Minimal mRNA and its use

Abstract

This invention provides a completely chemically synthesized RNA molecule that possesses a minimal structure useful for the expression of coding sequences. [Solution] The present invention provides an RNA molecule in which a completely chemically synthesized RNA has a general structure 5'-WXY-(coding sequence)-Z-3', where W is selected from the group consisting of a 5'-cap, a free 5'-triphosphate group, a free 5'-diphosphate group, a free 5'-monophosphate group, a free 5'-OH group, and chemically modified analogs of the 5'-cap, the free 5'-triphosphate group, the free 5'-diphosphate group, and the free 5'-monophosphate group; X is an optional 5'UTR sequence; Y is an optional start codon; and Z is selected from the group consisting of a free 3'-OH group, a stop codon, and an optional stop codon that leads to a poly(A) tail via a 3'UTR sequence.

Description

Technical Field

[0001] The present invention relates to a completely chemically synthesized RNA molecule (hereinafter referred to as "ChemRNA") having a minimum structure useful for the expression of a coding sequence. The ChemRNA of the present invention has a general structure of 5'-W-X-Y-(coding sequence)-Z-3', where W is selected from the group consisting of a 5'-cap, a free 5'-triphosphate group, a free 5'-diphosphate group, a free 5'-monophosphate group, a free 5'-OH group, and chemically modified analogs of the 5'-cap, the free 5'-triphosphate group, the free 5'-diphosphate group, and the free 5'-monophosphate group; X is an optional 5'UTR sequence; Y is an optional start codon; Z is connected to the coding sequence directly and is selected from the group consisting of a free 3'-OH group, a stop codon, and a stop codon connected to a poly(A) tail via an optional 3'UTR sequence. The present invention further relates to an RNA population in which at least 85% or more of the RNA population has the same chemical composition as that of the RNA of the present invention, and an RNA population containing the RNA of the present invention in which at least 1% of the RNA is present in a state one nucleotide shorter than the full-length RNA. The RNA and RNA population of the present invention are useful for the expression of an amino acid sequence encoded by a coding sequence in cells or organisms or in a cell-free expression system. The present invention further relates to a pharmaceutical composition, a vaccine containing the RNA or RNA population, and a diagnostic tool.

Background Art

[0002] Synthetic messenger RNA (mRNA) is being intensively developed as a vector for protein expression for vaccination and for therapeutic purposes such as the expression of proteins like cytokines and antibodies, replacement of defective or abnormal proteins in genetic diseases, or DNA repair using CRISPR-CAS. According to prior art, mRNA is produced in vitro by an enzymatic process. Typically, template DNA is transcribed to RNA by RNA polymerase (in vitro transcription mRNA; ivt mRNA), the DNA is degraded by DNase, and the mRNA is finally polyadenylated by poly(A) polymerase (Non-Patent Literature 1). Enzymatic synthesis of mRNA is efficient, robust, and allows for the production of large quantities of therapeutic mRNA. However, it has two significant drawbacks: (i) because it is produced through a biological process, the resulting RNA is usually classified by regulatory authorities as a gene therapy product; and (ii) enzymatic synthesis of mRNA usually requires an additional step of transcribing DNA to RNA and also requires the prior preparation of a DNA template, making the process indirect. For purposes such as anti-cancer vaccines, especially when mRNA vaccines aim to induce T-cell immunity against mutations, it is necessary to encode only a single epitope (short, about 3 to 8 amino acids), so the mRNA can be made relatively short. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2017 / 167910 [Non-patent literature]

[0004] [Non-Patent Document 1] Tusup et al. (2019) Chimia (Aarau) 73 (6), 391-394 [Non-Patent Document 2] Elfakess and Dikstein (2008) PLoS ONE 3 (8), e3094 [Non-Patent Document 3] Hinz et al. (2017) Methods in Mol. Biol. 1499, 203-222 [Non-Patent Document 4] Strenkowska et al. (2016) Nucleic Acids Research 44 (20), pages 9578-9590 [Non-Patent Document 5] Beaucage and Iyer (1992) Tetrahedron Vol. 48. No. 12, pp. 2223-2311 [Non-Patent Document 6] Beaucage and Reese (2009) Curr. Protoc. Nucleic Acid Chem. 38:2.16.1‐2.16.31 [Non-Patent Document 7] Sahin et al. (2017) Nature 547, 222-226 [Non-Patent Document 8] Sekine, et al. (1996) J. Org. Chem. 61, 4412-4422 [Overview of the project] [Problems that the invention aims to solve]

[0005] The fundamental technical problem of this invention is to provide mRNA that solves the above-mentioned problems encountered by enzymatically produced RNA. [Means for solving the problem]

[0006] The solution to the above technical problems is to provide embodiments of the present invention as defined in the claims, this specification, and the drawings. [Brief explanation of the drawing]

[0007] [Figure 1A]The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 18 hours of incubation, from OT1 mouse splenocytes transfected with the indicated agonist alone. [Figure 1B] The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 18 hours of incubation, by OT1 mouse splenocytes and B16 cells transfected with the indicated activators. [Figure 2A] The graph shows IFN-γ release, measured as IFN-γ (gamma, g) in the cell supernatant after 18 hours of incubation, released by OT1 mouse splenocytes transfected with the indicated agonist alone. [Figure 2B] The graph shows IFN-γ release, measured as IFN-γ (gamma, g) in the cell supernatant after 18 hours of incubation, released by OT1 mouse splenocytes and B16 cells transfected with the indicated activators. [Figure 3A]The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 18 hours of incubation, from OT1 mouse splenocytes transfected with the indicated agonist. The results shown are from untreated enzymatically polyadenylated RNA. The samples used are as follows: Capped 5n SIINFEKL:5'-N7-MeGppp,7mGppp-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); ppp 5n SIINFEKL:5'-ppp-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); p 5n SIINFEKL:5'-p-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); oligo:5'-N7-MeGppp,7mGppp-acaagAUGuuccaggaauuuguugacugggaaaacguuUAA-3' (SEQ ID NO:23). [Figure 3B] The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 18 hours of incubation, from OT1 mouse splenocytes transfected with the indicated agonists. The results shown are obtained using enzymatically polyadenylated RNA. The samples used are as follows: Capped 5n SIINFEKL:5'-N7-MeGppp,7mGppp-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); p 5n SIINFEKL:5'-p-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); OH 5n SIINFEKL:5'-OH-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); [Figure 4]The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 44-hour incubation, from OT1 mouse splenocytes transfected with the indicated agonist. The samples used are as follows: Cap 5n UTR:5'-ppp-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); UTR:5'-p-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); OH 5n UTR:5'-OH-caagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:24); Min SIINFEKL:5'-AUGAGUAUAAUCAACUUUGAAAAAACUG-3'(SEQ ID NO:25); No STOP:5'-ACAAGAUGAGUAUAAUCAACUUUGAAAAACUG-3'(SEQ ID NO:26); No AUG:5'-AGUAUAAUCAACUUUGAAAAACUG-3'(SEQ ID NO:27); Kif18b capped oligo:5'-N7-MeGppp,7mGppp-acaagAUGuuccaggaauuuguugacugggaaaacguuUAA-3' (SEQ ID NO:23). [Figure 5]The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 24-hour incubation, from OT1 mouse splenocytes transfected with the indicated activator. The samples used are as follows: Cap 5n UTR:5'-ppp-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); UTR:5'-p-acaagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:22); OH 5n UTR:5'-OH-caagAUGaguauaaucaacuuugaaaaacugUAA-3'(SEQ ID NO:24); Min SIINFEKL:5'-AUGAGUAUAAUCAACUUUGAAAAAACUG-3'(SEQ ID NO:25); No STOP:5'-ACAAGAUGAGUAUAAUCAACUUUGAAAAACUG-3'(SEQ ID NO:26); No AUG:5'-AGUAUAAUCAACUUUGAAAAACUG-3'(SEQ ID NO:27); Kif18b capped oligo:5'-N7-MeGppp,7mGppp-acaagAUGuuccaggaauuuguugacugggaaaacguuUAA-3' (SEQ ID NO:23). [Figure 6A] This graph shows the FACS analysis results of PMBC cultures from healthy donors after 7 days of incubation without RNA transfection. [Figure 6B] The graph shows the FACS analysis of PMBC cultures from healthy donors after transfection with Oligo Flu matrix (5'OH-AUGGGGAUUUUGGGGUUUGUGUUCACGCUC-3'; SEQ ID NO:28), which encodes the influenza virus epitope GILGFVFTL (SEQ ID NO:30) with methionine ligated to the front (5' side), and 7 days of incubation. [Figure 6C]RNA encoding the CMV epitope NLVPMVATV (SEQ ID NO:33) with methionine linked to the front (5' side), transfected with Oligo CMV pp65 (5’-OH AUGAACCUGGUGCCCAUGGUGGCUACGGUU-3’;SEQ ID NO:31), showing a graph of FACS analysis of PMBC cultures from healthy donors after 7-day incubation. [Figure 7A] Showing a graph of FACS analysis of PMBC cultures from healthy donors after 14-day incubation without RNA transfection. [Figure 7B] RNA encoding the influenza virus epitope GILGFVFTL (SEQ ID NO:30) with methionine linked to the front (5' side), transfected with Oligo Flu matrix(5’OH-AUGGGGAUUUUGGGGUUUGUGUUCACGCUC-3’;SEQ ID NO:28), showing a graph of FACS analysis of PMBC cultures from healthy donors after 14-day incubation. [Figure 7C] RNA encoding the CMV pp65 epitope MNLVPMVATV (SEQ ID NO:32) with methionine linked to the front (5' side), transfected with Oligo CMV pp65 (5’-OH AUGAACCUGGUGCCCAUGGUGGCUACGGUU-3’;SEQ ID NO:31), showing a graph of FACS analysis of healthy donors after 14-day incubation. [Figure 7D] A gating method on lymphocyteslyymph lymphocytes in forward scattering and side scattering, showing the gating method on CD3+ and CD4+ populations in the control culture without RNA transfection. [Figure 7E] Showing dot plot analysis of the culture without RNA after gating. [Figure 7F] Showing dot plot analysis of the culture transfected with Oligo Flu matrix RNA after gating. [Figure 7G]This is a dot plot analysis of cultures transfected with Oligo CMV pp65 RNA after gating. [Figure 8] The graph shows IL-2 release, measured as IL-2 in the cell supernatant after 40 hours of incubation, from OT1 mouse splenocytes transfected with the indicated drug. The samples are as follows: SIINFEKL-CF:5'-AUGAGUAUAAU[2'-FC]AA[2'-FC]UUUGAAAAA[2'-FC]UG-3' (where 2'-FC is 2'-fluorodeoxycytosine; SEQ ID NO:25); SIINFEKL:5'-AUGAGUAUAAUCAACUUUGAAAAACUG-3' (SEQ ID NO:25). [Modes for carrying out the invention]

[0008] Specifically, the present invention provides a completely chemically synthesized RNA (also referred to herein as "ChemRNA") having the structure of the following general formula (1). 5'-WXY-(code arrangement)-Z-3' (1) Here, W is selected from the group consisting of 5'-caps, free 5'-triphosphate group, free 5'-diphosphate group, free 5'-monophosphate group, free 5'-OH group, and chemically modified analogs of the 5'-caps, free 5'-triphosphate group, free 5'-diphosphate group, and free 5'-monophosphate group. X may or may not exist, and if it exists, it is a 5'UTR sequence. Y may or may not exist, and if it exists, it is the start codon. Z is selected from a group consisting of a free 3'-OH group, a stop codon, a stop codon that connects directly to the code sequence, and a stop codon that connects to the poly(A) tail, and optionally a stop codon that connects to the poly(A) tail via a 3'UTR sequence.

[0009] The preferred ChemRNA of the present invention has one of the structures related to the following formulas (2) to (61). [Formula 1] TIFF2026102892000002.tif235159TIFF2026102892000003.tif229152TIFF2026102892000004.tif55155Here, polyA is a poly(A) tail, stop is a stop codon. UTR is 5'UTR, triP is a free triphosphate group, diP is a free diphosphate group, mP is a free monophosphate group.

[0010] As defined herein, N7MeGppp is N7-methylguanosine triphosphate.

[0011] A particularly preferred chemRNA of the present invention is given by formula (58).

[0012] Other particularly preferred chemRNAs of the present invention include those of formula (3).

[0013] In a more preferred embodiment of the present invention, ChemRNA is RNA of formula (15).

[0014] In another preferred embodiment of the present invention, the ChemRNA has the structure shown in formula (39).

[0015] In a more preferred embodiment of the present invention, the ChemRNA has the structure of formula (51).

[0016] In a more preferred embodiment of the present invention, the ChemRNA has the structure shown in formula (61).

[0017] In one aspect of the present invention, the RNA has a 5'-cap, a 5'UTR, a start codon, a coding sequence, and a stop codon, as outlined in more preferred detail in formula (3). Generally, the RNA in this aspect of the present invention is defined by the following general structure, alternatively.

[0018] 5'-cap-5'UTR-(start codon)-(code sequence)-(termination codon)-3'

[0019] If present, the stop codon is preferably selected from UAA, UAG, and UGA.

[0020] If present, the RNA of the present invention preferably contains a relatively short 5'UTR sequence. Particularly preferred 5'UTR sequences for use in the present invention are selected from 5'UTR sequences not exceeding 10 nucleotides, more preferably 2 to 10 nucleotides, i.e., very preferred 5'UTR sequences for use in the present invention have a length of 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0021] Examples of preferred 5'UTR sequences used in the present invention are disclosed, for example, in Non-Patent Document 2. A very preferred 5'UTR sequence includes the 5'-AAG-3' sequence. More specifically, the 5'UTR sequence for RNA of the present invention comprises the 5'-AAG-3' motif, has a length of 5 nucleotides, and more preferably the 5'-AAG-3' motif is located directly before (on the 5' side of) the start codon. A preferred 5'UTR sequence used in the present invention is the 5'-ACAAG-3' sequence. In other embodiments using this motif, a particularly preferred 5'UTR sequence of 5, 6, 7, or 8 nucleotides in length, the 5'UTR sequence may also include this sequence, where it is preferred that the 5-nucleotide sequence 5'-ACAAG-3' is located directly before (on the 5' side of) the start codon.

[0022] In another aspect of the present invention, the 5'UTR is selected from the 5'UTR sequences disclosed in Patent Document 1. Specifically, the 5'UTR preferably includes or consists of the sequence 5'-CGCCACC-3', where the C nucleotide at position 6 (counting from the 5' end) may be substituted with an adenosine nucleotide, and / or the C nucleotide at position 7 (counting from the 5' end) may be substituted with a guanosine nucleotide, and / or the A nucleotide at position 5 may be substituted with a guanosine nucleotide. Particularly preferred 5'UTR sequences of this type, including such sequences, are selected from these sequences in which the 5'-CGCCACC-3' sequence is located directly before (towards the 5' end of) the start codon. In another preferred embodiment, the 5'UTR comprises the sequence 5'-CNGCCACC-3' where N is selected from A, C, G, and U, or consists of the sequence 5'-CNGCCACC-3' where N is selected from A, C, G, and U, where the C nucleotide at position 7 (counting from the 5' end) may be substituted with an A nucleotide, and / or the nucleotide at position 8 (counting from the 5' end) may be substituted with a G nucleotide, and / or the A nucleotide at position 6 (counting from the 5' end) may be substituted with a G nucleotide. This type of 5'UTR sequence, comprising such a sequence, is selected from sequences in which the sequence 5'-CNGCCACC-3' is located directly before (to the 5' end of) the start codon.

[0023] One of the remarkable discoveries of the present invention is that the RNAs disclosed and described herein, which are useful for the expression of coding sequences, do not require a 3' poly(A) tail. Therefore, in preferred embodiments of the RNA molecules disclosed herein, there is no poly(A) tail at the 3' end. In other embodiments of the present invention, the RNA contains a poly(A) tail at the 3' end. If a poly(A) tail is present, it is preferably relatively short. Preferred poly(A) tails have up to 30 nucleotides, such as 2 to 30 nucleotides, more preferably up to 20 nucleotides, such as 5 to 20 nucleotides, even more preferably up to 15 nucleotides, such as 5 to 15 nucleotides, and still even more preferably up to 10 nucleotides, such as 5 to 10 nucleotides. Particularly preferred lengths of the poly(A) tail are 5, 10, 15, 20, 25, and 30 nucleotides.

[0024] A further, even more surprising, discovery relating to the present invention is that preferred embodiments of ChemRNA do not require a 5'-cap structure, which is particularly useful in the expression of coding sequences in cells or organisms.

[0025] Furthermore, an even more remarkable discovery is that the ChemRNA of the present invention can lack a phosphate group at its 5' end (i.e., the 5' terminal group is OH) for usefulness in the expression of coding sequences.

[0026] A particularly surprising discovery of this invention is that ChemRNA does not require a start codon and / or even a stop codon to be useful in the expression of coding sequences.

[0027] Furthermore, due to their entirely chemical preparation process, the RNA and its population relating to the present invention may not be considered as gene therapy products (see Non-Patent Document 3), which would make the regulatory approval process much simpler and faster.

[0028] The preferred RNA of the present invention is an RNA oligonucleotide. The RNA oligonucleotide of the present invention preferably does not have a length of 200 nucleotides or more (i.e., is not composed of 200 nucleotides or more), more preferably has a maximum length of 100 nucleotides, more preferably a maximum length of 80 nucleotides, and even more preferably a maximum length of 70 nucleotides. Particularly preferred oligonucleotide RNAs of the present invention have a length of 24, 25, 26, 27, 28, 29, or 30 to 200 nucleotides, more preferably 24, 25, 26, 27, 28, 29, or 30 to 120 nucleotides, and even more preferably 24, 25, 26, 27, 28, 29, or 30 to 100 nucleotides.

[0029] It is also preferable that the RNA is single-stranded.

[0030] In other aspects of the present invention, the RNAs defined and disclosed herein may also be partially or completely double-stranded. The partially double-stranded RNAs of the present invention may consist only of single strands forming a double-stranded portion or region of the double-stranded structure by a self-complementary sequence portion in the single-stranded RNA forming a hairpin, or they may consist only of a portion or a region of the double-stranded structure. Therefore, in the case of the partially double-stranded RNAs of the present invention resulting from self-complementarity, such partially double-stranded RNAs of the present invention should be understood to also be single-stranded RNAs. Other partially double-stranded RNAs of the present invention consist of two or more double strands, typically having complementary sequences; therefore, although the RNA formulas of the present invention show only single strands, the sequences of strands that are fully or partially complementary to the strands shown in the various embodiments herein should be understood to be determined by the RNA base pair complementarity rules known in the art. The partially double-stranded RNA of the present invention, which is formed by two or more strands, typically double-stranded, can take any form, such as fluctuating double-stranded RNA, double-stranded RNA having one blunt end and one end with an overhang, or double-stranded RNA having two overhangs formed by the same strand. With respect to the present invention, double-stranded RNA is considered to be formed by three or more strands, such as in species where the double strands are complementary to different regions of a third RNA strand. In certain embodiments of the present invention, the RNA can also be a fully double-stranded RNA having two blunt ends. In certain embodiments, double-stranded RNA, specifically double-stranded RNA composed of two or more, preferably two individual strands, can function, for example, as a precursor for providing a single strand that codes for a peptide through a contained coding sequence.

[0031] The complete or partial double-stranded RNA of the present invention may provide RNA with further functionality. In a preferred embodiment, the double-stranded RNA of the present invention as defined above is thought to have a free 5' triphosphate attached to the blunt end single strand of the double-stranded RNA of the present invention so that it can function as a ligand for RIG-I. Other embodiments relate to RNA that can trigger TLRs, such as the double-stranded RNA of the present invention, which is 45 bp or longer, typically 50 bp or longer, and triggers TLR3.

[0032] The RNA of the present invention contains a coding sequence and is preferably useful for expressing the coding sequence in a cell-based in vitro or in vivo expression system, or in a cell-free in vitro expression system. For use in cell-free expression systems, the RNA of the present invention without a 5'-cap, or a first or second RNA population containing RNA such as the RNA of the present invention lacking a 5'-cap, are particularly preferred. According to the present invention, the RNA defined and disclosed herein is also referred to as “coding RNA”. The RNA of the present invention does not need to contain a 3' poly(A) tail and / or a 5'-cap and / or a start codon and / or a stop codon, but the RNA of the present invention is also referred to as “mRNA”.

[0033] The coding sequences of RNA molecules disclosed herein are not particularly limited. As outlined above, preferred coding sequences are selected such that the entire length of the RNA essentially encompasses the entire length of the RNA oligonucleotide boundaries. Preferred coding sequences encode 4 to 65 amino acids. Particularly preferred coding sequences for use in the present invention are relatively short and encode 4 to 40 amino acids. More preferred coding sequences encode amino acid sequences of 8 to 30 amino acids.

[0034] As outlined below in more detail, preferred peptides encoded by the coding sequence are preferably peptides derived from cancer or oncoproteins (also referred to herein as “cancer antigens”), such as epitopes, or preferably peptides derived from infectious agents such as viruses, bacteria, or fungi.

[0035] Each protein, polypeptide, or oligopeptide-related cancer or tumor-derived peptide is defined herein as a “cannabeptide” and, in a certain preferred embodiment, may have at least one amino acid sequence different from that of a non-cancer wild-type sequence.

[0036] A more preferred peptide encoded by the coding sequence contained in the RNA species of the present invention is a tissue peptide recognized by autoimmune cells.

[0037] Another advantage of the present invention is the ability to provide mRNA having site-specific chemical modifications at precise nucleotide positions, which is usually not possible when mRNA is prepared by enzymatic synthesis. For example, it is possible to provide a single nucleotide with specific chemical modifications (in the phosphate backbone, ribose, or base portion). In a preferred embodiment, the RNA has a chemical modification at a single nucleotide. Preferred chemical modifications are located at the 3' terminal nucleotide and / or the 5' terminal nucleotide.

[0038] Accordingly, according to a preferred embodiment of the present invention, the RNA comprises at least one chemical modification, i.e., at least one chemically modified nucleotide analogue. In this regard, “chemical modification” and “chemically modified nucleotide analogue” mean that the nucleotide is chemically modified compared to the corresponding standard (i.e., unmodified) nucleotides a, c, g, and u, respectively. The chemical modification may be the phosphate, ribose, or base portion of the nucleotide. As used throughout this specification, the term “nucleotide” is understood to mean “ribonucleotide” unless otherwise specified. The modification can be introduced during chemical synthesis or added to ChemRNA by enzymes, for example, from the methylase and deaminase families. Another preferred example of enzymatic modification is the addition of a poly(A) tail to the 3' end of a ChemRNA, preferably having a structure according to formula (3), (6), (9), (12), (15), (18), (21), (24), (27), (30), (33), (36), (39), (42), (45), (48), (51), (54), (57), or (60), particularly preferably having a structure according to formula (3), (15), (39), or (51), by incubation with a poly(A) polymerase, such as poly(A) polymerase from E. coli.

[0039] Compared to standard nucleotides, chemical modifications of nucleotide analogs can occur at the ribose, phosphate, and / or base moieties. For molecules requiring high stability, particularly RNA-degrading enzymes, modifications at the ribose and / or phosphate moieties are especially preferred.

[0040] Preferred examples of ribose-modified ribonucleotides are analogs in which the 2'-OH group is substituted with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, with R being a C1-C6 alkyl, alkenyl, or alkynyl group, and halo being F, Cl, Br, or I. More preferred nucleotide analogs are methylated and fluorinated nucleotide analogs, most preferably 2'-O methylated and 2'-F analogs.

[0041] As mentioned above, at least one modified ribonucleotide can be selected from analogs having chemical modifications to the base portion. Examples of such analogs include, but are not limited to, 5-aminoallyluridine, 6-azauridine, 8-azaadenosine, 5-bromouridine, 7-deazaadenosine, 7-deazaguanosine, and N 6 -Methyladenosine, 5-methylcytidine, pseudouridine, N 1 Methylpseudolidine, N 1 It contains methyladenosine, thymine, and 4-thiouridine.

[0042] An example of a backbone-modified ribonucleotide, in which the phosphate ester group between adjacent ribonucleotides is modified, is the phosphorothioate group.

[0043] A more preferred embodiment of the RNA according to the present invention, including modified nucleotide analogs, is selected from RNA in which the modification is at the 3' end.

[0044] A preferred modification is one of the modifications shown in the following table, where the most preferred position of each nucleotide analog is the 3' end (left column: name of the modified nucleotide analog, right column: abbreviation). [Table 1]

[0045] The RNA of the present invention may also contain a 5' cap or a free 5'-phosphate group, i.e., chemical analogs of free 5'-triphosphate, free 5'-diphosphate, or 5'-monophosphate, as included in the definition of the group W in formula (1). Typically, and preferably, an analog of the phosphate-containing 5' group is thiophosphate, and preferred thiophosphate contains one sulfur atom per phosphate group. A 5'-phosphate-containing group having two or more phosphates (i.e., free 5'-diphosphate groups, free 5'-triphosphate, or 5' cap) is understood to contain two or more phosphates, preferably two triphosphate moieties. The introduction of thiophosphate to each 5' cap and free 5'-phosphate group is known in the art. See, for example, Non-Patent Literature 4 for thiophosphate-containing 5' cap structures.

[0046] The RNA chemical synthesis protocol of the present invention is generally known in the art and is typically carried out by a solid-phase method based on the phosphate amidite method (see, for example, Non-Patent Documents 5 and 6).

[0047] A further subject of the present invention is a (first) RNA population, wherein at least 85%, preferably 90%, and more preferably 95% of the RNAs in the RNA population are (first) RNA populations having the same chemical structure as the RNA defined above, where the RNA may be understood as being completely chemically synthesized, or the general definition may be as described above without explicitly limiting it to "completely chemically synthesized".

[0048] Another aspect of the present invention is a different (second) RNA population comprising the full-length n-nucleotide RNA as defined herein, and at least 1% of RNA having a chemical composition of at least 95%, preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, and still more preferably at least 99%, that matches the chemical composition of full-length RNA, but having a length of (n-1) nucleotides. Here, the proportion of the chemical composition of (n-1) RNA matching that of full-length n RNA refers to the chemical composition of (n-1) nucleotides in full-length n RNA (i.e., the (n-1) nucleotide-length RNA present in at least 1% is one nucleotide shorter than full-length n RNA, but otherwise its nucleotide sequence matches the nucleotide sequence of full-length n RNA by at least 95%, preferably at least 96%, more preferably at least 97%, even more preferably 98%, and still more preferably at least 99%). In a more preferred embodiment, the RNA population further comprises at least 1% RNA having a length of (n-2) but whose chemical composition matches that of full-length RNA by at least 93%, preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, still more preferably at least 98%, and particularly preferably at least 99%, respectively, where the proportion of the chemical composition of the (n-2) RNA matching that of full-length RNA of length n refers to the chemical composition of (n-2) nucleotides in the full-length RNA of length n (i.e., the RNA having (n-2) nucleotides, present in an amount of at least 1%, is 2 nucleotides shorter than the full-length RNA of length n, but otherwise its nucleotide sequence matches that of full-length RNA of length n by at least 93%, preferably at least 95%, more preferably at least 96%, even more preferably 97%, still more preferably 98%, and particularly preferably at least 99%).In a more preferred embodiment, the RNA population further comprises at least 1% RNA having a length of (n-3) which is at least 93%, preferably at least 95%, more preferably at least 96%, even more preferably at least 97%, still more preferably at least 98%, and particularly preferably at least 99%, which is the same chemical composition as full-length RNA, where the proportion of the chemical composition of (n-3) RNA that matches that of full-length RNA of length n is the proportion of the chemical composition of (n-3) nucleotides in full-length RNA of length n (i.e., the RNA having (n-3) nucleotides, which is present in an amount of at least 1%, is only 3 nucleotides shorter than full-length RNA of length n, but otherwise its nucleotide sequence matches the nucleotide sequence of full-length RNA of length n by at least 90%, preferably at least 95%, more preferably at least 96%, even more preferably 97%, still more preferably 98%, and particularly preferably at least 98.5%). In this aspect of the present invention, RNA may also be defined and understood as being entirely chemically synthesized, or it may be defined as outlined above but without being explicitly limited to “fully chemically synthesized.” According to the present invention, all references to “n” in the second RNA population disclosed herein are understood to mean an integer, where “n” is at least 10, at least 20 in a given aspect of the present invention, at least 30 in another preferred aspect of the present invention, preferably from 20 to 200, more preferably from 30 to 200, even more preferably from 30 to 120, and still even more preferably from 30 to 100.

[0049] The present invention relates to a pharmaceutical composition comprising RNA as defined herein, or a primary RNA population as defined herein, or a secondary RNA population as defined herein, which may be combined as appropriate with one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Preferably, the pharmaceutical composition is in the form of a vaccine comprising RNA as defined herein, or a primary RNA population as defined herein, or a secondary RNA population as defined herein.

[0050] To further enhance the effect, the vaccine according to the present invention preferably comprises one or more adjuvants to achieve a synergistic effect of the vaccine. Here, “adjuvant” includes any compound that promotes an immune response. Various mechanisms are possible in this regard and depend on the various types of adjuvants. For example, compounds that enable DC maturation, such as lipopolysaccharides or CD40 ligands, form the first class of suitable adjuvants. In general, any factor that affects the immune system or cytokines of the type of “danger signal” (LPS, GP96, dsRNA, etc.), such as GM-CSF, can be used as an adjuvant that can enhance and / or influence the immune response in a controlled manner. CpG oligodeoxyribonucleotides can also be used as appropriate in this regard, although their side effects that occur in certain situations must be taken into consideration. Particularly preferred adjuvants are cytokines such as monokines, lymphokines, interleukins, and chemokines, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CFS, LT-α, or growth factors such as hGH. More well-known adjuvants include oils such as aluminum hydroxide, Freund's adjuvant, or Montanide®, most preferably Montanide® ISA51. Lipopeptides such as Pam3Cys are also particularly suitable for use as adjuvants in the vaccines and / or pharmaceutical compositions of the present invention.

[0051] In a preferred embodiment, the vaccine according to the present invention may also be used in conjunction with other therapeutic agents. The vaccine according to the present invention may be used in conjunction with other treatments such as chemotherapy agents for cancer patients, immune checkpoint inhibitors or combination therapies for HIV patients, or chloroquine, which is used as a drug for malaria infection and is known to improve cross-presentation.

[0052] The vaccine composition of the present invention is used in general vaccination, stimulating an immune response by introduction into a living organism. Here, RNA may be applied in a naked form (i.e., specifically, in an uncomplexed form), or contained in particles such as complexes with cationic ions, liposomes, or polymers, or contained in cells (e.g., by in vitro electroporation and subsequent adoptive immunotherapy, or by direct injection with needle-requiring or needle-free devices), the RNA or first or second RNA population disclosed herein. The vaccine composition of the present invention can be administered systemically, preferably by intravenous or subcutaneous injection, and can also be administered locally at sites where mRNA delivery is desired, such as injection into tumors, muscles, dermis, or lymph nodes. Other preferred routes of administration are nasal and oral administration. In another embodiment, antigen-presenting cells such as DCs (or precursor cell populations such as PMBCs from which DCs are first isolated or at least purified) are prepared (typically from a blood sample taken from the patient) from the patient to be treated, into which the RNA or RNA population of the present invention is introduced. If appropriate, after the incubation process, the RNA-carrying DCs are reintroduced to the patient, preferably by intravenous administration.

[0053] The vaccine according to the present invention is suitable for the treatment of cancer or tumors. Preferably, the RNA of the present invention, or the RNA in the first or second RNA population of the present invention, contains a coding sequence that encodes an epitope of a tumor-specific antigen (TSA). Specific examples of tumor antigens from which the epitopes encoded by the RNA / RNA population are derived include 707-AP, AFP, ART-4, BAGE, β-catenin / m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / neu, and HLA-A. *This includes 02:01-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, RAGE, RU1, RU2, SAGE, SART-1 or SART-3, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, and WT1. For specific sequences of tumor antigen-derived MHC-associated epitopes, see https: / / syfpeithi.de. Particularly preferred coding sequences in the RNA of this invention are HLA-A * 02:01 codes for an associated epitope, more specifically, KVLEYVIKV from MAGE-A1 (SEQ ID NO:1), FLWGPRALV from MAGE-A3 (SEQ ID NO:2), HLYQGCQVV from HER-2 / neu (SEQ ID NO:3) and YLVPQQGFFC (SEQ ID NO:4), and APDTRPAP from MUC1 (SEQ ID NO:5) and / or NLTISDUVSV (SEQ ID NO:6). In another preferred embodiment of the present invention, the RNA coding sequence codes for a tumor epitope comprising one or more mutations found in the tumor. Specific examples of preferred tumor epitopes of this type are described in Non-Patent Document 7, more specifically the epitopes found in the columns labeled “AAsequence,” “Predicted MHCI epitope,” and “Predicted MHC II epitope” in Supplementary Table 1 of that document, and “Amino acid sequence” in Supplementary Table 2, respectively, and these sequences are explicitly referenced herein. Tumor peptides are also examples of epitopes from the hypervariable loop of the TCR or immunoglobulin chain, particularly specific examples from chronic lymphoma or leukemia cells.

[0054] The vaccine according to the present invention can also be used against infectious diseases. Preferred epitopes encoded by the coding sequences of embodiments of the present invention include AIDS (HIV), hepatitis A, B, and C, herpes, shingles (chickenpox), rubella (rubella virus), yellow fever, dengue fever, etc., flaviviruses, influenza viruses, coronaviruses, hemorrhagic infections (Marburg or Ebola virus), legionellosis (legionella), gastric ulcers (Helicobacter), cholera (Vibrio), infections caused by Escherichia coli, staphylococcus, salmonella, streptococcus (tetanus), etc. These include infectious agents that cause bacterial infections, infections caused by protozoan pathogens such as malaria, sleeping sickness, leishmaniasis, and toxoplasmosis, namely infections caused by the malaria parasite, Trypanosoma, Leishmania, and Toxoplasma, respectively, and fungal infections such as those caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides imitis, Blastomyces dermatichidis, and Candida albicans. Preferred embodiments of the RNA of the present invention include, for example, HIV-1-derived epitopes selected from PLTFGWCYKL (SEQ ID NO: 7), SLYNTVATL (SEQ ID NO: 8), TLNAWVKVV (SEQ ID NO: 9), RGPGRAFVTI (SEQ ID NO: 10), AFHHVAREL (SEQ ID NO: 11), VLEWRFDSRL (SEQ ID NO: 12), ILKEPVHGV (SEQ ID NO: 13), VIYQYMDDL (SEQ ID NO: 14), KYTAFTIPSI (SEQ ID NO: 15), and KLTPLCVTL (SEQ ID NO: 16), or preferably HPV11-derived epitopes such as RLVTLKDIV (SEQ ID NO: 17), or TIHDIILECV (SEQ ID NO: 18), YMLDLQPETT (SEQ ID NO: 19), LLMGTLGIV (SEQ ID HLA-A from each pathogen, such as an HPV16-derived epitope selected from NO:20 or preferably TLGIVCPI (SEQ ID NO:21) *02:01- Encodes the present epitope. Further examples of preferred epitopes include influenza virus epitopes, more preferably influenza A and B subtypes, particularly influenza A-derived epitopes, and coronaviruses, more preferably SARS-CoV-1, SARS-CoV-2, and MERS-CoV-derived epitopes. An example of a preferred peptide, more preferably a pathogenic bacterial epitope, is a peptide, more preferably a Mycobacterium tuberculosis epitope. As for tumor antigens, many specific sequences of epitopes encoded by the RNA coding sequences according to the present invention are known to those skilled in the art and can be selected from databases available at https: / / syfpeithi.de.

[0055] As with all specific epitopes expressly disclosed herein, by reference to the literature and public epitope databases, it is understood that, in a given aspect, the RNA coding sequence of the present invention can encode a sequence containing a specific epitope sequence, specifically a specific MHC class I epitope sequence or a specific MHC class II epitope sequence. In other aspects, the RNA coding sequence of the present invention consists of a nucleotide sequence encoding such a specific epitope.

[0056] The vaccine according to the present invention may be used in combination with chloroquine, a pharmaceutical compound that increases cross-presentation and thus induces antigen-specific effector T cells.

[0057] In aspects of the present invention, specifically RNA, primary RNA populations, and secondary RNA populations are useful as pharmaceutical agents. In aspects of the present invention, specifically RNA, primary RNA populations, and secondary RNA populations are particularly useful in the treatment of cancer and tumors, and also in the treatment and / or prevention of infectious diseases such as infections caused by viral, prokaryotic, and fungal infectious agents.

[0058] The present invention also provides the use of the RNA and / or primary RNA populations and / or secondary RNA populations disclosed herein for the preparation of agents for the treatment of cancer and tumors. The present invention also provides the use of the RNA and / or primary RNA populations and / or secondary RNA populations disclosed herein for the preparation of agents for the treatment and / or prevention of infectious diseases.

[0059] The present invention further provides a method for treating cancer or tumor in a subject, comprising administering an effective amount of the pharmaceutical composition according to the present invention to a subject in need thereof.

[0060] The present invention further provides a method for treating and / or preventing an infection in a subject, comprising administering an effective dose of the vaccine according to the present invention to a subject in need thereof.

[0061] A further object of the present invention is a diagnostic kit comprising the RNA and / or primary RNA population and / or secondary RNA population of the present invention. Each of the RNAs preferably encodes a peptide of an infectious agent, such as a viral, bacterial, or fungal peptide. Preferred peptides are epitopes of such infectious agents. Specific examples of epitopes, and examples of preferred epitopes, are outlined above with respect to the vaccine of the present invention.

[0062] The diagnostic kit preferably further includes at least one transfection reagent, such as a liposome reagent, and / or an apparatus or apparatus component for performing a detection method and / or separation method (e.g., an electrode for electroporation).

[0063] The present invention further relates to a method for diagnosing the presence of infectious agents causing cancer, autoimmune diseases, infectious diseases, and / or diseases in a subject suspected of having and / or being infected by such infectious agents. Such a method includes stimulating a T cell population of a subject with at least one RNA and / or at least one primary RNA population and / or at least one secondary RNA population having a coding sequence encoding a peptide, preferably an epitope, of the cancer, the target tissue of the autoimmune disease, or the infectious agent; and detecting the presence of T cells specific to the peptide, preferably the epitope. In relation to the present invention, “T cell population” is a cell population of a subject containing T cells. A typical T cell population is a PMBC obtained from a subject.

[0064] The step of stimulating the T cells preferably includes transfecting the target cell population with at least one RNA and / or at least one primary RNA population and / or at least one secondary RNA population containing the peptide of the infectious agent, preferably a coding sequence encoding the epitope, and detecting the presence of T cells specific to the peptide, preferably the epitope. After transfection, the cells are typically incubated under appropriate conditions for a period of preferably 1 to 30 hours.

[0065] Detection of stimulated T cells typically involves FACS analysis of cultured T cells, preferably CD3+CD4+ or CD3+CD8+ T cells specific to the detected antigen. Alternatively, cytokine secretion from T cells can be used to assess whether they are stimulated by chemRNA-encoded peptides (e.g., ELISA or ELISpot to measure interleukin-2 (IL-2) or interferon-γ (IFN-γ) production).

[0066] Stimulation of T cells specific to a given antigen, preferably a cancer or tumor antigen, can also be used in methods for the treatment of tumors and cancer (and the use of RNA or RNA populations of the present invention), as already described. In a certain embodiment, T cells obtained from a subject suffering from cancer or a tumor, i.e., typically the T cell population described above, are transfected with a suitable cancer peptide, the positive T cells are detected and enriched, preferably by FACS, and the proliferated anti-cancer peptide-stimulated T cells are returned to the subject suffering from cancer or a tumor. It is preferable that the detected and enriched T cells be proliferated before being reinjected into the subject. Suitable proliferation techniques are known in the art. The methods described above can also be used to specifically stimulate regulatory T cells (Tregs) that can be used to control autoimmune diseases.

[0067] With respect to all and any of the applications, uses, and methods disclosed, specified, and described herein that use RNA having the structures defined herein, the present invention also covers such applications, uses, and methods. Herein, the RNA is enzymatically synthesized RNA having the same or substantially the same structure as the fully chemically synthesized RNA defined above, except that the RNA is prepared completely or almost enzymatically. Methods for the enzymatic synthesis of RNA are known in the art. Typically, RNA polymerases such as T7 or Sp6 RNA polymerase are used, and various protocols containing reagents as kits are commercially available from various vendors (e.g., New England Biolabs Inc. in Ipswich, Massachusetts, USA; Promega Corp. in Madison, Wisconsin, USA; and various other companies).

[0068] The present invention will be further described by the following non-limiting embodiments. [Examples]

[0069] Example 1

[0070] The following RNA oligonucleotides were chemically synthesized using standard oligonucleotide synthesis methods provided by the commercial supplier (Bio-Synthesis, Inc., Lewisville, Texas, USA). [Payment 10] TIFF2026102892000006.tif131705'-cap was chemically prepared based on the method described in Non-Patent Document 8. The resulting structure is shown below (start and stop codons are underlined). [Distribution 11] TIFF2026102892000007.tif13170 This code sequence encodes the MESIINFEKL amino acid sequence containing the SIINFEKL epitope in ovalbumin (positions 257 to 264 in ovalbumin; UniProt Acc. No. P01012).

[0071] In another embodiment, the sequence is the same as above, but 3'-(A) 20 We prepared fully chemically synthesized RNA with a tail (in this case, the start and stop codons are also underlined). [Distribution 12] TIFF2026102892000008.tif12170

[0072] In the comparative example ("SIINFEKL ChemRNA Poly-A"), the above RNA (SIINFEKL ChemRNA) lacking a poly-A tail was polyadenylated by incubating it with a commercially available enzyme (E. coli poly-A polymerase, catalog number M0276, from New England Biolabs Inc. in Ipswich, Massachusetts, USA) in the presence of ATP for 2 hours, according to the manufacturer's instructions.

[0073] The following RNAs were used as controls. Positive control: Enzymatically prepared mRNA encoding ovalbumin (Trilink Biotechnologies, LLC, San Diego, California, USA) Negative control: Enzymatically prepared mRNA encoding luciferase (prepared in the laboratory by the inventors).

[0074] RNA was prepared using the lipofectamine reagent MessengerMax (Thermo Fischer Scientific Corp., Waltham, Massachusetts, USA) by mixing 200 ng of RNA with 400 ng of MessengerMax, 20 ng of RNA with 40 ng of MessengerMax, or 20 ng of RNA with 40 ng of MessengerMax per cell culture well. The mixture was transfected according to the manufacturer's instructions for the MessengerMax reagent by adding 100,000 splenocytes per 100 μl culture well (splenocytes only) or 100,000 splenocytes and 50,000 B16 cells per 100 μl culture well (splenocytes and B16 cells) to either RAG2 KO C57Bl / 6 mouse OT1 splenocytes only or the said splenocytes and syngenic B16 cancer cells, respectively.

[0075] After 18 hours of incubation, IFN-γ and IL-2 in the culture supernatant were measured by ELISA using commercially available assays (ELISA MAX® Standard Set Mouse IFN-γ and ELISA MAX® Standard Set Mouse IL-2, both provided by BioLegend Inc. in San Diego, California, USA). Cytokines are produced and released into the culture medium by OT1 cells when T lymphocytes are activated, i.e., when they recognize the SIINFEKL peptide on H-2 Kb mouse class I molecules. The results are shown in Figure 1 (IL-2 release, A: splenocytes only, B: splenocytes and B16 cells) and Figure 2 (IFN-γ release, A: splenocytes only, B: splenocytes and B16 cells).

[0076] Figures 1 and 2 show that fully chemically synthesized RNA, SIINFEKL ChemRNA, induced potent releases of IL-2 and IFN-γ by OT1 cells. The produced signals were stronger than those of the positive control ovalbumin mRNA (enzymatically synthesized mRNA encoding full-length ovalbumin). Treatment of SIINFEKL ChemRNA with poly(A) polymerase did not improve the effect of the chemically synthesized oligonucleotide. Even more surprising is that the poly(A) tail is not required for a potent cytokine response.

[0077] Example 2

[0078] The following RNAs were prepared by chemical synthesis by commercial suppliers (BioSynthesis, Inc. in Lewisville, Texas, USA, or Microsynth AG in Balgach, Switzerland, respectively). As outlined in Example 1, the RNAs were capped where necessary. In the following sequences, capital letters indicate the start and stop codons, respectively. The construct contains a 5' UTR sequence (acaag) directly before the start codon (5' side).

[0079] [Distribution 13] TIFF2026102892000009.tif20167 This construct therefore has a 5' cap structure but does not contain a poly-A tail (see Example 1).

[0080] [Distribution 14] TIFF2026102892000010.tif18165 This construct lacks a 5' cap structure and a poly-A tail. This construct has a triphosphate group (indicated as 5'-ppp) at the 5' end.

[0081] [Distribution 15] TIFF2026102892000011.tif18165 This construct lacks a 5' cap structure and a poly-A tail. This construct has a monophosphate group (indicated as 5'-p) at the 5' end.

[0082] [Distribution 16] TIFF2026102892000012.tif24165 This construct lacks a 5' cap structure and even lacks a phosphate group at the C-5' of the 5' terminal ribose, and therefore only has a 5'OH group. Furthermore, this construct lacks a poly(A) tail.

[0083] [Distribution 17] TIFF2026102892000013.tif24165

[0084] Here, oligonucleotides are used as negative controls. Ovalbumin mRNA is used as a positive control. Splenocytes that have not been transfected with any RNA (alone) are used as a further negative control.

[0085] Mouse OT1 splenocytes (100 μl, 100,000 cells per well) were transfected with the oligonucleotides described above, except that 200 ng, 20 ng, and 5 ng of RNA were used per well, respectively.

[0086] In another experiment, RNA(a), (c), and (e) were polyadenylated as described in Example 1 and used for transfection in the quantities outlined above (as ChemRNA, without considering the additional weight from the poly-A tail addition).

[0087] These cells were incubated for 18 hours, and IL-2 in the culture supernatant was measured as described in Example 1.

[0088] The results are shown in Figure 3A (without polyadenylated RNA) and Figure 3B (polyadenylated constructs (a), (c), and (e)).

[0089] To our great surprise, all constructs (a) through (d) elicited higher IL-2 release by OT1 cells compared to full-length ovalbumin mRNA. Thus, the RNA of the present invention is expressed in splenocytes and results in strong IL-2 expression even without the 5' cap structure and poly-A tail. Even more surprisingly, this is true even when the RNA has only the 5' triphosphate or no phosphorylation at the 5' terminal nucleotide. As shown in Figure 3B, at small amounts of the tested constructs, the addition of a short poly-A tail resulted in slightly higher IL-2 compared to the respective unadenylated RNA.

[0090] Example 3

[0091] To further investigate RNAs that possess fewer of the necessary structures typically involved in inducing the IL-2 response in immune cells, additional constructs were synthesized by the commercial supplier (Microsynth AG, Balgach, Switzerland).

[0092] [Distribution 18] TIFF2026102892000014.tif18167 This construct lacks a cap or phosphate group and a poly(A) tail at the 5' end (i.e., OH), as well as a 5' UTR sequence and even a stop codon.

[0093] [Distribution 19] TIFF2026102892000015.tif18167 This construct corresponds to construct (f), except that it includes 5'UTR ACAAG.

[0094] [Payment 20] TIFF2026102892000016.tif18167 This construct (h) is the absolute minimal chemRNA because it consists only of the coding sequence of the indicated epitope (it does not have a cap structure or phosphate group at the 5' end, so the group that binds to the 5'C of the 5' terminal nucleotide is OH). It also does not have a standard start codon.

[0095] Constructs (a) through (h) were transfected into OT1 mouse splenocytes using the method outlined in Example 1, but with 200 ng, 20 ng, and 5 ng of RNA. After 44 hours of incubation, IL-2 levels in the culture supernatant were measured as outlined in Example 1. The results are shown in Figure 4. Completely unexpectedly, even constructs (f), (g), and (h) (i.e., coding sequences without start codons) resulted in significantly higher IL-2 release by OT1 cells than the negative control, and even higher than the positive control: ovalbumin mRNA.

[0096] Although the IL-2 measurement was performed after 24 hours, the above experiment was repeated, and the results obtained after 44 hours of incubation were confirmed. The results are shown in Figure 5. Interestingly, construct(f) (characterized by having only the start codon of the real mRNA and lacking other attributes) gave the highest IL-2 concentration at 200 ng after 24 hours of incubation.

[0097] Example 4

[0098] We further investigated whether the RNA of the present invention can be used for peptide expression, particularly for the expression of oligopeptides such as epitopes of infectious agents and epitopes of cancer peptides, and whether such peptides can be expressed in human cells.

[0099] As an example of a construct for viral peptide expression, the following construct was prepared by the commercial supplier (Microsynth AG, Balgach, Switzerland) using chemical synthesis.

[0100] [Delivery 21] TIFF2026102892000017.tif30166

[0101] [Delivery 22] TIFF2026102892000018.tif30166

[0102] Both constructs consist only of a start codon (without a cap at the 5' end and no phosphate group) and a coding sequence (without a stop codon and no poly-A tail).

[0103] PMBCs were isolated from the blood of healthy volunteer HLA-A2-positive donors. To start three cultures, 10 million PMBCs were used in 10 ml of complete medium each. For transfection, RNA was prepared by mixing 1 μg of RNA and 2 μg of MessengerMax lipofectamine reagent (Thermo Fischer Scientific Corp., Waltham, Massachusetts, USA) in 25 μl of OptiMEM medium. Each mixture was added to the respective PMBC culture. The third culture was treated with the same 50 μl mixture without added RNA. After one week of incubation, antibody and tetramer staining was performed on 2 ml of cell culture. FACS analysis was performed using the following settings. FACS: Gating to lymphocytes in FSC-SSC Phycoerythrin (PE):FLU Matrix Tetramer (Peptide: GILGFVFTL; HLA-A2 with SEQ ID NO: 30) Allophycocyanin (APC):CMV pp65 tetramer (peptide: NLVPMVATV; HLA-A2 with SEQ ID NO: 33)

[0104] The results are shown in Figure 6.

[0105] The experiment was extended to a total of two weeks of cell culture, an additional week prior to FACS analysis. On day 7, the transfection protocol was repeated, and the medium was replaced with fresh medium supplemented with 5 ng / ml recombinant human IL-2. This replacement with fresh medium supplemented with 5 ng / ml recombinant human IL-2 was also repeated on days 9 and 12.

[0106] The results are shown in Figure 7.

[0107] Experiments have shown that the uncapped ChemRNA of the present invention, lacking a 5' cap, 5' phosphate group, 5' UTR, stop codon, and poly-A tail, can be expressed by PMBCs and presented to human T cells.

[0108] The FACS analysis shown in Figure 6B (compared to culture without RNA transfection) clearly demonstrated the proliferation of FLU-specific T cells in PMBC cultures transfected with RNA encoding the FLU epitope after one week. This indicates that the RNA-encoded epitope was produced and presented to the T cells.

[0109] After two weeks, the signal in the cultures transfected with Oligo Flu matrix increased significantly (Figure 7B), and a positive signal was also detected in the cultures transfected with Oligo CMV pp65 RNA (Figure 7C). The signal in the cultures transfected with Oligo CMV pp65 RNA was weaker compared to the cultures transfected with Oligo Flu matrix RNA, which may be due to the cells being anerious or the peptide sequence being inappropriate.

[0110] Example 5

[0111] Furthermore, we investigated whether the RNA of the present invention can be used for the expression of peptides, particularly oligopeptides such as epitopes of infectious agents and epitopes of oncopeptides, and whether it can provide expression of such peptides, especially when modified nucleotides are included in the RNA sequence, particularly in the coding sequence.

[0112] The constructs used are as follows:

[0113] [Delivery 23] TIFF2026102892000019.tif18160 This construct corresponds to construct (f) of Example 2.

[0114] [Delivery 24] TIFF2026102892000020.tif30167

[0115] The following RNAs were used as controls. Positive control: Enzymatically prepared mRNA encoding ovalbumin (Trilink Biotechnologies, LLC, San Diego, California, USA) Negative control: Enzymatically prepared mRNA encoding luciferase (prepared in the laboratory by the inventors).

[0116] Mouse OT1 splenocytes (100 μl per well, 100,000 cells) were transfected with the oligonucleotides described above, except that 200 ng, 20 ng, and 0 ng (as a negative control) of RNA were used per well, respectively. After 40 hours of incubation, IL-2 levels in the culture supernatant were measured as outlined in Example 1. The results are shown in Figure 8.

[0117] As shown in Figure 8, the inclusion of 2'-FC in RNA does not interfere with the expression of the encoded peptide.

[0118] This invention demonstrates that various types of chemically synthesized RNAs, having coding sequences that preferably encode peptides, more preferably viral epitopes of infectious agents, or cancer antigen epitopes, but lacking one, more, or any messenger RNA-specific structural features, can be expressed by living cells, particularly mammalian cells. This is a completely unexpected discovery and opens up the possibility of using minimal chemRNAs for a variety of diagnostic and therapeutic applications.

[0119] Firstly, it is unexpected that 5'-capped RNA, possessing an AUG start codon in a non-Cossack context and lacking a poly-A tail, exhibited specific T cell stimulation (IL-2 production in transfected mouse OT1 splenocytes). Further research on the present invention has led to an even more surprising result: capping is not actually necessary for stimulating OT1 mouse splenocytes for IL-2 release. This even makes it possible to use RNA without a 5' phosphate group, instead possessing a 5' OH group. Further results of the present invention demonstrate that a 5' UTR is not absolutely necessary for coding sequence expression. While the presence of a start codon is desirable for optimizing coding sequence expression, a start codon is also not absolutely necessary. If the last nucleotide at the 3' end of the RNA is the last nucleotide of the coding sequence, then a stop codon is also not necessary.

[0120] [Sequence List] SEQUENCE LISTING <110> University of Zurich <120> Minimal messenger RNAs and uses thereof <130> P22-068JP1 <140> JP2022-513178 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Lys Val Leu Glu Tyr Val Ile Lys Val 1 5 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 His Leu Tyr Gln Gly Cys Gln Val Val 1 5 <210> 4 <211> 10 <212> PRT <213> Homo sapiens <400> 4 Tyr Leu Val Pro Gln Gln Gly Phe Phe Cys 1 5 10 <210> 5 <211> 8 <212> PRT <213> Homo sapiens <400> 5 Ala Pro Asp Thr Arg Pro Ala Pro 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Asn Leu Thr Ile Ser Asp Val Ser Val 1 5 <210> 7 <211> 10 <212> PRT <213> Human immunodeficiency virus type 1 <400> 7 Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu 1 5 10 <210> 8 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 8 Ser Leu Tyr Asn Thr Val Ala Thr Leu 1 5 <210> 9 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 9 Thr Leu Asn Ala Trp Val Lys Val Val 1 5 <210> 10 <211> 10 <212> PRT <213> Human immunodeficiency virus type 1 <400> 10 Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 1 5 10 <210> 11 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 11 Ala Phe His His Val Ala Arg Glu Leu 1 5 <210> 12 <211> 10 <212> PRT <213> Human immunodeficiency virus type 1 <400> 12 Val Leu Glu Trp Arg Phe Asp Ser Arg Leu 1 5 10 <210> 13 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 13 Ile Leu Lys Glu Pro Val His Gly Val 1 5 <210> 14 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 14 Val Ile Tyr Gln Tyr Met Asp Asp Leu 1 5 <210> 15 <211> 10 <212> PRT <213> Human immunodeficiency virus type 1 <400> 15 Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile 1 5 10 <210> 16 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 16 Lys Leu Thr Pro Leu Cys Val Thr Leu 1 5 <210> 17 <211> 9 <212> PRT <213> Human papillomavirus type 11 <400> 17 Arg Leu Val Thr Leu Lys Asp Ile Val 1 5 <210> 18 <211> 10 <212> PRT <213> Human papillomavirus type 16 <400> 18 Thr Ile His Asp Ile Ile Leu Glu Cys Val 1 5 10 <210> 19 <211> 10 <212> PRT <213> Human papillomavirus type 16 <400> 19 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 10 <210> 20 <211> 9 <212> PRT <213> Human papillomavirus type 16 <400> 20 Leu Leu Met Gly Thr Leu Gly Ile Val 1 5 <210> 21 <211> 8 <212> PRT <213> Human papillomavirus type 16 <400> 21 Thr Leu Gly Ile Val Cys Pro Ile 1 5 <210> 22 <211> 35 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 22 acaagaugag uauaaucaac uuugaaaaac uguaa 35 <210> 23 <211> 41 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide MFQEFVDWENV of mutated Kif18b <400> 23 acaagauguu ccaggaauuu guugacuggg aaaacguuua a 41 <210> 24 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 24 caagaugagu auaaucaacu uugaaaaacu guaa 34 <210> 25 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 25 augaguauaa ucaacuuuga aaaacug 27 <210> 26 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 26 acaagaugag uauaaucaac uuugaaaaac ug 32 <210> 27 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 27 aguauaauca acuuugaaaa acug 24 <210> 28 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding influenza virus epitope GILGFVFTL <400> 28 auggggauuu ugggguuugu guucacgcuc 30 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide comrising influenza virus M peptide GILGFVFTL <400> 29 Met Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 10 <210> 30 <211> 9 <212> PRT <213> Influenza virus <400> 30 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 <210> 31 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide MNLVPMVATV <400> 31 augaaccugg ugcccauggu ggcuacgguu 30 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide containing peptide NLVPMVATV of cytomegalo virus <400> 32 Met Asn Leu Val Pro Met Val Ala Thr Val 1 5 10 <210> 33 <211> 9 <212> PRT <213> cytomegalo virus <400> 33 Asn Leu Val Pro Met Val Ala Thr Val 1 5 <210> 34 <211> 11 <212> PRT <213> Homo sapiens <400> 34 Met Phe Gln Glu Phe Val Asp Trp Glu Asn Val 1 5 10 <210> 35 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> RNA encoding peptide SIINFEKL <400> 35 acaagaugga gaguauaauc aacuuugaaa aacuguaaaa aaaaaaaaaa aaaaaaaa 58

Claims

1. A completely chemically synthesized RNA having the structure of the following general formula (1): 5'-W-X-Y-(code arrangement)-Z-3' (1) Here, W is selected from the group consisting of 5'-cap, free 5'-triphosphate group, free 5'-diphosphate group, free 5'-monophosphate group, free 5'-OH group, and chemically modified analogs of the 5'-cap, free 5'-triphosphate group, free 5'-diphosphate group, and free 5'-monophosphate group. X may or may not exist, and if it exists, it is a 5'UTR array. Y may or may not exist, and if it exists, it is the start codon. Z is selected from a group consisting of a free 3'-OH group, a stop codon, a stop codon that connects directly to the code sequence, and a stop codon that connects to the poly(A) tail, and optionally a stop codon that connects to the poly(A) tail via a 3'UTR sequence.

2. RNA according to claim 1, characterized in that it has a structure selected from the group consisting of the following general formulas (2) to (61): [Formula 1] Here, PolyA is a poly(A) tail, stop is a stop codon, UTR is 5' UTR, triP is a free triphosphate group, diP is a free diphosphate group, mP is a free monophosphate group.

3. RNA according to claim 2, characterized in that it has a structure selected from the group consisting of general formulas (3), (15), (39), (51), and (58).

4. RNA according to any one of claims 1 to 3, wherein if an UTR is present, the UTR has a length of 2 to 10 nucleotides.

5. The RNA according to claim 4, characterized in that the UTR has a length of 2 to 5 nucleotides.

6. RNA according to claim 4 or 5, wherein the UTR has the sequence aag.

7. The RNA according to claim 6, wherein the UTR has the sequence acaag.

8. RNA according to any one of claims 1, 2, 4, 5, 6, 7, or 8, wherein, if a poly(A) tail is present, the poly(A) tail has a length of up to 30 nucleotides, preferably 5 to 30 nucleotides.

9. The RNA according to claim 8, characterized in that the poly(A) tail has a length of up to 20 nucleotides, preferably 5 to 20 nucleotides.

10. The RNA according to claim 9, characterized in that the poly(A) chain has a length of up to 10 nucleotides, preferably 5 to 10 nucleotides.

11. RNA according to any one of claims 1 to 10, characterized in that the coding sequence codes for up to 65 amino acids, preferably 4 to 40 amino acids.

12. RNA according to claim 11, characterized in that the coding sequence codes for 4 to 30 amino acids, preferably 8 to 20 amino acids.

13. RNA according to any one of claims 1 to 12, characterized in that the RNA is an RNA oligonucleotide.

14. RNA according to claim 13, characterized in that it consists of a maximum of 200 nucleotides, preferably a maximum of 120 nucleotides, and more preferably a maximum of 100 nucleotides.

15. RNA according to any one of claims 1 to 14, wherein the RNA comprises at least one chemical modification, preferably a chemical modification at one nucleotide, and more preferably at a terminal nucleotide.

16. RNA according to any one of claims 1 to 15, characterized in that the RNA is enzymatically modified.

17. RNA according to any one of claims 1 to 16, characterized in that the coding sequence encodes an infectious agent peptide, a cancer peptide, or a tissue peptide recognized by autoimmune cells.

18. RNA according to claim 17, characterized in that the infectious agent is selected from viruses, bacteria and fungi.

19. RNA according to claim 17, characterized in that the cancer peptide contains at least one amino acid that is different from the amino acid sequence of a non-cancer wild-type RNA.

20. RNA according to any one of claims 1 to 19, characterized in that the coding sequence of the RNA is expressed when the RNA is present in a cell, organism or cell-free expression system.

21. RNA according to any one of claims 1 to 20, characterized in that the RNA is single-stranded.

22. An RNA population characterized in that at least 85%, preferably at least 90%, and more preferably at least 95% of the RNA in the RNA population has the same chemical composition as the RNA described in claims 1 to 21.

23. An RNA population comprising RNA having the full length of n nucleotides as defined in claims 1 to 21, and at least 1% of RNA having a chemical composition of at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and even more preferably at least 99% of which matches the chemical composition of full-length RNA, but which has a length of (n-1) nucleotides as full-length RNA, wherein the proportion of the chemical composition of the (n-1) length RNA matching the chemical composition of the n length full-length RNA represents the chemical composition of (n-1) nucleotides in the n length full-length RNA.

24. An RNA population according to claim 22 or 23, characterized in that its chemical composition comprises at least 93%, preferably at least 95%, more preferably at least 97%, still more preferably at least 98%, and even more preferably at least 99%, RNA having a length of (n-2) as full-length RNA, wherein the proportion of the chemical composition of RNA of length (n-2) that matches the chemical composition of full-length RNA of length n means relating to the chemical composition of (n-2) nucleotides in full-length RNA of length n.

25. An RNA population according to claims 22 to 24, characterized in that its chemical composition comprises at least 90%, preferably at least 95%, more preferably at least 97%, still more preferably at least 98%, and even more preferably at least 98.5%, RNA having a length of (n-3) as full-length RNA, wherein the proportion of the chemical composition of the (n-3) length RNA that matches the chemical composition of the n length full-length RNA means relating to the chemical composition of (n-3) nucleotides in the n length full-length RNA.

26. A pharmaceutical composition comprising RNA according to any one of claims 1 to 21, or an RNA population according to any one of claims 22 to 25.

27. A diagnostic kit comprising RNA according to any one of claims 1 to 21, or an RNA population according to any one of claims 22 to 25.

28. A diagnostic kit according to claim 27, characterized in that the coding sequence codes for a peptide of an infectious agent.

29. A diagnostic kit according to claim 28, further comprising T cells specific to the infectious agent.

30. A vaccine comprising RNA according to any one of claims 1 to 21, or an RNA population according to any one of claims 22 to 25.

31. RNA according to any one of claims 1 to 21, or RNA population according to any one of claims 22 to 25, for use as a pharmaceutical.

32. Use of RNA according to any one of claims 1 to 21, or RNA population according to any one of claims 22 to 25, in a diagnostic process.

33. Use of RNA according to any one of claims 1 to 21, or RNA population according to any one of claims 22 to 25, for the expression of coding sequences in cell-free expression systems, cells, or organisms.

34. A method for expressing amino acid sequences in cells or organisms, A method for expressing an amino acid sequence in a cell or organism, comprising the step of introducing RNA according to any one of claims 1 to 21, or an RNA population according to any one of claims 22 to 25, into the cell or organism.

35. A method for expressing amino acid sequences in a cell-free expression system, A method for expressing an amino acid sequence in a cell-free expression system, comprising the step of incubating RNA according to any one of claims 1 to 21, or an RNA population according to any one of claims 22 to 25, in the presence of a cell-free expression system.

36. Use of RNA according to any one of claims 1 to 21, or RNA population according to any one of claims 22 to 25, in the treatment of cancer or tumors.

37. RNA or RNA population for use according to claim 36, characterized in that the treatment comprises vaccination of the cancer patient against the cancer the cancer patient has.

38. RNA for use in claim 36 or 37, wherein the RNA is defined in claim 19.

39. RNA according to any one of claims 1 to 21 or RNA population according to any one of claims 22 to 25 for use in the treatment and / or prevention of infectious diseases.

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