Therapeutic RNA

Abstract

The present invention provides a modified therapeutic mRNA comprising at least one nucleic acid sequence selected from at least one frameshifted nucleic acid sequence, at least one ribosome slippery sequence, and / or at least one other reading frame sequence, wherein the nucleic acid sequence comprises at least one synonymous mutation. Also provided are methods for producing the modified therapeutic mRNA, methods for reducing off-target immunogenicity to the therapeutic mRNA, and methods for reducing out-of-frame translation and / or increasing the translational fidelity of the therapeutic mRNA. The modified therapeutic mRNA for use as a drug is further provided.

Description

[Technical Field]

[0001] The present invention provides a method for producing RNA that has higher translation fidelity and, when translated, produces fewer out-of-frame products. The RNA produced by such a method and its use as a drug are also provided. [Background technology]

[0002] The effectiveness of mRNA therapeutics critically depends on the ability of the innate immune system to evade it and to robustly translate therapeutic proteins from exogenously introduced mRNA. While chemical modification of RNA has historically been used to evade nucleic acid sensors, conflicting reports exist regarding the levels of proteins resulting from the translation of modified mRNA.

[0003] The key features of therapeutic in vitro transcription (IVT) mRNA are that it contains modified ribonucleotides that have been shown to reduce innate immunogenicity, and that these ribonucleotides can further enhance mRNA stability; both of these are desirable characteristics for therapeutic use. 1,2 For example, clinically approved SARS-CoV-2 mRNA vaccines incorporate (N)1-methylpseudridine (1-methylψ), which has been shown to reduce IVT mRNA innate immunogenicity. 3-5 Some modified ribonucleotides, such as 5-methylcytidine (5-methyl C), are naturally occurring post-transcriptional mRNA modifiers in eukaryotes, while other modified ribonucleotides, such as 1-methylψ, are not. 6-10 .

[0004] Despite their widespread use, surprisingly little is known about how ribonucleotide modifications affect protein synthesis, particularly regarding the translation of therapeutic IVT mRNA. Certain ribonucleotide modifiers, such as inosine, can recode mRNA sequences. 155-methyl C has previously been shown to increase misleading during mRNA translation in prokaryotes, but its effect on mRNA translation fidelity in eukaryotes has not been investigated. 16 The effect of 5-methoxyU on translation fidelity has never been studied. Pseudouridine (ψ) is known to increase mRNA stop codon misreading in eukaryotes and may affect misreading during mRNA translation in prokaryotes. 16-18 While 1-methylψ is not thought to affect codon misleading, it has been shown to influence the rate of protein synthesis on mRNA and ribosome density, suggesting a direct effect on mRNA translation. 19-20 .

[0005] It is currently unclear which modified ribonucleotides affect mRNA translation fidelity, and most existing studies are limited to understanding the frequency of misreading at a given codon. mRNA codon misreading is only one type of post-transcriptional mechanism that can alter polypeptide sequences. To date, no studies have investigated the fundamental question of whether modified ribonucleotides can affect the maintenance of an accurate reading frame during the translation of synthetic transcripts.

[0006] Understanding these methods is crucial not only for increasing our knowledge of protein synthesis with modified mRNA in general, but also for the robust design and evaluation of novel mRNA-based therapeutics that utilize modified ribonucleotides within very different RNA sequences or therapeutic contexts.

[0007] There is a need for improved mRNA-based therapeutics. There is also a need for improved mRNA-based methods for producing mRNA therapeutics. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent and Trademark Publication No. 2015 / 0064235 [License 2] U.S. Patent and Trademark Publication No. 2008 / 0103745 [Non-licensed literature]

[0009] [Non-licensed Document 1] Moon, S., LNCS, 2004, 3036: 334-34 [Non-licensed Document 2] Hammell, AB, Genomic Res., 1999, 9: 417-427 [Non-licensed Document 3] Bekaert, M., Bioinformatics, 2003, 19: 327-335 [Non-licensed Document 4] Shah, AA, Bioinformatics, 2002, 18: 1046-1053 [Non-licensed Document 5] Moon S, Byun Y, Han K. FSDB: a frameshift signal database. Comput Biol Chem. 2007;31(4):298-302. Doi:10.1016 / j.compbiolchem.2007.05.004 [Non-licensed Document 6] Belew, Ashton T., et al. "PRFdb: a database of computationally predicted eukaryotic programmed-1 ribosomal frameshift signals." BMC genomics 9.1 (2008): 1-7.) [Non-licensed Document 7] Therapeutic mRNA Delivery. Mol Ther. 2019;27(4):710-728. doi:10.1016 / j.ymthe.2019.02.012 [Non-licensed Document 8] Qin S, Tang X, Chen Y, et al., mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166p. Published May 21, 2022. doi:10.1038 / s41392-022-01007-w [Non-Patent Document 9] Fuller, CW et al. (2009). The challenges of sequencing by synthesis. Nature biotechnology, 27(11), pp. 1013-1023 [Non-Patent Document 10] Patel, RK, & Jain, M. (2012). NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PloS one, 7(2), e30619 [Non-Patent Document 11] Feng et al., 2013 Cell Res. 23, pp. 1229-1232 [Non-Patent Document 12] Sander & Joung Nat. Biotechnol. 32, pp. 347-355 2014 [Overview of the project] [Problems that the invention aims to solve]

[0010] The inventors were interested in how modified ribonucleotides affect the fidelity of mRNA translation. They investigated how 5-methoxyuridine (5-methoxy U), 5-methyl C, and 1-methyl ψ affect IVT mRNA translation. 5-methoxy U, 5-methyl C, and 1-methyl ψ have been used in IVT mRNA in attempts to increase recombinant protein synthesis in vitro and for preclinical proof-of-concept studies of IVT mRNA-based therapies.11,12 1-Methyl-ψ is a ribonucleotide that has been incorporated into approved IVT mRNA-based SARS-CoV2 vaccines but also into mRNA-based human vaccines and therapies under development. 4,13,14 .

[0011] The inventors have revealed that 1-methyl-ψ is a modified ribonucleotide that significantly increases +1 ribosomal frameshifting during mRNA translation and that cellular immunity against the +1 frameshifted products can occur following vaccination with mRNA containing 1-methyl-ψ.

[0012] This is the first report that mRNA modification affects ribosomal frameshifting. Other ribonucleotide modification strategies, such as the incorporation of 5-methoxy-U, can significantly reduce the translation efficiency of IVT mRNA, which can limit clinical translation. The inventors have revealed that IVT mRNA contains few nucleotide insertions / deletions, which cannot be changed by the incorporation of 1-methyl-ψ. During the translation of 1-methyl-ψ mRNA, +1 ribosomal frameshifting is associated with ribosomal slippery sequences. Furthermore, the translation of mRNA containing 1-methyl-ψ results in ribosomal stalling. These new findings are particularly important for a fundamental understanding of how chemical ribonucleotide modifications affect mRNA translation and for designing and optimizing future mRNA-based therapeutics to avoid mistranslation events that can reduce efficacy or increase toxicity.

[0013] The inventors have also found that frameshift events can occur due to another reading frame that can lead to mistranslation events that can reduce the efficacy or increase the toxicity of mRNA-based therapeutics.

[0014] Based on our findings, we have been able to provide a novel method for producing mRNA for therapeutic use that generates lower levels of out-of-frame products by introducing synonymous mutations into mRNA. Accordingly, we have also been able to provide novel mRNA containing such synonymous mutations and novel mRNA for use in various therapeutic methods. [Means for solving the problem]

[0015] A first aspect of the present invention provides a modified therapeutic mRNA comprising at least one frameshift nucleic acid sequence that increases the frequency of out-of-frame translation of mRNA, wherein the at least one frameshift nucleic acid sequence comprises at least one synonymous mutation for reducing the frequency of out-of-frame translation of the modified therapeutic mRNA.

[0016] In a second embodiment, a modified therapeutic mRNA is provided, comprising at least one ribosome slippery sequence that increases the frequency of out-of-frame translation of the mRNA, wherein the at least one ribosome slippery sequence comprises at least one synonymous mutation for reducing the frequency of out-of-frame translation of the modified therapeutic mRNA.

[0017] A third embodiment provides a modified therapeutic mRNA comprising at least one different reading frame sequence encoding a different translation product from the in-frame translation product of the modified therapeutic mRNA, and a modified therapeutic mRNA comprising at least one synonymous mutation for introducing a stop codon (PTC) into the at least one different reading frame sequence.

[0018] In a fourth aspect, a nucleic acid is provided which encodes a modified therapeutic mRNA of any prior claim, optionally comprising a DNA template for in vitro transcription of the modified therapeutic mRNA.

[0019] A fifth aspect describes a method for producing a modified therapeutic mRNA in which the frequency of out-of-frame translation is reduced, a. A step of providing therapeutic mRNA or nucleic acid encoding therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of therapeutic mRNA or the nucleic acid encoding therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the at least one modified frameshift sequence comprises at least one synonymous mutation; A method including this is provided.

[0020] A sixth aspect describes a method for reducing off-target immunogenicity to therapeutic mRNA and / or its translation product, a. A step of providing therapeutic mRNA or nucleic acid encoding therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of therapeutic mRNA or the nucleic acid encoding therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the at least one modified frameshift sequence comprises at least one synonymous mutation; and d. The process of administering the modified therapeutic mRNA; A method including this is provided.

[0021] In the seventh aspect, a method for reducing out-of-frame translation of therapeutic mRNA, a. A step of providing therapeutic mRNA or nucleic acid encoding therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of therapeutic mRNA or the nucleic acid encoding therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the at least one modified frameshift sequence comprises at least one synonymous mutation; and d. The process of translating the modified therapeutic mRNA; A method including this is provided.

[0022] In the eighth aspect, a method for increasing the translational fidelity of therapeutic mRNA, A step of providing therapeutic mRNA or nucleic acid encoding therapeutic mRNA; A step of identifying at least one frameshift sequence within the nucleic acid sequence of therapeutic mRNA or nucleic acid encoding therapeutic mRNA; A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the at least one modified frameshift sequence comprises at least one synonymous mutation; and The process of translating modified therapeutic mRNA into a target that requires it; A method including this is provided.

[0023] In certain embodiments, at least one synonymous mutation increases the translation fidelity of the modified therapeutic mRNA when used.

[0024] In certain embodiments, out-of-frame translation includes a +1 frame shift, a +2 frame shift, a -1 frame shift, or a -2 frame shift.

[0025] In certain embodiments, at least one synonymous mutation reduces the off-target immunogenicity of the modified therapeutic mRNA and / or its translation product.

[0026] In a particular embodiment, at least one synonymous mutation causes an out-of-frame codon to be mutated into a non-congeneral amino acid.

[0027] In certain embodiments, off-target immunogenicity includes cellular immunogenicity.

[0028] In certain embodiments, at least one frameshift nucleic acid sequence causes ribosome arrest.

[0029] In a particular embodiment, at least one frameshift nucleic acid sequence comprises at least one ribosome slippery sequence.

[0030] In a particular embodiment, at least one ribosome slippery sequence is a. XXXYYYZ, where X is any nucleotide, Y is A or U, and Z is A, U, or C; b. PPPX, where X is any nucleotide and PPP is a trinucleotide repeat of any nucleotide; c.m1ψm1ψm1ψX, where X is any nucleotide and m1ψ is (N)1-methylpseuduridine; d. CUUAGG, CUUGAC, CAGCAG, or UCUGCGG; and / or e. A sequence or formula containing any one of (a) to (d) and a non-canonical nucleotide. Includes at least one array selected from.

[0031] In certain embodiments, PPPX can be decoded by the same isoreceptor tRNA.

[0032] In certain embodiments, X is (N)1-methylpseudridine or cytidine. For example, with respect to m1ψm1ψm1ψX, X may be (N)1-methylpseudridine or cytidine.

[0033] In a particular embodiment, at least one frameshift nucleic acid sequence comprises or further comprises at least one other reading frame sequence encoding an out-of-frame product different from the translation product of the modified therapeutic mRNA in-frame translation.

[0034] In certain embodiments, at least one synonymous mutation includes a synonymous mutation for introducing a stop codon (PTC) into one other reading frame sequence.

[0035] In certain embodiments, the modified therapeutic mRNA comprises at least one chemically modified ribonucleotide.

[0036] In certain embodiments, at least one chemically modified ribonucleotide comprises (N)1-methylpseuduridine.

[0037] In certain embodiments, the translation step includes a step of translating in vivo or in vitro.

[0038] In a particular embodiment, the in vivo translation step includes administering the modified therapeutic mRNA to a target that requires it.

[0039] In a particular embodiment, the identification step is the step of sequencing the modified therapeutic mRNA and / or the nucleic acid encoding the modified therapeutic mRNA; and / or A process for analyzing a modified therapeutic mRNA sequence and / or the nucleic acid encoding the modified therapeutic mRNA sequence. Includes.

[0040] In a ninth aspect, modified therapeutic mRNAs described herein are provided for use as drugs.

[0041] In a tenth aspect, a modified therapeutic mRNA described herein is provided for use as a vaccine.

[0042] Throughout this specification and the claims, the words “comprise” and “contain,” and their variations thereof, mean “comprise but not limited to,” and are not intended (and do not exclude) other parts, additives, components, integers, or processes.

[0043] Throughout this specification and the claims, the singular form includes the plural form unless otherwise specified in the context. In particular, where the indefinite article is used, the specification should be understood to assume both the singular and plural forms unless otherwise specified in the context.

[0044] Features, integers, characteristics, compounds, chemical parts, or groups described in conjunction with specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, provided they are not incompatible therewith.

[0045] Various aspects of the present invention are described in further detail below.

[0046] Embodiments of the present invention are further described below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0047] [Figure 1]Figure 1 shows how a +1 frameshift polypeptide is produced by translation of 1-methylψ modified mRNA. a. Structure of IVT mRNA transcripts used to explore protein synthesis fidelity. WT Fluc contains only the firefly luciferase (Fluc) coding sequence (in frame). Fluc+1FS and Fluc-1FS: The green segments represent the in-frame N-terminal Flic coding sequence, and the red or crimson segments represent the +1 frameshifted or -1 frameshifted Fluc C-terminal coding sequence, respectively. b. Luciferase activity produced by translation of WTFluc mRNA containing either the unmodified control (canonical nucleotide) or the indicated modified nucleotide, respectively. "**" P<0.01 (one-way ANOVA with Dunnett's test). c. Luciferase activity produced by translation of modified Fluc-1FS mRNA and unmodified control. d. Luciferase activity produced by translation of modified Fluc+1FS mRNA and unmodified control. "**" P<0.01 (One-way ANOVA with Dunnett's test). e. Luciferase activity in the solubilizer produced by 8 hours of transfection of HeLa cells with unmodified or 1-methylψFluc+1FS mRNA. "**" P<0.01 (Welsh's one-sided t-test). Western blot analysis of polypeptides produced by mRNA translation in fd (anti-FLAG epitope). All data are obtained from n=3 replicated experiments. d shows a single blot from n=3 replicated experiments. [Figure 2] Figure 2 shows the validation of WT Fluc, Fluc+1FS, and Fluc-1FS mRNA. a. WT Fluc, Fluc+1FS, and Fluc-1FS mRNA transcripts. b. Luciferase activity generated by translation of WT Fluc, Fluc-1FS, and Fluc+1FS mRNA. [Figure 3]Figure 3 shows that the +1 frameshifted product induces an off-target cellular immune response following modified mRNA vaccination. a. Depiction of the spike and +1FS product produced by 1-methylψ-modified spike mRNA translation. b. Splenocyte IFNγ ELISpot response from untreated (n=5) and BNT162b2-vaccinated (n=7) mice stimulated with +1FS spike peptide. c. Representative plots of PBMC IFNγ ELISpot response wells for two individuals vaccinated with BNT162b2 (top) or ChAdOx1 nCoV-19 (bottom). From left to right: in-frame spike response; +1FS spike response; control without peptide. d. Summary data and statistics on PBMC IFNγ ELISpot response from donors stimulated with +1FS spike peptide and vaccinated with ChAdOx1 nCoV-19 (n=19) or BNT162b2 (n=22). * P<0.05, Welsh's one-sided t-test. Undetected responses: BNT162b2 (14 / 22), ChAdOx1, nCoV-19 (15 / 19). [Figure 4] Figure 4 shows that the mistranslation of 1-methylψ-mRNA is due to a +1 ribosome frameshift and not a transcription error. a. Peptide coverage plot of purified high molecular weight polypeptide produced by translation of 1-methylψ Fluc+1FS mRNA, showing in-frame residues (top) and +1 frameshifted residues (bottom). "-10logPEP" is the mass spectral percolator score (only high-quality peptides are shown). b. Nucleotide deletions in unmodified (top) and 1-methylψ (bottom) Fluc+1FS mRNA, quantified by RNA-seq analysis. c. Nucleotide insertions in unmodified (top) and 1-methylψ (bottom) Fluc+1FS mRNA. [Figure 5] Figure 5 shows the correlation between nucleotide insertions or deletions in unmodified Fluc+1FS mRNA and 1-methylψFluc+1FS mRNA. The coordinates are (x, y), where x = relative frequency 5'-3' in unmodified mRNA and y = relative frequency 5'-3' in 1-methylψmRNA for a, nucleotide deletion or b, nucleotide insertion. [Figure 6] Figure 6 shows that the +1 ribosome frameshift is dependent on the mRNA slippery sequence and is associated with ribosome arrest during 1-methylψ-mRNA translation. a. SDS-PAGE autoradiograph of arrested [35S]-Met-peptidyl-tRNA produced by 30 minutes of translation of unmodified or 1-methylψ-Fluc mRNA. Samples were treated with RNAse to digest peptide-associated tRNA. Full-length Fluc is indicated by an arrow, and the intermediate elongated polypeptide is shown below. Arrested peptidyl-tRNA during the 1-methylψ-mRNA translation reaction is indicated by an asterisk. b. SDS-PAGE autoradiograph of arrested [35S]-Met-polypeptide produced by 30 minutes of translation of unmodified or 1-methylψ-Fluc mRNA, with or without 100 μM paromomycin (+PMN and -PMN, respectively). c. Figure showing strategies for mRNA slippery sequence mutagenesis. d. +1FS activity after 2 hours of translation of mutant mRNA or Fluv+1FS2 control mRNA. *P<0.05, (one-way ANOVA with Dunnett's test). e. Quantified by total mRNA translation over 2 hours for each Fluv+1FS mRNA and mutant mRNA, by [35S]-Met incorporation. [Figure 7]Figure 7 shows the interferon-gamma ELISpot response to in-frame spike peptide pools derived from mouse splenocytes and human PBMCs. a. Splenocyte IFNγ ELISpot response from untreated (n=5) and BNT162b2-vaccinated (n=7) mice stimulated with in-frame SARS-CoV-2 spike peptide. b. PBMC IFNγ ELISpot response from individuals stimulated with in-frame SARS-CoV-2 spike peptide and vaccinated with ChAdOx1 nCoV-19 (n=19) or BNT162b2 (n=22). All ChAdOx1 nCoV-19 and n=12 BNT162b2 responses were assayed using the entire in-frame SARS-CoV-2 spike peptide (spike pool), while n=10 BNT162b2 responses were assayed separately with in-frame SARS-CoV-2 spike peptides corresponding to the S1 / S2 region (S1+S1 pool). Undetected responses: ChAdOx1 nCoV-19 (4 / 19), BNT162b2 spike pool (4 / 12), BNT162b2 S1+S1 pool (0 / 10). [Figure 8] Figure 8 shows that sequence optimization suppresses ribosome frameshift. a) A representative example of +1PTC introduction in a protein-coding sequence. The WT and mutant sequences are synonymous with the encoded peptide KDHDIDYK (SEQ ID NO: 1) in the main frame (0). The synonymous C2A mutation (bold) introduces an amber stop codon into the mutant sequence, which codes for the PTC when translated in the +1 frame, preventing undesirable +1 peptide elongation. b) Levels of the optimized synonymous sequence (AntiFS) for +1 ribosome frameshift and +1PTC in the firefly luciferase slippery sequence mutation T208C. The levels are normalized to Fluc+1FS translation. [Figure 9]Figure 9 shows that synonymous mutations targeted at ribosome slippery sites reduce ribosome +1 frameshift with minimal impact on in-frame mRNA translation efficiency. a. Figure showing the predicted ribosome slippery sequence and its stop codon-adjacent RNA sequence. b. Western blot analysis (anti-FLAG) of polypeptides produced by translation of mRNA in Figure 6d and mRNA containing targeted mutations (U*187C / U*208C) at slippery sites B and C. [Figure 10] Figure 10 shows that ribosome +1 frameshift at additional (N)1-methylpseudolylated ribosome slippery sequences can be reduced by targeted synonymous mutations. a. Depiction of the IVT mRNA +1FS reporter. b. Table showing the read-through status of one expected stop codon and its respective RNA sequence from SEQ ID NO: 6 for six expected ribosome slippery sites (slippery sites 1-6) and ribosome slippery site (SX). c. Western blot analysis of translation reactions from 14 mRNAs containing slippery sites 1-6 or SX and their respective RNA sequence status from SEQ ID NO: 6, unmodified or containing (N)1-methylψ, within the reporter structure depicted in a. Myc tag expression shows in-frame mRNA, and FLAG tag expression shows +1 frame translation, which is a mistranslation indicating ribosome +1 frameshift. d. Targeted synonymous mutations at slippery sites 1-6 or SX reduce +1 frame mRNA translation while maintaining in-frame mRNA translation. The anti-Myc tag blots and anti-FLAG tag blots are displayed from n=2 repeated anti-Myc tag blots and n=2 repeated anti-FLAG tag blots. [Modes for carrying out the invention]

[0048] The patents, scientific and technical documents referenced herein clarify the knowledge available to those skilled in the art at the time of filing. All disclosures of published patents, published and pending patent applications, and other publications cited herein are incorporated by reference to the same extent as they are specifically and individually indicated to be incorporated by reference. In the event of any inconsistency, this disclosure shall prevail.

[0049] Various aspects of the present invention are described in further detail below.

[0050] Detailed explanation therapeutic mRNA Modified therapeutic mRNAs are provided herein. The term mRNA refers to an RNA molecule that codes for a protein. mRNA may also refer to ribonucleic acid (RNA) transcribed from a DNA sequence by the RNA polymerase enzyme and interacting with ribosomes to synthesize DNA-encoded proteins. Generally, mRNA is classified into two subclasses: pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5' capping, splicing, editing, and polyadenylation), and therefore may contain a 5' untranslated region (UTR), introns, and / or 3'UTR (such as polyadenylated sequences). Mature mRNA has been modified through post-transcriptional processing (e.g., splicing to remove introns and polyadenylated regions) and can interact with ribosomes to carry out protein synthesis. The specific nucleic acid sequence composition and length of mRNA depend on the protein encoded by the mRNA. Traditionally, the basic components of an mRNA molecule include at least a coding region, 5'UTR, 3'UTR, 5' cap, and poly-A tail. In vitro transcription (IVT) mRNA can function as mRNA but is distinguished from wild-type mRNA in terms of its functional and / or structural design features, which help overcome existing problems in effective polypeptide production using nucleic acid-based therapeutics. For example, IVT mRNA can be chemically modified. For instance, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G".

[0051] The therapeutic mRNA of the present invention is referred to as “modified.” In the context of the present invention, modified therapeutic mRNA is therapeutic mRNA that has been modified to introduce synonymous mutations as described herein. In some cases, the modified therapeutic mRNA of the present invention may further include additional modifiers such as chemically modified nucleotides and / or additional gene (nucleic acid sequence) modifiers.

[0052] The therapeutic mRNA of the present invention may contain ribonucleotides that do not exist in nature, such as naturally occurring ribonucleotides and / or chemically modified nucleotides (e.g., canonical nucleotides). In some examples, the modified therapeutic mRNA provided herein may contain at least one chemically modified ribonucleotide. In some examples, the chemically modified ribonucleotides may be selected from the group consisting of pseudoridine, N1-methylpseudridine (1-methylψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrolauridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine. Other representative chemical modifiers useful in mRNA described herein include those listed in U.S. Patent Application Publication No. 2015 / 0064235. The aforementioned patent documents are incorporated herein by reference.

[0053] In some examples, the modified therapeutic mRNAs provided herein contain at least one N1-methylpseuduridine.

[0054] The modified therapeutic mRNAs provided herein may be pre-mRNA or mature mRNA. In some cases, the modified therapeutic mRNAs provided herein may contain one or more features of pre-mRNA, but not all features of pre-mRNA. For example, the modified therapeutic mRNAs provided herein may contain polyadenylated sequences but not introns. In some cases, the modified therapeutic mRNAs provided herein may contain one or more features of mature mRNA, but not all features of mature mRNA.

[0055] "Therapeutic mRNA" refers to mRNA molecules that encode therapeutic proteins (e.g., in vitro transcription (IVT) mRNA). Therapeutic proteins mediate various effects in host cells or subjects to treat a disease or alleviate the signs and symptoms of a disease. For example, therapeutic proteins can replace deficient or abnormal proteins, enhance the function of endogenous proteins, provide cells with novel functions (e.g., inhibit or activate endogenous cellular activity), or act as delivery agents for alternative therapeutic compounds (e.g., antibody-drug conjugates). Therapeutic mRNA may be useful for the treatment or prevention of the following diseases and conditions: infectious diseases (bacterial, viral, parasitic infections, etc.), cell proliferation disorders (cancer, etc.), genetic disorders, inflammatory diseases, cardiovascular disorders, metabolic diseases, allergic diseases, neurodegenerative diseases, protein or enzyme deficiency diseases, and / or autoimmune diseases.

[0056] Examples of therapeutic mRNAs include Pfizer and BioNtech's BNT162b2 (Covid-19), Moderna's mRNA-1273 (Covid-19), mRNA-2416 (solid tumors or lymphoma), MRT5005 (cystic fibrosis), mRNA-2752 (solid tumors or lymphoma), AZD-8601 (heart failure), NY-ESO-1 (multiple myeloma, synovial sarcoma, melanoma), and CTX001. (β-thalassemia), SB-728mR-HSPC (HIV), SB-728mR-T (HIV), BNT163 (HSV2), BNT164 (tuberculosis), BNT165 (malaria), BNT167 (herpes zoster), BNT161 (influenza), BNT153 (undisclosed cancer), BNT152 (undisclosed cancer), BNT142 (undisclosed cancer), BNT141 (undisclosed cancer), BNT131 (undisclosed cancer), BNT122 (melanoma), colorectal cancer, These include BNT116 (non-small cell lung cancer), BNT115 (ovarian cancer), BNT113 (head and neck cancer), BNT112 (prostate cancer), BNT111 (melanoma), mRNA-1345 (multinuclear respiratory virus), mRNA-1010 (influenza), mRNA-1647 (cytomegalovirus), mRNA-4157 / V940 (melanoma), mRNA-3927 (propionic acidemia), mRNA-0184 (heart failure), and VX-522 (cystic fibrosis). The use of each mRNA therapy is indicated in parentheses.

[0057] Therapeutic mRNA molecules are generally synthesized in the laboratory (e.g., by in vitro transcription). mRNA can be isolated from tissues or cells by various methods. For example, total RNA extraction can be performed in cells or cell solubilates, and the extracted total RNA thus obtained can be purified (e.g., on a column containing oligo-dT beads) to obtain extracted mRNA. Alternatively, mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). IVT is a method that enables the synthesis of ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)) by template instruction (e.g., via an IVT DNA template). IVT generally relies on the manipulation of a DNA template containing a bacteriophage promoter sequence upstream of the sequence of interest, followed by transcription using the corresponding RNA polymerase. In vitro mRNA transcripts can be used as in vivo therapeutics, for example, to instruct ribosomes to express protein therapeutics within targeted tissues.

[0058] "In vitro transcription template (IVT)," as used herein, refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some examples, an IVT template encodes a 5' untranslated region, includes an open reading frame, and encodes a 3' untranslated region and a poly(A) tail. The specific nucleotide sequence composition and length of an IVT template depend on the target mRNA encoded by the template. IVT mature mRNA preparation involves several steps: linear DNA template acquisition, IVT, 5' capping, and poly(A) tail addition.

[0059] The "5' untranslated region (UTR)" is the region of mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by ribosomes) that does not code for either a protein or a peptide. IVT mRNA is performed by linearizing a plasmid DNA template or a PCR template requiring at least a promoter and the corresponding mRNA construct sequence. IVT mRNA can be performed by adding polymerase (T7, T3, or SP6), but requires additional capping. Uncapped mRNA is rapidly degraded by RNase to contain a 5'-ppp group, which causes greater immunostimulation, and treatment with phosphatase can reduce the undesirable potency. For capping IVT mRNA, two methods can be performed: co-transcriptional capping and post-transcriptional capping. A cap dinucleoside mixture containing four nucleoside triphosphatases (NTPs) is incorporated into the 5' of the RNA along with RNA polymerase during co-transcriptional capping. The co-transcriptional capping method has enabled coordinated transcription with mRNA capping. The poly(A) tail of IVT mRNA is typically encoded within the DNA template or bound to the IVT mRNA by enzymatic polyadenylation. The former may offer more precise control over the length of the poly(A) tail. After synthesis, IVT mRNA is mixed with RNA polymerase and the DNA template, and therefore may require purification of the IVT mRNA, including steps to remove immunostimulant contaminants, free ribonucleotides, short mRNA, and DNA template. Generally, DNase is used to degrade excess DNA template.The synthesized mRNA may be purified and isolated using a commercially available purification kit, followed by precipitation using ethanol or isopropanol, which removes most impurities and yields high-purity mRNA. The mRNA can then be precipitated using high-concentration LiCl or alcohol-based precipitation, chromatography (molecular exclusion chromatography, ion-exchange chromatography, or affinity chromatography using immobilized oligo-dT), or elution from a silica membrane column that removes proteins, free nucleotides, or other components but not dsRNA impurities. Reverse-phase HPLC can be used to remove dsRNA impurities from the transcription reaction solution.

[0060] The "3' untranslated region (UTR)" is the region of mRNA immediately downstream (i.e., 3') of a stop codon (i.e., the codon in the mRNA transcript that signals the end of translation) that does not code for either a protein or a peptide.

[0061] The "poly-A tail" is a region of mRNA located immediately downstream (i.e., 3') of the 3' UTR, for example, containing multiple consecutive adenosine monophosphates. The poly-A tail may contain 10 to 300 adenosine monophosphates. For example, the poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some cases, the poly-A tail contains 50 to 250 adenosine monophosphates. In the relevant biological context (e.g., in cells, in vivo, etc.), the poly-A tail functions to protect mRNA from enzymatic degradation, for example, in the cytoplasm, and assists in transcription termination, extrusion of mRNA from the nucleus, and translation. However, in some cases, the mRNA molecules provided herein do not contain a poly-A tail (such molecules are referred to as "tailless").

[0062] An "open reading frame" is a continuous sequence of DNA or RNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a protein or peptide.

[0063] Frameshift nucleic acid sequences The modified therapeutic mRNAs provided herein may contain at least one frameshift nucleic acid sequence. The term “frameshift nucleic acid sequence” is used to refer to any nucleic acid sequence, such as a template RNA or DNA sequence for IVT encoding the modified therapeutic mRNA of the present invention, which may cause or increase the possibility of ribosome frameshift (i.e., translation or ribosome frameshift). “Frameshift” is the way in which a ribosome moves to a reading frame (modified frame) of one or a few nucleotides at a site in mRNA. Frameshift can result in the production of multiple, unique proteins from a single mRNA. Proteins are translated by reading three nucleotides (codons) from the 5' to the 3' end, starting with the amino acid methionine as the start codon. Each codon is translated to a single amino acid. The code itself is degenerate, meaning that a particular amino acid can be identified by more than one codon. Due to the movement of any number of nucleotides not divisible by 3 in the reading frame, the subsequent codons are read differently from the codons in the intended or exact reading frame. This effectively alters the ribosome reading frame, resulting in the production of another polypeptide encoded by mRNA. Translation of a frameshifted codon may be called out-of-frame translation, and its product (i.e., another protein or polypeptide) may be called an out-of-frame product or protein, or another product or protein (i.e., a substitute for the product encoded by the unframeshifted (accurate or in-frame translated) open reading frame). A frameshifted nucleic acid sequence may result in another translated product, such as a cleaved protein (e.g., due to premature termination of translation), a different amino acid sequence from the amino acid sequence encoding the unframeshifted open reading frame, or a protein with an increased amino acid length from the desired product produced by translation in the accurate open reading frame (e.g., due to misreading or read-through of a stop codon).

[0064] The frameshift nucleic acid sequence may be any sequence known or predicted to result in a ribosome frameshift. Methods for identifying or predicting frameshift nucleic acid sequences are publicly known. For example, US20080103745A1 describes a model for predicting frameshifts. Other methods for predicting ribosome frameshifts include those described in Moon, S. et al., LNCS, 2004, 3036: pp. 334-334; Hammell, AB et al., Genomic Res., 1999, 9: pp. 417-427; Bekaert, M. et al., Bioinformatics, 2003, 19: pp. 327-335; and Shah, AA et al., Bioinformatics, 2002, 18: pp. 1046-1053.

[0065] Each frameshift can be a -2, -1, +1, or +2 frameshift. A "-1 frameshift" is a frameshift in which ribosomes move nucleotides upstream, and a "+1 frameshift" is a frameshift in which ribosomes move nucleotides downstream.

[0066] Known frameshift nucleic acid sequences that can cause a -1 or +1 frameshift include sequences UUUUGA (SEQ ID NO: 2), UCCUGA (SEQ ID NO: 3), or CCCUGA (SEQ ID NO: 4); a spacer component having a spacer containing 4 to 11 nucleotides; and / or a secondary structure that can be named a stem-loop or pseudoknot.

[0067] Known frameshift nucleic acid sequences that can cause a -1 or +1 frameshift include a site containing the sequence XXXYYYZ (SEQ ID NO: 5) in sequence, where X is any nucleotide, Y is A or U, and Z is A, U, or C; a space component having 4 to 11 nucleotides; and / or a secondary structural component that can be named a stem-loop or pseudoknot.

[0068] In some examples, the frameshift nucleic acid sequence may include the sequence PPPX (SEQ ID NO: 306), where X is any nucleotide and PPP is a trinucleotide repeat of any nucleotide. In some examples, the frameshift nucleic acid sequence may include the sequence m1ψm1ψm1ψX (SEQ ID NO: 307), where X is any nucleotide and m1ψ is (N)1-methylpseudolidine. In some examples, the frameshift nucleic acid sequence may include the sequences CUUAGG (SEQ ID NO: 308), CUUGAC (SEQ ID NO: 309), CAGCAG (SEQ ID NO: 310), or UCUGCGG (SEQ ID NO: 311).

[0069] In some cases, frameshift nucleic acid sequences may include sequences containing non-canonical amino acids. Non-canonical amino acids are non-proteinogenic amino acids that are naturally occurring in organisms or synthesized in the laboratory and are not found in the genetic code of naturally occurring organisms.

[0070] In some cases, the frameshift nucleic acid sequence may contain any combination of the sequences described above.

[0071] Frameshift nucleic acid sequences may, in some cases, be called ribosomal slippery sequences. Therefore, in some examples, the modified therapeutic mRNA of the present invention may contain one or more ribosomal slippery sequences.

[0072] A pseudoknot is a secondary RNA substructure containing two or more stem-loop motifs interposed by a stem. It is thought that pseudoknots or stem-loop structures in mRNA induce ribosome rest, ultimately leading to a frameshift.

[0073] In some cases, frameshift nucleic acid sequences can also cause ribosome arrest. In some cases, frameshift nucleic acid sequences are any sequence that can cause ribosome arrest. Sequences that cause ribosome arrest are known and may include mRNA secondary structures, sequences of rare or difficult-to-decipher codons, and sequences encoding codons that encode certain amino acids, such as proline, glycine, positively charged amino acids, and negatively charged amino acids.

[0074] Other frameshift nucleic acid sequences are known. For example, the frameshift nucleic acid sequences may be sequences identified in databases such as the FSDB (See Moon S, Byun Y, Han K. FSDB: a frameshift signal database. Comput Biol Chem. 2007;31(4):298~302. Doi:10.1016 / j.compbiolchem.2007.05.004) or the PRFdb (See Belew, Ashton T., et al. “PRFdb: a database of computationally predicted eukaryotic programmed-1 ribosomal frameshift signals.” BMC genomics 9.1 (2008): pp. 1~7.).

[0075] In some cases, the frameshift nucleic acid sequence is an alternative reading frame sequence. For example, the frameshift nucleic acid sequence may be a sequence that codes for a different reading frame at positions -2, -1, +1, or +2 from the exact open reading frame, resulting in the production of an alternative or out-of-frame protein. The alternative reading frame sequence may be a sequence that causes ribosome arrest, or may be located in close proximity to a sequence that causes ribosome arrest (for example, at a position of approximately -2 to +2 nucleotides from the sequence that causes ribosome arrest).

[0076] In some examples, the frameshift nucleic acid sequence may be considered a slippery sequence and may encode an alternative leading frame sequence and / or cause ribosome arrest. In some examples, the frameshift nucleic acid sequence includes a modified ribonucleotide, such as a chemically modified ribonucleotide as described herein. In some examples, the frameshift nucleic acid sequence includes (N)1-methylpseudolidine.

[0077] Throughout this description, the process of analyzing the sequence of therapeutic mRNA is referred to. It is understood that the analysis and / or sequencing may be performed on mRNA, RNA sequences, or on templates for the production of mRNA or RNA sequences. For example, analysis of a DNA template used for mRNA production (e.g., by IVT) may be performed to identify the DNA sequence encoding the frameshift nucleic acid sequence described herein after transcription.

[0078] Therefore, nucleic acids encoding the modified therapeutic mRNAs described herein are also provided herein. In some examples, DNA templates for in vitro transcription of the modified therapeutic mRNAs described herein are provided.

[0079] Out-of-frame and / or alternative translation products may be more immunogenic than in-frame translation products. Out-of-frame and / or alternative translation products may also have reduced efficacy compared to in-frame translation products. Frameshift nucleic acid sequences described herein may result in reduced efficiency and / or fidelity (accuracy to the intended translation product).

[0080] Synonymous mutation Without being constrained by theory, the inventors found that introducing one or more synonymous mutations into a frameshift nucleic acid sequence could help reduce out-of-frame translation, and therefore, the production of out-of-frame or alternative translation products.

[0081] A "synonymous mutation" in the mRNA described herein is a change compared to the reference sequence (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides compared to the reference sequence) and the change does not alter the encoded amino acid. For example, GGT, GGA, GGC, and GGG all encode glycine. Any change at the third position of a codon (e.g., A->G) results in the same amino acid incorporated into that position in the protein sequence.

[0082] The introduction of synonymous mutations may therefore result in a reduction in the level of out-of-frame translation products produced when the modified therapeutic mRNA described herein is translated, compared to therapeutic mRNA without synonymous mutations. Thus, synonymous mutations may result in an increase in translational fidelity compared to therapeutic mRNA without synonymous mutations. "Translational fidelity" refers to the precision of mRNA translation. For example, it is the translation of the desired protein (including amino acids encoded by in-frame codons) produced by translation occurring in the intended reading frame (i.e., accurate or in-frame translation). In some cases, synonymous mutations may increase translational efficiency. For example, synonymous mutations may reduce or prevent ribosome arrest, which may therefore allow for an increase in the rate of translation compared to therapeutic mRNA without synonymous mutations.

[0083] In some cases, synonymous mutations may introduce premature stop codons. A “premature stop codon” or “immature stop codon” is a stop codon in mRNA (before an endogenous or desired termination codon) resulting from a mutation (i.e., nucleic acid modification). A premature stop codon (PTC) may include one of three stop codons: UAA; UAG; or UGA. The modified therapeutic mRNAs provided herein may include immature stop codons encoded in a frameshifted reading frame. For example, if translated in frame, the translation product is not affected by synonymous mutations and the inserted PTC is not read as a PTC. However, if a frameshift occurs (i.e., a -1, -2, +1, or +2 frameshift), the frameshifted reading frame contains at least one codon encoding a PTC that terminates translation and produces a truncated protein that may be degraded if produced in the subject. Such synonymous mutations can interfere with out-of-frame translation and help destabilize mRNA undergoing out-of-frame translation.

[0084] Synonymous mutations may introduce out-of-frame codons (i.e., frameshifted codons) encoding non-homogeneous amino acids. aa-tRNAs that can participate in standard Watson-Crick interactions with the first two bases in the codon and form canonical or non-Watson-Crick pairs at the third or "fluctuating" position are called homogeneous-tRNAs. In contrast, tRNAs that do not meet these requirements are generally called near- and non-homogeneous-tRNAs.

[0085] Because synonymous mutations reduce out-of-frame translation and its products, the translation products of modified therapeutic mRNA containing synonymous mutations may have reduced immunogenicity compared to the translation products of mRNA without synonymous mutations. In some cases, the translation products may have reduced innate immunogenicity. In some cases, the translation products may have reduced cellular immunogenicity.

[0086] Nucleic acids encoding the modified therapeutic mRNAs described herein, including the synonymous mutations described herein, are also provided herein. In some examples, DNA templates for in vitro transcription of the modified therapeutic mRNAs described herein, including the synonymous mutations described herein, are provided.

[0087] Modifications and methods for producing modified therapeutic mRNAs can be applied to any known therapeutic mRNA. For example, one of the therapeutic mRNAs mentioned above is BNT162b2, Moderna's mRNA-1273, mRNA-2416, MRT5005, AZD-8601, NY-ESO-1, CTX001, SB-728mR-HSPC, and SB-728mR-T. For example, a therapeutic mRNA according to Sequence ID No. 6, modified to contain at least one synonymous mutation as described herein, is provided. In one example, a modified therapeutic mRNA according to Sequence ID No. 7 or a DNA template for IVT encoding a modified therapeutic mRNA according to Sequence ID No. 7 is provided.

[0088] Medical use Modified therapeutic mRNAs may be used for several in vitro and in vivo uses. All references to medical use, methods of treatment, and methods of manufacturing drugs using modified therapeutic mRNAs should be understood to relate to methods of treating subjects using the modified therapeutic mRNAs described herein. Thus, for example, modified therapeutic mRNAs described herein are provided for the manufacture of drugs to treat any of the disorders, conditions, or diseases described herein. For example, modified therapeutic mRNAs described herein are provided for use in treating any of the disorders, conditions, or diseases described herein. For example, modified therapeutic mRNAs described herein are provided for use in treating any of the disorders, conditions, or diseases described herein. For example, a method is provided for treating a subject requiring the modified therapeutic mRNAs described herein, comprising the step of administering them. For example, the method is a method for treating any of the disorders, conditions, or diseases described herein.

[0089] The modified therapeutic mRNAs described herein may be intended for use as drugs. For example, the modified therapeutic mRNAs described herein may be intended for use in methods of preventing or treating a disease or condition in a subject. The disease or condition being treated depends on the protein encoded by the modified therapeutic mRNA. Generally, the modified therapeutic mRNAs described herein may be intended for use in treating any disease or condition that may benefit from the administration of mRNA or the protein translated therefrom. Modified therapeutic mRNAs are intended or may be used in methods of treating infectious diseases (such as bacterial, viral, and parasitic infections), proliferative disorders (such as cancer), genetic disorders, inflammatory diseases, cardiovascular disorders, metabolic diseases, allergic diseases, neurodegenerative diseases, protein or enzyme deficiency disorders, and / or autoimmune diseases.

[0090] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating hereditary disorders. Hereditary disorders are congenital or acquired disorders caused by abnormalities in chromosomes or mitochondrial DNA, and examples include Down syndrome, Wilson's disease, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter syndrome, Apert syndrome, Crouzon syndrome, 22q11.2 deletion syndrome, Williams syndrome, Laurence Moon-Beadle syndrome, Prader-Willi syndrome, Angelman syndrome, Kallmann syndrome, Eycardi-Gutierre syndrome, Miller-Dieker syndrome, Rubinstein-Taybe syndrome, Cornelia de Lange syndrome, cat-meow syndrome, hyperfemale, hypermale, and mitochondrial diseases.

[0091] For example, the modified therapeutic mRNAs described herein may be intended for use in methods for treating protein or enzyme deficiency disorders. "Protein or enzyme deficiency disorder" means any disease or disorder associated with a subject that lacks one or more proteins or enzymes, or lacks sufficient activity of a protein or enzyme that causes symptoms and adverse effects in the subject.Examples of protein or enzyme deficiency disorders include Pompe disease, mucopolysaccharidosis types I, II, and VI, hemophilia A and B hyperhomocysteinemia, Danon disease, myoclonus renal failure syndrome, sialic acid storage disorders such as ISSD, Salah disease and moderate to severe Salah disease, Niemann-Pick disease C1 and C2, and cuclipid storage disorder type IV; neuronal ceroid lipofuscinosis includes ceroid lipofuscinosis type 1 (Hartia-Santaburi disease and INCL), neuronal ceroid lipofuscinosis type 2 (Yansky-Birshosky disease), and ceroid lipofuscinosis type 3 (Battenspirmey). Jajjögren's disease), waxy lipofuscinosis type 4 (Parry's disease and Kufus A and B), ceroid lipofuscinosis type 5 (late premature Finnish type), cereofuscinosis type 6 (Lake Cavano or Indian type), ceroid lipofuscinosis type 7 (Turkish type), ceroid lipofuscinosis type 8 (Northern epilepsy, epileptic psychiatry), ceroid lipofuscinosis type 9, ceroid lipofuscinosis type 10, ceroid lipofuscinosis type 11, ceroid lipofuscinosis type 12, ceroid lipofuscinosis type 13, ceroid lipofus This includes, but is not limited to, 14 types of thin deposition disorders; Hermanskiy-Padlak disease type 1, Hermanskiy-Padlak disease type 2, Hermanskiy-Padlak disease type 3, Hermanskiy-Padlak disease type 4, Hermanskiy-Padlak disease type 5, Hermanskiy-Padlak disease type 6, Hermanskiy-Padlak disease type 7, Hermanskiy-Padlak disease type 8, Hermanskiy-Padlak disease type 9, Grischelli syndrome 1 (Elejarde syndrome), Grischelli syndrome 2 (Chediak-Higashi disease), lysosome storage disorders, such as Hurler syndrome, Niemann-Pick disease, and Taysachs disease. Lysosomal organelle disorders, including but not limited to Gaucher disease, Fabry disease, or Krabbe disease; phenylketonuria; mitochondrial disorders; Fleetwright ataxia; peroxisome disorders, e.g., Zellweger syndrome or adrenoleukodystrophy; metal metabolism disorders, e.g., Wilson's disease or hemochromatosis; organic acid metabolism disorders, e.g., methylmalonic acidemia or propionic acidemia; urea cycle disorders, e.g., ornithine transcarbamylase deficiency or citrullinemia and / or β-thalassemia.Another example of an enzyme deficiency disorder is type 1 diabetes, which results from the patient's inability to produce insulin.

[0092] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating cardiovascular disorders. "Cardiovascular disease" or "cardiovascular disorder" means a disease affecting the heart, or the blood vessels, or both. For example, cardiovascular diseases include arrhythmias (atrial, ventricular, or both); atherosclerosis and its complications; angina pectoris; dyscardiac disorders; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysms; vasculitis, stroke; peripheral occlusive arteriovenous disease of the limbs, organs, or tissues; reperfusion injury following ischemia of the brain, heart, kidneys, or other organs or tissues; endotoxin, surgical, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vasoconstriction (including vasoconstriction associated with migraines); vascular abnormalities, dysfunction limited to a single organ or tissue.

[0093] For example, the modified therapeutic mRNAs described herein may be intended for use in methods for treating autoimmune diseases. “Autoimmune disease” refers to a disease or condition in which the immune system of a subject has an abnormal immune response to substances that do not normally elicit an immune response in healthy subjects. Examples of treatable autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, and autoimmune diseases. Retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathy, Barlow's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, scarring pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsacki's disease - Myocardiitis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, herpetiform dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), lupus supraclavicularis, Dressler syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrous alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis) Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes zoster of pregnancy, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoprotein, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes mellitus), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD),Lupus (SLE), Lyme disease, chronic Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mohren's ulcer, Muchahabermann's disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular scarring pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (Streptococcus-associated childhood autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Personage Turner syndrome, ciliary body squamous cellulitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, and III polyglandular autoimmune syndromes, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, Original biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell fistula, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, semen and testicular autoimmunity, systemic These include rigidity syndrome, subacute bacterial endocarditis (SBE), Suzak syndrome, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes mellitus, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous skin disease, vitiligo, or Wegener's granulomatosis (i.e., granulomatosis with polyangiitis (GPA)).

[0094] For example, the modified therapeutic mRNAs described herein may be intended for use in methods for treating neurodegenerative diseases. “Neurodegenerative disease” refers to a disease or condition in which the function of the nervous system is impaired. Examples of neurodegenerative diseases that can be treated include Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia with telangiectasia, Batten disease (also known as Szpilmayer-Voigt-Sjögren-Batten disease), bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Streusler-Scheinker disease, Huntington's disease, HIV-related dementia, Kennedy disease, and Krabbe disease. These include Kuru disease, Lewy body dementia, Machad-Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbach disease, Pick's disease, primary lateral sclerosis, prion diseases, Refsum disease, Sandhoff disease, Schilder's disease, subacute combined degeneration of the spinal cord following pernicious anemia, schizophrenia, spinocerebellar ataxia (multiple types with varying characteristics), spinal muscular atrophy, Steele-Richardson-Olsewski disease, or spinal fistula.

[0095] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating metabolic disorders. A “metabolic disorder” is a disease or condition in which the metabolism or metabolic system of interest (e.g., the function of storing or utilizing energy) is impaired. Examples of metabolic disorders that can be treated include diabetes mellitus (e.g., type 1 or type 2), obesity, metabolic syndrome, or mitochondrial disorders (e.g., mitochondrial dysfunction or abnormal mitochondrial function).

[0096] For example, the modified therapeutic mRNAs described herein may be intended for use in methods for treating inflammatory diseases. “Inflammatory disease” means a disease or condition characterized by abnormal inflammation (for example, an increased level of inflammation compared to a control, such as a healthy person without the disease). Examples of inflammatory diseases include traumatic brain injury, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile-onset diabetes mellitus, type 1 diabetes mellitus, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjögren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behçet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves' ophthalmopathy, inflammatory bowel disease, Addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, graft rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

[0097] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating cancer. The modified therapeutic mRNAs provided herein may help induce an effective tumor-responsive T cell response against a tumor. When intended for use in treating cancer, the modified therapeutic mRNAs encode tumor-associated epitopes. The modified therapeutic mRNAs described herein may effectively help generate a population of immune cells, particularly CD+8 effector T cells (also known as cytotoxic T lymphocytes (CTLs)). Immune cells induced by administration of the modified therapeutic mRNAs described herein may respond to the epitope, or multiple epitopes, translated from the modified therapeutic mRNAs described herein. These immune cells then receive immune stimulation to kill cancer cells that present the same or similar epitopes. Such modified therapeutic mRNAs intended for use in treating cancer may be called "cancer vaccines" or "cancer immunotherapy vaccines."

[0098] Pharmaceutical uses and methods for treating cancer may include administering a therapeutically effective amount of the modified therapeutic mRNA described herein to a subject in need.

[0099] The medicinal uses and methods of the treatments described herein may be used in the treatment of a wide range of cancers. Tumors may be of mesenchymal or epithelial origin. Cancers include those of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tongue, lip, larynx, kidney, bladder, prostate, brain, and blood cells. The medicinal uses and methods of the treatments described herein may be used in the treatment of solid tumors.

[0100] Appropriately, cancers treated by the pharmaceutically acceptable use or method of the treatment described herein may be solid tumors selected from, but not limited to, the group consisting of pancreatic ductal adenocarcinoma, pancreatic cancer; breast cancer; melanoma; non-small cell lung cancer; small cell lung cancer; nasopharyngeal cancer; hepatocellular carcinoma; colorectal cancer; esophageal cancer; gastric cancer; anal cancer; small intestine cancer; mesothelioma; kidney cancer, renal cell carcinoma; bladder cancer; prostate cancer; ovarian cancer; vulvar cancer; cervical cancer; penile cancer; uveal melanoma; retinoblastoma; sarcoma; osteosarcoma; glioblastoma; adrenocortical carcinoma; neuroblastoma; Wilms' tumor; endometrial cancer; and thyroid cancer.

[0101] With respect to cancer, the terms “treatment” and “to treat” should be interpreted as encompassing therapies performed to prevent, slow, or reduce undesirable physiological changes or impairments, such as growth, development, or metastasis of cancer. Beneficial or desired outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the disease (i.e., a non-worsening disease), delay or slowing of disease progression, destaging of the tumor (e.g., a change from a resectable boundary to a modifiable boundary in the case of surgical resection), recovery or mitigation of the disease state, and remission (partial or total).

[0102] The treatment may result in a longer survival compared to the survival predicted without treatment. Alternatively, or even more, the treatment may provide patients with an improved standard of living compared to the standard of living predicted without treatment.

[0103] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating allergic diseases. “Allergic disease” refers to a condition caused by hypersensitivity of the immune system to typically harmless substances in the environment. Allergic diseases include, but are not limited to, asthma, hypersensitivity lung disease, rhinitis, rhinoconjunctivitis, sinusitis, atopic eczema, contact dermatitis, allergic conjunctivitis (intermittent and persistent), vernal keratoconjunctivitis (hay fever), atopic keratoconjunctivitis, giant papillary conjunctivitis, urticaria, angioedema, hypersensitivity pneumonitis, eosinophilic bronchitis, vasculitis, hypersensitivity vasculitis, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Wegner granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, temporal arteritis, celiac disease, mastocytosis, and anaphylaxis.

[0104] In particular, the modified therapeutic mRNAs described herein may encode proteins containing allergic epitopes. In most cases, allergic epitopes are clearly epitopes from or derived from allergens that cause allergic symptoms or allergic reactions in a subject.

[0105] In some cases, the modified therapeutic mRNAs described herein that encode proteins containing allergenic epitopes may be intended for use in methods of allergic immunotherapy (AIT). In some cases, this is subcutaneous allergic immunotherapy (SCIT). Modified therapeutic mRNAs for use in AIT and / or SCIT may be called allergy vaccines.

[0106] Generally, AIT (Assisted Immune Therapy) involves administering an allergen to a patient to treat their allergy to that allergen, i.e., reducing the current or future immune response, such as the allergen-specific IgE response and / or histamine release by mast cells and / or granulocytes induced by the allergen, and / or the appearance of clinical symptoms of the allergy. Immunotherapy is routinely performed by repeatedly administering a single dose or increasing dose of the allergen to the patient in need, thereby obtaining an adaptive immune response in the patient with reduced hypersensitivity to the allergen.

[0107] During AIT or SCIT, increasing doses of the allergen or allergen epitope are administered, followed by maintenance doses for several years, with the aim of inducing immunological changes that lead to symptom recovery during treatment and sustained desensitization (immune tolerance) from AIT or SCIT.

[0108] Typically, at the initiation of AIT or SCIT, subjects receive an increasing dose of the allergen or allergen epitope at weekly intervals over several weeks to months under closely monitored medical supervision. The gradual dose escalation allows for better treatment tolerance and reduces the risk of severe hypersensitivity reactions associated with allergen administration.

[0109] For example, the modified therapeutic mRNAs described herein may be intended for use in methods of treating infectious diseases. “Infectious disease” means a disease resulting from infection. Infection is a condition caused by the invasion of an organism or foreign pathogen (i.e., an infectious pathogen). Infectious pathogens include, but are not limited to, bacteria, fungi, viruses, viroids, nematodes (e.g., parasitic worms such as roundworms and pinworms), arthropods (e.g., mites, fleas, lice, ticks), and macroparasites (e.g., tapeworms). Common infectious diseases include bacterial and viral infections. When the modified therapeutic mRNAs described herein are intended for use in methods of treating infectious diseases, they may contain epitopes or antigens from or derived from pathogens that cause infectious diseases.

[0110] The modified therapeutic mRNAs described herein may be particularly useful for the prevention or treatment of infectious diseases caused by intracellular pathogens. For example, viruses (e.g., CMV, HIV, Covid-19, SARS virus such as coronavirus), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protists (e.g., Toxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., Plasmodium vivax)). These include Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale, and Plasmodium malariae. The compositions and formulations described herein may reduce humoral responses and increase cell-mediated responses to immunogenic immunomodulators containing epitopes derived from such intracellular pathogens.

[0111] In relation to infectious diseases, “treatment” means any administration of the modified therapeutic mRNA described herein that partially or completely reduce, restore, mitigate, suppress, delay, reduce the severity and / or the incidence of one or more symptoms or characteristics of an infectious disease or predisposition to the disease. Such treatment may be in subjects showing no signs of the disease and / or only early signs of the disease. Alternatively or further, such treatment may be in subjects showing established signs of one or more of the disease. Thus, in relation to infectious diseases, “to treat” means vaccination of the subject. “Prevention” means delaying the onset of an infectious disease. Prevention may be considered complete when the onset of the infectious disease or disorder is delayed for a predetermined period of time.

[0112] In some cases, the modified therapeutic mRNAs described herein may be used as vaccines. Therefore, in some cases, immunogenic compositions comprising the described modified therapeutic mRNAs are provided herein. In some cases, the composition is a vaccine composition. The terms “immunogenic composition,” “immunological composition,” and “immunogenic or immunological composition” refer to compositions that induce an immune response to an antigen or immunogen after administration to a subject. The terms “vaccine” and “vaccine composition” refer to compositions that induce a protective immune response to or effectively protect against a target antigen; for example, a protective immune response to a targeted antigen or immunogen after administration to a subject.

[0113] In some cases, the method of preventing and / or treating infectious diseases is vaccination. "Vaccination" means, for example, administering a modified therapeutic mRNA described herein with the aim of inducing an immune response to a disease-causing pathogen. Vaccination may be administered before, during, and / or after exposure to a disease-causing pathogen, and in some cases, before, during, and / or immediately after exposure to the pathogen. In some cases, vaccination may involve multiple doses of the modified therapeutic mRNA described herein, appropriately spaced apart.

[0114] In some cases, modified therapeutic mRNA may be intended for use in viral vaccination. For example, it may be intended for use in target vaccination against viruses of the Retroviridae, Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Bunyaviridae, Flaviviridae, Filoviridae, Togaviridae, Picornaviridae, Caliciviridae, and Coronavirusidae families. Examples of such viruses include, but are not limited to, adenoviruses, rhinoviruses, hepatitis, immunodeficiency viruses, polio, measles, Ebola, coxsackievirus, rhinovirus, West Nile virus, smallpox, encephalitis, yellow fever, dengue fever, influenza (including human, avian, and swine), lassa virus, lymphocytic choriomeningitis, Junin virus, machuppo virus, Guanalito virus, hantavirus, Rift Valley fever, lacrosse virus, California encephalitis, Crimean-Congo virus, Marburg virus, Japanese encephalitis, Kasanur forest virus, Venezuelan horse encephalitis, Eastern horse encephalitis, Western horse encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Puntatoro virus, Takalibe virus, and pachindae virus.

[0115] In some cases, the virus is influenza (including human, avian, and swine) or severe acute respiratory syndrome (SARS) virus. In some cases, the virus is a coronavirus. In some cases, the virus is COVID-19.

[0116] As used herein, the terms “treat,” “treating,” and “treatment” are generally interpreted to include therapeutic interventions performed for the purpose of preventing the onset of a condition, disorder, or symptom (e.g., allergic diseases, infectious diseases, etc.) or altering their pathological state. Thus, “treatment” includes both therapeutic treatments and prophylactic or preventative measures (such as vaccination) whose purpose is to prevent or slow the progression of a targeted condition, disorder, or symptom. Accordingly, “treatment” includes, for example, a reduction, delay, or inhibition of disease symptoms by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to before treatment.

[0117] As used herein, the term “subject” generally refers to an individual, for example, a person who has or is at risk of having a particular condition, disorder, or symptom. A subject may also be a patient, i.e., a subject who requires treatment according to the present invention. A subject may have previously received treatment for the condition, disorder, or symptom. Alternatively, a subject may not have received any treatment prior to treatment according to the present invention.

[0118] The modified therapeutic mRNAs described herein can generally be administered to a subject by any conventional route, including injection, or by progressive infusion over time. Administration may be, for example, intramuscular, intravascular, intracavitary, intracerebral, intrafocal, rectal, subcutaneous, intradermal, epidural, subarachnoid, and transdermal.

[0119] The procedures and methods of medical use described herein may provide the modified therapeutic mRNA described herein to the recipient via any appropriate route of administration.

[0120] Modified therapeutic mRNA can be administered via any preferred route of administration. Modified therapeutic mRNA, or its medical use, may utilize a route of administration selected from the group consisting of intravenous (iv); subcutaneous (sc); intramuscular (im); intradermal (id); sublingual (sl); and intranasal administration.

[0121] Those skilled in the art can determine the appropriate form of the modified therapeutic mRNA of the present invention for use in a desired administration route.

[0122] In some cases, the modified therapeutic mRNA described may be administered via a route selected from intratumor, inhalation, or intracardiac administration.

[0123] The modified therapeutic mRNAs described herein are intended for administration in effective doses. “Effective dose” is the amount, alone or in combination with additional doses, that produces the desired (therapeutic or non-therapeutic) response. The effective dose used depends, for example, on the therapeutic (or non-therapeutic) purpose, the route of administration, and the patient / subject's condition. For example, the appropriate dose of the modified therapeutic mRNA of the present invention for a given patient / subject is determined by the attending physician (or the person administering the composition) taking into account various factors known to modify the action of the modified therapeutic mRNA of the present invention, such as the severity and type of disease, body weight, sex, diet, time and route of administration, other drugs, and other relevant clinical factors. Dosage and schedule may be modified according to specific conditions, disorders, or symptoms of the patient / subject's overall condition. The effective dose may be determined by in vitro or in vivo methods.

[0124] composition In some cases, the modified therapeutic mRNAs described herein are formulated as compositions. In some cases, the modified therapeutic mRNAs described herein may be provided as part of a pharmaceutical formulation or composition. Conveniently, such formulations may be administered to human subjects (as described elsewhere herein) who require them.

[0125] A composition containing the modified therapeutic mRNA described may include a delivery agent specifically designed for the administration and delivery of the modified therapeutic mRNA to a target. For example, the composition may include modified therapeutic mRNA encapsulated in lipids, polymers, or dendrimers. In some examples, the composition may include cell-penetrating peptides that are or may not be ligated to the modified therapeutic mRNA or its translation product, or that may be ligated during use (i.e., covalently or noncovalently). For example, the modified therapeutic mRNA may be delivered to target tissue or cells by the use of lipid nanoparticles, autologous T cells, CAR-T cells, plasmid DNA, modified CD34+hHSPC, cytotoxic T lymphocytes, or T cells. For example, see Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019;27(4):710-728. doi:10.1016 / j.ymthe.2019.02.012 and Qin S, Tang X, Chen Y et al., mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. Published May 21, 2022. doi:10.1038 / s41392-022-01007-w.

[0126] The pharmaceutical formulations and compositions described herein may include, together with pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers, the modified therapeutic mRNA described herein.

[0127] The compositions and formulations may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, suitable carriers, adjuvants, and other immunostimulants such as cytokines, as well as other therapeutic agents or compounds optionally.

[0128] As used herein, “pharmaceutically acceptable” means a substance that is not biologically or otherwise undesirable, i.e., that can be administered to an individual together with selected modified therapeutic mRNA without causing any undesirable biological effects or interacting in a harmful manner with any other components of the pharmaceutical preparation containing it.

[0129] Excipients are natural or synthetic substances formulated together with the active ingredient (e.g., the modified therapeutic mRNA provided herein) and are included for the purpose of increasing the volume of the formulation or to impart therapeutic enhancement to the active ingredient in the final dosage form, such as by promoting drug absorption or solubility. Excipients can also be useful in the manufacturing process to assist in the handling of the active ingredient, such as by promoting powder flowability or non-adhesion, and to assist in vitro stability, such as preventing denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Suitable excipients are therefore readily identifiable by those skilled in the art. Examples of suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0130] An adjuvant is an agent and / or immunologist that modifies the effects of other agents in a pharmaceutical formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore readily identifiable by those skilled in the art. As merely an example, a pharmaceutical formulation may contain an adjuvant selected from the group consisting of AS03; AddaS03; AS04; MF59; AddaVax; Poly I:C; R848; Cpg; virus-like particles; virosomals; MPL; and flagellin proteins.

[0131] A diluent is a chemical used to dilute a substance. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore readily identifiable by those skilled in the art.

[0132] The carrier is harmless to the recipient at the dosage and concentration used and is compatible with the other components of the formulation. The term "carrier" means an organic or inorganic component, whether natural or synthetic, that binds to the active ingredient and facilitates its application. Pharmaceutically acceptable carriers are well known in the art. Suitable carriers are therefore readily identifiable by those skilled in the art.

[0133] Method of preparation and use Methods for producing the modified therapeutic mRNA described herein are also provided herein. The methods include a step of providing the therapeutic mRNA. The step of providing the therapeutic mRNA may be performed passively or actively. That is, the therapeutic mRNA may be provided by a third party. For example, the therapeutic mRNA may be purchased or obtained from a third party who actively produced the mRNA. In some cases, the step of providing the therapeutic mRNA may include a step of synthesizing or purifying the therapeutic mRNA. Methods for synthesizing mRNA and therapeutic mRNA are known in the art. For example, therapeutic mRNA may be synthesized using methods such as IVT, as described above. Other methods, such as those described in Qin S, Tang X, Chen Y et al., mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. Published 2022 May 21. doi:10.1038 / s41392-022-01007-w, are known to those skilled in the art.

[0134] In some cases, the nucleic acid template encoding the described therapeutic mRNA may be provided or prepared. For example, the nucleic acid template may be a DNA template suitable for transcription of the therapeutic mRNA.

[0135] Next, the provided therapeutic mRNA is analyzed to identify the frameshift nucleic acid sequence within the therapeutic mRNA nucleic acid sequence. The step of identifying the frameshift nucleic acid sequence can be carried out by any suitable method. If the mRNA sequence is unknown, the identification step may include the step of determining the mRNA sequence.

[0136] RNA sequencing may involve the use of amplification (PCR)-based methods (reverse transcription PCR (RT-PCR) and quantitative reverse transcription PCR (qRT-PCR)) or methods such as RNAseq (also known as next-generation sequencing, second-generation sequencing, or large-scale parallel sequencing). DNA sequencing methods are well known and include methods similar to those used to determine mRNA sequences.

[0137] Next-generation sequencing (NGS) (second-generation sequencing or large-scale parallel sequencing; Mardis, ER (2008)). Because many NGS technologies are available, there are slight differences in methodologies for RNA sequencing. The following is a general explanation of how RNA sequencing using NGS works. Total RNA is extracted from the target sample using a general RNA extraction method. The RNA sample can be enriched using a post-extraction method. Next, complementary DNA (cDNA) is synthesized using the extracted RNA. The cDNA is then used as a template for RNA sequencing. NGS uses a variant of sequencing through synthetic (SBS) chemistry (Fuller, CW et al., (2009). The challenges of sequencing by synthesis. Nature biotechnology, 27(11), pp. 1013-1023). Using cDNA as a template, new nucleotide fragments known as reads are synthesized base by base, and each incorporated base is recorded during sequencing (Fuller, 2009). The data output from RNA sequencing is a list of all generated reads and their sequences (Fuller, 2009 and Metzker, 2010). This data is then subjected to quality control (Patel, RK, & Jain, M. (2012). NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PloS one, 7(2), e30619).

[0138] If the therapeutic mRNA sequence is known, the identification process may include the use of sequence analysis software capable of identifying the frameshift nucleic acid sequence described above. In some cases, the mRNA sequence may be analyzed manually by referring to the frameshift nucleic acid sequences already described in the art.

[0139] After the frameshift nucleic acid sequences are identified, the therapeutic mRNA is modified to contain synonymous mutations in one or more of the identified frameshift nucleic acid sequences. mRNA sequence modification can be achieved by any known nucleic acid modification method, such as random mutagenesis, chemical mutagenesis, site-directed mutagenesis, or gene editing techniques. In some cases, the modification is introduced into a template for the production of the modified therapeutic mRNA. For example, the modification is introduced into a DNA template used for mRNA production (e.g., IVT).

[0140] Therefore, IVT templates encoding the modified therapeutic mRNA described herein are also provided herein. For example, DNA molecules encoding the modified therapeutic mRNA described herein are also provided herein.

[0141] Editing and / or mutagenesis techniques are well known in the art. Similarly, introduction can be accomplished in any manner known in the art, including gene transfer, transgenicity, or site-specific nuclease (SDN) use. In particular, modifications to DNA sequences are introduced via site-specific nucleases (SDNs). More specifically, SDNs are selected from transcription activators such as meganucleases, zinc fingers, effector nuclease systems (TALENs), or clustered and regularly arranged short palindromic sequence repeat (CRISPPR) systems. SDNs are also called “genome editing,” or genome editing with designed nucleases (GEEN). This is a type of genetic manipulation in which DNA is inserted, deleted, or replaced using a designed nuclease that creates site-specific double-strand breaks (DSBs) at desired locations in the DNA. The induced double-strand breaks are repaired through non-homologous end joining (NHEJ) or homologous recombination (HR) to obtain the targeted mutation (“editing”). In particular, SDN may include techniques such as meganucleases, zinc finger nucleases (ZFNs), effector-based nucleases (TALENs) (Feng et al., 2013, Cell Res. 23, pp. 1229-1232; Sander & Joung Nat. Biotechnol. 32, pp. 347-355, 2014), and transcription activators such as clustered, regularly arranged short palindromic sequence repeats (CRISPR-Cas) systems. Gene editing can also be achieved by SDN-2. SDN-2 is similar to SDN and also provides a small nucleotide template complementary to the cleavage region. The template contains one or more sequence modifications to the DNA, which are incorporated to create mutations in the target DNA.

[0142] After one or more synonymous mutations are introduced into mRNA or into a template for mRNA production, mRNA containing one or more synonymous mutations is produced. For example, mRNA is produced using a method such as IVT as described herein.

[0143] The modified therapeutic mRNAs provided herein may be intended for use as translation templates for in vitro or ex vivo production of proteins. The increased translational fidelity and / or efficiency provided by the introduction of the synonymous mutations described herein may provide an improved method of in vitro translation, and therefore an improved method of in vitro protein synthesis. Given the properties of the modified therapeutic mRNAs described herein (e.g., reduced out-of-frame translation, reduced immunogenicity, improved translational fidelity and / or improved translational efficiency), the modified therapeutic mRNAs may offer a number of different uses and methods.

[0144] For example, a method for reducing off-target immunogenicity to a modified therapeutic mRNA and / or its translation product is provided herein, comprising the step of preparing the modified therapeutic mRNA described herein. After preparation, the modified therapeutic mRNA is administered to a subject requiring it as described herein.

[0145] After administration, a modified therapeutic mRNA containing one or more synonymous mutations is translated within the subject. The translation product contains lower levels of out-of-frame translation products, resulting in lower overall immunogenicity compared to therapeutic mRNA that does not contain synonymous mutations.

[0146] For example, a method for reducing out-of-frame translation of modified therapeutic mRNA is provided herein. The method comprises the step of preparing modified therapeutic mRNA containing one or more synonymous mutations as described herein. After the preparation of modified therapeutic mRNA containing one or more synonymous mutations, the mRNA is translated. Including one or more synonymous mutations can result in a reduction in arrests and a reduction in the level of out-of-frame translation (and therefore, the production of out-of-frame translation products). Such a method can also increase translation fidelity. Thus, a method for increasing the translation fidelity of modified therapeutic mRNA containing one or more synonymous mutations as described herein is also provided.

[0147] In some cases, translation of modified therapeutic mRNA containing one or more synonymous mutations can be performed in vitro, ex vivo, or in vivo.

[0148] "Ex vivo" generally refers to activities performed outside of living organisms, such as experiments or measurements, in or on living tissues, preferably in an artificial environment outside of the organism, with minimal alteration to the natural state.

[0149] The use of in vitro translation has a variety of applications, including rapid identification of gene products (e.g., proteomics), identification of mutation sites through the synthesis of cleaved gene products, protein folding studies, and the incorporation of modified or non-natural amino acids for functional studies. All of these can be enhanced by the use of modified mRNA as described herein.

[0150] Methods for in vitro translation are well known in the art. For example, the method may include the use of an in vitro translation system such as rabbit reticulocyte lysate, wheat malt extract, or an E. coli-free cell line.

[0151] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in the invention. Any methods and materials similar to or equivalent to those described herein may be useful in carrying out the invention, but preferred methods and materials are described herein. Thus, the terms defined immediately below are more fully explained by referring to the specification as a whole. Furthermore, where used herein, the singular terms “a,” “an,” and “the” include plural references unless the context otherwise clearly indicates. Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxyl direction. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, for these methods, protocols, and reagents may vary depending on the context in which they are used by those skilled in the art.

[0152] Aspects of the present invention are demonstrated by the following non-limiting embodiments. [Examples]

[0153] summary In vitro transcription (IVT) mRNA is a modality that can combat human diseases, as exemplified by its use as a vaccine for SARS-CoV-2. IVT mRNA is transfected into target cells, translated into recombinant proteins, and the bioactivity or immunogenicity of the encoded proteins exerts the intended therapeutic effect.1~3 Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to reduce their innate immunity. 4,5 However, its effect on mRNA translation fidelity has not been fully investigated. Here, we demonstrate that incorporation of (N)1-methylpseudridine into mRNA results in a +1 ribosome frameshift in vitro and in vivo, and that cellular immunization against the +1 frameshifted product from BNT162b2 mRNA translation occurs in mice and humans after vaccination. The observed +1 ribosome frameshift is a result of (N)1-methylpseudridine-induced ribosome arrest during IVT mRNA translation, and the frameshift occurred at a slippery sequence. However, it is demonstrated that synonymous mutations targeting such slippery sequences provide an effective strategy to reduce the production of the frameshifted product. Overall, these data increase our understanding of how modified ribonucleotides affect mRNA translation fidelity and shed light on potential off-target effects for future such therapeutics, even though no adverse events have been reported from mRNA-based SARS-CoV-2 vaccines in humans, indicating the need for further sequence optimization.

[0154] Materials and methods ethics statement Animal experiments were authorized by the UK Home Office in accordance with Animal Science Procedures 1986 (License PP6047951) and approved by the Cambridge University Local Ethics Committee. Human sample collection and analysis were conducted in accordance with the principles of Good Clinical Practice standards and the NIHR National Bioresources approved protocol. Samples were collected with written informed consent from all research participants under the NIHR National Bioresources-Research Tissue Bank (NBR-RTB) Ethics (REC:17 / EE / 0025) and from the PITCH study. PITCH is a sub-investigation of the SIREN study, which was approved by the Berkshire Research Ethics Board, Health Research 250 Authority (IRAS ID 284460, REC reference 20 / SC / 0230), and PITCH was approved as a sub-investigation on December 2, 2020. SIREN is registered with ISRCTN (Triak ID:252ISRCTN11041050). Some participants were recruited under collaborative research protocols. In Liverpool, some participants were recruited under the "Human Immune Response to Acute Viral Infection" study (16 / NW / 0170), approved by the North West-Liverpool Central Research Ethics Committee on March 8, 2016, and amended on September 14, 2020, and May 4, 2021. In Oxford, participants were recruited under the GI Biobank Study 16 / YH / 0247, approved by the Research Ethics Committee (REC) at the Yorkshire & The Humber-Sheffield Research Ethics Committee on July 29, 2016, and amended for this purpose on June 8, 2020. The studies were conducted in accordance with all applicable ethical regulations for studies involving human participants, and in accordance with the Helsinki Declaration (2008) Principles and Harmonization International Conference (ICH) Good Clinical Practice (GCP) guidelines. Written informed consent was obtained for all participants enrolled in the studies.

[0155] Plasmid and mRNA synthesis Phusion high-fidelity DNA polymerase reagent was obtained from New England Biolabs (Ipswich, USA). In-frame WTFluc template DNA was prepared by Xbal digest of pUCK100Fluc containing an 80nt poly(A) tail. 30 Fluc+1FS and Fluc-1FS template DNAs were prepared by overlap-extension PCR of pUCK100Fluc using FlucFLAG_F, Fluc-1FS_R for Fluc-1FS NFluc, Fluc-1FS_F for Fluc-1FS CFluc, or FlucFLAG_F, Fluc+1FS_R, Fluc+1FS_F for Fluc+1FS NFluc, and Fluc_R for Fluc+1FS CFluc. The PCR products were reinserted into pUCK100 using Ncol and Nhel, and linear template DNAs were prepared by Xbal digest. In Figure 10, A206G, T187C, and T208C mRNAs, as well as all mRNAs, were transcribed from custom genes subcloned in pUC57T7 (Genscript Biotech Corporation, New Jersey, USA), and linear template DNAs were prepared by BamHI digest or Xbal digest. Fluc+1FS2 mRNA was subcloned in pUC57T7 and prepared from Fluc+1FS template DNA linearized by BamHI. * 187C / U *208C template DNA was prepared by overlap-extension PCR and reinsertion into the pUC57 site. In vitro transcription was performed using the TranscriptAid T7 high-yield transcription kit (Thermo Scientific K0441). UTP and CTP were substituted where necessary with 5-methoxyUTP, (N)1-methylpseudoUTP, or 5-methylCTP. Modified nucleotides were obtained from Trilink Biotechnologies (San Diego, USA). Transcripts were 5'-capped using the Vaccina Capping System (NEB M2080S) and purified by phenol / chloroform extraction and G50 size exclusion. Transcripts were quantified using a Nanodrop ND2000 spectrophotometer (Thermo Scientific) and stored at -80°C.

[0156] RNA gel electrophoresis The samples were heated in formamide / bromophenol blue / xylene cyanol dye at 95°C for 3 minutes, cooled on ice for 2 minutes, and dissolved on a 1% agarose formaldehyde MOPS acetate gel at 90V for 90 minutes. The gels were stained in 0.5 μg / ml ethidium bromide for 1 hour, immersed in distilled water for 1 hour, and visualized by UV transmitted illumination.

[0157] Cell culture and mRNA transfection HeLa cells were donated by the Proudfoot Laboratory at Oxford University. The cells were grown in DMEM (Gibco 41966029) supplemented with 10% FBS at 37°C and 5% CO2. Approximately 16 hours before transfection, the cells were placed in 6-well plates in a 0.2 × 10⁶ well. 6Cells were seeded at 1 / ml. Ten minutes prior to transfection, the medium was changed to OptiMEM (Gibco 31985062), and the cells were then transfected with 4 pmol of Fluc+1FS mRNA / Lipofectamine-2000 (Invitrogen 11668019). After 4 hours of transfection, OptiMEM was replaced with DMEM, and the cells were cultured for a further 4 hours before being lysed in passive lysis buffer (Promega E1941). The lysates were centrifuged (10,000 g, 5 min), and luciferase activity was determined from the supernatant using a luciferase assay system (Promega E4550) and a GloMax multi-well plate luminometer (Promega).

[0158] In vitro translation IVT mRNA was translated using the Flexi® rabbit reticulocyte lysate system with nuclease-treated RRL (Promega L4540). For simultaneous translation labeling, 0.33 μl of translation grade [ 35 S]-methionine (Hartman Analytic KSM-01) and 0.67 μl of amino acid-minus methionine (Promega L996A) were used per 15 μl reaction. Unlabeled products were prepared with 1 μl of total (unlabeled) amino acids (Promega L4461). The amount of IVT mRNA was 50 nM, and paromomycin (Sigma Aldrich P9297) was included where indicated at 100 μM. Creatine phosphate (Roche 10621714001), creatine kinase (Roche 21778721), potassium acetate (Sigma Aldrich P1190), and magnesium acetate (Sigma Aldrich M5661) were included at 10 mM, 25 μg / ml, 50 mM, and 0.5 mM, respectively. 36The reaction was incubated at 30°C for the instructed time, then transferred to ice, to which 10 μl of RNase A / T1 / benzonase was added and incubated for 10 minutes. Luciferase activity was determined using a luciferase assay system (Promega E4550) and measured using a GloMax multiwell plate luminometer (Promega). For Western blotting, 2× reduced LDS PAGE buffer was mixed with each sample and heated at 70°C for 10 minutes. The cooled samples were dissolved on NuPAGE® 12%, Bis-Tris, 1.0 mm, Mini Protein Gels (Invitrogen NP0342BOX). In the analysis in Figure 10, the samples were dissolved on Novex 10 to 20%, Trisine, 1.0 mm, Mini Protein Gels (Invitrogen EC66255BOX). The lysates were transferred to a nitrocellulose membrane and searched using anti-FLAG M2 antibody (Sigma Aldrich F1804), anti-Myc tag antibody [9E10] (AbCam Ab32), and anti-mouse-HRP antibody (Dako P0447), and detected on Clarity Western ECL substrate (Bio-Rad 1705060).

[0159] Peptide LC-MS / MS analysis IVT mRNA was translated as described above. After RNA digestion, the translation product was immunoprecipitated overnight at 4°C using anti-FLAG magnetic agarose beads (Pierce). The beads were washed twice in PBS and once in water, eluted in LDS PAGE buffer, and dissolved on NuPAGE® 4-12%, Bis-Tris, 1.5 mm, Mini Protein Gel (NP0335BOX). The gel was stained with Coomassie dye, and the range between approximately 60 kDa and 75 kDa (Precision Plus Protein® All Blue Prestained Protein Standard, Bio-rad) was excised. 37The samples were processed for mass spectrometry analysis as described above. Briefly, the excised gel sections were cut into 1 mm pieces and placed in 1.5 ml microtubes. Coomassie staining was removed by incubation with a mixture of 25 mM ammonium bicarbonate and acetonitrile (2:1) and 25 mM ammonium bicarbonate. Each 15-minute incubation at 37°C was repeated until the gel pieces were completely discolored. A first incubation was performed at 60°C for 60 minutes with fresh 10 mM final concentration dithiothreitol in 25 mM ammonium bicarbonate, then the solution was changed to 60 mM final concentration iodoacetamide in 25 mM ammonium bicarbonate, and the cysteine ​​reduction and alkylation were performed by incubation for an additional 45 minutes at room temperature in the dark. After dehydrating the gel pieces with acetonitrile, trypsin solution was added (10 ng / μL in 25 mM ammonium bicarbonate) until the gel pieces were completely recovered. Digestion was performed at 37°C for 16 hours. Trypsin inactivation was performed by adding formic acid to a final concentration of 1% (v / v). Subsequently, the peptides were extracted by sequential incubation in water vs. acetonitrile vs. formic acid (50:49:1% (v / v)) and (80:19:1). The extracted peptides were pooled, completely dried, and resuspended in water vs. acetonitrile (97:3% (v / v)) with 0.1% (v / v) TFA for mass spectral analysis.

[0160] Mass spectroscopy Ingel digestion was analyzed using an Ultimate 3000 RSLC® nanosystem (Thermo Scientific, Hemel Hempstead) coupled to an Orbitrap Eclipse® mass spectrometer (Thermo Scientific). Samples were packed onto a trap column (Thermo Scientific, PepMap100, C18, 300 μm x 5 mm) using partial loop injection at a flow rate of 15 μL / min for 3 minutes with 0.1% (v / v) FA in 3% acetonitrile. The peptides were separated on an analytical column (Easy-Spray C18 7μm x 500mm 2μm column) at a flow rate of 300 nL / min. The gradient was measured from 97% A (0.1% formic acid) 3% B (80% acetonitrile, 0.1% formic acid) to 25% B over 50 minutes, then to 40% B over another 6 minutes, then to 90% B over another 2 minutes, remaining at 90% for 12 minutes, after which the percentage of B was reduced to 3%, and the column was re-equilibriumized for 15 minutes before the next injection. Data were obtained using two FAIMS cv's (-50V, -70V). For each FAIMS experiment (maximum cycle time of 1.5 seconds per experiment), data were obtained in data-dependent mode. MS1 consisted of a 120,000 resolution full-scan MS scan (AGC set to 100% (4e5 ions), maximum packing time 50 minutes) using the mass range 380–1500 m / z. The intensity MS2 trigger threshold was set to 5.0e3, and the experiment used a 40-second dynamic exclusion window to avoid repeated selection of peptides representing MSMS. MS / MS was performed on an orbitrap using 30,000 resolutions (AGC set to 100% (5e4 ions), maximum packing time 54 minutes). 32% HCD collision energy was used to break down peptides, and an isolation window of 1.2 was used.

[0161] Proteome Discoverer v2.5 Analysis Raw data was submitted and processed using Proteome Discoverer v2.5 (Thermo Fisher Scientific). The raw files were submitted for database searches using Proteome Discoverer with SequestHF against a Homo sapiens database containing human protein sequences from UniProt / Swiss-Prot, including firefly luciferase and common contaminant proteins (several human keratins, BSA, and porcine trypsin). Spectral identification was performed using the following parameters: MS precision, 10 p.pm; MS / MS precision, 0.02 Da; up to two false cleavage sites allowed; cysteine ​​carbamide methylation; and methionine oxidation as a variable modification. An interactive workflow was used during the processing steps. After the initial Sequest HT search, an inferior scoring node was used, and spectra with lower confidence levels were resubmitted for a second Sequest HT search using additional dynamic corrections (N,Q deamide; N-terminal pyroglutamate; methionine elimination and acetylation). Peptides were assigned to their respective reading frames from Fluc+1FS mRNA by testing. A percolator node was used to estimate the false discovery rate, and only rank 1 peptide identifications with high confidence (FDR < 1%) were accepted.

[0162] RNA-seq analysis RNA-seq libraries were prepared from 1 μg of IVT mRNA using the NextFlex Rapid Directional RNA-seq Kit 2.0 (Perkin Elmer) according to the manufacturer's protocol. The libraries were amplified by 6 PCR cycles and purified by PAGE. Sequencing was performed using Illumina MiSeq (1 x 150 cycles V3) at the Biochemical DNA Sequencing Facility, University of Cambridge. Reads were aligned with STAR 2.7.4a. 38Insertions and deletions per reference nucleotide were filtered for partial alignment and located from high-quality reads (QC score > 35) normalized to read depth. Insertion / deletion plots show the mean mutation frequency for n=3 repeat RNA-seq experiments.

[0163] SDS-PAGE Autoradiography IVT mRNA was translated in nuclease-treated RRL (Promega), and the product was co-translationally labeled as described above for 30 minutes. For peptidyl-tRNA analysis, two sets of samples were coated, to which 2.5 μl of RNase A / T1 / benzonase or water was added and incubated for a further 10 minutes to obtain RNase+ / - samples. 2X LDS PAGE buffer was mixed with each sample and heated at 70°C for 10 minutes. The cooled samples were lysed on NuPAGE® 12%, Bis-Tris, 1.0 mm, Mini Protein Gels (Invitrogen NP0342BOX). The lysed gels were fixed in 10% methanol / acetic acid for 45 minutes and dried at 80°C for 2 hours using a Fisher gel drying system. Images were obtained by autoradiography using a Typhoon FLA 9000 and storage phosphor screen (GE Healthcare).

[0164] Built-in [ 35 S]-methionine quantification IVT mRNA was translated in nuclease-treated RRL (Promega), and the product was co-translationally labeled for 2 hours as described above. 35S]-Met incorporation was assayed according to the manufacturer's protocol. Briefly, after RNA digestion, the reaction product was incubated in 1M NaOH for 10 minutes. Polypeptides were precipitated on 5% TCA and collected on a Whatman fiberglass filter, which was washed three times with 5% TCA and once with acetone. The dry filters were immersed in 2 ml of EcoScint liquid scintillation cocktail (National Diagnostics) and counted on a Tri-Carb 4910 TR liquid scintillation counter (PerkinElmer). Incorporated [ 35 The S]-Met compound was determined for each reaction from the cpm of precipitated polypeptide per cpm of unwashed filter (total cpm).

[0165] Mouse immunization C57BL / 6J mice (wild type, WT) were purchased from Charles Rivers Laboratory. Mice were either intramuscularly injected with two doses of 10 μg BNT162b2 or left untreated. Spleens were obtained 8 days post-vaccination, and cell suspensions were prepared. Briefly, spleens were crushed with a syringe plunger and filtered through a 70 μm cell strainer. Red blood cells were lysed in RBC lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) prior to counting and cryopreservation for the ELISpot assay.

[0166] IFNγ ELISpot The human IFN-γ ELISpot assay was performed using the human IFN-γ ELISpot PLUS kit (ALP) (MabTech 3420-4APT) as previously described. 39 The in-frame spike peptide pool and the +1FS spike peptide pool were obtained from Mimotopes (Melbourne, Australia) (SEQ ID NOs. 10-290). The spike S1+S2 peptide pool was as previously described. 39In short, cryopreserved PBMCs were thawed in RPMI1640 medium supplemented with 1% (v / v) penicillin / streptomycin (Sigma) containing 0.01% (v / v) benzonase nuclease (Merck). The PBMCs were washed and then incubated in RPMI1640 medium, 10% (v / v) human serum, and 1% (v / v) penicillin / streptomycin at 37°C and 5% CO2 for 1–2 hours. Pre-coated IFNγ ELISpot 96-well plates (MabTech 3420-4APT-2) were washed three times with PBS and blocked in RPMI1640 medium / 10% (v / v) human AB serum / 1% (v / v) penicillin / streptomycin for 45 minutes. The duplicated peptide pool was seeded at 4 μg / ml, 50 μL per well, and DMSO (Sigma) was used as a negative control at an equivalent concentration to the peptide. 200,000 cells were added to 50 μl and incubated for 18–24 hours. The cells were discarded, and the plates were washed with PBS 0.05% (v / v) Tween (Sigma) and incubated with IFNγ detection antibody (clone 7-B6-1, 1 μg / ml) at room temperature for 2–4 hours. The washed plates were then incubated with streptavidin alkaline phosphatase antibody (1 μg / ml) for 1–2 hours. The plates were then washed, and chromogenic development was performed using a one-step NBT / BCIP substrate solution. 50 μl of filtered NBT / BCIP was added to each well at room temperature for 5 minutes, after which development was stopped with cold water. The plates were dried at room temperature for approximately 48 hours. Spots were quantified using the AID iSpot spectral EliSpot reader (software version 7.0, Autoimmun Diagnostika, Strassberg). The mean spot count in the background well was subtracted from that in the test well, and the value was expressed as spot-forming units (SFU) per million cells. The mouse IFNγ ELISpot assay was performed using lyophilized splenocytes thawed as described above and incubated in RPMI1640 medium / 10% FBS only.Peptide stimulation and downstream processing were performed using a pre-coated mouse IFN-γELISpot PLUS kit (ALP) (MabTech 3321-4APT-2) as described above.

[0167] Data Availability Statement Mass spectrometry data is provided in Table 1 and is available in the PRIDE Partner Repository. 40 The data was deposited with the ProteomeXchange Consortium via Accession PXD039483. Reviewer login details are: username: reviewer_pxd039483@ebi.ac.uk and password: JAQVmZq3. RNA-seq reads and processed files are available in NCBI Gene Expression Omnibus (Accession GSE223044). Reviewer token: ajqbgkiavtghlkt. Additional data is available from the corresponding author upon reasonable request.

[0168] Mass spectrometry data have been deposited under MassIVE ID MSV000093074. RNA-seq reads and processed files are available in NCBI Gene Expression Omnibus (accession GSE223044). Additional data are available from figshare (https: / / doi.org / 10.6084 / m9.figshare.24271744). The following acceptance numbers were used for mass spectral analysis: UP000001811 and P08659 (UniProt). Source data is available at https: / / www.nature.com / articles / s41586-023-06800-3#Sec23.

[0169] Code Availability Statement The script for handling the alignment is on GitHub 41 It is available from.

[0170] (Method references) TIFF2026514077000002.tif115156

[0171] Results and Discussion A key characteristic of therapeutic IVT mRNA is that it contains modified ribonucleotides, which have been shown to reduce innate immunogenicity and further increase mRNA stability. Both of these characteristics are advantageous for therapeutic use. 1,2 For example, clinically approved SARS-CoV-2 mRNA vaccines incorporate (N)1-methylpseudridine (1-methylψ), which has been shown to reduce the innate immunogenicity of IVT mRNA. 3~5 Some modified ribonucleotides, such as 5-methylcytidine (5-methyl C), are naturally occurring post-transcriptional mRNA modifiers in eukaryotes, while other modified ribonucleotides, such as 1-methylψ, are not. 6~10 .

[0172] The effects of 5-methoxyuridine (5-methoxy U), 5-methyl C, and 1-methyl ψ on IVT mRNA translation were investigated. 5-methoxy U, 5-methyl C, and 1-methyl ψ have been utilized in IVT mRNA for attempts to enhance recombinant protein synthesis in vitro and for preclinical proof-of-concept studies of IVT mRNA-based therapies. 11,12 As mentioned, 1-methylψ is a ribonucleotide that has been incorporated into approved IVT mRNA-based SARS-CoV-2 vaccines, as well as mRNA-based human vaccines and therapies currently under development. 4,13,14 .

[0173] Despite their widespread use, surprisingly little is known about how ribonucleotide modifications affect protein synthesis, particularly regarding the translation of therapeutic IVT mRNA. This example investigates how modified ribonucleotides affect the fidelity of mRNA translation for several reasons. Certain ribonucleotide modifications can recode mRNA sequences, such as inosine. 15 While 5-methyl C has previously been shown to increase misleading during mRNA translation in prokaryotes, its effect on eukaryotic mRNA translation fidelity has not been investigated. 16 The effect of 5-methoxyU on translation fidelity has not been investigated. Pseudouridine (ψ) is known to increase mRNA stop codon misreading in eukaryotes and may affect misreading during prokaryotic mRNA translation. 16~18 While 1-methylψ is not thought to affect codon misleading, it has been shown to influence protein synthesis rate and ribosome density on mRNA, suggesting a direct effect on mRNA translation. 19,20 .

[0174] It is currently unclear which modified ribonucleotides affect mRNA translation fidelity, and most existing studies are limited to understanding the frequency of misreading at a given codon. mRNA codon misreading is only one type of post-transcriptional mechanism that can alter polypeptide sequences. To date, no studies have investigated the fundamental question of whether modified ribonucleotides can influence the maintenance of the correct reading frame during translation of synthetic transcripts. Understanding these methods is of great importance to increase our knowledge of protein synthesis with modified mRNA in general, but it is also essential for the robust design and evaluation of novel mRNA-based therapeutics that utilize modified ribonucleotides within very different RNA sequences or therapeutic contexts.

[0175] To investigate how ribonucleotide modifications affect reading frame maintenance during mRNA translation, we designed and synthesized IVT mRNAs reporting out-of-frame protein synthesis (Figure 1a). These mRNAs encode the N-terminal segment (NFluc) and the immediately downstream complementary C-terminal segment (CFluc) of firefly luciferase. CFluc is encoded in the -1 reading frame in Fluc-1FS and in the 1+ reading frame in Fluc+1FS. The Fluc-1FS and Fluc+1FS mRNAs are designed to produce catalytically inactive (cleaved) NFluc when translated normally. However, if the ribosome moves out-of-frame during translation, it is possible to produce an extended polypeptide containing residues from both in-frame NFluc and out-of-frame CFluc, and this polypeptide has increased catalytic activity.

[0176] Unmodified Fluc-1FS and Fluc+1FS mRNAs were synthesized, and these mRNAs contained canonical ribonucleotides, which were translated in vitro. It was confirmed that Fluc-1FS and Fluc+1FS mRNAs produced catalytically inactive NFluc (Figure 2). In comparison, unmodified WTFluc mRNA, while containing the complete in-frame firefly luciferase coding sequence, produced the expected active protein (Figure 2). Next, mRNAs containing 5-methoxy U, 5-methyl C, 1-methyl ψ, 5-methoxy U and 5-methyl C, or 1-methyl ψ and 5-methyl C were synthesized and translated. Translation of WTFluc mRNA was not significantly affected by 1-methyl ψ or 5-methyl variants alone, but was reduced by the incorporation of both ribonucleotides into a single transcript (Figure 1b). Incorporation of 5-methoxy U alone, or in combination with 5-methyl C, significantly reduced the translation of WTFluc mRNA (Figure 1b). Ribonucleotide modification did not appear to affect the ribosome-1 frameshift (Figure 1c). However, the incorporation of 1-methylψ significantly increased the ribosome-+1 frameshift (Figure 1d). HeLa cells transfected with 1-methylψ Fluc+1FS mRNA repeated the results from in vitro translation (Figure 1e). Based on these findings, the inventors concluded that IVT mRNA containing 1-methylψ or 5-methylC exhibits translation efficiency similar to unmodified mRNA, but 1-methylψ significantly increases the ribosome-+1 frameshift during mRNA translation.

[0177] The significant increase in ribosome +1 frameshift observed during 1-methylψ mRNA translation was intriguing, and it was inferred that a better understanding of the translation product would complement reporter assay data and help explain how the +1 frameshifted product originates. To address these aspects, polypeptides produced during IVT mRNA translation were explored by Western blotting. Translation of unmodified Fluc+1FS mRNA produced the expected in-frame cleaved product, which was also true for 5-methylC mRNA (Figure 1f). Translation of 1-methylψ mRNA produced the expected in-frame product, but with a higher molecular weight and two additional bands (Figure 1f). These products were proposed to be +1 frameshifted polypeptides. It was also confirmed that 1-methylψ / 5-methylC-, 5-methoxyU-, and 5-methoxyU / 5-methylC-mRNAs are relatively poor mRNA templates for protein synthesis (Figure 1f).

[0178] 1-methylψ is also used in clinically approved SARS-CoV-2 mRNA vaccines. 3,4 Since 1-methylψ increased the +1 ribosome frameshift during translation in vitro, it was investigated whether this occurs in vivo for BNT162b2, a SARS-CoV-2 mRNA vaccine containing 1-methylψ. It was hypothesized that the +1 ribosome frameshift during recombinant antigen mRNA translation leads to the presentation of the +1 frameshifted product to T cells, which can induce an off-target cellular immune response (Figure 3a). Antigen presentation from mistranslation of endogenous tumor mRNA has been shown to occur, for example, in vivo. 21To address this possibility, mice were vaccinated with BNT162b2, and the mouse T cell response to the in-frame SARS-CoV-2 spike protein was quantified, with the +1 frameshifted product predicted by the interferon-gamma ELISpot assay. The response to the +1 frameshifted spike peptide was significantly increased in vaccinated mice compared to untreated mice (Figure 3b). These data suggest that the +1 frameshifted product encoded by BNT162b2 spike mRNA is a T cell antigen for inbred mice, and that off-target immunity to this antigen can be detected following vaccination.

[0179] The interferon-gamma ELISpot response was compared to the predicted +1 frameshifted SARS-CoV-2 spike protein product in 22 individuals vaccinated with BNT162b2, and these responses were compared to those in 19 individuals vaccinated with ChAdOx1 nCoV-19, another SARS-CoV-2 vaccine that shares the same spike protein antigen as BNT162b2 but is not translated from 1-methylψ mRNA. 22 A significantly higher interferon-gamma response to the +1 frameshifted antigen was detected in the BNT162b2 vaccine group compared to ChAdOx1 nCoV-19 (Figure 3C). During SARS-CoV-2 virus replication, a programmed -1 ribosome frameshift occurred spontaneously during ORF1a / b translation. 23 These data cannot be the result of natural SARS-CoV-2 infection for the following reasons: Firstly, frameshift activity is not known to occur during SARS-CoV-2 spike subgenome mRNA translation (which in itself is a major discovery). Secondly, the -1 frameshift (and not the +1 frameshift) is limited to a single programmed site in ORF1a / b. 23Thirdly, the +1 frameshifted peptide is expected to be derived from the BNT162b2 mRNA sequence, not from the wild-type virus's S gene sequence. Instead, these data suggest that human vaccination with 1-methylψ mRNA can induce cellular immunity against peptide antigens produced by the +1 ribosome flake shift.

[0180] To provide further mechanical insight into the +1 ribosome frameshift during translation of 1-methylψ mRNA and to identify potential frameshift sites / sequences, large-scale in vitro translation of 1-methylψ Fluc+1FS mRNA was performed, the major putative +1 frameshifted polypeptide was purified, and liquid chromatography-serial mass spectrometry was performed. From this single polypeptide, six in-frame peptides and nine peptides derived from the mRNA+1 frame were identified (Figure 4a and Table 1). All in-frame peptides were located in the N-terminal region, and the +1 frameshifted peptides were located downstream (Figure 4a). These data indicated that the extended polypeptide is actually a chimeric polypeptide consisting of an in-frame N-terminal residue and a +1 frameshifted C-terminal residue.

[0181] Errors during protein synthesis, including frameshifts, may be the result of DNA mutations or transcription errors. 24Therefore, faithful translation of inaccurate mRNA sequences can produce inaccurate proteins. In vitro transcripts are presumed to be accurate RNA copies of template DNA, and their accuracy can be assessed by the fidelity of the RNA polymerase used. However, using canonical substrates rNTPs instead of modified nucleotides may increase transcription errors. To address this possibility, high-throughput RNA sequencing was performed on unmodified and 1-methylψFluc+1FS mRNA, and nucleotide insertions and deletions in each population of IVT mRNA were quantified. The nucleotide deletion profiles for each mRNA were very similar (Figures 4b and 5), as were the nucleotide insertions (Figure 4c), suggesting little site-specific difference. The overall frequencies of insertions and deletions were low, with no significant difference between unmodified and 1-methylψ mRNA (Table 2), which is supported by recent findings. 25 In summary, it was concluded that the frameshifted product of 1-methylψ mRNA translation was not due to transcriptional errors, but rather to a post-transcriptional mechanism: the true ribosome +1 frameshift.

[0182] Ribosome frameshift is a well-established phenomenon that occurs during the translation of many naturally occurring mRNAs. 24 Ribosome arrest is associated with several such mechanisms, and it was questioned whether the presence of 1-methylψ in IVT mRNA leads to ribosome arrest during translation. 26-29 To do this, obviously intermediate peptidyl-tRNAs produced during translation of unmodified or 1-methylψWT Fluc mRNA were assayed, which are a result of ribosome arrest. 30 Translation of unmodified mRNA did not clearly produce observable peptidyl-tRNA, but several stable peptidyl-tRNA intermediates were detected during the translation of 1-methylψ mRNA (Figure 6a). Translation elongation of 1-methylψ mRNA was also slower than that of unmodified mRNA (Figure 4a), which is supported by previous findings. 20All reactions occurred within 30 minutes, and the translation of 1-methylψ-containing mRNA produced fewer full-length proteins, suggesting a slower elongation rate compared to unmodified mRNA, and a higher proportion of premature polypeptide products. These data strongly suggest ribosome arrest during translation of 1-methylψ-containing mRNA.

[0183] It was unclear whether 1-methylψ affected mRNA sequencing speed or another process during elongation. Similar to previous findings regarding "hungry" codons at the +1 frameshift site during translation of naturally occurring mRNA, it was hypothesized that slower sequencing of 1-methylψ codons during translation elongation could lead to ribosome arrest. 21,28 The molecular mechanism of ribosome arrest during 1-methylψ mRNA translation was investigated in detail using the aminoglycoside paromomycin. In very brief terms, during mRNA sequencing, congeneral aminoacyl-tRNA anticodon:codon interactions induce local conformational changes in 18S rRNA (in eukaryotes), followed by the formation of new peptide bonds, ribosomal subunit rotation, and subsequent ribosomal conformational changes, elongation factor 2 binding, and transposition to the next codon complete the elongation cycle. 31 Paromomycin binds to helix 44 of 18S rRNA in elongated ribosomes, altering its conformation at the decoding site, which inhibits translation, but also allows productive binding of close- and non-congenital aminoacyl-tRNAs to the 80S ribosome A site. 32 In doing so, paromomycin increases the misuptake of amino acids into the elongated polypeptide. 33It was hypothesized that ribosome arrest during 1-methylψ mRNA translation could be reduced by paromomycin if the slow decoding was due to altered aminoacyl-tRNA binding kinetics, because paromomycin-bound ribosomes can take up additional near- and non-homogeneous aminoacyl-tRNAs, effectively increasing the substrate aminoacyl-tRNA pool at the arrest site. Translation of 1-methylψ mRNA was slower than that of unmodified mRNA, and the proportion of premature polypeptide products was higher (Figures 6a and 6b). However, during 1-methylψ mRNA translation, polypeptide elongation was actually improved by the addition of paromomycin, and paromomycin was only repressive for unmodified mRNA translation (Figure 6b). Taken together, these data suggest that translation of 1-methylψ mRNA is prone to ribosome arrest, likely caused by altered aminoacyl-tRNA binding, and that this arrest can be rescued by increasing the incorporation of near- and non-homogeneous amino acids into the elongating polypeptide.

[0184] While there is no evidence that the frameshift product produced in humans from BNT162b2 vaccination (Figure 3) is associated with adverse events, it is important for the future use of mRNA technology that mRNA sequence designs be modified to accommodate reduced ribosome frameshift events. This is because this could limit its future use for applications requiring frequent administration, such as in vivo hormone production. In such cases, the major in-frame product is unlikely to induce an adaptive immune response, but the presentation of the +1 frameshift product may activate T cells that target host cells expressing recombinant hormones. It was inferred that if the 1+ ribosome frameshift site / sequence could be identified, it would be possible to modify the sequence to reduce such effects. As proof in principle, a reporter IVT mRNA system was used. LC-MS / MS analysis showed that translation of 1-methylψ mRNA resulted in the synthesis of the +1 frameshift product within the coding sequence region between the detected in-frame residue and the downstream +1 frameshifted residue (Figure 4a). The RNA sequences corresponding to this region were searched for determinants of ribosome frameshift based on a published mechanism, and three potential ribosome slippery sequences were identified from this mechanism (Figure 6c), all of which had the potential to be decoded by the same aminoacyl-tRNA at the in-frame codon or the nearest +1 frame codon. These were therefore hypothesized to function as sites for +1 ribosome frameshift (Figure 6c). These sites in 1-methylψFluc+1FS mRNA were synonymously mutated so that the in-frame amino acid remained unchanged, but the nearest +1 frame codon was mutated to a non-homogeneous amino acid, thus disrupting the ribosome slippery sequence, and therapeutic mRNA was translated to evaluate the contribution of each site to +1 ribosome frameshift (Figure 6c). Luciferase activity resulting from translation of A206G mRNA (slippery site A mutant) was not different from control levels, indicating that this site does not affect +1 ribosome frameshift (Figure 6d).However, both T187C mRNA and T208C mRNA (slippery site B and C mutants, respectively) significantly reduced the +1 ribosome frameshift (Figure 6d). Importantly, the translation efficiency of each mRNA was equal, suggesting that no mutations had a detrimental effect on mRNA translation, but only the +1 ribosome frameshift activity did (Figure 6e). Translation of mRNAs containing both T187C and T208C mutations (mutations at slippery sites B and C) did not produce any detectable frameshift activity (Figure 9). In summary, these data suggest that (N)1-methylpseudridine in limited mRNA sequences triggers the +1 ribosome frameshift, but that this problem can be mitigated with appropriate mRNA sequence design.

[0185] Mouse and human vaccination with BNT162b2 mRNA resulted in increased cellular immunogenicity against the +1 framepeptide (Figure 3). However, it remained unclear which ribosomal slippery sites in the BNT162b2 RNA sequence contributed to the +1 ribosomal frameshift during BNT162b2 mRNA translation, and how local RNA sequence contexts affected the efficiency of the ribosomal +1 frameshift. We identified six ribosomal slippery sites (slippery sites 1-6) in the BNT162b2 RNA sequence, as well as another element (sequence X) that we hypothesized could support the ribosomal +1 frameshift under certain conditions. The core motifs of slippery sites 1-6 were identical to slippery site B or slippery site C in Fluc+1FS mRNA, but (as expected) their local RNA sequence contexts were different.

[0186] The inventors individually assayed sequence X for BNT162b2 slippery sites 1-6 and +1 ribosome frameshift using a series of double-tagged reporter mRNAs (SpikeFS1-SpikeFS6 mRNA and SpikeSX mRNAs). Each mRNA coding sequence was constructed with an N-terminal 3xMyc-tag in-frame up to approximately 500-900 nucleotide sections of the BNT162b2 mRNA coding sequence containing the relevant ribosome slippery site or sequence X (Figure 10a). The 3xFLAG-tag is a 10-25 nucleotide inserted downstream of each ribosome slippery site or sequence X and is coded in the mRNA +1 frame (Figure 10a). Normal translation of each mRNA is expected to produce a polypeptide consisting of the N-terminal 3xMyc-tag and the SARS-CoV-2 spike protein portion. However, a ribosome +1 frameshift in the upstream region is predicted to produce a chimeric polypeptide containing a short +1 frame mutation including the N-terminal 3xMyc- tag, the SARS-CoV-2 spike protein portion, and the C-terminal 3xFLAG. Candidate ribosome slippery sites for each mRNA are shown in Figure 10b.

[0187] The inventors synthesized canonical ribonucleotides (unmodified) or the above mRNAs containing 1-methylψ, and assayed their translation products by Western blotting. By assaying the abundance of anti-FLAG reactive products, the inventors confidently identified +1 flame products from the translation of 1-methylpseudolylated SpikeFS1, SpikeSX, SpikeFS2, SpikeFS3, and SpikeFS6 mRNA (Figure 10c). These +1 flame products were not detected from the translation of unmodified mRNA equivalents. Interestingly, the translation of 1-methylψSpikeFS4 and SpikeFS5 did not produce significant levels of +1 flame products.

[0188] The inventors hypothesized that synonymous mutations in the BNT162b2 ribosome slippery sequence could be used to reduce or suppress the +1 ribosome frameshift, as previously demonstrated by synonymous mutations in slippery sites B and C in Fluc+1FS mRNA. To investigate this possibility, the inventors synonymously mutated candidate ribosome slippery sites in 1-methylpseudolylated SpikeFS1, SpikeFS2, SpikeFS3, SpikeFS6, and SpikeSX such that the most recent +1 frame codon-deciphering aminoacyl-tRNA is no longer a cognate of the major frame codon, i.e., T187C and T208C (U * 187C and U * As previously performed with 208C) mRNA, the ribosome slippery site was disrupted but the amino acid sequence of the polypeptide encoded in the mRNA major frame was not altered. Next, these mRNAs (AntiFS1, AntiFS2, AntiFS3, AntiFS6, and AntiSX) were translated and assayed for in-frame and +1-frame translation as before. As previously revealed, +1-frame products were detected by translation of 1-methylψ SpikeFS1, SpikeFS2, SpikeFS3, SpikeFS6, and SpikeSX mRNA (Figure 10d). However, translation of 1-methylψAntiFS1, AntiFS2, AntiFS3, AntiFS6, and AntiSX mRNA resulted in significantly reduced synthesis of +1-frame products, while supporting in-frame mRNA translation (Figure 10d). In summary, these data indicate that it is possible to search mRNA sequences for potential sites of ribosome +1 frameshift, that ribosome +1 frameshift occurs during translation of the vast majority of mRNAs containing these predicted sites, and that targeted mutations in these sites can significantly reduce ribosome +1 frameshift during mRNA translation.

[0189] conclusion It has been shown that 1-methylψ is a modified ribonucleotide that significantly increases the +1 ribosome frameshift during mRNA translation, and that cellular immunity to the +1 frameshifted product may occur following vaccination with mRNA containing 1-methylψ. To the best of our knowledge, this is the first report that mRNA modification affects ribosome frameshift. Other ribonucleotide modification strategies, such as the incorporation of 5-methoxyU, significantly reduce the translation efficiency of IVT mRNA, which may limit clinical translation. It has been shown that IVT mRNA contains very few nucleotide insertions / deletions, and this is not altered by 1-methylψ incorporation. The +1 ribosome frameshift during translation of 1-methylψ mRNA is affected by ribosome slippery sequences. Translation of mRNA containing 1-methylψ results in ribosome arrest. The arrest is likely caused by altered aminoacyl-tRNA binding, which explains why the +1 ribosome frameshift cannot occur during translation of unmodified mRNA, and both ribosome arrest and ribosome slippery sequences are important for productive +1 ribosome frameshift. The mechanical data presented herein are supported by previous findings on ribosome frameshift during translation of naturally occurring mRNA, which relate ribosome arrest and ribosome slippery sequences to the +1 frameshift. 21,26-29,34,35 The inventors have also revealed that targeted mutations in ribosome slippery sites can reduce the +1 ribosome frameshift while preserving in-frame mRNA translation. These novel findings are particularly important for a fundamental understanding of how ribonucleotide modifications affect mRNA translation and for designing and optimizing future mRNA-based therapeutics to avoid mistranslation events that can reduce efficiency and / or increase toxicity.

[0190] (Example 2) A second method to reduce out-of-frame translation events involved identifying and removing other sequences that supported out-of-frame protein synthesis while preserving the otherwise encoded protein product. This was achieved by disrupting another reading frame with a premature stop codon (PTC) within its own reading frame. The protein-coding sequence was designed so that it would not encounter a PTC unless out-of-frame protein synthesis was initiated. The PTC prevented peptide elongation in the other reading frame by terminating translation, reducing the +1 ribosome frameshift level to an undetectable level (Figure 8). The synthesis and presentation of out-of-frame peptides are a source of off-target cellular immunity (Figure 3) and a potential source of toxicity in future mRNA-based drugs. PTCs may further act to destabilize mRNA undergoing out-of-frame protein synthesis.

[0191] method T208C (SEQ ID NO: 8) and AntiFS (SEQ ID NO: 9) were transcribed using the TranscriptAid T7 high-yield transcription kit (Thermo Scientific), 5'-capped using the Vaccinia Capping System (NEB M2080S), and purified. Templates were prepared by BamHI digestion of custom plasmids (Genscript Biotech Corporation). The gene sequences for T208C and AntiFS are provided below. To assay the +1 ribosome frameshift, 50 nM IVT mRNA was translated with the previously described

[13] nuclease-treated RRL (Promega). Luciferase activity after 2 hours was assayed using the Luciferase Assay System (Promega).

[0192] (References for Example 2) TIFF2026514077000003.tif192157TIFF2026514077000004.tif93157

[0193] The reader's interest is directed towards all papers and documents filed concurrently with or prior to this specification relating to this application and made available for public inspection of this specification, the contents of all such papers and documents are incorporated herein by reference.

[0194] All of the features disclosed herein (including any accompanying claims, abstracts, and drawings) and / or all of the steps of any method or process disclosed herein may be combined in any combination except any combination in which at least some of the features and / or steps are mutually exclusive.

[0195] Each feature disclosed herein (including any accompanying claims, abstract, and drawings) may be replaced by another feature serving the same, equivalent, or similar purpose, unless expressly specified otherwise. Thus, unless expressly specified otherwise, each disclosed feature is only one example from an equivalent or similar feature in the general series.

[0196] The present invention is not limited to any of the details of the embodiments described herein. The present invention extends to any novel feature or any novel combination of features disclosed herein (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination of any step of any method or process so so disclosed.

[0197] (References) TIFF2026514077000005.tif212157TIFF2026514077000006.tif213157TIFF2026514077000007.tif182157

[0198] array

[0199] [Table 1A]

[0200] Table 1B

[0201]

Table 1C

[0202]

Table 1D

[0203] Table 1E

[0204] Table 1F

[0205]

Table 1G

[0206] Table 1H

[0207]

Table 1I

[0208]

Table 1J

[0209] Table 1K

[0210]

Table 1L

[0211] Table 2

[0212] Table 3

Claims

1. A modified therapeutic mRNA comprising at least one frameshift nucleic acid sequence that increases the frequency of out-of-frame translation of the mRNA, wherein the at least one frameshift nucleic acid sequence comprises at least one synonymous mutation for reducing the frequency of out-of-frame translation of the modified therapeutic mRNA.

2. A modified therapeutic mRNA comprising at least one ribosome slippery sequence that increases the frequency of out-of-frame translation of the mRNA, wherein the at least one ribosome slippery sequence comprises at least one synonymous mutation for reducing the frequency of out-of-frame translation of the modified therapeutic mRNA.

3. A modified therapeutic mRNA comprising at least one different reading frame sequence encoding a different translation product from the in-frame translation product of the modified therapeutic mRNA, wherein the modified therapeutic mRNA comprises at least one synonymous mutation for introducing a stop codon (PTC) into the at least one different reading frame sequence.

4. A method for producing modified therapeutic mRNA in which the frequency of out-of-frame translation is reduced, a. A step of providing therapeutic mRNA or nucleic acid encoding therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of therapeutic mRNA or the nucleic acid encoding therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the modified frameshift sequence comprises at least one synonymous mutation; A method that includes this.

5. A method for reducing off-target immunogenicity to therapeutic mRNA and / or its translation product, a. A step of providing therapeutic mRNA or nucleic acid encoding the therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of the therapeutic mRNA or the nucleic acid encoding the therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the modified at least one frameshift sequence comprises at least one synonymous mutation; and d. The process of administering the modified therapeutic mRNA to a target that requires it; A method that includes this.

6. A method for reducing out-of-frame translation of therapeutic mRNA and / or increasing the translational fidelity of therapeutic mRNA, a. A step of providing therapeutic mRNA or nucleic acid encoding the therapeutic mRNA; b. Identifying at least one frameshift sequence within the nucleic acid sequence of the therapeutic mRNA or the nucleic acid encoding the therapeutic mRNA; c. A step of producing a modified therapeutic mRNA comprising at least one modified frameshift sequence, wherein the modified at least one frameshift sequence comprises at least one synonymous mutation; and d. The step of translating the modified therapeutic mRNA; A method that includes this.

7. The modified therapeutic mRNA according to any one of claims 1 to 3, or the method according to any one of claims 4 to 6, wherein the at least one synonymous mutation increases the translation fidelity of the modified therapeutic mRNA when used.

8. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7, wherein the out-of-frame translation includes a +1 frame shift, a +2 frame shift, a -1 frame shift, or a -2 frame shift, or the method according to any one of claims 4 to 7.

9. The modified therapeutic mRNA according to any one of claims 1 to 3, 7, and 8, or the method according to any one of claims 4 to 8, wherein the at least one synonymous mutation reduces the off-target immunogenicity of the modified therapeutic mRNA and / or its translation product.

10. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7 to 9, wherein the at least one synonymous mutation causes an out-of-frame codon to be replaced with a non-homogeneous amino acid, or the method according to any one of claims 4 to 9.

11. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7 to 10, wherein the off-target immunogenicity includes cellular immunogenicity, or the method according to any one of claims 4 to 10.

12. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7 to 11, wherein the at least one frameshift nucleic acid sequence causes ribosome arrest, or the method according to any one of claims 4 to 11.

13. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7 to 12, wherein the at least one frameshift nucleic acid sequence comprises or further comprises at least one ribosome slippery sequence, or the method according to any one of claims 4 to 13.

14. The aforementioned at least one ribosome slippery sequence, a. XXXYYYZ, where X is any nucleotide, Y is A or U, and Z is A, U, or C; b. PPPX, where X is any nucleotide and PPP is a trinucleotide repeat of any nucleotide; c.m1ψm1ψm1ψX, where X is any nucleotide and m1ψ is (N)1-methylpseuduridine; d. CUUAGG, CUUGAC, CAGCAG, or UCUGCGG; and / or e. A sequence or formula containing any one of (a) to (d) and a non-canonical nucleotide. Includes at least one of the arrays selected from, The modified therapeutic mRNA or method according to claim 13.

15. The modified therapeutic mRNA according to any one of claims 1, 2, and 7-12, or the method according to any one of claims 4-14, wherein the at least one frameshift nucleic acid sequence comprises or further comprises at least one other reading frame sequence encoding an out-of-frame product different from the translation product of in-frame translation of the modified therapeutic mRNA.

16. The modified therapeutic mRNA or method according to claim 15, wherein the at least one synonymous mutation includes a synonymous mutation for introducing a stop codon (PTC) into another reading frame sequence.

17. The modified therapeutic mRNA according to any one of claims 1 to 3 and 7 to 16, wherein the modified therapeutic mRNA comprises at least one chemically modified ribonucleotide, or the method according to any one of claims 4 to 16.

18. The modified therapeutic mRNA or method according to claim 17, wherein the at least one chemically modified ribonucleotide comprises (N)1-methylpseudolidine.

19. A nucleic acid encoding a modified therapeutic mRNA according to any one of claims 1 to 18, wherein the nucleic acid optionally comprises a DNA template for in vitro transcription of the modified therapeutic mRNA.

20. A modified therapeutic mRNA according to any one of claims 1 to 18, for use as a drug; optionally, for use as a vaccine.

21. The method according to any one of claims 6 to 18, wherein the translation step includes a step of translating in vivo or in vitro.

22. The method according to any one of claims 6 to 18, wherein the in vivo translation step includes administering the modified therapeutic mRNA to a subject in need thereof.

23. The identification step is the step of sequencing the modified therapeutic mRNA and / or the nucleic acid encoding the modified therapeutic mRNA; and / or The process includes analyzing the modified therapeutic mRNA sequence and / or the nucleic acid encoding the modified therapeutic mRNA sequence. The method according to any one of claims 4 to 18 and 21 to 22.

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