Methods and compositions for localized expression of administered RNA

Abstract

Disclosed herein are methods for localized expression of an administered RNA molecule at a site of acute inflammation, methods for treating tissue or organ damage characterized by a site of acute inflammation in the tissue or organ in a subject, methods for treating a subject having a site of acute inflammation in an tissue or organ, methods for treating tissue damage in the heart or brain cause by ischemia in a subject, as well as medical preparations or kits suitable for administration and localized expression at a site of acute inflammation.

Description

[0001] METHODS AND COMPOSITIONS FOR LOCALIZED EXPRESSION OF ADMINISTERED RNA

[0002] Technical Field

[0003] The invention provides methods for localized expression of an administered RNA molecule at a site of acute inflammation, methods for treating tissue or organ damage characterized by a site of acute inflammation in the tissue or organ in a subject, methods for treating a subject having a site of acute inflammation in an tissue or organ, methods for treating tissue damage in the heart or brain cause by ischemia in a subject, as well as medical preparations or kits suitable for administration and localized expression at a site of acute inflammation. fiadcsround

[0004] Apart from their well-known ability to encode biologically active proteins, nucleic acids, such as DNA and RNA, have other remarkable properties that make them atractive therapeutic agents. Nucleic acidbased therapeutics are easy to manufacture and are relatively inexpensive. The use of RNA to deliver foreign genetic information into target cells offers an attractive alternative to DNA. The advantages of RNA include transient expression and non-transforming character. RNA does not require nucleus infiltration for expression and, moreover, cannot integrate into the host genome, thereby eliminating the risk of oncogenesis.

[0005] Acute inflammatory reactions, In particular in the heart or central nervous system, such as brain or spinal cord, represent a challenge for the treatment of various diseases and disorders with accompanying acute inflammation. Heart failure (HF) is a rapidly growing public health issue currently affecting an estimated 37 million patients globally (B. Ziaeian and G. C. Fonarow, Nat Rev Cardiol 13, 368-378 (2016)). A major cause for the development of HF is coronary heart disease and myocardial infarction (MI) (V. L. Roger, Circ Res 113, 646-659 (2013)). Lack of coronary blood supply induces a sterile acute inflammation that greatly impacts subsequent tissue repair and remodeling, such as at the site and area around the infarct. Monocytes and macrophages (mono-mac) comprise the most abundant cells in the infarcted myocardium, commonly Identified by flow cytometric analysis as CD45 and CDllb positive, Ly6G and lineage negative cells (F. K. Swirski and M. Nahrendorf, Science 339, 161-166 (2013)). At the peak of acute inflammation, a large number of mono-mac - about 700,000 - invade the heart (F. Leuschner et al., J Exp Med 209, 123-137 (2012)). A tissue residence time of approximately 20 hours in inflamed tissue indicates a constant cell turn-over (F. Leuschner eta!., J Exp Med 209, 123- 137 (2012)). Inflammatory Ly6Chighmonocytes infiltrate via Monocyte Chemotactic Protein 1 (MCP1) / C-C chemokine receptor type 2 (CCR2) to scavenge necrotic debris and promote proteolysis. A second phase of the monocytic response peaks around day 7 after MI with a predominance of Ly6Clow macrophages. These cells are thought to promote tissue repair by secreting anti-inflammatory and angiogenic factors, among them IL-10 and vascular endothelial growth factor (VEGF) (S. D. Prabhu and N. G. Frangogiannis, Circ Res 119, 91-112 (2016)). The exact cues inducing the phase switch remain poorly defined, but it appears well established that exaggerated or prolonged presence of pro- inflammatory mono-mac after MI results in impaired healing and worsening of left ventricular function (P. Panizzi etal, J Am Coll Cardiol 55, 1629-1638 (2010)).

[0006] Injuries or damages of the central nervous system, in both spinal cord and brain, are also known to be associated with acute inflammation and with infiltration of monocytes and macrophages (A. Alam et al., J Neuroinflammation 17, 328 (2020), A. D. Greenhalgh and S. David, J Neuroscience 34(18), 6316-6322 (2014), D. Han etal., Neurol Sci 41, 3589-3607 (2020), C. L. Hsieh etal., Eur J Immunol 43, 2010-2022 (2013), L. M. Milich etal., Acta Neuropathol 137, 785-797 (2019)).

[0007] Strictly local administration is sometimes not possible, e.g., for acute inflammatory loci not easily accessible to the operator or where injection poses risk to the patient. Furthermore, depending on the site of injection, spread of the administered agents to adjacent tissues or even the lymph or blood system cannot always be avoided.

[0008] It is often also problematic to systematically administer certain proteins as either their half-life is limited, they do not reach the site of acute inflammation, or systemic administration causes strong side effects resulting from either suppression or overstimulation of the immune response. Sometimes a local administration of proteins is not always suitable as even local administration is sometimes not sufficient to reach the site where the protein is needed for a therapeutic effect.

[0009] It Is therefore desirable to provide methods to locally express proteins at a site of acute inflammation and to provide medical preparations or kits, which avoid the above drawbacks. It is particularly desirable to employ the already existing endogenous machinery of response to acute inflammation for delivering a protein of interest to a site of acute inflammation.

[0010] Summit

[0011] The inventors have surprisingly found that it is possible to provide RNA formulations that allow expression of RNA molecules at a localized site of acute inflammation, in particular when these RNA formulations have been administered systemically.

[0012] The invention provides in a first aspect a method for localized expression of an RNA molecule at a site of acute inflammation present in a subject, wherein the method comprises administering to the subject the RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein.

[0013] In some embodiments, the site of acute inflammation can be a single localized site of acute inflammation. In some embodiments, the site of acute inflammation can be in a tissue or organ of the body. In some emodiments, the site of acute inflammation can be the entire tissue or organ, or the site of acute inflammation can be at a localized site (lesion) in the tissue or organ, or can be in only part of the tissue or organ. In some embodiments, the site of acute inflammation can be at a part in or site of the organ or tissue which has been injured or otherwise affected by a lack of oxygen, i.e., due to ischemia, for example, caused by an infarct or other insufficiency in blood supply to the tissue or organ. In some embodiments, the site of acute inflammation can be a lesion (tissue damage) in the heart or brain caused by a lack of oxygen. Thus, the site of acute inflammation can be at a site of injury or damage.

[0014] In some embodiments, the site of acute inflammation can be sterile.

[0015] In some embodiments, the organ can be heart, brain, spinal cord, lung, kidney, or liver.

[0016] In some embodiments, the tissue can be vascular tissue or nerve tissue.

[0017] In some embodiments, the tissue can be nerve tissue of the peripheral nervous system.

[0018] In some embodiments, the injury or damage can be caused by ischemia, blunt force trauma, chemical bum, or radiation bum.

[0019] In some embodiments, the injury or damage can be caused by blunt force trauma.

[0020] In some embodiments, the injury or damage can result in a reduction in or loss of function of the organ or tissue.

[0021] In some embodiments, the injury or damage can be caused by ischemia.

[0022] In some embodiments, the ischemia can be in the heart caused by coronary heart disease or myocardial infarction. Preferably, the ischemia is in the heart caused by myocardial infarction.

[0023] In some embodiments, the ischemia can be in the brain caused by a stroke.

[0024] In some embodiments, the site of acute inflammation can be characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

[0025] In some embodiments, the site of acute inflammation can be characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation. The site absent the acute inflammation can be in the tissue or organ in which the site of acute inflammation is present. The site absent the acute inflammation can be in the same tissue or organ in another subject not having the acute inflammation. Preferably, the another subject is of the same species as the subject having the site of acute inflammation. The site absent the acute inflammation can be the same (local) site in the same tissue or organ in another subject that does not have the acute inflammation.Tthe type of immune cell can be selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. In some embodiments, the T lymphocytes can be cytotoxic T cells or regulatory T cells.

[0026] In some embodiments, the site of acute inflammation can be characterized by the infiltration of and / or increased presence / n umber of macrophages, neutrophils, T cells, B cells and / or natural killer cells. Preferably, the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages.

[0027] In some embodiments, the site of acute inflammation can be characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Ghighand / or CD14+and / or CD16+and / or CD68+and / or CCR2+and / or MHC class IF immune cells.

[0028] In some embodiments, the administered RNA molecule is present in, preferably taken up by, monocytes and / or macrophages in the subject. Preferably, the monocytes and / or macrophages transport the administered RNA to the site of acute inflammation. In an embodiment, the RNA molecule is taken up by monocytes and / or macrophages at the site of acute inflammation. Preferably, the macrophages are characterized by a higher expression of F4 / 80 and / or a higher expression of MHC II compared to monocytes and the monocytes are characterized by a lower F4 / 80 expression and / or a lower expression of MHC II compared to macrophages.

[0029] In some embodiments, the RNA molecule can be administered systemically and / or is administered locally to the site of acute inflammation. Preferably, systemic administration is by subcutaneous injection, intravenous administration, or intraarterial administration. Preferably, local administration is via a catheter or by injection to the site of acute inflammation, preferably via a catheter. Preferably, local administration is via a catheter in an artery. Preferably, the artery is a coronary artery.

[0030] In some embodiments, the method can comprise administering two RNA molecules, a first and a second RNA molecule, to the subject, wherein the second RNA molecule comprises at least one open reading frame encoding for at least one protein different from that encoded by the first RNA molecule.

[0031] In some embodiments, the second RNA molecule can be administered systemically and / or can be administered locally to the site of acute inflammation. Preferably, systemic administration is by subcutaneous injection, intravenous administration, or intraarterial administration, in particular by intravenous administration. Preferably, local administration is intrathecal, intracerebroventricular, intraparenchymal or via a catheter or by injection at or adjacent to the site of acute inflammation. Preferably, local administration is via a catheter or by injection at or adjacent to the site of acute inflammation. Preferably, local administration is via a catheter in an artery. Preferably, the artery is a coronary artery.

[0032] In some embodiments, the second RNA molecule can be administered in the same manner as the first RNA molecule.In an alternative embodiment, the second RNA molecule can be administered in a different manner as the first RNA molecule. Preferably, the second RNA molecule is administered locally and the first RNA is administered systemically.

[0033] In some embodiments, the first RNA molecule can be administered before the second RNA molecule. Preferably, the first RNA molecule is administered at least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days before the second RNA molecule. Preferably, the first RNA molecule is administered at least 2 days before the second RNA molecule.

[0034] In some embodiments, the second RNA molecule can be administered before the first RNA molecule. Preferably, the second RNA molecule is administered at least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days before the first RNA molecule. Preferably, the second RNA molecule is administered at least 2 days before the first RNA molecule.

[0035] In some embodiments, the first and second RNA molecules can be administered at least 48 hours / 2 days apart from each other.

[0036] In some embodiments, the first and the second RNA molecules can be administered concurrently. In some embodiments, the first and the second RNA molecules can be administered on the same day. In some embodiments, the first and the second RNA molecules can be administered not more than within 6 hours of each other.

[0037] In some embodiments the first and / or second RNA molecule can be administered 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours after the site of acute inflammation appeared, preferably after the tissue or organ damage appeared.

[0038] In some embodiments, the individual RNA molecules can be administered separately on different days and / or all of the RNA molecules can be administered concurrently on the same day. For example, on day 1 a first RNA molecule can be administered separately, and on day 3 and day 5 the first and a second RNA molecule can be administered concurrently, and on day 7 the second RNA molecule can be administered separately.

[0039] In some embodiments, the method can comprise multiple administrations of the RNA molecule(s). Preferably, administration is repeated at least once, at least twice, at least thrice, at least four times, at least five times. Preferably, the administration is repeated at least thrice. In some embodiments, the RNA molecule(s) can be a replicable or non-replicable RNA molecule.

[0040] In some embodiments, the RNA molecule(s) can be mRNA, e.g., linear or circular mRNA.

[0041] In some embodiments, the RNA molecule(s) can comprise one or more of the following elements: a 5'- cap, a 5' UTR, a 3' UTR and / or a poly A structure. Preferably, the 5'-cap is a cap analog. Preferably, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1. Preferably, the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2. Preferably, the poly A structure is an interrupted poly A structure. The poly A structure can comprise the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3.

[0042] In some embodiments, the RNA molecule(s) comprise the nucleotide sequence of any one of SEQ ID NOs 39 to 42, wherein "-CDS-" as shown in the sequences represents a nucleotide sequence encoding the protein to be expressed (e.g., "-CDS-” may be replaced with a sequence encoding IL-10 (such as any one of SEQ ID NOs 4-7; or "-CDS-" may be replaced with a sequence encoding VEGF-A (such as any one of SEQ ID NOs 11-14 and 43).

[0043] In some embodiments, the RNA molecule(s) does not comprise one or more of the following elements: a 5'-cap, a 5' UTR, a 3' UTR and / or a poly A structure.

[0044] In some embodiments, the RNA molecule(s) can be non-immunogenic.

[0045] In some embodiments, the RNA molecule(s) can comprise a nucleotide modification. Preferably, the modification is the substitution of one or more U residues with pseudouridine, Nl-methylpseudoruridine or 5-methyluridine. The one or more substituted U residues can be Nl-methylpseudouridine.

[0046] In some embodiments, at least 50%, at least 70%, at least 90% at least 99% or 100% of the U residues in the RNA molecule(s) can be substituted.

[0047] In some embodiments, the protein encoded by the RNA molecule(s) can provide a therapeutic effect or benefit.

[0048] In some embodiments, the protein encoded by the RNA molecule(s) can enhance healing at the site of acute inflammation.

[0049] In some embodiments, the protein encoded by the RNA molecule(s) can decrease inflammation at the site of acute inflammation. In some embodiments, the protein encoded by the RNA molecule(s) can enhance organ or tissue function. Preferably, the organ is the heart or brain.

[0050] In some embodiments, the protein encoded by the RNA molecule(s) can be an immune system modulator. Preferably, the immune system modulator is a cytokine or a functional fragment or variant thereof.

[0051] In some embodiments, the cytokine can be an anti-inflammatory cytokine. In some embodiments, the immune system modulator is interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, IL- 18 binding protein, IL-19, IL-35, IL-36 receptor antagonist, IL-37, IL-38 or TGF-p, or a functional fragment or variant thereof. Preferably, the immune system modulator is IL-4, IL-10, or IL-13, or a functional fragment or variant thereof, preferably IL-10 or a functional fragment or variant thereof. In some embodiments, the cytokine is a functionally equivalent molecule that binds to and activates its receptor.

[0052] In some embodiments, the protein encoded by the RNA molecule(s) can increase neurogenesis, neuronal paterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis at or adjacent to the site of acute inflammation. Preferably the protein encoded by the RNA molecule(s) that increases neurogenesis, neuronal paterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, and / or glial growth is selected from the group consisting of Insulin-like growth factor 1 (IGF 1), Agrin, Meteorin-Like Protein (METRNL), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), placental growth factor (PGF), granulocyte / macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF), or a functional fragment or variant thereof.

[0053] In some embodiments, the protein encoded by the RNA molecule(s) can increase angiogenesis at or adjacent to the site of acute inflammation. Preferably the protein encoded by the RNA molecule(s) that increases angiogenesis is selected from the group consisting of Insulin-like growth factor 1 (IGF1), Agrin, Meteorin-Like Protein (METRNL), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), placental growth factor (PGF), granulocyte / macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF), or a functional fragment or variant thereof. The protein encoded by the RNA molecule(s) that increases neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis can be VEGF, agrin, or METRNL, or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof. Preferably, the VEGF is VEGF-A, VEGF-B, VEGF-C, or VEGF-D.

[0054] In some embodiments, the protein encoded by the RNA molecule(s) that increases angiogenesis can be VEGF, agrin, or METRNL, or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof. Preferably, VEGF is VEGF-A, VEGF-B, VEGF-C, or VEGF-D. In some embodiments, VEGF can be VEGF164 or VEGFiss.

[0055] In some embodiments, the protein encoded by the RNA molecule(s) can be a human protein.

[0056] In some embodiments, the protein encode by the RNA molecule(s) is substantially not expressed at sites other than the site of acute inflammation. Preferably, the expression at the sites other than the site of acute inflammation differs by a factor of 2, 3, 4, 5, 10, 20, 30, 100 or more.

[0057] In some embodiments, the subject can be administered two RNA molecules, the first RNA molecule encodes IL-10 or a functional fragment or variant thereof, and the second RNA molecule encodes VEGF- A or a functional fragment or variant thereof, wherein the first RNA can be administered systemically and the second RNA can be administered locally. Preferably, the systemic administration is by intravenous injection and the local administration is via a catheter in an artery. Preferably, the artery is in an organ in which the site of acute inflammation is present. Preferably, the organ is heart or brain.

[0058] In some embodiments, the RNA molecule(s) can be formulated in a composition with at least one lipid. Preferably the RNA molecule(s) and at least one lipid form particles. Preferably, the particles are lipid nanopartides (LNP), lipoplexes (LPX) or liposomes. Preferably, the particles are nanoparticles, in which:

[0059] (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and / or

[0060] (ii) the nanoparticles have a net negative charge and / or

[0061] (iii) the zeta potential of the nanoparticles Is 0 or less. Optionally, the nanoparticle can have or can also have a charge ratio of positive charges to negative charges in the nanoparticles can be 1.4:1 or less.

[0062] In some embodiments, the charge ratio of positive charges to negative charges in the nanoparticles can be between 1:1 and 1:8, preferably between 1:1 and 1:4.

[0063] In some embodiments, the at least one lipid can be a cationic lipid. Preferably, the lipid comprises a cationic head group.

[0064] In some embodiments, the lipid can be a pH responsive lipid.

[0065] In some embodiments, the at least one lipid can be a PEGylated-lipid.

[0066] In some embodiments, the composition can further comprise at least one helper lipid.

[0067] In some embodiments, the helper lipid can be a neutral lipid.

[0068] In some embodiments, the at least one cationic lipid can comprise l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), l,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the at least one helper lipid can comprise l,2-di-(9Z-octadecenoyl)-sn-glycero- 3-phosphoethanolamine (DOPE), cholesterol (Choi), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0069] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one helper lipid can be from 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1.

[0070] In some embodiments, the nanopartides can be lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0071] In some embodiments, the nanoparticles can be lipoplexes comprising DODMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0072] In some embodiments, the nanopartides can be lipoplexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0073] In some embodiments, the nanoparticles can be lipoplexes comprising DODMA:Cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

[0074] In some embodiments, the nanopartides can be lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0075] In some embodiments, the nanoparticles can be lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0076] In some embodiments, the nanoparticles can be lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In some embodiments, the particles can be LNPs, which are complexed with and / or encapsulate the RNA molecule(s).

[0077] In some embodiments, the particles can be vesicles encapsulating the RNA molecule, preferably unilamellar liposomes.

[0078] In some embodiments, the RNA molecule(s) can be formulated in a composition comprising a polyalkyleneimine. Preferably, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the RNA molecules (N:P ratio) is 2.0 to 15.0, preferably 6.0 to 12.0.

[0079] In some embodiments, the ionic strength of the composition can be 50 mM or less, preferably wherein the concentration of monovalent cationic ions is 25 mM or less and the concentration of divalent cationic ions is 20 pM or less.

[0080] In some embodiments, the particles formed can be polyplexes.

[0081] In some embodiments, the polyalkyleneimine can comprise the following general formula (I): wherein

[0082] R is H, an acyl group or a group comprising the following general formula (II): wherein R1 is H or a group comprising the following general formula (III): n, m, and I are independently selected from integers from 2 to 10; and p, q, and r are integers, wherein the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5-102 to 107 Da, preferably 5000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da. Preferably, n, m, and I can be independently selected from 2, 3, 4, and 5, preferably from 2 and 3. Preferably, Ri is H. Preferably, R is H or an acyl group. In some embodiments, the polyalkyleneimine can comprise polyethylenimine and / or polypropylenimine, preferably polyethylenimine.

[0083] In some embodiments, at least 92% of the N atoms in the polyalkyleneimine can be protonatable.

[0084] In another embodiment the invention provides a method for treating tissue or organ damage characterized by a site of acute inflammation in the tissue or organ in a subject, the method comprising administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one therapeutically active protein.

[0085] In some embodiments of the method for treating tissue or organ damage, the RNA molecule can be formulated with a reagent capable of forming negatively charged particles with the RNA molecule.

[0086] In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

[0087] In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation.

[0088] In some embodiments of the method for treating tissue or organ damage, the type of immune cell can be selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. Preferably, the T lymphocytes can be cytotoxic T cells or regulatory T cells.

[0089] In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

[0090] In some embodiments of the method for treating tissue or organ damage, the administered RNA molecule is present in, preferably taken up by, monocytes and / or macrophages in the subject. Preferably, the monocytes and / or macrophages (e.g,, peripheral monocytes and / or macrophages) transport the administered RNA to the site of acute inflammation (e.g., when the monocytes and / or macrophages infiltrate the site of acute inflammation). In an embodiment, the RNA molecule is taken up by monocytes and / or macrophages at the site of acute inflammation. Preferably, the macrophages are characterized by a higher expression of F4 / 80 and / or a higher expression of MHC II compared to monocytes and the monocytes are characterized by a lower F4 / 80 expression and / or a lower expression of MHC II compared to macrophages.

[0091] In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be characterized by the infiltration of and / or increased presence / number of macrophages. In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Ghi9hand / or CD14+and / or CD16+and / or CD68+and / or CCR2+and / or MHC class II+immune cells.

[0092] In some embodiments of the method for treating tissue or organ damage, the site of acute inflammation can be sterile.

[0093] In some embodiments of the method for treating tissue or organ damage, the therapeutically active protein can have an anti-inflammatory effect.

[0094] In some embodiments of the method for treating tissue or organ damage, the therapeutically active protein can induce neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis, preferably angiogenesis.

[0095] In some embodiments of the method for treating tissue or organ damage, the method can further comprise administering a second RNA molecule, which second RNA molecule comprises at least one open reading frame encoding for at least one different therapeutically active protein. Preferably one therapeutically active protein is IL-4, IL- 10 or IL-13, preferably IL-10, or a functional fragment or variant thereof and the other therapeutically active protein is VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof. Preferably the RNA molecule encoding IL-4, IL-10, or IL-13 is administered systemically and the RNA molecule encoding VEGF, agrin, or METRNL is administered locally.

[0096] In another embodiment, the invention provides a method for treating a subject having a site of acute inflammation in an tissue or organ, wherein the method comprises administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein having a regenerative or anti-inflammatory effect.

[0097] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the RNA molecule can be formulated with a reagent capable of forming negatively charged particles with the RNA molecule.

[0098] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

[0099] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation. Preferably, the type of immune cell can be selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. Preferably, the T lymphocytes can be, in particular are, cytotoxic T cells or regulatory T cells.

[0100] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

[0101] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be characterized by the infiltration of and / or increased presence / number of macrophages.

[0102] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Gh'9hand / or CD14+and / or CD16+and / or CD68+and / or CCR2+and / or MHC class IF immune cells.

[0103] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the administered RNA molecule is present in, preferably taken up by, monocytes and / or macrophages in the subject. Preferably, the monocytes and / or macrophages transport the administered RNA to the site of acute inflammation. In an embodiment, the RNA molecule is taken up by monocytes and / or macrophages at the site of acute inflammation. Preferably, the macrophages are characterized by a higher expression of F4 / 80 and / or a higher expression of MHC II compared to monocytes and the monocytes are characterized by a lower F4 / 80 expression and / or a lower expression of MHC II compared to macrophages.

[0104] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be sterile.

[0105] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the site of acute inflammation can be a site of tissue damage or injury in the tissue or organ, preferably in the heart or brain.

[0106] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the tissue damage or injury in the heart can be caused by ischemia due to a myocardial infarction.

[0107] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the tissue damage or injury in the heart can be caused by a surgical intervention.

[0108] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the tissue damage or injury in the brain can be caused by ischemia due to a stroke. In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the tissue damage or injury in the brain can be caused by a traumatic injury, such as blunt force trauma.

[0109] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the protein can be IL-4, IL-10, or IL-13 or a functional fragment or variant thereof, preferably IL-10 or a functional fragment or variant thereof.

[0110] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the method can further comprise administering a second RNA molecule, which second RNA molecule comprises at least one open reading frame encoding at least one protein having regenerative effect. Preferably, the regenerative effect is induction of neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis, preferably angiogenesis. Preferably, the protein is Agrin, VEGF, or METRNL, or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof.

[0111] In some embodiments of the method for treating a subject having a site of acute inflammation in an tissue or organ, the RNA molecule encoding IL-4, IL-10, or IL-13 is administered systemically and the RNA molecule encoding Agrin, VEGF, or METRNL is administered locally.

[0112] In another aspect, the invention provides a method for treating tissue damage in the heart or brain cause by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10 or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF or a functional fragment or variant thereof; where each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule; and wherein the first RNA molecule is administered systemically and the second RNA molecule is administered locally. Preferably wherein the systemic administration is by intravenous administration and the local administration is via a catheter, preferably in an artery, in the heart or brain at or adjacent to the site of tissue damage.

[0113] In an embodiment, in which the subject has a myocardial infarction, the subject can be administered an RNA molecule encoding IL-10 or a functional fragment or variant thereof on day 1; on day 3 and on day 5 the subject is administered an RNA molecule encoding IL- 10 or a functional fragment or variant thereof and an RNA molecule encoding VEGF-A or a functional fragment or variant thereof (or a single RNA molecule encoding both); and on day 7 the subject is administered an RNA molecule encoding VEGF-A or a functional fragment or variant thereof.

[0114] In an embodiment, in which the subject has a myocardial infarction, the subject can be administered an RNA molecule encoding VEGF-A or a functional fragment or variant thereof on day 1; on day 3 and on day 5 the subject is administered an RNA molecule encoding IL-10 or a functional fragment or variant thereof and an RNA molecule encoding VEGF-A or a functional fragment or variant thereof (or a single RNA molecule encoding both); and on day 7 the subject is administered an RNA molecule encoding IL- 10 or a functional fragment or variant thereof.

[0115] In another aspect, the invention provides a medical preparation or kit comprising an RNA molecule or a composition as described herein.

[0116] In an embodiment, the invention provides a medical preparation or kit comprising a first RNA molecule comprising at least one open reading frame coding for IL-4, IL-10, or IL-13 or a functional fragment or variant thereof, preferably IL-10 or a functional fragment or variant thereof, and a second RNA molecule comprising at least one open reading frame coding for Agrin, VEGF, or METRNL or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof.

[0117] In some embodiments of the medical preparation or kit, the first and / or second RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule(s).

[0118] In some embodiments of the medical preparation or kit, the first and second RNA molecules are in separate containers.

[0119] In an embodiment, the invention provides a medical preparation or kit comprising: (i) a first lipoplex formulation comprising an RNA molecule comprising at least one open reading frame coding for IL-4, IL-10, or IL-13 or a functional fragment or variant thereof, preferably IL-10 or a functional fragment or variant thereof, and (ii) a second lipoplex formulation comprising a second RNA molecule comprising at least one open reading frame coding for Agrin, VEGF, or METRNL or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof.

[0120] In an embodiment, the invention provides a medical preparation or kit comprising a first RNA molecule comprising at least one open reading frame coding for IL-4, IL-10, or IL-13 or a functional fragment or variant thereof, preferably IL-10 or a functional fragment or variant thereof, and a second RNA molecule comprising at least one open reading frame coding for Agrin, VEGF, or METRNL or a functional fragment or variant thereof, preferably VEGF or a functional fragment or variant thereof, for use as a medicament.

[0121] In some embodiments of the medical preparation or kit, the medical preparation is for use in a method for treating tissue damage in the heart or brain cause by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10 or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF or a functional fragment or variant thereof; where each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule; and wherein the first RNA molecule is administered systemically and the second RNA molecule is administered locally.

[0122] In another aspect, the invention provides an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein, for use in a method according to the invention.

[0123] Detailed Description of the Invention

[0124] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0125] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kblbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0126] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g,, Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook eta / . eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0127] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0128] In particular embodiments, the methods of the present invention can be combined with embodiments of other methods of the present invention or embodiments of medical preparations disclosure herein.

[0129] The term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein preferably means + / - 10 % of the numerical value or range recited or claimed.

[0130] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or dearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise dearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-daimed element essential to the practice of the invention.

[0131] Unless expressly specified otherwise, the term "comprising" is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising". It is, however, contemplated as a specific embodiment of the present invention that the term "comprising" encompasses the possibility of no further members being present, i.e„, for the purpose of this embodiment "comprising" is to be understood as having the meaning of "consisting of".

[0132] Indications of relative amounts of a component characterized by a generic term are meant to refer to the total amount of all specific variants or members covered by said generic term. If a certain component defined by a generic term is specified to be present in a certain relative amount, and if this component is further characterized to be a specific variant or member covered by the generic term, it is meant that no other variants or members covered by the generic term are additionally present such that the total relative amount of components covered by the generic term exceeds the specified relative amount; more preferably no other variants or members covered by the generic term are present at all.

[0133] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention was not entitled to antedate such disclosure.

[0134] Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0135] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement by about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%. "Physiological pH" as used herein refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0136] As used in the present disclosure, "% w / v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).

[0137] As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

[0138] As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0139] As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like material.

[0140] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.

[0141] The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, Ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum Z is taken over all the different kinds of ions (i) in solution.

[0142] According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of the nucleic acid. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.

[0143] "Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.

[0144] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably.

[0145] The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.

[0146] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.

[0147] In the present specification, a structural formula of a compound may represent a certain isomer of said compound. It is to be understood, however, that the present invention includes all isomers such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers and the like which occur structurally and isomer mixtures and is not limited to the description of the formula. "Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical (spatial) positioning of the functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers which are non-superimposable mirrorimages of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is denoted by the prefix (±). "Diastereomers" are stereoisomers which are non- superimposable and which are not mirror-images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneo"sly and rever"ibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; ie., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol- tautomerism. "Conformers" are stereoisomers that can be interconverted just by rotations about formally single bonds, and include — in particular — those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0148] The term "nef'charge" refers to the charge on a whole object, such as a compound or particle.

[0149] An ion having an overall net positive charge is a cation, while an ion having an overall net negative charge is an anion. Thus, according to the invention, an anion is an ion with more electrons than protons, giving it a net negative charge; and a cation is an ion with fewer electrons than protons, giving it a net positive charge.

[0150] Terms as "charged" "net charge", "negatively charged", "positively charged" "neutral", with reference to a given compound or particle, refer to the electric net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0151] The term "protein" according to the invention relates to a molecule with peptide bonds comprising a sequence of amino acids. This molecule can be produced by translation of an RNA molecule that encodes such molecule. The term also relates to peptides, oligopeptides, polypeptides, full length modified or wild-type proteins and fragments and variants of full length modified or wild-type proteins. The term "protein" also relates to those moleucles that have post-translational modifications.

[0152] The term "nucleic acid" according to the invention also comprises a chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids containing non-natural nucleotides and nucleotide analogs. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In general, a nucleic acid molecule or a nucleic acid sequence refers to a nucleic acid which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the invention, nucleic acids comprise genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared and chemically synthesized molecules. According to the invention, a nucleic acid may be in the form of a single-stranded or double-stranded and linear or covalently closed circular molecule.

[0153] According to the invention "nucleic add sequence" refers to the sequence of nucleotides in a nucleic add, e.g.; a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). The term may refer to an entire nucleic acid molecule (such as to the single strand of an entire nucleic add molecule) or to a part (e.g. a fragment) thereof.

[0154] According to the present invention, the term "RNA" or "RNA molecule" relates to a molecule which comprises ribonucleotide residues and which is preferably entirely or substantially composed of ribonucleotide residues. The term "ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. The term "RNA" comprises double-stranded RNA, single stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs, particularly analogs of naturally occurring RNAs. According to the invention, RNA may be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule to which no complementary nucleic acid molecule (typically no complementary RNA molecule) is associated. Single-stranded RNA may contain self-complementary sequences that allow parts of the RNA to fold back and to form secondary structure motifs including without limitation base pairs, stems, stem loops and bulges. Single-stranded RNA can exist as minus strand [(-) strand] or as plus strand [(+) strand]. The (+) strand is the strand that comprises or encodes genetic information. The genetic information may be for example a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand may serve directly as template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, (+) strand and (-) strand are two separate RNA molecules, and both these RNA molecules associate with each other to form a double-stranded RNA ("duplex RNA").

[0155] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the "duration of expression" of the RNA. It can be expected that RNA having a long half-life will be expressed for an extended time period.

[0156] The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a particular period of time.

[0157] "Fragment", with reference to a nucleic acid sequence, relates to a part of a nucleic acid sequence, f.e.; a sequence which represents the nucleic acid sequence shortened at the 5'- and / or 3'-end(s). Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention those fragments of RNA molecules are preferred which retain RNA stability and / or translational efficiency.

[0158] "Fragment", with reference to an amino add sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. The term "variant" with respect to, for example, nucleic acid and amino acid sequences, according to the invention includes any variants, in particular mutants, viral strain variants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic add sequences, wherein a degenerate nucleic acid according to the invention is a nucleic acid that differs from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. A virus homolog is a nucleic acid or amino acid sequence with a different virus of origin from that of a given nucleic acid or amino acid sequence.

[0159] In some embodiments, a fragment or variant of an amino acid sequence (peptide or protein) is a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino add sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino add sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0160] Nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions and / or insertions in comparison with the reference nucleic acid. Deletions include removal of one or more nucleotides from the reference nucleic acid. Addition variants comprise 5'- and / or 3'-terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. In the case of substitutions, at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted in its place (such as transversions and transitions). Mutations include abasic sites, crosslinked sites, and chemically altered or modified bases. Insertions include the addition of at least one nucleotide into the reference nucleic add. According to the invention, "nucleotide change" can refer to single or multiple nucleotide deletions, additions, mutations, substitutions and / or insertions in comparison with the reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a deletion of a single nucleotide, the addition of a single nucleotide, the mutation of a single nucleotide, the substitution of a single nucleotide and / or the insertion of a single nucleotide, in comparison with the reference nucleic acid. According to the invention, a nucleic acid variant can comprise one or more nucleotide changes in comparison with the reference nucleic acid.

[0161] Variants of specific nucleic acid sequences preferably have at least one functional property of said specific sequences and preferably are functionally equivalent to said specific sequences, e.g., nucleic acid sequences exhibiting properties identical or similar to those of the specific nucleic acid sequences.

[0162] As described below, some embodiments of the present invention are characterized, inter alia, by nucleic acid sequences that are homologous to other nucleic add sequences. These homologous sequences are variants of other nucleic acid sequences.

[0163] Preferably the degree of identity between a given nucleic acid sequence and a nucleic acid sequence which is a variant of said given nucleic acid sequence or between a given amino acid sequence of a protein and an amino add sequence which is a variant of said given amino acid sequence will be at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of identity is preferably given for a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In preferred embodiments, the degree of identity is given for the entire length of the reference nucleic acid sequence.

[0164] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino add substitutions. "Sequence identity" between two amino acid or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.

[0165] The term "% identical" is intended to refer, in particular, to a percentage of amino acids or nucleotides which are identical in an optimal alignment between two sequences to be compared, with said percentage being purely statistical, and the differences between the two sequences may be randomly distributed over the entire length of the sequence and the sequence to be compared may comprise additions or deletions in comparison with the reference sequence, in order to obtain optimal alignment between two sequences. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2:482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sei. USA 85:2444 or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0166] Percentage identity is obtained by determining the number of identical positions in which the sequences to be compared correspond, dividing this number by the number of positions compared and multiplying this result by 100.

[0167] For example, the BLAST program "BLAST 2 sequences" which is available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi may be used.

[0168] A nucleic acid is "capable of hybridizing" or "hybridizes" to another nucleic add if the two sequences are complementary with one another. A nucleic acid is "complementary" to another nucleic add if the two sequences are capable of forming a stable duplex with one another. According to the invention, hybridization is preferably carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et a!., Editors, 2ndEdition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F.M. Ausubel et at, Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2 x SSC at room temperature and then in 0.1-0.5 x SSC / 0.1 x SDS at temperatures of up to 68°C.

[0169] A percent complementarity Indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or "fully complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. Most preferably, the degree of complementarity according to the invention is 100%.

[0170] The term "derivative" comprises any chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate. The term "derivative" also comprises nucleic acids which contain nucleotides and nucleotide analogs not occurring naturally. Preferably, a derivatization of a nucleic acid increases its stability. A "nucleic add sequence which is derived from a nucleic acid sequence" refers to a nucleic acid which is a variant of the nucleic acid from which it is derived. Preferably, a sequence which is a variant with respect to a specific sequence, when it replaces the specific sequence in an RNA molecule retains RNA stability and / or translational efficiency.

[0171] "nt" is an abbreviation for nucleotide; or for nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0172] According to the invention, the term "codon" refers to a base triplet in a coding nucleic acid that specifies which amino add will be added next during protein synthesis at the ribosome.

[0173] The terms "transcription" and "transcribing" relate to a process during which a nucleic acid molecule with a particular nucleic acid sequence (the "nucleic acid template") is read by an RNA polymerase so that the RNA polymerase produces a single-stranded RNA molecule. During transcription, the genetic information in a nucleic acid template is transcribed. The nucleic acid template may be DNA; however, e.g.; in the case of transcription from an alphaviral nucleic add template, the template is typically RNA. Subsequently, the transcribed RNA may be translated into protein. According to the present invention, the term "transcription" comprises vitro transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term "vector". The cloning vectors are preferably plasmids. According to the present invention, RNA preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA,

[0174] The single-stranded nucleic add molecule produced during transcription typically has a nucleic acid sequence that is the complementary sequence of the template.

[0175] According to the invention, the terms "template" or "nucleic acid template" or "template nucleic acid" generally refer to a nucleic add sequence that may be replicated or transcribed.

[0176] "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence, where appropriate as part of a complete RNA molecule, which is a transcription product of a template nucleic add sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0177] "3' end of a nucleic acid" refers according to the invention to that end which has a free hydroxy group. In a diagrammatic representation of double-stranded nucleic acids, in particular DNA, the 3' end is always on the right-hand side. "5' end of a nucleic acid" refers according to the invention to that end which has a free phosphate group. In a diagrammatic representation of double-strand nucleic acids, in particular DNA, the 5' end is always on the left-hand side.

[0178] 5' end 5'— P-NNNNNNN-OH-3' 3' end

[0179] 3'-HO-NNNNNNN-P— 5'

[0180] "Upstream" describes the relative positioning of a first element of a nucleic acid molecule with respect to a second element of that nucleic acid molecule, wherein both elements are comprised in the same nucleic acid molecule, and wherein the first element is located nearer to the 5' end of the nucleic acid molecule than the second element of that nucleic acid molecule. The second element is then said to be "downstream" of the first element of that nucleic acid molecule. An element that is located "upstream" of a second element can be synonymously referred to as being located "5"' of that second element. For a double-stranded nucleic acid molecule, indications like "upstream" and "downstream" are given with respect to the (+) strand.

[0181] According to the invention, "functional linkage" or "functionally linked" relates to a connection within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if it influences transcription of said coding sequence. Functionally linked nucleic acids are typically adjacent to one another, where appropriate separated by further nucleic acid sequences, and, in particular embodiments, are transcribed by RNA polymerase to give a single RNA molecule (common transcript).

[0182] In particular embodiments, a nucleic acid is functionally linked according to the invention to expression control sequences which may be homologous or heterologous with respect to the nucleic acid.

[0183] The term "expression control sequence" comprises according to the invention promoters, ribosomebinding sequences and other control elements which control transcription of a gene or translation of the derived RNA. In particular embodiments of the invention, the expression control sequences can be regulated. The precise structure of expression control sequences may vary depending on the species or cell type but usually includes 5'-untranscribed and 5"- and 3'-untranslated sequences involved in initiating transcription and translation, respectively. More specifically, 5'-untranscribed expression control sequences include a promoter region which encompasses a promoter sequence for transcription control of the functionally linked gene. Expression control sequences may also include enhancer sequences or upstream activator sequences. An expression control sequence of a DNA molecule usually includes 5'-untranscribed and 5'- and 3'-untranslated sequences such as TATA box, capping sequence, CAAT sequence and the like.

[0184] The nucleic add sequences specified herein, in particular transcribable and coding nucleic acid sequences, may be combined with any expression control sequences, in particular promoters, which may be homologous or heterologous to said nucleic acid sequences, with the term "homologous" referring to the fact that a nucleic acid sequence is also functionally linked naturally to the expression control sequence, and the term "heterologous" referring to the fact that a nucleic acid sequence is not naturally functionally linked to the expression control sequence.

[0185] A transcribable nucleic acid sequence, in particular a nucleic acid sequence coding for a peptide or protein, and an expression control sequence are "functionally" linked to one another, if they are covalently linked to one another in such a way that transcription or expression of the transcribable and in particular coding nucleic acid sequence is under the control or under the influence of the expression control sequence. If the nucleic acid sequence is to be translated into a functional peptide or protein, induction of an expression control sequence functionally linked to the coding sequence results in transcription of said coding sequence, without causing a frame shift in the coding sequence or the coding sequence being unable to be translated into the desired peptide or protein.

[0186] The term "promoter" or "promoter region" refers to a nucleic acid sequence which controls synthesis of a transcript, e.g. a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. The promoter region may include further recognition or binding sites for further factors involved in regulating transcription of said gene. A promoter may control transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible" and initiate transcription in response to an inducer, or may be "constitutive" if transcription is not controlled by an inducer. An inducible promoter is expressed only to a very small extent or not at all, if an inducer is absent. In the presence of the inducer, the gene is "switched on" or the level of transcription is increased. This is usually mediated by binding of a specific transcription factor.

[0187] The term "core promoter" refers to a nucleic acid sequence that is comprised by the promoter. The core promoter is typically the minimal portion of the promoter required to properly initiate transcription. The core promoter typically includes the transcription start site and a binding site for RNA polymerase.

[0188] A "polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymeric molecule from monomeric building blocks. An "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. A "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxy ribonucleotide building blocks. For the case of DNA polymerases and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule. Typically, an RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, which is either a DNA molecule (in that case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP), or is an RNA molecule (in that case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0189] An "RNA-dependent RNA polymerase" or "RdRP", is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of aiphaviral RNA-dependent RNA polymerase, sequential synthesis of (-) strand complement of genomic RNA and of (+) strand genomic RNA leads to RNA replication. RNA-dependent RNA polymerase is thus synonymously referred to as "RNA replicase" or simply "replicase". In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Typical representatives of viruses encoding an RNA-dependent RNA polymerase are alphaviruses.

[0190] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The RNA molecule that is synthesized may be, e.g., identical or complementary to the template RNA molecule. In general, RNA replication may occur via synthesis of a DNA intermediate, or may occur directly by RNA-dependent RNA replication mediated by an RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication does not occur via a DNA intermediate, but is mediated by a RNA-dependent RNA polymerase (RdRP): a template RNA strand (first RNA strand) - or a part thereof - serves as template for the synthesis of a second RNA strand that is complementary to the first RNA strand or to a part thereof. The second RNA strand - or a part thereof - may in turn optionally serve as a template for synthesis of a third RNA strand that is complementary to the second RNA strand or to a part thereof. Thereby, the third RNA strand is identical to the first RNA strand or to a part thereof. Thus, RNA- dependent RNA polymerase is capable of directly synthesizing a complementary RNA strand of a template, and of indirectly synthesizing an identical RNA strand (via a complementary intermediate strand).

[0191] According to the invention, the term "gene" refers to a particular nucleic acid sequence which Is responsible for producing one or more cellular products and / or for achieving one or more intercellular or intracellular functions. More specifically, said term relates to a nucleic acid section (typically DNA; but RNA in the case of RNA viruses) which comprises a nucleic acid coding for a specific protein or a functional or structural RNA molecule.

[0192] An "isolateted molecule" as used herein, is intended to refer to a molecule which is substantially free of other molecules such as other cellular material. The term "isolated nucleic acid" means according to the invention that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel- electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic add available to manipulation by recombinant techniques.

[0193] The term "vector" is used here in its most general meaning and comprises any intermediate vehicles for a nucleic acid which, for example, enable said nucleic acid to be introduced into prokaryotic and / or eukaryotic host cells and, where appropriate, to be integrated into a genome. Such vectors are preferably replicated and / or expressed in the cell. Vectors comprise plasmids, phagemids, virus genomes, and fractions thereof. The term "recombinant" in the context of the present invention means "made through genetic engineering". Preferably, a "recombinant object" such as a recombinant cell in the context of the present invention is not occurring naturally.

[0194] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but that may be discovered and / or isolated in the future from a natural source.

[0195] According to the invention, the term "expression" is used in its most general meaning and comprises production of RNA and / or protein. It also comprises partial expression of nucleic acids. Furthermore, expression may be transient or stable. With respect to RNA, the term "expression" or "translatinon" relates to the process in the ribosomes of a cell by which a strand of coding RNA (e.g. messenger RNA) directs the assembly of a sequence of amino acids to make a peptide or protein.

[0196] According to the invention, the term "mRNA" means "messenger-RNA" and relates to a transcript which is typically generated by using a DNA template and encodes a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein coding region, a 3'-UTR, and a poly(A) sequence. mRNA may be generated by in vitro transcription from a DNA template. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available. According to the invention, mRNA may be modified by stabilizing modifications and capping.

[0197] According to the invention, the term "primary structure", with reference to a nucleic acid molecule, refers to the linear sequence of nucleotide monomers.

[0198] According to the invention, the term "secondary structure", with reference to a nucleic acid molecule, refers to a two-dimensional representation of a nucleic acid molecule that reflects base pairings; e.g.,' in the case of a single-stranded RNA molecule particularly intramolecular base pairings. Although each RNA molecule has only a single polynucleotide chain, the molecule is typically characterized by regions of (intramolecular) base pairs. According to the invention, the term "secondary structure" comprises structural motifs including without limitation base pairs, stems, stem loops, bulges, loops such as interior loops and multi-branch loops. The secondary structure of a nucleic add molecule can be represented by a two-dimensional drawing (planar graph), showing base pairings (for further details on secondary structure of RNA molecules, see Auber eta / ., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structure of certain RNA molecules is relevant in the context of the present invention.

[0199] According to the invention, secondary structure of a nucleic acid molecule, particularly of a singlestranded RNA molecule, is determined by prediction using the web server for RNA secondary structure prediction (htp: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predictl / Predictl.html).

[0200] Preferably, according to the invention, "secondary structure", with reference to a nucleic acid molecule, specifically refers to the secondary structure determined by said prediction. The prediction may also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0201] According to the invention, a "base pair" is a structural motif of a secondary structure wherein two nucleotide bases associate with each other through hydrogen bonds between donor and acceptor sites on the bases. The complementary bases, A: U and G:C, form stable base pairs through hydrogen bonds between donor and acceptor sites on the bases; the A:U and G:C base pairs are called Watson-Crick base pairs. A weaker base pair (called Wobble base pair) is formed by the bases G and U (G:U). The base pairs A:U and G:C are called canonical base pairs. Other base pairs like G:U (which occurs fairly often in RNA) and other rare base-pairs (e.g. A:C; U:U) are called non-canonical base pairs.

[0202] According to the invention, "nucleotide pairing" refers to two nucleotides that associate with each other so that their bases form a base pair (canonical or non-canonical base pair, preferably canonical base pair, most preferably Watson-Crick base pair).

[0203] According to the invention, the terms "stem loop" or "hairpin" or "hairpin loop", with reference to a nucleic add molecule, all interchangeably refer to a particular secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule, such as single-stranded RNA. The particular secondary structure represented by the stem loop consists of a consecutive nucleic acid sequence comprising a stem and a (terminal) loop, also called hairpin loop, wherein the stem is formed by two neighbored entirely or partially complementary sequence elements; which are separated by a short sequence (e.g. 3-10 nucleotides), which forms the loop of the stem-loop structure. The two neighbored entirely or partially complementary sequences may be defined as, e.g., stem loop elements stem 1 and stem 2. The stem loop is formed when these two neighbored entirely or partially reverse complementary sequences, e.g. stem loop elements stem 1 and stem 2, form base-pairs with each other, leading to a double stranded nucleic acid sequence comprising an unpaired loop at its terminal ending formed by the short sequence located between stem loop elements stem 1 and stem 2. Thus, a stem loop comprises two stems (stem 1 and stem 2), which - at the level of secondary structure of the nucleic acid molecule - form base pairs with each other, and which - at the level of the primary structure of the nucleic acid molecule - are separated by a short sequence that is not part of stem 1 or stem 2. For illustration, a two-dimensional representation of the stem loop resembles a lollipop-shaped structure. The formation of a stem-loop structure requires the presence of a sequence that can fold back on itself to form a paired double strand; the paired double strand is formed by stem 1 and stem 2. The stability of paired stem loop elements is typically determined by the length, the number of nucleotides of stem 1 that are capable of forming base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides of stem 2, versus the number of nucleotides of stem 1 that are not capable of forming such base pairs with nucleotides of stem 2 (mismatches or bulges). According to the present invention, the optimal loop length is 3-10 nucleotides, more preferably 4 to 7, nucleotides, such as 4 nucleotides, 5 nucleotides, 6 nucleotides or 7 nucleotides. If a given nucleic acid sequence is characterized by a stem loop, the respective complementary nucleic acid sequence is typically also characterized by a stem loop. A stem loop is typically formed by single-stranded RNA molecules.

[0204] According to the invention, "disruption" or "disrupt", with reference to a specific secondary structure of a nucleic add molecule (e.g., a stem loop) means that the specific secondary structure is absent or altered. Typically, a secondary structure may be disrupted as a consequence of a change of at least one nucleotide that is part of the secondary structure. For example, a stem loop may be disrupted by change of one or more nucleotides that form the stem, so that nucleotide pairing is not possible.

[0205] According to the invention, "compensates for secondary structure disruption" or "compensating for secondary structure disruption" refers to one or more nucleotide changes in a nucleic acid sequence; more typically it refers to one or more second nucleotide changes in a nucleic add sequence, which nucleic acid sequence also comprises one or more first nucleotide changes, characterized as follows: while the one or more first nucleotide changes, in the absence of the one or more second nucleotide changes, cause a disruption of the secondary structure of the nucleic acid sequence, the co-occurrence of the one or more first nucleotide changes and the one or more second nucleotide changes does not cause the secondary structure of the nucleic acid to be disrupted. Co-occurrence means presence of both the one or more first nucleotide changes and of the one or more second nucleotide changes. Typically, the one or more first nucleotide changes and the one or more second nucleotide changes are present together in the same nucleic acid molecule. In a specific embodiment, one or more nucleotide changes that compensate for secondary structure disruption is / are one or more nucleotide changes that compensate for one or more nucleotide pairing disruptions. Thus, in one embodiment, "compensating for secondary structure disruption" means "compensating for nucleotide pairing disruptions", i.e. one or more nucleotide pairing disruptions, for example one or more nucleotide pairing disruptions within one or more stem loops. The one or more one or more nucleotide pairing disruptions may have been introduced by the removal of at least one initiation codon. Each of the one or more nucleotide changes that compensates for secondary structure disruption is a nucleotide change, which can each be independently selected from a deletion, an addition, a substitution and / or an insertion of one or more nucleotides. In an illustrative example, when the nucleotide pairing A:U has been disrupted by substitution of A to C (C and U are not typically suitable to form a nucleotide pair); then a nucleotide change that compensates for nucleotide pairing disruption may be substitution of U by G, thereby enabling formation of the C:G nucleotide pairing. The substitution of U by G thus compensates for the nucleotide pairing disruption. In an alternative example, when the nucleotide pairing A:U has been disrupted by substitution of A to C; then a nucleotide change that compensates for nucleotide pairing disruption may be substitution of C by A, thereby restoring formation of the original A:U nucleotide pairing. In general, in the present invention, those nucleotide changes compensating for secondary structure disruption are preferred which do neither restore the original nucleic acid sequence nor create novel AUG triplets. In the above set of examples, the U to G substitution is preferred over the C to A substitution.

[0206] According to the invention, the term "tertiary structure", with reference to a nucleic acid molecule, refers to the three-dimensional structure of a nucleic acid molecule, as defined by the atomic coordinates.

[0207] According to the invention, a nucleic acid such as RNA, e.g., mRNA, may encode a peptide or protein. Accordingly, a transcribable nucleic add sequence or a transcript thereof may contain an open reading frame (ORF) encoding a peptide or protein.

[0208] According to the invention, the term "nucleic acid encoding a peptide or protein" means that the nucleic acid, if present in the appropriate environment, preferably within a cell, can direct the assembly of amino acids to produce the peptide or protein during the process of translation. Preferably, coding RNA according to the invention is able to interact with the cellular translation machinery allowing translation of the coding RNA to yield a peptide or protein.

[0209] According to the invention, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and up to preferably 50, preferably 100 or preferably 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino adds, but the terms "peptide", "polypeptide" and "protein" are used herein usually as synonyms.

[0210] The terms "peptide" and "protein" comprise, according to the invention, substances which contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also comprise substances containing bonds such as ester, thioether or disulfide bonds.

[0211] According to the invention, the terms "initiation codon" and "start codon" synonymously refer to a codon (base triplet) of an RNA molecule that is potentially the first codon that is translated by a ribosome. Such codon typically encodes the amino acid methionine in eukaryotes and a modified methionine in prokaryotes. The most common initiation codon in eukaryotes and prokaryotes is AUG. Unless specifically stated herein that an initiation codon other than AUG is meant, the terms "initiation codon” and "start codon", with reference to an RNA molecule, refer to the codon AUG. According to the invention, the terms "initiation codon" and "start codon" are also used to refer to a corresponding base triplet of a deoxyribonucleic acid, namely the base triplet encoding the initiation codon of an RNA. If the initiation codon of messenger RNA is AUG, the base triplet encoding the AUG is ATG. According to the invention, the terms "initiation codon" and "start codon" preferably refer to a functional initiation codon or start codon, i.e., to an initiation codon or start codon that is used or would be used as a codon by a ribosome to start translation. There may be AUG codons in an RNA molecule that are not used as codons by a ribosome to start translation, e.g., due to a short distance of the codons to the cap. These codons are not encompassed by the term functional initiation codon or start codon.

[0212] According to the invention, the terms "start codon of the open reading frame" or "initiation codon of the open reading frame" refer to the base triplet that serves as initiation codon for protein synthesis in a coding sequence, e.g., in the coding sequence of a nucleic acid molecule found in nature. In an RNA molecule, the start codon of the open reading frame is often preceded by a 5' untranslated region (5'- UTR), although this is not strictly required.

[0213] According to the invention, the terms "native start codon of the open reading frame" or "native initiation codon of the open reading frame" refer to the base triplet that serves as initiation codon for protein synthesis in a native coding sequence. A native coding sequence may be, e.g., the coding sequence of a nucleic acid molecule found in nature. In some embodiments, the present invention provides variants of nucleic acid molecules found in nature, which are characterized in that the native start codon (which is present in the native coding sequence) has been removed (so that it is not present in the variant nucleic acid molecule).

[0214] According to the invention, "first AUG" means the most upstream AUG base triplet of a messenger RNA molecule, preferably the most upstream AUG base triplet of a messenger RNA molecule that is used or would be used as a codon by a ribosome to start translation. Accordingly, "first ATG" refers to the ATG base triplet of a coding DNA sequence that encodes the first AUG. In some instances, the first AUG of a mRNA molecule is the start codon of an open reading frame, Ze., the codon that is used as start codon during ribosomal protein synthesis.

[0215] According to the invention, the terms "comprises the removal" or "characterized by the removal" and similar terms, with reference to a certain element of a nucleic acid variant, mean that said certain element is not functional or not present in the nucleic acid variant, compared to a reference nucleic acid molecule. Without limitation, a removal can consist of deletion of all or part of the certain element, of substitution of all or part of the certain element, or of alteration of the functional or structural properties of the certain element. The removal of a functional element of a nucleic acid sequence requires that the function is not exhibited at the position of the nucleic acid variant comprising the removal. For example, an RNA variant characterized by the removal of a certain initiation codon requires that ribosomal protein synthesis is not initiated at the position of the RNA variant characterized by the removal. The removal of a structural element of a nucleic add sequence requires that the structural element is not present at the position of the nucleic acid variant comprising the removal. For example, a RNA variant characterized by the removal of a certain AUG base triplet, Ze., of a AUG base triplet at a certain position, may be characterized, e.g., by deletion of part or all of the certain AUG base triplet (e.g., AAUG), or by substitution of one or more nucleotides (A, U, G) of the certain AUG base triplet by any one or more different nucleotides, so that the resulting nucleotide sequence of the variant does not comprise said AUG base triplet. Suitable substitutions of one nucleotide are those that convert the AUG base triplet into a GUG, CUG or UUG base triplet, or into a AAG, ACG or AGG base triplet, or into a AUA, AUC or AUU base triplet. Suitable substitutions of more nucleotides can be selected accordingly.

[0216] The term "autologous" is used to describe anything that is derived from the same subject. For example, "autologous cell" refers to a cell derived from the same subject. The term "allogeneic" is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. The term "syngeneic" Is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues or cells. The term "heterologous" is used to describe something consisting of multiple different elements. As an example, the Introduction of one individual's cell into a different individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

[0217] The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic add described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or the body of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic adds greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example, Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

[0218] According to various embodiments of the present disclosure, a nucleic acid such as RNA encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g., express the encoded peptide or polypeptide intracellularly (e.g. in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on the surface.

[0219] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. The term "monocyte" refers to a type of leukocyte that can differentiate into macrophages or dendritic cells. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0220] The term "disease" (also referred to as"disorder" herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

[0221] The term "inflammation" is to be understood to refer to a complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair. Inflammation is not strictly associated with an infectious disease, but can be a response thereto. Inflammation can also have other origins, such as traumatic injury or other diseases, such as cancer, ischemia or autoimmune diseases. The term "acute inflammation" is to be understood herein to refer to an inflammatory response that persists for hours to days and reaches a peak within days, and is characterized by the influx of a large number of peripheral immune cells (e.g., monocytes and / or macrophages) within hours and days after the injury to tissue, in which the number of influxed peripheral immune cells then decreases over time, e.g., over a number of days. Acute inflammation may be caused, for example, by injury or tissue damage, e.g., blunt force trauma, an ischemic event, myocardial infarction, or a stroke. Acute inflammation therefore differs from chronic inflammation, which may be understood to refer to an inflammatory response that persists over a longer time period, e.g., weeks, months or even years, and in which peripheral immune cells may be recruited continuously to the inflammatory site and tissue resident immune cells may display exaggerated local proliferation. Typically, chronic inflammation exhibits a large number of peripheral immune cells at the site of chronic inflammation that does not decrease over time, e.g., over weeks and months. Also, acute inflammation, as understood in the context of the present invention, is different from and is not a part of systemic inflammation since it is localized at one or more specific sites in the subject rather than throughout the subject in a non-site-specific manner. In an embodiment, there may be more than one site of acute inflammation in subject, but not such that there is systemic inflammation throughout the subject. In an embodiment, inflammation does not persist at the site of acute inflammation for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In an embodiment, inflammation persists at the site of acute inflammation for 1 to 30, 5 to 25, or 10 to 20 days.

[0222] The term "sterile" in connection with inflammation is to be understood to refer to an inflammation that is not associated with an infectious disease. Preferably this means that no other organism than the subject having the site of acute inflammation is involved in the occurrence of the inflammation. Preferably a sterile inflammation Is not caused by one or more pathogens and / or an infectious disease and is preferably characterized by the absence of pathogens.

[0223] The term "therapeutic treatment" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0224] The terms "prophylactic treatment" of"preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

[0225] The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g, mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other nonmammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease (e.g., cancer, infectious diseases) but may or may not have the disease, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In some embodiments of the present disclosure, the "individual" or "subject" is a "patient". The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0226] A medical preparation, in particular kit, described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the compositions of the invention or be shipped together with a container which contains the compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0227] The following provides specific and / or preferred variants of the individual features of the invention. The present invention also contemplates as particularly preferred embodiments those embodiments, which are generated by combining two or more of the specific and / or preferred variants described for two or more of the features of the present invention.

[0228] Site of acute inflammation

[0229] According to the invention; the site of acute Inflammation is located at and localized to a specific site in the subject. In an embodiment, acute systemic inflammation is not encompassed in the present invention since it is found throughout the subject in a non-site-specific manner. A site of acute inflammation is typically characterized by an inflammatory response that persist for hours to days and reaches a peak within days, and is characterized by the influx of a large number of peripheral immune cells within hours and days after an injury to tissue. In an embodiment, the site of acute inflammation is a single localized site of acute inflammation, which is to be understood as a site of acute inflammation which is coherent and restricted to only one site within the body of the subject. In an embodiment, the site of acute inflammation is one or more localized sites of acute inflammation, which are to be understood as being coherent sites within the body of the subject. Preferably, the acute inflammation is sterile.

[0230] In an embodiment, the localized acute inflammation at a specific site of a subject can be determined using imaging techniques, such as PET scan, or by indirect signs, such as by edema as measured, for example by MRI or ultrasound. Without being bound by theory, the signal intensity in imaging techniques can enable the skilled person to approximate the grade of inflammation.

[0231] In an embodiment, the site of acute inflammation is characterized by the infiltration of immune cells compared to the site absent the acute inflammation. Preferably the immune cells are selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. In particular the site of acute inflammation is characterized by an infiltration of macrophages and / or monocytes compared to the site absent the acute inflammation.

[0232] In an embodiment, the site of acute inflammation is characterized by the presence of immune cells, preferably compared to the site absent the acute inflammation. Preferably the immune cells are selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. In particular the site of acute inflammation is characterized by the presence of macrophages and / or monocytes, preferably compared to the site absent the acute inflammation.

[0233] In an embodiment, the site of acute inflammation is characterized by the absence of immune cells, preferably specific immune cells, in particular selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0234] In some embodiments, the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

[0235] In some embodiments, the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages.

[0236] In some embodiments, the site of acute inflammation, preferably in the heart or brain, is characterized by the infiltration of and / or increased presence / number of CD45+and / or CDllb+and / or Ly6G (Lymphocyte antigen 6 complex locus G6D)hi9hand / or CD14+and / or CD16+and / or CD68+and / or CCR2+and / or MHC class IF immune cells.

[0237] Without being bound by theory, the macrophages or their precursors, in particular monocytes, originate preferably from the spleen, wherein they or their precursors can take up the RNA molecule administered to the subject, preferably systemically administered, and move to the site of acute inflammation, where they express the protein encoded by the RNA molecule. A negative charge of the particles can help the particles to preferably be taken up by macrophages or their precursors, in particular monocytes.

[0238] Infiltration, presence or absence of immune cells can be tested with conventional methods, such as flow cytometry, histology or immune-fluorescence microscopy. In an embodiment, the presence or absence of immune cells can be determined with a biopsy of the site of acute inflammation and subsequent flow cytometry, histology or immune-fluorescence microscopy, preferably histology.

[0239] The location of the site of acute inflammation is not particularly limited. The site of acute inflammation can be in a tissue or organ. In particular, it can be in an organ, in particular in the heart, brain, spinal cord, lung, kidney or liver, or it can be in tissue, such as a muscle, a peripheral nerve or a joint. In preferred embodiments, the site of acute inflammation is in a tissue, which is vascular tissue or nerve tissue, in particular nerve tissue in the peripheral nervous system. In particularly preferred embodiments, the site of acute inflammation is in the heart. In particularly preferred embodiments, the site of acute inflammation is in the brain. In some embodiments, the organ or tissue is not an organ or tissue of the immune system, e.g., the spleen or thymus.

[0240] The site of acute inflammation can preferably be a site of injury or damage in a tissue or organ, as described herein. In some embodiments, the injury or damage results in a reduction in or loss of function of the organ or tissue. In some embodiments, the injury or damage is in or on the surface of an organ, preferably in an organ. In some embodiments, the injury or damage is in or on the surface of an internal organ. In some embodiments, the injury or damage can be in an organ, in particular in the heart, brain, spinal cord, lung, kidney or liver, or it can be in tissue, such as a muscle, a peripheral nerve or a joint. In preferred embodiments, the injury or damage is in a tissue, which is vascular tissue or nerve tissue. In particularly preferred embodiments, injury or damage Is in the heart. In particularly preferred embodiments, the injury or damage is in the brain.

[0241] The cause of inflammation is not particularly limited. In some embodiments, the injury or damage can be caused by a disease, preferably by ischemia, or by a trauma, preferably by force or by chemicals or by radiation. Preferably the injury or damage is caused by ischemia, blunt force trauma, chemical burn, or radiation burn.

[0242] In some embodiments the injury or damage is caused by a trauma. In these particular embodiments, the injury or damage is caused by force, preferably blunt force, which does not penetrate the skin. For example, the injury or damage can be a contusion or a traumatic brain injury or concussion, preferably a traumatic brain injury.

[0243] In a preferred embodiment the injury or damage is caused by ischemia. The term "ischemia" is to be understood to refer to a restriction of blood supply to any tissue, muscle group or organ. In preferred embodiments, the ischemia is in an organ, in particular the heart or brain. In a preferred embodiment, the injury or damage is caused by ischemia in the heart, which is caused by coronary heart disease or myocardial infarction, preferably myocardial infarction.

[0244] In a preferred embodiment, the injury or damage is caused by ischemia in the brain, which is caused by a stroke.

[0245] In some embodiments, the injury or damage is due to a surgical intervention. In an embodiment, the surgical intervention was in the heart. In some embodiments, the tissue injury or damage is due to Left Anterior Descending (LAD) surgery.

[0246] Administration

[0247] In some embodiments of the present invention, the RNA molecule is administered to the subject systemically. The terms "administered systemically" or "systemic administration", which are used interchangeably herein, relate to the administration of the composition into the circulatory system of the subject. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In preferred embodiments, the systemic administration is by parenteral administration, in particular subcutaneous administration, intravenous administration or intraarterial administration. Preferably, systemic administration is intravenous or intraarterial administration, preferably intravenous. Particularly suitable for systemic administration are negatively charged lipoplex particles.

[0248] In some embodiments, the RNA molecule is administered locally, which preferably means directly at the site of acute inflammation or adjacent to it. Throughout this application, this type of administration is also termed "local administration". In preferred embodiments, the local administration is via direct application of the composition, e.g., with a needle and syringe, at or adjacent to the site of acute inflammation. In a preferred embodiment, the local administration is via a catheter. In a preferred embodiment, the local administration is via a catheter in an artery, most preferred via a catheter into the heart of the subject. In some embodiments, the local administration is via a catheter into a coronary artery of the subject.

[0249] In a preferred embodiment, the local administration is intrathecal, intracerebroventricular or intraparenchymal, into the brain or spinal cord.

[0250] In some embodiments, the local administration is not during open heart surgery. Preferably local administration is not via a needle and syringe, in particular not directly into the ventricles of the heart.

[0251] In some embodiments, the method for localized expression of an RNA molecule at a site of acute inflammation comprises administering two RNA molecules, a first and a second RNA molecule. The route of administration, systemic or local administration, can be the same or can differ for the two RNA molecules. For example, while one RNA molecule is administered systemically, the other RNA molecule is administered locally. In some embodiments the two RNA molecules are administred the same way, systemic or locally. In other embodiments, the two RNA molecules are administered differently, e.g., the first RNA is administered systemically, while the second RNA is administered locally.

[0252] The systemic administration in these embodiments can be by parenteral administration, in particular subcutaneous administration, intravenous administration or intraarterial administration. Preferably, systemic administration is intravenous or intraarterial administration, preferably intravenous. The local administration in these embodiments can be via a catheter or by injection at or adjacent to the site of acute inflammation. Preferably the local administration is via a catheter in an artery, preferably a coronary artery. The local administration in these embodiments can also be intrathecal, intracerebroventricular or intraparenchymal. Preferably the local administration is into the brain via intracerebroventricular or intraparenchymal administration. Preferably the local administration is into the spinal coard via intrathecal administration.

[0253] In some embodiments, the two RNA molecules are administered concurrently or at the same time, preferably in the same composition. In a preferred embodiment, concurrently is to be understood to mean that the maximal time span between two administrations is 45 min or less, preferably 30 min or less, preferably 10 min or less.

[0254] In some embodiments, the first or the second RNA molecule is administered before the other. Preferably the first or second RNA is administered at least 1 h, at least 6 h, at least 12 h, at least 24 h, at least 2 d, at least 3 d, before the second or first RNA molecule. In a preferred embodiment, the first or second RNA molecule is administered at least 2 d before the second or first RNA molecule.

[0255] In some embodiments, the method comprises multiple administrations of the RNA molecule(s). In preferred embodiments, administration is repeated at least once, at least twice, at least thrice, at least four times, at least five times. In preferred embodiments, the first RNA molecule and the second RNA molecule are administered at the same interval or at different intervals. For example, the first RNA molecule can be administered once and the second RNA molecule can be administered at least two, preferably at least three times.

[0256] In some mebodiments, the first and second RNA molecules are administered multiple times, wherein the first and second RNA molecules are administered concurrently and individually. For example, on day one only the first RNA molecule is administered and on day three, i.e., 2 days after the first RNA molecule is administered, the first and second RNA molecules are administered concurrently.

[0257] In a preferred embodiment, on day one only the first RNA molecule is administered, on day three and five the first and second RNA molecules are administered concurrently and on day 7 only the second RNA molecule is administered.

[0258] The route of administration in these embodiments can depend on the respective RNA molecule or the needs of the subject. For example, the first RNA molecule can each time be administered systemically, whereas the second RNA molecule can each time be administered locally.

[0259] RNA molecule

[0260] The following embodiments about the RNA molecule according to the invention can apply to both, the first and / or second RNA molecule as described herein. In some embodiments, the RNA described herein is single-stranded RNA that may be translated into the respective protein upon entering cells, e.g., cells of a subject. In addition to wildtype or codon-optimized sequences encoding an amino add sequence comprising the amino acid sequence of a protein having biological activity, e.g., a pharmaceutically active protein such as antigen sequence (protein comprising an epitope), the RNA may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5'-cap, 5' UTR, 3' UTR, poly(A) structure). In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA molecule is an mRNA containing one or more or all of these elements. In some embodiments, beta-S-ARCA(Dl) (m27'2'~°GppSpG) or m273'" °Gppp(mi2'’°)ApG may be utilized as specific capping structure at the 5’-end of the RNA. As 5‘-UTR sequence, the 5'-UTR sequence of the human alpha-globin mRNA, optionally with an optimized 'Kozak sequence' to increase translational efficiency may be used. As 3'-UTR sequence, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) placed between the coding sequence and the poly(A)-tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used. These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017 / 060314, herein incorporated by reference). Alternatively, the 3'-UTR may be two re-iterated 3’-UTRs of the human beta-globin mRNA. Furthermore, a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues may be used. This poly(A)-tail sequence was designed to enhance RNA stability and translational efficiency.

[0261] The RNA described herein may encode an amino acid sequence comprising the amino acid sequence of a protein having biological activity, e.g., a pharmaceutically active protein such as for angiogenesis. The encoded amino acid sequence may comprise amino acid sequences other than the amino acid sequence of a protein having biological activity. Such other amino acid sequences may support the function or activity of the protein having biological activity. In some embodiments, such other amino acid sequences comprise an amino acid sequence enhancing antigen processing and / or presentation. Alternatively, or additionally, such other amino acid sequences comprise an amino acid sequence which breaks immunological tolerance.

[0262] In some embodiments of the present disclosure, the RNA molecule is "replicon RNA" or "replicon RNA molecule" or simply a "replicon", in particular "self-replicating RNA" or "self-amplifying RNA" or "replicable RNA molecule". A replicon RNA molecule is an RNA that is able to be replicated by an RNA- dependent RNA polymerase (replicase) by virtue of comprising nucleotide sequences that can be recognized by the replicase such that the RNA is replicated. The replicon does not necessarily encode the replicase, such that replicons can be replicated in cis (by the encoded replicase; also called a "cis- replicon") or in trans (by a replicase provided in another manner, e.g., a separate replicase encoding nucleic acid, such as an mRNA; also called "trans-replicon"). In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et a / ., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et a / ., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, f.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non- structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic add molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0263] The replicons of the present invention are not particle-forming. This means that, following inoculation of a host cell by a replicon of the present invention, the host cell does not produce virus particles, such as next generation virus particles. In one embodiment, the RNA replicon according to the invention is completely free of genetic information encoding any virus structural protein, e.g., alphavirus structural protein, such as core nucleocapsid protein C, envelope protein P62, and / or envelope protein El. Preferably, the replicon according to the present invention does not comprise a virus packaging signal, e.g., an alphavirus packaging signal. For example, the alphavirus packaging signal comprised in the coding region of nsP2 of SFV (White et a / ., 1998, J. Virol. 72:4320-4326) may be removed, e.g. by deletion or mutation. A suitable way of removing the alphavirus packaging signal includes adaptation of the codon usage of the coding region of nsP2. The degeneration of the genetic code may allow to delete the function of the packaging signal without affecting the amino acid sequence of the encoded nsP2.

[0264] In an embodiment, the RNA described herein may have modified nucleotides / nucleosides / backbone modifications. The term "RNA modification" as used herein may refer to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.

[0265] In this context, a modified RNA molecule as defined herein may contain nucleotide analogues / modifications, e.g., backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in an RNA molecule as defined herein are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the RNA molecule as defined herein. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the RNA molecule. In this context, nucleotide analogues or modifications are preferably selected from nucleotide analogues, which are applicable for transcription and / or translation.

[0266] Sugar Modifications: The modified nucleosides and nucleotides, which may be incorporated into a modified RNA molecule as described herein, can be modified in the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy" -2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), -0(CH2CH2 0)nCH2CH2 OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, wherein the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and 0. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA molecule can include nucleotides containing, for instance, arabinose as the sugar.

[0267] Backbone Modifications: The phosphate backbone may further be modified in the modified nucleosides and nucleotides, which may be incorporated into a modified RNA molecule as described herein. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate estere, hydrogen phosphonates, phosphoroam idates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene -phosphonates).

[0268] Base Modifications: The modified nucleosides and nucleotides, which may be incorporated into a modified RNA molecule as described herein can further be modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

[0269] In particular embodiments of the present invention, the nucleotide analogues / modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'- triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'- deoxy-cytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'- fluorothymidine-5'-triphosphate, 2'-0-methyl inosine-5'-triphosphate 4-thio-uridine-5'-triphosphate, 5- aminoallylcytidine-5’-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'- triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'- deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl- 2' -deoxycytidine-5' -tri-phosphate, 5-propynyl-2'- deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6- chloropurineriboside-5’-triphosphate, 7-deaza-adenosine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole- riboside-5'-triphosphate, Nl-methyladenosine-5'-triphosphate, Nl-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, N6-methylguanosine-5'- triphosphate, pseudo-uridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'- triphosphate. Particular preference may be given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7- deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate. In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 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- thiouridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl- pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l- deaza-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4- methoxy-2-thio-pse udou rid ine.

[0270] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-th io-cytid ine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0271] In other embodiments, modified nucleosides include 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-diamino-purine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyljadenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methyl-thio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2- methylthio-adenine, and 2-methoxy-adenine. In other embodiments, modified nucleosides include inosine, l-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-gua nosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0272] In some embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group. In specific embodiments, a modified nucleoside is 5'-0-(l-thiophosphate)-adenosine, 5'-0-(l-thiophosphate)-cytidine, 5'-0-(l- thiophosphate)-guanosine, 5'-0-(l-thiophosphate)-uridine or 5'-0-(l-thiophosphate)-pseudouridine.

[0273] In further embodiments, a modified RNA may comprise nucleoside modifications selected from 6-aza- cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-iiridine, 5-iodo-uridine, Nl- methyl-pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy- uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl- guanosine, 5-methyl-cytdine, 8-oxo-gua nosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2-amino-6- chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0274] In certain preferred embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0275] The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

[0276] O

[0277] The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

[0278] UTP (uridine 5'-triphosphate) has the following structure:

[0279] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0280] "Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0281] Another exemplary modified nucleoside is Nl-methyl-pseudouridine (mW), which has the structure:

[0282] Nl-methyl-pseudo-UTP has the following structure:

[0283] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:

[0284] In certain preferred embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine. In certain preferred embodiments, RNA comprises a modified nucleoside in place of at least one uridine.

[0285] In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

[0286] In certain preferred embodiments, the modified nucleoside is independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (m1ψ ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ ). In some embodiments, the modified nucleoside comprises Nl-methyl-pseudouridine (mlψ ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ψ ), Nl-methyl-pseudouridine (m1ψ ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ ) and Nl-methyl-pseudouridine (mlψ ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ ) and 5-methyl- uridine (m5U). In some embodiments, the modified nucleosides comprise Nl-methyl-pseudouridine (mlψ ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ ), Nl-methyl-pseudouridine (m1ψ ), and 5-methyl-uridine (m5U).

[0287] In certain preferred embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (rim5s2U), 5-methylaminomethyl-uridine (mnmsU), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnmsU), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(im5s2U), l-taurinomethyl-4-thio- pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mxs4ψ ), 4-thio-l- methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ ), 2-thio-l-methyl-pseudouridine, 1-methyl-l- deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4- methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methy!-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ip), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ipm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2 / -O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl- urldine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5- [3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.

[0288] In an embodiment, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine such as those described above. For example, in one embodiment, in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ ), Nl-methyl-pseudouridine (mini), and 5-methyl-uridine (m5U). In an embodiment, the RNA comprises 5-methylcytidine and Nl-methyl-pseudouridine (mlψ ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (mlψ ) in place of each uridine.

[0289] RNA molecules according to the invention may optionally be characterized by the following features: hAg-Kozak: 5’-UTR sequence of the human alpha-globin mRNA with an optimized 'Kozak sequence' to increase translational efficiency.

[0290] Glycine-serine linker (GS): Sequences coding for short linker peptides predominantly consisting of the amino adds glycine (G) and serine (S), as commonly used for fusion proteins.

[0291] FI element: The 3'-UTR is a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression. Preferably the FI element is the FI element according to SEQ ID NO: 2.

[0292] A30L70: A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells. Preferably the poly(A)-tail is the poly(A)-tail according to SEQ ID NO: 3. RNA molecules according to the invention may optionally be characterized by further features, e.g., by a 5'-cap, a 5'-UTR, a 3'-UTR, a poly(A) sequence, and / or adaptation of the codon usage for optimized translation and / or stabilization of the RNA molecule, as detailed below.

[0293] Cap

[0294] In some embodiments, the RNA molecule according to the present invention comprises a 5'-cap.

[0295] The terms "5'-cap", "cap", "5'-cap structure", "cap structure" are used synonymously to refer to a dinucleotide that is found on the 5' end of some eukaryotic primary transcripts such as precursor messenger RNA. A 5'-cap is a structure wherein a (optionally modified) guanosine is bonded to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate linkage (or modified triphosphate linkage in the case of certain cap analogs). The terms can refer to a conventional cap or to a cap analog.

[0296] "RNA which comprises a 5'-cap" or "RNA which is provided with a 5'-cap" or "RNA which is modified with a 5'-cap" or "capped RNA" refers to RNA which comprises a 5'-cap. For example, providing an RNA with a 5'-cap may be achieved by in vitro transcription of a DNA template in presence of said 5'-cap, wherein said 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post- transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus. In capped RNA, the 3' position of the first base of a (capped) RNA molecule is linked to the 5' position of the subsequent base of the RNA molecule ("second base") via a phosphodiester bond.

[0297] In one embodiment, the RNA molecule comprises a 5'-cap. In one embodiment, the RNA molecule does not comprise a 5'-cap.

[0298] The term "conventional 5'-cap" refers to a naturally occurring 5'-cap, preferably to the 7- methylguanosine cap. In the 7-methylguanosine cap, the guanosine of the cap is a modified guanosine wherein the modification consists of a methylation at the 7-position.

[0299] In the context of the present invention, the term "5'-cap analog" refers to a molecular structure that resembles a conventional 5'-cap, but is modified to possess the ability to stabilize RNA if attached thereto, preferably in vivo and / or in a cell. A cap analog is not a conventional 5'-cap.

[0300] For the case of eukaryotic mRNA, the 5'-cap has been generally described to be involved in efficient translation of mRNA: in general, in eukaryotes, translation is initiated only at the 5‘ end of a messenger RNA (mRNA) molecule, unless an internal ribosomal entry site (IRES) is present. Eukaryotic cells are capable of providing an RNA with a 5'-cap during transcription in the nucleus: newly synthesized mRNAs are usually modified with a 5'-cap structure, e.g.; when the transcript reaches a length of 20 to 30 nucleotides. First, the 5‘ terminal nucleotide pppN (ppp representing triphosphate; N representing any nucleoside) is converted in the cell to 5' GpppN by a capping enzyme having RNA 5'-triphosphatase and guanylyltransferase activities. The GpppN may subsequently be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form the mono-methylated m7GpppN cap. In one embodiment, the 5'-cap used in the present invention is a natural 5'-cap.

[0301] In the present invention, a natural 5'-cap dinucleotide is typically selected from the group consisting of a non-methylated cap dinucleotide (G(5')ppp(5’)N; also termed GpppN) and a methylated cap dinucleotide ((m7G(5')ppp(5')N; also termed m7GpppN), m7GpppN (wherein N is G) is represented by the following formula:

[0302] Capped RNA of the present invention can be prepared in vitro, and therefore, does not depend on a capping machinery in a host cell. The most frequently used method to make capped RNAs in vitro is to transcribe a DNA template with either a bacterial or bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a cap dinucleotide such as m7G(5')ppp(5')G (also called m7GpppG). The RNA polymerase initiates transcription with a nucleophilic attack by the 3'-OH of the guanosine moiety of m7GpppG on the a-phosphate of the next templated nucleoside triphosphate (pppN), resulting in the intermediate rrfGpppGpN (wherein N is the second base of the RNA molecule). The formation of the competing GTP-initiated product pppGpN is suppressed by setting the molar ratio of cap to GTP between 5 and 10 during in vitro transcription.

[0303] In preferred embodiments of the present invention, the 5'-cap (if present) is a 5'-cap analog. These embodiments are particularly suitable if the RNA is obtained by in vitro transcription, e.g. is an in vitro transcribed RNA (IVT-RNA). Cap analogs have been initially described to facilitate large scale synthesis of RNA transcripts by means of in vitro transcription. For messenger RNA, some cap analogs (synthetic caps) have been generally described to date, and they can all be used in the context of the present invention. Ideally, a cap analog is selected that is associated with higher translation efficiency and / or increased resistance to in vivo degradation and / or increased resistance to in vitro degradation.

[0304] Preferably, a cap analog is used that can only be incorporated into an RNA chain in one orientation. Pasquinelli et at., 1995, RNA J. 1:957-967) demonstrated that during in vitro transcription, bacteriophage RNA polymerases use the 7-methylguanosine unit for initiation of transcription, whereby around 40-50% of the transcripts with cap possess the cap dinucleotide in a reverse orientation (J.e., the initial reaction product is Gpppm7GpN). Compared to the RNAs with a correct cap, RNAs with a reverse cap are not functional with respect to translation of a nucleic acid sequence into protein. Thus, it is desirable to incorporate the cap in the correct orientation, i.e., resulting in an RNA with a structure essentially corresponding to m7GpppGpN etc. It has been shown that the reverse integration of the cap- dinucleotide is inhibited by the substitution of either the 2'- or the 3'-OH group of the methylated guanosine unit (Stepinski eta!., 2001, RNA J. 7:1486-1495; Peng et a!., 2002, Org. Lett. 24:161-164). RNAs which are synthesized in presence of such "anti reverse cap analogs" are translated more efficiently than RNAs which are in vitro transcribed in presence of the conventional 5'-cap m7GpppG. To that end, one cap analog in which the 3' OH group of the methylated guanosine unit is replaced by OCH3 is described, e.g., by Holtkamp eta!., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is a suitable cap dinucleotide according to the present invention:

[0305] In an embodiment, the RNA molecule of the present invention is essentially not susceptible to decapping. This is important because, in general, the amount of protein produced from synthetic mRNAs introduced into cultured mammalian cells is limited by the natural degradation of mRNA. One in vivo pathway for mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase, which contains a regulatory subunit (Dcpl) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the a and p phosphate groups of the triphosphate bridge. In the present invention, a cap analog may be selected or present that is not susceptible, or less susceptible, to that type of cleavage. A suitable cap analog for this purpose may be selected from a cap dinucleotide according to formula (I):

[0306] wherein R1is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl,

[0307] R2and R3are independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R2and R3together form O-X-O, wherein X is selected from the group consisting of optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3), and

[0308] C(CH3)2, or R2is combined with the hydrogen atom at position 4' of the ring to which R2is attached to form -O-CHz- or -CH2-O-,

[0309] R5is selected from the group consisting of S, Se, and BH3,

[0310] R4and R6are independently selected from the group consisting of 0, S, Se, and BH3, n is 1, 2, or 3.

[0311] Preferred embodiments for R1, R2, R3, R4, Rs, R6are disclosed in WO 2011 / 015347 Al and may be selected accordingly in the present invention.

[0312] For example, in an embodiment, the RNA molecule of the present invention comprises a phosphorothioate-cap-analog. Phosphorothioate-cap-analogs are specific cap analogs in which one of the three non-bridging O atoms in the triphosphate chain is replaced with an S atom, i.e., one of R4, R5or R6in Formula (I) is S. Phosphorothioate-cap-analogs have been described by Kowalska eta / ., 2008, RNA, 14:1119-1131, as a solution to the undesired decapping process, and thus to increase the stability of RNA in vivo. In particular, the substitution of an oxygen atom for a sulphur atom at the betaphosphate group of the 5'-cap results in stabilization against Dcp2. In that embodiment, which is preferred in the present invention, R5in Formula (I) is S; and R4and R6are O. In a further embodiment, the RNA molecule of the present invention comprises a phosphorothioate- cap-analog wherein the phosphorothioate modification of the RNA 5'-cap is combined with an "antireverse cap analog" (ARCA) modification. Respective ARCA-phosphorothioate-cap-analogs are described in WO 2008 / 157688 A2, and they can all be used in the RNA molecule of the present invention. In that embodiment, at least one of R2or R3in Formula (I) is not OH, preferably one among R2and R3is methoxy (OCH3), and the other one among R2and R3is preferably OH. In a preferred embodiment, an oxygen atom is substituted for a sulphur atom at the beta-phosphate group (so that R5in Formula (I) is S; and R4and R6are 0). It is believed that the phosphorothioate modification of the ARCA ensures that the a, p, and y phosphorothioate groups are precisely positioned within the active sites of capbinding proteins in both the translational and decapping machinery. At least some of these analogs are essentially resistant to pyrophosphatase Dcpl / Dcp2. Phosphorothioate-modified ARCAs were described to have a much higher affinity for eIF4E than the corresponding ARCAs lacking a phosphorothioate group.

[0313] A respective cap analog that is particularly preferred in the present invention, Ze, m2Z2'“°GppspG, is termed beta-S-ARCA (WO 2008 / 157688 A2; Kuhn et aL, 2010, Gene Ther. 17:961-971). Thus, in one embodiment of the present invention, the RNA of the present invention is modified with beta-S-ARCA. beta-S-ARCA is represented by the following structure:

[0314] In general, the replacement of an oxygen atom for a sulphur atom at a bridging phosphate results in phosphorothioate diastereomers which are designated DI and D2, based on their elution pattern in HPLC. Briefly, the DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta- S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta- S-ARCA(D2)) and thus exhibits a shorter retention time. Determination of the stereochemical configuration by HPLC is described in WO 2011 / 015347 Al.

[0315] In a first particularly preferred embodiment of the present invention, RNA molecule of the present invention is modified with the beta-S-ARCA(D2) diastereomer. The two diastereomers of beta-S-ARCA differ in sensitivity against nucleases. It has been shown that RNA carrying the D2 diastereomer of beta- S-ARCA is almost fully resistant against Dcp2 cleavage (only 6% cleavage compared to RNA which has been synthesized in presence of the unmodified ARCA 5'-cap), whereas RNA with the beta-S-ARCA(Dl) 5'-cap exhibits an intermediary sensitivity to Dcp2 cleavage (71% cleavage). It has further been shown that the increased stability against Dcp2 cleavage correlates with increased protein expression in mammalian cells. In particular, it has been shown that RNAs carrying the beta-S-ARCA(D2) cap are more efficiently translated in mammalian cells than RNAs carrying the beta-S-ARCA(Dl) cap. Therefore, in one embodiment of the present invention, RNA molecule of the present invention is modified with a cap analog according to Formula (I), characterized by a stereochemical configuration at the P atom comprising the substituent R5in Formula (I) that corresponds to that at the Pp atom of the D2 diastereomer of beta-S-ARCA. In that embodiment, R5in Formula (I) is S; and R4and R6are 0. Additionally, at least one of R2or R3in Formula (I) is preferably not OH, preferably one among R2and R3is methoxy (0CH3), and the other one among R2and R3is preferably OH.

[0316] In a second particularly preferred embodiment, the RNA molecule of the present invention is modified with the beta-S-ARCA(Dl) diastereomer. This embodiment is particularly suitable for transfer of capped RNA into immature antigen presenting cells. It has been demonstrated that the beta-S-ARCA(Dl) diastereomer, upon transfer of respectively capped RNA into immature antigen presenting cells, is particularly suitable for increasing the stability of the RNA, increasing translation efficiency of the RNA, prolonging translation of the RNA, increasing total protein expression of the RNA, and / or increasing the immune response against an antigen or antigen peptide encoded by said RNA (Kuhn etak, 2010, Gene Ther. 17:961-971). Therefore, in an alternative embodiment of the present invention, the RNA molecule of the present invention is modified with a cap analog according to Formula (I), characterized by a stereochemical configuration at the P atom comprising the substituent R5in Formula (I) that corresponds to that at the Pp atom of the Di diastereomer of beta-S-ARCA. Respective cap analogs and embodiments thereof are described in WO 2011 / 015347 Al and Kuhn et ak, 2010, Gene Ther. 17:961-971. Any cap analog described in WO 2011 / 015347 Al, wherein the stereochemical configuration at the P atom comprising the substituent R5corresponds to that at the Pp atom of the DI diastereomer of beta-S- ARCA, may be used in the present invention. Preferably, R5in Formula (I) is S; and R4and R6are O. Additionally, at least one of R2or R3in Formula (I) is preferably not OH, preferably one among R2and R3is methoxy (OCH3), and the other one among R2and R3is preferably OH.

[0317] In one embodiment, the RNA molecule of the present invention is modified with a 5'-cap structure according to Formula (I) wherein any one phosphate group is replaced by a boranophosphate group or a phosphoroselenoate group. Such caps have increased stability both in vitro and in vivo. Optionally, the respective compound has a 2'-O- or 3'-O-alkyl group (wherein alkyl is preferably methyl); respective cap analogs are termed BHa-ARCAs or Se-ARCAs. Compounds that are particularly suitable for capping of mRNA include the p-BHa-ARCAs and p-Se-ARCAs, as described in WO 2009 / 149253 A2. For these compounds, a stereochemical configuration at the P atom comprising the substituent R5in Formula (I) that corresponds to that at the Pp atom of the DI diastereomer of beta-S-ARCA is preferred.

[0318] In one embodiment, the 5' cap can be a CleanCap supplied by Trilink Biotechnologies, San Diego, CA having the following structure:

[0319] In one embodiment, the 5' cap can be a CleanCap supplied by Trilink Biotechnologies, San Diego, CA having the following structure:

[0320] UTR

[0321] The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). A 3'-UTR, if present, is located at the 3' end of a gene, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does preferably not include the poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) tail (if present), e.g. directly adjacent to the poly(A) tail.

[0322] A 5'-UTR, if present, is located at the 5’ end of a gene, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'-cap.

[0323] 5'- and / or 3'-untranslated regions may, according to the invention, be functionally linked to an open reading frame, so as for these regions to be associated with the open reading frame in such a way that the stability and / or translation efficiency of the RNA comprising said open reading frame are increased.

[0324] In some embodiments, the RNA molecule according to the present invention comprises a 5'-UTR and / or a 3'-UTR.

[0325] UTRs are implicated in stability and translation efficiency of RNA. Both can be improved, besides structural modifications concerning the 5'-cap and / or the 3' poly(A)-tail as described herein, by selecting specific 5' and / or 3' untranslated regions (UTRs). Sequence elements within the UTRs are generally understood to influence translational efficiency (mainly 5'-UTR) and RNA stability (mainly 3 -UTR). It is preferable that a 5'-UTR is present that is active in order to increase the translation efficiency and / or stability of the RNA molecule. Independently or additionally, it is preferable that a 3'-UTR is present that is active in order to increase the translation efficiency and / or stability of the RNA molecule.

[0326] The terms "active in order to increase the translation efficiency" and / or "active in order to increase the stability", with reference to a first nucleic acid sequence (e.g. a UTR), means that the first nucleic acid sequence is capable of modifying, in a common transcript with a second nucleic acid sequence, the translation efficiency and / or stability of said second nucleic acid sequence in such a way that said translation efficiency and / or stability is increased in comparison with the translation efficiency and / or stability of said second nucleic acid sequence in the absence of said first nucleic add sequence.

[0327] In one embodiment, the RNA molecule comprises a 5'-UTR derived from a eukaryotic 5'-UTR and / or a 3'-UTR derived from a eukaryotic 3'-UTR.

[0328] A 5’-UTR according to the present invention can comprise any combination of more than one nucleic acid sequence, optionally separated by a linker. A 3'-UTR according to the present invention can comprise any combination of more than one nucleic acid sequence, optionally separated by a linker.

[0329] The term "linker" according to the invention relates to a nucleic add sequence added between two nucleic acid sequences to connect said two nucleic acid sequences. There is no particular limitation regarding the linker sequence.

[0330] A 3'-UTR typically has a length of 200 to 2000 nucleotides, e.g. 500 to 1500 nucleotides. The 3'- untranslated regions of immunoglobulin mRNAs are relatively short (fewer than about 300 nucleotides), while the 3'-untranslated regions of other genes are relatively long. For example, the 3’-untranslated region of tPA is about 800 nucleotides in length, that of factor VIII is about 1800 nucleotides in length and that of erythropoietin is about 560 nucleotides in length. The 3'-untranslated regions of mammalian mRNA typically have a homology region known as the AAUAAA hexanucleotide sequence. This sequence is presumably the poly(A) attachment signal and is frequently located from 10 to 30 bases upstream of the poly(A) atachment site. 3'-untranslated regions may contain one or more inverted repeats which can fold to give stem-loop structures which act as barriers for exoribonucleases or interact with proteins known to increase RNA stability (e.g. RNA-binding proteins).

[0331] The human beta-globin 3'-UTR, particularly two consecutive identical copies of the human beta-globin 3'-UTR, contributes to high transcript stability and translational efficiency (Holtkamp eta!., 2006, Blood 108:4009-4017). Thus, in one embodiment, the RNA molecule according to the present invention comprises two consecutive identical copies of the human beta-globin 3'-LTTR. Thus, it comprises in the 5' -> 3' direction: (a) optionally a 5'-UTR; (b) an open reading frame; (c) a 3'-UTR; said 3'-UTR comprising two consecutive identical copies of the human beta-globin 3'-UTR, a fragment thereof, or a variant of the human beta-globin 3'-UTR or fragment thereof.

[0332] In an embodiment, the RNA molecule according to the present invention comprises a 3'-UTR which is active in order to increase translation efficiency and / or stability, but which is not the human beta-globin 3'-UTR, a fragment thereof, or a variant of the human beta-globin 3'-UTR or fragment thereof.

[0333] In an embodiment, the RNA molecule according to the present invention comprises a 5'-UTR which is active in order to increase translation efficiency and / or stability.

[0334] Poly(A) sequence

[0335] In some embodiments, the RNA molecule according to the present invention comprises a 3'-poly(A) sequence. The terms "3'-poly(A) sequence" "poly (A) tail", "poly(A) sequence" and "polyA structure" are used synonymously throughout this application.

[0336] According to the invention, in one embodiment, a poly(A) sequence comprises or essentially consists of or consists of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100 and preferably up to 500, preferably up to 400, preferably up to 300, preferably up to 200, and in particular up to 150, A nucleotides, and in particular about 120 A nucleotides. In this context "essentially consists of" means that most nucleotides in the poly(A) sequence, typically at least 50 %, and preferably at least 75 % by number of nucleotides in the "poly(A) sequence", are A nucleotides (adenylate), but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), C nucleotides (cytidylate). In this context "consists of" means that all nucleotides in the poly(A) sequence, i.e. 100 % by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0337] Indeed, it has been demonstrated that a 3' poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the 3’ poly(A) sequence (Holtkamp eta / ., 2006, Blood, vol. 108, pp. 4009-4017).

[0338] The present invention provides for a 3' poly(A) sequence to be attached during RNA transcription, i.e. during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassete.

[0339] In a preferred embodiment of the present invention, the 3' poly(A) cassete present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT). Such random sequence may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005004 Al. Any poly(A) cassete disclosed in WO 2016 / 005004 Al may be used in the present invention. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency.

[0340] Consequently, in a preferred embodiment of the present invention, the 3' poly(A) sequence contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides in length.

[0341] Codon usage

[0342] In general, the degeneracy of the genetic code will allow the substitution of certain codons (base triplets coding for an amino add) that are present in an RNA sequence by other codons (base triplets), while maintaining the same coding capacity (so that the replacing codon encodes the same amino add as the replaced codon). In some embodiments of the present invention, at least one codon of an open reading frame comprised by an RNA molecule differs from the respective codon in the respective open reading frame in the species from which the open reading frame originates. In that embodiment, the coding sequence of the open reading frame is said to be "adapted" or "modified". The coding sequence of an open reading frame comprised by the RNA molecule may be adapted.

[0343] For example, when the coding sequence of an open reading frame is adapted, frequently used codons may be selected: WO 2009 / 024567 Al describes the adaptation of a coding sequence of a nucleic acid molecule, involving the substitution of rare codons by more frequently used codons. Since the frequency of codon usage depends on the host cell or host organism, that type of adaptation is suitable to fit a nucleic acid sequence to expression in a particular host cell or host organism. Generally, speaking, more frequently used codons are typically translated more efficiently in a host cell or host organism, although adaptation of all codons of an open reading frame is not always required.

[0344] For example, when the coding sequence of an open reading frame is adapted, the content of G (guanylate) residues and C (cytidylate) residues may be altered by selecting codons with the highest GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames were reported to have the potential to reduce immune activation and to improve translation and half-life of RNA (Thess eta / ., 2015, Mol. Ther. 23:1457-1465).

[0345] DNA

[0346] The present invention also provides a DNA comprising a nucleic acid sequence encoding the RNA molecule(s) according to the present invention.

[0347] Preferably, the DNA is double-stranded.

[0348] In a preferred embodiment, the DNA is a plasmid. The term "plasmid", as used herein, generally relates to a construct of extrachromosoma I genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0349] The DNA of the present invention may comprise a promoter that can be recognized by a DNA-dependent RNA-polymerase. This allows for transcription of the encoded RNA in vivo or in vitro, e.g. of the RNA of the present invention. IVT vectors may be used in a standardized manner as template for in vitro transcription. Examples of promoters preferred according to the invention are promoters for SP6, T3 or T7 polymerase.

[0350] In one embodiment, the DNA of the present invention is an isolated nucleic acid molecule.

[0351] Methods of preparing RNA

[0352] The RNA molecule according to the present invention may be obtainable by in vitro transcription. In wW-transcribed RNA (IVT-RNA) is of particular interest in the present invention. IVT-RNA is obtainable by transcription from a nucleic add molecule (particularly a DNA molecule). The DNA molecule(s) of the present invention are suitable for such purposes, particularly if comprising a promoter that can be recognized by a DNA-dependent RNA-polymerase.

[0353] RNA according to the present invention can be synthesized in vitro. This allows to add cap-analogs to the in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly-(dT) sequence on the DNA template. Alternatively, capping and poly(A) tail addition can be achieved enzymatically after transcription.

[0354] The in vitro transcription methodology is known to the skilled person. For example, as mentioned in WO 2011 / 015347 Al, a variety of in vitro transcription kits is commercially available. In some embodiments, the RNA molecule or RNA, e.g., RNA comprising at least one open reading frame encoding for at least on peptide or protein, used in the present disclosure is non-immunogenic.

[0355] The term "non-immunogenic RNA" (such as "non-immunogenic mRNA") as used herein refers to RIMA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non- immunogenic, i.e.f than would have been induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non- immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription. In certain embodiments, non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and / or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.

[0356] For rendering the non-immunogenic RNA (especially mRNA) non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m3U), 5- methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio- uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl- uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl- pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnmsU), 5- carboxymethylaminomethyl-2-thio-iiridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio- uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4- thio-pseudouridine (m1s4i|j), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ ), 2-thio-l- methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ ), 5- (isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio- uridine, 2'-0-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (msUm), 2'-O-methyl-pseudouridine (qim), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (ψ ), Nl-methyl-pseudouridine (mliy) or 5-methyl-uridine (m5U), in particular Nl-methyl-pseudouridine.

[0357] In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0358] During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. Formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP may be added once or several times during synthesis of mRNA. Also, dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using £. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0359] As the term is used herein, "remove" or "removal" refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the nonseparated mixture of first and second substances.

[0360] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, e.g., dsRNA (especially mRNA) is removed from non-immunogenic RNA , such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, , at least 99.993%,, at least 99.994%, , at least 99.995%, at least 99.996%, , at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0361] Various methods can be used to determine the amount of dsRNA. For example, a sample may be contacted with dsRNA-specific antibody and the amount of antibody binding to RNA may be taken as a measure for the amount of dsRNA in the sample. A sample containing a known amount of dsRNA may be used as a reference.

[0362] For example, RNA may be spoted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCI, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For detection of dsRNA, the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected. In some embodiments, the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10-fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0363] In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non- immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by a 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor. The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non- immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0364] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

[0365] Protein

[0366] According to the invention, RNA used in the present disclosure comprises a nucleic acid sequence encoding a protein, e.g., a protein that provides for therapeutic effect. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (Le., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0367] In some embodiments, RNA used in the present disclosure comprises a nucleic acid sequence encoding a protein, e.g., a protein that provides for therapeutic effect, and is capable of expressing said protein, in particular if transferred into a cell or subject. Thus, in some embodiments, the nucleic acid used in the present disclosure contains a coding region (open reading frame (ORF)) encoding a protein, e.g., a protein that provides for therapeutic effect. In this respect, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon. In some embodiments, RNA used in the present disclosure comprises a nucleic acid sequence encoding more than one protein, e.g., two, three, four or more proteins. According to the present disclosure, the term "therapeutically active protein", ” protein provides for therapeutic effect", " protein provides for therapeutic benefit", " protein with therapeutic benefit" or "protein with therapeutic effect" means a protein that can be used in the treatment of an individual where the expression of a protein would be of benefit, e.g., in ameliorating the symptoms of a disease. Preferably, a protein with therapeutic effect has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease. In some embodiments, a protein with therapeutic effect has a positive or advantageous effect on the condition or disease state of an individual when administered to the individual in a therapeutically effective amount. A protein with therapeutic effect may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease. The term "protein provides for therapeutic effect" includes entire proteins, and can also refer to fragments thereof with therapeutic effect. It can also include variants and / or analogs of a protein with therapeutic effect.

[0368] Specific examples of proteins with therapeutic effect include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genomic engineering proteins, and blood proteins.

[0369] According to the present disclosure, the term "healing at the site of acute inflammation" means the inflammation at the site of acute inflammation has been removed, preferably completely removed, and / or tissue or organ damage is decreased or removed.

[0370] According to the present disclosure, the term "decreases inflammation at the site of acute inflammation" means that the intensity or the volume of the inflammation are decreased. This can preferably be measured by measuring levels of biomarkers in the blood or in the tissue or organ with the site of acute inflammation or by imaging techniques such as MRI or PET scan or fluorescence microscopy.

[0371] In some embodiments, the protein encoded by the RNA molecule enhances tissue or organ function, which is to be understood in that, in case of an impaired function of the tissue or organ due to a site of acute inflammation compared to the normal function of tissue or organ without a site of acute inflammation, the function of thetissue or organ is restored.

[0372] Without being bound by theory, systemic administration of the RNA molecule(s) as described herein leads to a preferential uptake of the RNA molecule(s) by immune cells, preferably monocytes and / or macrophages in case the RNA molecule(s) are formed as negatively charged particles, and subsequent movement of these immune cells to the site of acute inflammation, and / or local administration of the RNA molecule(s) and subsequent uptake of the RNA molecule(s) by immune cells, preferably macrophages and / or monocytes, results in localized expression of the protein(s) at the site of acute inflammation, which leads to healing at the site of acute inflammation and / or a decrease of inflammation.

[0373] Particularly preferred proteins encoded by the RNA molecule are immune system modulators, preferably a cytokine or a functional fragment or variant thereof. According to the present disclosure an immune system modulator is a protein, which can increase or decrease the activity of the immune system, in particular of immune cells. Particularly preferred are immune system modulators that decrease the activity of the immune system, e.g. an anti-inflammatory immune system modulator.

[0374] The term "cytokines" relates to proteins which have a molecular weight of about 5 to 60 kDa and which participate in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, when released, cytokines exert an effect on the behavior of cells around the place of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs).

[0375] In some embodiments, the protein encoded by the RNA molecule is an Interleukin as an antiinflammatory cytokine.

[0376] Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins. According to the disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal, preferably from human or mouse, most preferably from human.

[0377] Preferred Interleukins are interleukin (IL)-l receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13, IL-18 binding protein, IL-19, IL-35, IL-36 receptor antagonist, IL-37, IL-38. Particularly preferred is IL-10 or a functional fragment or variant thereof. In some embodiments, the protein encoded by the RNA molecule(s) is IL-10, IL-4 or IL-13, preferably IL-10, or a functional fragment or variant thereof.

[0378] In some embodiments, the IL-10 can be encoded by the nucleic acid sequence of any one of SEQ ID NOs: 4 to 7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. In some embodiments the RNA molecule can comprise a sequence encoding IL-10, wherein the sequence encoding IL-10 is selected from any one of SEQ ID NOs 4-7, wherein each thymine (T) is replaced with a uracil (U) or variant thereof, such as pseudouridine or 1-methylpseudouridine.

[0379] In some embodiments, the IL-10 encoded by the RNA molecule(s) comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9. The IL-10 preferably further comprises a signal peptide sequence. The signal peptide sequence may be any signal peptide sequence that is capable of directing secretion of the protein in the target subject. The signal peptide sequence is preferably an endogenous human signal peptide. The signal peptide sequence is typically located N- terminally of the IL-10 protein sequence. The signal peptide sequence may comprise or consist essentially of the sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the IL-10 encoded by the RNA molecule(s) comprises the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 10. In some preferred embodiments the IL-10 encoded by the RNA molecule(s) has the amino add sequence of SEQ ID No: 8, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 8.

[0380] In some embodiments, the protein encoded by the RNA molecule(s) is a protein that is having an effect on angiogenesis, neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis at or adjacent to the site of acute inflammation.

[0381] In some embodiments, the protein encoded by the RNA molecule(s) is a protein that is increasing angiogenesis, neurogenesis, neuronal paterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth and / or angiogenesis at or adjacent to the site of acute inflammation.

[0382] In some embodiments, the protein encoded by the RNA molecule(s) increases angiogenesis at or adjacent to the site of acute inflammation. According to the present disclosure, "angiogenesis" Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis continues the growth of the vasculature by processes of sprouting and spliting.

[0383] Proteins known to increase angiogenesis include of insulin-like growth factor 1 (IGF1), Agrin, Meteorin- Like Protein (METRNL), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), placental growth factor (PGF), granulocyte / macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Particularly preferred as a protein encoded by an RNA molecule according to the present disclosure is VEGF or a functional fragment or variant thereof.

[0384] VEGF is a sub-family of growth factors, the platelet-derived growth factor family of cystine-knot growth factors and comprises in mammals five members: VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF- C and VEGF-D. In some embodiments, the protein encoded by the RNA molecule(s) is VEGF, agrin or METRNL, or a functional fragment or variant thereof. In some embodiments, the protein encoded by the RNA molecule(s) is VEGF-A, most preferably VEGF-Aies.

[0385] In some embodiments the VEGF functional variant is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 11 to 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto.

[0386] In some embodiments, the VEGF can be encoded by the nucleic acid sequence of any one of SEQ ID NOs: 11 to 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to any one of SEQ ID NOs: 11 to 14. In some embodiments the RNA molecule can comprise a sequence encoding VEGF-A, wherein the sequence encoding VEGF-A is selected from any one of SEQ ID NOs 11 to 14, wherein each thymine (T) is replaced with a uracil (U) or variant thereof, such as pseudouridine or 1-methylpseudouridine.

[0387] In some embodiments, the VEGF, or functional variant thereof, encoded by the RNA molecule(s) comprises the amino add sequence of SEQ ID NO: 16, or an amino add sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 16. The VEGF preferably further comprises a signal peptide sequence. The signal peptide sequence may be any signal peptide sequence that is capable of directing secretion of the protein in the target subject. The signal peptide sequence is preferably an endogenous human VEGF signal peptide. The signal peptide sequence is typically located N-terminally of the VEGF protein sequence. The signal peptide sequence may comprise or consist essentially of the sequence of SEQ ID NO: 17, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the VEGF, or functional variant thereof, encoded by the RNA molecule(s) comprises the amino add sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 17. In some embodiments the VEGF, or functional variant thereof, has the amino add sequence of SEQ ID NO: 15, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 15.

[0388] In some embodiments the VEGF functional variant is VEGFies, in particular VEGFws, encoded by the nucleic acid sequence of any one of SEQ ID NOs: 11 to 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of any one of SEQ ID NOs: 11 to 14.

[0389] In some embodiments the VEGF functional variant encoded by the RNA molecule(s) is VEGFiss, in particular VEGFies, and has the amino acid sequence of SEQ ID No: 15, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 15.

[0390] In some embodiments, the protein encoded by the RNA molecule is substantially not expressed at sites other than the site of acute inflammation. This is to be understood to refer to an increased expression compared to normal levels of the protein. E.g., the protein encoded by the RNA molecule can be endogenously expressed, however, the level is not higher than normally expected in a site without acute inflammation, whereas the expression at the site of acute Inflammation is increased due to the administration of the RNA molecule. Preferably the expresseion at sites other than the site of acute inflammation differs by a factor of 2, preferably 3, preferably 4, preferably 5, preferably 10, preferably 30, preferably 100 or more. Preferably the difference means that the expression is lower by a factor such as 2 at other sites than the site of acute inflammation. The level of expression of a protein can be measured by means known to the person skilled in the art such as western blot.

[0391] In some preferred embodiments, the protein encoded by the first RNA molecule is IL-10 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is VEGF, preferably VEGF-A, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL-10 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is agrin, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL-10 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is METRNL, or a functional variant or fragment thereof.

[0392] In some embodiments, the protein encoded by the first RNA molecule is IL-4 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is VEGF, preferably VEGF-A, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL-4 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is agrin, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL-4 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is METRNL, or a functional variant or fragment thereof.

[0393] In some embodiments, the protein encoded by the first RNA molecule is IL-13 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is VEGF, preferably VEGF-A, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL- 13 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is agrin, or a functional variant or fragment thereof. In some embodiments, the protein encoded by the first RNA molecule is IL-13 or a functional variant or fragment thereof and the protein encoded by the second RNA molecule is METRNL, or a functional variant or fragment thereof.

[0394] In such embodiments, the first and second RNA molecules can be administered systemically. Alternatively in such embodiments, preferably the first RNA is administered systemically, preferably by intravenous injection, and the second RNA molecule is administered locally, preferably via a catheter. In some embodiments, the catheter may be in an artery, and the artery may be in (or leading to) an organ in which the site of acute inflammation is present, preferably the heart or brain, preferably the heart. In preferred embodiments, the first and the second RNA molecules are formulated in lipoplexes, as further described herein.

[0395] Composition

[0396] In some embodiments, the RNA molecule as described herein, preferably the first and / or second RNA molecule, are formulated in a composition with at least one lipid or polymer, preferably a cationic or cationically charged lipid.

[0397] In some embodiments, the composition comprises an RNA molecule and a reagent capable of forming negatively charged particles with the RNA molecule. In some embodiments, the composition comprises the particles formed by the RNA molecule and reagent. Preferably the particles are lipoplex particles. The particles may be negatively charged.

[0398] In some embodiments, the composition comprises at least two RNA molecules, wherein the at least two RNA molecules are comprising different open reading frames encoding for different proteins.

[0399] In some embodiments, the composition comprises exactly two RNA molecules, wherein the exactly two RNA molecules are comprising different open reading frames encoding for different proteins.

[0400] A composition described herein may additionally comprise salts, buffers, or other components as further described below.

[0401] In some embodiments, a salt for use in the compositions described herein comprises sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmolality agent for preconditioning RNA prior to mixing with lipids. In some embodiments, the compositions described herein may comprise alternative organic or inorganic sate. Alternative salts include, without limitation, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0402] Generally, compositions for storing RNA particles such as for freezing RNA particles comprise low sodium chloride concentrations, or comprises a low ionic strength. In some embodiments, the sodium chloride is at a concentration from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.

[0403] According to the present disclosure, the RNA particle compositions described herein have a pH suitable for the stability of the RNA particles and, in particular, for the stability of the RNA. Without wishing to be bound by theory, the use of a buffer system maintains the pH of the particle compositions described herein during manufacturing, storage and use of the compositions. In some embodiments of the present disclosure, the buffer system may comprise a solvent (in particular, water, such as deionized water, in particular water for injection) and a buffering substance. The buffering substance may be selected from 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)propane- 1,3-diol (Tris), acetate, and histidine. A preferred buffering substance is HEPES.

[0404] Compositions described herein may also comprise a cryoprotectant and / or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage, freezing, spray-drying and / or lyophilization, for example to reduce or prevent aggregation, particle collapse, RNA degradation and / or other types of damage.

[0405] In an embodiment, the cryoprotectant is a carbohydrate. The term "carbohydrate", as used herein, refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

[0406] In an embodiment, the cryoprotectant is a monosaccharide. The term "monosaccharide", as used herein refers to a single carbohydrate unit {e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose and the like.

[0407] In an embodiment, the cryoprotectant is a disaccharide. The term "disaccharide", as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose and the like.

[0408] The term "trisaccharide" means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose and melezitose. In an embodiment, the cryoprotectant is an oligosaccharide. The term "oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced.

[0409] In an embodiment, the cryoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide", as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1- 4 linkages or 1-6 linkages, to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, P cyclodextrin, or y cyclodextrin.

[0410] Other exemplary cyclic oligosaccharide cryoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety", as used herein refers to cyclodextrin (e.g., an a, P, or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. A cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.

[0411] Carbohydrate cryoprotectants, e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in an embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p-cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified p cyclodextrins).

[0412] An exemplary cryoprotectant is a polysaccharide. The term "polysaccharide", as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.

[0413] In some embodiments, RNA particle compositions may include sucrose. Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the compositions, thereby preventing RNA (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition. In some embodiments, RNA particle compositions may include alternative cryoprotectants to sucrose. Alternative stabilizers include, without limitation, trehalose and glucose. In a specific embodiment, an alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.

[0414] A preferred cryoprotectant is selected from the group consisting of sucrose, trehalose, glucose, and a combination thereof, such as a combination of sucrose and trehalose. In a preferred embodiment, the cryoprotectant is sucrose.

[0415] Some embodiments of the present disclosure contemplate the use of a chelating agent in an RNA composition described herein. Chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, transdiaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid. In some embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is EDTA disodium dihydrate. In some embodiments, the EDTA is at a concentration from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM or from about 0.25 mM to about 1 mM.

[0416] In an alternative embodiment, the RNA particle compositions described herein do not comprise a chelating agent.

[0417] Terms such as "stability" or "desired storage stability" as used herein may refer to physicochemical stability of the product, e.g., Tris / sucrose finished product, in unopened thawed vials for up to 24 hours at 30 °C, and in syringes for up to 24 hours at 2-8 °C and 12 hours at 30 °C. Such terms may refer to shelf-life for the product of 6 months or more when stored at -90 to -60 °C.

[0418] Particles

[0419] In some embodiments, the RNA molecule(s) is forming particles with at least one lipid or polymer, preferably a cationic or cationically charged lipid.

[0420] In some embodiments, the particles formed are lipoplex particles, preferably negatively charged lipoplex particles. Without being bound by theory the RNA particles (e.g., the RNA-lipoplex partides) specifically target an immune cell, in particular in the spleen, which immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, preferably cytotoxic T cells or regulatory T cells, and B lymphocytes. Preferably the immune cell is a monocyte or macrophage. In some embodiments, the RNA particles (e.g., RNA-LPX particles) target the spleen, in particular professional antigen presenting cells, preferably monocytes and / or macrophages or their precursors.

[0421] In some embodiments, the particles are nanoparticles, in which:

[0422] (i) the number of positive charges in the nanopartides does not exceed the number of negative charges in the nanopartides and / or

[0423] (ii) the nanoparticles have a net negative charge and / or

[0424] (iii) the zeta potential of the nanoparticles is 0 or less. Optionaly, the nanoparticles can have or can also have a charge ratio of positive charges to negative charges in the nanoparticles can be 1.4:1 or less.

[0425] In some embodiments, the charge ratio of positive charges to negative charges in the nanoparticles is between 1:1 and 1:8, preferably between 1:1 and 1:4.

[0426] The spleen targeting RNA particles described herein at physiological pH preferably have a net negative charge such as a charge ratio of positive charges to negative charges from about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0,

[0427] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" preferably includes nanopartides.

[0428] An "RNA particle" can be used to deliver RNA to a target site of interest (e.g., cell, tissue, organ, and the like). An RNA particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid or lipid-like material. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material combines together with the RNA to form aggregates, and this aggregation results in colloidally stable particles.

[0429] In one embodiment, the particles formed in the composition of the present invention are nanopartides. In that embodiment, the composition according to the present invention comprises RNA in the form of nanoparticles. Nanoparticles can be obtained by various protocols and with various complexing compounds. Lipids, polymers, oligomers, or amphiphiles are typical constituents of nanoparticulates.

[0430] As used herein, the term "nanoparticle" refers to any particle having a diameter making the particle suitable for systemic, in particular parenteral, administration, of, in particular, nucleic acids, typically a diameter of 1000 nanometers (nm) or less. In one embodiment, the nanopartides have an average diameter in the range of from about 50 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm such as about 150 nm to about 200 nm. In one embodiment, the nanopartides have a diameter in the range of about 200 to about 700 nm, about 200 to about 600 nm, preferably about 250 to about 550 nm, in particular about 300 to about 500 nm or about 200 to about 400 nm. In one embodiment, the average diameter is between about 50 to 150 nm, preferably, about 60 to 120 nm. In one embodiment, the average diameter is less than 50 nm.

[0431] Lipid

[0432] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0433] As used herein, the term "hydrophobic" refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least 6 carbon atoms. The hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution. The term "hydrocarbon" includes alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogens in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0434] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety which may have six to thirty, typically six to twenty, often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0435] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon carbon double bond in which the total carbon atoms may be six to thirty, typically six to twenty often six to eighteen.

[0436] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon carbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen. Alkynyl groups can optionally have one or more carbon carbon double bonds.

[0437] As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head atached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is Insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwiterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0438] The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" relates to substances, in particular amphiphilic substances, that structurally and / or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. Examples of lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function- spacer-sterol constructs (FSS) as well as artificial amphipathic molecules. Lipids are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble. Traditional surfactant monomers are generally cone shaped. The hydrophilic head groups tend to occupy more molecular space than the linear alkyl chains. In some embodiments, surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid" is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or dearly contradicted by context.

[0439] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, / .&, sterol-containing metabolites such as cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0440] Faty acids, or faty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 cartons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty add category are the fatty esters and faty amides.

[0441] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues atached to glycerol via a giycosidic linkage. The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer),

[0442] Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide- linked fatty acid. The faty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0443] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0444] Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coll is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0445] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0446] According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwiterionic form at a selected pH. In some embodiments, the lipid included in the composition comprising the RNA molecule is a reagent capable of forming negatively charged particles with the RNA molecule.

[0447] In some embodiments, the lipid forms particles with the RNA molecule.

[0448] In some embodiments, the particles formed from the RNA molecules and lipid are lipid nanoparticles (LNP), lipoplexes (LPX) or liposomes. Preferably, the particles are LPX particles.

[0449] In general, a lipoplex (LPX) is obtainable from mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids. In some embodiments, the lipid phase comprises liposomes.

[0450] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.

[0451] In some embodiments, liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

[0452] In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with RNAs. In some embodiments, formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA-lipoplexes. In some embodiments, these formulations are characterized by their poor encapsulation of the RNA and incomplete entrapment of the RNA.

[0453] In some embodiments, an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acid (especially mRNA) results in complexation and spontaneous formation of nucleic add lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and / or DODMA, and additional lipids, such as DOPE. In some embodiments, an RNA (especially mRNA) lipoplex particle is a nanopartide. In general, a lipid nanoparticle (LNP) is obtainable from direct mixing of RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.

[0454] Preferably, the at least one lipid comprises at least one cationic lipid, preferably is a cationic lipid. As used herein, a "cationic lipid" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

[0455] In some embodiments, the particles are RNA encapsulated in a vesicle. Such formulation is a particular composition comprising particles according to the invention. A vesicle is a lipid bilayer rolled up into a spherical shell, enclosing a small space and separating that space from the space outside the vesicle. Typically, the space inside the vesicle is an aqueous space, i.e., comprises water. Typically, the space outside the vesicle is an aqueous space, i.e., comprises water. The lipid bilayer is formed by one or more lipids (vesicle-forming lipids). The membrane enclosing the vesicle is a lamellar phase, similar to that of the plasma membrane. The vesicle according to the present invention may be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. When encapsulated in a vesicle, the RNA is typically separated from any external medium. Thus, it is present in protected form, functionally equivalent to the protected form in, e.g., a natural alphavirus. Suitable vesicles are particles, particularly nanoparticles, as described herein.

[0456] For example, RNA may be encapsulated in a liposome. In that embodiment, the composition is or comprises a liposome formulation. In some embodiments, the particles are liposomes encapsulating the RNA molecule(s). Encapsulation within a liposome will typically protect RNA from RNase digestion. It is possible that the liposomes include some external RNA (e.g. on their surface), but at least half of the RNA (and ideally all of it) is encapsulated within the core of the liposome.

[0457] Liposomes are microscopic lipidic vesicles often having one or more bilayers of a vesicle-forming lipid, such as a phospholipid, and are capable of encapsulating a drug, e.g. RNA. Different types of liposomes may be employed in the context of the present invention, including, without being limited thereto, multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multivesicular vesicles (MV), and large multivesicular vesicles (LMV) as well as other bilayered forms known in the art. The size and lamellarity of the liposome will depend on the manner of preparation. There are several other forms of supramolecular organization in which lipids may be present in an aqueous medium, comprising lamellar phases, hexagonal and inverse hexagonal phases, cubic phases, micelles, reverse micelles composed of monolayers. These phases may also be obtained in the combination with DNA or RNA, and the interaction with RNA and DNA may substantially affect the phase state. Such phases may be present in nanoparticulate RNA formulations of the present invention.

[0458] Liposomes may be formed using standard methods known to the skilled person. Respective methods include the reverse evaporation method, the ethanol injection method, the dehydration-rehydration method, sonication or other suitable methods. Following liposome formation, the liposomes can be sized to obtain a population of liposomes having a substantially homogeneous size range.

[0459] In a preferred embodiment of the present invention, the RNA is present in a liposome which includes at least one cationic lipid. Respective liposomes can be formed from a single lipid or from a mixture of lipids, provided that at least one cationic lipid is used. Preferred cationic lipids have a nitrogen atom which is capable of being protonated; preferably, such cationic lipids are lipids with a tertiary amine group. A particularly suitable lipid with a tertiary amine group is l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA). In one embodiment, the RNA according to the present invention is present in a liposome formulation as described in WO 2012 / 006378 Al: a liposome having a lipid bilayer encapsulating an aqueous core including RNA, wherein the lipid bilayer comprises a lipid with a pKa in the range of 5.0 to 7.6, which preferably has a tertiary amine group. Preferred cationic lipids with a tertiary amine group include DLinDMA (pKa 5.8) and are generally described in WO 2012 / 031046 A2. According to WO 2012 / 031046 A2, liposomes comprising a respective compound are particularly suitable for encapsulation of RNA and thus liposomal delivery of RNA. In one embodiment, the RNA according to the present invention is present in a liposome formulation, wherein the liposome includes at least one cationic lipid whose head group includes at least one nitrogen atom (N) which is capable of being protonated, wherein the liposome and the RNA have a N:P ratio of between 1:1 and 20:1. According to the present invention, "N:P ratio" refers to the molar ratio of nitrogen atoms (N) in the cationic lipid to phosphate atoms (P) in the RNA comprised in a lipid containing particle (e.g. liposome), as described in WO 2013 / 006825 Al. The N:P ratio of between 1: 1 and 20:1 is implicated in the net charge of the liposome and in efficiency of delivery of RNA to a vertebrate cell.

[0460] In one embodiment, the RNA according to the present invention is present in a liposome formulation that comprises at least one lipid which includes a polyethylene glycol (PEG) moiety, wherein RNA is encapsulated within a PEGylated liposome such that the PEG moiety is present on the liposome's exterior, as described in WO 2012 / 031043 Al and WO 2013 / 033563 Al.

[0461] In one embodiment, the RNA according to the present invention is present in a liposome formulation that does not comprise a lipid which includes a polyethylene glycol (PEG) moiety.

[0462] In one embodiment, the RNA according to the present invention is present in a liposome formulation, wherein the liposome has a diameter in the range of 60-180 nm, as described in WO 2012 / 030901 Al.

[0463] In other embodiments, the RNA according to the present invention is present in the form of an emulsion. Emulsions have been previously described to be used for delivery of nucleic acid molecules, such as RNA molecules, to cells. Preferred herein are oil-in-water emulsions. The respective emulsion particles comprise an oil core and a cationic lipid. More preferred are cationic oil-in-water emulsions in which the RNA according to the present invention is complexed to the emulsion particles. The emulsion particles comprise an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged RNA, thereby anchoring the RNA to the emulsion particles. In an oil-in-water emulsion, emulsion particles are dispersed in an aqueous continuous phase. For example, the average diameter of the emulsion particles may typically be from about 80 nm to 180 nm. In one embodiment, the composition of the present invention is a cationic oil-in-water emulsion, wherein the emulsion particles comprise an oil core and a cationic lipid, as described in WO 2012 / 006380 A2. The RNA according to the present invention may be present in the form of an emulsion comprising a cationic lipid wherein the N:P ratio of the emulsion is at least 4:1, as described in WO 2013 / 006834 Al. The RNA according to the present invention may be present in the form of a cationic lipid emulsion, as described in WO 2013 / 006837 Al. In particular, the composition may comprise RNA complexed with a particle of a cationic oil-in-water emulsion, wherein the ratio of oil / lipid is at least about 8:1 (mole:mole).

[0464] In other embodiments, the particles according to the invention comprises RNA in the format of a lipoplex. The term, "lipoplex" or "RNA lipoplex" refers to a complex of lipids and nucleic adds such as RNA. Lipoplexes can be formed of cationic (positively charged) liposomes and the anionic (negatively charged) nucleic acid. The cationic liposomes can also include a neutral "helper" lipid. In the simplest case, the lipoplexes form spontaneously by mixing the nucleic acid with the liposomes with a certain mixing protocol, however various other protocols may be applied. It is understood that electrostatic interactions between positively charged liposomes and negatively charged nucleic acid are the driving force for the lipoplex formation (WO 2013 / 143555 Al). In one embodiment of the present invention, the net charge of the RNA lipoplex particles is close to zero or negative. It is known that electro-neutral or negatively charged lipoplexes of RNA and liposomes lead to substantial RNA expression in spleen dendritic cells (DCs) after systemic administration and are not associated with the elevated toxicity that has been reported for positively charged liposomes and lipoplexes (cf. WO 2013 / 143555 Al). Therefore, in one embodiment of the present invention, the composition according to the invention comprises RNA in the format of nanopartides, preferably lipoplex nanopartides, in which (i) the number of positive charges in the nanopartides does not exceed the number of negative charges in the nanoparticles and / or (ii) the nanopartides have a net negative charge and / or (iii) the charge ratio of positive charges to negative charges in the nanopartides is 1.4:1 or less and / or (iv) the zeta potential of the nanoparticles is 0 or less. As described in WO 2013 / 143555 Al, zeta potential is a scientific term for electrokinetic potential in colloidal systems. In the present invention, (a) the zeta potential and (b) the charge ratio of the cationic lipid to the RNA in the nanoparticles can both be calculated as disclosed in WO 2013 / 143555 Al. In summary, particles which are nanoparticulate lipoplex formulations with a defined particle size, wherein the net charge of the partides is negative, as disclosed in WO 2013 / 143555 Al, are preferred particles in the context of the present invention. In other embodiments, the lipoplexes are obtained according to a method as disclosed in WO 2019 / 077053 Al. According to WO 2019 / 077053 Al, lipoplexes can be obtained by adding liposome colloid with a solution comprising RNA. The liposome colloid, according to WO 2019 / 077053 Al, can be obtained by a method comprising injecting a lipid solution in ethanol into an aqueous phase to produce the liposome colloid, wherein the concentration of at least one of the lipids in the lipid solution corresponds to or is higher than the equilibrium solubility of the at least one lipid in ethanol. A particularly preferred method of producing a liposome colloid comprises injecting a lipid solution comprising DOTMA and DOPE in a molar ratio of about 2:1 in ethanol into water stirred at a stirring velocity of about 150 rpm to produce the liposome colloid, wherein the concentration of DOTMA and DOPE in the lipid solution is about 330 mM.

[0465] In other embodiments, the lipoplexes are RNA lipoplex particles according to WO 2020 / 069632 Al comprising RNA, and at least one cationic lipid and at least one additional lipid, sodium chloride at a concentration of about 10 mM or less, a stabilizer at a concentration of more than about 10% weight by volume percent (% w / v) and less than about 15% weight by volume percent (% w / v), and a buffer. Preferably the lipoplexes according to the invention are RNA lipoplex particles comprising DOTMA and DOPE in a molar ratio of about 2:1, wherein the ratio of positive charges to negative charges in the composition is about 1.3: 2.0, sodium chloride at a concentration of about 8.2 mM, sucrose at a concentration of about 13% (w / v), HEPES at a concentration of about 5 mM with a pH of about 6.7, and EDTA at a concentration of about 2.5 mM, as described in WO 2020 / 069632 Al.

[0466] In some embodiments, the composition comprises nanoparticles formed by the RNA molecule and reagent, which are lipid nanopartides (LNPs). The LNP may comprise any lipid capable of forming a particle to which the nucleic acid molecules are atached, or in which the nucleic acid molecules are encapsulated.

[0467] In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

[0468] In one embodiment, the LNP does not comprise a PEGylated lipid.

[0469] In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0470] In one embodiment, the LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid. In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.

[0471] In one embodiment, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43 mol percent. In one embodiment, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.

[0472] In one embodiment, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugated lipid.

[0473] In one embodiment, the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0474] In one embodiment, the mol percent is determined based on total mol of lipid present in the lipid nanopartide.

[0475] In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In one embodiment, the neutral lipid is DSPC.

[0476] In one embodiment, the steroid is cholesterol.

[0477] In one embodiment, the polymer conjugated lipid is a PEGylated lipid. In one embodiment, the PEGylated lipid has the following structure. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0478] R12and R13are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In one embodiment, R12and R13are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In one embodiment, w has a mean value ranging from 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R12and R13are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

[0479] In one embodiment, the PEGylated lipid is DMG-PEG 2000, e.g., having the following structure:

[0480] In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (III): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, - C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, - (C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, - OC(=O)NRa- or -NRaC(=O)O- or a direct bond;

[0481] G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0482] G3is C1-C24 alkylene, C1-C24 alkenylene, Cs-Cs cycloalkylene, C3-C8 cycloalkenylene;

[0483] Rais H or C1-C12 alkyl;

[0484] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0485] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;

[0486] R4is C1-C12 alkyl;

[0487] R5is H or Ci-Ce alkyl; and x is 0, 1 or 2.

[0488] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0489] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

[0490] R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0491] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0492] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (HID): wherein y and z are each independently integers ranging from 1 to 12.

[0493] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-. In some different embodiments of Formula (III), the lipid has one of the following structures (HIE) or (IIIF):

[0494] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

[0495] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0496] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0497] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0498] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted.

[0499] In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene. In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: wherein:

[0500] R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0501] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0502] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures:

[0503] In some of the foregoing embodiments of Formula (III), R3is OH, CN, -C(=O)OR4, -OC(=O)R4or - NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0504] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below. Representative Compounds of Formula (III)

[0505]

[0506] In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid and a PEGylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

[0507] In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 50 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount from about 5 to about 15 mole percent. In one embodiment, the steroid is present in the LNP in an amount from about 35 to about 45 mole percent. In one embodiment, the PEGylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent.

[0508] In some embodiments, the LNP comprises compound III- 3 in an amount from about 40 to about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol in an amount from about 35 to about 45 mole percent, and ALC-0159 in an amount from about 1 to about 10 mole percent.

[0509] In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mote percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent. In various different embodiments, the cationic lipid has one of the structures set forth in the table below,

[0510] In some embodiments, the LNP comprises a cationic lipid shown in the above table, e.g., a cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is DMG-PEG 2000. In one embodiment, the LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid).

[0511] In one embodiment, the cationic lipid has the following structure:

[0512] The N / P ratio is preferably at least about 4. In some embodiments, the N / P ratio ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P ratio is about 6.

[0513] LNPs described herein may have an average diameter that in one embodiment ranges from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

[0514] In some preferred embodiments, the RNA molecule(s) are formulated in RNA lipoplex (RNA-LPX) particles. In some embodiments, such RNA lipoplex particles target the spleen.

[0515] In some embodiments, RNA lipoplex particles target immune cells, preferably monocytes and / or macrophages.

[0516] The spleen targeting RNA lipoplex particles described herein at physiological pH preferably have a net negative charge such as a charge ratio of positive charges to negative charges from about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2.2.0, about 1.1:2.0, or about 1:2.0.

[0517] In one embodiment, the nanoparticles can further comprise at least one helper lipid, wherein, for example, the helper lipid can be a neutral lipid. In various embodiments, the at least one cationic lipid can comprise l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleyloxy-3- dimethylaminopropane (DODMA), and / or l,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In various embodiments, the at least one helper lipid can comprise l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol (Choi), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In various embodiments, the molar ratio of the at least one cationic lipid to the at least one helper lipid can be from 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In one embodiment, the nanoparticles can be lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In an embodiment, the nanoparticles can be lipoplexes comprising DODMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In an embodiment, the nanoparticles can be lipoplexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In an embodiment, the nanoparticles are lipoplexes comprising DODMA:Cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

[0518] In one embodiment, the nanoparticles can be lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In an embodiment, the nanopartides can be lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2. In an embodiment, the nanoparticles can be lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0519] Polymer

[0520] In some embodiments, the RNA is formulated in a composition comprising a polymer, preferably a polyalkyleneimine.

[0521] In some embodiments, the polymer, preferably polyaklyeneimine, included in the composition comprising the RNA molecule is a reagent capable of forming negatively charged particles with the RNA molecule.

[0522] In some embodiments, the particles formed from the RNA molecules and the polymer, preferably polyaklyeneimine, are polymer-based polyplexes. Given their high degree of chemical flexibility, polymers are commonly used materials for nanopartidebased delivery. Typically, cationic polymers are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethylenimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic add delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(p-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0523] A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.

[0524] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0525] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0526] In certain embodiments, polymer may be protamine or polyalkyleneimine. In some embodiments, the reagent comprises a polyalkyleneimine, preferably is a polyalkyleneimine.

[0527] The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0528] According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0529] In one embodiment, the polyalkyleneimine comprises polyethylenimine and / or polypropyleneimine, preferably polyethylenimine. A preferred polyalkyleneimine is polyethylenimine (PEI). The average molecular weight of PEI is preferably 0.75-102to 107Da, preferably 1000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

[0530] Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethylenimine (PEI).

[0531] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In one embodiment, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0532] Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0533] In an embodiment, the RNA is formulated in a composition comprising a polyalkyleneimine, and, for example, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphor atoms (P) in the RNA molecules (N:P ratio) can be 2.0 to 15.0, preferably 6.0 to 12.0 and / or the ionic strength of the composition can be 50 mM or less, preferably wherein the concentration of monovalent cationic ions can be 25 mM or less and the concentration of divalent cationic ions can be 20 p M or less.

[0534] In an embodiment, the particles formed can be polyptexes. In an embodiment, the polyalkyleneimine can comprise the following general formula (I): wherein

[0535] R is H, an acyl group or a group comprising the following general formula (II): wherein Ri is H or a group comprising the following general formula (III): n, m, and I are independently selected from integers from 2 to 10; and p, q, and r are integers, wherein the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5-102to 107Da, preferably 5000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da. In an embodiment, n, m, and I can be independently selected from 2, 3, 4, and 5, preferably from 2 and 3 and / or Ri can be H. In an embodiment, R can be H or an acyl group.

[0536] In an embodiment, the polyalkyleneimine can comprise polyethylenimine and / or polypropylenimine, preferably polyethylenimine. In an embodiment, at least 92% of the N atoms in the polyalkyleneimine can be protonatable.

[0537] Methods of treatment

[0538] In a further aspect, the present invention provides a method for treating tissue or organ damage characterized by a site of acute inflammation in the tissue or organ in a subject, the method comprising administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one therapeutically active protein.

[0539] The treatment of tissue or organ damage is not limited to a reversal of the damage, but also includes the relief or reduction of symptoms of tissue or organ damage or partial reversal of tissue or organ damage. The herein described methods of treatment lead preferably to a reduction in inflammation, which helps reliefing or reducing symptoms. In another aspect, the invention provides a method for treating a subject having a site of acute inflammation in a tissue or organ, wherein the method comprises administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein having a regenerative or anti-inflammatory effect.

[0540] The cause for the site of acute inflammation is not particularly limited, for example, the subject may suffer from a neurodegenerative, inflammatory or autoimmune disease, from a blunt force trauma or ischemia, inparticular myocardial infarction or stroke.The methods for treating according to the invention may comprise any method for localized expression of an RNA molecule at a site of acute inflammation as previously described.

[0541] In some embodiments, the RNA molecule administered in a method for treating as described herein is formulated with a reagent capable of forming negatively charged particles with the RNA molecule.

[0542] In some embodiments of the methods for treating, the site of acute inflammation is characterized by infiltration of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation. Preferably the infiltrating immune cells are monocytes and / or macrophages, in particular macrophages.

[0543] In some embodiments of the methods for treating, the site of acute inflammation is characterized by presence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation. Preferably the immune cells present are monocytes and / or macrophages, in particular macrophages. Preferably the presence of immune cells is to mean an increase in the number of immune cells as compared to the site absent the acute inflammation.

[0544] In some embodiments of the methods for treating, the site of acute inflammation is characterized by absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation. Preferably the absence of immune cells is to mean an decrease in the number of immune cells as compared to the site absent the acute Inflammation.

[0545] Preferably the type of immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. Preferably the T lymphocytes can be cytotoxic T cells or regulatory T cells.

[0546] In some embodiments of the methods for treating, the site of acute inflammation is sterile.

[0547] In some embodiments of the methods for treating, the site of acute inflammation is a site of tissue injury or damage in the tissue or organ of a patient, preferably in the heart, brain or spinal cord, preferably in the heart. The cause of tissue damage or injury in heart or brain can be caused by ischemia. In particular, the cause for ischemia in the heart is myocardial infarction or coronary heart disease, in particular myocardial infarction. In one embodiment, the cause for ischemia in the brain is a stroke.

[0548] In some embodiments, the methods for treating further comprise administering a second RNA molecule, which second RNA molecule comprises at least one open reading frame encoding at least one protein having regenerative effect. The regenerative effect can be angiogenesis, neurogenesis, neuronal paterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance and / or glial growth. The regenerative effect can preferably be angiogenesis. The protein encoded by the RNA molecule in these embodiments can preferably be VEGF or a functional fragment or variant thereof, preferably as described herein, in particular VEGF-A.

[0549] The mode of administration of the RNA molecule(s) in the methods for treating can be any of the modes described herein, in particular systemic or local administration. Preferably the systemic administration is by parenteral administration, in particular subcutaneous administration, intravenous administration or intraarterial administration. Preferably, systemic administration is intravenous or intraarterial administration, preferably intravenous. In some embodiments the two RNA molecules are administred the same way, systemic or locally. In other embodiments, the two RNA molecules are administered differently, e.g., the first RNA is administered systemically, while the second RNA is administered locally. The local administration is preferably via a catheter, preferably in an artery, preferably in the heart or brain.

[0550] Also, the timing of administration of two RNA molecules can be any of the herein described timings, e.g., the first or second RNA molecule is administered before the second RNA molecule. Preferably the first RNA molecule is administered at least 1 h, at least 6 h, at least 12 h, at least 24 h, at least 2 d, at least 3 d, before the second RNA molecule. Preferably the first RNA molecule is administered at least 2 d before the second RNA molecule.

[0551] In some embodiments, the methods for treating are methods for treating tissue injury or damage in an organ of a patient, preferably in the heart, brain or spinal cord, preferably in the heart, wherein the method comprises administering two RNA lipoplex formulations (preferably wherein the RNA lipoplexes in the formulations are negatively charged), wherein (i) the first RNA lipoplex formulation comprises an RNA molecule encoding IL-10, IL-4 or IL-13, preferably IL-10, or a functional fragment or variant thereof; and (ii) the second RNA lipoplex formulation comprises an RNA molecule encoding VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof. In some such embodiments, (i) the first RNA lipoplex formulation is administered systemically and the second RNA-lipoplex formulation is administered locally, as defined herein; (ii) the first RNA lipoplex formulation and the second RNA-lipoplex formulation are both administered systemically; (iii) the first RNA-lipoplex formulation and the second RNA-lipoplex formulation are both administered locally, as defined herein; or (iv) the first RNA-lipoplex formulation is administered locally, as defined herein, and the second RNA- lipoplex formulation is administered systemically, as defined herein. In some embodiments, the preferred form of administration is (i).

[0552] In a further aspect, the invention provides a method for treating tissue damage in the heart or brain caused by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10, IL-4, or IL-13, preferably IL-10, or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof. In some embodiments, each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule. In preferred embodiments, each RNA molecule is formulated as RNA lipoplex particles, as described herein. Preferably, the first RNA molecule is administered systemically and the second RNA molecule is administered locally. The invention further provides a method for treating acute coronary syndrome (such as myocardial infarction), or stroke in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10, IL-4, or IL-13, preferably IL-10, or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof. In some embodiments, such methods typically treat the tissue damage in the heart or brain caused by ischemia, which is caused by the acute coronary syndrome or stroke in the subject.

[0553] In a further aspect, the invention provides a method for treating tissue damage in the heart or brain caused by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10 or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF or a functional fragment or variant thereof; where each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule; and wherein the first RNA molecule is administered systemically and the second RNA molecule is administered locally, wherein the first RNA molecule is administered first and, two days and four days after the first RNA molecules has been administered, the first and second RNA molecules are administered concurrently and six days after the first RNA molecules has been administered, the second RNA molecule is administered.

[0554] Without being bound by theory, the first RNA molecule encoding for IL-10 or a functional fragment or variant thereof is administered first to inhibit the inflammatory response and afterwards the second RNA molecule is co-administered for a regenerative effect, in particular for angiogenesis, neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance and / or glial growth.

[0555] In certain embodiments, additional treatments may be administered to a patient in combination with the treatments described herein. Such additional treatments include classical cancer therapy, e.g., radiation therapy, surgery, hyperthermia therapy and / or chemotherapy.

[0556] In some embodiments, the additional treatment can be surgery to restore blood flow, in particular after a myocardial infarction, e.g., bypass surgery, or stroke. In some embodiments, the additional treatment can be surgery to release pressure in the central nervous system or relief pressure on the central or peripheral nervous system. The additional treatment may include the administration of additional drugs or medical preparations.

[0557] Medical preparation or kit

[0558] An aspect of the invention is the provision of a medical preparation or kit comprising an RNA molecule or a composition as described herein.

[0559] An aspect of the invention is the provision of a medical preparation or kit comprising a first RNA molecule comprising at least one open reading frame coding for IL-10, IL-4, or IL-13, preferably IL-10, or a functional fragment or variant thereof, and a second RNA molecule comprising at least one open reading frame coding for VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof.

[0560] In some embodiments, the first RNA molecule is formulated in lipoplex particles, in an RNA lipoplex formulation, as described herein. In some embodiments, the first RNA molecule Is formulated with a reagent capable of forming negatively charged particles with the RNA molecule. In a preferred embodiment, the reagent comprises at least one lipid.

[0561] In some embodiments, the second RNA molecule is formulated in lipoplex particles, in an RNA lipoplex formulation, as described herein. In some embodiments, the second RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule. In a preferred embodiment, the reagent comprises at least one lipid. In some embodiments, the first and second RNA molecules are each formulated in lipoplex particles, in a first and a second RNA lipoplex formulations, as described herein. In some embodiments, the first and second RNA molecules are formulated with a reagent capable of forming negatively charged particles with the RNA molecules. In a preferred embodiment, the reagent comprises at least one lipid or polymer as described herein.

[0562] In some embodiments, the lipoplex formulation comprises a cationic or cationically ionizable lipid and a helper lipid. In some embodiments, the cationic or cationically ionizable lipid may comprise DOTMA, DODMA, and / or DOTAP. In some embodiments, the helper lipid may comprise DOPE, Choi, DOPC, and / or DSPC. In some embodiments, the RNA lipoplex formulation comprises lipoplex particles comprising DOTMA, DODMA or DOTAP (preferably DOTMA); and DOPE, Choi or DSPC (preferably DOPE); in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 (such as about 2:1) and wherein the charge ratio of positive charges in DOTMA, DODMA or DOTAP (preferably DOTMA) to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

[0563] Thus, in some embodiments, the invention further provides a medical preparation or kit comprising:

[0564] (i) a first RNA lipoplex formulation comprising RNA lipoplex particles comprising a first RNA molecule, comprising at least one open reading frame coding for IL-10, IL-4, or IL-13, preferably IL-10, or a functional fragment or variant thereof; optionally wherein the RNA lipoplex particles comprise DOTMA and DOPE, optionally in a molar ratio of 7:3 to 5:5 (such as about 2:1), and optionally wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4:2 to 1.1:2 (such as about 1.2:2); and

[0565] (ii) a second RNA lipoplex formulation comprising RNA lipoplex particles comprising a second RNA molecule comprising at least one open reading frame coding for VEGF, agrin, or METRNL, preferably VEGF, or a functional fragment or variant thereof; optionally wherein the RNA lipoplex particles comprise DOTMA and DOPE, optionally in a molar ratio of 7:3 to 5:5 (such as about 2:1), and optionally wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4:2 to 1.1:2 (such as about 1.2:2).

[0566] In some mebodiments, the first and second RNA molecule (or first and second RNA lipoplex formulations) are included in the medical preparation or kit in separate containers.

[0567] In some embodiments, the first and second RNA molecule (or first and second RNA lipoplex formulations) are included in the medical preparation or kit together in one container. In some embodiments, the medical preparation may be administered in the form of any suitable medical preparation.

[0568] In some embodiments, the medical preparation or kit is for therapeutic or prophylactic treatments, e.g., for use as a medicine. In some embodiments, the medical preparation or kit is for use in a method according to the invention.

[0569] In a further aspect, the invention provides a medical preparation or kit for use in any of the methods of treatment as described herein.

[0570] In a preferred embodiment, the embodiments relating to methods described herein relate to a medical preparation or kit described herein, too.

[0571] The term "medical preparation" relates to a composition comprising a therapeutically effective agent, according to the invention one or two RNA molecules, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said medical preparation is useful for treating, preventing, or reducing the severity of a disease by administration of said medical preparation to a subject.

[0572] The medical preparations or kits of the present disclosure may further comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, IPS, GP96, CpG oligodeoxy nucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-y, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0573] The medical preparations of the present disclosure may be in a storable form (e.g., in a frozen, spray- dried or lyophilized / freeze-dried form) or in a "ready-to-use form" (Ze, in a form which can be immediately administered to a subject, e.g., without any processing such as diluting). Thus, prior to administration of a storable form of a medical preparation, this storable form has to be processed or transferred into a ready-to-use or administrable form. Eg., a frozen medical preparation has to be thawed, or a freeze-dried medical preparation has to be reconstituted, e.g. by using a suitable solvent (e.g., deionized water, such as water for injection) or liquid (e.g., an aqueous solution).

[0574] The medical preparations according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

[0575] The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the medical preparation.

[0576] The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In some embodiments relating to the treatment of a particular disease, the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the medical preparationss described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the medical preparations described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0577] The medical preparation or kit of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the medical preparations of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0578] Suitable preservatives for use in the medical preparations of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0579] The term "excipient" as used herein refers to a substance which may be present in a medical preparation of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants. The term "diluent" relates a diluting and / or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and / or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

[0580] The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the medical preparation. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymere, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0581] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0582] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0583] In a further aspect of the invention, the medical preparation or kit according to the invention are used for the manufacture of a medicament for any of the herein disclosed methods.

[0584] Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

[0585] The description (including the following examples) is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims. DESCRIPTION OF THE FIGURES

[0586] Figure 1:

[0587] Standard RNA-LPX act Interferon a receptor (IFNAR)-dependent in macrophages and are immersed by mono-mac after myocardial infarction. (A) Expression of eGFP in eDCs, monocytes and macrophages in the presence (top row) or absence (bottom row) of the interferon-o / p receptor (IFNAR) 24 h after i.v. injection of IPX-formulated mRNAs and saline (control) (n=3). (B) For the evaluation of LPX-localization into infarcted myocardium, mice were injected intravenously with three different doses of Luc mRNA on day 2 after LAD ligation. Sham-operated animals served as controls. 24 h later, hearts were harvested and analyzed for luciferase activity. The luminescence was measured in relative light unit (RLU). n=3 per group. (C) Representative images of immunofluorescent labeling after administration of LPX- encapsulated enhanced green fluorescent protein (eGFP, green) mRNA and co-staining with CDllb (red). DAPI in blue. (D) FACS analysis for cell specific uptake of IPX into infarcted hearts on day 4 after MI. eGFP in green, Luc in black. n=3-5 mice per group. * indicates pcO.Ol.

[0588] Figure 2:

[0589] RNA-LPX-mediated overexpression of IL-10 after myocardial infarction. (A) IL- 10 blood kinetics 3, 6 and 24 h after application of different doses of IL-10 mRNA or rIL-10 in uninfarcted mice (n=3). (B to D) Time-line of the respective administration and analysis. On day 3 after MI, mice were injected with 40 pg IL-10 or Luc encapsuled with LPX. Additional groups included sham operated animals, injection of rIL-10 or Mono / Mac depleted animals (Clo-Lip). 24 hours after LPX administration, hearts were removed for analyses. (C) illustrates IL-10 expression on mRNA level as measured by quantitative PCR (q-PCR). (D) illustrates protein analysis by western blot of whole heart tissue. (E to H) CDllb-positive cells were isolated from the removed hearts. Expression levels of IL-10, IL-ip, IL-6, and TNF-a were analyzed by q-PCR. n=4-8 mice per group. One-way ANOVA analysis was applied for comparison of the different groups, *p<0.05; ** / K0.01.

[0590] Figure 3:

[0591] IL-10 mRNA accelerates the resolution of infarct inflammation and impacts STAT3 phosphorylation. (A) Representative dot blots illustrating the flow cytometric analysis of hearts from treatment of 40 pg Luc mRNA or 40 pg IL- 10 mRNA on day 3 after MI. Quantification of the percentage of Ly6ChighF4 / 80lowmono-mac (B) and Ly6lowF4 / 80highmacrophages (C) in the respective groups after previous gating through CDllb and lineage markers. n=8 per group. (D) Analysis of phosphorylated and total STATS after overexpression of IL-10 in monocytes / macrophages in vitro. (E) Ratio of phosphorylated STAT3 to total STAT3 in vitro. (F) Analysis of STAT3 after isolation of CD11-positive cells from hearts on day 3 after MI. (G) shows the respective quantification, n =3-4 mice per group. One-way ANOVA analysis was applied for comparison of the different groups, students't-test was performed for the analysis in B and C. * indicates p<0.05.

[0592] Figure 4:

[0593] IL-10 improves healing after myocardial infarction. (A) Experimental set-up indicating the different timeframes of 40 pg IL-10 mRNA administration. (B) Serum cardiac Troponin T (cTnT) levels on day 1 post MI. (C) heart to body weight ratio on day 28 after MI. (D) Ejection fraction (EF) measured by echocardiography on day 28 after MI. (E) Cardiac mRNA levels (relative fold change) of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), / 2-MHC (myosin heavy chain p) and collagen I 28 days after LAD ligation. (F to J). Treatment using a dose 4 pg of IL-10 mRNA per administration (timeframe according to the „early treatment" scheme illustrated in Figure 4A.). (F) Serum Troponin T levels on day 1 after MI. (G) Quantification of histological scar thickness. (H and I) illustrate echocardiographic measurements on day 28 after MI. Images in (J) depict representative strain analyses from the three groups of mice investigated. For histological analyses hearts were stained with Masson's trichrome. n=6-10 mice per group. One-way ANOVA or student's t test analyses were applied, n.s.: not significant, *p<0.05; ** / K0.01.

[0594] Figure 5:

[0595] Mono-mac targeted VEGF overexpression after myocardial infarction. (A) Transfection of macrophages with 2 pg of different VEGF-Isoforms and evaluation of VEGFiw expression by q-PCR. (B) ...

Claims

We claim:

1. A method for localized expression of an RNA molecule at a site of acute inflammation present in a subject, wherein the method comprises administering to the subject the RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein.

2. The method according to claim 1, wherein the site of acute inflammation is a single localized site of acute inflammation.

3. The method according to claim 1 or 2, wherein the site of acute inflammation is characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

4. The method according to any one of claims 1 to 3, wherein the site of acute inflammation is characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation.

5. The method according to claim 3 or 4, wherein the type of immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

6. The method according to claim 5, wherein the T lymphocytes can be cytotoxic T cells or regulatory T cells.

7. The method according to any one of claims 3 to 6, wherein the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

8. The method according to any one of claims 3 to 7, wherein the site of acute inflammation is characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Ghighand / or CD14+and / or CD16+and / or CD68+and / or CCR2+and / or MHC class II+immune cells.

9. The method according to any one of claims 1 to 8, wherein the site of acute inflammation is sterile.

10. The method according to any one of claims 1 to 9, wherein the site of acute inflammation is in a tissue or organ.

11. The method according to any one of claims 1 to 10, wherein the site of acute inflammation is a site of injury or damage in a tissue or organ.

12. The method according to claim 10 or 11, wherein the organ is heart, brain, spinal cord, lung, kidney, or liver.

13. The method according to claim 10 or 11, wherein the tissue is vascular tissue or nerve tissue.

14. The method according to any one of claims 11 to 13, wherein the injury or damage is caused by ischemia, blunt force trauma, chemical burn, or radiation burn.

15. The method according to any one of claims 11 to 14, wherein the injury or damage results in a reduction in or loss of function of the organ or tissue.

16. The method according to claim 14 or 15, wherein the injury or damage is caused by ischemia.

17. The method according to claim 16, wherein the ischemia is in the heart caused by coronary heart disease or myocardial infarction.

18. The method according to claim 16, wherein the ischemia is in the brain caused by a stroke.

19. The method according to any one of claims 1 to 18, wherein the RNA molecule is administered systemically and / or is administered locally to the site of acute inflammation.

20. The method according to claim 19, wherein systemic administration is by subcutaneous injection, intravenous administration, or intraarterial administration.

21. The method according to claim 19, wherein local administration is via a catheter or by injection at or adjacent to the site of acute inflammation.

22. The method according to claim 21, wherein local administration is via a catheter in an artery.

23. The method according to claim 22, wherein the artery is a coronary artery.

24. The method according to any one of claims 1 to 23, wherein the method comprises administering two RNA molecules, a first and a second RNA molecule, to the subject, wherein the second RNA molecule comprises at least one open reading frame encoding for at least one protein different from that encoded by the first RNA molecule.

25. The method according to claim 24, wherein the second RNA molecule is administered systemically and / or is administered locally to the site of acute inflammation.

26. The method according to claim 25, wherein systemic administration is by subcutaneous injection, intravenous administration, or intraarterial administration,27. The method according to claim 25, wherein local administration is via a catheter or by injection at or adjacent to the site of acute inflammation.

28. The method according to claim 27, wherein local administration is via a catheter in an artery.

29. The method according to claim 28, wherein the artery is a coronary artery.

30. The method according to any one of claims 24 to 29, wherein the second RNA molecule is administered in the same manner as the first RNA molecule.

31. The method according to any one of claims 24 to 29, wherein the second RNA molecule is administered in a different manner as the first RNA molecule.

32. The method according to claim 31 wherein the second RNA molecule is administered locally and the first RNA is administered systemically.

33. The method according to any one of claims 24 to 32, wherein the first RNA molecule is administered before the second RNA molecule.

34. The method according to claim 33, wherein the first RNA molecule is administered at least 1 h, at least 6 h, at least 12 h, at least 24 h, at least 2 d, at least 3 d, before the second RNA molecule.

35. The method according to any one of claims 24 to 32, wherein the second RNA molecule is administered before the first RNA molecule.

36. The method according to claim 35, wherein the second RNA molecule is administered at least 1 h, at least 6 h, at least 12 h, at least 24 h, at least 2 d, at least 3 d, before the first RNA molecule.

37. The method according to any one of claims 24 to 32, wherein the first and the second RNA molecules are administered concurrently.

38. The method according to any one of claims 1 to 37, wherein the method comprises multiple administrations of the RNA molecule(s).

39. The method according to claim 38, wherein administration is repeated at least once, at least twice, at least thrice, at least four times, at least five times.

40. The method according to any one of claims 1 to 39, wherein the RNA molecule(s) is a replicable or non-replicable RNA molecule.

41. The method according to any one of claims 1 to 39, wherein the RNA molecule(s) is linear or circular mRNA.

42. The method according to any one of claims 1 to 41, wherein the RNA molecule(s) comprises one or more of the following elements: a 5'-cap, a 5' UTR, a 3' UTR and / or a poly A structure.

43. The method according to claim 42, wherein the 5'-cap is a cap analog.

44. The method according to claim 42 or 43, wherein the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1.

45. The method according to any one of claims 42 to 44, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.

46. The method according to any one of claims 42 to 45, wherein the poly A structure is an interrupted poly A structure.

47. The method according to any one of claims 42 to 45, wherein the poly A structure comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 3.

48. The method according to any one of claims 1 to 41, wherein the RNA molecule(s) does not comprise one or more of the following elements: a 5'-cap, a 5' UTR, a 3' UTR and / or a poly A structure.

49. The method according to any one of claims 1 to 48, wherein the RNA molecule(s) is non- immunogenic.

50. The method according to any one of claims 1 to 49, wherein the RNA molecule(s) comprises a nucleotide modification.

51. The method according to claim 50, wherein the modification is the substitution of one or more U residues with pseudouridine, Nl-methylpseudoruridine or 5-methyluridine.

52. The method according to claim 51, wherein the one or more substituted U residues is Nl- methylpseudouridine.

53. The method according to claim 51 or 52, wherein at least 50%, at least 70%, at least 90% at least 99% or 100% of the U residues in the RNA molecule(s) are substituted.

54. The method according to any one of claims 1 to 53, wherein the protein encoded by the RNA molecule(s) provides a therapeutic effect or benefit.

55. The method according to any one of claims 1 to 54, wherein the protein encoded by the RNA molecule(s) enhances healing at the site of acute inflammation.

56. The method according to any one of claims 1 to 55, wherein the protein encoded by the RNA molecule(s) decreases inflammation at the site of acute inflammation.

57. The method according to any one of claims 1 to 56, wherein the protein encoded by the RNA molecule(s) enhances organ or tissue function.

58. The method according to claim 57, wherein the organ is the heart or brain.

59. The method according to any one of claims 1 to 58, wherein the protein encoded by the RNA molecule(s) is an immune system modulator.

60. The method according to claim 59, wherein the immune system modulator is a cytokine or a functional fragment or variant thereof.

61. The method according to claim 60, wherein the cytokine is an anti-inflammatory cytokine.

62. The method according to any one of claims 59 to 61, wherein the immune system modulator is interleukin (IL)-l receptor antagonist, IL-4, , IL-10, IL-11, IL-13, IL-18 binding protein, IL-19, IL-35, IL-36 receptor antagonist, IL-37, IL-38 or TGF-p, or a functional fragment or variant thereof.

63. The method according to claim 62, wherein the immune system modulator is IL- 4, IL-10, or IL-13, or a functional fragment or variant thereof.

64. The method according to claim 63, wherein the immune system modulator is IL-10 or a functional fragment or variant thereof.

65. The method according to any one of claims 60 to 64, wherein the cytokine is a functionally equivalent molecule that binds to and activates its receptor.

66. The method according to any one of claims 1 to 65, wherein the protein encoded by the RNA molecule(s) increases neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth or angiogenesis at or adjacent to the site of acute inflammation.

67. The method according to claim 66, wherein the protein encoded by the RNA molecule(s) that increases neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth or angiogenesis is selected from the group consisting of Insulin-like growth factor 1 (IGF1), Agrin, Meteorin-Like Protein (METRNL), basic fibroblast growth factor (bFGF), angiopoietin 1 (ANG1), placental growth factor (PGF), granulocyte / macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF), or a functional fragment or variant thereof.

68. The method according to claim 66 or 67, wherein the protein encoded by the RNA molecule(s) that increases neurogenesis, neuronal paterning, neuronal guidance, neuronal migration,neuroprotection, axon guidance, glial growth or angiogenesis is VEGF, agrin, or METRNL, or a functional fragment or variant thereof.

69. The method according to claim 68, wherein the protein encoded by the RNA molecule(s) that increases neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth or angiogenesis is VEGF.

70. The method according to claim 69, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C, or VEGF-D.

71. The method according to claim 69, wherein VEGF is VEGF164 or VEGFies.

72. The method according to any one of claims 1 to 71, wherein the protein encoded by the RNA molecule(s) is a human protein.

73. The method according to any one of claims 1 to 72, wherein the protein encoded by the RNA molecule(s) is substantially not expressed at sites other than the site of acute inflammation.

74. The method according to claim 73, preferably wherein the expression at the sites other than the site of acute inflammation differs by a factor of 2, 3, 4, 5, 10, 20, 30, 100 or more.

75. The method according to any one of claims 1 to 74, wherein the subject is administered two RNA molecules, the first RNA molecule encodes IL-10 or a functional fragment or variant thereof, and the second RNA molecule encodes VEGF-A or a functional fragment or variant thereof, wherein the first RNA is administered systemically and the second RNA is administered locally.

76. The method according to claim 75, wherein the systemic administration is by intravenous injection and the local administration is intrathecal, intracerebroventricular, intraparenchymal or via a catheter in an artery.

77. The method according to claim 76, wherein the artery is in an organ in which the site of acute inflammation is present.

78. The method according to claim 77, wherein the organ is heart or brain.

79. The method according to any one of claims 1 to 78, wherein the RNA molecule(s) is formulated in a composition with at least one lipid.

80. The method according to claim 79, wherein the RNA molecule(s) and at least one lipid form particles.

81. The method according to claim 80, wherein the particles are lipid nanopartides (LNP), or lipoplexes (LPX) or liposomes.

82. The method according to claim 80 or 81, wherein the partides are nanoparticles, in which:(i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or(ii) the nanoparticles have a net negative charge, and / or(iii) the zeta potential of the nanoparticles is 0 or less.

83. The method according to claim 82, wherein the charge ratio of positive charges to negative charges in the nanoparticles is between 1:1 and 1:8, preferably between 1:1 and 1:4.

84. The method according to any one of claims 79 to 83, wherein the at least one lipid is a cationic lipid.

85. The method according to claim 84, wherein the lipid comprises a cationic head group.

86. The method according to claim 83 or 84, wherein the lipid is a pH responsive lipid.

87. The method according to any one of claims 79 to 86, wherein the at least one lipid is a PEGylated-lipid.

88. The method according to any one of claims 79 to 87, wherein the composition further comprises at least one helper lipid.

89. The method according to claim 88, wherein the helper lipid is a neutral lipid.

90. The method according to any one of claims 84 to 89, wherein the at least one cationic lipid comprises l,2-di-O-octadecenyl-3-trimethylamrnonium propane (DOTMA), l,2-dioleyloxy-3- dimethylaminopropane (DODMA), and / or l,2-dio!eoyl-3-trimethylammonium-p’'opane (DOTAP).

91. The method according to any one of claims 84 to 90, wherein the at least one helper lipid comprises l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Choi), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

92. The method according to claim 82 or 83, wherein the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1.

93. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

94. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DODMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

95. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DODMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

96. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DODMA:Cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

97. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negativecharges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

98. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

99. The method according to any one of claims 82 to 92, wherein the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2 and even more preferably about 1.2:2.

100. The composition according to claim 80 or 81, wherein the particles are LNPs, which are complexed with and / or encapsulate the RNA molecule(s).

101. The method according to claim 80 or 81, wherein the particles are vesicles encapsulating the RNA molecule, preferably unilamellar liposomes.

102. A method for treating tissue or organ damage characterized by a site of acute inflammation in the tissue or organ in a subject, the method comprising administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one therapeutically active protein.

103. The method according to claim 102, wherein the RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule.

104. The method according to claim 102 or 103, wherein the site of acute inflammation is characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

105. The method according to any one of claims 102 to 104, wherein the site of acute inflammation is characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation.

106. The method according to claim 104 or 105, wherein the type of immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

107. The method according to claim 106, wherein the T lymphocytes can be cytotoxic T cells or regulatory T cells.

108. The method according to any one of claims 102 to 107, wherein the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

109. The method according to any one of claims 102 to 108, wherein the site of acute inflammation is characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Ghi9himmune cells.

110. The method according to any one of claims 102 to 109, wherein the site of acute inflammation is sterile.

111. The method according to claim 102 to 110, wherein the therapeutically active protein has an anti-inflammatory effect.

112. The method according to claim 102 to 111, wherein the therapeutically active protein induces neurogenesis, neuronal patterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth or angiogenesis.

113. The method according to any one of claims 102 to 112, which method further comprises administering a second RNA molecule, which second RNA molecule comprises at least one open reading frame encoding for at least one different therapeutically active protein.

114. The method according to claim 113, wherein one therapeutically active protein is IL-4, IL-10, or IL-13, or a functional fragment or variant thereof and the other therapeutically active protein is VEGF, agrin, or METRNL, or a functional fragment or variant thereof.

115. The method according to claim 114, wherein the RNA molecule encoding IL-4, IL- 10, or IL-13 is administered systemically and the RNA molecule encoding VEGF, agrin, or METRNL is administered locally.

116. A method for treating a subject having a site of acute inflammation in a tissue or organ, wherein the method comprises administering to the subject an RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein having a regenerative or anti-inflammatory effect.

117. The method according to claim 116, wherein the RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule.

118. The method according to claim 116 or 117, wherein the site of acute inflammation is characterized by the infiltration of and / or presence of and / or absence of one or more types of immune cells at the site of acute inflammation compared to the site absent the acute inflammation.

119. The method according to any one of claims 116 to 118, wherein the site of acute inflammation is characterized by an increase or decrease in the number of immune cells present at the site of acute inflammation compared to the site absent the acute inflammation.

120. The method according to claim 118 or 119, wherein the type of immune cell is selected from the group consisting of neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

121. The method according to claim 120, wherein the T lymphocytes can be cytotoxic T cells or regulatory T cells.

122. The method according to any one of claims 116 to 121, wherein the site of acute inflammation is characterized by the infiltration of and / or increased presence / number of macrophages, neutrophils, T cells, B cells and / or natural killer cells.

123. The method according to any one of claims 116 to 122, wherein the site of acute inflammation is characterized by the infiltration and / or increased amount of CD45+and / or CDllb+and / or Ly6Ghighimmune cells.

124. The method according to any one of claims 116 to 123, wherein the site of acute inflammation is sterile.

125. The method according to any one of claims 116 to 124, wherein the site of acute inflammation is a site of tissue damage or injury in the tissue or organ.

126. The method according to any one of claims 116 to 125, wherein the organ is heart or brain.

127. The method according to claim 126, wherein the tissue damage or injury in the heart is caused by ischemia due to a myocardial infarction.

128. The method according to claim 126, wherein the tissue damage or injury in the brain is caused by ischemia due to a stroke.

129. The method according to any one of claims 116 to 128, wherein the protein is IL-4, IL-10, or IL-13 or a functional fragment or variant thereof.

130. The method according to claim 129, wherein the protein is IL-10 or a functional fragment or variant thereof.

131. The method according to any one of claims 116 to 130, which method further comprises administering a second RNA molecule, which second RNA molecule comprises at least one open reading frame encoding at least one protein having regenerative effect-132. The method according to claim 131, wherein the regenerative effect is induction of neurogenesis, neuronal paterning, neuronal guidance, neuronal migration, neuroprotection, axon guidance, glial growth or angiogenesis.

133. The method according to claim 131 or 132, wherein the protein is Agrin, VEGF, or METRNL or a functional fragment or variant thereof.

134. The method according to claim 133, wherein the protein is VEGF or a functional fragment or variant thereof.

135. The method according to any one of claims 131 to 134, wherein the RNA molecule encoding IL-4, IL-10, or IL-13 is administered systemically and the RNA molecule encoding Agrin, VEGF, or METRNL is administered locally.

136. A method for treating tissue damage in the heart or brain caused by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-10 or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding VEGF or a functional fragment or variant thereof; where each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule; and wherein the first RNA molecule is administered systemically and the second RNA molecule is administered locally.

137. The method according to claim 136, wherein the systemic administration is by intravenous administration and the local administration is via a catheter in the heart or brain at or adjacent to the site of tissue damage.

138. A medical preparation or kit comprising a) a first RNA molecule comprising at least one open reading frame coding for IL-4, IL-10, or IL- 13, or a functional fragment or variant thereof, and b) a second RNA molecule comprising at least one open reading frame coding for Agrin, VEGF, or METRNL, or a functional fragment or variant thereof.

139. The medical preparation or kit according to claim 138, wherein the first RNA molecule comprises at least one open reading frame coding for IL-10 or a functional fragment or variant thereof and the second RNA molecule comprises at least one open reading frame coding for VEGF or a functional fragment or variant thereof.

140. The medical preparation or kit according to claim 138 or 139, wherein the first and / or second RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule(s).

141. The medical preparation or kit according to any one of claims 138 to 140, wherein the first and second RNA molecules are in separate containers.

142. The medical preparation or kit according to any one of claims 138 to 141 for use as a medicament.

143. The medical preparation according to any one of claims 138 to 142 for use in a method for treating tissue damage in the heart or brain cause by ischemia in a subject, wherein the method comprises administering to the subject (i) a first RNA molecule encoding IL-4, IL- 10, or IL-13, or a functional fragment or variant thereof, and (ii) a second RNA molecule encoding Agrin, VEGF, or METRNL, or a functional fragment or variant thereof; where each RNA molecule is formulated with a reagent capable of forming negatively charged particles with the RNA molecule; and wherein the first RNA molecule is administered systemically and the second RNA molecule is administered locally.

144. An RNA molecule, wherein the RNA molecule comprises at least one open reading frame encoding for at least one protein, for use in a method according to anyone of claims 1 to 137.

Images