Lipid-Based Formulations for Administration of RNA

Abstract

The present disclosure relates to a composition comprising RNA and cationic ionizable lipid for delivering RNA to target tissue after administration, particularly after parenteral administration.The present disclosure also relates to a method for delivering RNA to a cell of a subject or for treating or preventing a disease or disorder of a subject, comprising administering the composition of the present disclosure to the subject.Furthermore, the present disclosure also relates to a method for delivering a pharmacologic active peptide or protein to a subject or for treating or preventing a disease or disorder of a subject, comprising administering the RNA composition of the present disclosure to the subject, wherein the RNA codes for a pharmacologic active peptide or protein.

Description

[Technical Field]

[0001] The present disclosure relates to a composition comprising RNA and cationic ionizable lipids for delivering RNA to target tissue after administration, particularly after parenteral administration.The present disclosure also relates to a method for delivering RNA to cells of a subject or for treating or preventing a disease or disorder of a subject, comprising administering the composition of the present disclosure to a subject.In some embodiments, the RNA composition comprises single-stranded RNA, such as mRNA, encoding a target peptide or polypeptide, such as a pharmaceutically active peptide or polypeptide.The RNA is taken up by cells of the target tissue, and the RNA can be translated into the encoded peptide or protein to exhibit its physiological activity.Therefore, the present disclosure also relates to a method for delivering a pharmaceutically active peptide or protein to a subject or for treating or preventing a disease or disorder of a subject, comprising administering the RNA composition of the present disclosure to a subject, wherein the RNA encodes a pharmaceutically active peptide or protein. [Background technology]

[0002] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. The advantages of RNA include its transient expression and non-transforming properties. RNA does not require nuclear penetration for expression, and it cannot integrate into the host genome, eliminating the risk of tumorigenesis.

[0003] RNA can be delivered to targets via a variety of vehicles, primarily based on cationic polymers or lipids, which bind to RNA to form nanoparticles. The nanoparticles are intended to protect RNA from degradation, enable delivery of RNA to the target site, and facilitate cellular uptake and processing by target cells. The efficiency of RNA delivery depends in part on the molecular composition of the nanoparticles and can be affected by many parameters, including particle size, formulation, and charge or grafting with molecular moieties such as polyethylene glycol (PEG) or other ligands. Summary of the Invention [Problem to be solved by the invention]

[0004] It is necessary to provide a formulation for delivering biologically active RNA to target tissue, and the delivered RNA is efficiently translated into its encoded peptide or polypeptide.The present inventors have surprisingly found that the RNA particle formulation described herein meets the above requirements. [Means for solving the problem]

[0005] The present invention generally provides a composition comprising RNA and a cationic ionizable lipid having the structure of formula (I) shown below. The inventors have found that RNA formulations containing this cationic ionizable lipid of formula (I) can be produced in a robust and reproducible manner. They are suitable as RNA delivery systems and exhibit characteristics comparable to known systems.

[0006] The RNA formulations described herein are useful as RNA delivery vehicles for in vivo applications, such as pharmaceutical uses.

[0007] In a first aspect, the present invention provides a method for the preparation of a nucleic acid molecule comprising: (i) RNA; and (ii) a nucleic acid molecule of formula (I):

[0008] [ka]

[0009] (In the formula, R 1 and R 2 Each of the groups independently represents R 5 or -G 1 -L 1 -R 6 and R 1 and R 2 At least one of the is -G 1 -L 1 -R 6 and; R3 and R 4 Each of the 1-6 Alkyl, C 2-6 Alkenyl, aryl and C 3-10 cycloalkyl; R 5 and R 6 each is independently an acyclic hydrocarbyl group having at least 10 carbon atoms; G 1 and G 2 each independently represents an unsubstituted C 1-12 Alkylene or C 2-12 alkenylene; L 1 and L 2 each independently represents -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - and -NR a C(═O)O—; R a is H or C 1-12 is alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2) and a cationic ionizable lipid comprising the structure:

[0010] In some embodiments, each L 1 are independently -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NR a C(=O)- and -C(=O)NR a In some embodiments, each L 1 are independently —O(C═O)— or —(C═O)O—.

[0011] In some embodiments, L 2 -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR a C(=O)- and -C(=O)NR a In some embodiments, L 2 is -O(C=O)- or -(C=O)O-.

[0012] In some embodiments, R 5 is a straight chain alkyl or alkenyl group having at least 10 carbon atoms, preferably at least 14 carbon atoms, and more preferably at least 16 carbon atoms. In some embodiments, R 5 is a straight chain alkyl or alkenyl group having at least two carbon-carbon double bonds. In some embodiments, R 5 has the following structure:

[0013] [ka]

[0014] (In the formula,

[0015] [ka]

[0016] is R 5 represents the bond that connects it to the rest of the compound) It has.

[0017] In some embodiments, each G 1 are independently unsubstituted linear C 1-12 Alkylene or C 2-12 Alkenylene, preferably unsubstituted linear C 6-12 Alkylene or C 6-12 In some embodiments, each G is an alkenylene. 1 are independently unsubstituted linear C8-12 Alkylene or C 8-12 Alkenylene, e.g., unsubstituted linear C 6-10 Alkylene or C 6-10 In some embodiments, each G is an alkenylene. 1 is an unsubstituted straight chain C8 alkylene.

[0018] In some embodiments, each R 6 are independently linear hydrocarbyl groups having at least 10 carbon atoms. In some embodiments, each R 6 is R 6 through the internal carbon atoms of L 1 In some embodiments, each R 6 is, independently,

[0019] [ka]

[0020] (In the formula,

[0021] [ka]

[0022] is R 6 L 1 (represents a bond that connects to is selected from the group consisting of:

[0023] In some embodiments, R 1 and R 2 One of them is R 5 and the other is -G 1 -L 1 -R 6 is.

[0024] In some embodiments, R 1 and R 2 Each of the independently -G 1 -L 1 -R 6 is.

[0025] In some embodiments, R 3 and R 4 Each of the 1-6 Alkyl or C 2-6 Alkenyl, preferably C 1-4 Alkyl or C 2-4 In some embodiments, R is alkenyl. 3 and R 4 Each of the C groups, such as methyl or ethyl, 1-3 In some embodiments, R 3 and R 4 Each of is methyl.

[0026] In some embodiments, G 2 is the unsubstituted C 2-10 Alkylene or C 2-10 Alkenylene, preferably unsubstituted C 2-6 Alkylene or C 2-6 In some embodiments, G is an alkenylene. 2 is the unsubstituted C 2-4 Alkylene or C 2-4 Alkenylene, for example ethylene or trimethylene.

[0027] In some embodiments, m is 0, 1, 2, or 3. In some embodiments, m is 0. In some embodiments, m is 2.

[0028] In some embodiments, the cationic ionizable lipid has the following formula (IIIa) or (IIIb):

[0029] [ka]

[0030] (In the formula, R 3 and R 4 Each of the 1-4 Alkyl or C 2-4 is alkenyl; R 5 is a straight chain alkyl or alkenyl group having at least 16 carbon atoms; Each R 6 are independently linear hydrocarbyl groups having at least 10 carbon atoms, where R 6 is R 6 through the internal carbon atoms of L 1 is bound to; each G 1 are independently unsubstituted linear C 6-12 Alkylene or C 6-12 alkenylene; G 2 is the unsubstituted C 2-6 Alkylene or C 2-6 alkenylene; L 1 and L 2 each is independently —O(C═O)— or —(C═O)O—; and m is 0, 1, 2 or 3) It contains one of the following structures:

[0031] In some embodiments, R 3 and R 4 Each of the C groups, such as methyl or ethyl, 1-3 It is alkyl.

[0032] In some embodiments, R 5 is a straight chain alkyl or alkenyl group having at least 16 carbon atoms, the alkenyl group having at least two carbon-carbon double bonds.

[0033] In some embodiments, each G 1 are independently unsubstituted linear C 8-10 Alkylene or unsubstituted linear C 8-10 Alkenylene, for example, unsubstituted straight chain C8 alkylene.

[0034] In some embodiments, G 2 is the unsubstituted C 2-4 Alkylene or C2-4 Alkenylene, for example ethylene or trimethylene.

[0035] In some embodiments, m is 0. In some embodiments, m is 2.

[0036] In some embodiments, the cationic ionizable lipid has the following structures (IV-1), (IV-2), and (IV-3):

[0037] [ka]

[0038] Contains one of the following:

[0039] In some embodiments, the cationic ionizable lipid comprises about 20 mol% to about 80 mol%, preferably about 25 mol% to about 65 mol%, and more preferably about 30 mol% to about 50 mol% of the total lipid present in the composition. In some embodiments, the cationic ionizable lipid comprises about 40 mol% to about 50 mol% of the total lipid present in the composition. In some embodiments, the cationic or cationic ionizable lipid comprises about 55 mol% to about 65 mol% of the total lipid present in the composition.

[0040] In some embodiments, the composition further comprises a polymer-conjugated lipid.

[0041] In some embodiments, the polymer-conjugated lipid is a polysarcosine-conjugated lipid. Thus, in some embodiments, the composition further comprises at least one polysarcosine-conjugated lipid (such as two or more polysarcosine-conjugated lipids).

[0042] In some embodiments, the polysarcosine comprises between 2 and 200 sarcosine units, preferably between 5 and 100 sarcosine units, more preferably between 10 and 50 sarcosine units, more preferably between 15 and 40 sarcosine units, more preferably about 23 sarcosine units.

[0043] In some embodiments, the polysarcosine-linked lipid has the following general formula (V):

[0044] [ka]

[0045] where x is the number of sarcosine units. Includes the structure of

[0046] In some embodiments, the polysarcosine-linked lipid has the following general formula (VI):

[0047] [ka]

[0048] where one of R1 and R2 comprises a hydrophobic group, the other is a functional group that may comprise H, a hydrophilic group, or a targeting moiety, and x is the number of sarcosine units. In some embodiments, R1 is a functional group that may include H, a hydrophilic group, or a targeting moiety, and R2 includes one or two linear alkyl or alkenyl groups, each having at least 12 carbon atoms, preferably at least 14 carbon atoms.

[0049] In some embodiments, the polysarcosine-linked lipid has the following general formula (VII):

[0050] [ka]

[0051] where R is a functional group that may contain H, a hydrophilic group, or a targeting moiety, and x is the number of sarcosine units. It has the following structure.

[0052] In some embodiments, the polysarcosine-linked lipid has the following formula (VII-1):

[0053] [ka]

[0054] (wherein n is 23) It has the following structure.

[0055] In some embodiments, the polysarcosine-conjugated lipids comprise about 0.1 mol% to about 5 mol% of the total lipids present in the composition. In some embodiments, the polysarcosine-conjugated lipids comprise about 0.5 mol% to about 4.5 mol% of the total lipids present in the composition. In some embodiments, the polysarcosine-conjugated lipids comprise about 1 mol% to about 4 mol% of the total lipids present in the composition. In some embodiments, the polysarcosine-conjugated lipids comprise about 3 mol% to about 4 mol% of the total lipids present in the composition.

[0056] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid.

[0057] In some embodiments, the PEGylated lipid has the following general formula (VIII):

[0058] [ka]

[0059] (In the formula, R 12 and R 13are each independently a straight or branched alkyl, alkenyl, or alkynyl chain, each of the alkyl, alkenyl, and alkynyl chains independently containing 10 to 30 carbon atoms, optionally interrupted by one or more ester linkages, and w has an average value in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R 12 and R 13 Each of R is independently a straight alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms. 12 and R 13 are identical. In some embodiments, R 12 and R 13 Each of R is a linear alkyl chain containing 12 carbon atoms. 12 and R 13 Each of R is a linear alkyl chain containing 14 carbon atoms. 12 and R 13 Each of R is a linear alkyl chain containing 16 carbon atoms. 12 and R 13 In some embodiments, R 12 and R 13 is a linear alkyl chain containing 12 carbon atoms, and R 12 and R 13 and the other is a linear alkyl chain containing 14 carbon atoms. In some embodiments, z has an average value in the range of 40 to 50, for example, an average value of 45.

[0060] In some embodiments, the PEGylated lipid comprises about 1 mol% to about 10 mol% of the total lipid present in the composition. In some embodiments, the PEGylated lipid comprises about 1 mol% to about 5 mol% of the total lipid present in the composition. In some embodiments, the PEGylated lipid comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition.

[0061] In some embodiments, the composition further comprises one or more additional lipids, hi some embodiments, the one or more additional lipids are selected from the group consisting of phospholipids, steroids, and combinations thereof.

[0062] In some embodiments, the composition comprises a cationic ionizable lipid, a polymer-bound lipid, a phospholipid, and a steroid.

[0063] In some embodiments, the polymer-conjugated lipid is selected from a polysarcosine-conjugated lipid and a PEGylated lipid.

[0064] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidylphosphatidylcholine (D ... Dodecylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE). In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0065] In some embodiments, the phospholipids comprise from about 5 mol% to about 40 mol% of the total lipids present in the composition. In some embodiments, the phospholipids comprise from about 5 mol% to about 20 mol% of the total lipids present in the composition. In some embodiments, the phospholipids comprise from about 5 mol% to about 15 mol% of the total lipids present in the composition.

[0066] In some embodiments, the steroid comprises a sterol, such as cholesterol.

[0067] In some embodiments, the steroid comprises about 10 mol% to about 65 mol% of the total lipids present in the composition. In some embodiments, the steroid comprises about 20 mol% to about 60 mol% of the total lipids present in the composition. In some embodiments, the steroid comprises about 30 mol% to about 50 mol% of the total lipids present in the composition. In some embodiments, the steroid comprises about 25 mol% to about 35 mol% of the total lipids present in the composition.

[0068] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-conjugated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-1) or (IV-3), more preferably (IV-3), the polysarcosine-conjugated lipid has formula (VII-1), the phospholipid is DSPC, and the steroid is cholesterol.

[0069] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-conjugated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), the polysarcosine-conjugated lipid has formula (VII-1), the phospholipid is DOPE, and the steroid is cholesterol.

[0070] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid is one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-1) or (IV-3), more preferably (IV-3), the PEGylated lipid is DMG-PEG 2000, the phospholipid is DSPC, and the steroid is cholesterol.

[0071] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid is one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), the PEGylated lipid is DMG-PEG 2000, the phospholipid is DOPE, and the steroid is cholesterol.

[0072] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 30 mol% to about 50 mol of the total lipid present in the composition, the polysarcosine-linked lipid comprises about 1 mol% to about 4.5 mol%, e.g., about 3 mol% to about 4 mol%, of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol%, and the steroid comprises about 30 mol% to about 50 mol% of the total lipid present in the composition. In some embodiments, the cationic ionizable lipid comprises about 40 mol% to about 50 mol% of the total lipid present in the composition.

[0073] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-conjugated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 40 mol% to about 50 mol of the total lipid present in the composition, the polysarcosine-conjugated lipid comprises about 3 mol% to about 4 mol% of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid comprises about 35 mol% to about 45 mol% of the total lipid present in the composition. In some of these embodiments, the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-3), the polysarcosine-conjugated lipid has formula (VII-1), the phospholipid is DSPC, and the steroid is cholesterol. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0074] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-3), and comprises about 40 mol% to about 50 mol of the total lipid present in the composition; the polysarcosine-linked lipid has formula (VII-1) and comprises about 3 mol% to about 4 mol% of the total lipid present in the composition; the phospholipid is DSPC and comprises about 5 mol% to about 15 mol% of the total lipid present in the composition; and the steroid is cholesterol and comprises about 35 mol% to about 45 mol% of the total lipid present in the composition. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0075] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 55 mol% to about 65 mol% of the total lipid present in the composition, the polysarcosine-linked lipid comprises about 1 mol% to about 4.5 mol%, e.g., about 3 mol% to about 4 mol%, of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol%, and the steroid comprises about 25 mol% to about 35 mol% of the total lipid present in the composition. In some of these embodiments, the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), the polysarcosine-linked lipid has formula (VII-1), the phospholipid is DOPE, and the steroid is cholesterol. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0076] In some embodiments, the composition comprises a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), and comprises about 55 mol% to about 65 mol% of the total lipid present in the composition; the polysarcosine-linked lipid has formula (VII-1) and comprises about 1 mol% to about 4.5 mol%, e.g., about 3 mol% to about 4 mol%, of the total lipid present in the composition; the phospholipid is DOPE and comprises about 5 mol% to about 15 mol% of the total lipid present in the composition; and the steroid is cholesterol and comprises about 25 mol% to about 35 mol% of the total lipid present in the composition. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0077] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 30 mol% to about 50 mol% of the total lipid present in the composition, the PEGylated lipid comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid comprises about 30 mol% to about 50 mol% of the total lipid present in the composition. In some embodiments, the cationic ionizable lipid comprises about 40 mol% to about 50 mol% of the total lipid present in the composition.

[0078] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 40 mol% to about 50 mol of the total lipid present in the composition, the PEGylated lipid comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid comprises about 35 mol% to about 45 mol% of the total lipid present in the composition. In some of these embodiments, the cationic ionizable lipid is one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-3), the PEGylated lipid is DMG-PEG 2000, the phospholipid is DSPC, and the steroid is cholesterol. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0079] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-3), and comprises about 40 mol% to about 50 mol of the total lipid present in the composition; the PEGylated lipid is DMG-PEG 2000, and comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition; the phospholipid is DSPC, and comprises about 5 mol% to about 15 mol% of the total lipid present in the composition; and the steroid is cholesterol, and comprises about 35 mol% to about 45 mol% of the total lipid present in the composition. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0080] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid comprises about 55 mol% to about 65 mol% of the total lipid present in the composition, the PEGylated lipid comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition, the phospholipid comprises about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid comprises about 25 mol% to 35 mol% of the total lipid present in the composition. In some of these embodiments, the cationic ionizable lipid is one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), the PEGylated lipid is DMG-PEG 2000, the phospholipid is DOPE, and the steroid is cholesterol. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0081] In some embodiments, the composition comprises a cationic ionizable lipid, a PEGylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid has one of structures (IV-1), (IV-2), and (IV-3), preferably (IV-2), and comprises about 55 mol% to about 65 mol of the total lipid present in the composition; the PEGylated lipid is DMG-PEG 2000, and comprises about 1 mol% to about 2.5 mol% of the total lipid present in the composition; the phospholipid is DOPE, and comprises about 5 mol% to about 15 mol% of the total lipid present in the composition; and the steroid is cholesterol, and comprises about 25 mol% to about 35 mol% of the total lipid present in the composition. In some of these embodiments, the N / P value in the composition is at least about 4. In some embodiments, the N / P value is in the range of 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6. In some embodiments, the N / P value is about 8. In some embodiments, the N / P value is about 12.

[0082] In some embodiments, at least a portion of (i) the RNA, (ii) the cationic ionizable lipid, and, if present, (iii) one or more additional lipids, are present in a nanoparticle, such as a lipid nanoparticle (LNP).

[0083] In some embodiments, the nanoparticles (e.g., LNPs) have a size of about 30 nm to about 500 nm. In some embodiments, the nanoparticles have a size of about 40 nm to about 200 nm, e.g., about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0084] In some embodiments, the RNA is mRNA.

[0085] In some embodiments, the RNA comprises a modified nucleoside in place of uridine, hi some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0086] In some embodiments, the RNA has a codon-optimized coding sequence.

[0087] In some embodiments, the coding sequence has an increased G / C content compared to the wild-type coding sequence.

[0088] In some embodiments, the RNA (i) comprises modified nucleosides in place of uridine, (ii) has a codon-optimized coding sequence, and (iii) has a coding sequence with increased G / C content compared to the wild-type coding sequence. In some embodiments, the modified nucleosides are selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0089] In some embodiments, the RNA comprises at least one of a 5' cap, a 5' UTR, a 3' UTR, and a polyA sequence.

[0090] In some embodiments, the RNA includes all of a 5' cap, a 5' UTR, a 3' UTR, and a polyA sequence.

[0091] In some embodiments, the polyA sequence comprises at least 100 A nucleotides. In some embodiments, the polyA sequence is an interrupted sequence of A nucleotides.

[0092] In some embodiments, the 5' cap is a Cap 1 structure. In some embodiments, the 5' cap is a Cap 2 structure.

[0093] In some embodiments, the RNA encodes one or more peptides or proteins. In some embodiments, the one or more peptides or proteins are pharmaceutically active peptides or proteins. In some embodiments, the one or more peptides or proteins comprise epitopes for inducing an immune response against an antigen in a subject. In some embodiments, the one or more peptides or proteins are pharmaceutically active peptides or proteins and comprise epitopes for inducing an immune response against an antigen in a subject.

[0094] In some embodiments, the pharmaceutically active peptide or protein and / or antigen or epitope is derived from or is the SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0095] In some embodiments, the RNA comprises an open reading frame (ORF) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0096] In some embodiments, the SARS-CoV-2 S protein variant has a proline residue substitution at positions 986 and 987 of SEQ ID NO:11.

[0097] In some embodiments, the SARS-CoV-2 S protein variant has at least 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 12.

[0098] In some embodiments, the fragment comprises the receptor binding domain (RBD) of the SARS-CoV-2 S protein.

[0099] In some embodiments, (i) a fragment of the SARS-CoV-2 S protein or (ii) an immunogenic variant of the SARS-CoV-2 S protein has at least 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 11.

[0100] In a second aspect, the present invention provides a method for delivering RNA to cells in a subject, the method comprising administering to the subject a composition of the first aspect. In some embodiments of the second aspect, the composition is administered parenterally, for example intramuscularly.

[0101] In a third aspect, the present invention provides a method for delivering a pharmaceutically active peptide or protein to a subject, the method comprising administering to the subject the composition of the first aspect, wherein the RNA encodes the pharmaceutically active peptide or protein. In some embodiments of the third aspect, the composition is administered parenterally, for example intramuscularly.

[0102] In a fourth aspect, the present invention provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the first aspect, wherein delivery of RNA to cells of the subject is beneficial for treating or preventing the disease or disorder. In some embodiments of the fourth aspect, the composition is administered parenterally, for example intramuscularly.

[0103] In a fifth aspect, the present invention provides a method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of the first aspect, wherein the RNA encodes a pharmaceutically active peptide or protein, and delivery of the pharmaceutically active peptide or protein to the subject is beneficial for treating or preventing the disease or disorder. In some embodiments of the fifth aspect, the composition is administered parenterally, for example intramuscularly.

[0104] Further embodiments of the above aspects are described herein.

[0105] In some embodiments, the RNA described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is a single-stranded RNA (particularly mRNA) that can be translated into a respective protein upon entry into a cell, e.g., a recipient cell. In addition to a wild-type or codon-optimized sequence encoding an amino acid sequence that includes the amino acid sequence of a biologically active peptide or polypeptide, e.g., an antigenic sequence (epitope-containing peptide or polypeptide), the RNA may include one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum efficacy of the RNA with respect to stability and translation efficiency. In some embodiments, the RNA includes all of these elements. In some embodiments, the RNA encodes β-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O ) ApG may be used as a specific capping structure at the 5' end of the RNA drug substance. The 5'-UTR sequence may be the 5'-UTR sequence of human α-globin mRNA, which may have an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR sequence may be a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I), located between the coding sequence and the poly(A) tail, to ensure higher maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see International Publication No. 2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeat 3'-UTRs of human β-globin mRNA. Additionally, a poly(A) tail measuring 110 nucleotides in length may be used, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues. This poly(A) tail sequence was designed to increase RNA stability and translation efficiency.

[0106] An amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide, such as an antigen sequence, may contain amino acid sequences other than the amino acid sequence of the biologically active peptide or polypeptide. Such other amino acid sequences may support the function or activity of the biologically active peptide or polypeptide. In some embodiments, such other amino acid sequences include amino acid sequences that enhance antigen processing and / or presentation. Alternatively or additionally, such other amino acid sequences include amino acid sequences that disrupt immune tolerance. [Brief explanation of the drawings]

[0107] [Figure 1AB] Properties of RNA LNP formulations containing different cationic ionizable lipids of Formula (I) disclosed herein (i.e., lipids EA-405, HY-405, or HY-501) and polysarcosine-linked lipid C14pSar23 (abbreviated as "pSar" in the figure). (A) Particle size and PDI. (B) Zeta potential. [Figure 1CD] Properties of RNA LNP formulations containing different cationic ionizable lipids of Formula (I) disclosed herein (i.e., lipids EA-405, HY-405, or HY-501) and polysarcosine-linked lipid C14pSar23 (abbreviated as "pSar" in the figure). (C) Accessible RNA. (D) Free RNA (Lane 1: EA-405_pSar, Lane 2: HY-405_pSar, Lane 3: HY-501_pSar, Lane 4: EA-405-NP6_47.5%, Lane 5: HY-405-NP6_47.5%, Lane 6: HY-501-NP6_47.5%). [Figure 2](A) In vitro expression and (B) viability of RNA LNP formulations containing different cationic ionizable lipids (EA-405, HY-405, and HY-501) in a skeletal muscle cell line (C2C12), a hepatocellular carcinoma cell line (HepG2), and a mouse macrophage cell line (Raw cells). Firefly luciferase expression after 24 hours of incubation with 12.5, 25, and 50 ng / well of mRNA-loaded LNPs. [Figure 3A] (A) In vivo whole-body bioluminescence imaging (BLI) of animals (n = 3) at 6 hours (top row), 24 hours (middle row), and ex vivo BLI (bottom row). [Figure 3B] (B) In vivo luciferase expression in muscle regions 6 and 24 hours after transfection for LNP formulations containing different cationic ionizable lipids and C14pSar23. [Figure 4A] (A) Terminal complement complex (SC5b-9) formation after incubation of LNPs containing different cationic ionizable lipids and control substances with human serum. The horizontal dashed line indicates the level of SC5b-9 formation for PBS. [Figure 4B] (B) Hemolysis analysis after incubation (at neutral pH conditions) of human red blood cells with LNP formulations containing different cationic ionizable lipids and a control. [Figure 5A] Characterization of RNA LNP formulations containing HY-501 and C14pSar23. (A) Particle size and PDI. [Figure 5BC] Characterization of RNA LNP formulations containing HY-501 and C14pSar23. (A) Zeta potential. (B) Accessible RNA. (C) [Figure 5D] Characterization of RNA LNP formulations containing HY-501 and C14pSar23. (D) Free RNA. [Figure 6](A) Terminal complement complex (SC5b-9) formation after incubation of HY-501-containing LNP formulations and control substances with human serum. The horizontal dashed line indicates the level of SC5b-9 formation for PBS. (B) Hemolysis analysis after incubation of HY-501-containing LNP formulations with human whole blood (at neutral pH). [Figure 7] Quantification of S1 protein expression after transfection with C14pSar23 containing LNPs using HY-501 as the cationic lipid. (A) Variation of pSar mol% versus MFI (mean fluorescence intensity) across the cell population. Data were fitted using a second-order polynomial function. (B) Viability of all formulations tested. Mean values ​​(n=3) ± standard deviation are plotted. [Figure 8AB] Characterization of RNA LNP formulations containing HY-405 and C14pSar23. (A) Particle size and PDI. (B) Zeta potential. [Figure 8CD] Characterization of RNA LNP formulations containing HY-405 and C14pSar23. (C) Accessible RNA. (D) Free RNA. [Figure 9] (A) Terminal complement complex (SC5b-9) formation after incubation of LNP formulations and control substances with human serum. The horizontal dashed line indicates the level of SC5b-9 formation for PBS. The horizontal dashed line indicates the level of SC5b-9 formation for PBS. (B) Hemolysis analysis after incubation of HY405-containing LNP formulations with human whole blood (at neutral pH). [Figure 10] (A) Accessible RNA measured by Ribogreen assay. (B) Agarose gel electrophoresis images of LNP formulations containing HY501 or HY405 (first injection). [Figure 11] (A) Accessible RNA measured by Ribogreen assay. (B) Agarose gel electrophoresis images of LNP formulations containing HY501 or HY405 (second injection). [Figure 12]Quantification of S1 protein expression after transfection with LNP formulations containing HY405 or HY501. (A) MFI (mean fluorescence intensity) of the entire cell population. (B) Viability of all formulations tested. Mean values ​​(n=3) + / - standard deviation (SD) are plotted. [Figure 13] Quantification of S1 protein expression after transfection with LNP formulations containing BM, HY405, or HY501. MFI: mean fluorescence intensity across the entire cell population. Viability is the viability of all formulations tested. Mean values ​​(n=3) + / - SD are plotted. [Figure 14] Serological analysis was performed using anti-S1-ELISA to detect SARS-CoV-2 S protein-specific antibodies; after the first immunization, animals were bled weekly for serological analysis. [Figure 15] Graph showing pVNT50 values ​​of Group 2 (HY-501), Group 3 (HY-405) and control (PBS) at different time points (D29, D28, D35, D42, D49, D56). [Figure 16] Graph showing IFNγ secretion from splenocytes after restimulation with (A) a mix of overlapping peptides specific for the SARS-CoV-2 S protein, (B) a SARS-CoV-2 RBD peptide pool, and (C) an irrelevant peptide. [Figure 17] Physicochemical properties of EA-405, HY-501, and HY-405 LNP formulations, including (A) size and polydispersity index (PDI), (B) zeta potential, and (C) encapsulation efficiency, RNA accessibility, and free RNA. [Figure 18A] (A) Representative in vivo dorsal bioluminescence images (BLI) of BALB / c mice injected with 1 μg / leg of modified mRNA encoding luciferase via the intramuscular route over time. Relative luminescence scale is shown. [Figure 18BC]Representative (B) in vivo ventral bioluminescence images (BLI) and (C) ex vivo bioluminescence images (BLI) of BALB / c mice injected with 1 μg / leg of modified mRNA encoding luciferase via the intramuscular route over time. The relative luminescence scale is shown. In (C), organs are labeled from top to bottom as follows: heart, lungs, liver, spleen, and kidney. [Figure 19] Quantification of bioluminescence signals measured in BALB / c mice injected with 1 μg / leg LNP containing modified mRNA encoding luciferase 6 hours after injection: (A) in vivo in muscle, (B) in vivo in the liver region, (C) ex vivo in the spleen, and (D) ex vivo in organs. Data are expressed as total flux (photons / second) and represent the mean ± SD of three mice / group (n=3) for in vivo BLI and one mouse / group (n=1) for ex vivo BLI. [Figure 20] Physicochemical properties of LNP optimization with respect to N / P ratio (3-12). Bar graphs include size and charge of formulations containing (a) EA-405, (b) HY-501, and (c) HY-405. Encapsulation efficiency and RNA accessibility of formulations containing (d) EA-405, (e) HY-501, and (f) HY-405. Free RNA of formulations containing (g) EA-405, (h) HY-501, and (i) HY-405. [Figure 21] In vitro luciferase expression of (a) EA-405 in C2C12 cell line, (b) EA-405 in HepG2 cell line, (c) EA-405 in RAW cell line, (d) HY-501 in C2C12 cell line, (e) HY-501 in HepG2 cell line, (f) HY-501 in RAW cell line, (g) HY-405 in C2C12 cell line, (h) HY-405 in HepG2 cell line, and (i) HY-405 in RAW cell line using mRNA doses of 12.5 ng, 25 ng, and 50 ng. [Figure 22AB]Representative (A) in vivo dorsal bioluminescence images (BLI) and (B) in vivo ventral bioluminescence images (BLI) of BALB / c mice injected with 1 μg / leg of mod.mRNA-luciferase-containing LNPs with different N / P ratios via the intramuscular route over a 48-hour period. Relative luminescence scales are indicated where appropriate. [Figure 22C] (C) Representative ex vivo bioluminescence images (BLI) of BALB / c mice injected with 1 μg / leg of mod.mRNA-luciferase-containing LNPs with different N / P ratios via the intramuscular route over a 48-hour period. The relative luminescence scale is indicated where appropriate. In (C), organs are labeled from top to bottom as follows: heart, lungs, liver, spleen, and kidney. [Figure 23] BALB / c mice were injected with 1 μg / leg LNP (with different N / P ratios: 4, 6, and 12) containing luciferase-encoding mod. mRNA 6 hours after injection. (a) Quantification of bioluminescence signals measured in the muscle in vivo, (b) Quantification of bioluminescence signals measured in the liver region in vivo, (c) Quantification of bioluminescence signals measured in the spleen ex vivo, and (d) Quantification of bioluminescence signals measured in the organs ex vivo. Data are expressed as total flux (photons / second) and represent the mean ± SD of three mice / group (n=3) for in vivo BLI and one mouse / group (n=1) for ex vivo BLI. [Figure 24] Antibody titers (measured by ELISA) of EA-405, HY-501, and HY-405 formulations against BM and buffer control (PBS) at (a) day 14 after priming, (b) day 28 after boosting, and (c) day 42 as endpoint. (d) IFN-γ ELISpot analysis performed with total splenocytes harvested on day 42. [Figure 25] Physicochemical characterization of EA-405 LNPs (a) in terms of size and polydispersity index (PDI), (b) in terms of zeta potential, (c) in terms of encapsulated / accessible RNA, and (d) in terms of free RNA. [Figure 26](A) Representative in vivo dorsal bioluminescence images (BLI) of a BALB / c mouse injected with 1 μg / leg of mod.mRNA encoding luciferase via the intramuscular route at 6 hours and 24 hours. (B) Representative in vivo ventral bioluminescence images (BLI) of a BALB / c mouse injected with 1 μg / leg of mod.mRNA encoding luciferase via the intramuscular route at 6 hours. Relative luminescence scale is shown. [Figure 27] (a) Quantification of bioluminescent signal measured in muscle (6 and 24 hours after injection) (expressed as total in vivo flux (photons / second)), (b) quantification of bioluminescent signal measured in muscle ("kinetics" over 9 days) (expressed as total in vivo flux (photons / second)), (c) quantification of bioluminescent signal measured in liver (expressed as total in vivo flux (photons / second)), and (d) IFN-γ ELISpot analysis in BALB / c mice injected with 1 μg / leg of LNPs containing mod. mRNA encoding luciferase. Data represent the mean ± SD of three mice / group and two sites (n = 6) for in vivo BLI and the mean ± SD of three mice / group (n = 3) for liver and ELISpot. DETAILED DESCRIPTION OF THE INVENTION

[0108] Array Description The following table provides a list of the specific sequences referenced herein.

[0109] [Table 1] TIFF2025502796000014.tif185158TIFF2025502796000015.tif185158TIFF20255027960 00016.tif185158TIFF2025502796000017.tif185158TIFF2025502796000018.tif185158 TIFF2025502796000019.tif185158TIFF2025502796000020.tif185158TIFF20255027960 00021.tif185158TIFF2025502796000022.tif185158TIFF2025502796000023.tif186158

[0110] Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0111] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0112] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0113] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to imply the inclusion of a stated feature, element, member, integer, or step or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and to those that do not materially affect the basic and novel characteristics of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0114] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0115] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0116] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0117] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0118] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0119] In the context of the present disclosure, the term "about" indicates an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be understood by one of ordinary skill in the art, such specific deviations from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than artificial or engineered technical effects.

[0120] The 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, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0121] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0122] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0123] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0124] As used herein, terms such as "enhance" refer to the ability to cause an overall increase or enhancement in a level, for example, by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0125] As used herein, "physiological pH" refers to a pH of about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0126] As used in this disclosure, "% w / v" refers to weight-to-volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0127] As used in this disclosure, "wt. %" refers to weight percent, a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0128] As used in this 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.

[0129] As used in this disclosure, "mol % of 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 substances.

[0130] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Therefore, ionic strength, I, is calculated by the following equation:

[0131]

number

[0132] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is over all the different types of ions (i) in the solution.

[0133] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0134] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kilogram of solvent.

[0135] The term "lyophilize" or "lyophilization" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0136] The term "spray drying" refers to spray drying a substance by mixing a fluid and a (heated) gas that is atomized (atomized) in a vessel (spray dryer), and the solvent from the droplets formed evaporates, resulting in a dry powder.

[0137] The term "reconstitute" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0138] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0139] 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 exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0140] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of 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 include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0141] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0142] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0143] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0144] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used synonymously herein.

[0145] The term "biological activity" refers to a response of a biological system to a molecule. Such a biological system can be, for example, a cell or an organism. In some embodiments, such a response is therapeutically or pharmaceutically useful.

[0146] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of said structure.

[0147] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in reference to a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0148] With respect to an amino acid sequence (peptide or polypeptide), the term "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0149] As used herein, "variant" with respect to an amino acid sequence (peptide or polypeptide) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a native or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0150] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0151] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide or polypeptide) can include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0152] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also called N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions within the amino acid sequence that are not conserved between homologous peptides or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0153] In some embodiments, the degree of similarity, such as identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given for, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, for example, using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0154] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0155] The terms "% identical" and "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared.The percentage is purely statistical, and the differences between two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared.Comparing two sequences is usually carried out by comparing sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0156] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0157] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0158] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, for example at least 95%, at least 98 or at least 99% of the amino acid residues.

[0159] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.

[0160] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that contains 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 performing one or more functions of the parent molecule or sequence, e.g., capable of inducing an immune response. In some embodiments, alterations to 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, for example, the 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 that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0161] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0162] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0163] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient, or the cells may be present in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, and the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into the cell.RNA can be transfected into the cell to transiently express the encoded protein.

[0164] The present disclosure includes analogs of peptides or polypeptides. According to the present disclosure, a peptide or polypeptide analog is a modified form of the peptide or polypeptide from which it is derived, and retains at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the peptide or polypeptide, such as carbohydrates, lipids, and / or peptides or polypeptides. In some embodiments, a "peptide or polypeptide analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the peptides and polypeptides.

[0165] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0166] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0167] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0168] According to various embodiments of the present disclosure, nucleic acids such as RNAs encoding a peptide or polypeptide are taken up or introduced, i.e., transfected or transduced, into cells, which may be present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cells may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or polypeptide, and / or express it on their surface.

[0169] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide or polypeptide, the nucleic acid is capable of being expressed to produce said peptide or polypeptide when present in an appropriate environment, e.g., a cell.

[0170] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0171] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA), which can then be translated into peptides or polypeptides.

[0172] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0173] The medical preparations described herein, particularly kits, may include instruction materials or instructions. As used herein, "instruction materials" or "instructions" include publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instruction materials of the kits of the present disclosure may be, for example, attached to a container containing the composition / formulation of the present disclosure, or may be shipped together with a container containing the composition / formulation. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition will be used in conjunction with each other by the recipient.

[0174] A prodrug of a particular compound described herein is a compound that undergoes chemical conversion under physiological conditions to provide the particular compound upon administration to an individual. Furthermore, a prodrug can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with an appropriate enzyme or chemical reagent. Exemplary prodrugs are in vivo hydrolyzable esters (using alcohol or carboxy groups contained in the particular compound) or amides (using amino or carboxy groups contained in the particular compound). Specifically, any amino group contained in the particular compound that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0175] In this specification, the structural formula of a compound may represent a specific isomer of the compound. However, it should be understood that the present invention includes all isomers and isomeric mixtures, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, and tautomers, that arise structurally, and is not limited to the description of the formula. Also, in this specification, the structural formula of a compound may represent a specific salt and / or solvate of the compound. However, it should be understood that the present disclosure includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and / or solvate.

[0176] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert due to the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. One example of a tautomer is keto-enol tautomeric isomers. "Conformers" are stereoisomers that can formally be interconverted by rotation about a single bond alone, and include in particular those resulting in different three-dimensional forms of a (hetero)cyclic ring, such as the chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0177] The term "solvate," as used herein, refers to an addition complex of a substance dissolved in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, etc.), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), where the addition complex exists in crystalline or mixed crystal form. The amount of solvent in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solvent is water.

[0178] In an isotopically labeled compound, one or more atoms are replaced by corresponding atoms having the same number of protons but a different number of neutrons. For example, a hydrogen atom can be replaced with a deuterium or tritium atom. Exemplary isotopes that can be used in the present disclosure include deuterium, tritium, 11 C. 13 C. 14 C. 15 N, 18 F, 32 P,32 S, 35 S, 36 Cl, and 125 Contains I.

[0179] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which has a length dimension, the so-called Z 平均 , and results in a dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is determined by this Z 平均 Used synonymously with the value of

[0180] In some embodiments, the "polydispersity index," as mentioned in the definition of "mean diameter," is calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be considered as a measure of the size distribution of an ensemble of nanoparticles.

[0181] The "radius of gyration" of the particle around the axis of rotation (R g ) is the radial distance from the axis of rotation of the point at which the moment of inertia of a particle about a given axis is the same as its actual mass distribution, if the entire mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle's components from either its center of mass or a given axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at i For a polymer consisting of n mass elements (i=1, 2, 3, ..., n), R g is the s over all mass elements i 2 is the mass-averaged square root of and can be calculated as follows:

[0182]

number

[0183] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors

[0184]

number

[0185] , the structure function S is defined as:

[0186]

number

[0187] where N is the number of components (Guinier's law).

[0188] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle drags more water molecules with it as it moves through the solution. Since the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is determined by the Stokes-Einstein equation:

[0189]

number

[0190] (In the formula, k B is the Boltzmann constant, T is the temperature, η is the viscosity of the solvent, and D is the diffusion coefficient) The diffusion coefficient can be experimentally determined, for example, by using dynamic light scattering (DLS). Thus, one procedure for determining the hydrodynamic radius of a particle or particle population (e.g., the hydrodynamic radius of a particle contained in a sample or control composition disclosed herein, or the hydrodynamic radius of a particle peak obtained by subjecting such a sample or control composition to field-flow fractionation) is to measure the DLS signal of the particle or particle population (e.g., the DLS signal of a particle contained in a sample or control composition disclosed herein, or the DLS signal of a particle peak obtained by subjecting such a sample or control composition to field-flow fractionation).

[0191] As used herein, the expression "light scattering" refers to a physical process in which light is forced to deviate from a straight line trajectory by one or more paths due to local inhomogeneities in the medium through which it passes.

[0192] The term "UV" means ultraviolet and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than those of visible light but longer than X-rays.

[0193] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the radius of gyration (R) can be calculated by using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is calculated using the following formula:

[0194]

number

[0195] where c is the mass concentration of particles in the solvent (g / mL) and A2 is the second virial coefficient (mol mL / g 2 ), P(θ) is the form factor for the angular dependence of the scattered light intensity, and R θ is the excess Rayleigh ratio (cm -1 ) and K* is 4π 2 η o (dn / dc) 2 λ0 -4 N A -1 is an optical constant equal to , where η o is the refractive index of the solvent at the incident radiation (vacuum) wavelength, λ is the incident radiation (vacuum) wavelength (nm), and N A is Avogadro's number (mol -1 ) and dn / dc is the differential refractive index increment (mL / g) (See, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry plot is a graphical representation using the following terms or their reciprocals:

[0196]

number

[0197] (In the formula, c, R θ and K* is as defined above). Preferably, the Debye plot is calculated using the following term or its reciprocal:

[0198]

number

[0199] (In the formula, c, R θ and K* is as defined above). It is calculated using

[0200] As used herein, the term "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is incident on a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution strike the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0201] As used herein, the term "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and size of all macromolecules within a radius. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0202] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. The nucleic acid can exist as a single-stranded or double-stranded molecule, linear or covalently closed circular molecule. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) of DNA or in vitro transcription of RNA (e.g., using RNA polymerase); (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0203] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be thought of as a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0204] The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by their single-letter codes: U, A, T, C, and G, respectively. However, thymidine is more commonly designated "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they are designated as A, C, and G, while in DNA, they are designated as dA, dC, and dG.

[0205] Modified purine (A or G) or pyrimidine (C, T, or U) base moieties may, in some embodiments, be modified with one or more alkyl groups, e.g., one or more C 1-4 Modified by alkyl groups, e.g., one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil, and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0206] As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to either or both ends of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. In the present disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0207] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0208] The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to one or both ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such modified / modified nucleotides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0209] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (e.g., small activator RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0210] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed acellularly. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0211] mRNA According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0212] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0213] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0214] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide or polypeptide.

[0215] In some embodiments, preferably the mRNA encoding the peptide or polypeptide has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0216] As is well-established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 4 thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Additionally, various in vitro transcription kits are commercially available from, for example, Thermo Fisher Scientific (TranscriptAid™ T7 kit, MEGAscript™ T7 kit, MAXIscript™, etc.), New England BioLabs Inc. (HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit, etc.), Promega (RiboMAX™, HeLaScribe™, Riboprobe™ system, etc.), Jena Bioscience (SP6 or T7 transcription kit, etc.), and Epicentre (AmpliScribe™, etc.). To provide a modified mRNA, correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be made in and / or added to the mRNA after transcription.

[0217] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0218] In some embodiments of the present disclosure, the mRNA is a "replicon mRNA" or simply a "replicon," particularly a "self-replicating mRNA" or "self-amplifying mRNA." In certain embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (forming viral particles). Typically, two open reading frames (ORFs) are present in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together by a second ORF that is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). Following infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly like a messenger RNA to translate the open reading frame encoding the nonstructural polyprotein (nsP1-nsP4).Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus 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 the viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated in trans by the replicase must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0219] In some embodiments of the present disclosure, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) contains one or more modifications, for example, to increase its stability, and / or increase translation efficiency, and / or reduce immunogenicity and / or reduce cytotoxicity. For example, to increase the expression of the RNA (particularly mRNA), modifications can be made within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without changing the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include: 5' cap structure; extension or truncation of a naturally occurring poly(A) tail; modification of the 5' and / or 3' untranslated region (UTR), for example, introduction of a UTR not associated with the coding region of the RNA; substitution of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to change, preferably increase, the GC content of the RNA).

[0220] In some embodiments, the RNA (particularly mRNA) described herein comprises a 5' cap structure. In some embodiments, the mRNA does not have an uncapped 5'-triphosphate. In some embodiments, the RNA (particularly mRNA) may comprise a conventional 5' cap and / or a 5' cap analog. The term "conventional 5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp) linked via its triphosphate moiety to the 5' end of the next nucleotide of the mRNA (i.e., the guanosine is linked to the remainder of the mRNA via a 5'-5' triphosphate bond). The guanosine is N 7 can be methylated at the cap structure m 7 The term "5' cap analog" is based on the traditional 5' cap, but uses a 5' cap analog with a reverse orientation to avoid incorporation of the 5' cap analog. 7 These include 5' caps modified at either the 2' or 3' position of the guanosine structure (such 5' cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as phosphorothioate-modified 5' cap analogs at the β-phosphate (e.g., m2), as described in PCT / EP 2019 / 056502. 7,2’O G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Providing RNA (particularly mRNA) having a 5' cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, with the 5' cap structure being co-transcriptionally incorporated into the generated RNA (particularly mRNA) strand, or RNA (particularly mRNA) can be generated, for example, by in vitro transcription, and the 5' cap structure can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0221] In some embodiments, the RNA (particularly mRNA) is m2 7,2’OG(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O In some embodiments, the RNA encoding a peptide or polypeptide comprising an antigen or epitope comprises a 5' cap structure selected from the group consisting of m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer).

[0222] In some embodiments, RNA (particularly mRNA) comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present disclosure, the term "cap 0" refers to the structure "m 7 GpppN" where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "Cap 1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap 2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0223] The 5' cap analog beta-S-ARCA (β-S-ARCA) has the following structure:

[0224] [ka]

[0225] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). The HPLC is preferably analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably in a 5 μm, 4.6 x 250 mm format, is used for the separation, allowing a flow rate of 1.3 ml / min to be applied. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH 5.9, within 15 min. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0226] 5' cap analog m2, a building block of cap 1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG (also called ApG) has the following structure:

[0227] [ka]

[0228] An exemplary cap 0 mRNA, including β-S-ARCA, has the following structure:

[0229] [ka]

[0230] m2 7,3’O An exemplary cap 0 mRNA containing G(5')ppp(5')G and mRNA has the following structure:

[0231] [ka]

[0232] m2 7,3’-O Gppp(m1 2’-O ) An exemplary cap 1 mRNA containing ApG and mRNA has the following structure:

[0233] [ka]

[0234] As used herein, the term "poly A tail" or "poly A sequence" refers to a continuous or intermittent sequence of adenylate residues typically located at the 3' end of an mRNA molecule. Poly A tails or poly A sequences are known to those skilled in the art and can follow the 3'-UTR of an RNA (particularly an mRNA) described herein. A continuous poly A tail is characterized by consecutive adenylate residues. In nature, continuous poly A tails are typical. The RNA (particularly an mRNA) disclosed herein can have a poly A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0235] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of mRNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0236] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides of the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0237] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called a poly(A) cassette.

[0238] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 A1 may be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5-50 nucleotides, exhibit sustained propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and are still associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level. Consequently, in some embodiments, the poly-A tails included in the RNA (particularly mRNA) molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0239] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0240] In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail set forth in SEQ ID NO:8. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0241] In some embodiments, the RNA (particularly mRNA) described in the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" generally does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence. The incorporation of a 3'UTR into the 3' untranslated region of an RNA (preferably mRNA) molecule can result in improved translation efficiency. By incorporating two or more such 3'UTRs (preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)), a synergistic effect can be achieved. 3'UTRs can be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as α2-globin, α1-globin, or β-globin, e.g., β-globin, e.g., human β-globin gene or mRNA. For example, an RNA (e.g., an mRNA) can be modified by replacing or inserting an existing 3'-UTR with one or more, e.g., two, copies of a 3'-UTR from a globin gene, e.g., α2-globin, α1-globin, β-globin, e.g., β-globin, e.g., human β-globin.

[0242] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 6. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 7.

[0243] In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:6 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:6.

[0244] In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:7, 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:7.

[0245] The RNA (particularly mRNA) described herein can have modified ribonucleotides to increase its stability, and / or reduce its immunogenicity, and / or reduce its cytotoxicity.For example, in some embodiments, the uridine in the RNA (particularly mRNA) described herein is replaced by modified nucleoside (partially or completely, preferably completely).In some embodiments, the modified nucleoside is modified uridine.

[0246] In some embodiments, the modified uridine substituting for uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0247] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in the mRNA is 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), ), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5- Methylaminomethyluridine (mnm5U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm5U), 1-taurinomethyluridine 5-Taurinomethyl-2-thiouridine (m5s2U), 1-Taurinomethyl-4-thiopseudouridine), 5-Methyl-2-thiouridine (m5s2U), 1-Methyl-4-thiopseudouridine (m1s4Ψ), 4-Thio-1-methylpseudouridine, 3-Methylpseudouridine (m3Ψ), 2-Thio-1-methylpseudouridine, 1-Methyl-1-deazapseudouridine, 2-Thio-1-methyl-1-deazapseudouridine, Dihydrouridine (D), Dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudouridine The uridine may be any one or more of uridine (Ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine]uridine, or any other modified uridine known in the art.

[0248] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified." The term "mΨ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ-modified or mΨ-modified or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, which completely replaces uridine.

[0249] The codons of the RNA (particularly mRNA) used in the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which the peptide or polypeptide of interest is to be expressed with codons that are synonymous and frequently occurring in the cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (particularly mRNA) described in the present disclosure is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to a wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0250] The term "codon-optimized" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, the coding region can be codon-optimized for optimal expression in a subject treated with the RNA (especially mRNA) described herein. Codon optimization is based on the observation that translation efficiency is also determined by the different frequencies of occurrence of tRNA in cells. Therefore, the sequence of RNA (especially mRNA) can be modified so that codons for which frequently occurring tRNAs are available are inserted instead of "rare codons".

[0251] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of an RNA (especially an mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unaltered compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode the exact same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U nucleotides or contain a lower content of A and / or U nucleotides.

[0252] In various embodiments, the G / C content of the coding region of the RNA (particularly mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild-type RNA.

[0253] Combinations of the above-mentioned modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, alteration of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine), and codon optimization have a synergistic effect on increasing RNA (preferably mRNA) stability and translation efficiency. Thus, in some embodiments, RNA (particularly mRNA) described in the present disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, namely: (i) incorporation of a 5' cap structure; (ii) incorporation of a polyA sequence, unmasking of a polyA sequence; (iii) modification of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine); and (v) codon optimization.

[0254] Some aspects of the present disclosure include targeted delivery of the mRNA disclosed herein to specific cells or tissues. In some embodiments, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. When the administered RNA (particularly mRNA) encodes an antigen or epitope for inducing an immune response, targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred. In some embodiments, the target cells are spleen cells. In some embodiments, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen. The "lymphatic system" is part of the circulatory system and is an important part of the immune system, comprising a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0255] Lipid-based mRNA delivery systems have an inherent selectivity for the liver, and depending on the composition of the mRNA delivery system used, mRNA expression in the liver can be achieved.Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).In some embodiments, the target organ for mRNA expression is the liver, and the target tissue is liver tissue.Delivery to such target tissue is preferred, particularly when it is desired to have mRNA or encoded peptide or polypeptide present in this organ or tissue, and / or when it is desired to express large amounts of encoded peptide or polypeptide, and / or when it is desired or required to have the encoded peptide or polypeptide present in a systemic manner, particularly in a significant amount.

[0256] In some embodiments, after administration of the RNA (particularly mRNA) compositions / formulations described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is RNA (particularly mRNA) that encodes a peptide or polypeptide, and the RNA is translated by the target cell to produce the peptide or polypeptide. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in a lymph node. In some embodiments, the target cell is a cell in the lung. In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a NK cell. In some embodiments, the target cell is a monocyte. Therefore, the RNA (especially mRNA) compositions / preparations described herein can be used to deliver RNA to such target cells.

[0257] Pharmaceutically active peptides or polypeptides "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or RNA (especially mRNA), to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0258] In some embodiments, RNA (particularly mRNA) described in this disclosure comprises a nucleic acid sequence that encodes a peptide or polypeptide, such as a pharmaceutically active peptide or polypeptide.

[0259] In some embodiments, the RNA (particularly mRNA) described herein comprises a nucleic acid sequence encoding a peptide or polypeptide, preferably a pharmaceutically active peptide or polypeptide, and is capable of expressing said peptide or polypeptide, particularly when introduced into a cell or subject. Thus, in some embodiments, the RNA (particularly mRNA) described herein comprises a coding region (open reading frame (ORF)) encoding a peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with an initiation codon and ending with a termination codon. Such nucleic acids encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active nucleic acids." In particular, such RNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active RNAs," and such mRNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active mRNAs." In some embodiments, an RNA used in this disclosure comprises a nucleic acid sequence that encodes more than one peptide or polypeptide, for example, two, three, four or more peptides or polypeptides.

[0260] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used in the treatment of an individual in whom expression of the peptide or polypeptide would be beneficial, for example, to ameliorate symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or pathology. A pharmaceutically active peptide or polypeptide can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide or polypeptide" includes the entire peptide or polypeptide and can also refer to a pharmaceutically active fragment thereof. The term can also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0261] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, immunostimulants, such as 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, genome engineering proteins, and blood proteins. In some embodiments, pharmaceutically active peptides and polypeptides comprise substituted proteins.

[0262] An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components of the immune system, particularly immune effector cells. Immunostimulants can be pro-inflammatory (e.g., when treating infections or cancer) or anti-inflammatory (e.g., when treating autoimmune diseases).

[0263] In one embodiment, the immunostimulatory agent is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins, such as IL2, IL7, IL12, IL15, and IL23, colony-stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. In another embodiment, the immunostimulatory agent comprises an adjuvant-type immunostimulatory agent, such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins, such as CD80, CD86, and ICAM-1.

[0264] The term "cytokine" refers to proteins with molecular weights of approximately 5 to 60 kDa that are involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, upon release, cytokines affect the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to interferon receptors on the surface of the regulated cells, thereby preventing intracellular viral replication. Interferons can be classified into two types: Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factors (TNFs), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), and variants and derivatives thereof.

[0265] According to the present disclosure, cytokine can be naturally occurring cytokine or its functional fragment or variant.Cytokine can be human cytokine and can be derived from any vertebrate, particularly any mammal.One particularly preferred cytokine is interferon alpha.

[0266] Immunostimulatory agent can be provided to subject by administering to subject the RNA that encodes immunostimulatory agent in the formulation for selectively delivering RNA to liver or liver tissue.The delivery of RNA to such target organ or tissue is preferred, particularly when it is desired to express a large amount of immunostimulatory agent, and / or when it is desired or required that immunostimulatory agent is present in systemic, particularly in significant amount.

[0267] RNA delivery systems have an inherent selectivity for the liver. This relates to lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles.

[0268] The example of suitable immunostimulant for targeting liver is cytokine that is involved in the proliferation and / or maintenance of T cell.Example of suitable cytokine includes IL2 or IL7, its fragment and variant, and the fusion protein of these cytokines, fragment and variant, such as extended PK cytokine.

[0269] In another embodiment, the RNA encoding the immunostimulatory agent can be administered in a formulation for selective delivery of the RNA to the lymphatic system, particularly to secondary lymphoid organs, more particularly to the spleen. Delivery of the immunostimulatory agent to such target tissues is particularly preferred when the presence of the immunostimulatory agent in this organ or tissue is desirable (for example, when the immunostimulatory agent, such as a cytokine, is required to induce an immune response, particularly during T cell priming or for the activation of resident immune cells), but when the immunostimulatory agent is not desirable to be present systemically, especially in significant amounts (for example, because the immunostimulatory agent has systemic toxicity).

[0270] Examples of suitable immunostimulatory agents include cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0271] Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell releases interferons to enhance the antiviral defenses of nearby cells.

[0272] Based on the type of receptor through which the interferons signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

[0273] All type I interferons bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

[0274] The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, resulting in the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0275] IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs). They are primarily involved in innate immunity against viral infections. The genes responsible for their synthesis belong to 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

[0276] IFNβ proteins are produced in large quantities by fibroblasts. They are primarily involved in the innate immune response and have antiviral activity. Two types of IFNβ, IFNβ1 and IFNβ3, have been described. Both native and recombinant forms of IFNβ1 possess antiviral, antibacterial, and anticancer properties.

[0277] Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12. Additionally, type II interferons are released by cytotoxic T cells and T helper cells.

[0278] Type III interferons signal through a receptor complex consisting of IL10R2 (also known as CRF2-4) and IFNLR1 (also known as CRF2-12). Although more recently discovered than type I and type II IFNs, recent information has demonstrated the importance of type III IFNs in several types of viral or fungal infections.

[0279] In general, type I and type II interferons are responsible for regulating and activating the immune response.

[0280] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0281] According to the present disclosure, the interferon can be a naturally occurring interferon or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal.

[0282] Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on characteristic structural features. However, their amino acid sequence similarity is fairly weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

[0283] According to the present disclosure, the interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be a human interleukin and may be derived from any vertebrate, particularly any mammal.

[0284] The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulatory agent can be fused to an extended PK group that increases its circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of immunostimulatory agents, such as cytokines or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0285] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses, by way of example, the characteristics of a compound, including absorption, distribution, metabolism, and excretion by a subject. As used herein, a "prolonged PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of prolonged PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary prolonged PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, the entire contents of which are incorporated herein by reference. As used herein, an "extended PK" immunostimulant refers to an immunostimulatory moiety in combination with an extended PK group. In some embodiments, an extended PK immunostimulant is a fusion protein in which the immunostimulatory moiety is linked or fused to the extended PK group.

[0286] In certain embodiments, the serum half-life of the extended PK immunostimulant is increased compared to the immunostimulant alone (i.e., the immunostimulant not fused to an extended PK group). In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40, or 50 times longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0287] As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or polypeptide, to decrease by 50% in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Extended PK immunostimulants suitable for use herein are stabilized in vivo, and their half-life is increased, for example, by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally include the steps of administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound decreases by 50% compared to the initial level at the time of administration. Further details are provided, for example, in standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0288] In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0289] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a protein, such as a Therapeutic protein, particularly an immunostimulant. Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a Therapeutic protein is linked in-frame to a polynucleotide encoding albumin. Once part of an albumin fusion protein, the Therapeutic protein and albumin can be referred to as "portions," "regions," or "moieties," respectively, of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In highly preferred embodiments, the albumin fusion protein comprises at least one molecule of a Therapeutic protein (including, but not limited to, the mature form of a Therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In some embodiments, the albumin fusion protein is processed by host cells, such as hepatocytes, in the target organ of the administered RNA and secreted into the circulation. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA may include, but is not limited to, signal peptide cleavage; disulfide bond formation; proper folding; carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation); specific proteolytic cleavage; and / or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by RNA in an unprocessed form, particularly with a signal peptide at its N-terminus, and after secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the "processed form of the albumin fusion protein" refers to the albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0290] In preferred embodiments, albumin fusion proteins containing a therapeutic protein have higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period from when a therapeutic protein is administered in vivo and transported into the bloodstream, until the therapeutic protein is degraded and removed from the bloodstream to organs such as the kidneys or liver, and finally, when the therapeutic protein is removed from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be easily determined by common assays known in the art.

[0291] As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of an albumin fusion protein can be derived from a different animal than the therapeutic protein portion.

[0292] In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0293] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and fragments and variants thereof) as well as albumin (and fragments and variants thereof) from other species.

[0294] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a Therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the Therapeutic protein portion of the albumin fusion protein is extended or expanded compared to its plasma stability in the unfused state.

[0295] The albumin portion of the albumin fusion protein may comprise the full length of the albumin sequence, or may comprise one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise approximately 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0296] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0297] According to the present disclosure, the albumin can be a naturally occurring albumin or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal.

[0298] Preferably, the albumin fusion protein comprises albumin as the N-terminal moiety and a therapeutic protein as the C-terminal moiety. Alternatively, albumin fusion proteins comprising albumin as the C-terminal moiety and a therapeutic protein as the N-terminal moiety may also be used. In other embodiments, the albumin fusion protein has therapeutic proteins fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused to the N-terminus and the C-terminus are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused to the N-terminus and the C-terminus are different therapeutic proteins. In some embodiments, both different therapeutic proteins are cytokines.

[0299] In some embodiments, the therapeutic protein(s) are linked to albumin via a peptide linker(s). A peptide linker between the fusion moieties can provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety to bind to, for example, its cognate receptor. The peptide linker can be composed of amino acids so that it is flexible or more rigid. The linker sequence can be cleavable by protease or chemically.

[0300] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains of the native immunoglobulin. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, an Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides, e.g., comprising only the hinge, CH2, and CH3 domains.The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. As described herein, those skilled in the art will understand that any Fc domain can be modified such that its amino acid sequence differs from that of a native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0301] The Fc domain of the polypeptide described herein can be derived from different immunoglobulin molecules.For example, the Fc domain of the polypeptide can include a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0302] In certain embodiments, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are encompassed by the term "Fc domain" for purposes of this disclosure). The Fc domain does not comprise a variable region that binds to an antigen. Fc domains suitable for use in the present disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain can be derived from an immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

[0303] Furthermore, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0304] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains can be selected, including Fc domain sequences that lack specific effector functions and / or have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and appropriate Fc domain sequences (e.g., hinge, CH2 and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using techniques widely recognized in the art.

[0305] In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in U.S. Patent Application Nos. 2005 / 0287153, 2007 / 0003549, 2007 / 0178082, 2007 / 0269422, 2010 / 0113339, WO 2009 / 083804, and WO 2009 / 133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin binding protein, such as those disclosed in US Patent Application No. 2007 / 0178082, US Patent Application No. 2014 / 0220017, and US Patent Application No. 2017 / 0145062, the entire contents of which are incorporated herein by reference. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US Patent Application No. 2012 / 0094909, the entire contents of which are incorporated herein by reference. A method for producing a fibronectin-based scaffold domain protein is also disclosed in US Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0306] In certain embodiments, the extended PK immunostimulatory agent suitable for use according to the present disclosure can use one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., an extended PK portion and an immunostimulatory portion) in the linear amino acid sequence of a polypeptide chain. For example, a peptide linker can be used to link the immunostimulatory portion to the HSA domain.

[0307] For example, suitable linkers for fusing an extended PK group to an immunostimulatory agent are well known in the art. Exemplary linkers include glycine-serine polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0308] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides a method for treating a subject having a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject an RNA described herein that encodes the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) into a subject having a deficiency of such a protein. The term also refers to the introduction of a protein into a subject who requires or would benefit from the provision of a protein, e.g., a subject suffering from a protein deficiency. The term "a disorder characterized by a protein deficiency" refers to any disorder exhibiting a pathology caused by the absence or insufficient amount of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein deficiency may be involved in infection, immunosuppression, organ failure, glandular disorders, radiation damage, nutritional deficiency, poisoning, or other environmental or external insults.

[0309] The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction typically involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) relaying and amplification of the signal; and (vi) degradation of the hormone. Hormones differ from cytokines in that (1) they typically act at more consistent concentrations and (2) they are generally produced by specific cell types. In some embodiments, a "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0310] The term "adhesion molecule" refers to a protein located on the surface of a cell and involved in binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0311] Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cell signaling pathways, for example, transmembrane protein kinase pathways such as receptor tyrosine kinases (RTKs). Such regulation can result in cell growth, division, survival or differentiation, or apoptosis. Specific examples of integrins include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α L β2, α M β2, α IIb β3, α V β1, αV β3, α V β5, α V β6, α V β8, and α6β4.

[0312] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in cell recognition, binding, and / or adhesion processes. Molecules belonging to this superfamily share the characteristic of containing regions known as immunoglobulin domains or immunoglobulin folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD3-γ, CD3-δ, CD-3ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and the like.

[0313] The term "immunologically active compound" refers to any compound that alters the immune response, for example, by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and can also downregulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants. Specific examples of immunologically active compound include interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferon, integrin, addressin, selectin, homing receptor, and antigen, particularly tumor-associated antigen, pathogen-associated antigen (such as bacterial antigen, parasite antigen or virus antigen), allergen and autoantigen.Immunologically active compound can be vaccine antigen, that is, the antigen that inoculates to subject induces immune response.

[0314] In some embodiments, the RNA (particularly mRNA) described in this disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject. "Peptides or polypeptides comprising an epitope for inducing an immune response against an antigen in a subject" are also referred to herein as "vaccine antigens," "peptide antigens and protein antigens," or simply "antigens."

[0315] In some embodiments, the RNA (particularly mRNA) encoding the vaccine antigen is a single-stranded 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject to which the RNA is administered, e.g., antigen-presenting cells (APCs). Preferably, the RNA (i) contains structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence) optimized for maximum RNA potency with respect to stability and translation efficiency; (ii) is modified for optimized effectiveness of the RNA (e.g., increased translation efficiency, reduced immunogenicity, and / or reduced cytotoxicity) (e.g., by replacing naturally occurring nucleosides (particularly cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine) (partially or completely, preferably completely) and / or by codon optimization); or (iii) is both (i) and (ii).

[0316] In some embodiments, β-S-ARCA(D1) is utilized as a specific capping structure at the 5' end of the RNA. In some embodiments, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO:6 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:6. In some embodiments, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:7 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:7. In some embodiments, the poly(A) sequence is 110 nucleotides long and consists of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to enhance RNA stability and translation efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 8 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: 8. In some embodiments, the RNA comprises a modified nucleoside in place of uridine. In some embodiments, the modified nucleoside substituting (partially or completely, preferably completely) for uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine. In some embodiments, the RNA encoding the vaccine antigen has (a) a codon-optimized coding sequence, (b) a coding sequence whose G / C content is increased compared to the wild-type coding sequence, or (c) both (a) and (b).

[0317] In some embodiments, RNA encoding the vaccine antigen is expressed in cells of a subject to provide the vaccine antigen. In some embodiments, expression of the antigen is at the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, RNA encoding the vaccine antigen is transiently expressed in cells of a subject. In some embodiments, RNA encoding the vaccine antigen is administered systemically. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in no or essentially no expression of RNA encoding the vaccine antigen in the lung and / or liver. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen that is at least five times higher than the level of expression in the lung.

[0318] A vaccine antigen comprises an epitope for inducing an immune response against the antigen in a subject.Therefore, a vaccine antigen comprises an antigen sequence for inducing an immune response against the antigen in a subject.Such an antigen sequence can correspond to a target antigen or a disease-related antigen, for example, a protein or tumor antigen of an infectious agent (for example, a viral antigen or a bacterial antigen), or its immunogenic variant, or an immunogenic fragment or immunogenic variant of a target antigen or a disease-related antigen.Therefore, an antigen sequence can comprise at least one epitope of a target antigen or a disease-related antigen or its immunogenic variant.

[0319] Antigen sequences, e.g., epitopes, suitable for use in accordance with the present disclosure may typically be derived from the target antigen, i.e., the antigen to which an immune response is elicited. For example, the antigen sequence included within a vaccine antigen may be the target antigen or a fragment or variant of the target antigen.

[0320] The antigen sequence or its processing product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR or CAR, carried by an immune effector cell. In some embodiments, the antigen sequence is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigen sequence or fragment.

[0321] The vaccine antigens that can be provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen preferably result in the induction of an immune response in the subject to which the vaccine antigen is provided, e.g., stimulation, priming, and / or expansion of immune effector cells. The immune response, e.g., stimulated, primed, and / or expanded immune effector cells, is preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, the vaccine antigen can include a disease-associated antigen, or a fragment or variant thereof. In some embodiments, such a fragment or variant is immunologically equivalent to the disease-associated antigen.

[0322] In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that induces an immune response, e.g., stimulation, priming, and / or expansion of immune effector cells, where the immune response, e.g., stimulated, primed, and / or expanded immune effector cells, target an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, a fragment of a disease-associated antigen, or an antigen that is homologous to a disease-associated antigen or its fragment. When a vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen targeted by an antigen receptor of an immune effector cell. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can induce, e.g., stimulate, prime, and / or expand, an immune response against immune effector cells bearing an antigen receptor that binds to the antigen or cells expressing the antigen. Preferably, the vaccine antigen (analogous to a disease-associated antigen) provides a relevant epitope for binding by an antigen receptor present on immune effector cells. In some embodiments, the vaccine antigen or a fragment thereof (analogous to a disease-associated antigen) is expressed (optionally in the context of an MHC) on the surface of a cell, such as an antigen-presenting cell, to provide a relevant epitope for binding by the immune effector cell. The vaccine antigen may be a recombinant antigen.

[0323] In some embodiments of all aspects of the invention, RNA encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, which binding results in stimulation, priming, and / or expansion of the immune effector cells. An "antigen" according to the present disclosure encompasses any substance that elicits an immune response and / or any substance against which an immune response or immune mechanism, such as a cellular and / or humoral response, is directed. This also includes situations in which an immune response or immune mechanism is directed against one or more antigenic peptides, particularly when the antigen is processed into antigenic peptides and presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, such as a peptide or polypeptide, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen" can include molecules that contain at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0324] In some embodiments, the antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In some embodiments, the antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells).

[0325] The term "autoantigen" refers to an antigen that originates within a subject's body (i.e., an autoantigen may also be called an "autogen") and that produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen may be the cause of an "autoimmune disease."

[0326] According to the present disclosure, any suitable antigen that is a candidate for an immune response may be used, and the immune response may include a humoral or cellular immune response, or both. In some embodiments of the present disclosure, the antigen is presented by cells, such as antigen-presenting cells, in association with MHC molecules, resulting in an immune response against the antigen. The antigen may correspond to a naturally occurring antigen or a product derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. According to the present disclosure, the antigen may correspond to a naturally occurring product, such as a viral protein, or a portion thereof.

[0327] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0328] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is present primarily intracellularly or as a surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen derived from a virus, bacterium, unicellular organism, or parasite, e.g., a viral antigen such as a viral ribonucleoprotein or coat protein. In some embodiments, the antigen should be presented by an MHC molecule that leads to modulation, particularly activation of cells of the immune system, such as CD4+ and CD8+ lymphocytes, particularly through modulation of the activity of T cell receptors.

[0329] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, this term refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, α2-H-ferroprotein, and γ-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer and tumor or cancer cells in terms of type and / or expression level.

[0330] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0331] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0332] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can include a continuous or discontinuous portion of the protein and can be, e.g., about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0333] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer, for example, to an incompletely displayed form of an antigen that can elicit an immune response against the antigen or a cell that expresses or contains and presents the antigen. In some embodiments, these terms relate to an immunogenic portion of an antigen. In some embodiments, this is the portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100 amino acids in length, e.g., about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids in length; for example, an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

[0334] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response. MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can be effective.

[0335] Peptide and polypeptide antigens can be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0336] A peptide or polypeptide antigen can be any peptide or polypeptide that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or polypeptide.

[0337] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, when recognized by immune effector cells, vaccine antigens can, in the presence of appropriate costimulatory signals, induce the stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In connection with embodiments of the present disclosure, vaccine antigens can be presented or present on the surface of cells, such as, for example, antigen-presenting cells.

[0338] In some embodiments, the antigen is expressed in a diseased cell (such as a tumor cell or an infected cell).

[0339] In some embodiments, the antigen is presented by a diseased cell (such as a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell releases cytotoxic factors, such as perforin and granzymes.

[0340] In some aspects, the antigen is expressed on the surface of a diseased cell (such as a tumor cell or an infected cell). In some embodiments, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope within the extracellular domain. In some embodiments, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen present on a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen present on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0341] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation is fused to the antigenic peptide or polypeptide (antigen sequence) directly or via a linker. Thus, in some embodiments, the RNA described herein comprises at least one coding region that encodes the antigenic peptide or polypeptide and the amino acid sequence that enhances antigen processing and / or presentation.

[0342] In some embodiments, antigens for vaccination that may be administered in the form of RNA encoding same include naturally occurring antigens or fragments thereof, such as epitopes thereof.

[0343] Such amino acid sequences that enhance antigen processing and / or presentation are preferably, but not limited to, located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the C-terminus of the amino acid sequence that disrupts immune tolerance). The amino acid sequences that enhance antigen processing and / or presentation defined herein preferably improve antigen processing and presentation. In some embodiments, the amino acid sequences that enhance antigen processing and / or presentation defined herein include, but are not limited to, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3), particularly sequences comprising the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof.

[0344] In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO:2, 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:2, or a functional fragment of the amino acid sequence of SEQ ID NO:2 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:2. In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO:2.

[0345] Thus, in some embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation, preferably fused to the C-terminus of the antigenic peptide or polypeptide, more preferably an antigenic peptide or polypeptide described herein.

[0346] Additionally, a secretory sequence, such as a sequence comprising the amino acid sequence of SEQ ID NO: 1, can be fused to the N-terminus of the antigenic peptide or polypeptide.

[0347] Amino acid sequences derived from tetanus toxoid of Clostridium tetani can be used to overcome self-tolerance mechanisms to efficiently initiate immune responses to self-antigens by providing T cell help during priming.

[0348] Tetanus toxoid heavy chains bind indiscriminately to MHC class II alleles and induce CD4 + It is known that these antigens contain epitopes that can induce memory T cells. Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens inhibits CD4 T cell activation during priming. + It is known that tumor-associated antigens improve immune stimulation compared to the application of tumor-associated antigens alone by providing mediated T cell help. + To reduce the risk of stimulating T cells, the whole tetanus toxoid fragment C stimulates CD8 + The entire fragment C is not used because it is known to contain a T cell epitope. To ensure binding to as many MHC class II alleles as possible, two peptide sequences containing promiscuous helper epitopes were instead selected. Based on data from ex vivo studies, the known epitope p2 (QYIKANSKFIGITEL; TT 830-844 ;SEQ ID NO: 9) and p16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT 578-609 ; SEQ ID NO: 10) was selected. The p2 epitope has already been used in peptide vaccination in clinical trials to enhance anti-melanoma activity.

[0349] Preclinical data demonstrated that an RNA vaccine encoding both tumor antigens and promiscuous tetanus toxoid sequences stimulated CD8 responses to tumor antigens. +This has been shown to result in enhanced T cell responses and improved tolerance breaking. Immune monitoring data from patients vaccinated with vaccines containing sequences fused in-frame with tumor antigen-specific sequences reveals that selected tetanus sequences are able to induce tetanus-specific T cell responses in nearly all patients.

[0350] According to some embodiments, the immune tolerance-disrupting amino acid sequence is fused to the antigenic peptide or polypeptide either directly or via a linker, for example a linker having an amino acid sequence according to SEQ ID NO:4.

[0351] Such an amino acid sequence that breaks immune tolerance is preferably, but not limited to, located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence that enhances antigen processing and / or presentation, where the amino acid sequence that breaks immune tolerance and the amino acid sequence that enhances antigen processing and / or presentation may be fused directly or via a linker, for example a linker having an amino acid sequence according to SEQ ID NO: 5). An amino acid sequence that breaks immune tolerance as defined herein preferably improves T cell responses. In some embodiments, an amino acid sequence that breaks immune tolerance as defined herein includes, but is not limited to, a sequence derived from the helper sequences p2 and p16 from tetanus toxoid (P2P16), in particular a sequence comprising the amino acid sequence of SEQ ID NO: 3 or a functional variant thereof.

[0352] In some embodiments, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:3, 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:3, or a functional fragment of the amino acid sequence of SEQ ID NO:3 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:3. In some embodiments, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:3.

[0353] Below, embodiments of vaccine RNA are described, and certain terms used when describing elements thereof have the following meanings: Cap: m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O ) A 5' cap structure selected from the group consisting of ApG.

[0354] hAg-Kozak: 5'-UTR sequence of human α-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

[0355] sec / MITD: A fusion protein tag derived from sequences encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3) that has been shown to improve antigen processing and presentation. sec corresponds to a 78-bp fragment encoding a secretory signal peptide that directs translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also known as the MHC class I transport domain.

[0356] Antigen: The sequence encoding each vaccine antigen / epitope.

[0357] Glycine-serine linker (GS): A sequence encoding a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), as commonly used in fusion proteins.

[0358] P2P16: a sequence encoding a helper epitope derived from tetanus toxoid for breaking immune tolerance.

[0359] FI elements: The 3'-UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression.

[0360] 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 increase RNA stability and translation efficiency in dendritic cells.

[0361] In some embodiments, the vaccine RNA described herein has the following structure: Cap-hAg-Kozak-sec-GS(1)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 β-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 It has one of the following. In some embodiments, the vaccine antigens described herein have the structure: sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD It has.

[0362] In some embodiments, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO:6. In some embodiments, sec comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, P2P16 comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, MITD comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, GS(1) comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, GS(2) comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, GS(3) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, FI comprises the nucleotide sequence of SEQ ID NO:7. In some embodiments, A30L70 comprises the nucleotide sequence of SEQ ID NO:8.

[0363] In some embodiments, the sequence encoding the vaccine antigen / epitope comprises modified nucleosides that substitute (partially or fully, preferably fully) for uridine, and the modified nucleosides are selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine.

[0364] In some embodiments, the sequences encoding the vaccine antigens / epitopes are codon optimized.

[0365] In some embodiments, the G / C content of the sequence encoding the vaccine antigen / epitope is increased compared to the wild-type coding sequence.

[0366] In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B cell receptor binds to a natural epitope of the antigen.

[0367] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located on the outside of the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0368] "Cell surface" or "surface of a cell" is used according to its usual meaning in the art, and thus includes the outside of a cell that is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of a cell when it is located on the surface of the cell and is accessible to binding by, for example, an antigen-specific antibody added to the cell. In some embodiments, the antigen expressed on the surface of a cell is an integral membrane protein with an extracellular portion that can be recognized by a CAR.

[0369] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, for example, by binding to a molecule, such as an antibody, that is located on the outside of the cell. In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0370] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen it targets, particularly when presented on the surface of an antigen-presenting cell or diseased cell, such as a cancer cell, in association with an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0371] The term "target" refers to an agent, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In some embodiments, target cells are cells that express an antigen and present said antigen in association with class I MHC.

[0372] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by cells such as antigen-presenting cells. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0373] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0374] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0375] The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitor cells first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as coreceptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they act as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce immune responses associated with T cells or B cells. In some embodiments, the dendritic cells are splenic dendritic cells.

[0376] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, resulting in the degradation of the pathogen. Peptides derived from degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0377] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen or a peptide derived from the antigen, which is presented together with class I MHC on the surface of an antigen-presenting cell. + It means T cells.

[0378] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0379] As used herein, "activation" or "stimulation" refers to the state of cells, such as immune effector cells, such as T cells, that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0380] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, leading to differentiation into an effector cell, such as an effector T cell.

[0381] The term "expansion" refers to the process by which a particular entity multiplies. In some embodiments, the term is used in the context of an immunological response in which immune effector cells are stimulated by an antigen, causing proliferation and amplification of specific immune effector cells that recognize the antigen. In some embodiments, expansion results in differentiation of immune effector cells.

[0382] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the integrated body's response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, to the agent. An immune response is characterized by the development of antibodies against one or more antigens, as well as antigen-specific T lymphocytes, e.g., CD4, which can be detected in various in vitro proliferation or cytokine production tests. + and CD8 + T lymphocytes, e.g., CD8 + The reaction may include one or more responses selected from the group consisting of T lymphocyte expansion.

[0383] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, for example, the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / protective / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, such as a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV, or RSV)). "Inducing" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was some immune response against a particular antigen or pathogen before induction and that the immune response was enhanced after induction. Thus, "inducing an immune response" in this context also includes "enhancing an immune response." In some embodiments, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease state is ameliorated by inducing an immune response.

[0384] The terms "cellular immune response," "cellular response," "cellular immunity," or similar terms are intended to include a cellular response to cells characterized by expression of an antigen and / or presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, including diseased cells.

[0385] The term "humoral immune response" refers to the process in living organisms by which antibodies are produced in response to agents and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-bound receptors that bind to a single antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigen epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the amplified antibody response that occurs upon re-exposure to a particular antigen.

[0386] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxy terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0387] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, e.g., antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are mainly V L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domain, and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which constitute the different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0388] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons and, as described herein, relate to a procedure in which one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding same, are administered to an individual to stimulate an immune response against said one or more immunogens or antigens or against cells characterized by the presentation of said one or more immunogens or antigens.

[0389] "Cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigen on the surface of antigen-presenting cells" or similar expressions refer to cells, such as diseased cells, particularly tumor or infected cells, or antigen-presenting cells, that present antigens or antigenic peptides, either directly or after processing, in association with MHC molecules, such as MHC class I and / or MHC class II molecules. In some embodiments, the MHC molecules are MHC class I molecules.

[0390] The term "allergen" refers to a type of antigen that originates outside the subject's body (i.e., allergens are sometimes called "xenoantigens") and that causes the subject's immune system to mount an unusually vigorous immune response to repel a perceived threat that would otherwise be harmless to the subject. "Allergy" is a disease caused by such a vigorous immune response to allergens. Allergens are typically antigens that can stimulate type I hypersensitivity reactions in atopic individuals via an immunoglobulin E (IgE) response. Specific examples of allergens include those derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and house dust mite proteins (e.g., Der p 2).

[0391] The term "growth factor" refers to a molecule that can stimulate cell growth, proliferation, healing, and / or cell differentiation. Typically, growth factors function as signaling molecules between cells. The term "growth factor" includes specific cytokines and hormones that bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs) such as VEGFA, epidermal growth factor (EGF), insulin-like growth factors, ephrins, macrophage colony-stimulating factors, granulocyte colony-stimulating factors, granulocyte-macrophage colony-stimulating factors, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (an anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β)), and tumor necrosis factor alpha (TNF-α). In some embodiments, a "growth factor" is a peptide or polypeptide growth factor.

[0392] The term "protease inhibitor" refers to a molecule, particularly a peptide or polypeptide, that inhibits the function of a protease. Protease inhibitors can be classified by the protease they inhibit (e.g., aspartic acid protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors such as serpins). Specific examples of protease inhibitors include serpins such as alpha-1-antitrypsin, aprotinin, and bestatin.

[0393] The term "enzyme" refers to a macromolecular biological catalyst that promotes chemical reactions. Like any catalyst, enzymes are not consumed by the reactions they catalyze and do not alter the equilibrium of said reactions. Unlike many other catalysts, enzymes are much more specific. In some embodiments, enzymes are essential for a subject's homeostasis; for example, enzyme dysfunction (particularly reduced activity, which can be caused by either mutation, deletion, or reduced production) results in disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0394] The term "receptor" refers to a protein molecule that receives a signal (e.g., a chemical signal called a ligand) from outside the cell. Binding of a signal (e.g., a ligand) to a receptor triggers some kind of cellular response, such as the intracellular activation of a kinase. Receptors include transmembrane receptors (e.g., ion channel-linked (ionotropic) receptors, G protein-coupled (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (e.g., cytoplasmic and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to a growth factor.

[0395] The term "apoptosis regulator" refers to molecules, particularly peptides or polypeptides, that regulate apoptosis, i.e., activate or inhibit apoptosis. Apoptosis regulators can be divided into two broad classes: those that regulate mitochondrial function and those that regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) that maintain mitochondrial integrity by preventing the loss of mitochondrial membrane potential and / or the release of proapoptotic proteins such as cytochrome C into the cytosol. Proapoptotic proteins that promote the release of cytochrome C (e.g., BAX, BAK, BIM) also belong to this first class. The second class includes inhibitors of apoptosis proteins (e.g., XIAP) or proteins such as FLIP that block caspase activation.

[0396] The term "transcription factor" refers to a protein that regulates the rate of transcription of genetic information from DNA to messenger RNA, particularly by binding to specific DNA sequences. Transcription factors can regulate cell division, proliferation, and death throughout life, cell migration and organization during embryonic development, and / or in response to signals from outside the cell, such as hormones. Transcription factors typically contain at least one DNA-binding domain that binds to a specific DNA sequence adjacent to the gene regulated by the transcription factor. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0397] The term "tumor suppressor protein" refers to a molecule, particularly a peptide or polypeptide, that protects cells from a step on the path to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exhibit a dampening or inhibitory effect on cell cycle regulation and / or promote apoptosis. Their function may be one or more of the following: suppression of genes essential for cell cycle continuation; linking the cell cycle to DNA damage (cell division should not occur as long as damaged DNA is present in the cell); initiating apoptosis if damaged DNA cannot be repaired; suppression of metastasis (e.g., preventing tumor cell dispersal, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI / SNF (SWItch / Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppressor of tumorigenicity 5 (ST5), suppressor of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).

[0398] The term "structural protein" refers to a protein that imparts rigidity and stiffness to otherwise fluid biological components. Structural proteins are mostly fibrous (such as collagen and elastin), but can also be globular (such as actin and tubulin). Globular proteins are usually soluble as monomers but polymerize to form long fibers that can, for example, constitute the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin, and dynein) that can generate mechanical force, and surfactant proteins. Specific examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

[0399] The term "reprogramming factor" or "reprogramming transcription factor" relates to a molecule, particularly a peptide or polypeptide, which, when expressed in a somatic cell, optionally together with further agents such as further reprogramming factors, results in the reprogramming or dedifferentiation of said somatic cell into a cell with stem cell properties, particularly pluripotency. Particular examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0400] The term " genome engineering protein " refers to the protein that can insert DNA, delete DNA or replace DNA in the genome of interest.Specific examples of genome engineering protein include meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9).

[0401] The term "blood protein" refers to peptides or polypeptides present in a subject's plasma, particularly the plasma of a healthy subject. Blood proteins have diverse functions, such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), and protease inhibitors (e.g., α1-antitrypsin). Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte-colony stimulating factor (G-CSF), modified factor VIII, and anticoagulants.

[0402] Thus, in some embodiments, the pharmaceutically active peptide or polypeptide is selected from the group consisting of: (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2) and interleukin 10 (IL-11), more preferably EPO; (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunologically active compound, particularly an antigen such as a viral or bacterial antigen, for example an antigen of SARS-CoV-2; (v) a hormone, particularly vasopressin, insulin or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly alpha 1-antitrypsin; (viii) a protease inhibitor, preferably herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminida (xii) tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, for example, selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome engineering proteins, particularly clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

[0403] In some embodiments, the pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response in the subject against the one or more antigens or one or more epitopes, which may be therapeutic or partially or fully protective.

[0404] In some embodiments, the RNA encodes at least one epitope, e.g., at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.

[0405] In some embodiments, the target antigen is a tumor antigen, and the antigen sequence (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen that contains one or more amino acid changes.

[0406] Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as claudin 6, claudin 18.2, and claudin 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / MelanA, MC1R, myosin / m, MUC 1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0407] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are particularly interesting antigen-presenting cells for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutagenesis can provide multiple epitopes for personalized vaccines that can be encoded by the RNA (particularly mRNA) described herein, for example, as a single polypeptide in which the epitopes are optionally separated by linkers. In some embodiments of the present disclosure, the RNA (particularly mRNA) encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include RNA (particularly mRNA) encoding at least five epitopes (termed "pentatopes") and RNA (particularly mRNA) encoding at least ten epitopes (termed "decatopes").

[0408] In some embodiments, the antigen or epitope is derived from a pathogen-associated antigen, particularly a viral antigen.

[0409] In some embodiments, the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or its immunogenic variant. Thus, in some embodiments, the mRNA used in the present disclosure encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or its immunogenic variant.

[0410] In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. Thus, in some embodiments, the RNA (particularly mRNA) described in this disclosure encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus S protein or its immunogenic variant. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

[0411] Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They have the largest genomes (26–32 kb) of any known RNA virus and are phylogenetically divided into four genera (α, β, γ, and δ), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. While several human coronaviruses generally cause mild respiratory illness, the disease can be more severe in infants, the elderly, and immunocompromised individuals. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), which belong to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses have emerged from animal reservoirs into human populations within the past 15 years and caused outbreaks with high case fatality rates. An outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes atypical pneumonia (coronavirus disease 2019; COVID-19), has been raging in China since mid-December 2019, evolving into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the betacoronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV.

[0412] Generally, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in virus assembly, while the N protein is required for viral RNA synthesis. The key glycoprotein, S, is responsible for virus binding and entry into target cells. The S protein is synthesized in the producing cells as a single-chain inactive precursor that is cleaved into two noncovalently associated subunits, S1 and S2, by a furin-like host protease. The S1 subunit contains a receptor-binding domain (RBD) that recognizes host cell receptors. The S2 subunit contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate fusion of the viral membrane with the host cell membrane by undergoing a large conformational rearrangement. The S1 and S2 subunits trimerize to form a large pre-fusion spike.

[0413] The SARS-CoV-2 S precursor protein can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit contains the receptor-binding domain (RBD) that mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0414] In some embodiments, the antigen or epitope is derived from the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the RNA (particularly mRNA) described in this disclosure encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. Thus, in some embodiments, the encoded amino acid sequence comprises an epitope of the SARS-CoV-2 S protein or an immunogenic variant thereof for inducing an immune response against the coronavirus S protein, particularly the SARS-CoV-2 S protein, in a subject. In some embodiments, the RNA (particularly mRNA) is administered to provide (after expression by appropriate target cells) antigens, e.g., antibodies and / or immune effector cells, for induction of an immune response targeting the target antigen (coronavirus S protein, particularly the SARS-CoV-2 S protein) or its processing products. In some embodiments, the immune response induced in accordance with the present disclosure is a B cell-mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in some embodiments, the immune response induced in accordance with the present disclosure is a T cell-mediated immune response. In some embodiments, the immune response is an anti-coronavirus, particularly an anti-SARS-CoV-2, immune response.

[0415] The SARS-CoV-2 coronavirus full-length spike (S) protein consists of 1273 amino acids and has the following amino acid sequence:

[0416] TIFF2025502796000037.tif126158

[0417] For the purposes of this disclosure, the above sequence is considered the wild-type SARS-CoV-2 S protein amino acid sequence. The SARS-CoV-2 S protein position numbers shown herein relate to the amino acid sequence according to SEQ ID NO: 11 and the corresponding positions in SARS-CoV-2 S protein variants.

[0418] In a specific embodiment, the full-length spike (S) protein according to SEQ ID NO: 11 is modified to stabilize the prototypical pre-fusion conformation. Stabilization of the pre-fusion conformation can be obtained by introducing two consecutive proline substitutions at amino acid residues 986 and 987 in the full-length spike protein. Specifically, the spike (S) protein stabilized protein variant is obtained in such a way that the amino acid residue at position 986 is replaced with proline and the amino acid residue at position 987 is also replaced with proline. In one embodiment, the SARS-CoV-2 S protein variant in which the prototypical pre-fusion conformation is stabilized comprises the following amino acid sequence:

[0419] TIFF2025502796000038.tif134158

[0420] Those skilled in the art are aware of various spike variants and / or resources that document them.

[0421] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the present disclosure) encodes an amino acid sequence that comprises, consists essentially of, or consists of the spike (S) protein of SARS-CoV-2, a variant thereof, or a fragment thereof.

[0422] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or 12, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or 12, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or 12 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or 12. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or 12.

[0423] In some embodiments, the encoded amino acid sequence comprises, consists essentially of, or consists of the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof.

[0424] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 11, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 11. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 11.

[0425] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 11, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 11. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 11.

[0426] In some embodiments, the encoded amino acid sequence comprises, consists essentially of, or consists of the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof. The amino acid sequence of amino acids 327-528 of SEQ ID NO: 11, a variant thereof, or a fragment thereof is also referred to herein as the "RBD" or "RBD domain."

[0427] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO: 11, or an immunogenic fragment of the amino acid sequence of amino acids 327-528 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO: 11. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO: 11.

[0428] According to certain embodiments, the signal peptide is fused, directly or via a linker, to the SARS-CoV-2 S protein, its variants, or fragments thereof, i.e., antigenic peptide or protein. Thus, in some embodiments, the signal peptide is fused to the above-mentioned amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the above-encoded amino acid sequence.

[0429] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 11 or 12, 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: 11 or 12, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 11 or 12 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: 11 or 12. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 11 or 12.

[0430] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 11, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 11. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 11.

[0431] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 11, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 11. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 11.

[0432] According to certain embodiments, the trimerization domain is fused, either directly or via a linker, e.g., a glycine / serine linker, to the SARS-CoV-2 S protein, its variants, or fragments thereof, i.e., antigenic peptide or protein. Thus, in some embodiments, the trimerization domain is fused to the above-described amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) contained in the above-described encoded amino acid sequence (optionally fused to the above-described signal peptide).

[0433] Such trimerization domains are preferably, but not limited to, located at the C-terminus of antigenic peptides or proteins. The trimerization domains defined herein preferably enable trimerization of antigenic peptides or proteins encoded by RNA. Examples of trimerization domains defined herein include, but are not limited to, Foldon, the natural trimerization domain of T4 fibritin. The C-terminal domain of T4 fibritin (Foldon) is essential for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. In some embodiments, the trimerization domains defined herein include, but are not limited to, a sequence comprising the amino acid sequence of SEQ ID NO: 13 or a functional variant thereof.

[0434] In some embodiments, the trimerization domain comprises the amino acid sequence of amino acids 3-29 of SEQ ID NO: 13, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO: 13, or a functional fragment of the amino acid sequence of amino acids 3-29 of SEQ ID NO: 13 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO: 13. In some embodiments, the trimerization domain comprises the amino acid sequence of amino acids 3-29 of SEQ ID NO: 13.

[0435] In some embodiments, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 13, 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: 13, or a functional fragment of the amino acid sequence of SEQ ID NO: 13 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: 13. In some embodiments, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 13.

[0436] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14, 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: 14, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 14 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: 14. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 14.

[0437] According to certain embodiments, the transmembrane domain is fused directly or via a linker, e.g., a glycine / serine linker, to the SARS-CoV-2 S protein, its variant, or fragment thereof, i.e., the antigenic peptide or protein. Thus, in some embodiments, the transmembrane domain is fused to the above-described amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragment (antigenic peptide or protein) contained in the above-described encoded amino acid sequence (optionally fused to the above-described signal peptide and / or trimerization domain). Such a transmembrane domain is preferably, but not limited to, located at the C-terminus of the antigenic peptide or protein. Preferably, such a transmembrane domain, if present, is located at the C-terminus of the trimerization domain. In some embodiments, the trimerization domain is located between the SARS-CoV-2 S protein, its variant, or fragment thereof, i.e., the antigenic peptide or protein, and the transmembrane domain. The transmembrane domain defined herein preferably enables anchoring of the RNA-encoded antigenic peptide or protein to the cell membrane. In some embodiments, a transmembrane domain sequence as defined herein includes, but is not limited to, a transmembrane domain sequence of a SARS-CoV-2 S protein, particularly a sequence comprising the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 11, or a functional variant thereof. In some embodiments, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 11, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 11, or a functional fragment of the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 11 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 11. In some embodiments, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO:11.

[0438] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 15, 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, or an immunogenic fragment of 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. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 15.

[0439] In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 16, 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: 16, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 16 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: 16. In some embodiments, the encoded amino acid sequence comprises the amino acid sequence of SEQ ID NO: 16.

[0440] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is (i) a nucleotide sequence of SEQ ID NO: 17, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 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: 17 or the nucleotide sequence of SEQ ID NO: 17. 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 12, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12, 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: 12, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 12 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: 12. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) (i) comprises the nucleotide sequence of SEQ ID NO: 17 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12.

[0441] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is a nucleoside-modified messenger RNA (modRNA) and has (i) the nucleotide sequence of SEQ ID NO:17, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:17, 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:17 or the nucleotide sequence of SEQ ID NO:17. , 96%, 95%, 90%, 85%, or 80% identity, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12, 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: 12, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 12 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: 12. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 17 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 12.

[0442] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is (i) a nucleotide sequence of SEQ ID NO: 18, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 18, 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: 18 or the nucleotide sequence of SEQ ID NO: 18. 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14, 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: 14, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 14 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: 14. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) (i) comprises the nucleotide sequence of SEQ ID NO: 18, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14.

[0443] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is a nucleoside-modified messenger RNA (modRNA) and has (i) the nucleotide sequence of SEQ ID NO:18, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:18, 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:18 or the nucleotide sequence of SEQ ID NO:18. , 96%, 95%, 90%, 85%, or 80% identity, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14, 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: 14, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 14 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: 14. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 18 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 14.

[0444] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is (i) a nucleotide sequence of SEQ ID NO: 19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19, 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: 19 or the nucleotide sequence of SEQ ID NO: 19. 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 15, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 15, 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, or an immunogenic fragment of 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. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in compositions / formulations and / or used in methods of the disclosure) (i) comprises the nucleotide sequence of SEQ ID NO: 19 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 15.

[0445] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is a nucleoside-modified messenger RNA (modRNA) and has (i) the nucleotide sequence of SEQ ID NO:19, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:19, 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:19 or the nucleotide sequence of SEQ ID NO:19. , 96%, 95%, 90%, 85%, or 80% identity, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 15, 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, or an immunogenic fragment of 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. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 15.

[0446] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in a composition / formulation of the present disclosure and / or used in a method of the present disclosure) is (i) a nucleotide sequence of SEQ ID NO:20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, 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:20 or the nucleotide sequence of SEQ ID NO:20. 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 16, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 16, 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: 16, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 16 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: 16. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) (i) comprises the nucleotide sequence of SEQ ID NO: 20, and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 16.In some embodiments, the RNA is a nucleoside-modified messenger RNA (modRNA) and is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO:20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, 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:20 or a nucleotide sequence of SEQ ID NO:20. and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 16, 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: 16, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 16 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: 16. In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the disclosure) is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20 and / or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 16.

[0447] In some embodiments, the RNA (particularly mRNA) described herein (e.g., included in the compositions / formulations and / or used in the methods of the present disclosure) comprises BNT162b2. BNT162b2 is an mRNA vaccine for preventing COVID-19 and has shown greater than 95% efficacy in preventing COVID-19. The vaccine is made from a 5'-capped mRNA encoding the full-length SARS-CoV-2 spike glycoprotein (S) encapsulated in lipid nanoparticles (LNPs). The RNA may be presented as a product containing BNT162b2 as the active substance, as well as other components, including a cationic ionizable lipid (particularly a compound of Formula (I)); a polymer-bound lipid, preferably selected from the group consisting of sarcosinylated lipids and PEGylated lipids; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. The S protein sequence was selected based on the sequences of "SARS-CoV-2 isolate Wuhan-Hu-1": GenBank: MN908947.3 (complete genome) and GenBank: QHD43416.1 (spike surface glycoprotein). The active substance consists of a single-stranded 5' cap codon-optimized mRNA that translates into the SARS-CoV-2 spike antigen. The protein sequence contains two proline mutations that ensure the antigenically optimal prefusion conformation (P2 S). The RNA does not contain uridine; instead, a modified N1-methylpseudouridine is used for RNA synthesis. The RNA contains common structural elements optimized to mediate high RNA stability and translation efficiency. LNP protects the RNA from RNAse degradation, enabling transfection of host cells after intramuscular (IM) delivery. The mRNA is translated into the SARS-CoV-2 S protein in the host cell. The S protein is then expressed on the cell surface, where it induces an adaptive immune response. The S protein has been identified as a target for neutralizing antibodies against the virus and is therefore considered a suitable vaccine component. BNT162b2 is administered intramuscularly (IM) to adults in two 30 μg doses given 21 days apart.

[0448] In some embodiments, the RNA (particularly mRNA) described herein is a modified RNA, particularly a stabilized mRNA. In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0449] In some embodiments, the RNA (particularly mRNA) described herein includes modified nucleosides in place of uridine.

[0450] In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0451] In some embodiments, the RNA (particularly mRNA) described herein comprises a 5' cap. 7,3’-O Gppp(m1 2’-O ) ApG is used as a specific capping structure at the 5' end of mRNA.

[0452] In some embodiments, RNA (particularly mRNA) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO:6 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:6.

[0453] In some embodiments, RNA (particularly mRNA) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a 3'UTR comprising the nucleotide sequence of SEQ ID NO:7 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:7.

[0454] In some embodiments, RNA (particularly mRNA) encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises a polyA sequence.

[0455] In some embodiments, the polyA sequence comprises at least 100 nucleotides.

[0456] In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:8.

[0457] In some embodiments, the RNA (particularly mRNA) described herein is or should be formulated as a liquid, a solid, or a combination thereof.

[0458] In some embodiments, the RNA (particularly mRNA) described herein is or should be formulated for injection.

[0459] In some embodiments, the RNA (particularly mRNA) described herein is or should be formulated for intramuscular administration.

[0460] In some embodiments, the RNA (particularly mRNA) described herein is or should be formulated as a composition, eg, a pharmaceutical composition.

[0461] In some embodiments, the composition comprises a cationic ionizable lipid of formula (I) (or any one of formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3)).

[0462] In some embodiments, the composition comprises a cationic ionizable lipid of Formula (I) (or any one of Formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3)), and one or more additional lipids. In some embodiments, the one or more additional lipids are selected from polymer-bound lipids, neutral lipids, and combinations thereof. In some embodiments, the neutral lipids comprise phospholipids, steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination of polymer-bound lipids, phospholipids, and steroid lipids.

[0463] In some embodiments, the composition comprises a cationic ionizable lipid of Formula (I) (or any one of Formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3)); a polymer-conjugated lipid selected from the group consisting of a polysarcosine-conjugated lipid and a PEG-conjugated lipid disclosed herein; and cholesterol.

[0464] In some embodiments, the composition comprises a cationic ionizable lipid of Formula (I) (or any one of Formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3)); a polymer-conjugated lipid selected from the group consisting of a polysarcosine-conjugated lipid and a PEG-conjugated lipid disclosed herein; cholesterol; and a phospholipid. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0465] In some embodiments, the composition comprises a cationic ionizable lipid of formula (IV-1), (IV-2) or (IV-3); a polymer-bound lipid selected from the group consisting of DMG-PEG 2000 and C14pSar23; cholesterol; and a phospholipid. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0466] In some embodiments, the RNA is mRNA or saRNA.

[0467] In some embodiments, at least a portion of (i) the RNA, (ii) the cationic ionizable lipid, and, if present, (iii) one or more additional lipids, are present in a particle. In some embodiments, the particle is a nanoparticle, such as a lipid nanoparticle (LNP).

[0468] In some embodiments, the composition, particularly the pharmaceutical composition, is a vaccine.

[0469] In some embodiments, the composition, particularly the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0470] In some embodiments, the RNA and / or the composition, particularly the pharmaceutical composition, is a component of a kit.

[0471] In some embodiments, the kit further comprises instructions for using the RNA to induce an immune response to coronavirus in a subject.

[0472] In some embodiments, the kit further comprises instructions for using the RNA to therapeutically or prophylactically treat a coronavirus infection in a subject.

[0473] In some embodiments, the subject is a human.

[0474] In some embodiments, the coronavirus is a betacoronavirus.

[0475] In some embodiments, the coronavirus is a sarbecovirus.

[0476] In some embodiments, the coronavirus is SARS-CoV-2.

[0477] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, the amino acid sequence induces an immune response with specificity that reacts with the reference amino acid sequence. Thus, in some embodiments, a molecule immunologically equivalent to an antigen exhibits the same or essentially the same properties as the antigen targeted by T cells and / or exerts the same or essentially the same effect with respect to stimulating, priming, and / or expanding T cells.

[0478] In some embodiments, the RNA (particularly mRNA) described in the present disclosure, for example, the RNA encoding a vaccine antigen, is non-immunogenic.The RNA encoding an immunostimulator can be administered according to the present disclosure to provide an adjuvant effect.The RNA encoding an immunostimulator can be standard RNA or non-immunogenic RNA.

[0479] As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that, e.g., upon administration to a mammal, does not induce a response by the immune system or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and processing that render the non-immunogenic RNA non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors, and / or by limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA) during in vitro transcription, and / or by removing double-stranded RNA (dsRNA), e.g., after in vitro transcription. In certain embodiments, the non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription.

[0480] To render non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating modified nucleosides, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Modified nucleosides that suppress RNA-mediated activation of innate immune receptors are particularly preferred. In some embodiments, the modified nucleoside comprises the substitution 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 3-methyl-uridine (m3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 In certain embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0481] In some embodiments, the substitution of one or more uridines with nucleosides comprising modified nucleobases comprises substitution 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.

[0482] During the 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 the enzyme's unusual activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to the inhibition of protein synthesis. The formation of dsRNA can be limited during the synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis. Optionally, UTP can be added once or several times during mRNA synthesis. Also, dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, using Escherichia coli (E. coli) RNase III, which specifically hydrolyzes dsRNA but not ssRNA, thereby removing dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulose materials. In some embodiments, RNA preparation is contacted with cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material and do not allow ssRNA to bind to the cellulose material.Suitable methods for providing ssRNA are disclosed in, for example, WO2017 / 182524.

[0483] "Removing" or "removal," as used herein, refers to the characteristic of a population of a first substance, such as non-immunogenic RNA, that is separated from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, the population of the first substance that is characterized by the removal of the population of the second substance has a measurably lower content of the second substance compared to an unseparated mixture of the first substance and the second substance.

[0484] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, and dsRNA (particularly dsmRNA) is removed from non-immunogenic RNA, for example, 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 non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) does not contain or essentially does not contain dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises single-stranded nucleoside-modified RNA (particularly mRNA) and is substantially free of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (particularly 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 compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0485] Various methods can be used to determine the amount of dsRNA.For example, sample can be contacted with dsRNA specific antibody, and the amount of antibody that binds to RNA can be considered as the measure of the amount of dsRNA in sample.The sample that contains known amount of dsRNA can be used as reference.

[0486] For example, RNA can be spotted on a membrane, such as a nylon blotting membrane. For example, the membrane can be blocked in TBS-T buffer (20 mM Tris pH 7.4, 137 mM NaCl, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For detecting dsRNA, the membrane can be incubated with a dsRNA-specific antibody, such as a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). For example, after washing with TBS-T, the membrane can be incubated with a secondary antibody, such as HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, catalog number 715-035-150), and the signal provided by the secondary antibody can be detected.

[0487] In some embodiments, non-immunogenic RNA (especially mRNA) is translated in cells more efficiently than standard RNA having the same sequence. In some embodiments, translation is enhanced 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the factor is between 10-1000-fold. In some embodiments, the fold is 10-100 fold. In some embodiments, the fold is 10-200 fold. In some embodiments, the fold is 10-300 fold. In some embodiments, the fold is 10-500 fold. In some embodiments, the fold is 20-1000 fold. In some embodiments, the fold is 30-1000 fold. In some embodiments, the fold is 50-1000 fold. In some embodiments, the fold is 100-1000 fold. In some embodiments, the fold is 200-1000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0488] In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower natural immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced 3-fold. In some embodiments, the natural immunogenicity is reduced 4-fold. In some embodiments, the natural immunogenicity is reduced 5-fold. In some embodiments, the natural immunogenicity is reduced 6-fold. In some embodiments, the natural immunogenicity is reduced 7-fold. In some embodiments, the natural immunogenicity is reduced 8-fold. In some embodiments, the natural immunogenicity is reduced 9-fold. In some embodiments, the natural immunogenicity is reduced 10-fold. In some embodiments, the natural immunogenicity is reduced 15-fold. In some embodiments, the natural immunogenicity is reduced 20-fold. In some embodiments, the natural immunogenicity is reduced 50-fold. In some embodiments, the natural immunogenicity is reduced 100-fold. In some embodiments, the natural immunogenicity is reduced 200-fold. In some embodiments, the natural immunogenicity is reduced by 500-fold, in some embodiments, the natural immunogenicity is reduced by 1000-fold, in some embodiments, the natural immunogenicity is reduced by 2000-fold.

[0489] The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0490] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is a relatively nonspecific, immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0491] particle Nucleic acids, such as RNA, particularly mRNA, described herein, can be present in particles comprising (i) the nucleic acid and (ii) at least one cationic or cationic ionizable compound, such as a polymer or lipid, that complexes the nucleic acid. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged nucleic acids are responsible for particle formation. This results in complexation and spontaneous formation of nucleic acid particles. During the manufacturing process, when an aqueous solution of RNA is introduced into an ethanolic lipid mixture containing cationic ionizable lipids at a pH of, for example, 5, electrostatic interactions occur between the negatively charged RNA drug substance and the positively charged cationic ionizable lipids. This electrostatic interaction results in particle formation consistent with efficient encapsulation of the RNA drug substance. After RNA encapsulation, adjusting the medium surrounding the resulting RNA-LNP to, for example, pH 8 neutralizes the surface charge on the LNP. When all other variables are held constant, charge-neutral particles exhibit longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are rapidly removed by the reticuloendothelial system. Upon endosomal uptake, the low pH of the endosome renders the LNP fusogenic, allowing release of RNA into the cytosol of the target cell.

[0492] Various types of RNA-containing particles have previously been described as suitable for delivering RNA in a microparticulate form (see, e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA can physically protect the RNA from degradation and, depending on the specific chemical properties, assist in cellular uptake and endosomal escape.

[0493] In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or molecular complex, particularly a particle-forming compound. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellae) made of one or more amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may also contain additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. Thus, the particle may be a monolamellar or multilamellar structure in which the substance constituting one or more layers or lamellae contains one or more amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), optionally in combination with additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0494] " RNA particle " can be used to deliver RNA to the target site of interest (for example, cell, tissue, organ, etc.). RNA particle can be formed from lipids, including at least one cationic or cationic ionizable lipid. Without intending to be bound by any theory, it is believed that cationic or cationic ionizable lipid forms aggregates together with RNA, and this aggregation results in colloidally stable particles.

[0495] The RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0496] The lipoplexes (LPX) described herein can be obtained by mixing two aqueous phases: one containing RNA and one containing a lipid dispersion. In some embodiments, the lipid phase comprises liposomes.

[0497] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles, the lamellae of which comprise a lipid bilayer and the enclosed lumen of which comprises an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that they can form a lamellar (bilayer) phase in an aqueous environment to which the lipids in the mixture are applied as needed.

[0498] In some embodiments, liposomes comprise a single or multiple phospholipid bilayer surrounding an aqueous core (also referred to herein as an aqueous lumen). They can be prepared from materials with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids used to formulate liposomes designed for nucleic acid delivery are amphiphilic in nature, consisting of positively charged (cationic) amine head groups linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0499] In some embodiments, the lipoplexes are multilamellar liposome-based formulations formed upon electrostatic interaction between cationic liposomes and RNA. In some embodiments, the formed lipoplexes have a distinct internal arrangement of molecules resulting from the conversion of the liposomal structure to a compact RNA-lipoplex.

[0500] In some embodiments, the LPX particles comprise amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids, and RNA (particularly mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids) and negatively charged RNA (particularly mRNA) result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic or cationic ionizable amphiphilic lipids, such as the cationic ionizable lipid of formula (I), DOTMA and / or DODMA, and optionally additional lipids, such as DOPE or DSPC. In some embodiments, the RNA (particularly mRNA) lipoplex particles are nanoparticles.

[0501] Generally, lipid nanoparticles (LNPs) can be obtained by directly mixing RNA in an aqueous phase with lipids in a phase containing an organic solvent such as ethanol. In this case, lipids or lipid mixtures that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0502] In some embodiments, the LNP comprises or consists of a cationic / cationic ionizable lipid (particularly a cationic ionizable lipid of any one of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3) disclosed herein) and a helper lipid, such as a phospholipid, cholesterol, and / or a polymer-conjugated lipid (e.g., a polyethylene glycol (PEG) lipid or a sarcosinylated lipid). In some embodiments, in the RNA LNPs described herein, the RNA (particularly mRNA) is bound to a cationic ionizable lipid (particularly a cationic ionizable lipid of any one of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3) disclosed herein) that occupies the central core of the LNP. In some embodiments, the polymer-bound lipid, together with the phospholipid, forms the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, charged and uncharged forms of cholesterol and cationic ionizable lipids (particularly cationic ionizable lipids of any one of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (IIa), (IIb), (IIIa), (IIIb), (IV-1), (IV-2), and (IV-3) disclosed herein) may be distributed throughout the LNP.

[0503] In some embodiments, the RNA (e.g., mRNA) described herein may be non-covalently associated with the particles described herein. In embodiments, the RNA (e.g., mRNA) may be attached to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or may be contained within the particle (encapsulated RNA (especially encapsulated mRNA)).

[0504] In some embodiments, the particles described herein (e.g., LNP and LPX) have a diameter in the range of about 10 to about 2000 nm, e.g., at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or up to about 1900 nm (e.g., up to about 1800 nm, up to about 1700 nm, up to about 1600 nm, up to about 1500 nm, up to about 1400 nm, up to about 1300 nm, up to about 1200 nm, up to about 1400 nm, up to about 1500 nm, up to about 1600 nm, up to about 1700 nm, up to about 1800 nm, up to about 1900 nm, or up to about 2000 nm). about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, about 20 to about 1500 nm, for example, about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about In some embodiments, the particles described herein (e.g., LNP and LPX) have a size (e.g., diameter) ranging from about 40 nm to about 200 nm, e.g., from about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0505] In some embodiments, the particles described herein (e.g., LNP and LPX) may have a particle size of about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm , about 50nm to about 250nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 600nm, about 100nm to about 500nm, about 100nm to about 450nm, about 100nm to about 400nm, about 100nm to about 350nm, about 100nm to about 300nm, about 100nm to about 250nm, Approximately 100nm to approximately 200nm, approximately 150nm to approximately 1000nm, approximately 150nm to approximately 800nm, approximately 150nm to approximately 700nm, approximately 150nm to approximately 600nm, approximately 150nm to approximately 500nm , about 150nm to about 450nm, about 150nm to about 400nm, about 150nm to about 350nm, about 150nm to about 300nm, about 150nm to about 250nm, about 150nm to about 200nm In some embodiments, the particles described herein (e.g., LNP and LPX) have an average diameter in the range of about 40 nm to about 200 nm, e.g., about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 150 nm.

[0506] In some embodiments, the particle described herein is nanoparticle.The term " nanoparticle " refers to the nano-sized particle that comprises nucleic acid (particularly mRNA) described herein and at least one cationic or cationic ionizable lipid, and the three external dimensions of the particle are all nanoscale, that is, at least about 1 nm and less than about 1000 nm.Preferably, the size of the particle is its diameter.

[0507] The RNA particles (particularly mRNA particles) described herein may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the nucleic acid particles may exhibit a polydispersity index ranging from about 0.01 to about 0.4 or from about 0.1 to about 0.3.

[0508] The N / P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. This correlates with the charge ratio, since nitrogen atoms are usually positively charged (depending on the pH) and phosphate groups are negatively charged. If charge balance exists, the N / P ratio is pH dependent. Because positively charged nanoparticles are thought to favor transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. In that case, the RNA is considered fully bound to the nanoparticles.

[0509] The RNA particles (particularly mRNA particles) described herein can be prepared using a wide range of methods, which may include obtaining a colloid from at least one cationic or cationic ionizable lipid and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0510] The term "colloid," as used herein, refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be called a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the suspension as a whole.

[0511] For the preparation of colloids containing at least one cationic or cationic-ionizable lipid, suitably adapted methods conventionally used to prepare liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

[0512] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to produce a liposome dispersion. Further miniaturization steps may also be included.

[0513] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure results in a milky...

Claims

1. (i) RNA; (ii) a compound of formula (I): 【Chemistry 1】 (In the formula, R １ and R ２ Each of R ５ Or -G １ -L １ -R ６ and R １ and R ２ At least one of is -G １ -L １ -R ６ and R ３ and R ４ Each of the groups is independently １－６ Alkyl, C ２－６ Alkenyl, aryl and C ３－１０ cycloalkyl; R ５ and R ６ each is independently an acyclic hydrocarbyl group having at least 10 carbon atoms; G １ and G ２ Each of １－１２ Alkylene or C ２－１２ alkenylene; L １ and L ２ Each of the groups independently represents -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, or -S(O) ｘ -, -S-S-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ --, --NR ａ C(=O)NR ａ -, -OC(=O)NR ａ - and -NR ａ C(═O)O—; R ａ is H or C １－１２ is alkyl; m is 0, 1, 2, 3 or 4; and x is 0, 1 or 2. and a cationic ionizable lipid comprising the structure A composition comprising:

2. Each L １ are independently -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)- and -C(=O)NR ａ -, preferably each L １ The composition of claim 1 , wherein is independently -O(C═O)- or -(C═O)O-.

3. L ２ is -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)- and -C(=O)NR ａ -, preferably L ２ The composition according to claim 1 or 2, wherein is -O(C=O)- or -(C=O)O-.

4. R ５ The composition of any one of claims 1 to 3, wherein is a straight chain alkyl or alkenyl group having at least 10 carbon atoms, preferably at least 14 carbon atoms, more preferably at least 16 carbon atoms.

5. R ５ The composition of any one of claims 1 to 4, wherein is a linear alkyl or alkenyl group having at least two carbon-carbon double bonds.

6. R ５ has the following structure: 【Chemistry 2】 (In the formula, 【Chemistry 3】 is R ５ represents the bond that attaches it to the rest of the compound) The composition according to any one of claims 1 to 5, having the formula:

7. Each G １ is independently an unsubstituted linear C １－１２ Alkylene or C ２－１２ Alkenylene, preferably unsubstituted linear C ６－１２ Alkylene or C ６－１２ Alkenylene, more preferably unsubstituted linear C ８－１２ Alkylene or C ８－１２ Alkenylene or unsubstituted linear C ６－１０ Alkylene or C ６－１０ Alkenylene, for example unsubstituted linear C ８ The composition of any one of claims 1 to 6, wherein the alkylene is an alkylene.

8. Each R ６ The composition of any one of claims 1 to 7, wherein is independently a straight chain hydrocarbyl group having at least 10 carbon atoms.

9. Each R ６ But, R ６ via the internal carbon atom of １ The composition according to any one of claims 1 to 8, wherein the

10. Each R ６ But independently, 【Chemistry 4】 (In the formula, 【Chemistry 5】 is R ６ L １ represents the bond that bonds to The composition according to any one of claims 1 to 9, selected from the group consisting of:

11. R １ and R ２ One of them is R ５ and the other is -G １ -L １ -R ６ The composition according to any one of claims 1 to 10,

12. R １ and R ２ Each of the groups is independently -G １ -L １ -R ６ The composition according to any one of claims 1 to 10,

13. R ３ and R ４ Each of the groups independently represents C １－６ Alkyl or C ２－６ Alkenyl, preferably C １－４ Alkyl or C ２－４ alkenyl, more preferably C １－３ A composition according to any one of claims 1 to 12, wherein the alkyl is alkyl, for example methyl or ethyl.

14. G ２ is unsubstituted C ２－１０ Alkylene or C ２－１０ Alkenylene, preferably unsubstituted C ２－６ Alkylene or C ２－６ Alkenylene, more preferably unsubstituted C ２－４ Alkylene or C ２－４ A composition according to any one of claims 1 to 13, wherein the alkenylene is, for example, ethylene or trimethylene.

15. A composition according to any one of the preceding claims, wherein m is 0, 1, 2 or 3, preferably 0 or 2.

16. The cationic ionizable lipid has the following formula (IIIa) or (IIIb): 【Chemistry 6】 (In the formula, R ３ and R ４ Each of the groups is independently １－４ Alkyl or C ２－４ alkenyl, more preferably C １－３ alkyl, for example methyl or ethyl; R ５ is a straight chain alkyl or alkenyl group having at least 16 carbon atoms, said alkenyl group preferably having at least two carbon-carbon double bonds; Each R ６ are independently linear hydrocarbyl groups having at least 10 carbon atoms; R ６ is R ６ via the internal carbon atom of １ is bound to; Each G １ are independently unsubstituted linear C ６－１２ Alkylene or C ６－１２ Alkenylene, for example unsubstituted linear C ８－１０ Alkylene or C ８－１０ Alkenylene, for example unsubstituted linear C ８ is alkylene; G ２ is unsubstituted C ２－６ Alkylene or C ２－６ Alkenylene, preferably unsubstituted C ２－４ Alkylene or C ２－４ alkenylene, for example ethylene or trimethylene; L １ and L ２ each is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2 or 3, preferably 0 or 2. The composition of any one of claims 1 to 15, comprising one of the following structures:

17. The cationic ionizable lipid has the following structures (IV-1), (IV-2) and (IV-3): 【Chemistry 7】 17. The composition of claim 1 or 16, comprising one of:

18. 18. The composition of any one of claims 1 to 17, wherein the cationic ionizable lipid comprises from about 20 mol% to about 80 mol%, preferably from about 25 mol% to about 65 mol%, more preferably from about 30 mol% to about 50 mol%, such as from about 40 mol% to about 50 mol%, or from about 55 mol% to about 65 mol% of the total lipid present in the composition.

19. The composition of any one of claims 1 to 18, further comprising at least one polysarcosine-linked lipid.

20. 20. The composition of claim 19, wherein the polysarcosine comprises from 2 to 200 sarcosine units, preferably from 5 to 100 sarcosine units, more preferably from 10 to 50 sarcosine units, more preferably from 15 to 40 sarcosine units, more preferably about 23 sarcosine units.

21. The polysarcosine-bound lipid has the following general formula (V): 【Chemistry 8】 where x is the number of sarcosine units.

21. The composition of claim 19 or 20, comprising the structure:

22. The polysarcosine-linked lipid has the following general formula (VI): 【Chemistry 9】 (In the formula, R １ and R ２ one of which contains a hydrophobic group and the other is a functional group which may contain H, a hydrophilic group, or a targeting moiety, and x is the number of sarcosine units. The composition of any one of claims 19 to 21, having the structure:

23. R １ is a functional group which may contain H, a hydrophilic group, or a targeting moiety; R ２ 23. The composition of claim 22, wherein contains one or two straight chain alkyl or alkenyl groups each having at least 12 carbon atoms, preferably at least 14 carbon atoms.

24. The polysarcosine-linked lipid has the following general formula (VII): 【Chemistry 10】 where R is H, a hydrophilic group, or a functional group that may include a targeting moiety, and x is the number of sarcosine units. The composition of any one of claims 19 to 23, having the structure:

25. The polysarcosine-linked lipid has the following formula: 【Chemistry 11】 (wherein n is 23) The composition of any one of claims 19 to 24, having the structure:

26. 26. The composition according to any one of claims 19 to 25, wherein the polysarcosine-linked lipid comprises from about 0.1 mol% to about 5 mol%, preferably from about 0.5 mol% to about 4.5 mol%, more preferably from about 1 mol% to about 4 mol%, for example from about 3 mol% to about 4 mol% of the total lipid present in the composition.

27. The composition of any one of claims 1 to 18, further comprising a pegylated lipid.

28. The PEGylated lipid has the following general formula (VIII): 【Chemistry 12】 (In the formula, R １２ and R １３ each is independently a straight or branched alkyl, alkenyl or alkynyl chain, each of said alkyl, alkenyl and alkynyl chains independently containing from 10 to 30 carbon atoms, optionally interrupted by one or more ester linkages, and w has an average value in the range of 30 to 60.

28. The composition of claim 27 having the structure: or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

29. R １２ and R １３ 29. The composition of claim 28, wherein each of is independently a linear alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

30. R １２ and R １３ The composition of claim 28 or 29, wherein are identical.

31. R １２ and R １３ The composition of claim 30, wherein each of is a linear alkyl chain containing 12 carbon atoms.

32. R １２ and R １３ The composition of claim 30, wherein each of is a linear alkyl chain containing 14 carbon atoms.

33. R １２ and R １３ 31. The composition of claim 30, wherein each of is a linear alkyl chain containing 16 carbon atoms.

34. R １２ and R １３ The composition of claim 28 or 29, wherein:

35. R １２ and R １３ is a linear alkyl chain containing 12 carbon atoms, R １２ and R １３ The composition of claim 34, wherein the other is a linear alkyl chain containing 14 carbon atoms.

36. A composition according to any one of claims 28 to 35, wherein z has an average value in the range of 40 to 50, for example an average value of 45.

37. 37. The composition of any one of claims 27 to 36, wherein the pegylated lipid comprises from about 1 mol% to about 10 mol%, preferably from about 1 mol% to about 5 mol%, more preferably from about 1 mol% to about 2.5 mol% of the total lipid present in the composition.

38. 38. The composition of any one of claims 1 to 37, wherein the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably the composition comprises said cationic ionizable lipid; a polymer-linked lipid, preferably selected from polysarcosine-linked lipids and pegylated lipids; a phospholipid; and a steroid.

39. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DPC), ... phatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 39. The composition of claim 38, wherein the phosphatidylethanolamine is selected from the group consisting of: dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE).

40. 40. The composition of claim 38 or 39, wherein the phospholipid comprises from about 5 mol% to about 40 mol%, preferably from about 5 mol% to about 20 mol%, more preferably from about 5 mol% to about 15 mol% of the total lipid present in the composition.

41. A composition according to any one of claims 38 to 40, wherein the steroid comprises a sterol, such as cholesterol.

42. 42. The composition of any one of claims 38 to 41, wherein the steroid comprises from about 10 mol% to about 65 mol%, preferably from about 20 mol% to about 60 mol%, more preferably from about 30 mol% to about 50 mol% or from about 25 mol% to about 35 mol% of the total lipids present in the composition.

43. The composition of any one of claims 38 to 42, comprising a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid constitutes about 30 mol% to about 50 mol%, for example about 40 mol% to about 50 mol%, of the total lipid present in the composition, the polysarcosine-linked lipid constitutes about 1 mol% to about 4.5 mol% of the total lipid present in the composition, the phospholipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid constitutes about 30 mol% to about 50 mol% of the total lipid present in the composition.

44. The composition of any one of claims 38 to 42, comprising a cationic ionizable lipid, a polysarcosine-linked lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid constitutes about 55 mol% to about 65 mol% of the total lipid present in the composition, the polysarcosine-linked lipid constitutes about 1 mol% to about 4.5 mol% of the total lipid present in the composition, the phospholipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid constitutes about 25 mol% to about 35 mol% of the total lipid present in the composition.

45. 43. The composition of any one of claims 38 to 42, comprising a cationic ionizable lipid, a pegylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid constitutes about 30 mol% to about 50 mol%, for example about 40 mol% to about 50 mol%, of the total lipid present in the composition, the pegylated lipid constitutes about 1 mol% to about 2.5 mol% of the total lipid present in the composition, the phospholipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid constitutes about 30 mol% to about 50 mol% of the total lipid present in the composition.

46. 43. The composition of any one of claims 38 to 42, comprising a cationic ionizable lipid, a pegylated lipid, a phospholipid, and a steroid, wherein the cationic ionizable lipid constitutes about 55 mol% to about 65 mol% of the total lipid present in the composition, the pegylated lipid constitutes about 1 mol% to about 2.5 mol% of the total lipid present in the composition, the phospholipid constitutes about 5 mol% to about 15 mol% of the total lipid present in the composition, and the steroid constitutes about 25 mol% to about 35 mol% of the total lipid present in the composition.

47. 47. The composition of any one of claims 1 to 46, wherein at least a portion of (i) the RNA, (ii) the cationic ionizable lipid, and, if present, (iii) the one or more further lipids, are present in a nanoparticle, such as a lipid nanoparticle (LNP).

48. 48. The composition of claim 47, wherein the nanoparticles have a size of about 30 nm to about 500 nm.

49. The composition of any one of claims 1 to 48, wherein the RNA is mRNA.

50. 50. The composition of any one of claims 1 to 49, wherein the RNA (i) comprises a modified nucleoside instead of uridine, preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U); (ii) has a codon-optimized coding sequence; and / or (iii) has a coding sequence with an increased G / C content compared to a wild-type coding sequence.

51. The composition of any one of claims 1 to 50, wherein the RNA comprises at least one, and preferably all, of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

52. 52. The composition of claim 51, wherein the polyA sequence comprises at least 100 A nucleotides, and the polyA sequence is preferably an interrupted sequence of A nucleotides.

53. 53. The composition of claim 51 or 52, wherein the 5' cap is a cap1 or cap2 structure.

54. 54. The composition of any one of claims 1 to 53, wherein the RNA encodes one or more peptides or proteins, preferably said one or more peptides or proteins are pharma- ceutically active peptides or proteins and / or comprise an epitope for inducing an immune response against an antigen in a subject.

55. 55. The composition of claim 54, wherein the pharma- ceutical active peptide or protein and / or the antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

56. 56. The composition of claim 54 or 55, wherein the RNA comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

57. 57. The composition of claim 55 or 56, wherein the SARS-CoV-2 S protein mutant has a proline residue substitution at positions 986 and 987 of SEQ ID NO:

11.

58. 58. The composition of any one of claims 55-57, wherein the SARS-CoV-2 S protein variant has at least 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO:11 or the amino acid sequence of amino acids 17-1273 of SEQ ID NO:

12.

59. 59. The composition of any one of claims 55 to 58, wherein the fragment comprises the receptor binding domain (RBD) of the SARS-CoV-2 S protein.

60. 60. The composition of claim 59, wherein (i) the SARS-CoV-2 S protein or (ii) the fragment of an immunogenic variant of the SARS-CoV-2 S protein has at least 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:

11.

61. 61. A method for delivering RNA to a cell of a subject, comprising administering to the subject a composition according to any one of claims 1 to 60.

62. 61. A method for delivering a pharma- ceutically active peptide or protein to a subject, comprising administering to the subject a composition according to any one of claims 1 to 60, wherein the RNA encodes the pharma-ceutically active peptide or protein.

63. 61. A method for treating or preventing a disease or disorder in a subject, comprising administering to a subject a composition according to any one of claims 1 to 60, wherein delivery of said RNA to cells of said subject is beneficial in treating or preventing said disease or disorder.

64. 61. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition according to any one of claims 1 to 60, wherein the RNA encodes a pharma- ceutical active peptide or protein, and wherein delivery of the pharma-ceutical active peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

65. The method of any one of claims 61 to 64, wherein the subject is a mammal.

66. 66. The method of claim 65, wherein the mammal is a human.

67. A composition according to any one of claims 1 to 60 for use in therapy.

68. A composition according to any one of claims 1 to 60 for use in inducing an immune response in a subject.

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