CN119464283A

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Publication number: CN119464283A
Application number: CN202411091666.2A
Authority: CN
Inventors: 郜鹏; 燕化远; 英博
Original assignee: Suzhou Aibo Biotechnology Co ltd
Current assignee: Suzhou Aibo Biotechnology Co ltd
Priority date: 2023-08-11
Filing date: 2024-08-09
Publication date: 2025-02-18
Also published as: WO2025036272A1

Abstract

The present disclosure relates to a modified RNA molecule with enhanced stability. In particular, the 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 N1vpppN pU, each of N1 and N2 is independently a nucleotide other than uridine. And, the present disclosure also relates to methods of producing the same, and pharmaceutical compositions comprising the same.

Description

Capped mRNA and method of making same

Technical Field

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

Reference to electronic sequence Listing

The contents of the electronic sequence Listing (P2024 TC2948.Xml; size: 133,721 bytes; date of creation: day 23, 7, 2024) are incorporated herein by reference in their entirety.

Background

RNA molecules produced by in vitro transcription offer the potential for valuable and highly desirable pharmaceutical compositions by allowing the delivery of genetic material capable of translating into a protein of interest to patient cells. In some aspects, the existing methods are limited by the immunostimulatory nature of the in vitro transcribed RNA molecules, resulting in inefficient expression of the protein of interest within the transfected cells. Methods and compositions addressing these and other needs are provided herein.

Disclosure of Invention

In some aspects, provided herein is a modified RNA molecule, wherein the modified RNA molecule comprises a5 'cap, wherein the RNA molecule is not self-replicable, and wherein at least one uridine in the molecule is a modified uridine except for a first 5' uridine in the molecule.

In some embodiments, the 5' end of the modified RNA molecule has the 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.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of N1pppN2pU or N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein at least one uridine in the molecule other than the first 5' uridine is a modified uridine.

In some embodiments, at least about 50% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of NlpppN pU or N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is modified.

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 the sequence of m⁷ GpppApU or m⁷ GvpppApU. In some embodiments, A is modified, preferably 2' -O-methylated.

In some embodiments, at least about 50% of the uridine in the molecule other than the first 5' uridine is m1 ψ (1-methyl-pseudouridine). In some embodiments, all uridine in the molecule except the first 5' uridine is m1 ψ.

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

In some embodiments, the coding sequence encodes a therapeutic payload comprising a compound capable of eliciting an immunity against one or more conditions or diseases of interest. In some embodiments, the condition of interest is associated with or caused by a pathogen infection, such as coronavirus (e.g., 2019-nCoV), influenza, measles, human Papilloma Virus (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 the cells. In some embodiments, the coding sequence encodes a tumor-associated antigen (TAA) characteristic of cancer or an antigenic fragment or epitope thereof.

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 a rabies virus antigen. In some embodiments, the coding sequence encodes a respiratory syncytial virus antigen. In some embodiments, the coding sequence encodes a varicella-zoster virus antigen.

In some embodiments, the molecule further comprises at least one modified G, C or a.

In some embodiments, the molecule has a modified backbone. In some embodiments, the modified backbone comprises at least one phosphorothioate linkage.

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

In some embodiments, the LNP comprises i) about 30 to 55 mole percent cationic lipid, ii) about 5 to 40 mole percent phospholipid, iii) about 20 to 50 mole percent sterol, and iv) polymer conjugated lipid.

In other aspects, provided 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, wherein the 5' cap has the sequence of N1pppN2pU or N1vpppN pU, 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, the in vitro transcription mixture comprises a modified uridine.

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, 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 the sequence of N1pppN pU or N1vpppN pU, 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 a modified uridine, and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

In some embodiments, all uridine in the molecule except the first 5' uridine are modified.

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 the sequence of N1pppN pU or N1vpppN pU, wherein U is an unmodified uridine, and wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine, and wherein all uridine in the molecule except the first 5' uridine is modified.

In some embodiments, the modified uridine is m1ψ. In some embodiments, all uridine in the modified RNA molecule except the first 5' uridine is m1ψ.

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 the sequence of m⁷ GpppApU or m⁷ GvpppApU. In some embodiments, A is modified, preferably 2' -O-methylated. In some embodiments, the 5' cap has the sequence of m⁷GpppA_m pU or m⁷GvpppA_m pU.

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

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

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

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 performing the methods disclosed herein, wherein the 5' cap has the sequence of N1pppN2pU or N1vpppN pU, 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 other aspects, provided herein are methods of reducing innate immune stimulation of an RNA molecule comprising using a 5 'cap having the sequence of N1pppN pU or N1vpppN pU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine.

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 the sequence of m⁷ GpppApU or m⁷ GvpppApU. In some embodiments, the 5' cap has the sequence of m⁷GpppA_m pU or m⁷GvpppA_m pU. In some embodiments, the method comprises modifying all uridine in the RNA molecule except the first 5' uridine. In some embodiments, the method comprises modifying all but the first 5' uridine in the RNA molecule to m1ψ.

Drawings

FIG. 1 shows expression of mRNA encoding eGFP protein in BHK-21 cell lines synthesized with (G^m7)ppp(A^m2) G and (G^m7)ppp(A^m2) U, respectively.

FIG. 2 shows in vitro expression of mRNA synthesized by (G^m7)ppp(A^m2) U and (G^m7)vppp(A^m2) U in HEK-293T cells.

FIG. 3 shows in vitro expression of mRNA synthesized by (G^m7)ppp(A^m2) U and (G^m7)vppp(A^m2) U in A549 cells.

Detailed Description

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 was individually incorporated by reference. If the definition set forth herein is contrary to or inconsistent with the definition set forth in the patents, applications, published applications and other publications, which are incorporated by reference herein, the definition set forth herein takes precedence over the definition set forth herein by reference.

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

I. Overview of the invention

In some aspects, provided herein are modified RNA molecules, wherein the modified RNA molecule comprises a5 'cap, and wherein at least one uridine in the molecule is a modified uridine, except for the first 5' uridine in the molecule. In some embodiments, the modified RNA molecule is not self-replicable.

Also provided are methods 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 the sequence of N1pppN2pU or N1vpppN pU, 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. Compositions, formulations, and kits for use according to the provided methods are also provided.

Currently, RNA molecules produced by in vitro transcription and transfected into cells typically stimulate innate immune receptors such as endosomal and cytoplasmic Pattern Recognition Receptors (PRRs), including toll-like receptors 3, 7 and 8 (Ma, x. And Hur, s.drug Metabolism pharmacokinet.2022.44, 100450). These toll-like receptors are capable of sensing single-and double-stranded RNAs and generating immune responses through intracellular signaling cascades. Activation of immune responses in cells transfected with in vitro transcribed RNA molecules is a major limitation of current gene therapy approaches.

The application provides novel methods for in vitro mRNA synthesis using specific uridine-containing caps (e.g., m⁷GpppA_m pU caps, m⁷GvpppA_m pU caps) and mRNA produced by such methods. The m⁷GpppA_m pU cap is traditionally used for self-replicating RNAs based on their function in sense strand RNA viruses such as Venezuelan Equine Encephalitis Virus (VEEV), semliki Forest Virus (SFV) and Sindbis Virus (SIN). The inventors for the first time used such cap structures for in vitro transcription of non-replicable mRNA. Surprisingly, it was found that a one-step synthesis using the m⁷GpppA_m pU cap can achieve a higher capping rate than an mRNA synthesis method using the m⁷GpppA_m pG cap. The non-replicable mRNA thus produced (except for the first uridine) may be fully uridine modified, resulting in reduced innate immune stimulation when delivered to cells.

Accordingly, in one aspect the present invention provides 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, wherein the 5' cap has the sequence of m⁷GpppA_m pU or m⁷GvpppA_m pU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises modified uridine, and wherein all uridine in the modified RNA molecule except the first 5' uridine is modified.

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-replicable, and wherein at least one uridine in the molecule is a modified uridine except for a first 5' uridine in the molecule.

Kits and compositions (e.g., pharmaceutical compositions) comprising modified RNA molecules for performing the methods described herein are also provided.

II. Definition of

For the purposes of explaining the present specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. To the extent that any definition set forth below conflicts with any document incorporated herein by reference, the definition shall govern.

The term "about" as used herein refers to the usual error range for the corresponding value as readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments directed to the value or parameter itself.

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 merely for convenience and brevity and should not be interpreted as a strict limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, a range of values is provided, and it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. If the stated range includes one or both of the limitations, ranges not including one or both of the limitations are also included in the claimed subject matter. This applies to the breadth of any range.

"Cap" as used herein refers to a non-extendable trinucleotide that, when incorporated at the 5' end of an RNA transcript, facilitates translation or localization, and/or prevents degradation of the RNA transcript.

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 analogs thereof, 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).

The term "ribonucleotide" or "nucleotide" as used herein refers to a compound consisting of a nucleobase linked to the C-carbon of ribose or an analog thereof. Ribose or the like may be substituted or unsubstituted.

As used herein, "nucleotide triphosphates" refers to nucleotides having a triphosphate group at the 5' position.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" as used herein refer to single-or double-stranded polymers of nucleotide monomers, including Ribonucleotides (RNA) and 2' -Deoxyribonucleotides (DNA) joined by internucleotide phosphodiester linkages. The polynucleotide may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or of chimeric mixtures thereof.

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

As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to substances that are not biologically or otherwise undesirable, e.g., the substances may be incorporated into a pharmaceutical composition for administration to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which they are contained. The pharmaceutically acceptable carrier or excipient preferably meets the required criteria for toxicological and manufacturing testing and/or is contained in inactive ingredient guidelines established by the U.S. food and drug administration.

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

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.

The term "ionizable lipid" as used herein 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 instance of embodiments contemplated with the term "ionizable lipid", as well as with "cationic lipid", as if all embodiments were specifically and individually listed.

The term "polymer conjugated lipid" as used herein refers to a lipid comprising a polymer moiety. The term encompasses pegylated lipids, including pegylated phosphatidylethanolamine, pegylated phosphatidic acid, pegylated ceramide, pegylated dialkylamine, pegylated diacylglycerol, and pegylated dialkylglycerol. The term also encompasses mixtures of polymer conjugated lipids, which may contain two or more polymer conjugated lipids. In each case of embodiments contemplated with the term "polymer conjugated lipids", also "pegylated lipids" are contemplated as if all embodiments were specifically and individually listed.

The term "lipid stabilizing agent" as used herein refers to a component of the 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 a liquid ordered phase of the lipid membrane in the LNP. See, for example, albertsen, H.C. et al, section 3.3.1, month "The role of lipid components in lipid nanoparticles for vaccines and gene therapy."Adv Drug Deliv Rev.2022, 9; 188:114416. Compounds useful as lipid stabilizers include sterols, corticosteroids, vitamins and other compounds that contain a steroid core.

The term "alkyl" as used herein refers to a chain of carbon atoms wherein all bonds between carbon atoms in the alkyl group are single bonds. The term includes straight and branched chains (e.g., the term includes n-propyl and isopropyl).

The term "Cx-Cy alkyl" as used herein refers to alkyl substituents 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.

The term "alkylene" as used herein refers to an alkyl chain attached to other chemical groups in 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.

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

The term "Cx-Cy alkenyl" as used herein refers to alkenyl substituents 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.

The term "alkenylene" as used herein refers to an alkenyl chain attached to other chemical groups at least two positions. "Cx-Cyalkenylene" refers to an alkenylene substituent having at least x carbon atoms and no more than y carbon atoms.

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

The term "Cx-Cy alkynyl" as used herein refers to alkynyl substituents having at least x carbon atoms and no more than y carbon atoms in the alkynyl chain.

The term "cycloalkyl" as used herein refers to a cyclic group of carbon atoms, wherein all bonds between carbon atoms are single bonds. The term "Cx-Cy cycloalkyl" refers to cycloalkyl substituents 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 attached to at least two other chemical groups.

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 is a double bond. The term "Cx-Cy cycloalkenyl" refers to cycloalkenyl substituents having at least x carbon atoms and no more than y carbon atoms.

The term "aryl" as used herein refers to a cyclic group having a carbon atom with at least one double bond. The term "Cx-Cy aryl" refers to arylene substituents 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.

The term "heterocycloalkyl" as used herein refers to a ring radical in which all bonds between ring atoms 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.

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 groups include, but are not limited to, pyridyl and furyl.

The term "carbocycle" as used herein refers to a substituent that may be cycloalkyl or aryl. Also, the term "heterocycle" refers to a substituent that may be a heterocycloalkyl or heteroaryl group.

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

The term "optionally substituted" as used herein means that the indicated substituents may be substituted or unsubstituted. The term substituted refers to the modification of another chemical moiety of the indicated substituent by substitution of one H atom. For example, ethanol is one example of ethane substituted with OH. In some embodiments, the optionally substituted group is optionally substituted with chloro, fluoro, bromo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C10 aryl, or 5-or 6-membered heteroaryl.

The terms "individual," "subject," and "patient" are used interchangeably herein to describe a mammal, including a human. In some embodiments, the individual is in need of treatment, e.g., the individual may have been diagnosed with or suspected of having cancer.

It is to be understood that embodiments of the invention described herein include embodiments that "consist of and/or" consist essentially of.

Reference herein to "about" a value or parameter includes (and describes) a variation on that value or parameter itself. For example, a description referring to "about X" includes a description of "X". In some embodiments, the term "about" a certain value or parameter refers to a range within 20% of either direction of the value or parameter.

As used herein, reference to "not" a certain value or parameter generally means and describes "different from" a certain value or parameter.

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

III preparation method

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

A. in vitro transcription

The modified RNAs described herein can be synthesized by in vitro transcription of an appropriate DNA template. The promoter used to control transcription may be a promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained, for example, by cloning a nucleic acid, in particular 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.

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 synthesized RNA. In some embodiments, the template DNA has a purity of greater than about 90%, greater than about 95%, or greater than about 99%. Plasmid purification methods are well known in the art and include miniprep, mesoprep and miniprep plasmid separations. In some embodiments, the plasmid DNA is largely in supercoiled form and is free of contaminating rnases, proteins, RNAs, and salts.

In some embodiments, the DNA template for in vitro transcription comprises fully 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 linearization of the plasmid DNA, the template DNA is further purified by phenol/chloroform extraction.

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 phage polymerase promoter.

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 the sequence of N1pppN pU, 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, 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 the sequence of N1vpppN pU, 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, the in vitro transcription of the modified RNA includes a capping step to produce a modified RNA comprising a 5' cap having the sequence of N1pppN pU, 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, 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 the sequence of m⁷ GpppApU. In some embodiments, A is modified, preferably 2' -O-methylated. In some embodiments, the 5' cap has the sequence of m⁷GpppA_m PU.

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 the sequence of N1vpppN pU, 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, 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 the sequence of m⁷ GvpppApU. In some embodiments, A is modified, preferably 2' -O-methylated. In some embodiments, the 5' cap has the sequence of m⁷GvpppA_m pU.

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 adenosines, guanines, cytidines, and uracils. 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, and in some embodiments, the in vitro transcription mixture comprises modified uridine. In some embodiments, the modified uridine comprises ψ or m1 ψ. In some embodiments, the modified uridine is m1ψ.

In some embodiments, between about 1% and about 99% of the uridine bases in the modified RNA molecules 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 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 uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 90% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 95% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 99% of the uridine in the molecule other than the first 5' uridine is modified.

In some embodiments, all uridine in the molecule except the first 5' uridine is m1 ψ.

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 the sequence of N1pppN2pU or N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

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 the sequence of N1pppN2pU or N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein all uridine in the molecule except the first 5' uridine is modified.

In a preferred embodiment, the method of in vitro transcription of 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 m1 ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

In a preferred embodiment, the method of in vitro transcription of 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 m1 ψ), and wherein all uridine in the molecule except the first 5' uridine are modified.

In a preferred embodiment, the method of in vitro transcription of 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 the sequence of m⁷ GpppApU or m⁷ GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

In a preferred embodiment, the method of in vitro transcription of 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 the sequence of m⁷ GpppApU or m⁷ GvpppApU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein all uridine in the molecule except the first 5' uridine are modified.

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 the sequence of m⁷GpppA_m pU or m⁷GvpppA_m pU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

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 the sequence of m⁷GpppA_m pU or m⁷GvpppA_m pU, wherein U is an unmodified uridine, wherein the in vitro transcription mixture comprises a modified uridine (preferably m1 ψ), and wherein all uridine in the molecule except the first 5' uridine is modified.

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, SP 3RNA polymerase, SP6 RNA polymerase, VSW-3RNA polymerase, or variants thereof. In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase or variant thereof. In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase.

Also provided herein are methods of isolating modified RNA molecules. In some embodiments, the modified RNA molecules are isolated by chemical, column, and/or gel based methods. Or in some embodiments, the modified RNA molecules are isolated by a bead-based method. In some embodiments, the bead-based method isolates the modified RNA molecule by binding of the poly (a) tail. In some embodiments, the methods of isolating modified RNA molecules described herein are used to clean RNA and separate it from components such as DNA templates, unincorporated nucleotides, or RNA modifying enzymes.

Modified RNA molecules

Also provided herein are RNA molecules produced by the methods described herein. Thus, in certain aspects, provided herein are modified RNA molecules comprising a 5 'cap, wherein at least one uridine in the molecule is a modified uridine, except for the first 5' uridine in the molecule. In some embodiments, the modified RNA molecule is not self-replicable.

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 nitrogen-containing base. In some embodiments, the modified RNA molecule comprises at least one modification 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 m1 ψ (1-methyl-pseudouridine).

In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN pU, 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 molecules 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 uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 90% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 95% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 99% of the uridine in the molecule other than the first 5' uridine is modified. In some embodiments, all uridine in the molecule except the first 5' uridine are modified.

The triphosphate bond of NlvpppN pU is vinyl modified compared to the triphosphate bond of NlpppN pU. For example, N1vpppN pU contains a vinyl modified triphosphate bond comprising the following moieties.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of N1pppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of NlpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of N1vpppN pU, wherein U is an unmodified uridine, wherein each of N1 and N2 is independently a modified or unmodified nucleotide other than the modified or unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is modified.

In some embodiments, the modified RNA molecule is involved 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).

In some embodiments, the modified RNA molecule comprises a non-coding element. In some embodiments, the non-coding elements include a 5' 7-methyl-GTP cap, a 5' untranslated region (UTR), and a 3' UTR. In some embodiments, the mRNA comprises a 5' utr. The 5' utrs provided herein can be recognized by a ribosome, allowing the ribosome to bind and initiate translation of mRNA (e.g., translation of a coding sequence and/or nucleic acid encoding an mRNA signal peptide). In some embodiments, the 5' utr is located upstream of the mRNA coding sequence. In some embodiments, the 5' utr is between about 5 and 1400 nucleotides in length. In some embodiments, the length of the 5' utr is between about 5 to 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.

In some embodiments, the mRNA comprises a 5'utr and a 3' utr, e.g., any of the 5'utr and 3' utr provided herein. In some embodiments, the 5'utr and the 3' utr are from the same species. In some embodiments, the 5'utr and the 3' utr are not from the same species. In some embodiments, the 5'utr is synthetic, while the 3' utr is not synthetic. In some embodiments, the 5'utr is not synthetic, whereas the 3' utr is synthetic.

In some embodiments, the mRNA comprises a poly (a) sequence (e.g., a polyadenylation sequence). The poly (A) sequence consists of a plurality of consecutive adenosine monophosphates. In some embodiments, the poly (a) sequence is critical for 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 poly (a) sequence is about 50 nucleotides or more in length, e.g., about 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides or more. In some embodiments, the poly (a) sequence is about 150 nucleotides or less in length, e.g., about 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 50 nucleotides or less. In some embodiments, the poly (a) sequence is about 105 nucleotides in length.

In some embodiments, the modified RNA molecule is not self-replicable. In some embodiments, the modified RNA molecule degrades in the cell over time. In some embodiments, mRNA degradation in the cell occurs through mRNA decay, AU-rich elements (ARE) in the 3' utr, destabilizing elements in the protein coding region, nonsense-mediated mRNA decay (NMD), and/or micrornas (mirnas). In some embodiments, the modified RNA molecule has a measurable half-life in a cell. In some embodiments, the modified RNA molecule has a half-life in the cell of between about 0 hours and about 24 hours. In some embodiments, the modified RNA molecule has a half-life in the cell of between 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 modified RNA molecule has a half-life in a cell of about 10 hours.

In some embodiments, the modified RNA molecule can be transfected into a cell for translation within the cell. Methods of transfection are known to those skilled in the art and include microinjection, chemical treatment, and electroporation. In some embodiments, the cells to be transfected include any patient cell in need of expression of 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.

A.5' cap

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

In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1pppN pU, 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, m⁷GpppA_m pU has the formula,

In some embodiments, the modified RNA molecule comprising a 5' cap comprising the sequence of m⁷GpppA_m pU exhibits improved RNA stability compared to the unmodified RNA.

In some embodiments, the 5' end of the modified RNA molecule has a sequence of N1vpppN pU, 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, the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein a is modified or unmodified. The triphosphate bond of GvpppApU is vinyl modified compared to the triphosphate bond of GpppApU. In some embodiments, a is 2' -O-methylated. In some embodiments, the modified RNA molecule has a sequence of m⁷ GvpppApU at the 5' end. In contrast to the triphosphate bond of m⁷ GpppApU, the triphosphate bond of m⁷ GvpppApU is vinyl modified. In some embodiments, the 5' end of the modified RNA molecule has the sequence of m⁷ GvpppApU, wherein a is modified or unmodified. In some embodiments, a is 2' -O-methylated. In some embodiments, the modified RNA molecule has a sequence of m⁷GvpppA_m pU at the 5' end. The triphosphate bond of m⁷GvpppA_m pU is vinyl modified compared to the triphosphate bond of m⁷GpppA_m pU. 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.

In some embodiments, the modified RNA molecule comprising a 5' cap comprising the sequence of m⁷GvpppA_m pU exhibits improved RNA stability compared to the unmodified RNA. In addition, the double bond structure makes the molecular conformation more stable, making nuclease more difficult to recognize and hydrolyze.

In some embodiments, m⁷GvpppA_m pU has the formula,

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, 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 in the molecule other than the first 5' uridine is a modified uridine.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GpppApU, 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.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷GpppA_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.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷GpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of m⁷GpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of m⁷GpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, 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 in the molecule other than the first 5' uridine is a modified uridine.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is not self-replicable, wherein the 5' end of the modified RNA molecule has a sequence of GvpppApU wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GvpppApU, 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.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GvpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GvpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷ GvpppApU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷GvpppA_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.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has a sequence of m⁷GvpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine are modified.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of m⁷GvpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is ψ.

In some embodiments, the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self replicable, wherein the 5' end of the modified RNA molecule has the sequence of m⁷GvpppA_m pU, wherein U is an unmodified uridine, and wherein all uridine in the molecule except the first 5' uridine is m1 ψ.

In some embodiments, "significantly lower immunostimulatory" refers to a detectable decrease in immunostimulatory. In another embodiment, the term refers to a fold reduction in immunostimulatory. In some embodiments, "significantly less immunostimulatory" refers to a decrease to an effective amount of the modified RNA molecule that can be administered to a cell without eliciting a detectable immune response. In some embodiments, the term refers to a decrease to allow repeated administration of the modified RNA molecule without eliciting an immune response sufficient to detectably reduce expression of the recombinant protein. In another embodiment, this reduction allows for repeated administration of the modified RNA molecule without eliciting an immune response sufficient to eliminate the expression of the detectable recombinant protein. Methods for determining immunostimulatory properties are well known in the art and include, for example, measuring secretion of cytokines (e.g., IL-12, IFN- α, TNF- α, RANTES, MIP-1α or β, IL-6, IFN- β or IL-8), measuring expression of DC activation markers (e.g., CD83, HLA-DR, CD80 and CD 86), or measuring the ability to act as adjuvants to adaptive immune responses. In some embodiments, the immunostimulatory properties of the modified RNA molecules are measured by a RIG-I activation assay.

B. Modification

The RNA molecule may comprise one or more modifications in addition to the first uridine (e.g., uridine introduced via the 5' cap). 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.

In some embodiments, the modified RNA molecule may comprise a modified base form. In some embodiments, purines and pyrimidines other than those commonly found in nature may be used. In some embodiments, the modified base is selected from the group consisting of m⁵ C (5-methylcytidine), m⁵ U (5-methyluridine), m⁶ A (N6-methyladenosine), S² U (2-thiouridine), a nucleotide sequence that is complementary to the nucleotide sequence of the modified base, Psicose (pseudouridine), um (2 '-O-methyluridine), hi¹ A (1-methyladenosine), hi² A (2-methyladenosine), am (2' -O-methyladenosine), ms²m⁶ A (2-methylthio-N6-methyladenosine), i⁶ A (N6-isopentenyl adenosine), ms²i⁶ A (2-methylthio-N6-isopentenyl adenosine), io⁶ A (N6- (cis-hydroxyisopentenyl) adenosine), ms²io⁶ A (2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine), g⁶ A (N6-glycylcarbamoyladenosine), t⁶ A (N6-threonyl carbamoyladenosine), ms²t⁶ A (2-methylsulfanyl-N6-threonyl carbamoyladenosine), m⁶t⁶ A (N6-methyl-N6-threonyl carbamoyladenosine), Im6A (N6-hydroxy N-valylcarbamoyladenosine), ms²hn⁶ A (2-methylsulfanyl-N6-hydroxy N-valylcarbamoyladenosine), ar (p) (2 '-O-ribosyl adenosine (phosphoric acid)), I (inosine), hi^l I (1-methyl inosine), hi¹ Im (1, 2' -O-dimethyl inosine), m³ C (3-methylcytidine), cm (2' -O-methylcytidine), S² C (2-thiocytidine), ac⁴ C (N4-acetylcytidine), f⁵ C (5-formylcytidine), m⁵ Cm (5, 2 '-O-dimethylcytidine), ac⁴ Cm (N4-acetyl-2' -O-methylcytidine), k² C (Lai Xiding), hi¹ G (1-methylguanosine), m² G (N2-methylguanosine), m⁷ G (7-methylguanosine), gm (2 '-O-methylguanosine), m²² G (N2, N2-dimethylguanosine), m² Gm (N2, 2' -O-dimethylguanosine), and, m²² Gm (N2, 2 '-O-trimethylguanosine), gr (p) (2' -O-ribosyl guanosine (phosphoric acid)), yW (Huai Dinggan), O² yW (peroxy Huai Dinggan), OHyW (hydroxy Huai Dinggan), OHyW (under-modified hydroxy Huai Dinggan), imG (astragaloside), mimG (methyl astragaloside), Q (astragaloside), oQ (epoxy-braided glycoside), galQ (galactosyl-braided glycoside), manQ (mannosyl-braided glycoside), preQ0 (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (gulurinoside), D (dihydrouridine), m5Um (5, 2' -O-dimethyluridine), S⁴ U (4-thiouridine), m⁵s² U (5-methyl-2-thiouridine), and the like, S² Um (2-thio-2' -O-methyluridine), acp³ U (3- (3-amino-3-carboxypropyl) uridine), ho⁵ U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo⁵ U (uridine 5-hydroxyacetic acid), mcmo5U (uridine 5-glycolate), chm⁵ U (5- (carboxyhydroxymethyl) uridine), mchm⁵ U (5- (carboxyhydroxymethyl) uridine methyl), mcm⁵ U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2' -O-methyluridine), mcm⁵s² U (5-methoxycarbonylmethyl-2-thiouridine), nmVU (5-aminomethyl-2-thiouridine), mcm5U (5-methylaminomethyluridine), mcm⁵s² U (5-methylaminomethyl-2-thiouridine), mcm⁵se² U (5-methylaminomethyl-2-selenouride), and, ncm⁵ U (5-carbamoylmethyluridine), ncm⁵ U (5-carbamoylmethyl-2 '-O-methyluridine), cmnm⁵ U (5-carboxymethylaminomethyluridine), cmnm⁵ U (5-carboxymethylaminomethyl-2' -0-methyluridine), cmnm⁵s² U (5-carboxymethylaminomethyl-2-thiouridine), m⁶ A (N6, N6-dimethyladenosine), im (2 '-O-methylainosine), m⁴ C (N4-methylcytidine), m⁴ Cm (N4, 2' -O-dimethylcytidine), hm⁵ C (5-hydroxymethylcytosine), m³ U (3-methyluridine), cm⁵ U (5-carboxymethyluridine), m⁶ Am (N5, 2' -O-dimethyladenosine), m⁶ 2Am (N6, N6, O-2 t-trimethyladenosine), m^2,7G(N², 7-dimethylguanosine), m^2,2,7 G (N2, N2, 7-trimethylguanosine), m³ Um (3, 2' -O-dimethyluridine), m⁵ D (5-methyldihydrouridine), f⁵ Cm (5-formyl-2 ' -O-methylcytidine), hi¹ Gm (1, 2' -O-dimethylguanosine), m¹ Am (1, 2' -O-dimethyl adenosine), τm⁵ U (5-taurine methyl uridine), tm⁵s² U (5-taurine methyl-2-thiouridine)), imG-14 (4-demethylaya glycoside), imG² (iso-ya glycoside) and ac⁶ A (N6-acetyl adenosine).

In some embodiments, the modified base comprises pseudouridine (5-ribosyl uracil) (ψ). In some embodiments, pseudouridine alters base pairing interactions, thereby affecting RNA secondary structure and mRNA encoding. In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m 1 ψ). In some embodiments, when ψ and m1 ψ are present in vitro transcribed mRNA within a host cell, the immunostimulatory properties of the modified RNA are reduced.

In some embodiments, between about 0.5% and 99% of uridine in the modified RNA molecule is 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 uridine in the modified RNA molecule is 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 uridine in the molecule other than the first 5' uridine is modified. In some embodiments, at least about 50% of uridine in the molecule other than the first 5' uridine is ψ. In some embodiments, at least about 50% of uridine in the molecule other than the first 5' uridine is m1ψ.

In some embodiments, all uridine in the molecule except the first 5' uridine are modified. In some embodiments, all but the first 5' uridine in the molecule is ψmodified. In some embodiments, all uridine in the molecule except the first 5' uridine is m1 ψ.

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, alkylphosphonate, phosphoramidate, boranephosphorate, and/or morpholino.

C. Coding sequence

In some embodiments, the modified RNA molecule comprises an open reading frame comprising 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.

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.

In some embodiments, the open reading frame encodes a recombinant protein. In some embodiments, the recombinant protein is selected from the group consisting of an enzyme, a structural protein, a transport protein, a receptor, a hormone, an antibody, a transcription factor, a growth factor, a cytokine, a motor protein, a structural protein, and a chaperone. 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.

In some embodiments, the open reading frame encodes a therapeutic payload comprising a compound capable of eliciting an immunity to one or more conditions or diseases of interest. In some embodiments, the condition of interest is associated with or caused by infection with a pathogen, such as coronavirus (e.g., 2019-nCoV), influenza, measles, human Papilloma Virus (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 the cells. In some embodiments, the open reading frame encodes a tumor-associated antigen (TAA) characteristic of cancer or an antigenic fragment or epitope thereof.

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 sequences set forth in SEQ ID NOs 1-2. In some embodiments, the open reading frame encoding the reporter protein comprises the sequences set forth in SEQ ID NOS.18-21 or 31-34.

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 SARS-COV-2 obronate BA.4 and BA.5 Receptor Binding Domains (RBDs). In some embodiments, the open reading frame encoding SARS-COV-2BA.4/BA.5RBD comprises the sequences set forth in SEQ ID NO. 3-4.

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 sequences set forth in SEQ ID NOs 5-6. In some embodiments, the open reading frame encoding the rabies virus antigen comprises the sequences set forth in SEQ ID NOS 15-17 or 28-30.

In some embodiments, the open reading frame encodes a Respiratory Syncytial Virus (RSV) antigen. In some embodiments, the open reading frame encoding an antigen of respiratory syncytial virus comprises the sequences set forth in SEQ ID NOS.7-8. In some embodiments, the open reading frame encoding an antigen of a respiratory syncytial virus comprises the sequences set forth in SEQ ID NOS 9-11 or 22-24.

In some embodiments, the open reading frame encodes a Varicella Zoster Virus (VZV) antigen. In some embodiments, the open reading frame encoding an antigen of a respiratory syncytial virus comprises the sequences set forth in SEQ ID NOS 12-14 or 25-27.

In some embodiments, the open reading frame comprises a coding sequence comprising modifications to the ribose backbone and/or the nitrogenous base. In some embodiments, the coding sequence further comprises at least one modification 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 linkage.

In some embodiments, the invention provides methods of inducing translation of a recombinant protein by 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.

V. pharmaceutical composition

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 into a cell. In some embodiments, the pharmaceutically acceptable carrier comprises a Lipid Nanoparticle (LNP), a lipid complex, or a liposome. 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, the liposome is an artificial vesicle having at least one lipid bilayer.

Lipid Nanoparticle (LNP)

In some embodiments, the pharmaceutical composition comprises LNP. In some embodiments, the modified RNA is formulated in an LNP, such as those described in international publication No. WO2012170930, incorporated herein by reference in its entirety. In some embodiments, the granularity of the LNP may be increased and/or decreased. The change in particle size may help counteract a biological response, such as (but not limited to) inflammation, or may increase the biological effect of the mRNA when administered to an individual.

In some embodiments, the LNP comprises from about 30 mole% to about 55 mole% cationic lipid. In some embodiments, the LNP comprises greater than about 30 mole% cationic lipid, for example greater than about 35 mole%, 40 mole%, 45 mole%, 50 mole%, 55 mole% or more of any of the cationic lipids. In some embodiments, the LNP comprises less than about 55 mole% cationic lipid, for example less than about any of 50 mole%, 45 mole%, 40 mole%, 35 mole%, 30 mole% or less cationic lipid.

In some embodiments, the LNP comprises about 5 mole% to about 40 mole% phospholipids. In some embodiments, the LNP comprises greater than about 5 mole% phospholipid, e.g., greater than about 10 mole%, 15 mole%, 20 mole%, 25 mole%, 30 mole%, 35 mole%, 40 mole% or more of any one of the phospholipids. In some embodiments, the LNP comprises less than about 40 mole% phospholipids, for example less than about 35 mole%, 30 mole%, 25 mole%, 20 mole%, 15 mole%, 10 mole%, 5 mole% or less of any of the phospholipids.

In some embodiments, the LNP comprises about 20 mole% to about 50 mole% sterols. In some embodiments, the LNP comprises greater than about 20 mole% sterols, for example, greater than any of about 25 mole%, 30 mole%, 35 mole%, 40 mole%, 45 mole%, 50 mole% or more sterols. In some embodiments, the LNP comprises less than about 50 mole% sterols, for example, less than about 45 mole%, 40 mole%, 35 mole%, 30 mole%, 25 mole%, 20 mole% or less sterols of any of the following.

In some embodiments, the LNP comprises a cationic lipid, a phospholipid, a sterol, and a polymer conjugated lipid, such as any of the cationic lipids, phospholipids, sterols, and polymer conjugated lipids described herein. In some embodiments, the LNP comprises i) from about 30 mole% to about 55 mole% cationic lipid, ii) from about 5 mole% to about 40 mole% phospholipid.

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

In some embodiments, the LNPs disclosed herein further comprise a therapeutic payload. The payload may be any substance or compound having a therapeutic or prophylactic effect. In some embodiments, the therapeutic payload is a small molecule, cytotoxin, radioactive ion, chemotherapeutic compound, vaccine, or a compound that elicits an immune response.

B. Cationic lipids

In one embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles described herein is a cationic lipid described in international patent publication No. WO2021204175, which is incorporated herein by reference in its entirety.

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

or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

G1 and G2 are each independently a bond, C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of the alkylene or alkenylene groups-CH 2-is optionally replaced by-O-;

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;

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;

r1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

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

r3 is-N (R4) R5;

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;

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;

x is 0, 1 or 2, and

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

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

Is a single bond or a double bond;

r1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

G4 is a bond, C1-C23 alkylene, C2-C23 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenyl;

r3 is-N (R4) R5;

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

x is 0, 1 or 2, and

In one embodiment, the compound is a compound of formula (01-I-B), (01-I-B'), (01-I-B "), (01-I-C), (01-I-D), or (01-I-E):

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

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 a C2-C4 alkylene group. In one embodiment, G3 is C2 alkylene. In one embodiment, G3 is C4 alkylene.

In one embodiment, R3 has one of the following structures:

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

In one embodiment, the compound is a compound of table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 1:

In one embodiment, the cationic lipids contained in the compositions, nanoparticle compositions, or nanoparticles provided herein are the cationic lipids 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):

g1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of said G1 and G2-CH 2-is optionally replaced by-O-, -C (=o) O-or OC (=o) -;

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);

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);

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

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

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

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

R3 is-N (R4) R5, -OR6 OR-SR 6;

r4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4-to 8-membered heterocycloalkyl;

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

r6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C10 aryl;

x is 0, 1 or 2, and

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

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

Each L1 is independently -OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SRl、-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);

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

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

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

R3 is-N (R4) R5, -OR6 OR-SR 6;

x is 0, 1 or 2, and

In one embodiment, the compound is A compound of formulA (02-V-A), (02-V-B), (02-V-C), (02-V-D), (02-V-E), (02-V-F):

wherein z is an integer from 2 to 12;

x0 is an integer from 1 to 11;

y0 is an integer from 1 to 11;

x1 is an integer from 0 to 9;

y1 is an integer from 0 to 9;

x2 is an integer from 2 to 5;

x3 is an integer from 1 to 5;

x4 is an integer from 0 to 3;

y2 is an integer from 2 to 5;

y3 is an integer from 1 to 5, and

Y4 is an integer from 0 to 3;

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, each L1 is independently-OR 1, -OC (=o) R1, OR-C (=o) OR1, and each L2 is independently-OR 2, -OC (=o) R2, OR-C (=o) OR2. In one embodiment, R1 and R2 are independently a linear C6-C10 alkyl group or-R7-CH (R8) (R9), wherein R7 is a C0-C5 alkylene group, and R8 and R9 are independently a C2-C10 alkyl group or a C2-C10 alkenyl group.

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

wherein z is an integer from 2 to 12;

y is an integer from 2 to 12;

x0 is an integer from 1 to 11;

x1 is an integer from 0 to 9;

x2 is an integer from 2 to 5;

x3 is an integer from 1 to 5, and

X4 is an integer from 0 to 3;

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, z is an integer from 2 to 6. In one embodiment, z is2, 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.

In one embodiment, each L1 is independently-OR 1, -OC (=o) R1 OR-C (=o) OR1, and L2 is-OC (=o) R2 OR-C (=o) OR2, -NRdC (=o) R2 OR-C (=o) nrenf. In one embodiment, R1 is a straight chain 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. 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 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.

In one embodiment, the compound is a compound of table 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof. Table 2:

in one embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles described herein is a cationic lipid described in international patent publication No. WO2022152109, which is incorporated herein by reference in its entirety.

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

g1 and G2 are each independently a bond, C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of the groups-CH 2-in the G1 and G2 groups are optionally replaced by-O-;

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;

Each L2 is independently -OC(＝O)R2、-C(＝O)OR2、-OC(＝O)0R2、-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)、-(C6-C10 arylene) -R2, - (6 to 10 membered heteroarylene) -R2, - (4 to 8 membered heterocyclylene) -R2, or R2;

r1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

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

g3 is C2-C12 alkylene or C2-C12 alkenylene, wherein a portion or all of the alkylene or alkenylene is optionally replaced with C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4-to 8-membered heterocyclylene, C6-C10 arylene, or 5-to 10-membered heteroarylene;

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

R4 is C1-C12 alkyl or C3-C8 cycloalkyl;

x is 0, 1 or 2;

n is 1 or 2;

m is 1 or 2, and

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

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

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

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

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

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

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

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

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

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 a C2-C6 alkylene group.

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 a substituted C1-C12 alkyl group. In one embodiment, R4 is-CH 2CH2OH.

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) nrenf. In one embodiment, R1, R2, rc and Rf are each independently a linear C6-C18 alkyl group, a linear C6-C18 alkenyl group or-R7-CH (R8) (R9), wherein R7 is a C0-C5 alkylene group, and R8 and R9 are each independently a C2-C10 alkyl group or a C2-C10 alkenyl group. In one embodiment, R1, R2, rc and Rf are each independently a linear C7-C15 alkyl group, a linear C7-C15 alkenyl group or-R7-CH (R8) (R9), wherein R7 is a C0-C1 alkylene group, and R8 and R9 are each independently a C4-C8 alkyl group or a C6-C10 alkenyl group. In one embodiment, ra, rb, rd and Re are each independently H.

In one embodiment, the compound is a compound of table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 3:

In one embodiment, the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in international patent publication No. WO2022247755A1, which is incorporated herein by reference in its entirety.

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

g1 and G2 are each independently a bond, C2-C12 alkylene or C2-C12 alkenylene;

r1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

ra, rb, rd and Re are each independently H, C C24 alkyl or C2C 24 alkenyl;

rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

r0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl or 4-to 8-membered heterocycloalkyl;

g3 is C2-C12 alkylene or C2-C12 alkenylene;

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

x is 0, 1 or 2;

s is 0 or 1, and

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.

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

r1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

g3 is C2-C12 alkylene or C2-C12 alkenylene;

G4 is C2-C12 alkylene or C2-C12 alkenylene;

R3 is-N (R4) R5 OR-OR 6;

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

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

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of formula (04-IV):

Or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G3 is a C2-C4 alkylene group. In one embodiment, G4 is a C2-C4 alkylene group.

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-CH 2CH2OH.

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) nrenf OR R2. In one embodiment, R1 and R2 are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In one embodiment, R1 and R2 are each independently-R7-CH (R8) (R9), wherein R7 is C1-C5 alkylene and R8 and R9 are each 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 straight chain 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 a C2-C10 alkyl group.

In one embodiment, the compound is a compound of table 4, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof. Table 4:

C. Kit for detecting a substance in a sample

In some embodiments, the kit further comprises modified nucleotides. 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 m1 ψ.

Methods for reducing innate immune stimulation

Provided herein are methods of reducing innate immune stimulation of RNA molecules.

In some embodiments, described herein are methods of reducing innate immune stimulation of an RNA molecule comprising using a 5 'cap having the sequence of N1pppN pU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-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.

In some embodiments, m⁷GpppA_m pU has the formula,

In some embodiments, described herein are methods of reducing innate immune stimulation of an RNA molecule comprising using a 5 'cap having the sequence of N1vpppN pU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-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.

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 the sequence of m⁷ GvpppApU. In some embodiments, the 5' cap has the sequence of m⁷ GvpppApU, wherein a is modified or unmodified. In some embodiments, a is 2' -O-methylated. In some embodiments, the 5' cap has the sequence of m⁷GvpppA_m pU. In some embodiments, the 5' cap comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 21 or 34.

In some embodiments, m⁷GvpppA_m pU has the formula,

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 uridine in the RNA molecule. In some embodiments, the method comprises modifying at least about 50% of the uridine in the RNA molecule, except for the first 5' uridine. In some embodiments, the method comprises modifying at least about 90% of the uridine in the RNA molecule, except for the first 5' uridine. In some embodiments, the method comprises modifying at least about 95% of the uridine in the RNA molecule, except for the first 5' uridine. In some embodiments, the method comprises modifying at least about 99% of the uridine in the RNA molecule, except for the first 5' uridine.

In some embodiments, the method comprises modifying all uridine in the RNA molecule except the first 5' uridine.

In some embodiments, the method comprises modifying between about 1% and about 99% of the uridine bases in the RNA molecule to be 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 uridine in the RNA molecule to be ψ. In some embodiments, the method comprises modifying at least about 50% of uridine in the RNA molecule other than the first 5' uridine to be ψ. In some embodiments, the method comprises modifying at least about 90% of uridine in the RNA molecule other than the first 5' uridine to be ψ. In some embodiments, the method comprises modifying at least about 95% of uridine in the RNA molecule other than the first 5' uridine to be ψ. In some embodiments, the method comprises modifying at least about 99% of uridine in the RNA molecule other than the first 5' uridine to be ψ. In some embodiments, the method comprises modifying all but the first 5' uridine in the RNA molecule to be ψ.

In some embodiments, the method comprises modifying between about 1% and about 99% of the uridine bases in the 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 uridine in the RNA molecule to m1ψ. In some embodiments, the method comprises modifying at least about 50% of uridine in the RNA molecule other than the first 5' uridine to m1ψ. In some embodiments, the method comprises modifying at least about 90% of uridine in the RNA molecule other than the first 5' uridine to m1ψ. In some embodiments, the method comprises modifying at least about 95% of uridine in the RNA molecule other than the first 5' uridine to m1ψ. In some embodiments, the method comprises modifying at least about 99% of uridine in the RNA molecule other than the first 5' uridine to m1ψ. In some embodiments, the method comprises modifying all but the first 5' uridine in the RNA molecule to m1ψ.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a5 'cap having a sequence of GpppApU and modifying at least one uridine in the RNA molecule other than a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

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 uridine in the RNA molecule except for a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

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 uridine in the RNA molecule except for the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

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 uridine in the RNA molecule except for the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GpppApU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GpppApU and modifying all uridine in the RNA molecule except for a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GpppApU and modifying all uridine in the RNA molecule except the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GpppApU and modifying all uridine in the RNA molecule except the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GpppA_m pU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GpppA_m pU and modifying all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GpppA_m pU and modifying all uridine in the RNA molecule except the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GpppA_m pU and modifying all uridine in the RNA molecule except the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

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 in the RNA molecule other than a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

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 uridine in the RNA molecule except for a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a5 'cap having a sequence of GvpppApU and modifying all uridine in the RNA molecule except for the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

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 uridine in the RNA molecule except for the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GvpppApU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GvpppApU and modifying all uridine in the RNA molecule except for a first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GvpppApU and modifying all uridine in the RNA molecule except the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷ GvpppApU and modifying all uridine in the RNA molecule except the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GvpppA_m pU and modifying at least one uridine in the RNA molecule other than the first 5' uridine to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GvpppA_m pU and modifying all but the first 5' uridine in the RNA molecule to produce an RNA molecule, wherein the RNA molecule is non-self-replicable, and wherein U is an unmodified uridine.

In some embodiments, the method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GvpppA_m pU and modifying all uridine in the RNA molecule except the first 5' uridine to be ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, a method of reducing innate immune stimulation of an RNA molecule comprises using a 5 'cap having the sequence of m⁷GvpppA_m pU and modifying all uridine in the RNA molecule except the first 5' uridine to m1 ψ to produce an RNA molecule, wherein the RNA molecule is non-self replicable, and wherein U is an unmodified uridine.

In some embodiments, the methods of producing an RNA molecule for reducing innate immune stimulation of the RNA molecule as described above include methods of in vitro transcription of DNA molecules into modified RNA molecules disclosed herein.

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

It is to be understood that any embodiment of a compound provided herein as set forth above, and any particular substituent and/or variable of a compound provided herein as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of a compound to form embodiments not specifically set forth above. Furthermore, where a list of substituents and/or variables is listed for any particular group or variable, it is to be understood that each individual substituent and/or variable may be deleted from a particular embodiment and/or claim and that the remaining list of substituents and/or variables is to be considered within the scope of embodiments provided herein.

It is to be understood that in this specification, combinations of the various substituents and/or variables depicted are permissible only if such contributions result in stable compounds.

Advantageous effects

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

(1) Capping efficiency will increase;

(2) RIG-I activation will be reduced, i.e. the immune stimulation is significantly reduced, resulting in reduced innate immune stimulation upon delivery to cells:

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

(4) Antigen specific IgG titers will be enhanced.

Examples

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

Example 1 in vitro transcription preparation of mRNA and detection of various indicators

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

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

Plasmid linearization DNA plasmid templates containing DNA encoding eGFP, SARS-COV-2 Omikovia 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, washing twice with 70% ethanol, and re-suspending in nuclease free water.

In vitro transcription

The purified linear plasmid was used as a template for in vitro transcription reactions using T7 RNA polymerase. In vitro transcription was performed by adding m⁷GpppA_m pU or m⁷GpppA_m pG, respectively, to synthesize mRNA having Cap1 "Cap" structure, and using m1 ψ instead of uridine to synthesize mRNA.

For m7GpppAmpU, the ATP/GTP/CTP/m1 ψ concentrations were 5mM each, the m7GpppAmpU concentration was 4mM, the 10X reaction buffer [400mM Tris-HCl (pH 7.9), 100mM DTT,20mM spermidine, 0.02% Triton X-100,270mM magnesium acetate ] concentration was 1X, the DNA template concentration was 50. Mu.g/mL, the murine RNase inhibitor concentration was 1 unit/. Mu.L, the yeast inorganic pyrophosphatase concentration was 0.002 unit/. Mu.L, and the T7 RNA polymerase concentration was 5 unit/. Mu.L. For m7GpppAmpG, the ATP/GTP/CTP/m1 ψ concentrations were 5mM each, the m7GpppAmpG concentration was 4mM, the 10X reaction buffer [400mM Tris-HCl (pH 7.9), 100mM DTT,20mM spermidine, 0.02% Triton X-100,270mM magnesium acetate ] concentration was 1X, the DNA template concentration was 50. Mu.g/mL, the murine RNase inhibitor concentration was 1 unit/. Mu.L, the yeast inorganic pyrophosphatase concentration was 0.002 unit/. Mu.L, and the T7 RNA polymerase concentration was 5 unit/. Mu.L.

The reaction was run for 2.5h followed by DNase I digestion (DNase I was added to the IVT reaction at a final concentration of 2 units/. Mu.L, incubated for 30 minutes at 37 ℃). And then immediately usedMRNA was purified using RNA purification kit (NEB, # T2050S), UV quantified using a NanoDrop One, analyzed for purity using a fragment analyzer, and cap rate detected using LC-MS.

As a result, as shown in Table 5, the cap rates of mRNAs synthesized on the basis of m⁷GpppA_m pU were all higher than those of mRNAs synthesized on the basis of m⁷GpppA_m pG, and the purities were substantially the same.

Table 5:

RIG-I activation studies were performed using mRNA synthesized from two cap analogues:

Reporter cell lines overexpressing RIG-I were 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℃in 5% CO 2. Prior to transfection, cells were seeded at a density of 20,000 cells/well in 96-well plates (in 100 μl of medium) until cell fusion reached 80% or more for transfection and 250ng mRNA was transfected using Lipofectamine 2000 (Invitrogen, catalog No. 11668-019). mu.L of cell supernatant was taken 24 hours after transfection. Cell supernatants were collected into new 96-well plates, with 10 μl of detection reagent QUANTI-Luc^TM added to each well. OD values at 560nm were read using Molecular Devices plate reader and data analyzed.

As a result, as shown in Table 6, the RIG-I activation of mRNA synthesized based on m⁷GpppA_m pU was lower than that of mRNA synthesized based on m⁷GpppA_m pG.

Table 6:

Cytotoxicity studies were performed using mRNA synthesized by two cap analogues:

HEK-293T and A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37℃in 5% CO2 and seeded in 96-well plates at 100. Mu.l cell suspension per well (5000 cells/well). Plates were pre-incubated in a humidified incubator for 24 hours (37 ℃,5% co 2) and then 10 μl of each mRNA was added to the wells. Plates were incubated in the incubator for 24 hours, 10. Mu.l of CCK-8 solution (catalog number: RM02823, abclonal) was added to each well of the plates, and then the plates were incubated in the incubator on a shaker for 1-4 hours. Mix gently. The absorbance at 450nm was then measured using an enzyme label.

As a result, as shown in Table 7, there was no significant difference in toxicity of the two mRNAs to the two cell lines.

Table 7:

example 2 rabies virus-based mRNA vaccine examples

This example describes the in vitro transcription of rabies virus-based mRNA vaccines.

MRNA encoding rabies virus antigen (designated mRNA5 (SEQ ID NO: 5), mRNA6 (SEQ ID NO: 6), respectively) was prepared according to the method of example 1 using m⁷GpppA_m pG and m⁷GpppA_m pU, respectively, both of which were synthesized by replacing uridine with m1 ψ. The lipid mixture (comprising 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), ionizable lipid, PEG-lipid, and cholesterol) was then dissolved in ethanol and then mixed with the mRNA solution in a T-mixer. The formulation was then concentrated to the desired concentration by Tangential Flow Filtration (TFF) membranes and filtered through a 0.22 μm filter to give the final product (designated LNP1, LNP2, respectively) which was stored at 2-8 ℃ until use. The finished product was used in experimental animals, and each preparation (0.1. Mu.g/50. Mu.L/mouse) was injected intramuscularly at a single point on day 0 to the right hind limb of BALB/c mice, 10 animals per group (except for 5 animals in PBS group), and whole blood was collected on day 13 after administration to measure serum levels of RABV-specific IgG titer.

Results:

as shown in table 8, the IgG titer level of the LNP2 group was higher than that of the LNP1 group.

Table 8:

example 3 respiratory syncytial virus-based mRNA vaccine examples

This example describes the in vitro transcription of an mRNA vaccine based on respiratory syncytial virus.

MRNA encoding respiratory syncytial virus antigen (designated mRNA7 (SEQ ID NO: 7), mRNA8 (SEQ ID NO: 8)) was prepared using m⁷GpppA_m pG and m⁷GpppA_m pU, respectively, according to the procedure of example 1, both of which were synthesized with m1 ψ instead of uridine. The lipid mixture (comprising 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), ionizable lipid, PEG-lipid, and cholesterol) was then dissolved in ethanol and then mixed with the mRNA solution in a T-mixer. The formulation was then concentrated to the desired concentration by Tangential Flow Filtration (TFF) membranes and filtered through a 0.22 μm filter to give the final product (designated LNP3, LNP4 respectively) which was stored at 2-8 ℃ until use. The finished product was used in experimental animals, on day 0, with single point intramuscular injection of each formulation (0.1 μg/50 μl/mouse) into the right hind limb of BALB/c mice, 5 animals with PBS,6 animals with LNP3, and 10 animals with LNP4. Whole blood was collected on day 13 post-administration to measure serum levels of RSV-specific IgG.

As a result, the LNP4 group had higher IgG titer levels than the LNP3 group, as shown in Table 9.

Table 9:

* Below the detection limit

Example 4 expression in BHK-21 cell line

MRNA encoding eGFP was synthesized using (G^m7)ppp(A^m2) G and (G^m7)ppp(A^m2) U, respectively, and both were synthesized using m1 ψ instead of uridine (i.e., mRNAl (SEQ ID NO: 1), mRNA2 (SEQ ID NO: 2), respectively) according to the method of example 1. 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℃under 5% CO 2. 24 hours prior to transfection, BHK-21 cells were seeded in 96-well plates (in 100. Mu.L of medium) at a density of 10,000 cells per well. 25ng mRNA was transfected with TransIT (Mirus bio, catalog number MIR 2250), and cells were harvested 24 hours later and resuspended in PBS. The data were analyzed by a Molecular Devices plate reader for detection at 488/507nm using GRAPHPADPRISM, and the results are shown in FIG. 1, indicating that the expression levels of eGFP in BHK-21 were similar on both mRNAs and that there was no significant difference between the groups.

Example 5 in vitro transcription preparation of mRNAs with similar Cap Structure and detection of the respective mRNAs

Purified linear plasmids encoding RSV, RABV, VZV antigen and eGFP protein, respectively, were used as templates for in vitro transcription reactions using T7 RNA polymerase. Linear templates were prepared and purified as described in example 1. mRNA having Cap1 "Cap" structure was synthesized by in vitro transcription with addition of (G^m7)ppp(A^m2)U、(G^m7,3'OMe)ppp(A^m2)U、(G^m7)ppp(A^m2) m1 ψ or (G^m7)vppp(A^m2) U, respectively. At the same time, mRNAs were synthesized with either m1ψ or uridine.

ATP/GTP/CTP/m1 ψ concentrations were 12.5mM each, (G^m7)ppp(A^m2) U or (G^m7,3'OMe)ppp(A^m2) U or (G^m7)ppp(A^m2) m1 ψ or (G^m7)vppp(A^m2) U concentration was 12.5mM, 10X reaction buffer [400mM Tris-HCl (pH 7.9), 100mM DTT,20mM spermidine, 0.02% Triton X-100,270mM magnesium acetate ] concentration was 1X, DNA template concentration was 50. Mu.g/mL, murine RNase inhibitor concentration was 1 unit/. Mu.L, yeast inorganic pyrophosphatase concentration was 0.0005 unit/. Mu.L, T7 RNA polymerase concentration was 5 units/. Mu.L.

The IVT reaction was run for 2.5h followed by DNase I digestion (DNase I was added to the IVT reaction at a final concentration of 2 units/. Mu.L and incubated for 30 minutes at 37 ℃). Table 11 provides specific information about mRNA, such as the structure of different cap analogs. Cap analogue products such as (G^m7)ppp(A^m2)U、(G^m7,3'OMe)ppp(A^m2)U、(G^m7)ppp(A^m2) m1 ψ or (G^m7)vppp(A^m2) U are commercially available, the preparation methods of which are known in the art. For example, (G^m7)vppp(A^m2) U is prepared as per CN116143855a or purchased from Jiangsu Synthgene biotechnology co. And then immediately usedMRNA was purified using RNA purification kit (NEB, # T2050S), UV quantified using NanoDrop One, purity analyzed using a fragment analyzer, cap rate detected using LC-MS, and RIG-I activation analyzed using HEK Lucia RIG-I cells from Invivogen (catalog number hkl-hrigi).

Results from the analysis of table 10:

(1) Yield: yield of mRNA synthesized based on (G^m7)ppp(A^m2) U was higher than that based on (G^m7,3'OMe)ppp(A^m2) U or (G^m7)ppp(A^m2) m1 ψ, with good agreement between 4 different mRNA samples. And the yield based on (G^m7)ppp(A^m2) U mRNA was substantially the same as the yield based on (G^m7)vppp(A^m2) U-encoding mRNA when encoding eGFP.

(2) Purity mRNA synthesized based on (G^m7)ppp(A^m2) U was higher than mRNA synthesized based on (G^m7,3'OMe)ppp(A^m2) U or (G^m7)ppp(A^m2) m1 ψ, with good agreement between 4 different mRNA samples. When uridine or m1 ψ modification is used, the purity of the mRNA encoding eGFP synthesized based on (G^m7)ppp(A^m2) U is substantially the same as the purity of the mRNA encoding eGFP synthesized based on (G^m7)vppp(A^m2) U.

(3) Capping rate-capping rate of mRNA synthesized based on all of these cap analogs was as high as 100%.

(4) RIG-I activation when uridine was replaced with m1ψ, RIG-I activation of mRNA based on (G^m7)ppp(A^m2) U was lower than that of mRNA based on (G^m7,3'OMe)ppp(Am²) U or (G^m7)ppp(A^m2) m1ψ, with good agreement between 4 different samples. In the case of uridine or m1 ψ modification, the RIG-I activation of mRNA encoding eGFP based on (G^m7)ppp(A^m2) U synthesis was substantially the same as the RIG-I activation of mRNA encoding eGFP based on (G^m7)vppp(A^m2) U synthesis.

Table 10:

Table 11:

Example 6 in vitro expression of mRNA synthesized from (G^m7)ppp(A^m2) U and (G^m7)vppp(A^m2) U

MRNA synthesized based on (G^m7)ppp(A^m2) U and (G^m7)vppp(A^m2) U and having uridine or m1 ψ modification was prepared as described in example 5. HEK-293T and A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37℃in 5% CO2 and seeded in 96-well plates at 100. Mu.l cell suspension per well (5000 cells/well). Plates were pre-incubated in a humidified incubator for 24 hours (37 ℃,5% co 2) and then 100ng of each mRNA was added to the wells. Portions of the cells were collected at 24 hours and 48 hours, respectively, and washed three times with PBS. The eGFP protein expression level was assessed using a flow cytometer. As shown in FIGS. 2-3, mRNA synthesized based on (G^m7)ppp(A^m2) U and (G^m7)vppp(A^m2) U had substantially the same expression level when uridine or m1 ψ modification was used.

Sequence(s)

In the following sequences, "ψ" means "m1 ψ (1-methyl-pseudouridine)", unless otherwise indicated. Abbreviations are for clarity of the sequence. The other abbreviations are as follows,

"M⁷ G" and "G^m7" =7-methylguanosine (may also be abbreviated "m 7G");

"a_m" and "a^m2" =2' -O-methyladenosine;

"ppp" = triphosphate bond;

"vppp" = vinyl modified triphosphate bond;

"G^m7,3'OMe" =3' -O-methylated m7G.

SEQ ID NO:10(MRNA 10)

SEQ ID NO:11(MRNA 11)

SEQ ID NO:12(MRNA 12)

SEQ ID NO:13(MRNA 13)

SEQ ID NO:14(MRNA 14)

SEQ ID NO:15(MRNA 15)

SEQ ID NO:16(MRNA 16)

SEQ ID NO:17(MRNA 17)

SEQ ID NO:18(MRNA 18)

SEQ ID NO:19(MRNA 19)

SEQ ID NO:20(MRNA 20)

SEQ ID NO:21(MRNA 21)

SEQ ID NO:22(MRNA 22)

SEQ ID NO:23(MRNA 23)

SEQ ID NO:24(MRNA 24)

SEQ ID NO:25(MRNA 25)

SEQ ID NO:26(MRNA 26)

SEQ ID NO:27(MRNA 27)

SEQ ID NO:28(MRNA 28)

SEQ ID NO:29(MRNA 29)

SEQ ID NO:30(MRNA 30)

SEQ ID NO:31(MRNA 31)

SEQ ID NO:32(MRNA 32)

SEQ ID NO:33(MRNA 33)

SEQ ID NO:34(MRNA 34)

Claims

Translated fromChinese

1.一种修饰的RNA分子，其中所述修饰的RNA分子包含5’帽，其中所述RNA分子是不可自我复制的，其中所述修饰的RNA分子的5’端具有N1pppN2pU或N1vpppN2pU的序列，其中U为未修饰的尿苷，其中N1和N2中的每一者独立地为除修饰或未修饰的尿苷以外的修饰或未修饰的核苷酸，并且其中所述分子中除第一个5’尿苷以外的至少一个尿苷为修饰的尿苷。1. A modified RNA molecule, wherein the modified RNA molecule comprises a 5' cap, wherein the RNA molecule is non-self-replicating, wherein the 5' end of the modified RNA molecule 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, and wherein at least one uridine other than the first 5' uridine in the molecule is a modified uridine.

2.如权利要求1所述的修饰的RNA分子，其中所述修饰的RNA分子的5’端具有GpppApU或GvpppApU的序列，其中A为修饰或未修饰的。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.如权利要求1或2所述的修饰的RNA分子，其中所述修饰的RNA分子的5’端具有m⁷GpppApU或m⁷GvpppApU的序列。The modified RNA molecule according to claim 1 or 2, wherein the 5' end of the modified RNA molecule has a sequence of^m7GpppApU or^m7GvpppApU .

4.如权利要求2或3所述的修饰的RNA分子，其中所述A为修饰的，优选被2’-O-甲基化。4. The modified RNA molecule of claim 2 or 3, wherein said A is modified, preferably 2'-O-methylated.

5.如权利要求1-4中任一项所述的修饰的RNA分子，其中所述修饰的RNA分子的5’端具有m⁷GpppA_mpU或m⁷GvpppA_mpU的序列。The modified RNA molecule according to any one_of claims 1 to 4, wherein the 5' end of the modified RNA molecule has a sequence_of^m7GpppAmpU or^m7GvpppAmpU .

6.如权利要求1-5中任一项所述的修饰的RNA分子，其中所述分子中除第一个5’尿苷以外的所有尿苷均为修饰的，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,

优选地，所述分子中除第一个5’尿苷以外的所有尿苷均为m1Ψ。Preferably, all uridines except the first 5' uridine in the molecule are m1Ψ.

7.如权利要求1-6中任一项所述的修饰的RNA分子，其包含含有编码序列的开放阅读框，7. The modified RNA molecule of any one of claims 1 to 6, comprising an open reading frame containing a coding sequence,

优选地，所述编码序列编码eGFP、SARS-COV-2BA.4/BA.5RBD、狂犬病病毒抗原、呼吸道合胞病毒抗原或水痘-带状疱疹病毒抗原。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.如权利要求1-7中任一项所述的修饰的RNA分子，其中所述分子还包含至少一个修饰的G、C或A。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.如权利要求1-8中任一项所述的修饰的RNA分子，其中所述分子具有修饰的主链,9. The modified RNA molecule of any one of claims 1 to 8, wherein the molecule has a modified backbone,

优选地，所述修饰的主链包含至少一个硫代磷酸酯键。Preferably, the modified backbone comprises at least one phosphorothioate bond.

10.一种包含权利要求1-9中任一项所述的修饰的RNA的药物组合物，其还包含药学上可接受的载体。10. A pharmaceutical composition comprising the modified RNA of any one of claims 1 to 9, further comprising a pharmaceutically acceptable carrier.

11.如权利要求10所述的药物组合物，其中所述药学上可接受的载体为LNP，11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable carrier is LNP,

优选地，所述LNP包含阳离子脂质，Preferably, the LNP comprises a cationic lipid,

更优选地，所述LNP包含：i)约30至55摩尔％的阳离子脂质；ii)约5至40摩尔％的磷脂；iii)约20至50摩尔％的固醇；以及iv)聚合物缀合的脂质。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.

12.一种将DNA分子体外转录为修饰的RNA分子的方法，其包括将所述DNA分子与包含依赖DNA的RNA聚合酶、修饰的和/或未修饰的核苷酸和5’帽的体外转录混合物组合，其中所述修饰的RNA分子是非可自我复制的，其中所述5’帽具有N1pppN2pU或N1vpppN2pU的序列，其中U为未修饰的尿苷，其中N1和N2中的每一者独立地为除修饰或未修饰的尿苷以外的修饰或未修饰的核苷酸，其中所述体外转录混合物包含修饰的尿苷，并且其中所述修饰的RNA分子中除第一个5’尿苷以外的至少一个尿苷为修饰的尿苷。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 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 contains modified uridine, and wherein at least one uridine other than the first 5' uridine in the modified RNA molecule is a modified uridine.

13.如权利要求12所述的方法，其中所述5’帽具有GpppApU或GvpppApU的序列，其中A为修饰或未修饰的。13. The method of claim 12, wherein the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified.

14.如权利要求12或13所述的方法，其中所述5’帽具有m⁷GpppApU或m⁷GvpppApU的序列。14. The method of claim 12 or 13, wherein the 5' cap has a sequence of^m7GpppApU or^m7GvpppApU .

15.如权利要求13或14所述的方法，其中所述A为修饰的，优选被2’-O-甲基化。15. The method of claim 13 or 14, wherein A is modified, preferably 2'-O-methylated.

16.如权利要求12-15中任一项所述的方法，其中所述5’帽具有m⁷GpppA_mpU或m⁷GvpppA_mpU的序列。16. The method of any one_{of claims 12-15, wherein the 5' cap has a sequence of m7GpppAmpU}_or^m7GvpppAmpU^.

17.如权利要求12-16中任一项所述的方法，其中所述修饰的RNA分子中除第一个5’尿苷以外的所有尿苷均为修饰的。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.如权利要求12-17中任一项所述的方法，其中所述修饰的尿苷为m1Ψ，18. The method of any one of claims 12 to 17, wherein the modified uridine is m1Ψ,

优选地，所述修饰的RNA分子中除第一个5’尿苷以外的所有尿苷均为m1Ψ。Preferably, all uridines except the first 5' uridine in the modified RNA molecule are m1Ψ.

19.如权利要求12-18中任一项所述的方法，其中所述依赖DNA的RNA聚合酶为T7 RNA聚合酶或其变体。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.如权利要求12-19中任一项所述的方法，其还包括分离所述修饰的RNA分子。20. The method of any one of claims 12-19, further comprising isolating the modified RNA molecule.

21.一种通过权利要求12-20中任一项所述的方法产生的修饰的RNA分子。21. A modified RNA molecule produced by the method of any one of claims 12-20.

22.一种试剂盒，其包含：1)依赖DNA的RNA聚合酶；2)修饰的和/或未修饰的核苷酸，和3)5’帽，以及4)用于执行权利要求12-20中任一项所述的方法的说明书，其中所述5’帽具有N1pppN2pU或N1vpppN2pU的序列，其中U为未修饰的尿苷，并且其中N1和N2中的每一者独立地为除修饰或未修饰的尿苷以外的修饰或未修饰的核苷酸。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.

23.一种降低RNA分子的先天免疫刺激的方法，其包括使用具有N1pppN2pU或N1vpppN2pU的序列的5’帽，并修饰所述RNA分子中除第一个5’尿苷以外的至少一个尿苷以产生RNA分子，其中所述RNA分子是不可自我复制的，其中U为未修饰的尿苷，其中N1和N2中的每一者独立地为除修饰或未修饰的尿苷以外的修饰或未修饰的核苷酸，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,

优选地，所述5’帽具有GpppApU或GvpppApU的序列，其中A为修饰或未修饰的，Preferably, the 5' cap has a sequence of GpppApU or GvpppApU, wherein A is modified or unmodified,

优选地，所述5’帽具有m⁷GpppApU或m⁷GvpppApU的序列，Preferably, the 5' cap has a sequence of m⁷ GpppApU or m⁷ GvpppApU,

优选地，所述5’帽具有m⁷GpppA_mpU或m⁷GvpppA_mpU的序列，Preferably, the 5' cap has a sequence_of^m7GpppAmpU or_m7GvpppAmpU^,

优选地，所述方法包括修饰所述RNA分子中除第一个5’尿苷以外的所有尿苷，Preferably, the method comprises modifying all uridines in the RNA molecule except the first 5' uridine,

更优选地，所述方法包括将所述RNA分子中除第一个5’尿苷以外的所有尿苷修饰为m1Ψ。More preferably, the method comprises modifying all uridines except the first 5' uridine in the RNA molecule to m1Ψ.