best minimum free energy, indicating more persistent expression. Taken together, these findings could be the explanation for the inferior onset expression of the 2x3’ UTR compared to the 5’UTR and the persistent expression at later time points of mRNA constructs containing 3’ UTRs. In accordance with protein half-lives, longer half-life values were obtained for mRNAs stabilized with UTRs. This was observed in both cell lines with cell specific differences most likely affecting the absolute values. In A549 cells, mRNA half-lives for the constructs with UTRs ranged from 13.0 h to 23.0 h as compared to 5.8 h for the control. In Huh7 cells, half-lives from 9.9 h to 13.6 h were measured for UTR-containing constructs, as opposed to a half-life of 7.8 h for the control mRNA. The half-life of the 3’UTR-stabilized mRNA in A549 cells is in good agreement with mRNA life times of similarly stabilized mRNAs that were reported previously (16,26). The fact that stability and decay kinetics of mRNA and protein differ in different cell types is most likely due to differences in the complex networks of interactions between mRNA and proteins which are very likely to be cell-type dependent. Taken together, our results in both A549 and Huh7 cells, independent of the analysis method (FC or single-cell analysis), suggest that sustained, high levels of protein expression can be induced by CYBA UTR stabilized mRNA. The choice of UTR combination depends on the need of the experiment of application. Where persistent protein expression with reduced mRNA decay is desired, mRNA stabilized with a 3' UTR alone might serve the purpose. However, the combination of 5'+3' UTR results in additional desirable features of early onset, high peak and cumulative protein expression. It is demonstrated here that single-cell analysis of mRNA-induced protein expression is a means to characterize and improve pharmacokinetic properties of mRNA constructs. Using this approach, it is possible to systematically assess the intracellular bioavailability of different mRNA constructs to identify sequences yielding sustained protein expression. Prolonged persistence of protein expression was found for constructs stabilized by UTR insertions using a single-cell model and FC analysis in two cell types. This finding is desired in case of developing mRNA therapeutics. Messenger RNA constructs with persistent protein expression over a period of time (AUC) is desirable and allows proper reduced dosing into a patient with a final therapeutic outcome. XIV. Additional materials and methods for the experiments concerning GLP-1 and GDF15 Design of sequences ^ Amino acid sequences for GDF15 and GDF15H6D (e.g. as depicted or comprised in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 12 or in SEQ ID NO: 14 or SEQ ID NO: 15, respectively). ^ For GLP1, sequence published by Parsons (loc. cit.) for the construct “EX4GLP1Gly8“ was selected. ^ GDF15 UTRs: Reference Sequence in NCBI (NM_004864.2) 5’UTR: Position in the reference sequence: 1-32 3’UTR: Position in the reference sequence: 960-1200 ^ Glucagon UTRs: Reference Sequence in NCBI (NM_002054.4) 5’UTR: Position in the reference sequence: 1-256 3’UTR: Position in the reference sequence: 800-1294 ^ Nucleic acid sequences for GDF15 and GDF15H6D were derived from amino acid sequence via reverse translation (Expasy). ^ Obtained nucleic acid sequence was codon optimized for expression in humans. ^ Final sequence to be synthesized was assembled in the following order: 1. 5‘cloning site 2. T7 promoter 3. 5‘UTR: natural from the gene (GDF15 or Glucagon), CYBA, human alpha globin 4. Kozak element 5. SOI (sequence of interest): Codon optimized GDF15 or GDF15H6D 6. 3‘UTR: natural from the gene, CYBA 7. 3‘ cloning site ^ Number of constructs for each sequence = 4 ^ ORF ^ ORF + natural 5‘ and 3‘UTRs ^ ORF + 5‘ and 3‘ CYBA-UTRs (“eth”; “ETH”) ^ ORF + 5‘ hAg-UTR ^ For each target (GDF15, GDF15H6D and GLP1), 3 different mRNA constructs containing the codon optimized coding region (ORF) were designed. ^ Natural 5‘ and 3‘ UTRs from the cDNA (nat) + ORF ^ CYBA 5‘ and 3‘ UTRs (ETH) + ORF ^ 5‘ human alpha globin (hAg) UTR + ORF ^ For most active constructs in vitro, STOP control was designed by converting ATG to TGA Modified/(SNIM®) RNA production ^ The 14 RNAs (4 for each target, including 2 STOP RNAs) were produced using modified RNA/SNIM®RNA Technology (e.g. WO 2011/012316). ^ All the modified RNA/SNIM®RNAs passed the Quality control criteria of: ^ Size – agarose gel electrophoresis ^ Integrity – lack of smearing on gel ^ Purity – ratios of 260/280 and 230/280 ≥ 1.8 ^ Concentration of each modified RNA/SNIM®RNA was adjusted to 1 mg/mL in water for injection. ^ modified RNA/SNIM®RNAs were stored at -80 ° C till further use. modified RNA/SNIM®RNA tested in the present study:
Figure imgf000106_0001
GLP1 ELISA For GLP1 quantification Glucagon-Like Peptide-1 (Active) ELISA Kit (Millipore) was used. ELISA was performed as described in the manufactures manual. Samples were diluted 1:5 or 1:2. Measurement was performed on a Tecan infinite 200Pro device (Excitation Wavelength: 355 nm; Emission Wavelength: 460 nm; Excitation Bandwidth: 9 nm; Emission Bandwidth: 20 nm; Gain: 62 Calculated From: B2 (100%); Number of Flashes: 25; Integration Time: 20 µs) GDF15 ELISA For GDF15 quantification Human GDF-15 Quantikine ELISA Kit (R&D Systems) was used. GDF15 ELISA was performed as described in the manufactures manual. Samples were diluted 1:100. Measurement was performed on a Tecan infinite 200Pro device (Mode: Absorbance; Measurement Wavelength: 450 nm; Bandwidth: 9 nm; Reference Wavelength: 540 nm; Bandwidth: 9 nm; Number of Flashes: 10) Statistical analysis If not described otherwise, statistical analysis was performed using Prism 6 for Windows (Version 6.05). One-way ANOVA and Dunnett's multiple comparisons test was applied. Statistical significance is indicated by 95% confidence intervals. * = significantly different with a confidence interval of 95 % (alpha = 0.05). Animal experiments All animal experiments were performed in accordance with German Animal Welfare Law (Tierschutzgesetz). Female adult Balb/c mice were housed in individually ventilated cages (IVC) in groups of n = 2 animals per cage. Animals were fed with mouse maintenance food (sniff GmbH, Germany) and water ad libidum. Test items (modified RNA/SNIM®RNA coding for GDF-15 and GLP-1 (functional and non-functional) and 2% sucrose as a control) were injected 6 hours before dawn into the lateral tail vein in a total volume of 150 µL using Insulin injection syringes (Microfine+ 1 mL, 0.33 mm (29G) x 12.7 mm). Weight of animals and food per cage was measured directly before and 24 hours after application of test item. Delta of weight and food was calculated in order to determine food consumption and weight development. Animals were anesthetized 24 hours after application through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0,05/5,0/0,5 mg/kg BW). Blood was taken from retrobulbar venous plexus and was collected in EDTA tubes (Microvette®500K3E, Sarstedt, Germany) supplemented with Protease-Inhibitor- Cocktail (20 µl per 800 µl whole blood). Blood samples were furthermore centrifuged for 5 min at 4 °C with 2000 g. Supernatant (plasma) was taken, transferred into 1,5 mL Eppendorf Tubes® and subsequently snap frozen and stored at -80 °C until further analysis. Statistical significances between groups were calculated using Mann-Whitney’s U-Test. P-values < 0,05 were considered as statistically significant. XV. In vitro testing of modified RNA/SNIM®RNAs concerning GLP-1 and GDF15 Overview Purpose & Tasks: ^ Targets: GLP-1 GDF-15, GDF-15H6D ^ Confirmation of protein expression of modified RNA/SNIM®RNAs produced in vitro. Characterization of protein expression of modified RNA/SNIM®RNAs produced in appropriate cell culture models. ^ Individual tasks of this work package comprise: ^ 1) Selection of an appropriate cellular model (HEK293) for each of the target proteins and optimization of modified RNA/SNIM®RNA transfection efficiency using firefly luciferase reporter modified RNA/SNIM®RNA. ^ 2) Expression and quantification of secreted proteins in each of the cellular systems (supernatants and lysates) via ELISA ^ 3) Analysis of expression kinetics (dose-dependent expression time-course over one week) Transfection into HEK293 cells ^ The produced modified RNA/SNIM®RNAs were tested for their functionality in HEK293 cells and the resulting protein expression was quantified in cell culture supernatants via ELISA. ^ HEK293 cells were transfected in 96-well plates using Lipofectamine2000 (Reagent to modified RNA/SNIM®RNA ratio 2 µL / 1 µg modified RNA/SNIM®RNA). Transfection example: 2x104 HEK293 cells were seeded / well in 96-well plates. At 24 h after seeding, cells were transfected using Lipofectamine2000 (Lipofectamine2000/mRNA ratio 2/1). mRNA was diluted in dH2O. Lipofectamine2000 was diluted in medium w/o serum and P/S and mixed by pipetting. mRNA solution was added to each Lipofectamine2000 solution and incubated for 5 min at RT. Medium w/o serum and P/S was provided for each formulation. Old media was removed from seeded cells and 80 µL fresh growth media was applied.20 µL of prepared lipoplex solution was transferred to the cell containing wells. To untransfected cells, 20 µL of media w/o serum and P/S was added. ^ For each target, the different constructs were compared with respect to: ^ Dose response ^ Kinetics of protein expression – 50 µL used for each measurement. Equal volume of fresh media added for each time point ^ ELISA Kits used in the present study: ^ EGLP-35K Glucagon-Like Peptide-1 (Active) ELISA Kit: Merck Millipore ^ Human GDF-15 Quantikine ELISA Kit: R&D Systems Dose response - GLP1 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.24 h after transfection supernatants were taken for analysis. Values of PBS treated control and modified eGFP-RNA/eGFP-SNIM®RNA (500 ng/well) treated samples were below the lower limit of quantification. hAg resulted in highest GLP1 expression for all doses compared (see Fig.13). Kinetics of protein expression - GLP1 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.50 µL of supernatant were taken for analysis at time points 24, 48 and 72 h. Mean +/- SEM of 3 experimental replicates (3 separately transfected wells) is shown. GLP1“hAg“ resulted in highest GLP1 expression at all the time points compared (see Fig.14). Lead candidate vs not translated (STOP) control RNA HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.24 h after transfection supernatant were taken for analysis. Mean +/- SEM of 3 experimental replicates (3 separately transfected wells) is shown. No translation of GLP1-STOP“hAg“ (see Fig.15). Dose response - GDF15 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected. Values of PBS treated control and eGFP-modified RNA/SNIM®RNA (500 ng/well) treated samples were below the lower limit of quantification. A rel. late time point (72h) was chosen to analyze potential differences between the stabilized (H6D) and not stabilized version of GDF-15 No significant differences at time point 72 h (see Fig.16). Kinetics of protein expression - GDF15 HEK2936 cells were seeded in 96-well plates.24 h after seeding cells were transfected.50 µL of supernatant were taken for analysis at time points 24, 48 and 72 h. Mean +/- SEM of 3 experimental replicates (3 separately transfected wells) is shown. At 24 h and 48 h, higher values for H6D mutants were observed compared to their corresponding wild type GDF-15 sequences, no significant differences at time point 72 h (see Fig.17). Summary for in vitro testings ^ GLP1“hAg“ with human-alpha-globin as a 5‘UTR could be identified as a lead candidate with respect to expression of GLP1. ^ For GDF15, H6D mutant sequences lead to higher GDF15 expression. ^ GDF15H6D“eth“/CYBA appears to be the lead candidate among the modified GDF15- RNA/GDF15-SNIM®RNAs compared in the present study. XVI. In vivo testing of modified RNA/SNIM®RNAs concerning GLP-1 and GDF15 Primary read-out of in vivo study: Reduction of food intake within 24 hours. Schematic overview:
Figure imgf000110_0002
Figure imgf000110_0001
Efficacy of modified RNA/SNIM®RNA coding for GDF-15 and GLP-1 was tested against two control groups (see below for a schematic overview).
Figure imgf000110_0003
Figure imgf000110_0004
If not indicated otherwise, hGDF15 WT (with 5´- and 3´- CYBA-UTRs) and hGLP-1 (with 5´-hAg- UTR) were used in the in vivo testings. modified RNA/SNIM®RNA containing WT sequence of GDF-15 (with 5´- and 3´- CYBA UTRs) resulted in highest plasma protein levels in vivo after 6 hours. Coding and non-coding modified GDF-15-RNA/GDF-15-SNIM®RNA was complexed to a lipid carrier (such as C12-(2-3-2)) and was injected intravenously into the tail vein of female adult Balb/c mice (n = 1). Blood was taken using BD™ P800 blood collecting tubes after 6 hours. Content of GDF-15 was determined using ELISA (R&D Systems). Data are shown in Fig.18. modified RNA/SNIM®RNAGDF-15 WT reduces food consumption and facilitates weight loss at already low doses. Data are shown in Fig.19. 1 mg/kg BW modified RNA/SNIM®RNAGDF-15 WT results in elevated GDF-15 plasma levels up to 24 hours post injection. Data are shown in Fig.20. modified RNA/SNIM®RNAGLP-1 (with 5´-hAg-UTR) reduces food consumption (36% vs Succrose, 19% vs STOP) and body weight. Data are shown in Fig.21. GLP-1 plasma levels are elevated against sucrose control, but not against STOP control. Data are shown in Fig.22. Both, GDF-15 and GLP-1 coding modified RNA/SNIM®RNA facilitate statistically significant reduction of food consumption. Dosis: 1 mg/kg BW, Reduction of food consumption within 24 hours Data are shown in Fig.23. Conclusions concerning the data underlying Figs.18 to 23: ^ GDF-15 shows physiological activity (reduction of food intake and weight loss) at a dosage of 0.5 mg/kg BW ^ GDF-15 and GLP-1 are superior against corresponding non-translated control modified RNA/SNIM®RNAs at a dosage of 1 mg/kg BW ^ GDF-15 levels are significantly elevated up to 24 hours post injection Normalizing food consumption to 100 g/mouse does not result in significant changes of results. Data are shown in Fig.24. Normalizing food consumption to 100 g/mouse does not result in significant changes of results. Data are shown in Fig.25. Application of 2% Sucrose solution does not alter food intake of test animals Data are shown in Fig.26. Inflammatory cytokines are within physiological ranges 24 hours after application of Test items. Data are shown in Fig.27. Application GLP-1 coding modified RNA/SNIM®RNA results in significantly increased IGF- 1 levels after 24 hours. Data are shown in Fig.28.
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The present invention refers the following nucleotide and amino acid sequences: SEQ ID NO: 1: Nucleotide sequence of the used 5’cyba-UTR (RNA; on DNA level, “U” will be “T”; Kozak sequence is included (underlined); additional 5´ C is included (underlined)) CCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCCGCCACC SEQ ID NO: 2: Nucleotide sequence of the used 3’cyba-UTR (RNA; on DNA level, “U” will be “T”) CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGAAGCCGGGA SEQ ID NO: 3: Nucleotide sequence of the used 5’hAg-UTR (RNA; on DNA level, “U” will be “T”; additional G is included (bold)) CUCUUCUGGUCCCCACAGACUCAGAGAGAACGCCACC SEQ ID NO: 4: Nucleotide sequence of EX4GLP1Gly8 (Parsons, Gene Therapy 14, 2007, 38-48) ATGAAGATCATCCTGTGGCTGTGTGTGTTCGGCCTGTTCCTGGCCACCCTGTTCCCCATCAGCTGGC AGATGCCCGTGGAGTCCGGCCTGTCCTCCGAGGACTCCGCCAGCTCCGAGAGCTTCGCCAAGCGC ATCAAGCGCCACGGCGAGGGCACCTTCACCAGCGACGTGAGCAGCTACCTGGAGGGCCAGGCCG CCAAGGAGTTCATCGCCTGGCTGGTGAAGGGCCGCGGCTGA SEQ ID NO: 5: Nucleotide sequence of EX4GLP1Gly8 (CO)-Hu (codon optimized sequence for humans (Hu)) ATGAAGATCATCCTGTGGCTGTGCGTGTTCGGCCTGTTCCTGGCCACCCTGTTCCCCATCAGCTGG CAGATGCCTGTGGAAAGCGGCCTGAGCAGCGAGGATAGCGCCAGCAGCGAGAGCTTCGCCAAGCG GATCAAGAGACACGGCGAGGGCACCTTCACCAGCGACGTGTCCAGCTACCTGGAAGGCCAGGCCG CCAAAGAGTTTATCGCCTGGCTCGTGAAGGGCAGAGGCTGA SEQ ID NO: 6: Nucleotide sequence of EX4GLP1Gly8 (CO)-Mu (codon optimized sequence for mouse (Mu)) ATGAAGATCATCCTGTGGCTGTGCGTGTTCGGCCTGTTCCTGGCCACCCTGTTCCCCATCAGCTGG CAGATGCCTGTGGAAAGCGGCCTGAGCAGCGAGGACAGCGCCAGCTCTGAGAGCTTCGCCAAGAG AATCAAGAGACACGGCGAGGGCACCTTCACCAGCGACGTGTCCTCTTACCTGGAAGGCCAGGCCG CCAAAGAGTTTATCGCCTGGCTCGTGAAGGGCAGAGGCTGA SEQ ID NO: 7: Amino acid sequence of human GLP1 (>sp|P01275|GLUC_HUMAN Glucagon OS=Homo sapiens GN=GCG PE=1 SV=3) MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDS RRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEV AIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK SEQ ID NO: 8: Amino acid sequence of mouse GLP1 (>sp|P55095|GLUC_MOUSE Glucagon OS=Mus musculus GN=Gcg PE=1 SV=1) MKTIYFVAGLLIMLVQGSWQHALQDTEENPRSFPASQTEAHEDPDEMNEDKRHSQGTFTSDYSKYLDSR RAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAI AEELGRRHADGSFSDEMSTILDNLATRDFINWLIQTKITDKK SEQ ID NO: 9: Nucleotide sequence of GDF15 WT (deduced mRNA sequence of amino acid sequence of GDF15 WT from reverse translate function of Expasy and human codon usage (standard format)) atgtacagaatgcagctgctgagctgcatcgccctgctgagcctggccctggtgaccaacagcgcccaccaccaccaccaccacgccagaaac ggcgaccactgccccctgggccccggcagatgctgcagactgcacaccgtgagagccagcctggaggacctgggctgggccgactgggtgctg agccccagagaggtgcaggtgaccatgtgcatcggcgcctgccccagccagttcagagccgccaacatgcacgcccagatcaagaccagcctg cacagactgaagcccgacaccgtgcccgccccctgctgcgtgcccgccagctacaaccccatggtgctgatccagaagaccgacaccggcgtg agcctgcagacctacgacgacctgctggccaaggactgccactgcatc SEQ ID NO: 10: Nucleotide sequence of GDF15 WT (CO)-Hu (codon optimized sequence for humans (Hu)) ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACCAATTCTGCC CACCACCACCATCACCACGCCCGGAATGGCGATCACTGTCCTCTGGGCCCTGGCCGGTGTTGCAGA CTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGAGA AGTGCAAGTGACCATGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCCAATATGCACGCCCA GATCAAGACCAGCCTGCACCGGCTGAAGCCCGATACAGTGCCTGCCCCTTGTTGCGTGCCCGCCA GCTACAACCCCATGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGACC TGCTGGCCAAGGACTGCCACTGCATC SEQ ID NO: 11: Nucleotide sequence of GDF15 WT (CO)-Mu (codon optimized sequence for mouse (Mu)) ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACAAACTCTGCC CACCACCACCATCACCACGCCAGAAACGGCGACCACTGTCCTCTGGGCCCTGGCAGATGCTGCAGA CTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGAGA AGTGCAAGTGACCATGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCTAATATGCACGCCCA GATCAAGACCAGCCTGCACAGACTGAAGCCCGACACCGTGCCTGCCCCTTGTTGTGTGCCTGCCAG CTACAACCCCATGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGACCT GCTGGCCAAGGACTGCCACTGCATC SEQ ID NO: 12: Amino acid sequence of GDF15 WT MYRMQLLSCIALLSLALVTNSAHHHHHHARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQ VTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCH CI bold: signal sequence, italic: his-tag, black: GDF15 Aa: A197-I308 SEQ ID NO: 13: Amino acid sequence of mouse GDF15 WT (>sp|Q9Z0J7|GDF15_MOUSE Growth/differentiation factor 15 OS=Mus musculus GN=Gdf15 PE=2 SV=2) MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLH ANQSREDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITR PLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHL ETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTP VVLMHRTDSGVSLQTYDDLVARGCHCA SEQ ID NO: 14: Nucleotide sequence of GDF15 H6D (deduced mRNA sequence of amino acid sequence of GDF15 H6D from reverse translate function of Expasy and human codon usage (standard format)) atgtacagaatgcagctgctgagctgcatcgccctgctgagcctggccctggtgaccaacagcgcccaccaccaccaccaccacgccagaaac ggcgacgactgccccctgggccccggcagatgctgcagactgcacaccgtgagagccagcctggaggacctgggctgggccgactgggtgctg agccccagagaggtgcaggtgaccatgtgcatcggcgcctgccccagccagttcagagccgccaacatgcacgcccagatcaagaccagcctg cacagactgaagcccgacaccgtgcccgccccctgctgcgtgcccgccagctacaaccccatggtgctgatccagaagaccgacaccggcgtg agcctgcagacctacgacgacctgctggccaaggactgccactgcatc SEQ ID NO: 15: Nucleotide sequence of GDF15 H6D (CO)-Hu (Codon optimized sequence for humans (Hu)) ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACCAATTCTGCC CACCACCACCATCACCACGCCCGGAATGGCGACGATTGTCCTCTGGGCCCTGGCCGGTGTTGCAG ACTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGAG AAGTGCAAGTGACCATGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCCAATATGCACGCCC AGATCAAGACCAGCCTGCACCGGCTGAAGCCCGATACAGTGCCTGCCCCTTGTTGCGTGCCCGCCA GCTACAACCCCATGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGACC TGCTGGCCAAGGACTGCCACTGCATC SEQ ID NO: 16: Nucleotide sequence of GDF15 H6D (CO)-Mu (codon optimized sequence for mouse (Mu)) ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACAAACTCTGCC CACCACCACCATCACCACGCCAGAAACGGCGACGACTGTCCTCTGGGCCCTGGCAGATGCTGCAG ACTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGAG AAGTGCAAGTGACCATGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCTAATATGCACGCCC AGATCAAGACCAGCCTGCACAGACTGAAGCCCGACACCGTGCCTGCCCCTTGTTGTGTGCCTGCCA GCTACAACCCCATGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGACC TGCTGGCCAAGGACTGCCACTGCATC SEQ ID NO: 17: Amino acid sequence of GDF15 H6D MYRMQLLSCIALLSLALVTNSAHHHHHHARNGDDCPLGPGRCCRLHTVRASLEDLGWADWVLSPREV QVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC HCI bold: signal sequence, italic: his-tag, black: GDF15 Aa: A197-I308 SEQ ID NO: 18: Nucleotide sequence of T7-5’hAg-EX4GLP1Gly8(CO)-Hu TAATACGACTCACTATAGGGAGACTCTTCTGGTCCCCACAGACTCAGAGAGAACGCCACCATGAAG ATCATCCTGTGGCTGTGCGTGTTCGGCCTGTTCCTGGCCACCCTGTTCCCCATCAGCTGGCAGATG CCTGTGGAAAGCGGCCTGAGCAGCGAGGATAGCGCCAGCAGCGAGAGCTTCGCCAAGCGGATCAA GAGACACGGCGAGGGCACCTTCACCAGCGACGTGTCCAGCTACCTGGAAGGCCAGGCCGCCAAAG AGTTTATCGCCTGGCTCGTGAAGGGCAGAGGCTGAGAATTC (bold: T7 promoter; italic: 5’hAg-UTR; underlined, italic & bold: Kozak element; italic/subscript: cloning site (BamHI)) SEQ ID NO: 19: Nucleotide sequence of T7-5’cyba-GDF15WT(CO)-Hu-3’cyba TAATACGACTCACTATAGGGAGACCGCGCCTAGCAGTGTCCCAGCCGGGTTCGTGTCGCCGCCAC CATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACCAATTCTGC CCACCACCACCATCACCACGCCCGGAATGGCGATCACTGTCCTCTGGGCCCTGGCCGGTGTTGCA GACTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGA GAAGTGCAAGTGACCATGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCCAATATGCACGCC CAGATCAAGACCAGCCTGCACCGGCTGAAGCCCGATACAGTGCCTGCCCCTTGTTGCGTGCCCGCC AGCTACAACCCCATGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGAC CTGCTGGCCAAGGACTGCCACTGCATCCCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCAC CTGCAATAAATGCAGCGAAGCCGGGAGAATTC (bold: T7 promoter; italic: 5’cyba-UTR; underlined, italic & bold: Kozak element; italic: 3’cyba- UTR; italic/subscript: cloning site (BamHI)) SEQ ID NO: 20: Nucleotide sequence
Figure imgf000118_0001
TCATCCTGTGGCTGTGCGTGTTCGGCCTGTTCCTGGCCACCCTGTTCCCCATCAGCTGGCAGTAGCC TGTGGAAAGCGGCCTGAGCAGCGAGGATAGCGCCAGCAGCGAGAGCTTCGCCAAGCGGATCAAGA GACACGGCGAGGGCACCTTCACCAGCGACGTGTCCAGCTACCTGGAAGGCCAGGCCGCCAAAGAG TTTATCGCCTGGCTCGTGAAGGGCAGAGGCTGAGAATTC (bold: T7 promoter; green/italic: 5’hAg-UTR; underlined, italic & bold: Kozak element; italic/subscript: cloning site (BamHI); italic/subscript: “STOP”) SEQ ID NO: 21: Nucleotide sequence of T7-5’cyba-GDF15WT(CO)-3’cyba-Stop TAATACGACTCACTATAGGGAGACCGCGCCTAGCAGTGTCCCAGCCGGGTTCGTGTCGCCGCCAC CTAGTACCGGTAGCAGCTGCTGAGCTGTATCGCCCTGCTGAGCCTGGCCCTCGTGACCAATTCTGCC CACCACCACCATCACCACGCCCGGAATGGCGATCACTGTCCTCTGGGCCCTGGCCGGTGTTGCAGA CTGCATACAGTGCGGGCCAGCCTGGAAGATCTGGGCTGGGCTGATTGGGTGCTGAGCCCCAGAGA AGTGCAAGTGACCTAGTGCATCGGCGCCTGCCCCAGCCAGTTCAGAGCCGCCAATATGCACGCCCA GATCAAGACCAGCCTGCACCGGCTGAAGCCCGATACAGTGCCTGCCCCTTGTTGCGTGCCCGCCA GCTACAACCCCTAGGTGCTGATCCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGACCT GCTGGCCAAGGACTGCCACTGCATCTGACCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCA CCTGCAATAAATGCAGCGAAGCCGGGAGAATTC (bold: T7 promoter; italic: 5’cyba-UTR; underlined, italic & bold: Kozak element; italic: 3’cyba- UTR; italic/subscript: cloning site; italic/subscript: “STOP”) SEQ ID NO: 22: Nucleotide sequence of T7 promoter; transcription starts at the underlined “G” TAATACGACTCACTATAGGGAGA SEQ ID NO: 23: Nucleotide sequence of used (partial) Kozak element (RNA; on DNA level, “U” will be “T”; “R” indicates a purine, preferably A or G, more preferably A) GCCRCC SEQ ID NO: 24: Nucleotide consensus sequence of vertebrate Kozak element (RNA; on DNA level, “U” will be “T”; “R” indicates any purine); the start codon is underlined GCCRCCAUG SEQ ID NO: 25: Genetic code of the human CYBA gene 5’-UTR GGCGGGGTTCGGCCGGGAGCGCAGGGGCGGCAGTGCGCGCCTAGCAGTGTCCCAGCCGGGTTCGTGTCGCC SEQ ID NO: 26: Genetic code of the human CYBA gene 3’-UTR CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTGCAATAAATGCAGCGAAGCCGGGA (underlined: insulin 3’UTR stability element (INS_SCE) bold: polyadenylation signal (PAS)) SEQ ID NO: 27: Genetic code of the hAg 5’-UTR without the 3’ partial Kozak element CTCTTCTGGTCCCCACAGACTCAGAGAGAAC SEQ ID NO: 28: Genetic code of the WT hAg 5’-UTR including the 3’ partial native Kozak element (bold & underlined) CTCTTCTGGTCCCCACAGACTCAGAGAGAACCCACC SEQ ID NO: 29: Nucleotide sequence encoding full length hCYBA >NM_000101.3 Homo sapiens cytochrome b-245 alpha chain (CYBA), mRNA GGCGGGGTTCGGCCGGGAGCGCAGGGGCGGCAGTGCGCGCCTAGCAGTGTCCCAGCCGGGTTCGTGTCGCCATGGGG CAGATCGAGTGGGCCATGTGGGCCAACGAGCAGGCGCTGGCGTCCGGCCTGATCCTCATCACCGGGGGCATCGTGGC CACAGCTGGGCGCTTCACCCAGTGGTACTTTGGTGCCTACTCCATTGTGGCGGGCGTGTTTGTGTGCCTGCTGGAGT ACCCCCGGGGGAAGAGGAAGAAGGGCTCCACCATGGAGCGCTGGGGACAGAAGTACATGACCGCCGTGGTGAAGCTG TTCGGGCCCTTTACCAGGAATTACTATGTTCGGGCCGTCCTGCATCTCCTGCTCTCGGTGCCCGCCGGCTTCCTGCT GGCCACCATCCTTGGGACCGCCTGCCTGGCCATTGCGAGCGGCATCTACCTACTGGCGGCTGTGCGTGGCGAGCAGT GGACGCCCATCGAGCCCAAGCCCCGGGAGCGGCCGCAGATCGGAGGCACCATCAAGCAGCCGCCCAGCAACCCCCCG CCGCGGCCCCCGGCCGAGGCCCGCAAGAAGCCCAGCGAGGAGGAGGCTGCGGTGGCGGCGGGGGGACCCCCGGGAGG TCCCCAGGTCAACCCCATCCCGGTGACCGACGAGGTCGTGTGACCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCAC CCACCTGCAATAAATGCAGCGAAGCCGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA SEQ ID NO:30: Nucleotide sequence encoding full length hAg 622 bp mRNA linear Homo sapiens hemoglobin subunit alpha 2 (HBA2), mRNA. ACCESSION NM_000517, VERSION NM_000517.4 1 cataaaccct ggcgcgctcg cgggccggca ctcttctggt ccccacagac tcagagagaa 61 cccaccatgg tgctgtctcc tgccgacaag accaacgtca aggccgcctg gggtaaggtc 121 ggcgcgcacg ctggcgagta tggtgcggag gccctggaga ggatgttcct gtccttcccc 181 accaccaaga cctacttccc gcacttcgac ctgagccacg gctctgccca ggttaagggc 241 cacggcaaga aggtggccga cgcgctgacc aacgccgtgg cgcacgtgga cgacatgccc 301 aacgcgctgt ccgccctgag cgacctgcac gcgcacaagc ttcgggtgga cccggtcaac 361 ttcaagctcc taagccactg cctgctggtg accctggccg cccacctccc cgccgagttc 421 acccctgcgg tgcacgcctc cctggacaag ttcctggctt ctgtgagcac cgtgctgacc 481 tccaaatacc gttaagctgg agcctcggta gccgttcctc ctgcccgctg ggcctcccaa 541 cgggccctcc tcccctcctt gcaccggccc ttcctggtct ttgaataaag tctgagtggg 601 cagcaaaaaa aaaaaaaaaa aa SEQ ID NO:31: Nucleotide sequence of extended 5’cyba-UTR (RNA; on DNA level, “U” will be “T”; Kozak element is lacking; additional 5´ C is included) GGAGCGCAGGGGCGGCAGUGCCGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC SEQ ID NO:32: Genetic code of the WT hAg 5’-UTR including the 3’ (partial) native Kozak element but having a “G” inserted at position 32 to obtain an optimal vertebrate Kozak element (bold & underlined) CTCTTCTGGTCCCCACAGACTCAGAGAGAACGCCACC SEQ ID NO:33: Nucleotide sequence of 3’ cloning site (BamHI restriction site) GAATTC SEQ ID NO:34: Nucleotide sequence of d2EGFP primer (forward primer) 5’-CAA CCA CTA CCT GAG CAC CC-3’ SEQ ID NO:35: Nucleotide sequence of d2EGFP primer (reverse primer) 5’-GTC CAT GCC GAG AGT GAT CC-3’ SEQ ID NO: 52 RNA sequence coding for the human GLP1 of SEQ ID NO: 7, codon optimized (hCO mRNA derived from SEQ ID NO: 7): ATGAAGTCCATCTACTTCGTGGCCGGCCTGTTCGTGATGCTGGTGCAAGGATCTTGGCAGCGGAGCCTGC AGGACACCGAGGAAAAGAGCAGAAGCTTCAGCGCCAGCCAGGCCGATCCTCTGTCTGACCCCGATCAGAT GAACGAGGACAAGAGACACAGCCAGGGCACCTTCACCAGCGACTACAGCAAGTACCTGGACTCTCGCAGA GCCCAGGACTTCGTGCAGTGGCTGATGAACACCAAGCGGAACCGGAACAATATCGCCAAGCGGCACGACG AGTTCGAGAGACATGCCGAGGGAACCTTTACCTCCGACGTGTCCAGCTACCTGGAAGGCCAGGCCGCCAA AGAGTTTATCGCCTGGCTCGTGAAAGGCAGAGGCAGAAGAGACTTCCCCGAAGAGGTGGCCATCGTGGAA GAACTCGGAAGAAGGCACGCCGACGGCAGCTTTAGCGACGAGATGAATACCATCCTGGACAACCTGGCCG CCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACCGGAAG SEQ ID NO: 53: RNA sequence coding for mouse GLP1 of SEQ ID NO: 8, human codon optimized (hCO mRNA derived from SEQ ID NO: 8): ATGAAGACCATCTACTTCGTGGCCGGCCTGCTGATCATGCTGGTGCAAGGATCTTGGCAGCACGCCCTGC AGGACACCGAGGAAAACCCTAGAAGCTTCCCCGCCTCTCAGACAGAGGCCCACGAAGATCCCGACGAGAT GAACGAGGACAAGAGACACAGCCAGGGCACCTTCACCAGCGACTACAGCAAGTACCTGGACTCTCGCAGA GCCCAGGACTTCGTGCAGTGGCTGATGAACACCAAGCGGAACCGGAACAATATCGCCAAGCGGCACGACG AGTTCGAGAGACATGCCGAGGGAACCTTTACCTCCGACGTGTCCAGCTACCTGGAAGGCCAGGCCGCCAA AGAGTTTATCGCCTGGCTGGTCAAAGGCAGAGGCAGAAGAGACTTCCCCGAGGAAGTGGCCATTGCCGAG GAACTCGGAAGAAGGCATGCCGACGGCAGCTTCTCCGATGAGATGAGCACCATCCTGGACAACCTGGCCA CCAGAGACTTCATCAACTGGCTGATCCAGACCAAGATCACCGACAAGAAG SEQ ID NO: 54: RNA sequence coding for mouse GDF15 WT derived from SEQ ID NO: 13, human codon optimized (hCO mRNA derived from SEQ ID NO: 13): ATGGCTCCTCCTGCTCTGCAAGCTCAACCTCCTGGTGGAAGCCAGCTGCGGTTCCTGCTGTTTCTGCTGC TGCTCCTGCTTCTGCTGAGCTGGCCTTCTCAAGGCGACGCTCTGGCTATGCCTGAGCAGAGGCCTTCTGG ACCTGAGAGCCAGCTGAATGCCGATGAGCTGAGAGGCAGATTCCAGGACCTGCTGTCTAGACTGCACGCC AACCAGTCCAGAGAGGACAGCAACAGCGAGCCCTCTCCTGATCCTGCTGTGCGGATCCTGTCTCCTGAAG TGCGGCTGGGATCTCACGGACAGCTGCTGCTGAGAGTGAACAGAGCCAGCCTGTCTCAGGGACTGCCTGA GGCCTATAGAGTGCATAGAGCCCTGTTGCTGCTGACCCCTACAGCCAGACCTTGGGACATCACCCGGCCT CTGAAGAGAGCACTGTCTCTGAGAGGCCCTAGAGCACCCGCTCTGAGACTGAGACTTACCCCTCCACCTG ACCTGGCCATGCTTCCTAGCGGAGGAACACAGCTGGAACTGCGCCTGAGAGTGGCTGCTGGCAGAGGCAG AAGATCTGCCCACGCTCACCCTAGAGATAGCTGTCCACTCGGCCCTGGCAGATGCTGTCACCTGGAAACA GTGCAGGCCACACTGGAAGATCTCGGCTGGAGTGACTGGGTGCTGAGCCCTAGACAGCTCCAGCTCTCTA TGTGCGTGGGCGAGTGCCCTCACCTGTACAGATCTGCCAATACTCACGCCCAGATCAAGGCCAGGCTGCA CGGACTGCAGCCTGATAAGGTTCCCGCTCCTTGCTGTGTGCCCAGCAGCTATACACCCGTGGTGCTGATG CACAGAACCGACAGCGGAGTGTCCCTGCAGACCTACGATGATCTGGTGGCCAGAGGCTGTCACTGTGCT SEQ ID NO: 55: RNA sequence coding for mouse GDF15 WT, derived from SEQ ID NO: 13, human codon optimized: ATGGCTCCTCCTGCTCTGCAAGCTCAACCTCCTGGTGGAAGCCAGCTGAGATTCCTGCTGTTTC TGCTGCTGCTCCTGCTTCTGCTGAGCTGGCCTTCTCAAGGCGACGCTCTGGCTATGCCTGAGCA GAGGCCTTCTGGACCTGAGTCTCAGCTGAACGCCGATGAGCTGAGAGGCAGATTCCAGGACCTG CTGTCTAGACTGCACGCCAACCAGTCCAGAGAGGACAGCAACAGCGAGCCCTCTCCTGATCCTG CTGTGCGGATCCTGTCTCCTGAAGTGCGGCTGGGATCTCACGGACAGCTGCTGCTGAGAGTGAA CAGAGCCAGCCTGTCTCAGGGACTGCCTGAGGCTTACAGAGTGCACAGGGCTCTGTTGCTGCTG ACCCCTACAGCCAGACCTTGGGACATCACCAGACCTCTGAAGAGAGCCCTGAGCCTGAGAGGAC CTAGAGCACCAGCTCTGAGACTGAGGCTGACACCTCCACCTGATCTGGCCATGCTTCCTAGCGG AGGCACACAGCTGGAACTGCGACTGAGAGTGGCTGCTGGCAGAGGCAGAAGATCTGCTCACGCT CACCCTAGAGACAGCTGCCCTCTTGGCCCTGGCAGATGCTGTCACCTGGAAACAGTGCAGGCCA CACTGGAAGATCTCGGCTGGAGTGACTGGGTGCTGAGCCCTAGACAGCTCCAGCTCTCTATGTG CGTGGGCGAGTGTCCCCACCTGTACAGATCTGCCAACACACACGCCCAGATCAAGGCCAGACTG CATGGCCTGCAGCCTGACAAAGTGCCTGCTCCTTGTTGCGTGCCCAGCAGCTATACACCCGTGG TGCTGATGCACAGAACCGACAGCGGAGTGTCCCTGCAGACATACGATGACCTGGTGGCCAGAGG CTGTCACTGTGCT SEQ ID NO: 56 RNA sequence coding for GDF15 H6D, derived from SEQ ID NO: 17 ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGCTGTCTCTGGCCCTGGTCACAAATTCTGCCCACC ACCATCACCACCACGCCAGAAACGGGGATGATTGTCCTCTCGGCCCTGGCAGATGCTGCAGACTGCATAC AGTTAGAGCCAGCCTGGAAGATCTCGGCTGGGCTGATTGGGTGCTGAGCCCCAGAGAAGTGCAAGTGACC ATGTGCATCGGCGCCTGTCCTAGCCAGTTCAGAGCCGCCAATATGCACGCCCAGATCAAGACCAGCCTGC ACAGACTGAAGCCCGACACAGTGCCTGCTCCATGTTGTGTGCCCGCCAGCTACAACCCCATGGTGCTGAT CCAGAAAACCGACACCGGCGTGTCCCTGCAGACCTACGATGATCTGCTGGCCAAGGACTGCCACTGCATC SEQ ID NO: 61 Protein sequence coding for human WT GDF15 >sp|Q99988|GDF15_HUMAN Growth/differentiation factor 15 OS=Homo sapiens OX=9606 GN=GDF15 PE=1 SV=3 MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLL TRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLS PTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGR RRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQ IKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

Claims

New PCT Patent Application Based on EP 22211336.7 Ethris GmbH Our Ref.: AF3725 PCT S3 CLAIMS 1. An RNA molecule comprising (a) a coding region coding for at least one of the regulators of energy homeostasis GLP- 1 and GDF-15, or an analogue thereof; and (b) upstream of said coding region one (or more) UTR(s) comprising (A) a 5’-UTR of cytochrome b-245 alpha polypeptide (CYBA), or a functional derivative of said 5’-UTR; or (B) a 5’-UTR of alpha globin (Ag), or a functional derivative of said 5’-UTR; and/or (c) downstream of said coding region one (or more) UTR(s) comprising a 3’-UTR of CYBA, or a functional derivative of said 3’-UTR.
2. The RNA molecule according to claim 1, wherein said UTR(s) as defined in claim 1(b) is/are located at the 5' end of the coding region as defined in claim 1(a).
3. The RNA molecule according to claim 1, wherein said UTR(s) as defined in claim 1(c) is/are located at the 3' end of the coding region as defined in claim 1(a).
4. The RNA molecule according to any one of claims 1 to 3, wherein said UTR(s) as defined in claim 1(b), in particular as defined in claim 1(b)(A), is/are located at the 5' end of the coding region as defined in claim 1(a) and wherein said UTR(s) as defined in claim 1(c) is/are located at the 3' end of the coding region as defined in claim 1(a).
5. The RNA molecule according to any one of claims 1 to 4, wherein said CYBA is human CYBA and/or said Ag is human Ag (hAg).
6. The RNA molecule according to any one of claims 1 to 5, wherein (i) said 5’-UTR of claim 1(b)(A) is or comprises the sequence as shown in SEQ ID NO:1 or SEQ ID NO:40; or (ii) said 5’-UTR of claim 1(b)(B) is or comprises the sequence as shown in SEQ ID NO:3; and/or (iii) said 3’-UTR of claim 1(c) is or comprises the sequence as shown in SEQ ID NO:2.
7. The RNA molecule according to any one of claims 1 to 5, wherein (i) said functional derivative of said 5’-UTR of claim 1(b)(A) is or comprises a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 or SEQ ID NO:40 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1 or SEQ ID NO:40, respectively; or (ii) said functional derivative of said 5’-UTR of claim 1(b)(B) is or comprises a sequence which shows 1 to 6 substitutions in comparison to SEQ ID NO:3 and which results in an RNA molecule having the same or a higher translation efficiency an RNA molecule comprising an UTR comprising SEQ ID NO:3; and/or (iii) said functional derivative of said 3’-UTR of claim 1(c) is or comprises a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2.
8. The RNA molecule according to any one of claims 1 to 7, wherein the RNA molecule comprises a poly-A tail at the 3' end.
9. The RNA molecule according to claim 8, wherein the poly-A tail has a length of at least 120 nucleotides.
10. The RNA molecule according to any one of claims 1 to 9, wherein said GLP-1 is human GLP-1 (hGLP-1).
11. The RNA molecule according to any one of claims 1 to 10, wherein said GLP-1, or analogue thereof, is encoded by SEQ ID NO:4, 5 or 6 or by SEQ ID NO: 52 or 53, or has the amino acid sequence as depicted in SEQ ID NO:7 or 8 or the amino acid sequence as depicted in SEQ ID NO:57, 58, 59, or 60.
12. The RNA molecule according to any one of claims 1 to 11, wherein said GDF-15 is human GDF-15 (hGDF-15).
13. The RNA molecule according to any one of claims 1 to 12, wherein said GDF-15 is wild- type (h)GDF-15 ((h)GDF-15 WT).
14. The RNA molecule according to any one of claims 1 to 13, wherein said GDF-15, or analogue thereof, is or is encoded by SEQ ID NOs:9, 10 or 11 or SEQ ID NO: 54, 55 or 56, or has the amino acid sequence as depicted in SEQ ID NO:12, 13 or 61.
15. The RNA molecule according to any one of claims 1 to 12, wherein said GDF-15, or analogue thereof, is the H6D mutant of (h)GDF-15 ((h)GDF-15 H6D).
16. The RNA molecule according to any one of claims 1 to 12 and 15, wherein said GDF-15, or analogue thereof, is encoded by SEQ ID NO:14, 15 or 16 or has the amino acid sequence as depicted in SEQ ID NO:17.
17. The RNA molecule according to any one of claims 1 to 16 which is selected from the group consisting of (i) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(A) and (c) and 2 to 7 and the coding region as defined in claim 13 or 14 (CYBA-GDF- 15 WT); (ii) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(B) and 2 to 7 and the coding region as defined in claim 10 or 11 (hAg-GLP-1); (iii) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(B) and 2 to 7 and the coding region as defined in claim 13 or 14 (hAg-GDF-15 WT); (iv) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(A) and (c) and 2 to 7 and the coding region as defined in claim 15 or 16 (CYBA-GDF- 15 H6D); (v) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(B) and 2 to 7 and the coding region as defined in claim 15 or 16 (hAg-GDF-15 H6D); and (vi) an RNA molecule comprising the UTR(s) as defined in any one of claims 1(b)(A) and (c) and 2 to 7 and the coding region as defined in claim 10 or 11 (CYBA-GLP- 1).
18. The RNA molecule according to any one of claims 1 to 17, wherein said coding region is codon optimized (for example for expression in mice or, preferably, in humans).
19. The RNA molecule according to any one of claims 1 to 18 which is a modified RNA molecule (e.g. SNIM®RNA molecule, like those as described in WO 2011/012316 or WO 2018/127382), e.g. wherein (about) 25% (e.g. ± 2, 3, 4 or 5 %) of the cytidine nucleotides are modified cytidine nucleotides (e.g. (5-)methylcytidines) and (about) 25% (e.g. ± 2, 3, 4 or 5 %) of the uridine nucleotides are modified uridine nucleotides (e.g. (2-)thiouridines).
20. A set of 2 or more RNA molecules as defined in any one of claims 1 to 19, wherein the coding region of one RNA molecule of said set codes for (the regulator of energy homeostasis) GLP-1, or analogue thereof, as defined in any one of claims 1, 10 and 11; and wherein the coding region of another RNA molecule of said set codes for (the regulator of energy homeostasis) GDF-15, or analogue thereof, as defined in any one of claims 1 and 12 to 16.
21. The set of 2 or more RNA molecule(s) of claim 20 which comprises one or more RNA molecule(s) of claim 17 which comprise(s) a coding region for (the regulator of energy homeostasis) GLP-1, or analogue thereof, and one or more RNA molecule(s) of claim 17 which comprise(s) a coding region for (the regulator of energy homeostasis) GDF-15, or analogue thereof.
22. A nucleic acid molecule encoding the RNA molecule of any one of claims 1 to 19 or the set of RNA molecules of claim 20 or 21; or a set of 2 or more nucleic acid molecules encoding the 2 or more RNA molecules, respectively, of the set of 2 or more RNA molecules of claim 20 or 21.
23. A vector comprising the nucleic acid molecule of claim 22; or a set of 2 or more vectors comprising the 2 or more nucleic acid molecules, respectively, of the set of 2 or more nucleic acid molecules of claim 22.
24. A host cell comprising the vector or the set of two or more vectors of claim 23; or a set of 2 or more host cells comprising the 2 or more vectors, respectively, of the set of 2 or more vectors of claim 23.
25. A pharmaceutical composition comprising the RNA molecule or set of RNA molecules according to any one of claims 1 to 21, the nucleic acid molecule or the set of nucleic acid molecules according to claim 22, the vector or the set of vectors according to claim 23 or the host cell or the set of host cells according to claim 24, and optionally a pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25 for use in an RNA-based therapy.
27. The pharmaceutical composition according to claim 25 or 26 for use (i) as an anorectic; (ii) in body weight control, in particular in decreasing (aberrant) body weight; and/or (iii) in the treatment or prevention of a metabolic disorder.
28. Method for (i) decreasing food intake; (ii) restraining appetite; (iii) controlling body weight, in particular decreasing (aberrant) body weight; and/or (iv) treating or preventing a metabolic disorder, said method comprising the step of administering a pharmaceutically active amount of the pharmaceutical composition according to claim 25 or 26 to a patient in need thereof.
29. The pharmaceutical composition or the method according to claim 27 or 28, wherein said metabolic disorder is selected from the group consisting of (i) obesity, in particular abdominal obesity; (ii) diabetes mellitus, in particular type II diabetes mellitus; (iii) insulin resistance; and/or (iv) metabolic syndrome.
30. The pharmaceutical composition or the method according to claim 29, wherein said metabolic syndrome (is a set of symptoms which) includes obesity (in particular abdominal obesity), hypertension, cardiovascular disease, elevated fasting plasma glucose, dyslipidemia, and/or an enhanced inflammatory state.
31. A kit comprising the RNA molecule or the set of RNA molecules according to any one of claims 1 to 21, the nucleic acid molecule or the set of nucleic acid molecules according to claim 22, the vector or the set of vectors according to claim 23 or the host cell or the set of host cells according to claim 24.
32. Use of one or more UTR(s) as defined in any one of claims 1 to 7, in particular as defined in claim 1(b), more particular as defined in claim 1(b)(A), and/or one or more UTR(s) as defined in claim 1(c), for increasing the efficiency of translating a coding region of an RNA molecule into at least one of the regulators of energy homeostasis GLP-1 and GDF-15 as defined in any one of claims 1 and 10 to 16 encoded by said coding region.
33. The RNA molecule, the set of RNA molecules, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition, the method, the kit or the use according to any one of claims 1 to 32, being combined with one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or with one or more liposomal transfection reagent(s), or being combined with the use of one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or with one or more liposomal transfection reagent(s).
34. The pharmaceutical composition, the method, the kit or the use according to any one of claims 25 to 33, wherein said RNA molecule, set of RNA molecules, nucleic acid molecule, vector or host cell is (to be) administered together with one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or with one or more liposomal transfection reagent(s).
35. The pharmaceutical composition, the method, the kit or the use according to any one of claims 25 to 34, wherein said RNA molecule, set of RNA molecules, nucleic acid molecule or vector is complexed with one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or with one or more liposomal transfection reagent(s) (e.g. to form lipoplexes, like lipid nanoparticles (LNPs) or lipidoid nanoparticles (LiNPs)).
36. The pharmaceutical composition or the kit according to any one of claims 25 to 35 comprising one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or with one or more liposomal transfection reagent(s).
37. The RNA molecule, the set of RNA molecules, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition, the method, the kit or the use according to any one of claims 33 to 36, wherein said one or more oligomer(s), polymer(s) (and/)or lipidoid(s) and/or one or more liposomal transfection reagent(s) is/are selected from the group consisting of (i) Lipofectamine, e.g. LipofectamineTM2000 (more preferred; Invitrogene, CA, USA); (ii) DOGTOR (preferred; OzBiosciences, Marseille, France); (iii) C12-(2-3-2) (e.g. as disclosed above and/or in WO 2014/207231, WO 2015/128030, WO 2016/075154, Zhang (loc. cit.) and/or Jarzebinska (loc. cit.)) (i) DreamFectTM, preferably DreamFect GoldTM (DFTM/DF-GoldTM; OzBiosciences, Marseille, France); (ii) DPPC; (iii) DOPE; and (ii) PEG-lipids, e.g. PEG2k-lipids (e.g. DMPE-PEG (e.g. DMPE -PEG2k), DMG-PEG (e.g. DMG-PEG2k); 38. The RNA molecule, the set of RNA molecules, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition, the method, the kit or the use according to any one of claims 1 to 37, wherein said upstream 5’-UTR of CYBA or functional derivative thereof, or said upstream 5’-UTR of Ag or functional derivative thereof, is replaced by another functional UTR, e.g. by a minimal UTR (as, for example, disclosed in WO 2017/167910).
PCT/EP2023/084367 2022-12-05 2023-12-05 Regulator(s) of energy homeostasis-encoding rna molecule(s) with increased translation efficiency WO2024121160A1 (en)

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