Capped mRNA and method for preparing same

Abstract

The present disclosure relates to a modified RNA molecule with enhanced stability. Specifically, the present disclosure relates to a modified RNA molecule comprising a 5'cap wherein the RNA molecule is not self-replicable wherein the 5'end of the modified RNA molecule has a sequence of N1pppN2pU or N1vpppN2pU, each of N1 and N2 is independently a nucleotide other than uridine, and methods of producing the same, and pharmaceutical compositions comprising the same.

Description

Technical Field

[0001] The present disclosure relates in some aspects to methods of producing modified RNA molecules with enhanced stability.

[0002] References to Electronic Sequence Listings

[0003] The contents of the electronic sequence listing (P2024TC2948.xml; size: 133,721 bytes; creation date: July 23, 2024) are hereby incorporated by reference in their entirety. Background Art

[0004] RNA molecules produced by in vitro transcription provide the potential for valuable and highly desirable pharmaceutical compositions by allowing the genetic material that can be translated into a protein of interest to be delivered to patient cells. In some respects, existing methods are limited by the immunostimulatory properties of in vitro transcribed RNA molecules, resulting in inefficient expression of the protein of interest in transfected cells. Methods and compositions for addressing such and other needs are provided herein. Summary of the invention

[0005] In some aspects, provided herein is a modified RNA molecule, wherein the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, and wherein at least one uridine in the molecule other than the first 5' uridine in the molecule is a modified uridine.

[0006] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0007] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0008] In some embodiments, at least about 50% of the uridines in the molecule other than the first 5' uridine are modified. In some embodiments, all of the uridines in the molecule other than the first 5' uridine are modified.

[0009] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of NlpppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein all uridines in the molecule except the first 5' uridine are modified.

[0010] In some embodiments, the 5' end of the modified RNA molecule has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' end of the modified RNA molecule has m 7 GpppApU or m 7 The sequence of GvpppApU. In some embodiments, A is modified, preferably 2'-O-methylated.

[0011] In some embodiments, at least about 50% of the uridines in the molecule other than the first 5' uridine are mlΨ (1-methyl-pseudouridine). In some embodiments, all of the uridines in the molecule other than the first 5' uridine are mlΨ.

[0012] In some embodiments, the modified RNA molecule comprises an open reading frame comprising a coding sequence.

[0013] In some embodiments, the coding sequence encodes a therapeutic payload comprising a compound capable of inducing immunity to one or more target disorders or diseases. In some embodiments, the target disorder is associated with or caused by a pathogen infection, such as coronavirus (e.g., 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. In some embodiments, the coding sequence encodes a pathogenic protein characteristic of a pathogen or an antigenic fragment or epitope thereof. In some embodiments, the target disorder is associated with or caused by tumor growth (e.g., cancer) of a cell. In some embodiments, the coding sequence encodes a tumor-associated antigen (TAA) or an antigenic fragment or epitope thereof characteristic of a cancer.

[0014] In some embodiments, the coding sequence encodes eGFP. In some embodiments, the coding sequence encodes SARS-COV-2BA.4 / BA.5RBD. In some embodiments, the coding sequence encodes rabies virus antigen. In some embodiments, the coding sequence encodes respiratory syncytial virus antigen. In some embodiments, the coding sequence encodes varicella-zoster virus antigen.

[0015] In some embodiments, the molecule further comprises at least one modified G, C, or A.

[0016] In some embodiments, the molecule has a modified backbone. In some embodiments, the modified backbone comprises at least one phosphorothioate bond.

[0017] In other aspects, provided herein is a pharmaceutical composition comprising a modified RNA disclosed herein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is LNP. In some embodiments, the LNP comprises a cationic lipid.

[0018] In some embodiments, the LNP comprises: i) about 30 to 55 mole percent of a cationic lipid; ii) about 5 to 40 mole percent of a phospholipid; iii) about 20 to 50 mole percent of a sterol; and iv) a polymer-conjugated lipid.

[0019] In other aspects, provided herein is a method for in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0020] In some embodiments, the in vitro transcription mixture comprises modified uridine.

[0021] In some embodiments, at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0022] In some embodiments, the method comprises combining a DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides, and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine, and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0023] In some embodiments, all but the first 5' uridine in the molecule are modified.

[0024] In some embodiments, the method comprises combining a DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides, and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0025] In some embodiments, the modified uridine is mlΨ. In some embodiments, all uridines except the first 5' uridine in the modified RNA molecule are mlΨ.

[0026] In some embodiments, the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap has m 7 GpppApU or m 7 In some embodiments, A is modified, preferably 2'-O-methylated. In some embodiments, the 5' cap has m 7 GpppA m pU or m 7 Gvppp m The sequence of pU.

[0027] In some embodiments, the DNA-dependent RNA polymerase is T7 RNA polymerase or a variant thereof.

[0028] In some embodiments, the method further comprises isolating the modified RNA molecule.

[0029] In other aspects, provided herein are modified RNA molecules produced by the methods disclosed herein.

[0030] In other aspects, provided herein is a kit comprising: 1) a DNA-dependent RNA polymerase; 2) modified and / or unmodified nucleotides, and 3) a 5' cap, and 4) instructions for practicing the methods disclosed herein, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0031] In other aspects, provided herein is a method for reducing innate immune stimulation of an RNA molecule, comprising using a 5' cap having a sequence of N1pppN2pU or N1vpppN2pU, and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine.

[0032] In some embodiments, the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap has m 7 GpppApU or m 7 In some embodiments, the 5' cap has m 7 GpppA m pU or m 7 Gvppp m In some embodiments, the method comprises modifying all uridines except the first 5' uridine in the RNA molecule. In some embodiments, the method comprises modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown respectively by (G m7 )ppp(A m2 )G and (G m7 )ppp(A m2 ) Expression of synthesized mRNA encoding eGFP protein in BHK-21 cell line.

[0034] Figure 2 Shown by (G m7 )ppp(A m2 )U and (G m7 )vppp(A m2 )U synthesized mRNA was expressed in vitro in HEK-293T cells.

[0035] Figure 3 Shown by (G m7 )ppp(A m2 )U and (G m7 )vppp(A m2 )U synthesized mRNA was expressed in vitro in A549 cells. DETAILED DESCRIPTION

[0036] All publications, including patent documents, scientific articles, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over definitions incorporated herein by reference.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0038] I. Overview

[0039] In some aspects, provided herein are modified RNA molecules, wherein the modified RNA molecule comprises a 5' cap, and wherein at least one uridine in the molecule, other than the first 5' uridine in the molecule, is a modified uridine. In some embodiments, the modified RNA molecule is not self-replicating.

[0040] Also provided is a method for in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining a DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, and wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. Also provided are compositions, preparations and kits used according to the provided methods.

[0041] At present, RNA molecules produced by in vitro transcription and transfected into cells usually stimulate innate immune receptors, such as endosomal and cytoplasmic pattern recognition receptors (PRR), including toll-like receptors 3, 7 and 8 (Ma, X. and Hur, S. Drug Metabolism Pharmacokinet. 2022.44, 100450). These toll-like receptors can sense single-stranded and double-stranded RNA and produce immune responses through intracellular signal cascades. The activation of immune responses in cells transfected with in vitro transcribed RNA molecules is a major limitation of current gene therapy methods.

[0042] The invention of the present application provides a method for using a specific uridine-containing cap (e.g., m 7 GpppA m pU cap, m 7 Gvppp m A novel method for in vitro mRNA synthesis using pU capping and mRNA produced by such a method. 7 GpppAm pU caps have traditionally been used for self-replicating RNAs based on their function in positive-strand RNA viruses such as Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), and Sindbis virus (SIN). The inventors have for the first time used this type of cap structure for in vitro transcription of non-replicating mRNAs. Surprisingly, the use of m 7 GpppA m Compared with the pG-capped mRNA synthesis method, the m 7 GpppA m One-step synthesis of the pU cap can achieve higher capping rates. The resulting non-replicable mRNA (except for the first uridine) can be fully uridine-modified, resulting in reduced innate immune stimulation when delivered to cells.

[0043] Thus, one aspect of the present invention provides a method for in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has m 7 GpppA m pU or m 7 Gvppp m pU, wherein U is unmodified uridine, wherein the in vitro transcription mixture contains modified uridine, and wherein all uridines except the first 5' uridine in the modified RNA molecule are modified.

[0044] In another aspect, a modified RNA molecule is provided, wherein the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, and wherein at least one uridine in the molecule other than the first 5' uridine in the molecule is a modified uridine.

[0045] Also provided are kits for practicing the methods described herein and compositions (eg, pharmaceutical compositions) comprising the modified RNA molecules.

[0046] In other aspects, provided herein is a method for reducing innate immune stimulation of an RNA molecule, comprising using a 5' cap having a sequence of N1pppN2pU or N1vpppN2pU, and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine.

[0047] II. Definitions

[0048] For the purpose of interpreting this specification, the following definitions shall apply and, where appropriate, terms used in the singular shall also include the plural and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition shall prevail.

[0049] The term "about" as used herein refers to the usual error range for the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments for that value or parameter per se.

[0050] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as a strict limitation on the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, a certain range of values ​​is provided, so it should be understood that each insertion value between the upper and lower limits of the range and any other stated values ​​or insertion values ​​in the stated range are all included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, subject to any special exclusion restrictions in the stated range. If the stated range includes one or two restrictions, the range that does not include one or both of these restrictions is also included in the claimed subject matter. This applies to the breadth of any range.

[0051] As used herein, a "cap" refers to a non-extendable trinucleotide that, when incorporated into the 5' end of an RNA transcript, facilitates translation or localization, and / or protects the RNA transcript from degradation.

[0052] The term "nucleobase" as used herein refers to a nitrogen-containing heterocyclic moiety nucleobase. Non-limiting examples of suitable nucleobases include: adenine, cytosine, guanine, thymine, uracil or its analogs, for example, 5-propargyl-uracil, 2-thio-5-propargyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil, 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(8-aza-7-deazaadenine).

[0053] As used herein, the term "ribonucleotide" or "nucleotide" refers to a compound consisting of a nucleobase linked to the C-carbon of a ribose sugar or an analog thereof. The ribose sugar or analog may be substituted or unsubstituted.

[0054] As used herein, "nucleotide triphosphate" refers to a nucleotide having a triphosphate group at the 5' position.

[0055] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" as used herein refer to single-stranded or double-stranded polymers of nucleotide monomers, including ribonucleotides (RNA) and 2'-deoxyribonucleotides (DNA) linked by phosphodiester bonds between nucleotides. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or of chimeric mixtures thereof.

[0056] As used herein, "self-replicating" RNA refers to an RNA molecule that, when delivered to a vertebrate cell, is transcribed from itself by a self-generated antisense copy, resulting in the production of multiple daughter RNAs. Self-replicating RNA molecules are typically +-stranded molecules that can be directly translated after delivery to a cell, thereby providing a template for an RNA-dependent RNA polymerase to produce antisense and sense transcripts from the delivered RNA.

[0057] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a substance that is not biologically or otherwise undesirable, for example, the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicology and manufacturing testing and / or are included in the inactive ingredient guidelines established by the U.S. Food and Drug Administration.

[0058] As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable base, composition or vehicle used in the drug delivery process, which may have one or more components including but not limited to excipients, binders, diluents, solvents, fillers and / or stabilizers.

[0059] The term "lipid" as used herein refers to a group of compounds including, but not limited to, fats, sterols, waxes, fat-soluble vitamins, monoglycerides, diglycerides, sphingolipids, and phospholipids. In the context of the present disclosure, phospholipids, ionizable lipids, polymer-conjugated lipids, and lipid stabilizers are considered lipids.

[0060] As used herein, the term "ionizable lipid" refers to a lipid having a non-zero net charge at physiological pH. The term encompasses cationic lipids, including lipids having a partial positive charge at physiological pH. The term also encompasses mixtures of ionizable lipids, which may contain two or more ionizable lipids. In each case where an embodiment is envisioned using the term "ionizable lipid," it is also envisioned using "cationic lipid," as if all embodiments specifically and individually listed ionizable lipids and cationic lipids.

[0061] As used herein, the term "polymer-conjugated lipid" refers to a lipid comprising a polymer portion. The term encompasses PEGylated lipids, including PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, and PEGylated dialkylglycerols. The term also encompasses mixtures of polymer-conjugated lipids, which may contain two or more polymer-conjugated lipids. In each case where an embodiment is envisioned using the term "polymer-conjugated lipid," it is also envisioned using "PEGylated lipid," as if all embodiments specifically and individually listed polymer-conjugated lipids and PEGylated lipids.

[0062] The term "lipid stabilizer" as used herein refers to a component in a lipid nanoparticle that is believed to help stabilize the LNP structure. Without being bound by theory, it is believed that the lipid stabilizer component in the LNP helps promote the liquid ordered phase of the lipid membrane in the LNP. For example, see Section 3.3.1 of Albertsen, HC et al. "The role of lipid components in lipidnanoparticles for vaccines and gene therapy." Adv Drug Deliv Rev. 2022 September; 188: 114416. Compounds that can be used as lipid stabilizers include steroids, corticosteroids, vitamins, and other compounds containing a steroid core.

[0063] As used herein, the term "alkyl" refers to a chain of carbon atoms in which all bonds between carbon atoms in the alkyl group are single bonds. The term includes straight chains and branched chains (eg, the term includes n-propyl and isopropyl).

[0064] As used herein, the term "Cx-Cy alkyl" refers to an alkyl substituent having at least x carbon atoms and no more than y carbon atoms in the alkyl chain. For example, the term "C1-C3 alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, and isopropyl substituents.

[0065] As used herein, the term "alkylene" refers to an alkyl chain connected to other chemical groups at at least two positions. "Cx-Cy alkylene" refers to an alkylene substituent having at least x carbon atoms and no more than y carbon atoms in the alkylene chain. For example, the term "C1-C3 alkylene" includes, but is not limited to, methylene, ethylene, n-propylene, and isopropylene.

[0066] As used herein, the term "alkenyl" refers to a chain of carbon atoms having at least one double bond between two carbon atoms in the chain. The term includes straight chains and branched chains (eg, the term includes 1-propenyl and isopropenyl).

[0067] As used herein, the term "Cx-Cy alkenyl" refers to an alkenyl substituent having at least x carbon atoms and no more than y carbon atoms in the alkenyl chain. For example, the term "C2-C4 alkenyl" includes, but is not limited to, vinyl and 1-propenyl.

[0068] As used herein, the term "alkenylene" refers to an alkenyl chain attached to other chemical groups at at least two positions. "Cx-Cy alkenylene" refers to an alkenylene substituent having at least x carbon atoms and not more than y carbon atoms.

[0069] As used herein, the term "alkynyl" refers to a chain of carbon atoms having at least one triple bond between two carbon atoms in the chain. The term includes straight and branched chains (e.g., the term includes 1-propynyl and isopropynyl).

[0070] As used herein, the term "Cx-Cyalkynyl" refers to an alkynyl substituent having at least x carbon atoms and not more than y carbon atoms in the alkynyl chain.

[0071] The term "cycloalkyl" as used herein refers to a cyclic group of carbon atoms in which all bonds between the carbon atoms are single bonds. The term "Cx-Cy cycloalkyl" refers to a cycloalkyl substituent having at least x carbon atoms and no more than y carbon atoms. For example, the term "C6-C10 cycloalkyl" includes, but is not limited to, cyclohexyl and cyclooctyl. The term "cycloalkylene" has the same meaning as cycloalkyl, except that the cycloalkylene substituent is connected to at least two other chemical groups.

[0072] The term "cycloalkenyl" as used herein refers to a cyclic group of carbon atoms, wherein at least one bond between two carbon atoms in the cycloalkenyl group is a double bond. The term "Cx-Cycycloalkenyl" refers to a cycloalkenyl substituent having at least x carbon atoms and not more than y carbon atoms.

[0073] As used herein, the term "aryl" refers to a cyclic group of carbon atoms having at least one double bond. The term "Cx-Cy aryl" refers to an arylene substituent having at least x carbon atoms and no more than y carbon atoms. For example, the term "C6-C10 aryl" includes, but is not limited to, phenyl and naphthyl. The term "arylene" has the same meaning as aryl, except that the arylene substituent is attached to at least two other chemical groups.

[0074] As used herein, the term "heterocycloalkyl" refers to a cyclic radical in which all bonds between atoms in the ring are single bonds. The term "Cx-Cy heterocycloalkyl" refers to a heterocycloalkyl substituent having at least x atoms and no more than y atoms. For example, the term "C5-C6 heterocycloalkyl" includes, but is not limited to, pyrrolidinyl and 1,4-dioxanyl. The term "heterocycloalkylene" has the same meaning as heterocycloalkyl, except that the heterocycloalkylene substituent is attached to at least two other chemical groups.

[0075] The term "heteroaryl" as used herein refers to a cyclic radical having at least one double bond. The term "x- to y-membered heteroaryl" refers to a cyclic radical having at least x atoms and no more than y atoms. For example, 5- or 6-membered heteroaryl includes, but is not limited to, pyridyl and furyl.

[0076] As used herein, the term "carbocycle" refers to a substituent that can be a cycloalkyl or aryl group. Likewise, the term "heterocycle" refers to a substituent that can be a heterocycloalkyl or heteroaryl group.

[0077] Possible atoms constituting the rings in heterocycloalkyl and heteroaryl groups and derivatives thereof include, but are not limited to, carbon, nitrogen, oxygen and sulfur.

[0078] The term "optionally substituted" as used herein means that the indicated substituent can be substituted or unsubstituted. The term substituted refers to another chemical moiety that modifies the indicated substituent by replacing an H atom. For example, ethanol is an example of ethane substituted by OH. In some embodiments, the optionally substituted group is optionally substituted by chlorine, fluorine, bromine, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C10 aryl or 5- or 6-membered heteroaryl.

[0079] The terms "individual," "subject," and "patient" are used interchangeably herein to describe mammals, including humans. In some embodiments, the individual is in need of treatment, for example, the individual may have been diagnosed with or is suspected of having cancer.

[0080] It should be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0081] Reference herein to "about" a value or parameter includes (and describes) variations in the value or parameter itself. For example, a description referring to "about X" includes a description of "X". In some embodiments, the term "about" a value or parameter refers to a range within 20% in either direction of the value or parameter.

[0082] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter.

[0083] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0084] III. Preparation Method

[0085] Provided herein are methods of making the modified RNA molecules disclosed herein.

[0086] A. In vitro transcription

[0087] The modified RNA described herein can be synthesized by in vitro transcription of an appropriate DNA template. The promoter used to control transcription can be a promoter for any RNA polymerase. The DNA template used for in vitro transcription can be obtained, for example, by cloning nucleic acids, particularly cDNA and introducing it into an appropriate vector for in vitro transcription. In some embodiments, the RNA can have modified nucleosides, including, for example, pseudouridine and / or 1-methyl pseudouridine.

[0088] In some embodiments, suitable DNA templates for in vitro transcription include linearized plasmid DNA, PCR products, or synthetic DNA oligonucleotides. In some embodiments, the purity of the template DNA affects the transcription yield and the integrity of the synthetic RNA. In some embodiments, the template DNA has a purity greater than about 90%, greater than about 95%, or greater than about 99%. Plasmid purification methods are well known in the art and include small-scale preparation, medium-scale preparation, and large-scale preparation plasmid separation. In some embodiments, the plasmid DNA is mostly in supercoiled form and does not contain contaminating RNases, proteins, RNA, and salts.

[0089] In some embodiments, the DNA template for in vitro transcription includes completely linearized plasmid DNA. In some embodiments, plasmid linearization is achieved by the action of a restriction enzyme downstream of the DNA template to be transcribed. In some embodiments, the restriction enzyme produces a blunt end or a 5'-overhang. In some embodiments, after the plasmid DNA is linearized, the template DNA is further purified by phenol / chloroform extraction.

[0090] In some embodiments, the DNA template comprises a double-stranded T7 promoter region upstream of the sequence to be transcribed. In some embodiments, the promoter comprises an SP6, T7 or T3 promoter region. In some embodiments, the promoter is a bacteriophage polymerase promoter.

[0091] In some embodiments, described herein is a method of in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides, and a 5' cap, wherein the modified RNA molecule is non-self-replicable, and wherein the 5' cap has a sequence of N1pppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0092] In some embodiments, described herein is a method for in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides, and a 5' cap, wherein the modified RNA molecule is non-self-replicable, and wherein the 5' cap has a sequence of N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0093] In some embodiments, the in vitro transcription mixture comprises a reaction buffer, modified and / or unmodified nucleotides (e.g., modified and / or unmodified adenosine, guanosine, cytidine, or uridine), a 5' cap, a template DNA, a DNA-dependent RNA polymerase, and nuclease-free water. 1X reaction buffer may include, for example, [40mM Tris-HCl (pH 7.9-8.0), 10mM DTT, 2mM spermidine, 0.002% TritonX-100, 27mM magnesium acetate]. In some embodiments, the reaction buffer comprises Tris-HCl (pH 7.9-8.0) at a concentration between about 10mM and about 80mM. In some embodiments, the reaction buffer comprises Tris-HCl (pH 7.9-8.0) at a concentration of about 10mM, about 20mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, or about 80mM. In some embodiments, the reaction buffer comprises Tris-HCl (pH 7.9-8.0) at a concentration of about 40mM. In some embodiments, the reaction buffer comprises DTT at a concentration between about 1mM and about 30mM. In some embodiments, the reaction buffer comprises DTT at a concentration of about 1mM, about 5mM, about 10mM, about 20mM or about 30mM. In some embodiments, the reaction buffer comprises DTT at a concentration of about 10mM. In some embodiments, the reaction buffer comprises spermidine at a concentration between about 0.5mM and about 10mM. In some embodiments, the reaction buffer comprises spermidine at a concentration of about 0.5mM, about 1mM, about 2mM, about 3mM, about 5mM or about 10mM. In some embodiments, the reaction buffer comprises spermidine at a concentration of about 2mM. In some embodiments, the reaction buffer comprises Triton X-100 at a concentration between about 0.0005% and about 0.05%. In some embodiments, the reaction buffer comprises a concentration of about 0.0005%, about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04% or about 0.05% Triton X-100. In some embodiments, the reaction buffer comprises a concentration of about 0.0005% and about 0.005% Triton X-100. In some embodiments, the reaction buffer comprises a concentration of about 0.0005%, about 0.001%, about 0.002%, about 0.003%, about 0.004% or about 0.005% Triton X-100. In some embodiments, the reaction buffer comprises Triton X-100 at a concentration of about 0.002%.In some embodiments, the reaction buffer comprises magnesium acetate at a concentration between about 5mM and about 100mM. In some embodiments, the reaction buffer comprises magnesium acetate at a concentration of about 5mM, about 10mM, about 20mM, about 27mM, about 30mM, about 40mM, about 50mM, about 75mM or about 100mM. In some embodiments, the reaction buffer comprises magnesium acetate at a concentration of about 27mM. Magnesium chloride (MgCl2) can be used to replace magnesium acetate. Similar effects can be achieved by regulating the concentration of magnesium chloride.

[0094] In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is between about 1mM and about 100mM. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is about 1mM, about 5mM, about 10mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM or about 100mM. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is between about 1mM and about 50mM. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is about 1mM, about 5mM, about 10mM, about 12.5mM, about 30mM, about 40mM or about 50mM. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is about 10mM each. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is about 5mM each. In some embodiments, the final concentration of modified and / or unmodified nucleotides in the in vitro transcription mixture is about 12.5mM each. In some embodiments, modified and / or unmodified nucleotides are added to the in vitro transcription mixture at an equal ratio. In some embodiments, the 5' cap is added to the in vitro transcription mixture at a concentration between about 1mM and about 50mM. In some embodiments, the 5' cap is added to the in vitro transcription mixture at a concentration of about 1mM, about 2mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 12.5mM, about 20mM, about 30mM, about 40mM or about 50mM. In some embodiments, the 5' cap is added to the in vitro transcription mixture at a concentration of about 8mM. In some embodiments, the 5' cap is added to the in vitro transcription mixture at a concentration of about 4mM. In some embodiments, the 5' cap is added to the in vitro transcription mixture at a concentration of about 12.5 mM. In some embodiments, the in vitro transcription mixture is assembled at room temperature.

[0095] In some embodiments, the enzymatic capping step can occur co-transcriptionally. In some embodiments, the capping step occurs during in vitro transcription of the modified RNA molecule. In some embodiments, the RNA cap is incorporated at the 5' end by an RNA polymerase with loose substrate specificity, thereby directly producing each 5'-capped RNA. In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of N1pppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of GpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of m 7 GpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap included in the enzymatic capping step has m 7 GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap included in the enzymatic capping step has m 7 GpppApU, wherein A is 2′-O-methylated. In some embodiments, the 5′ cap included in the enzymatic capping step has m 7 GpppA m In some embodiments, the 5' cap included in the enzymatic capping step has a sequence of m 7 GvpppApU, wherein A is 2′-O-methylated. In some embodiments, the 5′ cap included in the enzymatic capping step has m 7 Gvppp m The sequence of pU.

[0096] In some embodiments, the in vitro transcription of the modified RNA includes a capping step to produce a modified RNA comprising a 5' cap having a sequence of N1pppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the capping step is enzymatic or synthetic. In some embodiments, the 5' cap has a sequence of GpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap has m 7In some embodiments, A is modified, preferably 2′-O-methylated. In some embodiments, the 5′ cap has m 7 GpppA m The sequence of PUs.

[0097] In some embodiments, the in vitro transcription of the modified RNA includes a capping step to produce a modified RNA comprising a 5' cap having a sequence of N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the capping step is enzymatic or synthetic. In some embodiments, the 5' cap has a sequence of GvpppApU, wherein A is modified or unmodified. In some embodiments, the 5' cap has m 7 In some embodiments, A is modified, preferably 2′-O-methylated. In some embodiments, the 5′ cap has m 7 Gvppp m The sequence of pU.

[0098] In some embodiments, the in vitro transcription mixture comprises modified adenosine, guanosine, cytidine or uridine. In some embodiments, the in vitro transcription mixture comprises unmodified adenosine, guanosine, cytidine or uridine. In some embodiments, the in vitro transcription mixture comprises modified adenosine, guanosine, cytidine and uridine. In some embodiments, the in vitro transcription mixture comprises unmodified adenosine, guanosine, cytidine and uridine. In some embodiments, the in vitro transcription mixture comprises unmodified adenosine, guanosine, cytidine and uridine. In some embodiments, the in vitro transcription mixture comprises unmodified adenosine, guanosine and cytidine. In some embodiments, the in vitro transcription mixture comprises modified uridine. In some embodiments, the modified uridine comprises Ψ or m1Ψ. In some embodiments, the modified uridine is m1Ψ.

[0099] In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecule are modified. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridines in the RNA molecule are modified. In some embodiments, at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 90% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 95% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 99% of the uridines other than the first 5' uridine in the molecule are modified.

[0100] In some embodiments, all but the first 5' uridine in the molecule are modified.

[0101] In some embodiments, all uridines except the first 5' uridine in the molecule are m1Ψ.

[0102] In some embodiments, a method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine (preferably m1Ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0103] In some embodiments, a method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides, and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine (preferably m1Ψ), and wherein all uridines in the molecule except the first 5' uridine are modified.

[0104] In a preferred embodiment, the method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of GpppApU or GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1Ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0105] In a preferred embodiment, a method for in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of GpppApU or GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises modified uridines (preferably m1Ψ), and wherein all uridines in the molecule except the first 5' uridine are modified.

[0106] In a preferred embodiment, the method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has m 7 GpppApU or m 7 The sequence of GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1Ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0107] In a preferred embodiment, the method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has m 7 GpppApU or m 7 The sequence of GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1Ψ), and wherein all uridines except the first 5' uridine in the molecule are modified.

[0108] In a more preferred embodiment, the method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has m 7 GpppA m pU or m 7 Gvppp m pU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1Ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

[0109] In a more preferred embodiment, the method of in vitro transcribing a DNA molecule into a modified RNA molecule comprises combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has m 7 GpppA m pU or m 7 Gvppp m pU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture contains a modified uridine (preferably m1Ψ), and wherein all uridines except the first 5' uridine in the molecule are modified.

[0110] In some embodiments, the in vitro transcription mixture comprises a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase is T7 RNA polymerase, SP3 RNA polymerase, SP6 RNA polymerase, VSW-3 RNA polymerase, or a variant thereof. In some embodiments, the DNA-dependent RNA polymerase is T7 RNA polymerase or a variant thereof. In some embodiments, the DNA-dependent RNA polymerase is T7 RNA polymerase.

[0111] In some embodiments, the in vitro transcription reaction includes an incubation step. In some embodiments, the incubation time is between about 5 minutes and about 360 minutes. In some embodiments, the incubation time is about 20 minutes, about 40 minutes, about 60 minutes, about 80 minutes, about 100 minutes, about 120 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 180 minutes, about 200 minutes, about 220 minutes, about 240 minutes, about 260 minutes, about 280 minutes, about 300 minutes, about 320 minutes, about 340 minutes or about 360 minutes. In some embodiments, the incubation time is greater than about 360 minutes. In some embodiments, the incubation time is between about 5 minutes and about 140 minutes. In some embodiments, the incubation time is about 20 minutes, about 40 minutes, about 60 minutes, about 80 minutes, about 100 minutes, about 120 minutes or about 140 minutes. In some embodiments, the incubation time is greater than about 140 minutes. In some embodiments, incubation is carried out at a temperature between about 30°C and about 42°C. In some embodiments, incubation is carried out at about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C. In some embodiments, incubation is carried out at a temperature between about 35°C and about 40°C. In some embodiments, incubation is carried out at about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the incubation step includes incubating at 37°C for 2 hours. In some embodiments, incubation is carried out in a water bath, a heating block, a dry air incubator, or a PCR instrument.

[0112] In some embodiments, the product of the in vitro transcription reaction is evaluated to detect RNA purity and capping efficiency. In some embodiments, RNA concentration is quantified by ultraviolet absorbance. In some embodiments, RNA concentration is determined by measuring the ultraviolet absorbance at a wavelength of 260nm. In some embodiments, before quantifying RNA concentration by ultraviolet absorbance, free nucleotides in the transcription reaction are removed. In some embodiments, the transcription product of the in vitro transcription reaction is analyzed by gel electrophoresis. In some embodiments, the length, integrity and / or quantity of the transcript are analyzed by running aliquots of the transcription reaction on appropriate denaturing agarose gels or polyacrylamide gels. In some embodiments, transcripts greater than 0.3kb can be run on agarose gels. In some embodiments, transcripts less than about 0.3kb are run on denaturing polyacrylamide gels. In some embodiments, the percentage of polyacrylamide in the polyacrylamide gel is between about 5% and about 15%.

[0113] Also provided herein is a method for separating modified RNA molecules. In some embodiments, modified RNA molecules are separated by methods based on chemistry, columns and / or gels. Alternatively, in some embodiments, modified RNA molecules are separated by methods based on beads. In some embodiments, modified RNA molecules are separated by methods based on beads through the combination of poly (A) tails. In some embodiments, the method for separating modified RNA molecules described herein is used to clean up RNA and separate it from components such as DNA templates, unincorporated nucleotides or RNA modification enzymes.

[0114] IV. Modified RNA Molecules

[0115] Also provided herein are RNA molecules produced by the methods described herein. Thus, in some aspects, provided herein are modified RNA molecules comprising a 5' cap, wherein except for the first 5' uridine in the molecule, at least one uridine in the molecule is a modified uridine. In some embodiments, the modified RNA molecule is non-self-replicating.

[0116] In some embodiments, the modified RNA molecule comprises the nitrogenous bases guanine, uracil, adenine and cytosine. In some embodiments, the modified RNA molecule comprises a modified nitrogenous base. In some embodiments, the modified RNA molecule comprises at least one modified G, C or A. In some embodiments, the modified RNA molecule comprises at least one modified U. In some embodiments, the modified U comprises Ψ (pseudouridine). In some embodiments, the modified U comprises m1Ψ (1-methyl-pseudouridine).

[0117] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, about 1% to about 99% of the uridine bases in the modified RNA molecule are modified. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the uridine in the RNA molecule is modified. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 90% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 95% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are modified. In some embodiments, all of the uridines in the molecule except the first 5' uridine are modified.

[0118] In some embodiments, the 5' end of the modified RNA molecule has a sequence of NlpppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecule are Ψ. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridine in the RNA molecule is Ψ. In some embodiments, at least about 50% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 90% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 95% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are Ψ. In some embodiments, all of the uridines in the molecule except the first 5' uridine are Ψ.

[0119] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecule are m1Ψ. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridine in the RNA molecule is m1Ψ. In some embodiments, at least about 50% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 90% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 95% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are mlΨ. In some embodiments, all of the uridines in the molecule except the first 5' uridine are mlΨ.

[0120] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, about 1% to about 99% of the uridine bases in the modified RNA molecule are modified. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the uridine in the RNA molecule is modified. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 90% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 95% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are modified. In some embodiments, all of the uridines in the molecule except the first 5' uridine are modified.

[0121] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecule are Ψ. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridine in the RNA molecule is Ψ. In some embodiments, at least about 50% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 90% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 95% of the uridines in the molecule other than the first 5' uridine are Ψ. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are Ψ. In some embodiments, all of the uridines in the molecule except the first 5' uridine are Ψ.

[0122] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine. In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecule are m1Ψ. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridine in the RNA molecule is m1Ψ. In some embodiments, at least about 50% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 90% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 95% of the uridine in the molecule other than the first 5' uridine is m1Ψ. In some embodiments, at least about 99% of the uridines in the molecule except the first 5' uridine are mlΨ. In some embodiments, all of the uridines in the molecule except the first 5' uridine are mlΨ.

[0123] Compared with the triphosphate bond of NlpppN2pU, the triphosphate bond of NlvpppN2pU is vinyl modified. For example, N1vpppN2pU contains a vinyl modified triphosphate bond, including the following parts.

[0124]

[0125] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of N1pppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0126] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of NlpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0127] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0128] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0129] In some embodiments, the modified RNA molecule participates in protein synthesis. In some embodiments, the modified RNA molecule is mRNA. In some embodiments, the modified RNA molecule comprises a protein encoding open reading frame (ORF).

[0130] In some embodiments, the length of the modified RNA molecule is at least 20 nucleotides. In some embodiments, the length of the modified RNA molecule is between about 20 nucleotides and about 8000 nucleotides. In some embodiments, the length of the RNA molecule is at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1000 nucleotides, about 1500 nucleotides, about 2000 nucleotides, about 3000 nucleotides, about 4000 nucleotides, about 6000 nucleotides, about 7000 nucleotides or about 8000 nucleotides.

[0131] In some embodiments, the RNA molecule of modification comprises non-coding elements. In some embodiments, non-coding elements include 5'7-methyl-GTP cap, 5'untranslated region (UTR) and 3'UTR. In some embodiments, mRNA comprises 5'UTR. 5'UTR provided herein can be recognized by ribosomes, thereby allowing ribosomes to bind and start the translation of mRNA (for example, the translation of nucleic acids encoding sequence and / or encoding mRNA signal peptide). In some embodiments, 5'UTR is located upstream of the mRNA coding sequence. In some embodiments, the length of 5'UTR is between about 5 and 1400 nucleotides. In some embodiments, the length of 5'UTR is between about 5 and about 20, about 20 to about 40, about 40 to about 60, about 60 to about 80, about 80 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 400 or about 400 to about 500 nucleotides. In some embodiments, the 5'UTR is about 66 nucleotides in length.

[0132] In some embodiments, the mRNA comprises a 5'UTR and a 3'UTR, such as any of the 5'UTR and 3'UTR provided herein. In some embodiments, the 5'UTR and 3'UTR are from the same species. In some embodiments, the 5'UTR and 3'UTR are not from the same species. In some embodiments, the 5'UTR is synthetic and the 3'UTR is not synthetic. In some embodiments, the 5'UTR is not synthetic and the 3'UTR is synthetic.

[0133] In some embodiments, mRNA comprises a poly (A) sequence (e.g., a polyadenylation sequence). The poly (A) sequence is composed of a plurality of continuous adenosine monophosphates. In some embodiments, the poly (A) sequence is essential for the translation of mRNA. In some embodiments, the poly (A) sequence is located downstream of the coding sequence of the mRNA. In some embodiments, the poly (A) sequence is located downstream of the 3'UTR of the mRNA. In some embodiments, the length of the poly (A) sequence is about 50 nucleotides or longer, for example, about 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides or longer. In some embodiments, the length of the poly (A) sequence is about 150 nucleotides or shorter, for example, about 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 50 nucleotides or shorter. In some embodiments, the length of the poly (A) sequence is about 105 nucleotides.

[0134] In some embodiments, the modified RNA molecule is not self-replicable. In some embodiments, the modified RNA molecule is degraded in the cell over time. In some embodiments, the mRNA degradation in the cell is caused by mRNA decay, AU-rich elements (ARE) in 3′UTR, destabilizing elements in the protein coding region, nonsense-mediated mRNA decay (NMD) and / or microRNA (miRNA). In some embodiments, the modified RNA molecule has a measurable half-life in the cell. In some embodiments, the half-life of the modified RNA molecule in the cell is between about 0 hours and about 24 hours. In some embodiments, the half-life of the modified RNA molecule in the cell is about 0 hours to 1 hour, about 1 hour to 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 5 hours to about 7 hours, about 7 hours to about 9 hours, about 9 hours to about 11 hours, about 11 hours to about 15 hours, about 15 hours to about 20 hours, about 20 hours to about 24 hours. In some embodiments, the half-life of the modified RNA molecule in the cell is about 10 hours.

[0135] In some embodiments, the modified RNA molecule can be transfected into the cell for translation in the cell. Transfection methods are known to those skilled in the art, including microinjection, chemical treatment and electroporation. In some embodiments, the cells to be transfected include any patient cells that need to express the protein of interest. In some embodiments, the cells to be transfected include vascular endothelial cells, epidermal cells, bronchial endothelial cells, adipocytes, dermal fibroblasts, muscle cells and hematopoietic cells (e.g., T cells, B cells, dendritic cells, macrophages, etc.), germ cells or tissue culture cells.

[0136] A.5' cap

[0137] In some embodiments, the modified RNA molecule comprises a 5' cap.

[0138] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0139] In some embodiments, the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein A is modified or unmodified. In some embodiments, A is 2'-O-methylated. In some embodiments, the 5' end of the modified RNA molecule has m 7 In some embodiments, the 5' end of the modified RNA molecule has m 7GpppApU, wherein A is modified or unmodified. In some embodiments, A is 2′-O-methylated. In some embodiments, the 5′ end of the modified RNA molecule has m 7 GpppA m In some embodiments, the 5' end of the modified RNA molecule comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, or 8. In some embodiments, the 5' end of the modified RNA molecule comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 9, 12, 15, 18, 22, 25, 28, or 31. In some embodiments, the 5' end of the modified RNA molecule comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 12, 15, 18, 22, 25, 28, or 31.

[0140] In some embodiments, m 7 GpppA m pU has the following formula,

[0141]

[0142] In some embodiments, compared to unmodified RNA, the RNA comprising m 7 GpppA m RNA molecules modified with a 5' cap of a pU sequence exhibited improved RNA stability.

[0143] In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

[0144] In some embodiments, the 5' end of the modified RNA molecule has the sequence of GvpppApU, wherein A is modified or unmodified. Compared with the triphosphate bond of GpppApU, the triphosphate bond of GvpppApU is vinyl modified. In some embodiments, A is 2'-O-methylated. In some embodiments, the 5' end of the modified RNA molecule has m 7 The sequence of GvpppApU. 7 Compared with the triphosphate bond of GpppApU, m 7 The triphosphate bond of GvpppApU is vinyl modified. In some embodiments, the 5' end of the modified RNA molecule has m 7 GvpppApU, wherein A is modified or unmodified. In some embodiments, A is 2′-O-methylated. In some embodiments, the 5′ end of the modified RNA molecule has m 7Gvppp m pU sequence. 7 GpppA m Compared with the triphosphate bond of pU, m 7 Gvppp m The triphosphate bond of pU is vinyl modified. In some embodiments, the 5' end of the modified RNA molecule comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 21 or 34.

[0145] In some embodiments, compared to unmodified RNA, the RNA comprising m 7 Gvppp m The modified RNA molecule with the 5' cap of the pU sequence shows improved RNA stability. In addition, the double bond structure makes the molecular conformation more stable, making it more difficult for nucleases to recognize and hydrolyze.

[0146] In some embodiments, m 7 Gvppp m pU has the following formula,

[0147]

[0148] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0149] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0150] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0151] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0152] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GpppApU, wherein U is an unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0153] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0154] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0155] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0156] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 GpppA m pU, wherein U is an unmodified uridine, and wherein at least one uridine in the molecule other than the first 5' uridine is a modified uridine.

[0157] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 GpppA m pU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0158] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 GpppA mpU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0159] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 GpppA m pU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0160] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein U is an unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0161] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0162] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0163] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0164] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence of GvpppApU, wherein U is an unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

[0165] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7The sequence is GvpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0166] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GvpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0167] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 The sequence is GvpppApU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0168] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 Gvppp m pU, wherein U is an unmodified uridine, and wherein at least one uridine in the molecule other than the first 5' uridine is a modified uridine.

[0169] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 Gvppp m pU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are modified.

[0170] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 Gvppp m pU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are Ψ.

[0171] In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicating, wherein the 5' end of the modified RNA molecule has m 7 Gvppp m pU, wherein U is an unmodified uridine, and wherein all uridines except the first 5' uridine in the molecule are m1Ψ.

[0172] In some embodiments, the immunostimulatory activity of the modified RNA molecule comprising a 5' cap is significantly lower than that of the unmodified RNA molecule. In some embodiments, the immunostimulatory activity of the modified RNA molecule is at least 1 / 2 of that of the unmodified RNA molecule or the modified RNA molecule containing a different 5' cap (e.g., a 5' cap without unmodified uridine). In some embodiments, the immunostimulatory activity of the modified RNA molecule is about 1 / 2 to 1 / 20 of that of the unmodified RNA molecule or the modified RNA molecule containing a different 5' cap (e.g., a 5' cap without unmodified uridine). In some embodiments, the immunostimulatory activity of the modified RNA molecule is about 1.2, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 3, about 4, about 5, about 10, or about 20 times that of an unmodified RNA molecule or a modified RNA molecule containing a different 5' cap (e.g., a 5' cap without unmodified uridine). In some embodiments, the immunostimulatory activity of the modified RNA molecule is about 1.4 times that of an unmodified RNA molecule or a modified RNA molecule containing a different 5' cap (e.g., a 5' cap without unmodified uridine). In some embodiments, the immunostimulatory activity of the modified RNA molecule is about 1.9 times that of an unmodified RNA molecule or a modified RNA molecule containing a different 5' cap (e.g., a 5' cap without unmodified uridine).

[0173] In some embodiments, "significantly lower immunostimulation" refers to a detectable reduction in immunostimulation. In another embodiment, the term refers to a multiple reduction in immunostimulation. In some embodiments, "significantly lower immunostimulation" refers to a reduction to the extent that an effective amount of modified RNA molecules can be applied to cells without inducing a detectable immune response. In some embodiments, the term refers to a reduction to the extent that modified RNA molecules can be repeatedly administered without inducing an immune response that is sufficient to detectably reduce recombinant protein expression. In another embodiment, this reduction allows the modified RNA molecules to be repeatedly administered without inducing an immune response that is sufficient to eliminate detectable recombinant protein expression. The method for determining immunostimulation is well known in the art, and includes, for example, measuring the secretion of cytokines (e.g., IL-12, IFN-α, TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β or IL-8), measuring the expression of DC activation markers (e.g., CD83, HLA-DR, CD80 and CD86) or measuring the ability of an adjuvant as an adaptive immune response. In some embodiments, the immunostimulatory properties of the modified RNA molecules are measured by a RIG-I activation assay.

[0174] B. Modification

[0175] In addition to the first uridine (e.g., uridine introduced by the 5' cap), the RNA molecule may include one or more modifications. In some embodiments, the modification comprises a modified nucleoside. In some embodiments, the modified nucleoside is uridine (U). In some embodiments, the modified nucleoside is adenine (A). In some embodiments, the modified nucleoside is cytidine (C). In some embodiments, the modified nucleoside is guanine (G). In some embodiments, the nucleoside is a non-canonical nucleoside, such as inosine.

[0176] In some embodiments, the modified RNA molecule may include modified base forms. In some embodiments, purines and pyrimidines other than those commonly found in nature may be used. In some embodiments, the modified bases are selected from the group consisting of: 5 C(5-methylcytidine), m 5 U (5-methyluridine), m 6 A(N6-methyladenosine), S 2 U (2-thiouridine), Ψ (pseudouridine), Um (2′-O-methyluridine), Hi 1 A (1-methyladenosine), Hi 2 A (2-methyladenosine), Am (2′-O-methyladenosine), ms 2 m 6 A (2-methylthio-N6-methyladenosine), i 6 A(N6-isopentenyl adenosine), ms 2 i 6 A (2-methylthio-N6 isopentenyl adenosine), io 6 A(N6-(cis-hydroxyisopentenyl)adenosine), ms 2 io 6 A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g 6 A(N6-glycylcarbamoyladenosine), t 6 A(N6-threonylcarbamoyladenosine), ms 2 t 6 A (2-methylthio-N6-threonylcarbamoyl adenosine), m 6 t 6 A (N6-methyl-N6-threonylcarbamoyladenosine), Im6A (N6-hydroxynorvalylcarbamoyladenosine), ms 2 hn 6 A (2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine), Ar (p) (2′-O-ribosyl adenosine (phosphate)), I (inosine), Hi l I (1-methylinosine), Hi 1 Im (1,2'-O-dimethylinosine), m3 C(3-methylcytidine), Cm(2′-O-methylcytidine), S 2 C(2-thiocytidine), ac 4 C(N4-acetylcytidine), f 5 C(5-formylcytidine), m 5 Cm (5,2'-O-dimethylcytidine), ac 4 Cm (N4-acetyl-2'-O-methylcytidine), k 2 C (Lecidin), Hi 1 G (1-methylguanosine), m 2 G(N2-methylguanosine), m 7 G(7-methylguanosine), Gm(2′-O-methylguanosine), m 22 G(N2,N2-dimethylguanosine), m 2 Gm (N2, 2′-O-dimethylguanosine), m 22 Gm (N2, N2, 2′-O-trimethylguanosine), Gr (p) (2′-O-ribosylguanosine (phosphate)), yW (Huibutin), o 2 yW (peroxy hybutinoside), OHyW (hydroxy hybutinoside), OHyW* (undermodified hydroxy hybutinoside), imG (y-side), mimG (methyl y-side), Q (quercetin), oQ (epoxyquercetin), galQ (galactosyl-quercetin), manQ (mannosyl-quercetin), preQ0 (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaurine), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), S 4 U (4-thiouridine), m 5 s 2 U (5-methyl-2-thiouridine), S 2 Um (2-thio-2'-O-methyluridine), acp 3 U (3-(3-amino-3-carboxypropyl) uridine), ho 5 U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo 5 U (uridine 5-hydroxyacetic acid), mcmo5U (uridine 5-hydroxyacetic acid methyl ester), chm 5 U (5-(carboxyhydroxymethyl)uridine)), mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester), mcm 5 U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2′-O-methyluridine), mcm 5 s 2U (5-methoxycarbonylmethyl-2-thiouridine), nmVU (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine), mnm 5 s 2 U (5-methylaminomethyl-2-selenouridine), ncm 5 U (5-carbamoylmethyluridine), ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm 5 U (5-carboxymethylaminomethyluridine), cmnm 5 Um (5-carboxymethylaminomethyl-2′-0-methyluridine), cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine), m 6 2A (N6, N6-dimethyladenosine), Im (2′-O-methylinosine), m 4 C(N4-methylcytidine), m 4 Cm (N4, 2'-O-dimethylcytidine), hm 5 C(5-hydroxymethylcytidine), m 3 U (3-methyluridine), Cm 5 U (5-carboxymethyluridine), m 6 Am(N5,2′-O-dimethyladenosine), m 6 2Am(N6,N6,O-2t-trimethyladenosine), m 2，7 G(N 2 , 7-dimethylguanosine), m 2，2，7 G(N2,N2,7-trimethylguanosine), m 3 Um (3,2'-O-dimethyluridine), m 5 D(5-methyldihydrouridine), f 5 Cm (5-formyl-2'-O-methylcytidine), Hi 1 Gm (1,2'-O-dimethylguanosine), m 1 Am(1,2'-O-dimethyladenosine), τm 5 U (5-taurine methyluridine); Tm 5 s 2 U (5-taurine methyl-2-thiouridine)), imG-14 (4-demethylglucosidoside), imG 2 (isoglucosidase) and ac 6 A(N6-acetyladenosine).

[0177] In some embodiments, the modified base includes pseudouridine (5-ribosyluracil) (Ψ). In some embodiments, pseudouridine changes base pairing interactions, thereby affecting RNA secondary structure and mRNA coding. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1Ψ). In some embodiments, when Ψ and m1Ψ are present in in vitro transcribed mRNA in a host cell, the immunostimulatory properties of the modified RNA are reduced.

[0178] In some embodiments, between about 0.5% and 99% of the uridines in the modified RNA molecule are modified. In some embodiments, about 0.5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% of the uridines in the modified RNA molecule are modified. In some embodiments, at least one uridine other than the first 5' uridine in the molecule is a modified uridine. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are modified. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are Ψ. In some embodiments, at least about 50% of the uridines other than the first 5' uridine in the molecule are m1Ψ.

[0179] In some embodiments, all uridines except the first 5' uridine in the molecule are modified. In some embodiments, all uridines except the first 5' uridine in the molecule are Ψ-modified. In some embodiments, all uridines except the first 5' uridine in the molecule are m1Ψ.

[0180] In some embodiments, the modified RNA molecule comprises one or more modifications to the phosphate backbone and / or modifications to the sugar. In some embodiments, the modification is selected from the group consisting of MOE, 2'-OMe, LNA, GalNAc, 5' methylcytosine, phosphorothioate bonds, alkylphosphonate bonds, phosphoramidate bonds, boranophosphate bonds and / or morpholine rings.

[0181] C. Coding sequence

[0182] In some embodiments, the modified RNA molecule comprises an open reading frame containing a coding sequence. In some embodiments, the open reading frame encodes a recombinant protein. In some embodiments, the open reading frame encodes one or more recombinant proteins. In some embodiments, the open reading frame encodes a protein of interest. In some embodiments, the protein of interest is the only protein encoded in the coding sequence.

[0183] In some embodiments, the open reading frame encodes any known amino acid. In some embodiments, the open reading frame encodes an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine ​​and pyrrolysine.

[0184] In some embodiments, the open reading frame encodes a recombinant protein. In some embodiments, the recombinant protein is selected from the group consisting of: enzymes, structural proteins, transport proteins, receptors, hormones, antibodies, transcription factors, growth factors, cytokines, motor proteins, structural proteins and chaperones. In some embodiments, the enzyme is selected from the group consisting of: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, polymerases, kinases, phosphatases, proteases, lipases and amylases.

[0185] In some embodiments, the open reading frame encodes a therapeutic payload comprising a compound capable of inducing immunity to one or more target disorders or diseases. In some embodiments, the target disorder is associated with or caused by infection with a pathogen such as a coronavirus (e.g., 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. In some embodiments, the open reading frame encodes a pathogenic protein characteristic of a pathogen or an antigenic fragment or epitope thereof. In some embodiments, the target disorder is associated with or caused by tumor growth (e.g., cancer) of a cell. In some embodiments, the open reading frame encodes a tumor-associated antigen (TAA) or an antigenic fragment or epitope thereof characteristic of a cancer.

[0186] In some embodiments, the open reading frame encodes a reporter protein. In some embodiments, the reporter protein is a fluorescent protein. In some embodiments, the reporter protein is eGFP. In some embodiments, the open reading frame encoding the reporter protein comprises the sequence listed in SEQ ID NO: 1-2. In some embodiments, the open reading frame encoding the reporter protein comprises the sequence listed in SEQ ID NO: 18-21 or 31-34.

[0187] In some embodiments, the open reading frame encodes a viral protein. In some embodiments, the open reading frame encodes a protein from the SARS-COV-2 genome. In some embodiments, the open reading frame encodes the SARS-COV-2 Omicron BA.4 and BA.5 receptor binding domains (RBDs). In some embodiments, the open reading frame encoding the SARS-COV-2 BA.4 / BA.5 RBD comprises the sequence listed in SEQ ID NO:3-4.

[0188] In some embodiments, the open reading frame encodes a rabies virus (RABV) antigen. In some embodiments, the open reading frame encoding the rabies virus antigen comprises the sequence listed in SEQ ID NO: 5-6. In some embodiments, the open reading frame encoding the rabies virus antigen comprises the sequence listed in SEQ ID NO: 15-17 or 28-30.

[0189] In some embodiments, the open reading frame encodes a respiratory syncytial virus (RSV) antigen. In some embodiments, the open reading frame encoding a respiratory syncytial virus antigen comprises a sequence listed in SEQ ID NO: 7-8. In some embodiments, the open reading frame encoding a respiratory syncytial virus antigen comprises a sequence listed in SEQ ID NO: 9-11 or 22-24.

[0190] In some embodiments, the open reading frame encodes a varicella-zoster virus (VZV) antigen.In some embodiments, the open reading frame encoding a respiratory syncytial virus antigen comprises the sequence set forth in SEQ ID NO: 12-14 or 25-27.

[0191] In some embodiments, the open reading frame comprises a coding sequence containing modifications to the ribose backbone and / or nitrogenous bases. In some embodiments, the coding sequence also comprises at least one modified G, C or A. In some embodiments, the modified RNA molecule comprises a modified backbone. In some embodiments, the modified backbone comprises at least one phosphorothioate bond.

[0192] In some embodiments, the present invention provides a method of inducing translation of a recombinant protein in a mammalian cell, comprising contacting the mammalian cell with an in vitro transcribed RNA molecule encoding the recombinant protein, thereby producing the recombinant protein in the transfected cell.

[0193] V. Pharmaceutical Compositions

[0194] In some embodiments, the modified RNA molecule is formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier includes any vehicle capable of delivering the modified RNA molecule to a cell. In some embodiments, the pharmaceutically acceptable carrier includes lipid nanoparticles (LNP), lipid complexes or liposomes. In some embodiments, the lipid complex is a nano-sized lipid-based material formed by spontaneous self-assembly of cationic liposomes and nucleic acids. In some embodiments, a liposome is an artificial vesicle having at least one lipid bilayer.

[0195] ALipid Nanoparticles (LNP)

[0196] In some embodiments, the pharmaceutical composition comprises LNP. In some embodiments, the modified RNA is formulated in LNP, such as those described in International Publication No. WO2012170930, which is incorporated herein by reference in its entirety. In some embodiments, the particle size of the LNP can be increased and / or reduced. The change in particle size may help to offset biological reactions, such as, but not limited to, inflammation, or may increase the biological effect of mRNA when applied to an individual.

[0197] In some embodiments, LNP comprises about 30 mol % to about 55 mol % of cationic lipids. In some embodiments, LNP comprises greater than about 30 mol % of cationic lipids, for example greater than about 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol % or more of any cationic lipids. In some embodiments, LNP comprises less than about 55 mol % of cationic lipids, for example less than about 50 mol %, 45 mol %, 40 mol %, 35 mol %, 30 mol % or still less of any cationic lipids.

[0198] In some embodiments, LNP comprises about 5 mol % to about 40 mol % of phospholipids. In some embodiments, LNP comprises greater than about 5 mol % of phospholipids, for example, greater than about 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol % or more of any phospholipids. In some embodiments, LNP comprises less than about 40 mol % of phospholipids, for example, less than about 35 mol %, 30 mol %, 25 mol %, 20 mol %, 15 mol %, 10 mol %, 5 mol % or still less of any phospholipids.

[0199] In some embodiments, LNP comprises the sterol of about 20 mol % to about 50 mol %.In some embodiments, LNP comprises the sterol greater than about 20 mol %, for example greater than about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol % or any one of more sterol.In some embodiments, LNP comprises the sterol less than about 50 mol %, for example less than about 45 mol %, 40 mol %, 35 mol %, 30 mol %, 25 mol %, 20 mol % or any one of less sterol.

[0200] In some embodiments, LNP comprises cationic lipid, phospholipid, sterol and polymer conjugated lipid, for example any cationic lipid, phospholipid, sterol and polymer conjugated lipid described herein.In some embodiments, LNP comprises i) about 30 mol % to about 55 mol % cationic lipid, ii) about 5 mol % to about 40 mol % phospholipid.

[0201] In some embodiments, the weight ratio of total lipids to modified RNA included in LNP is about 10: 1 to about 30: 1, such as any of the following: about 10: 1 to about 20: 1, about 15: 1 to about 25: 1, and about 20: 1 to about 30: 1. In some embodiments, the weight ratio of total lipids to modified RNA included in LNP is greater than about 10: 1, such as greater than any of the following: about 15.1, 20: 1, 25: 1, 30: 1 or greater. In some embodiments, the weight ratio of total lipids to modified RNA included in LNP is less than about 30: 1, such as less than any of the following: about 25: 1, 20: 1, 15: 1, 10: 1 or less. In some embodiments, the weight ratio of total lipids to mRNA can be adjusted according to other components of the pharmaceutical composition, the individual to be administered, and / or the route of administration. For example, the amount of mRNA in LNP can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0202] In some embodiments, the LNP disclosed herein further comprises a therapeutic payload. The payload can be any substance or compound with a therapeutic or preventive effect. In some embodiments, the therapeutic payload is a small molecule, a cytotoxin, a radioactive ion, a chemotherapeutic compound, a vaccine, or a compound that induces an immune response.

[0203] In some embodiments, LNP disclosed herein comprises nucleic acid. In some embodiments, nucleic acid is DNA. In some embodiments, DNA is catalytic DNA, plasmid DNA, aptamer or complementary DNA (cDNA). In some embodiments, nucleic acid is RNA. In some embodiments, RNA is messenger RNA (mRNA), antisense oligonucleotide, micro RNA (miRNA), miRNA inhibitor (e.g., antagomir or antimir), messenger RNA interference complementary RNA (micRNA), multivalent RNA, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), antisense RNA, transfer RNA (tRNA), asymmetric interference RNA (aiRNA), ribozyme, aptamer or carrier. In some embodiments, RNA is mRNA hybrid. In some embodiments, nucleic acid is mRNA. In some embodiments, mRNA encodes protein. In some embodiments, protein is antibody. In some embodiments, antibody is bispecific antibody. In some embodiments, LNP comprises RNAi agent or RNAi inducer.

[0204] B. Cationic lipids

[0205] In one embodiment, the cationic lipid contained in the composition, nanoparticle composition, or nanoparticle described herein is a cationic lipid described in International Patent Publication No. WO2021204175, which is incorporated herein by reference in its entirety.

[0206] In one embodiment, the cationic lipid is a compound of formula (01-I):

[0207]

[0208] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0209] G1 and G2 are each independently a bond, a C2-C12 alkylene group or a C2-C12 alkenylene group, wherein one or more -CH2- in the alkylene group or alkenylene group is optionally replaced by -O-;

[0210] L1 is -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), -(C6-C10 arylene)-R1, -(6- to 10-membered heteroarylene)-R1 or R1;

[0211] L2 is -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), -(C6-C10 arylene)-R2, -(6- to 10-membered heteroarylene)-R2 or R2;

[0212] R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

[0213] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0214] Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

[0215] G3 is C2-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0216] R3 is -N(R4)R5;

[0217] R4 is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4- to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3, or a portion of G3 together with the nitrogen to which they are attached form a cyclic moiety;

[0218] R5 is C1-C12 alkyl or C3-C8 cycloalkyl; or R4, R5 together with the nitrogen to which they are attached form a cyclic moiety;

[0219] x is 0, 1, or 2; and

[0220] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene and cyclic moiety is independently optionally substituted.

[0221] In one embodiment, the cationic lipid is a compound of formula (01-II):

[0222]

[0223] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0224] is a single bond or a double bond;

[0225] G1 and G2 are each independently a bond, a C2-C12 alkylene group or a C2-C12 alkenylene group, wherein one or more -CH2- in the alkylene group or alkenylene group is optionally replaced by -O-;

[0226] L1 is -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), -(C6-C10 arylene)-R1, -(6- to 10-membered heteroarylene)-R1 or R1;

[0227] L2 is -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), -(C6-C10 arylene)-R2, -(6- to 10-membered heteroarylene)-R2 or R2;

[0228] R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

[0229] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0230] Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

[0231] G4 is a bond, C1-C23 alkylene, C2-C23 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0232] R3 is -N(R4)R5;

[0233] R4 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4- to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3, or a portion of G3 together with the nitrogen to which they are attached form a cyclic moiety;

[0234] R5 is C1-C12 alkyl or C3-C8 cycloalkyl; or R4, R5 together with the nitrogen to which they are attached form a cyclic moiety;

[0235] x is 0, 1, or 2; and

[0236] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene and cyclic moiety is independently optionally substituted.

[0237] In one embodiment, the compound is of formula (01-IB), (01-I-B'), (01-IB"), (01-IC), (01-ID) or (01-IE):

[0238]

[0239] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0240] In one embodiment, G1 and G2 are each independently C3-C7 alkylene. In one embodiment, G1 and G2 are each independently C5 alkylene. In one embodiment, G3 is C2-C4 alkylene. In one embodiment, G3 is C2 alkylene. In one embodiment, G3 is C4 alkylene.

[0241] In one embodiment, R3 has one of the following structures:

[0242]

[0243]

[0244] In one embodiment, R1, R2, Rc and Rf are each independently a branched C6-C32 alkyl or a branched C6-C32 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently a branched C6-C24 alkyl or a branched C6-C24 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently a branched C6-C24 alkyl or a branched C6-C24 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently a-R7-CH(R8)(R9), wherein R7 is a C0-C5 alkylene, and R8 and R9 are independently a C2-C10 alkyl. In one embodiment, R1, R2, Rc and Rf are each independently a-R7-CH(R8)(R9), wherein R7 is a C0-C1 alkylene, and R8 and R9 are independently a C4-C8 alkyl.

[0245] In one embodiment, the compound is a compound in Table 1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0246] Table 1:

[0247]

[0248]

[0249]

[0250] In one embodiment, the cationic lipid contained in the composition, nanoparticle composition or nanoparticle provided herein is a cationic lipid described in International Patent Publication No. WO2023 / 138611, which is incorporated herein by reference in its entirety. In one embodiment, the cationic lipid is a compound of formula (02-I):

[0251]

[0252] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0253] G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more -CH2- in G1 and G2 are optionally replaced by -O-, -C(=O)O- or OC(=O)-;

[0254] each L1 is independently -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), -NRaP(=O)(ORb)(ORc);

[0255] each L2 is independently -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), -NRdP(=O)(ORe)(ORf);

[0256] R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0257] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0258] Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

[0259] G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein a part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0260] R3 is -N(R4)R5, -OR6 or -SR6;

[0261] R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0262] R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0263] R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C10 aryl;

[0264] x is 0, 1, or 2: and

[0265] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene and cycloalkenylene is independently optionally substituted.

[0266] In one embodiment, the cationic lipid is a compound of formula (02-II):

[0267]

[0268] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0269] G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more -CH2- in G1 and G2 are optionally replaced by -O-, -C(=O)O- or OC(=O)-;

[0270] each L1 is independently -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), -NRaP(=O)(ORb)(ORc);

[0271] each L2 is independently -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), -NRdP(=O)(ORe)(ORf);

[0272] R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0273] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0274] Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

[0275] G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein a part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0276] R3 is -N(R4)R5, -OR6 or -SR6;

[0277] R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0278] R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0279] R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C10 aryl;

[0280] x is 0, 1, or 2: and

[0281] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene and cycloalkenylene is independently optionally substituted.

[0282] In one embodiment, the compound is of formula (02-VA), (02-VB), (02-VC), (02-VD), (02-VE), (02-VF):

[0283]

[0284]

[0285] wherein z is an integer from 2 to 12;

[0286] x0 is an integer from 1 to 11;

[0287] y0 is an integer from 1 to 11;

[0288] x1 is an integer from 0 to 9;

[0289] y1 is an integer from 0 to 9;

[0290] x2 is an integer from 2 to 5;

[0291] x3 is an integer from 1 to 5;

[0292] x4 is an integer from 0 to 3;

[0293] y2 is an integer from 2 to 5;

[0294] y3 is an integer from 1 to 5; and

[0295] y4 is an integer from 0 to 3;

[0296] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0297] In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4 or 5. In one embodiment, x0 and y0 are independently 2 to 6. In one embodiment, x0 and y0 are independently 4 or 5. In one embodiment, x1 and y1 are independently 2 to 6. In one embodiment, x1 and y1 are independently 4 or 5. In one embodiment, x2 and y2 are independently an integer from 2 to 5. In one embodiment, x2 and y2 are independently 3 or 5. In one embodiment, x3 and y3 are both 1. In one embodiment, x4 and y4 are independently 0 or 1.

[0298] In one embodiment, each L1 is independently -OR1, -OC(=O)R1 or -C(=O)OR1, and each L2 is independently -OR2, -OC(=O)R2 or -C(=O)OR2. In one embodiment, R1 and R2 are independently linear C6-C10 alkyl or -R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl.

[0299] In one embodiment, the compound is of formula (02-VI-A), (02-VI-B), (02-VI-C), (02-VI-D), (02-VI-E) or (02-VI-F):

[0300]

[0301]

[0302] wherein z is an integer from 2 to 12;

[0303] y is an integer from 2 to 12;

[0304] x0 is an integer from 1 to 11;

[0305] x1 is an integer from 0 to 9;

[0306] x2 is an integer from 2 to 5;

[0307] x3 is an integer from 1 to 5; and

[0308] x4 is an integer from 0 to 3;

[0309] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0310] In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4 or 5. In one embodiment, x0 is 4 or 5. In one embodiment, x1 is 4 or 5. In one embodiment, x2 is an integer from 2 to 5. In one embodiment, x2 is 3 or 5. In one embodiment, x3 is 0 or 1. In one embodiment, y is an integer from 2 to 6. In one embodiment, y is 5.

[0311] In one embodiment, each L1 is independently -OR1, -OC(=O)R1 or -C(=O)OR1, and L2 is -OC(=O)R2 or -C(=O)OR2, -NRdC(=O)R2 or -C(=O)NReRf. In one embodiment, R1 is a straight chain C6-C10 alkyl or -R7-CH(R8)(R9), wherein R7 is a C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, R2 and Rf are each independently a straight chain C6-C18 alkyl, C6-C18 alkenyl or -R7-CH(R8)(R9), wherein R7 is a C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, Rd and Re are each independently H.

[0312] In one embodiment, the compound is a compound in Table 2, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof. Table 2:

[0313]

[0314] In one embodiment, the cationic lipid contained in the composition, nanoparticle composition, or nanoparticle described herein is a cationic lipid described in International Patent Publication No. WO2022152109, which is incorporated herein by reference in its entirety.

[0315] In one embodiment, the cationic lipid is a compound of formula (03-I):

[0316]

[0317] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0318] G1 and G2 are each independently a bond, a C2-C12 alkylene group or a C2-C12 alkenylene group, wherein one or more -CH2- in G1 and G2 are optionally replaced by -O-;

[0319] each L1 is independently -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), NRaP(=O)(ORb)(ORc), -(C6-C10 arylene)-R1, -(6- to 10-membered heteroarylene)-R1, -(4- to 8-membered heterocyclylene)-R1, or R1;

[0320] Each L2 is independently -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReR f, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), NRdP(=O)(ORe)(ORf), -(C6-C10 arylene)-R2, -(6- to 10-membered heteroarylene)-R2, -(4- to 8-membered heterocyclylene)-R2, or R2;

[0321] R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0322] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0323] Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

[0324] G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein a part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4- to 8-membered heterocyclylene, C6-C10 arylene or 5- to 10-membered heteroarylene;

[0325] R3 is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4-8 membered heterocyclyl, C6-C10 aryl or 5-10 membered heteroaryl; or R3, G1 or a portion of G1 together with the nitrogen to which they are attached form a cyclic moiety; or R3, G3 or a portion of G3 together with the nitrogen to which they are attached form a cyclic moiety;

[0326] R4 is a C1-C12 alkyl group or a C3-C8 cycloalkyl group;

[0327] x is 0, 1, or 2;

[0328] n is 1 or 2;

[0329] m is 1 or 2; and

[0330] wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0331] In one embodiment, the compound is of formula (03-II-A):

[0332]

[0333] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0334] In one embodiment, the compound is of formula (03-II-B):

[0335]

[0336] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0337] In one embodiment, the compound is of formula (03-II-C):

[0338]

[0339] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0340] In one embodiment, the compound is of formula (03-II-D):

[0341]

[0342] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0343] In one embodiment, G1 and G2 are each independently C2-C12 alkylene. In one embodiment, G1 and G2 are each independently C5 alkylene. In one embodiment, G3 is C2-C6 alkylene.

[0344] In one embodiment, R3 is C1-C12 alkyl, C2-C12 alkenyl or C3-C8 cycloalkyl. In one embodiment, R3 is C3-C8 cycloalkyl. In one embodiment, R3 is unsubstituted. In one embodiment, R4 is substituted C1-C12 alkyl. In one embodiment, R4 is -CH2CH2OH.

[0345] In one embodiment, L1 is -OC(=O)R1, -C(=O)OR1, -NRaC(=O)R1 or -C(=O)NRbRc, and L2 is -OC(=O)R2, -C(=O)OR2, -NRdC(=O)R2 or -C(=O)NReRf. In one embodiment, R1, R2, Rc and Rf are each independently a linear C6-C18 alkyl, a linear C6-C18 alkenyl or -R7-CH(R8)(R9), wherein R7 is a C0-C5 alkylene, and R8 and R9 are independently a C2-C10 alkyl or a C2-C10 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently a linear C7-C15 alkyl, a linear C7-C15 alkenyl or -R7-CH(R8)(R9), wherein R7 is a C0-C1 alkylene, and R8 and R9 are independently C4-C8 alkyl or C6-C10 alkenyl. In one embodiment, Ra, Rb, Rd and Re are each independently H.

[0346] In one embodiment, the compound is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0347] Table 3:

[0348]

[0349]

[0350] In one embodiment, the cationic lipid contained in the particle or composition provided herein is a cationic lipid described in International Patent Publication No. WO2022247755A1, which is incorporated herein by reference in its entirety.

[0351] In one embodiment, the cationic lipid is a compound of formula (04-I):

[0352]

[0353] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0354] G1 and G2 are each independently a bond, a C2-C12 alkylene group or a C2-C12 alkenylene group;

[0355] L1 is -OC(=O)R1, -C(=O)OR1, -OC(=O)OR1, -C(=O)R1, -OR1, -S(O)xR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, -NRaC(=O)OR1, -SC(=S)R1, -C(=S)SR1, -C(=S)R1, -CH(OH)R1, -P(=O)(ORb)(ORc), -(C6-C10 arylene)-R1, -(6- to 10-membered heteroarylene)-R1 or R1;

[0356] L2 is -OC(=O)R2, -C(=O)OR2, -OC(=O)OR2, -C(=O)R2, -OR2, -S(O)xR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf, -NRdC(=O)OR2, -SC(=S)R2, -C(=S)SR2, -C(=S)R2, -CH(OH)R2, -P(=O)(ORe)(ORf), -(C6-C10 arylene)-R2, -(6- to 10-membered heteroarylene)-R2 or R2;

[0357] R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

[0358] Ra, Rb, Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;

[0359] Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

[0360] R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0361] G3 is C2-C12 alkylene or C2-C12 alkenylene;

[0362] R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0363] R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0364] x is 0, 1, or 2;

[0365] s is 0 or 1; and

[0366] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene and heteroarylene is independently optionally substituted.

[0367] In one embodiment, the cationic lipid is a compound of formula (04-III):

[0368]

[0369] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0370] R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

[0371] R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0372] G3 is C2-C12 alkylene or C2-C12 alkenylene;

[0373] G4 is C2-C12 alkylene or C2-C12 alkenylene;

[0374] R3 is -N(R4)R5 or -OR6;

[0375] R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4- to 8-membered heterocycloalkyl;

[0376] R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4-8 membered heterocycloalkyl; or R4, R5 together with the nitrogen to which they are attached form a cyclic moiety;

[0377] R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C10 aryl; and

[0378] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene and cyclic moiety is independently optionally substituted.

[0379] In one embodiment, the compound is of formula (04-IV):

[0380]

[0381] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0382] In one embodiment, G3 is C2-C4 alkylene. In one embodiment, G4 is C2-C4 alkylene.

[0383] In one embodiment, R0 is C1-C6 alkyl. In one embodiment, R3 is -OH. In one embodiment, R3 is -N(R4)R5. In one embodiment, R4 is C3-C8 cycloalkyl. In one embodiment, R4 is unsubstituted. In one embodiment, R5 is -CH2CH2OH.

[0384] In one embodiment, L1 is -OC(=O)R1, -C(=O)OR1, -C(=O)R1, -C(=O)NRbRc or R1; and L2 is -OC(=O)R2, -C(=O)OR2, -C(=O)R2, -C(=O)NReRf or R2. In one embodiment, R1 and R2 are each independently a branched C6-C24 alkyl or a branched C6-C24 alkenyl. In one embodiment, R1 and R2 are each independently -R7-CH(R8)(R9), wherein R7 is a C1-C5 alkylene group, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, R1 is a straight chain C6-C24 alkyl group, and R2 is a branched C6-C24 alkyl group. In one embodiment, R1 is a linear C6-C24 alkyl group, and R2 is -R7-CH(R8)(R9), wherein R7 is a C1-C5 alkylene group, and R8 and R9 are independently C2-C10 alkyl groups.

[0385] In one embodiment, the compound is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof. Table 4:

[0386]

[0387]

[0388] C. Kit

[0389] The present disclosure includes a kit comprising components for in vitro transcription of modified RNA disclosed herein. In some embodiments, the kit comprises 1) a DNA-dependent RNA polymerase; 2) modified and / or unmodified nucleotides (e.g., modified and / or unmodified adenosine, guanosine, cytidine, or uridine), and 3) a 5' cap, and 4) instructions for performing the methods disclosed herein. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has a sequence of N1pppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has m 7 GpppApU sequence, wherein A is modified or unmodified. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has m 7 GpppApU sequence, wherein A is 2'-O-methylated. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has m 7 GpppA m In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has a sequence of N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has a sequence of m 7 GvpppApU sequence, wherein A is modified or unmodified. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has m 7 GvpppApU sequence, wherein A is 2'-O-methylated. In some embodiments, the kit further comprises a 5' cap, wherein the 5' cap has m 7 Gvppp m The sequence of pU.

[0390] In some embodiments, the kit further comprises a modified nucleotide. In some embodiments, the kit further comprises at least one modified G, C or A. In some embodiments, the kit further comprises a modified uridine. In some embodiments, the kit further comprises a modified uridine, wherein the modified uridine comprises Ψ. In some embodiments, the kit further comprises a modified uridine, wherein the modified uridine comprises m1Ψ.

[0391] VI. Methods of reducing innate immune stimulation

[0392] Provided herein are methods for reducing innate immune stimulation of RNA molecules.

[0393] In some embodiments, described herein are methods of reducing innate immune stimulation of an RNA molecule comprising using a 5' cap having a sequence of N1pppN2pU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine.

[0394] In some embodiments, the 5' cap has a sequence of GpppApU, wherein A is modified or unmodified. In some embodiments, A is 2'-O-methylated. In some embodiments, the 5' cap has a sequence of m 7 In some embodiments, the 5' cap has m 7 In some embodiments, the 5' cap has m 7 GpppA m In some embodiments, the 5' cap comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, or 8. In some embodiments, the 5' cap comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 9, 12, 15, 18, 22, 25, 28, or 31. In some embodiments, the 5' cap comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 12, 15, 18, 22, 25, 28, or 31.

[0395] In some embodiments, m 7 GpppA m pU has the following formula,

[0396]

[0397] In some embodiments, described herein are methods of reducing innate immune stimulation of an RNA molecule comprising using a 5' cap having a sequence of N1vpppN2pU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine.

[0398] In some embodiments, the 5' cap has a sequence of GvpppApU, wherein A is modified or unmodified. In some embodiments, A is 2'-O-methylated. In some embodiments, the 5' cap has m 7In some embodiments, the 5' cap has m 7 In some embodiments, A is 2'-O-methylated. In some embodiments, the 5' cap has m 7 Gvppp m In some embodiments, the 5' cap comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 21 or 34.

[0399] In some embodiments, m 7 Gvppp m pU has the following formula,

[0400]

[0401] In some embodiments, the method comprises modifying between about 1% and about 99% of the uridine bases in the RNA molecule. In some embodiments, the method comprises modifying about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridine in the RNA molecule. In some embodiments, the method comprises modifying at least about 50% of the uridine in the RNA molecule except the first 5' uridine. In some embodiments, the method comprises modifying at least about 90% of the uridine in the RNA molecule except the first 5' uridine. In some embodiments, the method comprises modifying at least about 95% of the uridine in the RNA molecule except the first 5' uridine. In some embodiments, the method comprises modifying at least about 99% of the uridine in the RNA molecule except the first 5' uridine.

[0402] In some embodiments, the methods include modifying all but the first 5' uridine in the RNA molecule.

[0403] In some embodiments, the method comprises modifying between about 1% and about 99% of the uridine bases in an RNA molecule to Ψ. In some embodiments, the method comprises modifying about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridines in an RNA molecule to Ψ. In some embodiments, the method comprises modifying at least about 50% of the uridines in an RNA molecule other than the first 5' uridine to Ψ. In some embodiments, the method comprises modifying at least about 90% of the uridines in an RNA molecule other than the first 5' uridine to Ψ. In some embodiments, the method comprises modifying at least about 95% of the uridines in an RNA molecule other than the first 5' uridine to Ψ. In some embodiments, the method comprises modifying at least about 99% of the uridines in an RNA molecule other than the first 5' uridine to Ψ. In some embodiments, the method comprises modifying all uridines in an RNA molecule other than the first 5' uridine to Ψ.

[0404] In some embodiments, the method comprises modifying between about 1% and about 99% of the uridine bases in an RNA molecule to m1Ψ. In some embodiments, the method comprises modifying about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the uridines in an RNA molecule to m1Ψ. In some embodiments, the method comprises modifying at least about 50% of the uridines in an RNA molecule other than the first 5' uridine to m1Ψ. In some embodiments, the method comprises modifying at least about 90% of the uridines in an RNA molecule other than the first 5' uridine to m1Ψ. In some embodiments, the method comprises modifying at least about 95% of the uridines in an RNA molecule other than the first 5' uridine to m1Ψ. In some embodiments, the method comprises modifying at least about 99% of the uridines in an RNA molecule other than the first 5' uridine to m1Ψ. In some embodiments, the method comprises modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ.

[0405] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GpppApU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0406] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GpppApU and modifying all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0407] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GpppApU and modifying all uridines except the first 5' uridine in the RNA molecule to Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0408] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GpppApU and modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0409] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppApU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0410] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppApU and modifies all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0411] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppApU and modify all but the first 5' uridine in the RNA molecule to Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0412] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppApU and modify all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0413] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppA mpU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0414] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppA m pU and modifies all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0415] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppA m pU and modify all but the first 5' uridine in the RNA molecule to Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0416] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GpppA m pU and modify all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0417] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GvpppApU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0418] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GvpppApU and modifying all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0419] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GvpppApU and modifying all uridines except the first 5' uridine in the RNA molecule to Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0420] In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5' cap having a sequence of GvpppApU and modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0421] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GvpppApU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0422] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GvpppApU and modifies all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0423] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GvpppApU and modify all uridines except the first 5' uridine in the RNA molecule to Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0424] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 GvpppApU and modify all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0425] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 Gvppp m pU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0426] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 Gvppp mpU and modifies all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0427] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 Gvppp m pU and modify all but the first 5' uridine in the RNA molecule to ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0428] In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 7 Gvppp m pU and modify all uridines except the first 5' uridine in the RNA molecule to m1Ψ to produce an RNA molecule, wherein the RNA molecule is not self-replicable and wherein U is an unmodified uridine.

[0429] In some embodiments, the method for producing an RNA molecule for reducing innate immune stimulation of the RNA molecule as described above comprises a method for in vitro transcribing a DNA molecule into a modified RNA molecule disclosed herein.

[0430] In other aspects, use of a modified RNA molecule, pharmaceutical composition, method, modified RNA molecule or kit disclosed herein for reducing innate immune stimulation of RNA delivery is provided.

[0431] It should be understood that any embodiment of the compound provided herein as stated above, and any specific substituent and / or variable of the compound provided herein as stated above can be independently combined with other embodiments and / or substituents and / or variables of the compound to form the above embodiments not specifically stated. In addition, in the case of listing the substituent and / or variable list of any specific group or variable, it should be understood that each individual substituent and / or variable can be deleted from the specific embodiment and / or claim, and the remaining substituent and / or variable list will be considered to be within the scope of the embodiment provided herein.

[0432] It is understood that in the present specification, combinations of substituents and / or variables in the various formulae depicted are permissible only if such contributions result in stable compounds.

[0433] Beneficial Effects

[0434] In one aspect, the modified RNA molecules, pharmaceutical compositions thereof, and uses of the modified RNA molecules produced by the methods and kits herein will achieve at least one of the following:

[0435] (1) Capping efficiency will be improved;

[0436] (2) RIG-I activation will be reduced, i.e. immune stimulation will be significantly reduced, resulting in reduced innate immune stimulation when delivered to cells:

[0437] (3) Low toxicity, high yield and high purity will be achieved;

[0438] (4) Antigen-specific IgG titers will be enhanced.

[0439] Example

[0440] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0441] Example 1: Preparation of mRNA by in vitro transcription and detection of various indicators

[0442] The scope of the present disclosure is not intended to be limited to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. According to the description and teachings herein, various modifications to the compositions and methods will become apparent. Such changes may be practiced without departing from the true scope and spirit of the present disclosure, and such changes are intended to fall within the scope of the present disclosure.

[0443] This example describes the in vitro transcription of modified RNA molecules and the analysis of mRNA purity, 5' capping efficiency, and immunostimulatory properties.

[0444] Plasmid linearization: DNA plasmid templates containing DNA encoding eGFP, SARS-COV-2 Omicron BA.4 / 5RBD were linearized using restriction endonucleases. Purification was performed by adding 1 / 10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, washed twice with 70% ethanol, and resuspended in nuclease-free water.

[0445] In vitro transcription

[0446] The purified linear plasmid was used as a template for in vitro transcription using T7 RNA polymerase. 7 GpppA m pU or m 7 GpppA m pG was used for in vitro transcription to synthesize mRNA with Cap1 "cap" structure and m1Ψ was used instead of uridine to synthesize mRNA.

[0447] For m7GpppAmpU, the concentrations of ATP / GTP / CTP / m1Ψ were 5 mM each; the concentration of m7GpppAmpU was 4 mM; the concentration of 10X reaction buffer [400 mM Tris-HCl (pH 7.9), 100 mM DTT, 20 mM spermidine, 0.02% Triton X-100, 270 mM magnesium acetate] was 1X; the concentration of DNA template was 50 μg / mL; the concentration of mouse RNase inhibitor was 1 unit / μL; the concentration of yeast inorganic pyrophosphatase was 0.002 unit / μL; and the concentration of T7 RNA polymerase was 5 units / μL. For m7GpppAmpG, the concentrations of ATP / GTP / CTP / m1Ψ were 5 mM each; the concentration of m7GpppAmpG was 4 mM; the concentration of 10X reaction buffer [400 mM Tris-HCl (pH 7.9), 100 mM DTT, 20 mM spermidine, 0.02% Triton X-100, 270 mM magnesium acetate] was 1X; the concentration of DNA template was 50 μg / mL; the concentration of mouse RNase inhibitor was 1 unit / μL; the concentration of yeast inorganic pyrophosphatase was 0.002 unit / μL; and the concentration of T7 RNA polymerase was 5 units / μL.

[0448] The reaction was allowed to proceed for 2.5 h, followed by DNase I digestion (DNase I was added to the IVT reaction at a final concentration of 2 units / μL and incubated at 37°C for 30 min). mRNA was purified using RNA purification kit (NEB, #T2050S), quantified by UV using NanoDrop One, analyzed for purity using a fragment analyzer, and examined for capping efficiency using LC-MS.

[0449] Results: As shown in Table 5, based on m 7 GpppA m The capping rate of mRNA synthesized by pU was higher than that based on m 7 GpppA m The capping rate of the mRNA synthesized by pG and the purity are basically the same.

[0450] Table 5:

[0451]

[0452] RIG-I activation studies were performed using mRNA synthesized with two cap analogs:

[0453] A reporter cell line overexpressing RIG-I was used to detect differences in RIG-I mRNA secretion. HEK Lucia RIG-I cells were purchased from Invivogen (catalog number hkl-hrigi), and the cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Before transfection, cells were seeded in 96-well plates (in 100 μl of culture medium) at a density of 20,000 cells / well until cell fusion reached 80% or more for transfection, and 250 ng of mRNA was transfected using Lipofectamine 2000 (Invitrogen, catalog number 11668-019). 50 μL of cell supernatant was taken 24 hours after transfection. The cell supernatant was collected into a new 96-well plate, and 10 μL of detection reagent QUANTI-Luc was added to each well. TM The OD values ​​at 560 nm were read using a Molecular Devices plate reader and the data were analyzed.

[0454] Results: As shown in Table 6, based on m 7 GpppA m RIG-I activation of mRNA synthesized by pU is lower than that of m 7 GpppA m RIG-I activation of mRNA synthesized by pG.

[0455] Table 6:

[0456]

[0457]

[0458] Cytotoxicity studies using mRNA synthesized with two cap analogs:

[0459] HEK-293T and A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and seeded in 96-well plates, 100 μl cell suspension per well (5000 cells / well). The plate was pre-incubated in a humidified incubator for 24 hours (37°C, 5% CO2), and then 10 μl of each mRNA was added to the well. The plate was incubated in an incubator for 24 hours, 10 μl CCK-8 solution (Catalog Number: RM02823, abclonal) was added to each well of the plate, and then the plate was incubated on a shaker in an incubator for 1-4 hours. Mix gently. The absorbance at 450 nm was then measured using an enzyme marker.

[0460] Results: As shown in Table 7, there was no significant difference in the toxicity of the two mRNAs to the two cell lines.

[0461] Table 7:

[0462]

[0463] Example 2: Example of mRNA vaccine based on rabies virus

[0464] This example describes the in vitro transcription of a rabies virus-based mRNA vaccine.

[0465] According to the method of Example 1, m 7 GpppA m pG and m 7 GpppA m pU prepared mRNA encoding rabies virus antigens (named mRNA5 (SEQ ID NO: 5) and mRNA6 (SEQ ID NO: 6), both of which were synthesized with m1Ψ instead of uridine. Then, the lipid mixture (containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ionizable lipids, PEG-lipids and cholesterol) was dissolved in ethanol and then mixed with the mRNA solution in a T-type mixer. The preparation was then concentrated to the desired concentration by a tangential flow filtration (TFF) membrane and filtered through a 0.22 μm filter to obtain the finished products (named LNP1 and LNP2, respectively), which were stored at 2-8°C until use. The finished products were used in experimental animals, and each preparation (0.1 μg / 50 μL / mouse) was injected intramuscularly at a single point in the right hind limb of BALB / c mice on day 0, with 10 animals in each group (except 5 animals in the PBS group), and whole blood was collected on day 13 after administration to measure the serum level of RABV-specific IgG titer.

[0466] result:

[0467] As shown in Table 8, the IgG titer level of the LNP2 group was higher than that of the LNP1 group.

[0468] Table 8:

[0469]

[0470] Example 3: Example of mRNA vaccine based on respiratory syncytial virus

[0471] This example describes the in vitro transcription of a respiratory syncytial virus-based mRNA vaccine.

[0472] According to the method of Example 1, m 7 GpppA m pG and m 7 GpppA mpU prepared mRNA encoding respiratory syncytial virus antigens (named mRNA7 (SEQ ID NO: 7), mRNA8 (SEQ ID NO: 8) respectively), both of which were synthesized with m1Ψ instead of uridine. Then, the lipid mixture (containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), ionizable lipids, PEG-lipids and cholesterol) was dissolved in ethanol and then mixed with the mRNA solution in a T-type mixer. The preparation was then concentrated to the desired concentration by a tangential flow filtration (TFF) membrane and filtered through a 0.22μm filter to obtain the finished products (named LNP3 and LNP4 respectively), which were stored at 2-8°C until use. The finished products were used in experimental animals, and each preparation (0.1μg / 50μL / mouse) was injected intramuscularly at a single point in the right hind limb of BALB / c mice on day 0, 5 animals were injected with PBS, 6 animals were injected with LNP3, and 10 animals were injected with LNP4. Whole blood was collected on the 13th day after administration to measure the serum level of RSV-specific IgG.

[0473] Results: As shown in Table 9, the IgG titer level in the LNP4 group was higher than that in the LNP3 group.

[0474] Table 9:

[0475]

[0476] *Below detection limit

[0477] Example 4: Expression in BHK-21 cell line

[0478] According to the method of Example 1, (G m7 )ppp(A m2 )G and (G m7 )ppp(A m2)U synthesized the mRNA encoding the eGFP protein, and both were synthesized with m1Ψ instead of uridine (i.e., mRNA1 (SEQ ID NO: 1) and mRNA2 (SEQ ID NO: 2), respectively). BHK-21 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS; Fisher Scientific, Inc.), 100U / ml penicillin and 100mg / ml streptomycin at 37°C and 5% CO2. 24 hours before transfection, BHK-21 cells were seeded in 96-well plates at a density of 10,000 cells per well (in 100 μL culture medium). 25 ng of mRNA was transfected with TransIT (Mirusbio, catalog number MIR 2250), and the cells were collected 24 hours later and resuspended in PBS. The values ​​at 488 / 507 nm were read by a Molecular Devices plate reader for detection, and the data were analyzed using Graphpadprism9. The results are shown in the figure. Figure 1 As shown, the expression levels of eGFP in BHK-21 were similar for both mRNAs and had no significant difference between the groups.

[0479] Example 5: Preparation of in vitro transcription of mRNAs with similar cap structures and detection of each mRNA

[0480] Purified linear plasmids encoding RSV, RABV, VZV antigens and eGFP protein were used as templates for in vitro transcription using T7 RNA polymerase. The linear templates were prepared and purified as described in Example 1. m7 )ppp(A m2 )U、(G m7，3’OMe )ppp(A m2 )U、(G m7 )ppp(A m2 )m1Ψor(G m7 )vppp(A m2 )U is used for in vitro transcription to synthesize mRNA with Cap1 "cap" structure. At the same time, m1Ψ or uridine is used to synthesize mRNA.

[0481] The concentrations of ATP / GTP / CTP / m1Ψ were 12.5 mM each; (G m7 )ppp(A m2 )U or (G m7,3'OMe )ppp(A m2 )U or (G m7 )ppp(A m2 )m1Ψor(G m7 )vppp(A m2)U concentration was 12.5 mM; 10X reaction buffer [400 mM Tris-HCl (pH 7.9), 100 mM DTT, 20 mM spermidine, 0.02% Triton X-100, 270 mM magnesium acetate] concentration was 1X; DNA template concentration was 50 μg / mL; mouse RNase inhibitor concentration was 1 unit / μL; yeast inorganic pyrophosphatase concentration was 0.0005 unit / μL; T7 RNA polymerase concentration was 5 units / μL.

[0482] The IVT reaction was performed for 2.5 h, followed by DNase I digestion (DNase I was added to the IVT reaction at a final concentration of 2 units / μL and incubated at 37°C for 30 min). Table 11 provides detailed information about the mRNA, such as the structure of different cap analogs. Cap analog products such as (G m7 )ppp(A m2 )U、(G m7，3’OMe )ppp(A m2 )U、(G m7 )ppp(A m2 )m1Ψor(G m7 )vppp(A m2 )U are all commercially available, and their preparation methods are known in the art. For example, (G m7 )vppp(A m2 )U can be prepared according to CN116143855A, or purchased from Jiangsu Synthgene Biotechnology Co., Ltd. Then use immediately mRNA was purified using an RNA purification kit (NEB, #T2050S), quantified by UV using NanoDrop One, analyzed for purity using a fragment analyzer, detected for capping efficiency using LC-MS, and analyzed for RIG-I activation using HEK Lucia RIG-I cells purchased from Invivogen (Catalog No. HKL-HRIG).

[0483] From the analysis results in Table 10:

[0484] (1) Yield: Based on (G m7 )ppp(A m2 The yield of mRNA synthesized by )U is higher than that based on (G m7，3'OM e)PPP(A m2 )U or (G m7 )ppp(A m2 )m1Ψ synthesized mRNA yields, the four groups of different mRNA samples have good consistency. And when encoding eGFP based on (G m7 )ppp(A m2The yield of )U mRNA is similar to that based on (G m7 )vppp(A m2 )U encoding mRNA yields were essentially the same.

[0485] (2) Purity: Based on (G m7 )ppp(A m2 The purity of mRNA synthesized by )U is higher than that based on (G m7，3'OMe )ppp(A m2 )U or (G m7 )ppp(A m2 )m1Ψ-synthesized mRNA had good consistency among the four groups of mRNA samples. m7 )ppp(A m2 The purity of the mRNA encoding eGFP synthesized by )U was similar to that based on (G m7 )vppp(A m2 )U had basically the same purity of the mRNA encoding eGFP synthesized.

[0486] (3) Capping efficiency: The capping efficiency of mRNA synthesized based on all these cap analogs was equally high, up to 100%.

[0487] (4) RIG-I activation: When m1Ψ replaces uridine, based on (G m7 )ppp(A m2 )U-based RIG-I activation was lower than that based on (G m7，3'OM e) PPP(Am 2 )U or (G m7 )ppp(A m2 )m1ψ mRNA RIG-I activation, there is good consistency among the four different samples. When using uridine or m1Ψ modification, based on (G m7 )ppp(A m2 RIG-I activation of eGFP-encoding mRNA synthesized by U and based on (G m7 )vppp(A m2 )U synthesized mRNA encoding eGFP has basically the same RIG-I activation.

[0488] Table 10:

[0489]

[0490]

[0491]

[0492] Table 11:

[0493]

[0494] Embodiment 6: by (G m7 )ppp(A m2 )U and (G m7 )vppp(A m2 In vitro expression of mRNA synthesized by

[0495] As described in Example 5, a (G m7 )ppp(A m2 )U and (G m7 )vppp(A m2 )U synthesized and had uridine or m1Ψ modifications. HEK-293T and A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and seeded in 96-well plates with 100 μl of cell suspension per well (5000 cells / well). The plates were preincubated in a humidified incubator for 24 hours (37°C, 5% CO2), and then 100 ng of each mRNA was added to the wells. Part of the cells were collected at 24 hours and 48 hours, respectively, and washed three times with PBS. The eGFP protein expression level was evaluated by flow cytometry. Figure 2-3 As shown, when uridine or m1Ψ modification is used, based on (G m7 )ppp(A m2 )U and (G m7 )vppp(A m2 )U synthesized mRNA has basically the same expression level.

[0496] sequence

[0497] In the following sequences, "Ψ" means "m1Ψ (1-methyl-pseudouridine)" unless otherwise specified. The abbreviations are for clarity of the sequences. Other abbreviations are as follows,

[0498] “m 7 G" and "G m7 ” = 7-methylguanosine (also abbreviated as “m7G”);

[0499] “A m ” and “A m2 ” = 2'-O-methyladenosine;

[0500] "ppp" = triphosphate bond;

[0501] "vppp" = vinyl-modified triphosphate bond;

[0502] “G m7,3'OMe = 3'-O-methylated m7G.

[0503] SEQ ID NO:1(MRNA 1)

[0504]

[0505] SEQ ID NO:2(MRNA 2)

[0506]

[0507] SEQ ID NO:3(MRNA 3)

[0508]

[0509] SEQ ID NO:4(MRNA 4)

[0510]

[0511] SEQ ID NO:5(MRNA 5)

[0512]

[0513] SEQ ID NO:6(MRNA 6)

[0514]

[0515] SEQ ID NO:7(MRNA 7)

[0516]

[0517] SEQ ID NO:8(MRNA 8)

[0518]

[0519] SEQ ID NO:9(MRNA 9)

[0520]

[0521] SEQ ID NO:10(MRNA 10)

[0522]

[0523] SEQ ID NO:11(MRNA 11)

[0524]

[0525] SEQ ID NO:12(MRNA 12)

[0526]

[0527] SEQ ID NO:13(MRNA 13)

[0528]

[0529] SEQ ID NO:14(MRNA 14)

[0530]

[0531] SEQ ID NO:15(MRNA 15)

[0532]

[0533] SEQ ID NO:16(MRNA 16)

[0534]

[0535] SEQ ID NO:17(MRNA 17)

[0536]

[0537]

[0538] SEQ ID NO:18(MRNA 18)

[0539]

[0540] SEQ ID NO:19(MRNA 19)

[0541]

[0542]

[0543] SEQ ID NO:20(MRNA 20)

[0544]

[0545] SEQ ID NO:21(MRNA 21)

[0546]

[0547] SEQ ID NO:22(MRNA 22)

[0548]

[0549]

[0550] SEQ ID NO:23(MRNA 23)

[0551]

[0552]

[0553] SEQ ID NO:24(MRNA 24)

[0554]

[0555] SEQ ID NO:25(MRNA 25)

[0556]

[0557] SEQ ID NO:26(MRNA 26)

[0558]

[0559] SEQ ID NO:27(MRNA 27)

[0560]

[0561] SEQ ID NO:28(MRNA 28)

[0562]

[0563] SEQ ID NO:29(MRNA 29)

[0564]

[0565] SEQ ID NO:30(MRNA 30)

[0566]

[0567] SEQ ID NO:31(MRNA 31)

[0568]

[0569] SEQ ID NO:32(MRNA 32)

[0570]

[0571] SEQ ID NO:33(MRNA 33)

[0572]

[0573] SEQ ID NO:34(MRNA 34)

[0574]

Claims

1. A modified RNA molecule, wherein the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicative, wherein the 5' end of the modified RNA molecule has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

2. The modified RNA molecule of claim 1, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified.

3. The modified RNA molecule of claim 1 or 2, wherein the 5' end of the modified RNA molecule has m 7 GpppApU or m 7 The sequence of GvpppApU.

4. The modified RNA molecule according to claim 2 or 3, wherein said A is modified, preferably 2'-O-methylated.

5. The modified RNA molecule of any one of claims 1 to 4, wherein the 5' end of the modified RNA molecule has m 7 GpppA m pU or m 7 Gvppp m The sequence of pU.

6. The modified RNA molecule of any one of claims 1 to 5, wherein all uridines except the first 5' uridine in the molecule are modified, Preferably, all uridines except the first 5' uridine in the molecule are m1Ψ.

7. The modified RNA molecule of any one of claims 1 to 6, comprising an open reading frame containing a coding sequence, Preferably, the coding sequence encodes eGFP, SARS-COV-2BA.4 / BA.5RBD, rabies virus antigen, respiratory syncytial virus antigen or varicella-zoster virus antigen.

8. The modified RNA molecule of any one of claims 1 to 7, wherein the molecule further comprises at least one modified G, C or A.

9. The modified RNA molecule of any one of claims 1 to 8, wherein the molecule has a modified backbone, Preferably, the modified backbone comprises at least one phosphorothioate bond.

10. A pharmaceutical composition comprising the modified RNA of any one of claims 1 to 9, further comprising a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable carrier is LNP, Preferably, the LNP comprises a cationic lipid, More preferably, the LNP comprises: i) about 30 to 55 mol% of a cationic lipid; ii) about 5 to 40 mol% of a phospholipid; iii) about 20 to 50 mol% of a sterol; and iv) a polymer-conjugated lipid.

12. A method for in vitro transcribing a DNA molecule into a modified RNA molecule, comprising combining the DNA molecule with an in vitro transcription mixture comprising a DNA-dependent RNA polymerase, modified and / or unmodified nucleotides and a 5' cap, wherein the modified RNA molecule is non-self-replicable, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine, and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

13. The method of claim 12, wherein the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified.

14. The method of claim 12 or 13, wherein the 5' cap has m 7 GpppApU or m 7 The sequence of GvpppApU.

15. The method of claim 13 or 14, wherein said A is modified, preferably 2'-O-methylated.

16. The method of any one of claims 12-15, wherein the 5' cap has m 7 GpppA m pU or m 7 Gvppp m The sequence of pU.

17. The method of any one of claims 12-16, wherein all uridines except the first 5' uridine in the modified RNA molecule are modified.

18. The method of any one of claims 12 to 17, wherein the modified uridine is m1Ψ, Preferably, all uridines except the first 5' uridine in the modified RNA molecule are m1Ψ.

19. The method of any one of claims 12-18, wherein the DNA-dependent RNA polymerase is T7 RNA polymerase or a variant thereof.

20. The method of any one of claims 12-19, further comprising isolating the modified RNA molecule.

21. A modified RNA molecule produced by the method of any one of claims 12-20.

22. A kit comprising: 1) a DNA-dependent RNA polymerase; 2) modified and / or unmodified nucleotides, and 3) a 5' cap, and 4) instructions for performing the method of any one of claims 12-20, wherein the 5' cap has a sequence of N1pppN2pU or N1vpppN2pU, wherein U is unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than modified or unmodified uridine.

23. A method of reducing innate immune stimulation of an RNA molecule comprising using a 5' cap having a sequence of N1pppN2pU or N1vpppN2pU and modifying at least one uridine other than the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is not self-replicable, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than a modified or unmodified uridine, Preferably, the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified, Preferably, the 5' cap has m 7 GpppApU or m 7 The sequence of GvpppApU, Preferably, the 5' cap has m 7 GpppA m pU or m 7 Gvppp m The sequence of pU, Preferably, the method comprises modifying all uridines in the RNA molecule except the first 5' uridine, More preferably, the method comprises modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ.

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