Circular RNA vaccines and methods of use thereof

Abstract

This application provides circular RNA (circRNA) encoding therapeutic peptides (e.g., antigenic peptides, functional proteins, receptor proteins, or target proteins). In some embodiments, this application provides a circRNA vaccine against coronaviruses such as SARS-CoV-2. In some embodiments, the circRNA vaccine comprises a circRNA encoding a nucleic acid sequence of an antigenic peptide comprising the spike (S) protein of a coronavirus or a fragment thereof. Methods for treating or preventing diseases or conditions using circRNA or combinations thereof are also provided.

Description

Technical field

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims the priority of the international patent application PCT / CN2021 / 074998 filed on February 3, 2021 and the international patent application PCT / CN2020 / 110486 filed on August 21, 2020, the contents of which are incorporated by reference in their entirety Incorporated into this article. [Submit sequence listing as ASCII text file]

[0003] The following content submitted as an ASCII text file is incorporated herein by reference in its entirety: Computer Readable Form of the Sequence Listing (CRF) (file name: 165392000242SEQLIST.TXT, date of record: August 18, 2021, size: 247,489 bytes). [field]

[0004] The present application relates to circular RNAs (circRNAs) encoding therapeutic polypeptides, such as circRNA vaccines against coronaviruses, and methods of use thereof.

Prior technology

[0005] COVID-19 is a severe global public health emergency caused by coronavirus infection caused by the SARS-CoV-2 virus. Currently, no effective drugs or vaccines are available. Therefore, there is an urgent need to develop safe and effective vaccines against coronavirus (such as SARS-CoV-2) infection. Vaccines generally fall into two broad categories: vaccines that contain whole viruses (live attenuated or inactivated vaccines), or vaccines that contain parts of viruses, which can be recombinant protein or DNA or RNA based. Vaccines based on whole viruses have several disadvantages, including the need to handle large quantities of infectious virus during vaccine production for inactivated vaccines and the need for extensive safety testing of live attenuated vaccines. Recombinant protein-based vaccines are also limited by the ability to produce recombinant proteins globally, while DNA-based vaccines face difficulties related to the safe delivery of DNA and the effectiveness of generating immune responses (Amanat, F. and Krammer, F. SARS-CoV- 2 Vaccines: Status Report. (2020) Immunity52, 583–589).

[0006] The development of RNA-based vaccines offers a potential avenue for the development of immunogenic vaccines without the need to handle infectious virus during production. RNA molecules are considered significantly safer than DNA vaccines because RNA is more prone to degradation. They are quickly cleared from the organism, unable to integrate into the genome and affect the gene expression of the cell in an uncontrollable manner. RNA vaccines are also less likely to cause serious side effects, such as the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol. 2010, article ID623687). Transfection with RNA requires only insertion into the cytoplasm of the cell, which is easier to achieve than insertion into the nucleus.

Content of invention

[0007] The application provides a circRNA encoding a polypeptide (such as a therapeutic polypeptide), and a treatment method using the circRNA. In some embodiments, the application provides novel circular RNA (circRNA)-based vaccines against coronaviruses (eg, SARS-CoV-2). Optionally, the SARS-CoV-2 infection is caused by a SARS-CoV-2 variant (eg, the B.1.351 or B.1.617.2 variant). Also provided are methods for producing a circRNA vaccine and methods for using the circRNA vaccine to treat or prevent coronavirus infection.

[0008] One aspect of the present application provides a circular RNA (circRNA) comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and targeting proteins. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide.

[0009] In some embodiments according to any one of the above-mentioned circRNAs, the circRNA further comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding a therapeutic polypeptide.

[0010] In some embodiments according to any one of the above-mentioned circRNAs, the circRNA further comprises an internal ribosome entry site (IRES) sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence and a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments according to any of the above-mentioned circRNAs comprising an IRES sequence, the circRNA further comprises a polyAC or polyA sequence located at the 5' end of the IRES sequence.

[0011] In some embodiments according to any one of the above-mentioned circRNAs, the circRNA also comprises an m6A modification motif sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence, and a nucleic acid sequence encoding a therapeutic polypeptide.

[0012] In some embodiments according to any one of the above-mentioned circRNAs, the nucleic acid also encodes a signal peptide (SP) fused to the N-terminus of the therapeutic polypeptide. In some embodiments, the SP is the SP of human tissue plasminogen promoter (tPA), or the SP of human IgE immunoglobulin (eg, the sequence set forth in SEQ ID NO: 16). In some embodiments, the SP is that of a human IgE immunoglobulin (eg, the sequence set forth in SEQ ID NO: 17).

[0013] In some embodiments according to any one of the above-mentioned circRNAs, the circRNA further comprises: a 3' outer 3' that can be recognized by a 3' catalytic group I intron fragment that is flanked by the 5' end of the nucleic acid sequence encoding the antigenic polypeptide exon sequence, and a 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 21, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 22.

[0014] In some embodiments according to any one of the above-mentioned circRNAs, the circRNAs are circularized in vitro.

[0015] In some embodiments according to any one of the above-mentioned circRNAs, the therapeutic polypeptide is used to treat or prevent infection. In some embodiments, the infection is a viral (eg, coronavirus) infection. In some embodiments, the coronavirus is selected from the group consisting of SARS-CoV, MERS-COV, and SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2.

[0016] One aspect of the present application provides a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is an antigenic polypeptide. In some embodiments, the circRNA is any one of the circRNAs described above.

[0017] One aspect of the present application provides a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is a receptor protein. In some embodiments, the circRNA is any one of the circRNAs described above. In some embodiments, the therapeutic polypeptide is a soluble receptor comprising the extracellular domain of a naturally occurring receptor. In some embodiments, the receptor is the ACE2 receptor. In some embodiments, the receptor is a high affinity mutant ACE2 receptor.

[0018] In some embodiments, there is provided a composition comprising a plurality of circRNAs according to any one of the above-mentioned circRNAs encoding receptor proteins, wherein the receptor proteins corresponding to the plurality of circRNAs are different from each other. In some embodiments, multiple circRNAs target multiple (individual) coronavirus strains, such as SARS-CoV-2.

[0019] One aspect of the present application provides a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is a targeting protein. In some embodiments, the circRNA is any one of the circRNAs described above. In some embodiments, the targeting protein is an antibody. In some embodiments, the antibody is a neutralizing antibody, eg, a neutralizing antibody that targets a coronavirus, such as SARS-CoV-2. In some embodiments, the targeting protein is a therapeutic antibody.

[0020] In some embodiments, there is provided a composition comprising a plurality of circRNAs according to any one of the above-mentioned circRNAs encoding targeting proteins, wherein the targeting proteins corresponding to the plurality of circRNAs are different from each other. In some embodiments, the targeting protein is a neutralizing antibody. In some embodiments, multiple circRNAs target multiple coronavirus strains, such as SARS-CoV-2.

[0021] One aspect of the present application provides a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is a functional protein. In some embodiments, the circRNA is any one of the circRNAs described above. In some embodiments, the functional protein is a tumor suppressor, such as p53 or PTEN. In some embodiments, the functional protein is an enzyme, such as OTC, FAH or IDUA. In some embodiments, the functional protein is selected from the group consisting of DMD, COL3A1, BMPR2, AHI1, FANCC, MYBPC3, and IL2RG. In some embodiments, the therapeutic polypeptide comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) identical to an amino acid sequence selected from the group consisting of Sequence: SEQ ID NO: 18-25.

[0022] One aspect of the present application provides a circular RNA (circRNA), which comprises a nucleic acid sequence encoding an antigenic polypeptide, which comprises a coronavirus spike (Spike, S) protein or a fragment thereof. In some embodiments, the circRNA is any one of the circRNAs described above. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the S protein or fragment thereof comprises a D614G mutation.

[0023] In some embodiments according to any one of the above-mentioned circRNAs encoding antigenic polypeptides, the antigenic polypeptides comprise at least about 80% (for example, at least about 85%, 90%, 95%, Amino acid sequences with 98% or more, or 100%) sequence identity: SEQ ID NOs: 8-10 and 62-63. In some embodiments, the circRNA comprises at least about 80% (for example, at least about 85%, 90%, 95%, 98% or more, or 100% of the nucleic acid sequence selected from SEQ ID NO: 11-15 and 64). %) sequence identity of nucleic acid sequences.

[0024] In some embodiments according to any one of the above-mentioned circRNA encoding an antigenic polypeptide, the antigenic polypeptide comprises a receptor binding domain (RBD) of the S protein. In some embodiments, the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises amino acids that are at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) identical to the amino acid sequence of SEQ ID NO: 2 sequence.

[0025] In some embodiments according to any one of the above-mentioned circRNAs encoding antigenic polypeptides, the antigenic polypeptides comprise a receptor binding domain (RBD) of the S protein, wherein the RBD comprises an amino acid sequence having at least about Amino acid sequences that are 80% (eg, at least about 85%, 90%, 95%, 98%, 99% or more, or 100%) identical.

[0026] In some embodiments according to any one of the above-mentioned circRNA comprising RBD, the antigenic polypeptide further comprises a multimerization domain. In some embodiments, the multimerization domain is the C-terminal Foldon (Fd) domain of T4 fibrin, or the GCN4-based isoleucine zipper domain. In some embodiments, the multimerization domain comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more) of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. Multiple, or 100%) identical amino acid sequences. In some embodiments, the RBD is fused to the multimerization domain via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 5.

[0027] In some embodiments according to any one of the above-mentioned circRNAs encoding antigenic polypeptides, the antigenic polypeptides comprise the S2 region of the S protein. In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the one or more mutations include K986P and V987P. In some embodiments, the S2 region comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0028] In some embodiments according to any one of the above-mentioned circRNAs encoding antigenic polypeptides, the antigenic polypeptides comprise amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage. In some embodiments, the one or more mutations that inhibit S protein cleavage comprise a deletion of amino acid residues 681-684, wherein the numbering is based on SEQ ID NO: 1.

[0029] In some embodiments, there is provided a composition comprising a plurality of circRNAs according to any one of the above-mentioned circRNAs encoding antigenic polypeptides, wherein the antigenic polypeptides corresponding to the plurality of circRNAs are different from each other. In some embodiments, multiple circRNAs target multiple coronavirus strains, such as SARS-CoV-2.

[0030] In some embodiments, a circRNA vaccine is provided, comprising one or more circRNAs according to any one of the above-mentioned circRNAs encoding antigenic polypeptides.

[0031] In some embodiments, a pharmaceutical composition is provided, which comprises a circRNA according to any one of the above-mentioned circRNAs and a pharmaceutically acceptable carrier.

[0032] In some embodiments according to any one of the above-mentioned circRNA vaccine or pharmaceutical composition, the circRNA vaccine or pharmaceutical composition further comprises a transfection reagent. In some embodiments, the transfection reagent is polyethyleneimine (PEI) or lipid nanoparticles (LNP). In some embodiments, the LNP comprises MC3-lipid, DSPC, cholesterol and PEG2000-DMG. In some embodiments, the circRNA or pharmaceutical composition is not formulated with a transfection reagent.

[0033] Other aspects of the present application provide a method for treating or preventing individual coronavirus infection, comprising administering to the individual an effective amount of any one of the above-mentioned circRNA vaccines. In some embodiments, the infection is a SARS-CoV-2 infection. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual.

[0034] Another aspect of the present application provides a method for treating or preventing an individual's disease or condition, comprising administering an effective amount of any of the above-mentioned circRNAs or any of the above-mentioned pharmaceutical compositions to the individual. In some embodiments, wherein the circRNA encodes an antigenic polypeptide, a receptor or a targeting protein (eg, an antibody), the disease or condition is an infection, such as a viral infection. In some embodiments, the disease or condition is one associated with insufficient levels and / or activity of a naturally occurring protein corresponding to the therapeutic polypeptide. In some embodiments, the disease or condition is an inherited genetic disease associated with one or more mutations in a protein corresponding to a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is TP53 or PTEN, and the disease or condition is cancer. In some embodiments, the therapeutic polypeptide is OTC and the disease is ornithine transcarbamylase deficiency. In some embodiments, the therapeutic polypeptide is FAH and the disease is tyrosinemia. In some embodiments, the therapeutic polypeptide is DMD and the disease is Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, or familial dilated cardiomyopathy . In some embodiments, the therapeutic polypeptide is IDUA and the disease or condition is mucopolysaccharidosis type I (MPSI). In some embodiments, the therapeutic polypeptide is COL3A1 and the disease or condition is Ehlers-Danlos syndrome. In some embodiments, the therapeutic polypeptide is AHI1 and the disease or condition is Joubert syndrome. In some embodiments, the therapeutic polypeptide is BMPR2 and the disease or condition is pulmonary hypertension or pulmonary veno-occlusive disease. In some embodiments, the therapeutic polypeptide is FANCC and the disease or condition is Fanconi anemia. In some embodiments, the therapeutic polypeptide is MYBPC3 and the disease or condition is primary familial hypertrophic cardiomyopathy. In some embodiments, the therapeutic polypeptide is IL2RG, and the disease or condition is X-linked severe combined immunodeficiency. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual.

[0035] Other aspects of the present application provide linear RNA capable of forming any of the circRNAs provided herein.

[0036] In some embodiments, a linear RNA can be circularized by autocatalysis of a group I intron comprising a 5' catalytic group I intron segment and a 3' catalytic group I intron segment. In some embodiments, the linear RNA comprises: a 3' catalytic group I intronic segment flanking the 5' end of a 3' exon sequence recognized by a group I intron, and a The 5' catalytic group I intron fragment flanks the 3' end of the 5' exon sequence. In some embodiments, the 3' catalytic group I intron fragment comprises the sequence of SEQ ID NO: 28 and the 5' catalytic group I intron fragment comprises the sequence of SEQ ID NO: 29. In some embodiments, the linear RNA further comprises: 5' homologous sequences flanking the 5' end of the 3' catalytic group I intron segment, and 5' homologous sequences flanking the 3' end of the 5' catalytic group I intron segment 3' homologous sequence. In some embodiments, the 5' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 23, and the 3' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 24.

[0037] In some embodiments, the linear RNA can be circularized by a ligase (eg, RNA ligase). In some embodiments, the ligase is selected from the group consisting of T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2). In some embodiments, the linear RNA comprises: a 5' junction sequence located at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' junction sequence located at the 3' end of the nucleic acid sequence encoding the circRNA, wherein the 5' junction sequence and the 3' junction sequence can be Linked to each other by ligases.

[0038] One aspect of the present application provides a nucleic acid construct comprising a nucleic acid sequence encoding any of the above-mentioned linear RNAs. In some embodiments, the nucleic acid construct comprises a T7 promoter operably linked to the nucleic acid sequence encoding said linear RNA.

[0039] One aspect of the present application provides a method for producing circRNA, comprising: (a) making any of the above-mentioned linear RNAs in the 5' catalytic group I intron fragment and the 3' catalytic group I Autocatalytic conditions of sub-containing fragments to provide circularized RNA products, wherein the linear RNA comprises: 3' catalytic I flanking the 5' end of the 3' exon sequence recognized by group I introns a group I intron fragment, and a 5' catalytic group I intron fragment flanking the 3' end of the 5' exon sequence recognized by the group I intron; and (b) isolating the circularized RNA product, Thus providing a circRNA vaccine.

[0040] One aspect of the present application provides a method for producing circRNA, comprising: (a) contacting any of the above-mentioned linear RNAs with a single-stranded adapter nucleic acid, wherein the linear RNA comprises: encoding the circRNA The 5' junction sequence at the 5' end of the nucleic acid sequence, and the 3' junction sequence at the 3' end of the nucleic acid sequence encoding the circRNA; and wherein the single-stranded adapter nucleic acid comprises from the 5' end to the 3' end: and 3' a first sequence complementary to the linker sequence, and a second sequence complementary to the 5' linker sequence, and wherein the 5' linker sequence and the 3' linker sequence hybridize to the single stranded adapter nucleic acid to provide a double stranded nucleic acid intermediate comprising Single-strand break between the 3' end of the 5' linker sequence and the 5' end of the 3' linker sequence; (b) ligation of the intermediate to the RNA under conditions that allow ligation of the 5' linker sequence to the 3' linker sequence enzyme contacting to provide a circularized RNA product; and (c) isolating the circularized RNA product to provide a circRNA vaccine.

[0041] One aspect of the present application provides a method for producing circRNA, comprising: (a) making any of the above-mentioned linear RNAs with RNA ligase under conditions that allow the 5' junction sequence to be connected to the 3' junction sequence contacting to provide a circularized RNA product, wherein the linear RNA comprises: a 5' junction sequence at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' junction sequence at the 3' end of the nucleic acid sequence encoding the circRNA; and (b) Isolating the circularized RNA product, thereby providing circular RNA.

[0042] In some embodiments according to any one of the above-mentioned methods for producing a circRNA vaccine, the method also includes: obtaining linear RNA by in vitro transcription of a nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA.

[0043] In some embodiments according to any of the methods for producing a circRNA vaccine described above, the method further comprises purifying the circular RNA product.

[0044] Also provided are compositions, kits, and articles of manufacture for use in any of the above methods.

Implementation

[0047] In summary, the present invention... .

[0048] The application provides a circRNA encoding a therapeutic polypeptide such as an antigenic polypeptide, a functional protein, a receptor protein or a targeting protein (such as an antibody). In some embodiments, the present application provides a novel vaccine against coronaviruses (such as SARS-CoV-2 virus) based on circular RNA (circRNA). In some embodiments, the circRNA vaccine encodes an antigenic polypeptide comprising the spike protein of a coronavirus (such as SARS-CoV-2) or a fragment thereof. Unlike other types of coronavirus vaccines, the circRNA vaccine described here does not require the handling of large numbers of infectious particles during production. Furthermore, the circRNA vaccines described herein may provide enhanced stability and efficacy compared to linear RNA vaccines. For example, given their circular nature, circRNAs are particularly stable compared with many linear RNAs because they are resistant to exo-degradation by exosomal ribonuclease complexes. In some embodiments, the circRNA in the circRNA vaccine disclosed herein can undergo rolling circle translation by the ribosomes in the individual to which the vaccine has been administered, thereby producing a large number of antigenic polypeptides. The production of such circRNA vaccines can be performed using a variety of methods, such as chemical ligation, enzymatic catalysis, or ribozyme autocatalysis. The circRNA vaccine described here provides a platform for the rapid development of vaccines against emerging coronavirus strains. In addition, circular RNAs can be rapidly produced in large quantities in vitro without any nucleotide modification, which is quite different from classic mRNA vaccines. Our data demonstrate that exemplary circRNAs and encapsulated circRNA-LNP complexes are highly thermostable at 4°C or room temperature for 7–14 days. Due to their specific properties, circRNAs have potential in biomedical applications.

[0049] I. Definition

[0050] Terms used herein are as commonly used in the art unless otherwise defined below.

[0051] The terms "polynucleotide", "nucleic acid", "nucleotide sequence" and "nucleic acid sequence" are used interchangeably. They refer to polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0052] The term "vaccine" is understood to relate to an immunologically active pharmaceutical preparation. In certain embodiments, the vaccine induces adaptive immunity when administered to a host. Vaccine formulations may also contain pharmaceutical carriers, which may be designed for the particular mode in which the vaccine is intended to be administered.

[0053] The terms "group I intron" and "group I catalytic intron" are used interchangeably to refer to a self-splicing ribozyme that catalyzes its own excision from an RNA precursor. The group I intron consists of two fragments, the 5' catalytic group I intron fragment and the 3' catalytic group I intron fragment, which retain their folding and catalytic functions (i.e., self-splicing activity). In its natural environment, the 5' catalytic group I intronic segment is flanked at its 5' end by a 5' exon containing the 5' catalytic group I intronic segment recognized by the 5' catalytic group I intronic segment. ' exon sequence; and the 3' catalytic group I intron fragment is flanked at its 3' end by a 3' exon containing the 3' exon sequence. As used herein, the terms "5' exon sequence" and "3' exon sequence" are labeled according to the order of the exon relative to the group I intron in its natural environment, e.g., as shown in Figure 1A Show.

[0054] The term "therapeutic polypeptide" refers to a polypeptide having a therapeutic effect. Therapeutic polypeptides may be naturally occurring proteins or engineered functional variants thereof, including functional fragments, derivatives having one or more mutations (e.g., insertions, deletions, substitutions, etc.) to the amino acid sequence of naturally occurring proteins, As well as fusion proteins comprising naturally occurring proteins or fragments thereof. A therapeutic polypeptide can also be an engineered protein that has no naturally occurring counterpart. A therapeutic polypeptide can have a single polypeptide chain or multiple polypeptide chains.

[0055] The term "antigenic polypeptide" refers to a polypeptide that can be used to trigger the immune system of a mammal to produce antibodies specific for the polypeptide or a portion thereof. Antigenic polypeptides described herein include naturally occurring proteins, protein domains, and short peptide fragments derived from naturally occurring proteins. An antigenic polypeptide may comprise one or more known epitopes of a naturally occurring protein. Antigenic polypeptides may contain carrier proteins or multimerized proteins to enhance immunogenicity.

[0056] The term "functional protein" refers to a naturally occurring protein, a functional variant thereof, or an engineered derivative thereof, which plays a role in the treatment of a genetic disease or condition. A disease or condition may be caused in whole or in part by a change, such as a mutation, in a wild-type, naturally occurring protein that corresponds to a functional protein.

[0057] The term "targeting protein" refers to a polypeptide that specifically binds to a target molecule. Targeting proteins described herein include antibody-based and non-antibody-based binding proteins or target binding portions thereof.

[0058] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures including, but not limited to: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. As used herein, the term "antigen-binding fragment" refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2 , bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabodies (ds diabodies), single chain Fv (scFv), scFv dimers (bivalent diabodies), consisting of one or more CDRs Multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain the complete antibody structure.

[0059] As used herein, the terms "specifically bind", "specifically recognize" and "specific for" refer to a measurable and reproducible interaction, such as the binding between a target and a targeting moiety. For example, a targeting moiety that specifically recognizes a target (which may be an epitope) is a targeting moiety (such as an antibody) that binds to that target with a higher affinity, affinity Combined force, easier and / or longer duration. In some embodiments, the extent of binding of the targeting moiety to an unrelated molecule is less than about 10% of the binding of the targeting moiety to the target, as measured by, for example, radioimmunoassay (RIA). In some embodiments, a targeting moiety that specifically binds a target has a dissociation constant (KD) of: ≤10-5M, ≤10-6M, ≤10-7M, ≤10-8M, ≤10-9M, ≤ 10-10M, ≤10-11M or ≤10-12M. In some embodiments, specific binding can include, but does not require exclusive binding. The binding specificity of a targeting moiety can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western Blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™, and peptide scanning.

[0060] The term "functional variant" of a reference protein refers to a variant polypeptide derived from the reference protein or a portion thereof, and which variant has substantially the same activity (e.g., target binding or enzymatic activity) as the reference protein. ). "Essentially the same activity" refers to an activity level of at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the activity of the reference protein.

[0061] As used herein, the term "introducing / introduction" refers to the introduction of one or more polynucleotides (such as circRNA) or one or more constructs comprising vectors described herein, one or more Multiple transcripts are delivered to host cells. The methods of the present application can use many delivery systems, including but not limited to: virus, liposome, electroporation, microinjection and conjugation, to achieve the introduction of the circRNA or construct described herein into the host cell. Nucleic acids can be introduced into mammalian cells or target tissues using conventional viral-based and non-viral-based gene transfer methods. Such methods can be used to administer nucleic acids encoding circRNAs of the present application to cells or host organisms in culture. Non-viral vector delivery systems include: DNA plasmids, RNA (such as transcripts of the constructs described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles (such as liposomes). Viral vector delivery systems include DNA and RNA viruses with either episomal or integrated genomes for delivery to host cells.

[0062] As used herein, "operably linked", when referring to a first nucleic acid sequence that is operably linked to a second nucleic acid sequence, refers to when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence Case. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription of the coding sequence. Likewise, if the signal peptide affects extracellular secretion of the polypeptide, the coding sequence for the signal peptide is operably linked to the coding sequence for the polypeptide. Generally, operably linked nucleic acid sequences are contiguous, and when necessary to join two protein coding regions, the open reading frames are aligned.

[0063] As used herein, "complementarity" refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, out of 10 10, which are approximately 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" means at least about 70%, 75%, 80% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides , 85%, 90%, 95%, 97%, 98%, 99% or 100% complementarity, or refers to two nucleic acids that hybridize under stringent conditions.

[0064] As used herein, "treatment (treating)" is a method used to obtain beneficial or desired results, including clinical results. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reduction of one or more symptoms caused by the disease, reduction of the extent of the disease, stabilization of the disease (e.g., prevention of or delay the progression of the disease), prevent or delay the spread of the disease, prevent or delay the onset or recurrence of the disease, delay or slow the progression of the disease, improve the disease state, provide disease relief (whether partial or total), reduce the need for treatment of the disease The dosage of one or more other drugs can delay disease progression, improve quality of life, and / or prolong survival. "Treatment" also includes reducing the pathological consequences of a disease. The methods of the present application contemplate any one or more of these therapeutic aspects.

[0065] The terms "individual," "subject," and "patient" are used interchangeably herein to describe mammals, including humans. In some embodiments, the individual is a human. In some embodiments, the individual is a rodent, such as a mouse. In some embodiments, the individual has a genetic disease or condition. In some embodiments, the individual has a coronavirus infection. In some embodiments, the individual is at risk of contracting a coronavirus. In some embodiments, the individual is in need of treatment.

[0066] As understood in the art, "effective amount" refers to the amount of the composition sufficient to produce the desired therapeutic effect (for example, stimulating antibody production and increasing immunity against one or more coronaviruses, reducing severity or duration, stabilization of severity, or resolution of one or more symptoms of viral infection). For therapeutic use, beneficial or desired results include, for example, reduction of one or more symptoms caused by the disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological manifestations that arise during disease development type, improve the quality of life of those with the disease, reduce the dose of other drugs needed to treat the disease, enhance the effect of another drug, slow the progression of the disease, and / or prolong the life of the patient. In some embodiments, an effective amount of a therapeutic agent prolongs survival (including overall survival and progression-free survival); causes an objective response (including complete or partial response); ameliorates the disease or condition to some extent. or multiple signs or symptoms; and / or improve the subject's quality of life. In some embodiments, the effective amount is a prophylactically effective amount, which is sufficient to prevent or reduce the severity of one or more future symptoms of coronavirus infection when administered to individuals susceptible and / or infectable with the coronavirus the amount of the composition. For prophylactic use, beneficial or desired outcomes include, for example, outcomes such as elimination or reduction of risk, lessening the severity of future disease, or delaying the onset of disease (e.g., delaying biochemical, histological, and / or behavioral symptoms of disease, its complications and intermediate pathological phenotypes in the future development of the disease).

[0067] As used herein, the term "wild type" is a term of art understood by the skilled person to refer to the typical form of an organism, strain, gene or characteristic, as it occurs in nature, as distinct from a mutant or variant form.

[0068] The present disclosure provides several types of polynucleotide or polypeptide-based compositions, including variants and derivatives. These include, for example, substitutions, insertions, deletions and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that has been modified and / or altered in any way relative to a reference or starting molecule.

[0069] Accordingly, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications relative to a reference sequence (particularly the polypeptide sequences disclosed herein) are included within the scope of the present disclosure. For example, a sequence tag or an amino acid, such as one or more lysines, can be added to the peptide sequence (eg, at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification or localization. Lysine can be used to increase peptide solubility or to allow biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted to provide a truncated sequence. Certain amino acids (eg, C-terminal residues or N-terminal residues) may alternatively be deleted depending on the use of the sequence, eg, expression of the sequence as part of a larger sequence that is soluble or attached to a solid support.

[0070] The term "identity" refers to the overall relatedness between polymeric molecules, eg, between polynucleotide molecules (eg, DNA molecules and / or RNA molecules) and / or between polypeptide molecules. The calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second nucleic acid sequences ( gap) for optimal alignment, and sequences that are not identical can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%. Then, the nucleotides at corresponding nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules at that position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences, and the determination of percent identity between two sequences, can be accomplished using a mathematical algorithm. For example, to determine percent identity between two nucleic acid sequences, methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0), which uses the PAM 120 Weighted residue table with a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the NWSgapdna.CMP matrix using the GAP program in the GCG software suite. Methods commonly used to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); Incorporated herein by reference. Techniques to determine identity are incorporated into publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG suite of programs (Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0071] "Percent (%) amino acid sequence identity" with respect to a polypeptide sequence identified herein is defined as the difference between the candidate sequence and the compared polypeptide after alignment of the sequences considering any conservative substitutions as part of the sequence identity. Amino Acid Residues The percentage of amino acid residues that are identical. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, values ​​of % amino acid sequence identity were generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1 ):113, 2004, each of which is incorporated herein by reference in its entirety for all purposes).

[0072] The terms "non-naturally occurring" or "engineered" are used interchangeably and denote human intervention. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component in and with which it is naturally associated in nature as found in nature.

[0073] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts), and / or by which it is subsequently translated from the transcribed mRNA into a peptide, polypeptide or protein process. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include mRNA splicing in eukaryotic cells.

[0074] As used herein, the term "polypeptide" or "peptide" encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, and all other Type of modified protein (for example, by phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamination, ADP-ribosylation, proteins produced by PEGylation, biotinylation, etc.).

[0075] As used herein, the term "administered simultaneously" refers to the first therapy and the second therapy in the combination therapy administered at a time interval of not more than about 15 minutes, such as not more than about any of 10, 5 or 1 minute. When the first and second therapies are administered simultaneously, the first and second therapies can be contained in the same composition (e.g., a composition comprising the first and second therapies) or in separate compositions (e.g., the second One therapy is in one composition and the second therapy is contained in another composition).

[0076] As used herein, the term "sequential administration" refers to the first therapy and the second therapy in the combination therapy, in more than about 15 minutes (such as in more than about 20, 30, 40, 50, 60 minutes or more minutes) either) at intervals of administration. Either the first therapy or the second therapy may be administered first. The first and second therapies are contained in separate compositions which may be contained in the same or different packages or kits.

[0077] As used herein, the term "concurrent administration" means that in a combination therapy the administration of the first therapy and the administration of the second therapy overlap with each other.

[0078] The term "pharmaceutical composition" refers to a preparation in a form that permits the biological activity of the active ingredients contained therein to be effective and does not contain substances that are unacceptably toxic to the subject to which the preparation is administered. Additional components.

[0079] "Pharmaceutically acceptable carrier" refers to one or more ingredients in a pharmaceutical preparation that are non-toxic to the subject, rather than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to: buffers, excipients, stabilizers, cryoprotectants, tonicity agents, preservatives, and combinations thereof. A pharmaceutically acceptable carrier or excipient, preferably meeting the required standards of toxicology and manufacturing testing and / or included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration or other state / federal governments or documented in US Pharmacopoeia or other recognized pharmacopoeia for use in mammals, especially humans.

[0080] The term "package insert" is used to refer to instructions normally included in commercial packages of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or information about the use of such therapeutic products. WARNING INFORMATION.

[0081] An "article of manufacture" is any article (e.g., a package or container) or kit comprising at least one reagent, such as a medicament, for use in the treatment of a disease or condition (e.g., a coronavirus infection), or to specifically detect Probes for the biomarkers described above. In certain embodiments, an article of manufacture or a kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0082] It is to be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0083] Reference herein to "about" a value or parameter includes (and describes) variations involving that value or parameter itself. For example, description referring to "about X" includes description of "X."

[0084] As used herein, reference to "not" a value or parameter generally means and describes "except for" the value or parameter. For example, the method is not used to treat Type X diseases, ie the method is used to treat diseases other than Type X.

[0085] As used herein, the term "about X-Y" has the same meaning as "about X to about Y".

[0086] As used herein and in the appended claims, the singular forms "a / an" or "the" include plural referents unless the context clearly dictates otherwise.

[0087] As used herein, the term "and / or", phrases such as "A and / or B" are intended to include: both A and B; A or B; A (alone); and B (alone) . Likewise, as used herein, the term "and / or", such phrases as "A, B, and / or C", is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0088] II. therapeutic circular RNA

[0089] The application provides circular RNAs (circRNAs) encoding polypeptides, such as therapeutic polypeptides, including any of the therapeutic polypeptides described in section A. "Therapeutic Polypeptides" below.

[0090] In some embodiments, a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide is provided, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and targeting proteins.

[0091] In some embodiments, the circRNA is stable for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 when stored at 4°C or at room temperature. sky. In some embodiments, the circRNA is stable for at least 7 days when stored at 4°C or room temperature. In some embodiments, the circRNA is stable for at least 14 days when stored at 4°C or room temperature. In some embodiments, the circRNA is stable for at least 30 days when stored at 4°C. In some embodiments, the circRNA degrades less than 40% after 14 days of storage at room temperature.

[0092] In some embodiments, the application provides a circRNA comprising: (a) a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and targeting protein, and (b) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the nucleic acid sequence also encodes a SP (eg, human tPA or IgE SP) fused to the N-terminus of the therapeutic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence and a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide, and can be identified by a The 5' exon sequence flanking the 3' end of the nucleic acid sequence catalyzes the recognition of the group I intron fragment. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected.

[0093] In some embodiments, the application provides a circRNA comprising: (a) a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and targeting protein; (b) an IRES sequence, wherein the IRES sequence is operably linked to a nucleic acid sequence encoding a therapeutic polypeptide; and (c) an in-frame 2A peptide operably linked to the 3' end of the nucleic acid sequence encoding a therapeutic polypeptide coding sequence. In some embodiments, the nucleic acid sequence also encodes a SP (eg, human tPA or IgE SP) fused to the N-terminus of the therapeutic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, a nucleic acid sequence encoding a therapeutic polypeptide, and an in-frame 2A peptide coding sequence. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the therapeutic polypeptide, and can be identified by the nucleic acid sequence encoding the therapeutic polypeptide. The 5' catalytic group I intronic fragment recognizes the 5' exon sequence flanking the 3' end of the sequence. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected.

[0094] In some embodiments, the application provides a circRNA comprising: (a) a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and a targeting protein; and (b) an m6A modification motif sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the nucleic acid sequence also encodes a SP (eg, human tPA or IgE SP) fused to the N-terminus of the therapeutic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence, and a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the therapeutic polypeptide, and can be identified by the nucleic acid sequence encoding the therapeutic polypeptide. The 5' catalytic group I intronic fragment recognizes the 5' exon sequence flanking the 3' end of the sequence. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected.

[0095] In some embodiments, the application provides a circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the antigenic polypeptide is a protein or fragment thereof of an infectious agent. In some embodiments, the infectious agent is a virus. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is selected from the group consisting of SARS-CoV, MERS-COV, and SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. The circRNA can comprise any of the circRNA expression and / or circularization elements described in Section B "Other circRNA expression and circularization elements" below.

[0096] In some embodiments, the application provides a circRNA comprising a nucleic acid sequence encoding a receptor protein. In some embodiments, the receptor protein is a soluble receptor comprising the extracellular domain of a naturally occurring receptor. In some embodiments, the receptor protein is a receptor for an infectious agent (eg, a virus such as a coronavirus). In some embodiments, the receptor is an ACE2 receptor, such as a soluble ACE2 receptor. In some embodiments, the receptor is a high affinity mutant ACE2 receptor. The circRNA can comprise any of the circRNA expression and / or circularization elements described in Section B "Other circRNA expression and circularization elements" below.

[0097] In some embodiments, the application provides a circRNA comprising a nucleic acid sequence encoding a targeting protein. In some embodiments, the targeting protein is an antibody. In some embodiments, the antibody is a neutralizing antibody, eg, a neutralizing antibody that targets a coronavirus, such as SARS-CoV-2. In some embodiments, the targeting protein is a therapeutic antibody. The circRNA can comprise any of the circRNA expression and / or circularization elements described in Section B "Other circRNA expression and circularization elements" below.

[0098] In some embodiments, the present application provides circular RNA vaccines for treating or preventing coronaviruses.

[0099] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS-CoV, MERS- COV or SARS-CoV-2) spike (S) protein or a fragment thereof. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises the S protein of SARS-CoV-2 or its fragment . In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises: (a) coronavirus (for example, SARS- CoV, MERS-COV or SARS-CoV-2) S protein or a fragment thereof; and (b) a multimerization domain. In some embodiments, the multimerization domain is the C-terminal Foldon (Fd) domain of T4 fibrin that mediates trimerization of T4 fibrin. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises the amino acid sequence set forth in SEQ ID NO: 3-4. In some embodiments, the multimerization domain is fused to the RBD domain of the S protein via a peptide linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises the S of coronavirus (for example, SARS-CoV2) The receptor binding domain (RBD) of the protein. In some embodiments, the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises the amino acid sequence of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises: (a) coronavirus (for example, SARS- CoV, MERS-COV or SARS-CoV-2) the RBD of the S protein fragment; and (b) the multimerization domain. In some embodiments, the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the multimerization domain is the C-terminal Foldon (Fd) domain of T4 fibrin that mediates trimerization of T4 fibrin. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 3-4. In some embodiments, the multimerization domain is fused to the RBD domain of the S protein via a peptide linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises the S of coronavirus (for example, SARS-CoV2) The S2 region of the protein. In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises the full-length S protein of SARS-CoV-2 Amino acid residues 2-1273, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 and 62-63. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises: (a) the nucleotide sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises coronavirus (for example, SARS- CoV-2) S protein or a fragment thereof; and (b) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is operably linked to a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: IRES sequence, Kozak sequence, SP and the nucleic acid sequence of coding antigenic polypeptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15.

[0107] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises: (a) a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS- CoV-2) S protein or a fragment thereof; (b) an IRES sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide; and (c) is operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide The in-frame 2A peptide coding sequence. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, an SP, a nucleic acid sequence encoding an antigenic polypeptide, and an in-frame 2A peptide coding sequence. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15.

[0108] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises: (a) a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS- CoV-2) S protein or a fragment thereof; and (b) an m6A modification motif sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modified motif sequence, a Kozak sequence, a SP and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15.

[0109] The present application further provides a mixture composition comprising a plurality of circRNAs, each circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, a receptor protein of an infectious agent, or a targeting protein (for example, an antibody such as a neutralizing antibody). In some embodiments, multiple circRNAs encode antigenic polypeptides different from each other, such as different mutants of antigenic polypeptides (eg, S protein or fragments thereof). In some embodiments, multiple circRNAs encode different receptor proteins from each other, such as different mutants of a receptor protein (eg, ACE2). In some embodiments, multiple circRNAs encode different targeting proteins from each other, such as different antibodies (eg, neutralizing antibodies).

[0110] A. Therapeutic polypeptides

[0111] In some aspects, provided herein are circRNAs comprising a therapeutic polypeptide. In some embodiments, a therapeutic polypeptide is an antigenic polypeptide, a functional protein, a receptor protein, or a targeting protein (eg, an antibody).

[0112] In some embodiments, the nucleic acid sequence may be codon optimized. A codon optimized sequence may be a sequence in which codons in a polynucleotide encoding a polypeptide have been substituted to increase expression, stability and / or activity of the polypeptide. Factors affecting codon optimization include, but are not limited to, one or more of the following: (i) a change in codon bias between two or more organisms or genes or a synthetically constructed bias table, (ii) an organism , a change in the degree of codon bias within a gene or group of genes, (iii) a systematic change in a codon including its context, (iv) a change in the codon according to which it decodes a tRNA, (v) a change in the codon according to which it decodes a tRNA, either overall or in a triplet Codon changes in GC% of a position, (vi) changes in the degree of similarity to a reference sequence (e.g., a naturally occurring sequence), (vii) changes in codon frequency cutoffs, (viii) structure of mRNA transcribed from a DNA sequence properties, (ix) prior knowledge about the function of the DNA sequence on which the set of codon substitutions is based, and / or (x) systematic variation in the set of codons per amino acid. In some embodiments, codon-optimized polynucleotides can minimize ribozyme collisions and / or structural interference between the restricted expression sequence and the IRES.

[0113] i. Antigenic polypeptide

[0114] The circular RNA vaccines described herein comprise a circular RNA (circRNA) encoding an antigenic polypeptide. In some embodiments, the antigenic polypeptide comprises the spike (S) protein or fragment thereof of coronavirus, such as any S protein or fragment thereof described in the subsection "Spike protein or fragment thereof" below. In some embodiments, the antigenic polypeptide comprises a multimerization domain, such as a native multimerization domain of the S protein, or an exogenous multimerization domain. Suitable multimerization domains are described in the subsection "Mulmerization domains" below. The S protein or a fragment thereof can be fused to the multimerization domain via a peptide linker, such as any of the peptide linkers described in the "Peptide Linker" section below.

[0115] An antigenic polypeptide comprises at least one epitope recognized by a T cell receptor (TCR). In some embodiments, the antigenic polypeptide is a full-length protein or a fragment thereof, or an antigenic fusion protein that can trigger an immune response in a subject. In some embodiments, the antigenic polypeptide is a short peptide of no more than 100 amino acids in length. The antigenic polypeptide may be a naturally derived peptide fragment from a protein antigen containing one or more epitopes, or an artificially designed peptide having one or more native epitope sequences, wherein a peptide linker may optionally be placed adjacent to the epitope. between bit sequences. In some embodiments, an antigenic polypeptide comprises a single epitope of an antigenic protein. In some embodiments, the antigenic polypeptide comprises any of about 1, 2, 3, 4, 5, 10 or more epitopes from a single antigenic protein. In some embodiments, the antigenic polypeptide comprises epitopes from a plurality (eg, 2, 3, 4, 5, 10 or more) of different antigenic proteins. In some embodiments, the antigenic polypeptide comprises a major histocompatibility complex (MHC) class I restricted epitope. In some embodiments, the antigenic polypeptide comprises an MHC class II restricted epitope. In some embodiments, the antigenic polypeptide comprises an MHC class I-restricted epitope and an MHC class II-restricted epitope.

[0116] In some embodiments, the antigenic polypeptide is an antigenic protein from a pathogen, such as a bacterium or a virus, or a fragment or variant thereof. In some embodiments, the antigenic polypeptide is an antigenic protein or fragment of a coronavirus (such as SARS-CoV2, including variants thereof).

[0117] In some embodiments, the antigenic polypeptide is an antigenic protein of a self-antigen, or a fragment or variant thereof, such as an antigen involved in a disease or condition. In some embodiments, the antigenic polypeptide is a tumor antigenic peptide. Tumor antigen peptide sequences are known in the art and can be found in public repositories such as the Cancer Antigen Peptide Database (van der Bruggen P et al. (2013) "Peptide database: T cell-defined tumor antigens." Cancer Immunity. URL: caped.icp.ucl.ac.be). The coding RNA sequence in the linear RNA or circRNA described herein can encode any known tumor antigen peptide or a combination thereof. In some embodiments, the antigenic polypeptide comprises an epitope of a tumor-associated antigen (TAA). In some embodiments, the antigenic polypeptide comprises an epitope of a tumor-specific antigen. In some embodiments, the antigenic polypeptide comprises an epitope of a neoantigen, ie, a newly acquired and expressed antigen present in an individual's tumor cells.

[0118] In some embodiments, the amino acid sequence of one or more epitope peptides is predicted based on the sequence of an antigenic protein (including neoantigens) using a bioinformatics tool for T cell epitope prediction. Exemplary bioinformatics tools for T cell epitope prediction are known in the art, see, e.g., Yang X. and Yu X. (2009) "An introduction to epitope prediction methods and software" Rev. Med. Virol .19(2): 77-96. In some embodiments, the sequence of the antigenic protein is known in the art, or available in public repositories. In some embodiments, the sequence of an antigenic protein, including a neoantigen, is determined by sequencing a sample (eg, a tumor sample) of the individual being treated.

[0119] In some embodiments, the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of a coronavirus (such as SARS-CoV, MERS-COV or SARS-CoV-2 virus). In some embodiments, the antigenic polypeptide is full length S protein. In some embodiments, the antigenic polypeptide is a fragment of a naturally occurring S protein. In some embodiments, the antigenic polypeptide comprises the Spike (S) protein of SARS-CoV-2 or a fragment thereof.

[0120] In some embodiments, the antigenic polypeptide comprises a variant of the S protein or a fragment thereof of a coronavirus. In some embodiments, the antigenic polypeptide comprises a naturally occurring variant of the S protein or fragment thereof of a coronavirus (eg, SARS-CoV-2). Variants of the SARS-CoV-2 genome have been described. See, eg, Forster et al. (2020). Phylogenetic network analysis of SARS-CoV-2 genomes. PNAS 117 (17) 9241-9243, which is incorporated herein by reference in its entirety. In some embodiments, the antigenic polypeptide comprises a variant of the S protein or a fragment thereof that confers a fitness advantage to the coronavirus, such as enhanced infectivity. In some embodiments, the antigenic polypeptide comprises the S protein or fragment thereof of SARS-CoV-2 with a D614G mutation. In some embodiments, the antigenic polypeptide is capable of eliciting an immune response against different strains and variants of coronavirus (such as SARS-CoV-2 variants) in an individual. In some embodiments, the antigenic polypeptide is capable of eliciting an immune response against a particular strain or variant of coronavirus in an individual.

[0121] In some embodiments, the antigenic polypeptide comprises the receptor binding domain (RBD) of the S protein of a coronavirus (eg, SARS-CoV2). In some embodiments, the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2. amino acid sequence. In some embodiments, the RBD comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 63. amino acid sequence.

[0122] In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein of a coronavirus (eg, SARS-CoV2). In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 6 amino acid sequence. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises K986P and V987P mutations. In some embodiments, the S2 region comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7 amino acid sequence.

[0123] In some embodiments, the antigenic polypeptide comprises the RBD and S2 regions of the S protein of a coronavirus (eg, SARS-CoV2). In some embodiments, the antigenic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 1 amino acid sequence.

[0124] In some embodiments, the antigenic polypeptide comprises a spike (S) protein fragment and a multimerization domain of a coronavirus (e.g., SARS-CoV, MERS-COV or SARS-CoV-2), which may be operable ground to the S protein fragment. In some embodiments, the multimerization domain is the C-terminal Foldon (Fd) domain of T4 fibrin that mediates trimerization of T4 fibrin. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises the amino acid sequence set forth in SEQ ID NO: 3 or 4. In some embodiments, the multimerization domain is fused to the S protein fragment via a peptide linker. In some embodiments, the antigenic polypeptide comprises the RBD domain of the S protein fused to the multimerization domain by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 5.

[0125] In some embodiments, the antigenic polypeptide comprises the Spike (S) protein or fragment thereof of SARS-CoV-2 fused to a multimerization domain. In some embodiments, the antigenic polypeptide comprises a fusion to the C-terminal Foldon (Fd) domain (e.g., SEQ ID NO: 3) of T4 fibrin that mediates trimerization of T4 fibrin (e.g., SEQ ID NO: 4) S protein fragment. In some embodiments, the antigenic polypeptide comprises an S protein fragment fused to a GCN-4-based isoleucine zipper domain. In some embodiments, the antigenic polypeptide comprises the receptor binding domain (RBD) of the S protein of SARS-CoV-2 fused to the multimerization domain by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 5.

[0126] The antigenic polypeptide may comprise a signal peptide (SP). In some embodiments, SP is fused to the N-terminus of the S protein or a fragment thereof. In non-limiting examples, the signal peptide is the signal sequence and propeptide from human tissue plasminogen promoter (tPA), the signal sequence from human IgE immunoglobulin, or the signal peptide sequence of MHC I. In some embodiments, the signal peptide can promote the secretion of the antigenic polypeptide encoded by the circRNA vaccine.

[0127] In some embodiments, the circRNA comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA does not include a stop codon at the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the in-frame 2A peptide coding sequence replaces the stop codon. In some embodiments, the circRNA does not contain a stop codon and the number of nucleotides that make up the RNA is a multiple of three. In some embodiments, a circRNA that does not have a stop codon and the number of nucleotides that make up the RNA is a multiple of three allows rolling circle translation of the circRNA. In some embodiments, the 2A peptide coding sequence allows rolling circle translation of the circRNA. In some embodiments, the 2A peptide allows cleavage of a polypeptide generated by rolling circle translation into monomeric polypeptide sequences. In a non-limiting example, the 2A peptide coding sequence encodes a P2A or T2A peptide, such as the sequence shown in SEQ ID NO: 44 or 45.

[0128] Also provided is a circRNA comprising a nucleic acid sequence encoding any of the antigenic polypeptides described herein. A nucleic acid sequence encoding an antigenic polypeptide may be codon-optimized. In some embodiments, the circRNA comprises a nucleic acid having at least about 80% (for example, at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with a nucleic acid sequence selected from the group Sequences: SEQ ID NO: 11-15 and SEQ ID NO: 48-49.

[0129] Spike protein or fragment thereof. The circRNA vaccine described herein comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and wherein said antigenic polypeptide comprises coronavirus (for example, SARS-CoV-2, MERS-CoV or SARS-coronavirus) Spike (S) protein or a fragment thereof. The sequence of the S protein of coronavirus is known in the art, including for example, NCBI RefSeq ID: YP_009047204.1 (MERS-CoV), GenBank accession number: AAT74874 (SARS-CoV), or NCBI RefSeq ID: YP_009724390 (SARS-CoV) CoV-2, provided as SEQ ID NO: 1 of the present application).

[0131] In some embodiments, the S protein or fragment thereof comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises a deletion of amino acid residues 681-684. In some embodiments, the S protein or fragment thereof comprises at least one point mutation in the S2 region, such as K986P, V987P, F817P, A892P, A899P or A942P mutations or combinations thereof. In some embodiments, the S protein or fragment thereof comprises a protein selected from A222V, E406W, K417N, K417T, N439K, L452R, L452Q, L455N, L478K, E484K, Q493F, F490S, N501Y, A570D, D614G, P681H, A701V, T716I, At least one mutation of S982A or a combination thereof. In some embodiments, the S protein or fragment thereof comprises an N501Y point mutation. In some embodiments, the S protein or fragment thereof comprises K417N, E484K and / or N501Y point mutations. In some embodiments, the S protein or fragment thereof comprises the E484K point mutation. In some embodiments, the S protein or fragment thereof comprises K417T, E484K and N501Y point mutations. In some embodiments, the S protein of SARS-CoV-2 or a fragment thereof comprises the K986P and V987P point mutations, alone or in combination with a deletion of amino acid residues 681-684. In some embodiments, the S protein or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 1-2, SEQ ID NO: 6-10 or SEQ ID NO: 63. In some embodiments, the S protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the S protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 63.

[0132] In some embodiments, the S protein or fragment thereof is α (B.1.1.7), β (B.1.351, B.1.351.2, B.1.351.3), δ (B.1.617.2 , AY.1, AY.2, AY.3) or γ (P.1, P.1.1, P.1.2) S protein or fragments thereof. In some embodiments, the S protein or fragment thereof comprises two, three, four, five or more mutations selected from the group consisting of: T19R, V70F, T95I, G142D, E156-, F157-, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R and D950N, wherein amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises an RBD comprising one, two or three or more mutations selected from the group consisting of K417N, L452R and T478K, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises two, three, four, five or more mutations selected from the group consisting of deletion of residue 69, deletion of residue 70, deletion of residue 144, E484K , S494P, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H and K1191N, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises an RBD domain comprising one, two or three mutations selected from the group consisting of E484K, S494P and N501Y, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises one, two, three, four, five or more mutations selected from the group consisting of D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G and A701V, wherein amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises an RBD comprising one, two or three mutations selected from K417N, E484K and N501Y, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises one, two, three, four, five or more mutations selected from the group consisting of: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y and T1027I, wherein amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises an RBD domain comprising one, two or three mutations selected from K417T, E484K and N501Y, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises an RBD domain comprising one, two or three mutations selected from the group consisting of E484K, N501Y and L452R, wherein amino acid numbering is based on SEQ ID NO: 1 .

[0133] In some embodiments, the S protein or fragment thereof comprises the N-terminal domain (NTD) of the S protein of a coronavirus (eg, SARS-CoV-2, MERS-CoV, or SARS-CoV).

[0134] In some embodiments, the S protein or fragment thereof comprises about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98%, or An amino acid sequence with more sequence identity, or an amino acid sequence having any of the sequences shown in SEQ ID NO: 1-2, SEQ ID NO: 6-10 and SEQ ID NO: 62-63.

[0135] RBD domain

[0136] In some embodiments, the S protein or fragment thereof comprises the receptor binding domain (RBD) of the S protein. In some embodiments, the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the RBD comprises a sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 . In some embodiments, the RBD comprises a sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 63 . In some embodiments, the RBD is linked to a multimerization domain. In some embodiments, the RBD is fused to the multimerization domain via a flexible peptide linker.

[0137] S2 area

[0138] In some embodiments, the S protein or fragment thereof comprises the S2 region of the S protein. In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises the K986P and V987P mutations, eg, as in the sequence set forth in SEQ ID NO:7. In some embodiments, the S2 region comprises a single point mutation, such as a K986P, V987P, F817P, A892P, A899P, or A942P mutation. In some embodiments, the S2 region comprises a combination of point mutations, including K986P, V987P, F817P, A892P, A899P, or A942P. In some embodiments, the S2 region comprises the wild-type sequence of the coronavirus S protein, such as the sequence of SEQ ID NO: 6, or has about 80%, at least 85%, at least about 90% of the amino acid sequence of SEQ ID NO: 6 , at least about 95%, at least about 98% or more sequence identity.

[0139] Multimerization domain

[0140] In some embodiments, the antigenic polypeptide further comprises a multimerization domain, such as a dimerization domain, a trimerization domain, or a domain that mediates formation of higher order multimers. In some embodiments, the multimerization domain is a trimerization domain. In a non-limiting example, the multimerization domain comprises a C-terminal Foldon (Fd) domain of T4 fibrin, wherein the C-terminal Foldon domain is a domain that mediates trimerization of T4 fibrin, such as SEQ ID NO: 3 shows the amino acid sequence. In another example, the multimerization domain comprises a GCN4-based isoleucine zipper (IZ) domain based on the trimerization domain of the GCN4 transcription initiation factor from Saccharomyces cerevisiae, such as SEQ ID NO : Amino acid sequence shown in 4. In some embodiments, the multimerization domain shares about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98%, or More sequence identity. In some embodiments, the GCN4 IZ domain or the T4 fibrin Fd domain can be modified to reduce their immunogenicity according to techniques known in the art. For example, the GCN4 IZ domain can be modified with N-linked glycosylation sites to reduce its immunogenicity (Sliepen et al. Immunosilencing a Highly Immunogenic Protein Trimerization Domain. The Journal of Biol. Chem. Vol. 290, No. 12, pp. 7436–7442). In some embodiments, the multimerization domain is fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the multimerization domain is fused to the C-terminus of the S protein or a fragment thereof.

[0141] ii. Targeting protein

[0142] In some embodiments, the therapeutic polypeptide is a targeting protein. In some embodiments, the targeting protein is an antibody or antigen-binding fragment thereof.

[0143] In some embodiments, the therapeutic polypeptide is an antibody. In some embodiments, the therapeutic polypeptide is a neutralizing antibody, ie, an antibody that blocks the interaction between a protein and its binding partner. In some embodiments, the antibody inhibits the activity of the protein, eg, by blocking the binding of the protein to a binding partner. In some embodiments, the targeting protein is a therapeutic antibody. In some embodiments, the antibody is a checkpoint inhibitor, eg, an antibody inhibitor of CTLA-4, PD-1, or PD-L1. In some embodiments, the antibody may be an antibody directed against a viral protein or a receptor that binds a viral protein.

[0144] The antibody may be an antigen-binding fragment of an antibody, such as a part or fragment of a whole or whole antibody having fewer amino acid residues than a whole or whole antibody, which is capable of binding antigen or interacting with the whole antibody (i.e., a derived antigen-binding fragment). intact antibody) competes for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to: Fab', F(ab')2Fv, single-chain Fv (scFv), single-chain Fab, diabody, single domain antibody (sdAb, nanobody), Camel Ig, Ig NAR , F(ab)'3 fragment, bi-scFv, (scFv)2 minibody, diabody, triabody, quadruple diabody, disulfide bond stabilized Fv protein ("dsFv"). In some embodiments, the neutralizing antibody can be a genetically engineered antibody, such as a chimeric antibody (eg, a humanized murine antibody), a heteroconjugated antibody (eg, a bispecific antibody), or an antigen-binding fragment thereof.

[0145] In some embodiments, the antibody is a neutralizing antibody that binds a viral protein. In some embodiments, the antibody is a neutralizing antibody that binds a viral protein receptor. In some embodiments, the antibody binds to a receptor required for viral entry into the cell (eg, the ACE2 receptor). In some embodiments, the antibody is a neutralizing antibody (nAb) that binds to the S protein of the coronavirus and prevents or reduces its ability to infect cells. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the nAb is a monoclonal antibody (mAb), a functional antigen-binding fragment (Fab), a single chain variable fragment (scFv), or a single domain antibody (VHH or Nanobody).

[0146] In some embodiments, the nAb binds to the RBD of the S protein of a coronavirus. In some embodiments, the nAb binds the NTD of the S protein of a coronavirus. In some embodiments, the nAB binds to the S2 region of the S protein of a coronavirus. In some embodiments, the nAb binds to the S1 / S2 proteolytic cleavage site of the coronavirus S protein. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, binding of the nAb to the S protein interferes with the interaction of the RBD of the S protein with the ACE2 receptor. In some embodiments, the nAb binds to the ACE2 binding site of the RBD. In some embodiments, binding of the nAb to the S protein interferes with S2-mediated membrane fusion. In some embodiments, binding of the nAb to the S protein interferes with viral entry into the host cell.

[0147] In some embodiments, the nAb binds an S protein comprising one or more mutations. In some embodiments, the nAb binds to an S protein or fragment thereof comprising at least one point mutation in the S2 region, such as a K986P, V987P, F817P, A892P, A899P, or A942P mutation, or a combination thereof. In some embodiments, the nAb binds to an S protein or fragment thereof comprising at least one point mutation selected from the group consisting of: A222V, E406W, K417N, K417T, N439K, L452R, L452Q, L455N, L478K, E484K, Q493F, F490S, N501Y , A570D, D614G, P681H, A701V, T716I, S982A, or combinations thereof. In some embodiments, the nAb binds the S protein or fragment thereof comprising the N501Y point mutation. In some embodiments, the nAb binds the S protein or fragment thereof comprising the K417N, E484K, and N501Y point mutations. In some embodiments, the nAb binds an S protein or fragment thereof comprising the E484K point mutation. In some embodiments, the nAb binds the S protein or fragment thereof comprising the K417T, E484K, and N501Y point mutations. In some embodiments, the nAb binds the S protein of SARS-CoV-2 or a fragment thereof comprising the K986P and V987P point mutations, alone or in combination with a deletion of amino acid residues 681-684. In some embodiments, the binding of the nAb to the S protein with any combination of mutations described above (eg, K417N, K417T, E484K, and / or N501Y) interferes with the interaction of the RBD of the S protein with the ACE2 receptor. In some embodiments, binding of the nAb to the S protein of any combination of mutations described above (eg, K417N, K417T, E484K, and / or N501Y) interferes with S2-mediated membrane fusion. In some embodiments, binding of the nAb to the S protein of any combination of mutations described above (eg, K417N, K417T, E484K, and / or N501Y) interferes with viral entry into the host cell.

[0148] Exemplary nAbs for binding and neutralizing the S protein of SARS-CoV-2 have been described, for example, in Barnes, C.O. et al. SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature 588, 682- 687 (2020), and Chinese patent application CN111690058A, the contents of which are incorporated herein by reference in their entirety.

[0149] In some embodiments, the nAb comprises a sequence selected from SEQ ID NO: 26-33. In some embodiments, the nAb comprises at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%) of a sequence selected from SEQ ID NO: 26-33. , at least 95%, at least 96%, at least 97%, or at least 99%) amino acid sequence identity.

[0150] In some embodiments, the antibody is an antibody directed against the S protein of SARS-CoV-2. In some embodiments, the antibody comprises at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence having 96%, at least 97%, or at least 99%, or 100%) amino acid sequence identity. In some embodiments, the antibody comprises at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence having 96%, at least 97%, or at least 99%, or 100%) amino acid sequence identity. In some embodiments, the antibody comprises at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least A sequence having 96%, at least 97%, or at least 99%, or 100%) amino acid sequence identity.

[0151] In some embodiments, the targeting protein is not an antibody. Examples of non-antibody-based targeting proteins include, but are not limited to: lipocalins, anticalins (artificial antibody analog proteins derived from human lipocalins), "T-body", peptides (such as BICYCLETM peptides), affibodies (antibody mimics composed of α-helices, such as triple-helix bundles), peptibodies (peptide-Fc fusions), DARPins (designed ankyrin repeat proteins, engineered Antibody Analog Protein), Affimer, Avimer, Knottin (Protein Structural Motif Containing 3 Disulfide Bridges), Monomer, Affinity Clamp, Extracellular Domain, Receptor Extracellular Domain, Receptor, Cytokines , ligands, immune cytokines, and Centryin. See, eg, Vazquez-Lombardi, Rodrigo, et al. Drug discovery today 20.10 (2015): 1271-1283.

[0152] iii. Soluble receptors

[0153] In some embodiments, the therapeutic polypeptide is a soluble receptor. Soluble receptors (sometimes referred to as soluble receptor decoys or "traps") may comprise all or part of the extracellular domain of the receptor protein. In some embodiments, the nucleotide sequence encoding all or part of the extracellular domain of the receptor protein is operably linked to a signal peptide for secretion from the cell.

[0154] In some embodiments, the soluble receptor comprises the extracellular domain of a naturally occurring receptor. In some embodiments, the soluble receptor variant comprises an engineered variant of the extracellular domain of a naturally occurring receptor, such as a variant comprising one or more mutations in the extracellular domain. In some embodiments, the soluble receptor comprises one or more mutations that increase the affinity of the soluble receptor for its ligand compared to the affinity of the naturally occurring receptor for its ligand.

[0155] In some embodiments, the soluble receptor is a fusion protein comprising one or more other protein domains operably linked to the extracellular domain of the receptor or a variant thereof. In some embodiments, the soluble receptor comprises the Fc domain of an immunoglobulin (Ig), such as a human immunoglobulin. In some embodiments, the soluble receptor comprises the Fc domain of human IgGl.

[0156] In some embodiments, the soluble receptor comprises an extracellular domain of a signaling receptor, and the soluble receptor can reduce or inhibit the activity of the signaling pathway by blocking the binding between the endogenous receptor and its ligand.

[0157] In some embodiments, the soluble receptor is a receptor that binds a viral protein and / or mediates viral entry. In some embodiments, the soluble receptor is a soluble ACE2 receptor. In some embodiments, the therapeutic polypeptide is a soluble ACE2 receptor variant capable of binding the S protein of a coronavirus. In some embodiments, soluble ACE2 may have a great advantage over antibodies due to resistance to escape mutations. Viruses with escape mutations in sACE2 should have limited binding affinity for the native ACE2 receptor on the cell surface, resulting in attenuated or eliminated virulence.

[0158] In some embodiments, the ACE2 receptor fragment is designed to have a higher affinity for the S protein of the coronavirus. In some embodiments, the soluble ACE2 receptor variant is capable of binding the S protein of coronavirus and blocking or reducing the binding of the S protein to the endogenous ACE2 receptor. In some embodiments, the soluble ACE2 receptor variant binds to the receptor binding domain (RBD) of the S protein. In some embodiments, the ACE2 receptor variant has enzymatic activity. In other embodiments, the ACE2 receptor variant is enzymatically inactive.

[0159] In some embodiments, the soluble ACE2 receptor variant comprises the soluble extracellular domain of wild-type (WT) human recombinant ACE2 (APN01). APN01 has been found to be safe in healthy volunteers and a small cohort of patients with acute respiratory distress syndrome because the intrinsic angiotensin-converting activity of ACE2 is not required for viral entry. APN01 is currently undergoing a Phase 2 clinical trial in Europe for the treatment of SARS-CoV-2 (NCT04335136). In some embodiments, the soluble ACE2 receptor variant comprises one or more mutations in the extracellular domain of human ACE2. In some embodiments, soluble ACE2 receptor variants are engineered by affinity maturation to have increased binding affinity for the RBD of the S protein. For example, the nucleotide sequence encoding the ACE2 wild-type ectodomain can be subjected to one or more rounds of random mutation and cell sorting to identify ACE2 variants with higher affinity for the RBD of the S protein wild-type ACE2.

[0160] In some embodiments, the soluble ACE2 receptor variant comprises at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%) of SEQ ID NO: 34 or 35 , at least 94%, at least 95%, at least 96%, at least 97%, or at least 99%, or 100%) amino acid sequence identity.

[0161] In some embodiments, the soluble ACE2 receptor variant is a fusion protein, eg, a fusion of an extracellular ACE2 receptor domain to the Fc region of a human IgGl.

[0162] In some embodiments, the soluble ACE2 receptor variant has a KD for the RBD of the S protein of about 15-20 nM. In some embodiments, the soluble ACE2 receptor variant has a KD for the RBD of the S protein of less than 15 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 500 pM, less than 250 pM, less than 200 pM, or less than 150 pM.

[0163] Soluble ACE2 receptor variants have been described, for example, in Haschke M et al., Clin Pharmacokinet. 2013 Sep;52(9):783-92; Glasgow A et al., Proceedings of the National Academy of Sciences Nov 2020, 117 (45) 28046-28055; and Higuchi Y. et al., bioRxiv 2020. 09.16.299891, the contents of which are hereby incorporated by reference in their entirety.

[0164] iv. Functional protein

[0165] In some embodiments, a therapeutic polypeptide can be any polypeptide capable of being expressed by a target cell (e.g., a human or mouse cell) to produce (and in some cases, secrete) a functional enzyme or protein, such as For example, as disclosed in International Application No. PCT / US2010 / 058457 and WO2020237227, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a therapeutic polypeptide can be engineered for secretion by operably linking a signal peptide to the amino terminus of the therapeutic polypeptide. For example, in some embodiments, upon expression of one or more therapeutic polynucleotides by a target cell, production of a functional enzyme or protein (e.g., urea cyclase or lysosomal enzyme) that is deficient in the subject can be observed. Storage disease-associated enzymes).

[0166] In some embodiments, the therapeutic polypeptide comprises a protein, such as IDUA, OTC, FAH, miniDMD, DMD, p53, PTEN, COL3A1, BMPR2, AHI1, FANCC, MYBPC3, ILRG2, or ARG1, wherein the absence of a functional protein pertaining to a disease or condition.

[0167] In some embodiments, a therapeutic polypeptide comprises a protein (eg, a lysosomal enzyme), wherein a deficiency of the protein is associated with a lysosomal storage disorder.

[0168] In some embodiments, a therapeutic polypeptide comprises a protein (eg, an enzyme), wherein a deficiency of the protein is associated with a metabolic disorder. In some embodiments, the therapeutic polypeptide comprises urea cyclase (eg, ARG1).

[0169] In some embodiments, a therapeutic polypeptide comprises a protein (eg, p53 or PTEN), wherein a deficiency of the protein is associated with cancer. In some embodiments, a therapeutic polypeptide comprises a tumor suppressor.

[0170] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%) of the amino acid sequence of wild-type mouse IDUA protein (e.g., SEQ ID NO: 18) , 98% or more, or 100%) amino acid sequence identity.

[0171] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0172] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%) of the amino acid sequence of a wild-type mouse OTC protein (e.g., SEQ ID NO: 20). , 98% or more, or 100%) amino acid sequence identity.

[0173] In some embodiments, the therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%) of the amino acid sequence of wild-type mouse FAH protein (e.g., SEQ ID NO: 21 ). , 98% or more, or 100%) amino acid sequence identity.

[0174] In some embodiments, the therapeutic polypeptide comprises at least about 80% (for example, at least about 85%, 90%, 95%, 98% of the amino acid sequence (for example, SEQ ID NO: 22) of the human miniDMD protein. % or more, or 100%) of amino acid sequences that are identical.

[0175] In some embodiments, a therapeutic polypeptide comprises at least about 80% (for example, at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0176] In some embodiments, the therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0177] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0178] In some embodiments, the therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0179] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0180] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0181] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0182] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0183] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0184] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0185] In some embodiments, a therapeutic polypeptide comprises at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) amino acid sequence identity.

[0186] v. Peptide linker

[0187] In some embodiments, multiple domains in a therapeutic polypeptide (e.g., multiple domains of the Spike protein or a fragment thereof) can be fused to each other, or comprise domains fused to each other via a peptide linker (e.g., antigenic polypeptide domain and carrier protein or multimerization domain). In some embodiments, the antigenic polypeptide is a domain of the coronavirus S protein fused to the multimerization domain via a peptide linker. Flexible peptide linkers such as glycine linkers, glycine-serine linkers, and linkers comprising other amino acids are known in the art (for example, suitable peptide linkers are described in: Chen et al. in Fusion Protein Linkers: Property, Design and Functionality. Adv. Drug Deli Rev. 2013 October 15; 65(10): 1357–1369). Peptide linkers can also be designed computationally. The peptide linker can be 1-10, 10-20, 20-30, 30-40, 40-50, or any length greater than 50 amino acids. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:5.

[0188] B. Other circRNA expression and circularization elements

[0189] The circRNAs of the circRNA vaccines described herein comprise one or more other expression elements that promote circRNA expression and / or circularization.

[0190] In some embodiments, the circRNA comprises a Kozak sequence operably linked to a nucleic acid sequence encoding an antigenic polypeptide comprising the spike (S) protein or its fragment. In some embodiments, Kozak sequences are used as protein translation initiation sites.

[0191] In some embodiments, the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising a spike (S) protein or a fragment thereof of a coronavirus (for example, SARS-CoV-2), and the nucleic acid sequence can be Operably linked to an internal ribosome entry site (IRES). In a non-limiting example, the IRES sequence may be: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus or CSFV virus. See, eg, Searching for IRES. RNA. 2006 Oct; 12(10): 1755-1785, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the IRES sequence is that of a cell. In some embodiments, the IRES sequence is followed by a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence.

[0192] In some embodiments, the polyA sequence or polyAC spacer is located 5' to the IRES. In some embodiments, the polyA or polyAC sequence is located between the 5' end of the IRES and the exon-exon splice junction. The internal polyA sequence or polyAC spacer can be 1-500 nucleotides in length (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450 or 500 nucleotides). In some embodiments, the polyA sequence or polyAC sequence may range in length from 10-70, 20-60, or 30-60 nucleotides. In some embodiments, the circRNA comprises a polyAC sequence as shown in SEQ ID NO: 37 at the 5' end of the IRES sequence. In some embodiments, no polyA sequence or polyAC sequence is placed 5' to the IRES sequence. Without being bound by any theory or hypothesis, the addition of an internal polyA sequence or polyAC spacer preceding the IRES sequence may help maintain a functional secondary structure of the IRES element for efficient protein translation initiated by the IRES. In some embodiments, the polyA sequence or polyAC spacer increases expression of the RNA construct.

[0193] In some embodiments, the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising a spike (S) protein or a fragment thereof of a coronavirus (for example, SARS-CoV-2), and the nucleic acid sequence can be Operably linked to the m6A (N6-methyladenosine) modification motif sequence. The m6A modified sequence may comprise an m6A consensus sequence. M6A consensus sequences are known in the art, e.g. the consensus sequence identified by Ke et al. 2017 (m6A mRNA modification is deposited in nascent pre-mRNA, does not require splicing but does specify cytoplasmic turnover. Genes & Dev. 2017. 31: 990-1006), and can be downloaded from GEO (GSE86336). In some embodiments, the m6A modification motif sequence comprises the sequence shown in SEQ ID NO: 38. In some embodiments, the m6A modification motif sequence is followed by a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide.

[0194] In some embodiments, the circRNA also comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment located at the 5' end flank of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence located at the encoding antigenic polypeptide The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence. In some embodiments, the 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 46, and the 5' catalytic group I intron fragment sequence comprises the nucleic acid sequence of SEQ ID NO: 47.

[0195] In some embodiments, the group I catalytic intron of the T4 bacteriophage Td gene is bisected in a manner that preserves structural elements critical for ribozyme folding. Exon fragment 2 is then joined to the upstream of exon fragment 1, and a nucleic acid sequence comprising a sequence encoding an antigenic polypeptide comprising a spike of a coronavirus is inserted between the exon-exon junctions. Spike (S) protein or a fragment thereof. In some embodiments, an antigenic polypeptide comprising an IRES or m6A sequence, a Kozak sequence, a signal peptide coding sequence, an S protein comprising coronavirus or a fragment thereof, and a stop codon or frame are inserted between the exon-exon junctions Sequence of the inner 2A peptide sequence.

[0196] In some embodiments, the circRNA comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are ligated by a ligase (for example, T4 RNA enzymes) linked to each other.

[0197] C. Exemplary therapeutic circRNA

[0198] i. Exemplary circRNAs for expressing therapeutic polypeptides

[0199] In some embodiments, the application provides a nucleic acid sequence comprising an encoding therapeutic polypeptide (such as any therapeutic polypeptide described in Section A above) and further comprising an internal ribosome entry site (IRES) sequence or m6A modification Motif sequence circular RNA (circRNA), wherein the IRES or m6A modified motif sequence is operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the therapeutic polypeptide (eg, antigenic polypeptide, soluble receptor, or antibody). In a non-limiting example, the signal peptide is human tissue plasminogen promoter (tPA) or an IgE signal peptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide, and can be identified by a The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40.

[0200] In some embodiments, the circular RNA (circRNA) provided by the application comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence and a therapeutic polypeptide encoding nucleic acid sequence. In some embodiments, the circRNA further comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the therapeutic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide, and can be identified by a The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence.

[0201] In some embodiments, the circular RNA (circRNA) provided by the application comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an internal ribosome entry site (IRES) sequence, a Kozak sequence and A nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide, and can be identified by a The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence.

[0202] In some embodiments, the circular RNA (circRNA) provided by the application comprises a nucleic acid sequence encoding a therapeutic polypeptide, and also comprises an in-frame 2A operably linked to the 3' end of the nucleic acid sequence encoding a therapeutic polypeptide Peptide coding sequence (eg, instead of a stop codon). In some embodiments, the circRNA also comprises an internal ribosome entry site (IRES) sequence or m6A modification motif sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. In some embodiments, the nucleic acid sequence also encodes a SP (eg, human tPA or IgE SP) fused to the N-terminus of the therapeutic polypeptide for secretion of the therapeutic polypeptide.

[0203] In some embodiments, the circular RNA (circRNA) provided by the application comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an internal ribosome entry site (IRES) sequence or a m6A modification base Sequence sequence, Kozak sequence, nucleic acid sequence encoding therapeutic polypeptide and in-frame 2A peptide coding sequence. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide, and can be identified by a The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence.

[0204] ii. Exemplary circRNA vaccine In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises the spike of coronavirus (for example, SARS-CoV2) Spike (S) protein or a fragment thereof, and also includes an internal ribosome entry site (IRES) sequence or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof. In a non-limiting example, the signal peptide is human tissue plasminogen promoter (tPA) or an IgE signal peptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence.

[0206] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS-CoV-2) The spike (S) protein or fragment thereof, and an internal ribosome entry site (IRES) sequence or m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof. In a non-limiting example, the signal peptide is human tissue plasminogen promoter (tPA) or an IgE signal peptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence.

[0207] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS-CoV-2) The spike (S) protein or fragment thereof, and also includes an internal ribosome entry site (IRES) or m6A modification motif sequence, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the signal peptide is, for example, human tissue plasminogen promoter (tPA) or an IgE signal peptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide, wherein said antigenic polypeptide comprises the spike (S) protein or fragment thereof of coronavirus (such as SARS-CoV-2) . In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5' end to 3' end: m6A modification motif sequence, Kozak sequence, a nucleic acid sequence encoding a signal peptide (SP) and a nucleic acid sequence encoding an antigenic polypeptide, wherein the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of a coronavirus (eg, SARS-CoV-2). In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0210] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS-CoV-2) The receptor binding domain (RBD) and multimerization domain of the spike (S) protein (eg, the C-terminal domain of T4 fibrin that mediates T4 fibrin trimerization). In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) sequence or an m6A modification sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof (eg, human tPA or IgE signal peptide). In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and described antigenic polypeptide comprises coronavirus (for example, derives from SARS-CoV- 2 wild-type or B.1.351 / 501Y.V2 variant) of the receptor-binding domain (RBD) and multimerization domain of the spike (S) protein (e.g., T4 fibers that mediate T4 fibrin trimerization C-terminal domain of the protein). In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) sequence or an m6A modification sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof (eg, human tPA or IgE signal peptide). In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide, wherein said antigenic polypeptide comprises coronavirus (for example, derived from wild-type SARS-CoV-2, or variants such as SARS - B.1.351 or B.1.617.2 variants of CoV-2). In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a polyAC sequence as shown in SEQ ID NO: 37 at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: m6A modified motif sequence, Kozak sequence, a nucleic acid sequence encoding a signal peptide (SP) and a nucleic acid sequence encoding an antigenic polypeptide comprising a coronavirus (for example, derived from wild-type SARS-CoV-2, or variants such as B of SARS-CoV-2 .1.351 or B.1.617.2 variant) of the receptor binding domain (RBD) of the Spike (S) protein. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES)) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide comprising coronavirus (for example, derived from wild-type SARS-CoV-2, or variants such as SARS - the receptor binding domain (RBD) and multimerization domain (e.g., the RBD that mediates T4 fibrin trimerization) of the Spike (S) protein of CoV-2 B.1.351 or B.1.617.2 variants) C-terminal domain of T4 fibrin). In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 63.

[0215] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and the antigenic polypeptide comprises a coronavirus (for example, SARS-CoV-2) The receptor binding domain (RBD) and S2 region of the spike (S) protein and the multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates T4 fibrin trimerization). In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof (eg, human tPA or IgE signal peptide). In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES)) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide, wherein said antigenic polypeptide comprises coronavirus (for example, derived from wild-type SARS-CoV-2, or variants such as The B.1.351 or B.1.617.2 variants of SARS-CoV-2) the receptor binding domain (RBD) and S2 region of the spike (S) protein and the multimerization domain (e.g., mediates T4 fibrin C-terminal domain of trimerized T4 fibrin). In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0217] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and said circRNA comprises such nucleic acid sequence, and it comprises from 5' end to 3' end: m6A modified motif sequence, Kozak sequence, a nucleic acid sequence encoding a signal peptide (SP), and a nucleic acid sequence encoding an antigenic polypeptide, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or a variant such as SARS-CoV-2 B.1.351 or B.1.617.2 variants) of the receptor binding domain (RBD) and S2 region of the spike (S) protein and the multimerization domain (e.g., T4 fibers that mediate T4 fibrin trimerization C-terminal domain of the protein). In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0218] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising the full-length S protein of SARS-CoV-2 Amino acid residues 2-1273, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0219] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and said circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and said antigenic polypeptide comprises the full-length S protein of SARS-CoV-2 Amino acid residues 2-1273, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the circRNA also includes an internal ribosome entry site (IRES) or m6A modification motif sequence, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the signal peptide is, for example, human tissue plasminogen promoter (tPA) or an IgE signal peptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES)) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide, which comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the Numbering is based on SEQ ID NO: 1. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: m6A modified motif sequence, Kozak Sequence, nucleic acid sequence encoding signal peptide (SP) and nucleic acid sequence encoding antigenic polypeptide comprising amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0222] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and said circRNA comprises the nucleic acid sequence of coding antigenic polypeptide, and said antigenic polypeptide comprises the spike (S) of SARS-CoV-2 A protein or a fragment thereof, wherein the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises K986P and V987P mutations. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of the S protein or a fragment thereof (eg, human tPA or IgE signal peptide). In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and said circRNA comprises the nucleic acid sequence shown in any one of SEQ ID NO: 11-15 or SEQ ID NO: 48-49.

[0223] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and also comprises a 3' end operably with the nucleic acid sequence encoding an antigenic polypeptide The in-frame 2A peptide coding sequence of connection, wherein said antigenic polypeptide comprises the spike (S) protein of coronavirus (for example, SARS-CoV-2) or its fragment. In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: internal ribosome entry site ( IRES)) sequence, Kozak sequence, nucleic acid sequence encoding signal peptide (SP), nucleic acid sequence encoding antigenic polypeptide, wherein said antigenic polypeptide comprises coronavirus S protein or its fragment and 2A peptide encoding sequence in frame. In some embodiments, the IRES sequence is: an IRES sequence of a CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and described circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: m6A modified motif sequence, Kozak Sequence, nucleic acid sequence encoding signal peptide (SP), nucleic acid sequence encoding antigenic polypeptide, wherein said antigenic polypeptide comprises coronavirus S protein or its fragment and 2A peptide coding sequence in frame. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0226] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising a coronavirus (for example, derived from wild-type SARS- CoV-2, or variants such as the B.1.351 or B.1.617.2 variants of SARS-CoV-2) spike (S) protein or fragments thereof, also comprising the 3' end of the nucleic acid sequence encoding the antigenic polypeptide can be An operably linked in-frame 2A peptide coding sequence. In some embodiments, the circRNA further comprises an m6A modification motif sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0227] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and also comprises a nucleic acid sequence 3' end operably linked with an encoding antigenic polypeptide The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or variants such as B.1.351 or B.1.617.2 variants of SARS-CoV-2 ) of the spike (S) protein or a fragment thereof. In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the 3' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence comprises the nucleic acid sequence of SEQ ID NO: 40.

[0228] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and is operably linked to the 3' end of the nucleic acid sequence encoding an antigenic polypeptide. The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or a variant such as the B.1.351 or B.1.617.2 variant of SARS-CoV-2 ) receptor binding domain (RBD) of the spike (S) protein. In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0229] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and also comprises a nucleic acid sequence 3' end operably linked with an encoding antigenic polypeptide The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or a variant such as the B.1.351 or B.1.617.2 variant of SARS-CoV-2 ) receptor binding domain (RBD) of the spike (S) protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosome entry site (IRES) or an m6A modification motif sequence, wherein the IRES or m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0230] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and also comprises a nucleic acid sequence 3' end operably linked to an encoding antigenic polypeptide The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or a variant such as the B.1.351 or B.1.617.2 variant of SARS-CoV-2 ) receptor binding domain (RBD) of the spike (S) protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal Foldon domain of T4 fibrin, or the isoleucine zipper domain based on GCN4). In some embodiments, circRNA also comprises internal ribosome entry site (IRES) sequence (for example, the IRES sequence of CVB3 virus, EV71 virus, EMCV virus, PV virus or CSFV virus) or m6A modification motif sequence, wherein IRES or The m6A modification motif sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0231] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and also comprises a nucleic acid sequence 3' end operably linked with an encoding antigenic polypeptide The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises a coronavirus (for example, derived from wild-type SARS-CoV-2, or a variant such as the B.1.351 or B.1.617.2 variant of SARS-CoV-2 ) of the receptor binding domain (RBD) and S2 region of the spike (S) protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD domain has at least 90% (eg, at least 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the RBD domain comprises the amino acid sequence of SEQ ID NO: 63.

[0232] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises a circRNA, and the circRNA comprises a nucleic acid sequence encoding amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein The numbering is based on SEQ ID NO: 1, further comprising an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the antigenic polypeptide further comprises a multimerization domain. In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0233] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises a circRNA, and the circRNA comprises a nucleic acid sequence encoding amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein The numbering is based on SEQ ID NO: 1, further comprising an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0234] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises a circRNA, and the circRNA comprises a nucleic acid sequence encoding amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein The numbering is based on SEQ ID NO: 1, further comprising an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the antigenic polypeptide further comprises a multimerization domain. In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0235] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and is operably linked to the 3' end of the nucleic acid sequence encoding an antigenic polypeptide. The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises the receptor binding domain (RBD) and the S2 region of the spike (S) protein of coronavirus (eg, SARS-CoV-2). In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0236] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and is operably linked to the 3' end of the nucleic acid sequence encoding an antigenic polypeptide. The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises the receptor binding domain (RBD) and the S2 region of the spike (S) protein of coronavirus (eg, SARS-CoV-2). In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0237] In some embodiments, the circular RNA (circRNA) vaccine provided by the application comprises circRNA, and the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide, and is operably linked to the 3' end of the nucleic acid sequence encoding an antigenic polypeptide. The in-frame 2A peptide coding sequence, wherein the antigenic polypeptide comprises the receptor binding domain (RBD) and the S2 region of the spike (S) protein of coronavirus (eg, SARS-CoV-2). In some embodiments, the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA further comprises an internal ribosomal entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide (for example, the IRES of CVB3 virus, EV71 virus, EMCV virus, PV virus, or CSFV virus sequence). In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof, and a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0238] In some embodiments, the circRNA vaccine provided comprises a nucleic acid sequence comprising from the 5' end to the 3' end: IRES sequence, Kozak sequence, SP, nucleic acid sequence encoding antigenic polypeptide, and 2A peptide in frame coding sequence. In some embodiments, the circRNA vaccine further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0239] In some embodiments, the circRNA vaccine provided comprises a nucleic acid sequence comprising from the 5' end to the 3' end: polyA or polyAC sequence, IRES sequence, Kozak sequence, SP, nucleic acid sequence encoding antigenic polypeptide and In frame 2A peptide coding sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end.

[0240] In some embodiments, the provided circular RNA (circRNA) comprises a nucleic acid sequence comprising from the 5' end to the 3' end: m6A modified motif sequence, Kozak sequence, SP, nucleic acid encoding antigenic polypeptide sequence, and the 2A peptide coding sequence in frame. In some embodiments, the antigenic polypeptide comprises the receptor binding domain (RBD) of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin).

[0241] In some embodiments, the provided circular RNA (circRNA) comprises a nucleic acid sequence comprising from the 5' end to the 3' end: m6A modified motif sequence, Kozak sequence, SP and nucleic acid encoding antigenic polypeptide sequence. In some embodiments, the antigenic polypeptide comprises the receptor binding domain (RBD) and the S2 region of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin).

[0242] In some embodiments, the provided circular RNA (circRNA) comprises a nucleic acid sequence comprising from the 5' end to the 3' end: m6A modified motif sequence, Kozak sequence, SP and the sequence encoding the antigenic polypeptide . In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, the C-terminal domain of T4 fibrin that mediates trimerization of T4 fibrin). In some embodiments, the circRNA comprises an in-frame 2A peptide coding sequence following the antigenic polypeptide.

[0243] III. Methods of treatment

[0244] The circRNAs and compositions derived herein can be used to treat or prevent diseases or conditions in individuals, including but not limited to genetic diseases (such as hereditary genetic diseases, metabolic diseases and cancer) and infections (such as viral infections such as coronavirus infections ). In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual.

[0245] In some embodiments, a method for treating or preventing a disease or condition in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the antigenic polypeptide is a protein or fragment thereof of an infectious agent, such as a virus, eg, a coronavirus. In some embodiments, the infectious agent is SARS-CoV-2. In some embodiments, the antigenic polypeptide is the S protein or a fragment thereof. In some embodiments, the disease or condition is a coronavirus infection. In some embodiments, the method comprises administering an effective amount of a mixture composition comprising multiple circRNAs encoding different antigenic polypeptides.

[0246] In some embodiments, a method for treating or preventing a disease or condition in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding a functional protein. In some embodiments, the functional protein is an enzyme, receptor, ligand, signaling molecule, or transcription factor. In some embodiments, the disease or condition is a metabolic disease. In some embodiments, the disease or condition is a lysosomal storage disorder. In some embodiments, the disease or condition is cancer.

[0247] In some embodiments, a method for treating or preventing a disease or condition in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding a receptor protein. In some embodiments, the receptor protein is a receptor for an infectious agent, such as a virus, eg, a coronavirus. In some embodiments, the receptor protein is a soluble receptor, such as soluble ACE2 receptor.

[0248] In some embodiments, a method for treating or preventing an individual's disease or condition is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding a targeting protein (such as an antibody). In some embodiments, the targeting protein is a neutralizing antibody. In some embodiments, the targeting protein is a therapeutic antibody. In some embodiments, the targeting protein specifically binds an infectious agent such as a virus, eg, a coronavirus.

[0249] In some embodiments, the application provides a circRNA for treating or preventing a disease or condition in an individual.

[0250] In some embodiments, the application provides a circRNA vaccine for treating or preventing individual coronavirus (eg, SARS-CoV, MERS-COV or SARS-CoV-2) infection.

[0251] In some embodiments, the application provides the use of a circRNA comprising a nucleic acid sequence encoding a therapeutic polypeptide in the preparation of a medicament for treating or preventing an individual's disease or condition.

[0252] In some embodiments, the application provides the use of a circRNA vaccine comprising circRNA in the preparation of a vaccine for treating or preventing individual coronavirus infection, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, wherein the The antigenic polypeptide comprises the Spike (S) protein of a coronavirus (eg, SARS-CoV-2) or a fragment thereof.

[0253] A. Treatment of Genetic Diseases or Conditions

[0254] The circRNAs described herein can be used to treat genetic diseases or conditions associated with mutations or defects in naturally occurring proteins corresponding to therapeutic polypeptides encoded by circRNAs. In some embodiments, the disease or condition is one associated with insufficient levels and / or activity of a naturally occurring protein corresponding to a therapeutic polypeptide. In some embodiments, the disease or condition is an inherited genetic disease associated with one or more mutations in a naturally occurring protein corresponding to the therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a wild-type protein or a functional variant thereof (eg, a functional fragment, fusion protein, or mutant).

[0255] In some aspects, the application provides methods and compositions for using a circRNA expressing a therapeutic polypeptide to treat a disease or condition associated with a deficiency of a functional protein such as an enzyme (eg, IDUA). In some embodiments, a therapeutic polypeptide comprises a nucleotide sequence encoding a protein or derivative thereof. In some embodiments, the circRNA is capable of expressing a functional protein or a functional derivative thereof capable of restoring the function of a protein associated with a disease or condition. In some embodiments, e.g., up to 8, 12, 16, 24, 30, 36 or 40 h after administration of the circRNA, compared to the endogenous wild-type protein of the cell or organism (e.g., mouse or human), the The circRNA can restore 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the protein activity.

[0256] In some embodiments, a therapeutic polypeptide can be any polypeptide capable of being expressed by a target cell (e.g., a human or mouse cell) to produce (and in some cases, secrete) a functional enzyme or protein, such as For example, disclosed in International Application No. PCT / US2010 / 058457. In some embodiments, a therapeutic polypeptide can be engineered for secretion by operably linking a signal peptide to the amino terminus of the therapeutic polypeptide. For example, in some embodiments, following expression of one or more therapeutic polynucleotides by a target cell, production of a functional enzyme or protein (e.g., urea cyclase or lysozyme) that is deficient in the subject can be observed. storage disease-associated enzymes).

[0257] Examples of disease-associated mutations that can be treated by the methods of the present application include, but are not limited to: TP53W53X (eg, 158G>A) associated with cancer, IDUAW402X associated with type I mucopolysaccharidosis (MPSI) ( For example, TGG>TAG mutation in exon 9), COL3A1W1278X associated with Ehlers-Danlos syndrome (eg, 3833G>A mutation), BMPR2W298X associated with primary pulmonary hypertension (eg, 893G>A ), AHI1W725X associated with Joubert syndrome (eg, 2174G>A), FANCCW506X associated with Fanconi anemia (eg, 1517G>A), MYBPC3W1098X associated with primary familial hypertrophic cardiomyopathy (eg , 3293G>A), and IL2RGW237X associated with X-linked severe combined immunodeficiency (eg, 710G>A). In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a monogenic disease. In some embodiments, the disease or condition is a polygenic disease.

[0258] In some embodiments, the disease or condition is a liver disease or condition. In some embodiments, the disease or condition is a disease or condition of the respiratory tract of the individual, such as a disease or condition of the lungs.

[0259] In some embodiments, a method for treating individual cancer is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding a tumor suppressor factor. In some embodiments, the tumor suppressor is TP53 (including functional variants thereof). In some embodiments, the tumor suppressor is PTEN (including functional variants thereof). In some embodiments, the tumor suppressor comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or any of 100%) sequence identity of amino acid sequences.

[0260] In some embodiments, a method for treating an individual's lysosomal storage disease is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding a lysosomal enzyme.

[0261] In some embodiments, a method of treating a liver disease or condition in an individual is provided, comprising administering to the individual an effective amount of a circRNA comprising a nucleic acid sequence encoding a liver protein (eg, an enzyme).

[0262] In some embodiments, a method for treating mucopolysaccharidosis type I (MPSI) in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding IDUA (including functional variants thereof). In some embodiments, the IDUA comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Amino acid sequences with any one of 100% sequence identity. In some embodiments, the IDUA comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Amino acid sequences with any one of 100% sequence identity.

[0263] In some embodiments, a method for treating individual ornithine transcarbamylase deficiency is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding OTC (including functional variants thereof). In some embodiments, the OTC comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences. In some embodiments, the OTC comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0264] In some embodiments, a method for treating individual tyrosinemia is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding FAH (including functional variants thereof). In some embodiments, the FAH comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences. In some embodiments, the FAH comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0265] In some embodiments, a method of treating Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, or familial dilated cardiomyopathy in an individual is provided, comprising administering to the individual an effective amount comprising the DMD coded DMD ( circRNA including its functional variants, such as the nucleic acid sequence of mini DMD). In some embodiments, the DMD comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences. In some embodiments, the DMD comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0266] In some embodiments, a method of treating individual Ehlers-Danlos syndrome is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding COL3A1 (including functional variants thereof). In some embodiments, COL3A1 comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0267] In some embodiments, a method of treating Joubert's syndrome in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding AHI1 (including functional variants thereof). In some embodiments, AHI1 comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0268] In some embodiments, a method for treating individual pulmonary hypertension or pulmonary veno-occlusive disease is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding FANCC (including its functional variant). In some embodiments, FANCC comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0269] In some embodiments, a method for treating an individual's primary familial hypertrophic cardiomyopathy is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding MYBPC3 (including functional variants thereof). In some embodiments, MYBPC3 comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0270] In some embodiments, a method of treating individual X-linked severe syndrome immunodeficiency is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding IL2RG (including functional variants thereof). In some embodiments, IL2RG comprises at least about 80% (e.g., at least about 85%, 90%, 93%, 95%, 97%, 98%, or 99%, or Any one of 100%) sequence identity of amino acid sequences.

[0271] In some embodiments, the circRNA has a functional half-life of at least or at least about 20h, 24h, 30h or 36h. In some embodiments, the circRNA has a duration of therapeutic effect in a human cell of at least or at least about 20h, 24h, 30h, or 36h. In some embodiments, the duration of the therapeutic effect of the circRNA in human cells is greater than or equal to the duration of the therapeutic effect of an equivalent linear RNA comprising the same expression sequence. In some embodiments, the functional half-life of the circRNA in human cells is greater than or equal to the functional half-life of an equivalent linear RNA comprising the same expression sequence.

[0272] In some embodiments, the therapeutic polypeptide comprises IDUA, and the disease or condition is Heller's syndrome. In some embodiments, administration of the circRNA restores at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40% in a human or animal model with a mutation in IDUA compared to wild type , at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% alpha-l-iduronidase. In some embodiments, the catalytic activity of IDUA is increased 4-24h (eg, 4-8h, 8-12h, 12-16h, 16-20h, and / or 16-24h) after administration of a circRNA encoding IDUA. In some embodiments, the circRNA encoding IDUA has a functional half-life of at least or at least about 20h, 24h, 30h, or 36h.

[0273] B. Treatment or prevention of coronavirus infection

[0274] The application provides a method for treating or preventing an individual's coronavirus (for example, SARS-CoV-2) infection, comprising administering to the individual an effective amount of the circRNA of any embodiment described herein, wherein the circRNA encodes a coronavirus Antigenic polypeptides or receptor proteins (such as soluble receptors), or neutralizing antibodies that specifically bind to coronaviruses. In some embodiments, the coronavirus is SARS-CoV, MERS-COV or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the application provides a method for preventing or reducing the risk of coronavirus (for example, SARS-CoV-2) infection in an individual, comprising administering to the individual an effective amount of the circRNA of any of the above embodiments, wherein the circRNA encodes a coronavirus Antigenic polypeptides or receptor proteins of viruses (such as soluble receptors), or neutralizing antibodies that specifically bind to coronaviruses. In some embodiments, the method comprises administering a mixture composition comprising multiple circRNAs encoding different antigenic polypeptides, receptor proteins, or neutralizing antibodies. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual. In some embodiments, the circRNA is administered as a naked circRNA or as a pharmaceutical composition comprising a transfection reagent.

[0275] In some embodiments, a method of treating or preventing an individual's coronavirus (for example, SARS-CoV-2) infection is provided, comprising administering to the individual an effective amount of a drug comprising a nucleic acid sequence encoding a coronavirus receptor protein circRNA. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the receptor protein is a soluble receptor, such as soluble ACE2 receptor. In some embodiments, the method comprises administering an effective amount of a mixture composition comprising multiple circRNAs encoding different receptor proteins.

[0276] In some embodiments, a method of treating or preventing a coronavirus (e.g., SARS-CoV-2) infection in an individual is provided, comprising administering to the individual an effective amount comprising a protein encoding a neutralizing antibody that specifically binds to a coronavirus The nucleic acid sequence of circRNA. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the method comprises administering an effective amount of a cocktail composition comprising multiple circRNAs encoding different neutralizing antibodies.

[0277] In some embodiments, the application provides a method for treating or preventing a coronavirus infection in an individual, comprising administering to the individual an effective amount of any one of the circRNA vaccines in the embodiments described herein. In some embodiments, the coronavirus is SARS-CoV, MERS-COV or SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the application provides a method for preventing or reducing the risk of coronavirus (eg, SARS-CoV-2) infection in an individual, comprising administering to the individual an effective amount of the circRNA vaccine of any one of the above embodiments. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual. In some embodiments, the circRNA vaccine is administered as a naked circRNA or as a pharmaceutical composition comprising a transfection reagent.

[0278] In some embodiments, the application provides a method for treating or preventing an individual's coronavirus infection, comprising administering to the individual an effective amount of any one of the circRNA vaccines in the embodiments described herein. In some embodiments, the coronavirus is a wild-type strain of SARS-CoV-2 or a variant strain of SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, SARS-CoV-2 is α (B.1.1.7), β (B.1.351, B.1.351.2, B.1.351.3), δ (B. 1.617.2, AY.1, AY.2, AY.3) or gamma (P.1, P.1.1, P.1.2) variants. In some embodiments, the variant can be any variant described online at cdc.gov / coronavirus / 2019-ncov / variants / . In some embodiments, the application provides methods of preventing or reducing the risk of a coronavirus infection (e.g., SARS-CoV-2 infection, such as infection with any of the variant SARS-CoV-2 strains described herein) in an individual, comprising administering to the individual Administering an effective amount of the circRNA vaccine of any one of the above embodiments. In some embodiments, the circRNA vaccine encodes an S protein or a fragment thereof comprising one, two or three mutations selected from the group consisting of K417N, L452R and T478K, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or fragment thereof comprising one, two, three, four, five or more mutations selected from the group consisting of deletion of residue 69, residue 70 deletion, residue 144 deletion, E484K, S494P, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H and K1191N, where amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or a fragment thereof comprising one, two or three mutations selected from the group consisting of E484K, S494P and N501Y, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or fragment thereof comprising one, two, three, four, five or more mutations selected from the group consisting of: D80A, D215G, 241del, 242del , 243del, K417N, E484K, N501Y, D614G and A701V, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or a fragment thereof comprising one, two or three mutations selected from the group consisting of K417N, E484K and N501Y, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or fragment thereof comprising one, two, three, four, five or more mutations selected from the group consisting of: L18F, T20N, P26S, D138Y , R190S, K417T, E484K, N501Y, D614G, H655Y and T1027I, wherein the amino acid numbering is based on SEQ ID NO: 1. In some embodiments, the circRNA vaccine encodes an S protein or a fragment thereof comprising one, two or three mutations selected from the group consisting of K417T, E484K and N501Y.

[0279] In some embodiments, the application provides a method of treating or preventing a multi-strain coronavirus (e.g., multi-strain SARS-CoV-2) infection in an individual comprising administering to the individual an effective amount of any of the embodiments described herein. Scheme of circRNA vaccine. In some embodiments, the application provides methods of treating or preventing infection with multiple strains of coronavirus (e.g., multiple strains of SARS-CoV-2) in an individual comprising administering to the individual an effective amount of a multiple strain of any embodiment described herein. Different circRNA vaccines. In some embodiments, the method comprises administering to the individual a composition comprising multiple (e.g., two or more) circRNAs, wherein the first circRNA encodes the S protein or a fragment thereof of a first coronavirus strain, and the second Two circRNAs encode the S protein or its fragments of the second coronavirus strain. In some embodiments, at least one of the plurality of circRNAs encodes the S protein or a fragment thereof contained in the D614G, B.1.1.7 / 501Y.V1 variant of SARS-CoV-2 or the SARS-CoV-2 Mutations found in the B.1.351 / 501Y.V2 variant of CoV-2.

[0280] In some embodiments, the application provides a method for treating or preventing an individual's coronavirus infection, comprising administering to the individual an effective amount of a circRNA vaccine comprising a circRNA, the circRNA comprising a nucleic acid sequence encoding an antigen polypeptide, the antigen The polypeptide comprises the S protein of a coronavirus (eg, SARS-CoV-2) or a fragment thereof. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the antigenic polypeptide comprises the S protein or fragment thereof of SARS-CoV-2 with a D614G mutation. In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual.

[0281] In some embodiments, the application provides a method for treating or preventing an individual's coronavirus infection, comprising administering to the individual an effective amount of a circRNA vaccine comprising a circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, Wherein said antigenic polypeptide comprises the S protein of coronavirus (eg, SARS-CoV-2) or its fragment; and (b) IRES sequence, wherein said IRES sequence is operably linked with the nucleic acid sequence encoding antigenic polypeptide. In some embodiments, the circRNA further comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, circRNA comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: IRES sequence, Kozak sequence, SP and the nucleic acid sequence of coding antigenic polypeptide. In some embodiments, the circRNA also comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the antigenic polypeptide comprises the S protein or fragment thereof of SARS-CoV-2 with a D614G mutation. In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual. In some embodiments, the circRNA vaccine is administered by intramuscular (i.m) injection. In some embodiments, one or more doses of the circRNA vaccine are administered. In some embodiments, the interval between doses is about 2 weeks (eg, 12, 13, 14, 15, or 16 days). In some embodiments, the method comprises administering a first dose of the circRNA vaccine and administering a second dose of the circRNA vaccine 2 weeks later or about 2 weeks later.

[0282] In some embodiments, the application provides a method for treating or preventing an individual's coronavirus infection, comprising administering to the individual an effective amount of a circRNA vaccine comprising a circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, Wherein the antigenic polypeptide comprises a coronavirus (eg, SARS-CoV-2) S protein or a fragment thereof; and (b) an m6A modification motif sequence operably linked to a nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence also encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modified motif sequence, a Kozak sequence, a SP and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA also includes a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and can be identified by the nucleic acid sequence encoding the antigenic polypeptide. The 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end. In some embodiments, the circRNA also comprises a 5' junction sequence at the 5' end of the circRNA and a 3' junction sequence at the 3' end of the circRNA, wherein the 5' junction sequence and the 3' junction sequence are passed through a ligase (for example, T4 RNA ligase) interconnected. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (eg, a C-terminal Fd domain, or a GCN-4-based isoleucine zipper domain). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations that stabilize the prefusion conformation of the S protein (eg, K986P and V987P). In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (eg, deletion of amino acid residues 681-684). In some embodiments, the antigenic polypeptide comprises the S protein or fragment thereof of SARS-CoV-2 with a D614G mutation. In some embodiments, the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15. In some embodiments, the circRNA undergoes rolling circle translation by ribosomes in an individual. In some embodiments, the circRNA vaccine is administered by intramuscular (i.m) injection. In some embodiments, one or more doses of the circRNA vaccine are administered. In some embodiments, the interval between doses is about 2 weeks (eg, 12, 13, 14, 15, or 16 days). In some embodiments, the method comprises administering a first dose of the circRNA vaccine and administering a second dose of the circRNA vaccine 2 weeks later or about 2 weeks later.

[0283] C. Formulation and Administration

[0284] In some embodiments, the circRNA composition (eg, circRNA vaccine or pharmaceutical composition) for administration further comprises a transfection reagent. In non-limiting examples, the transfection reagent is polyethyleneimine (PEI) or lipid nanoparticles (LNP). Suitable lipid nanoparticles for circRNA administration have been described, for example, in L. M. & Garidel, P. Lipid-based nanoparticle formulations for small molecules and RNA drugs. 890Expert Opin Drug Deliv16, 1205-1226, doi:10.1080 / 17425247.2019. 1669558 (2019), U.S. Patent Application Publication Nos. US20200121809, US20200163878, US20190022247, and International Patent Application Publication No. WO2021 / 030701, the contents of which are incorporated herein by reference in their entirety. In some embodiments, LNPs are formed from the following lipid mixture: MC3-lipid:DSPC:cholesterol:PEG2000-DMG. In some embodiments, MC3-lipid:DSPC:cholesterol:PEG2000-DMG is mixed in a molar ratio of 50:10:38.5:1.5. Other examples of liposomes that can be used to administer circRNA compositions (e.g., circRNA vaccines or pharmaceutical compositions) for administration include: protamine, cationic nanoemulsions, modified dendrimer nanoparticles, protamine Proteoliposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipids sold under the trademark LIPOFECTAMINE Transfection reagents, non-liposomal transfection reagents sold under the trademark FUGENE, or any combination thereof, can be used as transfection reagents.

[0286] In some embodiments, liposome formulations can be influenced by, but not limited to, the following factors: selection of cationic lipid components, cationic lipid saturation, nature of pegylation, ratio of all components and Biophysical parameters (eg size). In some embodiments, liposome formulations comprise cationic lipids, cholesterol, and pegylated lipids. For example, liposomal formulations can comprise cationic lipids, dipalmitoylphosphatidylincholine, cholesterol and PEG-c-DMA. See, eg, Semple et al. Nature Biotech. 2010 28:172-176, herein incorporated by reference in its entirety. In some embodiments, liposome formulations may comprise: about 35% to about 45% cationic lipids, about 40% to about 50% cationic lipids, about 50% to about 60% cationic lipids, and / or Or about 55% to about 65% cationic lipid. In some embodiments, the ratio of lipid to RNA in liposomes can be from about 5:1 to about 20:1, from about 10:1 to about 25:1, from about 15:1 to about 30:1, and / or At least 30:1. Suitable liposomal formulations have been described eg in WO2020237227, the content of which is incorporated herein by reference in its entirety.

[0287] In some embodiments, the circRNA is not formulated with a transfection reagent. In some embodiments, the circRNA is delivered as naked RNA. In some embodiments, the circRNA is delivered by gene gun or by electroporation.

[0288] The circRNA composition (for example, circRNA vaccine or pharmaceutical composition) for administration can be injected into the vasculature by systemic injection, injected into the lymph nodes systemically, injected subcutaneously or in a depot, or administered by local injection in subject.

[0289] In some embodiments, the circRNA vaccines herein (eg, encoding the S protein of a coronavirus or a fragment thereof) are administered by intramuscular (i.m) injection. In some embodiments, one or more doses of the circRNA vaccine are administered. In some embodiments, two or more doses of the circRNA vaccine are administered. In some embodiments, the interval between doses is about 2 weeks (eg, 12, 13, 14, 15, or 16 days). In some embodiments, the method comprises administering a first dose of the circRNA vaccine and administering a second dose of the circRNA vaccine 2 weeks later or about 2 weeks later.

[0290] In some embodiments, the circRNA can be formulated in lipid nanoparticles, such as those described in International Publication No. WO2012170930, which is incorporated herein by reference in its entirety.

[0291] In some embodiments, synthetic nanocarriers can be formulated for controlled and / or sustained release of the circRNAs described herein. As a non-limiting example, synthetic nanocarriers for sustained release can be formulated by methods known in the art, as described herein and / or as described in International Publication No. WO2010138192 and US Publication No. US20100303850, each Incorporated herein by reference in its entirety.

[0292] In some embodiments, the circRNA can be formulated for controlled and / or sustained release, wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer. CYSC polymers are described in US Patent No. US 8,399,007, which is incorporated herein by reference in its entirety.

[0293] In some embodiments, synthetic nanocarriers can be formulated for use as vaccines. In some embodiments, synthetic nanocarriers can encapsulate at least one circRNA encoding at least one antigen. As a non-limiting example, synthetic nanocarriers may comprise at least one antigen and excipients for vaccine dosage forms (see International Publication No. WO2011150264 and US Publication No. US20110293723, each of which is incorporated herein by reference in its entirety). As another non-limiting example, a vaccine dosage form may include at least two synthetic nanocarriers and excipients with the same or different antigens (see International Publication No. WO2011150249 and US Publication No. US20110293701, each of which is incorporated herein by reference in its entirety) ). Vaccine formulations may be selected by methods described herein, known in the art, and / or described in International Publication No. WO2011150258 and US Publication No. US20120027806, each of which is incorporated herein by reference in its entirety).

[0294] In some embodiments, a synthetic nanocarrier may comprise at least one circRNA encoding at least one adjuvant. As a non-limiting example, an adjuvant may comprise dimethyl dioctadecyl ammonium bromide, dimethyl dioctadecyl ammonium chloride, dimethyl dioctadecyl ammonium phosphate, or dimethyl dioctadecyl ammonium Octylammonium acetate (DDA) and the non-polar fraction of total lipid extract of mycobacterium or a portion of said non-polar fraction (see, e.g., U.S. Patent No. US 8,241,610; which is incorporated by reference in its incorporated herein in its entirety). In another embodiment, a synthetic nanocarrier may comprise at least one circRNA and an adjuvant. As a non-limiting example, synthetic nanocarriers comprising adjuvants can be formulated by the methods described in International Publication No. WO2011150240 and US Publication No. US20110293700, each of which is incorporated herein by reference in its entirety.

[0295] In some embodiments, the circRNA is used as an adjuvant. For example, RNA sensing in the cytoplasm can trigger innate immunity, and innate immune signaling is known to promote adaptive immunity through multiple pathways. Therefore, a circRNA comprising an antigenic polypeptide or a second circRNA (eg, a circRNA that does not encode a polypeptide) can be used as an adjuvant to enhance the adaptive immune response against the antigenic polypeptide.

[0296] In some embodiments, a circRNA composition for administration (eg, a circRNA vaccine or pharmaceutical composition) can be administered intranasally. For example, circRNA vaccines can be administered intranasally, similar to the administration of live vaccines. In some embodiments, the circRNA can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in the art.

[0297] In some embodiments, the circRNA vaccine includes an adjuvant, which can enable the vaccine to elicit a higher immune response. As a non-limiting example, the adjuvant can be a submicron oil-in-water emulsion, which can elicit a higher immune response in the human pediatric population (see, e.g., adjuvanted adjuvant described in US Patent Publication No. US20120027813 and US Patent No. US8506966). vaccines, the contents of each of which are incorporated herein by reference in their entirety).

[0298] In some embodiments, the circRNA compositions of the present application can be administered together with other preventive or therapeutic compounds. As non-limiting examples, prophylactic or therapeutic compounds may be adjuvants or boosters. As used herein, when referring to a prophylactic composition such as a vaccine, the term "booster" refers to additional administration of the prophylactic composition. A booster (or booster vaccine) can be given after earlier administration of the prophylactic composition. The time between the initial administration of the prophylactic composition and the booster can be, but not limited to: 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min , 15 min, 20 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 1 day, 36h, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months , 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years Years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 or 99+ years.

[0299] IV. Preparation method

[0300] The application further provides nucleic acid constructs (for example, linear RNA and carrier, etc.) for preparing circRNA described herein, and methods for preparing circRNA, for example, by chemical linkage, enzymatic linkage or ribozyme autocatalysis of linear RNA . In some embodiments, circRNAs are prepared by circularizing linear RNAs in vitro.

[0301] Linear RNA and nucleic acid constructs encoding it

[0302] In some embodiments, the application provides a linear RNA capable of forming the circRNA of any of the above embodiments. In some embodiments, linear RNA can be circularized by chemical circularization methods using cyanogen bromide or similar condensing agents. In some embodiments, a linear RNA can be cyclized by autocatalysis of a group I intron comprising a 5' catalytic group I intron segment and a 3' catalytic group I intron segment. In some embodiments, linear RNA can be circularized by a ligase. In some embodiments, linear RNA can be circularized by T4 RNA ligase. In some embodiments, linear RNA can be circularized by DNA ligase. Suitable ligases include, but are not limited to: T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2).

[0303] In some embodiments, the application provides a linear RNA capable of forming the circRNA of any of the above embodiments, wherein the linear RNA can be circularized by self-catalysis of group I introns. In some embodiments, the group I intron comprises a 5' catalytic group I intron fragment and a 3' catalytic group I intron fragment. In some embodiments, the linear RNA comprises a 3' catalytic 3' flanking the 5' end of a 3' exon sequence recognizable by a 3' catalytic group I intronic segment (such as the sequence set forth in SEQ ID NO: 39). A group I intron fragment (such as the sequence shown in SEQ ID NO: 46), and a 5' exon located at the 5' catalytic group I intron fragment (such as the sequence shown in SEQ ID NO: 40) A 5' catalytic group I intron fragment flanking the 3' end of the subsequence (eg, the sequence shown in SEQ ID NO: 47).

[0304] In some embodiments, the linear RNA comprises from 5' to 3' end: 3' intron-IRES-Kozak-SP-spike-5' intron sequence. In some embodiments, the spike sequence comprises one of the sequences set forth in SEQ ID NOs: 11-15 and SEQ ID NOs: 48-49.

[0305] In some embodiments, the linear RNA comprises from 5' to 3' end: 3' intron-IRES-Kozak-SP-RBD-5' intron sequence. In some embodiments, the RBD sequence comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1.

[0306] In some embodiments, the linear RNA comprises from 5' to 3' end: 3' intron-IRES-Kozak-SP-nAb-5' intron sequence. In some embodiments, the nAb sequence encodes one of the amino acid sequences set forth in SEQ ID NO: 26-35. In some embodiments, the nAb sequence encodes the amino acid sequence of SEQ ID NO: 26. In some embodiments, the nAb sequence encodes the amino acid sequence of SEQ ID NO: 27. In some embodiments, the nAb sequence encodes the amino acid sequence of SEQ ID NO: 30.

[0307] In some embodiments, the linear RNA comprises from 5' to 3' end: 3' intron-IRES-Kozak-IDUA-5' intron sequence. In some embodiments, the IDUA sequence encodes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the IDUA sequence encodes the amino acid sequence of SEQ ID NO: 19.

[0308] In some embodiments, the linear RNA further comprises a 5' homologous sequence flanking the 5' end of the 3' catalytic group I intronic segment, and a sequence located at the 3' end of the 5' catalytic group I intronic segment Flanking 3' homologous sequences. In some embodiments, the linear RNA comprises from 5' to 3' end: 5' homology arm - 3' catalytic group I intron fragment - 3' exon sequence - IRES-Kozak-SP-antigen polypeptide ( For example, spike protein or fragment thereof) - 5' exon sequence - 5' catalytic group I intron fragment - 3' homology arm sequence. In some embodiments, the length of the homologous sequence can be: 1-100, 5-80, 5-60, 10-50, or 12-50 nucleotides. In some embodiments, the homologous sequences are about 20-30 nucleotides in length. In some embodiments, the 5' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 41, and the 3' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the homology arms increase the efficiency of RNA circularization by about 0-20%, by more than 20%, by more than 30%, by more than 40%, or by more than 50%.

[0309] In some embodiments, a nucleic acid construct comprising a nucleic acid sequence encoding a linear RNA is provided. In some embodiments, a T7 promoter is operably linked to a nucleic acid sequence encoding a linear RNA. In some embodiments, the T7 promoter comprises the sequence shown in SEQ ID NO: 43. In some embodiments, the T7 promoter is capable of driving transcription in vitro.

[0310] plasmid

[0311] In some embodiments, the application provides plasmids comprising the nucleotide sequences described herein. In some embodiments, the plasmid is obtained by cloning the sequence encoding the linearized RNA into a plasmid vector. Plasmids can be generated by techniques known in the art, such as Gibson cloning or cloning using restriction enzymes. In some embodiments, the plasmid vector includes an antibiotic expression cassette that allows the antibiotic to select bacteria expressing the plasmid. In some embodiments, provided plasmids can be purified from bacteria and used to generate linear circRNA constructs. Any plasmid vector suitable for in vitro transcription of linear RNA can be used.

[0312] In some embodiments, the plasmid is linearized prior to in vitro transcription of the linear RNA. In some embodiments, recombinant plasmids are linearized by restriction enzyme digestion. In some embodiments, recombinant plasmids are linearized by PCR amplification. In some embodiments, the method further comprises performing in vitro transcription using a linearized plasmid template. In some embodiments, in vitro transcription is driven by a T7 promoter.

[0313] Linear RNA circularized by chemical ligation

[0314] In some embodiments, there is provided a method for preparing the circRNA described herein, comprising: (a) chemically linking the 5' end and the 3' end of the linear RNA comprising the nucleic acid sequence encoding the circRNA; (b) The circularized RNA product is isolated to provide circRNA.

[0315] In some embodiments, the step of circularizing the linear RNA comprises a chemical cyclization method using cyanogen bromide or a similar condensing agent.

[0316] In some embodiments, linear RNA can be chemically circularized. In some chemistries, the 5'- and 3'-ends of nucleic acids (e.g., linear circular polyribonucleotides) include chemically reactive groups that, when brought close together, can be placed on the 5'-end of the molecule. A new covalent bond is formed between the end and the 3' end. The 5'-end can contain an NHS ester reactive group, and the 3'-end can contain a 3'-amino-terminated nucleotide, so that in organic solvents, the 3'-amino-terminated nucleotide at the 3'-end of a linear RNA molecule Nucleotides undergo nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'- / 3'-amide bond.

[0317] In some embodiments, the cyclization efficiency of the cyclization methods provided herein is: at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35% , at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the cyclization efficiency of the cyclization methods provided herein is at least about 40%.

[0318] Linear RNA self-catalyzed by ribozymes

[0319] In some embodiments, circRNA can be obtained by ribozyme self-catalyzed circularization of linear RNA. In some embodiments, linear RNA is circularized in vitro. In some embodiments, cyclization by ribozyme autocatalysis comprises: (a) placing the linear RNA in an autocatalyzed position in the promoter group I intron (or 5' and 3' catalytic group I intron fragments thereof) Conditions to provide a circularized RNA product; (b) isolating the circularized RNA product to provide a circular RNA.

[0320] In some embodiments, the method comprises the step of obtaining the linearized RNA by first cloning the sequence encoding the linearized RNA into a plasmid vector and then linearizing the recombinant plasmid. In some embodiments, recombinant plasmids are linearized by restriction endonuclease digestion. In some embodiments, recombinant plasmids are linearized by PCR amplification. In some embodiments, the method further comprises performing in vitro transcription using a linearized plasmid template. In some embodiments, in vitro transcription is driven by a T7 promoter. In some embodiments, the method further comprises purifying the linear RNA transcript. In some embodiments, linear RNA is purified by gel purification.

[0321] In some embodiments, the present application provides methods for circularizing linear RNA (eg, purified linear RNA) by ribozyme autocatalysis of group I introns. During the splicing process, the 3' hydroxyl of guanosine nucleotides participates in the transesterification reaction at the 5' splicing site. Half of the 5' intron is excised, and the free hydroxyl at the end of this intermediate undergoes a second transesterification at the 3' splice site, resulting in cyclization of the middle region and excision of the 3' intron. In some embodiments, the conditions to initiate autocatalysis of the group I intron or 5' and 3' catalytic group I intron fragments are the addition of GTP and Mg2+. In some embodiments, there is provided a step of circularizing the linear RNA by adding GTP and Mg2+ at 55°C for 15 min. In some embodiments, the method further comprises treating with RNase R to digest the linear RNA transcript. In some embodiments, the method further comprises isolating circular RNA (circRNA). In some embodiments, the step of isolating the circRNA comprises gel purifying the circRNA. In some embodiments, purified circRNA can be stored at -80°C.

[0322] In some embodiments, the efficiency of cyclization is: at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 32%, at least 34% , at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%. In some embodiments, the efficiency of cyclization is: about 40% to about 50%, or greater than 50%.

[0323] Linear RNA circularized by ligation

[0324] In some embodiments, circRNAs can be obtained by circularizing linear RNAs using a ligase, such as RNA ligase. In some embodiments, linear RNA is circularized in vitro. In some embodiments, linear RNA can be circularized by T4 RNA ligase. In some embodiments, the linear RNA comprises a 5' junction sequence located at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' junction sequence located at the 3' end of the nucleic acid sequence encoding the circRNA, wherein the 5' junction sequence and the 3' junction sequence Can be linked to each other by RNA ligase. In a non-limiting example, linear RNA can be circularized by ligases such as T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2). Linear RNA can be circularized in the presence or absence of single-stranded nucleic acid linkers such as splint DNA.

[0325] In some embodiments, the application provides a method for producing any of the above-mentioned circRNAs, comprising: (a) combining the above-mentioned 5' junction sequence comprising the 5' end of the nucleic acid sequence encoding the circRNA and the 5' end of the encoding circRNA Any linear RNA of the 3' junction sequence at the 3' end of the nucleic acid sequence is contacted with a single-stranded adapter nucleic acid comprising, from the 5' end to the 3' end: a first sequence complementary to the 3' junction sequence and A second sequence complementary to the 5' linker sequence, and wherein the 5' linker sequence and the 3' linker sequence hybridize to the single stranded adapter nucleic acid to provide a double stranded nucleic acid intermediate at the 3' end of the 5' linker sequence and the 5' end of the 3' linker sequence; (b) contacting the intermediate with RNA ligase under conditions that allow ligation of the 5' linker sequence to the 3' linker sequence to provide a circularized an RNA product; and (c) isolating the circularized RNA product, thereby providing a circular RNA.

[0326] In some embodiments, the methods described herein comprise circularizing linear RNA in vitro, comprising: (a) under conditions that allow ligation of the 5' linker sequence to the 3' linker sequence, the Any linear RNA of the 5' junction sequence at the 5' end of the nucleic acid sequence of the circRNA and the 3' junction sequence at the 3' end of the nucleic acid sequence encoding the circRNA is contacted with RNA ligase to provide a circularized RNA product; and (b) separating the circle cationized RNA products, thereby providing circular RNA.

[0327] In some embodiments, the method further comprises treating with RNase R to digest the linear RNA transcript. In some embodiments, the method further comprises isolating circular RNA (circRNA). In some embodiments, the step of isolating the circRNA comprises gel purifying the circRNA. In some embodiments, purified circRNA can be stored at -80°C.

[0328] In some embodiments, a DNA or RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., a linear RNA) to a 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) to Formation of new phosphodiester bonds. In an exemplary reaction, linear circular RNA was incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) at 37°C according to the manufacturer's protocol 1h. The ligation reaction can occur in the presence of linear nucleic acids capable of pairing with juxtaposed 5'- and 3'-region alkyl groups to facilitate the enzymatic ligation reaction. In some embodiments, the connection is a splint connection. For example, splint ligases, such as SPLINTR® Ligase, can be used for splint ligation. For splint ligation, single-stranded polynucleotides (splints), such as ssRNA, can be designed to hybridize to both ends of a linear polyribonucleotide so that the two ends can be juxtaposed when hybridized to a single-stranded splint. Therefore, splint ligase can catalyze the connection of the juxtaposed two ends of linear polyribonucleotides to generate circular polyribonucleotides.

[0329] In some embodiments, DNA or RNA ligases can be used for circular RNA synthesis. As a non-limiting example, the ligase can be a circular ligase or a circular ligase.

[0330] Purification of circRNA

[0331] In some embodiments, the methods provided herein for producing circRNA further comprise the step of purifying the circular RNA product. In non-limiting examples, the circRNA is purified by gel purification or by high performance liquid chromatography (HPLC). In some embodiments, agarose gel electrophoresis allows simple and efficient separation of circular splice products from linear precursor molecules, nicked loops, splice intermediates, and excised introns. In some embodiments, the method comprises purifying the circular RNA by chromatography, such as HPLC. In some embodiments, purified circular RNA can be stored at -80°C.

[0332] V. Pharmaceutical compositions, kits and articles of manufacture

[0333] The application further provides a pharmaceutical composition comprising any circRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by admixing a therapeutic agent described herein having the desired degree of purity, optionally with a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include: buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives agents, isotonic agents (such as sodium chloride), stabilizers, metal complexes (such as zinc-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0334] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical compositions are contained in multi-use vials. In some embodiments, the pharmaceutical composition is contained entirely in a container. In some embodiments, pharmaceutical compositions are cryopreserved.

[0335] This application also provides kits and articles of manufacture for use in any of the embodiments of the methods of treatment described herein. Kits and articles of manufacture may comprise any of the formulations and pharmaceutical compositions described herein.

[0336] In some embodiments, a kit is provided comprising any of the circRNAs described herein and instructions for treating or preventing a disease or condition (eg, coronavirus infection).

[0337] In some embodiments, a kit is provided comprising any of the circRNAs described herein and instructions for treating or preventing coronavirus infection.

[0338] In some embodiments, a kit is provided comprising any of the plasmids or linear RNAs described herein, and instructions for preparing any of the circRNAs. In some embodiments, a kit is provided comprising any of the plasmids, linear RNAs, or circRNAs described herein, and instructions for administering the circRNA.

[0339] Kits of the invention are presented in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. Kits can optionally provide additional elements such as buffers and explanatory information. Accordingly, the present application also provides articles of manufacture, including vials (eg, sealed vials), bottles, jars, flexible packaging, and the like.

[0340] Instructions pertaining to the use of the compositions generally include information regarding dosages, dosing regimens and routes of administration for the intended treatment. The container can be a unit dose, bulk package (eg, a multi-dose package), or a subunit dose. For example, a kit comprising a sufficient dose of a circRNA as disclosed herein may be provided to provide effective treatment of an individual or a number of individuals. In addition, kits containing sufficient doses of the circRNA may be provided to allow multiple administrations to an individual (e.g., in the case of a circRNA vaccine, an initial vaccine administration followed by a booster administration). The kit can also include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in a pharmacy (eg, hospital pharmacy and compounding pharmacy).

[0341] In some embodiments, the kit includes a delivery system. The delivery system can be a unit dose delivery system. The volume of solution or suspension delivered per dose can be: about 5 to about 2000 μl, about 10 to about 1000 μl, or about 50 to about 500 μl. The delivery system for these different dosage forms can be a unit dose or multi-dose packaged syringe, dropper bottle, plastic squeeze device, nebuliser, nebuliser or pharmaceutical aerosol. In some embodiments, a delivery system for any one of the circRNAs described herein is provided, including the circRNA and a device for delivering the circRNA.

[0342] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0343] VI. Exemplary Embodiments

[0344] In some aspects, the present application provides the following exemplary embodiments.

[0345] Embodiment 1: a circular RNA (circRNA), comprising a nucleic acid sequence encoding a therapeutic polypeptide, wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins and targeting proteins.

[0346] Embodiment 2: the circRNA as described in Embodiment 1, further comprising a Kozak sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide.

[0347] Embodiment 3: the circRNA as described in Embodiment 1 or 2, further comprising an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding a therapeutic polypeptide.

[0348] Embodiment 4: The circRNA of any one of embodiments 1-3, further comprising an internal ribosome entry site (IRES) sequence operably linked to the nucleic acid sequence encoding a therapeutic polypeptide. Embodiment 5: the circRNA as described in embodiment 4, wherein said IRES sequence is the IRES sequence selected from the group: the IRES sequence of CVB3 virus, EV71 virus, EMCV virus, PV virus and CSFV virus. Embodiment 6: the circRNA as described in embodiment 4 or 5, comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: IRES sequence, Kozak sequence and the nucleic acid sequence of encoding therapeutic polypeptide.

[0351] Embodiment 7: the circRNA as described in any one of Embodiments 4-6, further comprising a polyAC or polyA sequence positioned at the 5' end of the IRES sequence.

[0352] Embodiment 8: The circRNA as described in any one of Embodiments 1-3, further comprising an m6A modification motif sequence operably linked to a nucleic acid sequence encoding a therapeutic polypeptide. Embodiment 9: circRNA as described in embodiment 8, comprises such nucleic acid sequence, and it comprises from 5 ' end to 3 ' end: m6A modification motif sequence, Kozak sequence and the nucleic acid sequence of encoding therapeutic polypeptide.

[0354] Embodiment 10: The circRNA according to any one of embodiments 1-9, wherein said nucleic acid sequence also encodes a signal peptide (SP) fused to the N-terminus of said therapeutic polypeptide.

[0355] Embodiment 11: The circRNA of Embodiment 10, wherein the SP is the SP of human tissue plasminogen promoter (tPA) or human IgE immunoglobulin.

[0356] Embodiment 12: The circRNA as described in any one of Embodiments 1-11, further comprising: it can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding a therapeutic polypeptide The 3' exon sequence of the therapeutic polypeptide, and the 5' exon sequence recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the therapeutic polypeptide. Embodiment 13: circRNA as described in embodiment 12, wherein said 3 ' exon sequence comprises the nucleotide sequence of SEQ ID NO: 40, and said 5 ' exon sequence comprises SEQ ID NO: 41 nucleic acid sequence.

[0358] Embodiment 14: The circRNA of any one of Embodiments 1-13, wherein the therapeutic protein is used to treat or prevent infection.

[0359] Embodiment 15: The circRNA of Embodiment 14, wherein the infection is a viral infection.

[0360] Embodiment 16: The circRNA of Embodiment 15, wherein the virus is a coronavirus. Embodiment 17: circRNA as described in embodiment 16, wherein said coronavirus is selected from lower group: SARS-CoV, MERS-COV and SARS-CoV-2.

[0362] Embodiment 18: The circRNA of Embodiment 17, wherein the coronavirus is SARS-CoV-2.

[0363] Embodiment 19: The circRNA of any one of embodiments 1-18, wherein the therapeutic polypeptide is an antigenic polypeptide.

[0364] Embodiment 20: The circRNA of Embodiment 19, wherein the antigenic polypeptide comprises a coronavirus spike (S) protein or fragment thereof.

[0365] Embodiment 21: The circRNA of Embodiment 20, wherein the antigenic polypeptide comprises the receptor binding domain (RBD) of the S protein.

[0366] Embodiment 22: The circRNA of Embodiment 21, wherein the RBD comprises amino acid residues 319-542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1.

[0367] Embodiment 23: the circRNA of embodiment 22, wherein the RBD comprises the amino acid sequence of SEQ ID NO: 2.

[0368] Embodiment 24: The circRNA of any one of embodiments 21-23, wherein the antigenic polypeptide further comprises a multimerization domain.

[0369] Embodiment 25: The circRNA of Embodiment 24, wherein the multimerization domain is the C-terminal Foldon (Fd) domain of T4 fibrin that mediates T4 fibrin trimerization, or based on GCN -4 isoleucine zipper domain.

[0370] Embodiment 26: The circRNA of Embodiment 24, wherein the multimerization domain comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[0371] Embodiment 27: The circRNA of any one of embodiments 24-26, wherein the RBD is fused to the multimerization domain by a peptide linker.

[0372] Embodiment 28: The circRNA of Embodiment 27, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 5.

[0373] Embodiment 29: The circRNA according to any one of embodiments 20-28, wherein the antigenic polypeptide comprises the S2 region of the S protein.

[0374] Embodiment 30: the circRNA of embodiment 29, wherein the S2 region comprises amino acid residues 686-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1.

[0375] Embodiment 31: The circRNA of Embodiment 29, wherein the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein.

[0376] Embodiment 32: The circRNA of Embodiment 31, wherein the one or more mutations comprise K986P and V987P.

[0377] Embodiment 33: The circRNA according to any one of embodiments 29-32, wherein the S2 region comprises the amino acid sequence of SEQ ID NO: 6 or 7.

[0378] Embodiment 34: the circRNA according to any one of embodiments 20-23 and 29-33, wherein the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, Wherein the numbering is based on SEQ ID NO: 1.

[0379] Embodiment 35: The circRNA of Embodiment 34, wherein the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage.

[0380] Embodiment 36: The circRNA of Embodiment 35, wherein the one or more mutations comprise a deletion of amino acid residues 681-684.

[0381] Embodiment 37: The circRNA of Embodiment 20, wherein the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 and 62-63.

[0382] Embodiment 38: the circRNA as described in embodiment 20, wherein the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15 and 64.

[0383] Embodiment 39: The circRNA of any one of Embodiments 1-18, wherein the Therapeutic protein is a receptor protein.

[0384] Embodiment 40: The circRNA of Embodiment 39, wherein the therapeutic protein is a soluble receptor comprising the extracellular domain of a naturally occurring receptor.

[0385] Embodiment 41: The circRNA of Embodiment 39 or 40, wherein the receptor is the ACE2 receptor.

[0386] Embodiment 42: The circRNA of Embodiment 41, wherein the receptor is a high affinity mutant ACE2 receptor.

[0387] Embodiment 43: The circRNA of any one of Embodiments 1-18, wherein the therapeutic protein is a targeting protein.

[0388] Embodiment 44: The circRNA of Embodiment 43, wherein the targeting protein is an antibody.

[0389] Embodiment 45: The circRNA of Embodiment 44, wherein the antibody is a neutralizing antibody.

[0390] Embodiment 46: The circRNA of Embodiment 44, wherein the targeting protein is a therapeutic antibody.

[0391] Embodiment 47: The circRNA of any one of Embodiments 1-13, wherein the therapeutic protein is a functional protein.

[0392] Embodiment 48: The circRNA of Embodiment 47, wherein the functional protein is a tumor suppressor.

[0393] Embodiment 49: The circRNA of Embodiment 48, wherein the tumor suppressor is selected from the group consisting of: p53 and PTEN.

[0394] Embodiment 50: The circRNA of Embodiment 47, wherein the functional protein is an enzyme.

[0395] Embodiment 51: The circRNA of Embodiment 50, wherein the enzyme is selected from the group consisting of OTC, FAH and IDUA.

[0396] Embodiment 52: the circRNA as described in embodiment 47, wherein the functional protein is selected from the group consisting of DMD, COL3A1, BMPR2, AHI1, FANCC, MYBPC3 and IL2RG.

[0397] Embodiment 53: A composition comprising a plurality of circRNAs according to any one of embodiments 20-38, wherein the antigenic polypeptides corresponding to the plurality of circRNAs are different from each other.

[0398] Embodiment 54: A composition comprising a plurality of circRNAs according to any one of embodiments 39-42, wherein the receptor proteins corresponding to the plurality of circRNAs are different from each other.

[0399] Embodiment 55: A composition comprising a plurality of circRNAs according to any one of embodiments 43-46, wherein the targeting proteins corresponding to the plurality of circRNAs are different from each other.

[0400] Embodiment 56: The composition of any one of embodiments 53-55, wherein the plurality of circRNAs target a plurality of coronavirus strains.

[0401] Embodiment 57: A circRNA vaccine comprising the circRNA described in any one of Embodiments 20-38 or the composition described in Embodiment 53 or 56.

[0402] Embodiment 58: A pharmaceutical composition comprising the circRNA described in any one of Embodiments 1-52 and a pharmaceutically acceptable carrier. Embodiment 59: the circRNA vaccine as described in embodiment 57 or the pharmaceutical composition described in embodiment 58, also comprise transfection reagent. Embodiment 60: the circRNA vaccine or pharmaceutical composition as described in embodiment 59, wherein said transfection reagent is polyethyleneimine (PEI) or lipid nanoparticle (LNP), optionally wherein LNP comprises : MC3-lipids, DSPC, cholesterol and PEG2000-DMG.

[0405] Embodiment 61: The circRNA vaccine of Embodiment 57 or the pharmaceutical composition of Embodiment 58, wherein the circRNA is not formulated with a transfection reagent.

[0406] Embodiment 62: A method for treating or preventing individual infection, comprising administering to the individual an effective amount of the circRNA described in any one of the embodiments 20-46, any one of the embodiments 53-56 Composition, or the circRNA vaccine of any one of embodiments 57 and 59-61.

[0407] Embodiment 63: The method of embodiment 62, wherein the infection is a coronavirus infection.

[0408] Embodiment 64: The method of embodiment 63, wherein the infection is a SARS-CoV-2 infection, optionally a SARS-CoV-2 infection caused by a SARS-CoV-2 variant (e.g., B .1.351 variant).

[0409] Embodiment 65: A method of treating or preventing an individual's disease or condition, comprising administering to the individual an effective amount of the circRNA described in any one of embodiments 1-52, or any one of embodiments 58-61 The pharmaceutical composition described in the item.

[0410] Embodiment 66: The method of Embodiment 65, wherein the disease or condition is a disease or condition associated with insufficient protein levels and / or activity corresponding to the Therapeutic protein.

[0411] Embodiment 67: The method of Embodiment 66, wherein said disease or condition is an inherited genetic disease associated with one or more mutations in a protein corresponding to said Therapeutic protein.

[0412] Embodiment 68: The method of any one of embodiments 65-67, wherein: (i) the therapeutic polypeptide is TP53 or PTEN, and the disease or condition is cancer; (ii) treating The therapeutic polypeptide is OTC, and the disease is ornithine transcarbamylase deficiency; (iii) the therapeutic polypeptide is FAH, and the disease is tyrosinemia; (iv) the therapeutic polypeptide is DMD, and the disease is Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, or familial dilated cardiomyopathy; (v) the therapeutic polypeptide is IDUA, and the disease or condition is mucopolysaccharide storage type I (MPSI); (vi) the therapeutic polypeptide is COL3A1, and the disease or condition is Ehlers-Danlos syndrome; (vii) the therapeutic polypeptide is AHI1, and the disease or condition is Joubert syndrome; (viii) the therapeutic polypeptide is BMPR2, and the disease or condition is pulmonary hypertension or pulmonary veno-occlusive disease; (ix) the therapeutic polypeptide is FANCC, and the disease or condition is Fanconi anemia; (x) the therapeutic polypeptide is MYBPC3, and the disease or condition is primary familial hypertrophic cardiomyopathy; or (xi) the therapeutic polypeptide is IL2RG, and the The disease or condition is X-linked severe combined immunodeficiency.

[0413] Embodiment 69: The method according to any one of embodiments 62-68, wherein the circRNA undergoes rolling circle translation by ribosomes in the individual.

[0414] Embodiment 70: The method of Embodiment 66, wherein the disease or condition is an inherited genetic disease associated with one or more mutations in a protein corresponding to a Therapeutic protein.

[0415] Embodiment 71: A linear RNA capable of forming the circRNA of any one of Embodiments 1-52. Embodiment 72: The linear RNA of embodiment 71, wherein said linear RNA comprises a group I intron comprising a 5' catalytic group I intron fragment and a 3' A catalytic group I intron fragment, wherein the linear RNA can be circularized by autocatalysis of the group I intron. Embodiment 73: The linear RNA of Embodiment 72, comprising a 3' catalytic group I flanking the 5' end of a 3' exon sequence recognizable by a fragment of the 3' catalytic group I intron The intron segment, and the 5' catalytic group I intron segment flanking the 3' end of the 5' exon sequence recognizable by the 5' catalytic group I intron segment. Embodiment 74: The linear RNA of Embodiment 73, comprising 5' homologous sequences flanking the 5' end of the 3' catalytic group I intron fragment, and 5' catalytic group I intronic 3' homologous sequences flanking the 3' end of the subfragment. Embodiment 75: the linear RNA as described in embodiment 74, wherein 5 ' homologous sequence comprises the nucleotide sequence of SEQ ID NO: 41, and 3 ' homologous sequence comprises the nucleotide sequence of SEQ ID NO: 42.

[0420] Embodiment 76: The linear RNA of embodiment 71, wherein said linear RNA can be circularized by a ligase. Embodiment 77: The linear RNA of embodiment 76, wherein the ligase is selected from the group consisting of T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1) and T4 RNA ligase 2 (T4 Rnl2).

[0422] Embodiment 78: the linear RNA of embodiment 76 or 77, comprising a 5' junction sequence at the 5' end of the nucleic acid sequence encoding circRNA, and a 3' junction sequence at the 3' end of the nucleic acid sequence encoding circRNA, wherein The 5' linker sequence and the 3' linker sequence can be linked to each other by RNA ligase.

[0423] Embodiment 79: A nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA of any one of embodiments 70-78.

[0424] Embodiment 80: The nucleic acid construct of claim 79 comprising a T7 promoter operably linked to the nucleic acid sequence encoding the linear RNA.

[0425] Embodiment 81: A method of producing a circRNA comprising: (a) subjecting the linear RNA of any one of embodiments 71-75 to an initiating 5' catalytic group I intron fragment and a 3' catalytic and (b) isolating the circularized RNA product, thereby providing circRNA.

[0426] Embodiment 82: A method of producing a circRNA comprising: (a) contacting the linear RNA of any one of embodiments 76-78 with a single-stranded adapter nucleic acid derived from The 5' end to the 3' end comprises: a first sequence complementary to the 3' junction sequence and a second sequence complementary to the 5' junction sequence, and wherein the 5' junction sequence and the 3' junction sequence are compatible with the single-stranded adapter nucleic acid hybridize to provide a double-stranded nucleic acid intermediate comprising a single-strand break between the 3' end of the 5' junction sequence and the 5' end of the 3' junction sequence; contacting the intermediate with an RNA ligase under conditions of linker ligation to provide a circularized RNA product; and (c) isolating the circularized RNA product to provide a circRNA.

[0427] Embodiment 83: A method of producing a circRNA, comprising: (a) subjecting any one of embodiments 76-78 to a circularized RNA product under conditions that allow ligation of the 5' linker sequence to the 3' linker sequence The linear RNA described in item is contacted with RNA ligase; And (b) isolates the RNA product of circularization, thereby provides circRNA.

[0428] Embodiment 84: The method of any one of embodiments 80-82, further comprising obtaining the linear RNA by in vitro transcription of a nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA.

[0429] Embodiment 85: The method according to any one of embodiments 80-84, further comprising purifying the circularized RNA product.

[0430] Example

[0431] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therefrom will be suggested to those skilled in the art, which are to be included within the spirit and scope of the application and the present application. application within the scope of the accompanying embodiments.

[0432] Example 1: In vitro circRNA production by ligation

[0433] This example demonstrates the in vitro production of circular RNA (circRNA) by ligation.

[0434] Linear RNAs were designed such that they could be circularized to generate circRNAs comprising from 5' to 3': IRES-Kozak-SP-spike sequence, as shown in Figure 2A. The linear RNA is designed from 5' to 3' with: IRES sequence (SEQ ID NO: 53), Kozak sequence (SEQ ID NO: 36), signal peptide coding sequence (SEQ ID NO: 16 or SEQ ID NO: 17), and Spike protein coding sequence (SEQ ID NO: 15) with K986P / V987P and Δ681-684 modifications, followed by a TAA stop codon.

[0435] Standard laboratory methods and materials can be used to prepare linear RNAs that can be circularized to produce the circular RNAs (circRNAs) disclosed herein. A cDNA sequence encoding a linear RNA can be synthesized by de novo DNA synthesis. Synthetic nucleic acids can be ordered from synthetic nucleotide services such as GBLOCKS® (Integrated DNA Technologies). Nucleic acid sequences encoding linear RNA sequences can be cloned into a plasmid vector containing a T7 promoter with a multiple cloning site flanked by restriction sites such as the Xba1 restriction site. The resulting plasmid can be transformed into chemically competent Escherichia coli (E. coli.).

[0436] For this example, NEB DH5-alpha competent E. coli cells were used. 100 ng of plasmid was used for transformation according to NEB instructions. The protocol is as follows: 1. Thaw a tube of NEB 5-α competent E. coli cells on ice for 10 minutes. 2. Add 1-5μL containing 1pg-100ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix the cells and DNA. Do not vortex. 3. Place the mixture on ice for 30 min. Do not mix. 4. Heat shock at 42°C for exactly 30 seconds. Do not mix. 5. Place on ice for 5 minutes. Do not mix. 6. Pipette 950 μL of room temperature SOC into the mixture. 7. Place at 37°C for 60 minutes. Shake vigorously (250rpm) or spin. 8. Heat the option plate to 37°C. 9. Mix the cells well by flicking and inverting the tube.

[0437] Spread 50-100 μL of each dilution on a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36h, or at 25°C for 48h.

[0438] A single colony was then used to inoculate 5 ml of LB growth medium using appropriate antibiotics and allowed to grow (250 RPM, 37° C.) for 5 h. This was then used to inoculate 200 ml of medium and allowed to grow overnight under the same conditions. For isolation of plasmid (up to 850 mg), maxiprep was performed using the Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, CA) following the manufacturer's instructions.

[0439] To generate a linearized plasmid DNA template for in vitro transcription (IVT), a plasmid (an example of which is shown in Figure 2) is first linearized using a restriction enzyme such as Xbal. A typical Xbal restriction digest would include the following: plasmid 1.0mg 10x buffer 1.0mL; Xbal 1.5mL; dH20 up to 10mL; incubate at 37°C for 1h. If performed at lab scale (<5), reactions were cleaned up using the Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, CA) following the manufacturer's instructions. Products with greater load capacity, such as Invitrogen's standard PURELINK™ PCR kit (Carlsbad, CA), may require larger purifications. After cleanup, the linearized vector was quantified using NanoDrop and analyzed using agarose gel electrophoresis to confirm linearization.

[0440] Unmodified linear RNA was synthesized from the linearized plasmid by in vitro transcription using T7 RNA polymerase. Transcribed RNA was purified with the RNA Purification System (QIAGEN), treated with alkaline phosphatase (ThermoFisher Scientific, EF0652) according to the manufacturer's instructions, and purified again with the RNA Purification System.

[0441] Splint-ligated circRNAs are generated by treating transcribed linear RNA and DNA splints with T4 DNA ligase (New England Bio, Inc., M0202M), and circRNAs are isolated after enrichment with RNase R treatment . RNA quality was assessed by agarose gel or automated electrophoresis (Agilent).

[0442] Example 2: Group I ribozyme self-catalyzed in vitro circRNA production

[0443] This example demonstrates the in vitro production of circular RNA (circRNA) by group I ribozyme autocatalysis.

[0444] Linear RNAs are designed so that they can be circularized to produce circRNAs that contain from 5' to 3': 5' homology arms - 3' catalytic group I intron fragments - can be synthesized by 3' catalytic group I 3' exon sequence recognized by intron fragment (i.e. exon 2) - m6A modification motif - Kozak-SP-spike-2A peptide - 5' recognized by 5' catalytic group I intron fragment Exon sequence (ie exon 1) - 5' catalytic group I intron fragment - 3' homology arm, as shown in Figure 1C. Linear RNA is designed from 5' to 3' with: 5' homology arm (SEQ ID NO: 41), 3' catalytic group I intron sequence (SEQ ID NO: 46), 3' catalytic group I intron sequence The 3' exon sequence (SEQ ID NO: 39), m6A modification motif sequence (SEQ ID NO: 38), Kozak sequence (SEQ ID NO: 37), signal peptide coding sequence (SEQ ID NO : 16 or SEQ ID NO: 17), spike protein coding sequence (SEQ ID NO: 15) with K986P / V987P and Δ681-684 modification, 2A peptide coding sequence (SEQ ID NO: 44 or SEQ ID NO: 45) , the 5' exon sequence recognized by the 5' catalytic group I intron fragment (SEQ ID NO: 40), the 5' catalytic group I intron fragment (SEQ ID NO: 47) and the 3' homologous Arm (SEQ ID NO: 43).

[0445] Linear RNAs that can be circularized to generate the circular RNAs (circRNAs) disclosed herein can be prepared by the same method as described in Example 1 above.

[0446] Circular RNA is generated by ribozyme autocatalysis of group I introns. During the splicing process, the 3' hydroxyl of guanosine nucleotides participates in the transesterification reaction at the 5' splicing site. Half of the 5' intron is excised, and the free hydroxyl group at the end of the intermediate undergoes a second transesterification at the 3' splice site, resulting in cyclization of the middle region and excision of the 3' intron.

[0447] Unmodified linear mRNA or circRNA precursors were synthesized from linearized plasmid DNA templates by in vitro transcription using the T7 High Yield RNA Synthesis Kit (New England Biolabs). After in vitro transcription, the reaction was treated with DNase I (New England Biolabs) for 20 min. After DNase treatment, unmodified linear mRNA was column purified using the MEGAclear Transcription Clean-up kit (Ambion). The RNA was then heated to 70°C for 5 min and immediately placed on ice for 3 min before using mRNA cap-2'-O-methyltransferase (NEB) and vaccinia capping enzyme (NEB) according to the manufacturer's instructions Do capping. Poly-A tails were added to capped linear transcripts using E. coli PolyA polymerase (NEB) according to the manufacturer's instructions and fully processed mRNA was column purified. For circRNA, after DNase treatment, additional GTP was added to a final concentration of 2 mM, and the reaction was heated at 55°C for 15 min. The RNA is then column purified. In some cases, purified RNA was re-circularized: the RNA was heated to 70°C for 5 min and immediately placed on ice for 3 min before adding GTP to a final concentration of 2 mM and magnesium-containing buffer (50 mM Tris - HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5; New England Biolabs). The RNA was then heated to 55°C for 8 min before column purification. To enrich circRNAs, 20 µg of RNA was diluted in water (86 µL final volume), then heated at 65°C for 3 min, then cooled on ice for 3 min. Add 20 U RNase R and 10 µL 10× RNase R buffer (Epicenter), and incubate the reaction at 37°C for 15 min; add an additional 10 U RNase R halfway through the reaction. The RNase R digested RNA was column purified. RNA was separated on 2% E-gel EX agarose gels (Invitrogen) precast on E-gel iBase (Invitrogen) using the E-gel EX 1-2% protocol; ssRNA ladder (NEB) was used as standard.

[0448] For gel extraction, the band corresponding to the circRNA was excised from the gel and then extracted using the Zymoclean Gel RNA extraction kit (Zymogen). For HPLC, 30 µg of RNA was heated at 65°C for 3 min and then placed on ice for 3 min. RNA was run on an Agilent 1100 Series HPLC (Agilent) through a 4.6 x 300 mm size exclusion column (Sepax Technologies; part number: 215980P-4630) with a particle size of 5 μm and a pore size of 200 Å. RNA was run at a flow rate of 0.3 mL / min in RNase-free TE ss (10 mM Tris, 1 mM EDTA, pH: 6). RNA was detected by UV absorbance at 260 nm. The resulting RNA fractions were precipitated with 5M ammonium acetate, resuspended in water, and in some cases treated with RNase R as described above.

[0449] The resulting circRNA is shown in Figure 1C. Embodiment 3: Gel electrophoresis and RNase R resistance of circRNA

[0451] This example demonstrates the purity and endonuclease resistance of the purified circRNA.

[0452] First, using the circRNA backbone as described in Examples 1 and 2 above, a circRNA construct was designed comprising the nucleotide sequence encoding the RBD of the SARS-CoV-2 Spike protein.

[0453] Briefly, linear RNAs are designed to be circularized to generate circRNAs, which contain from 5' to 3': 5' homology arms - 3' catalytic group I intron segments - can be generated by 3 'The 3' exon sequence recognized by the catalytic group I intronic fragment (i.e. exon 2) - IRES-Kozak-SP-RBD-TAA stop codon - can be recognized by the 5' catalytic group I intronic fragment The 5' exon sequence (ie, exon 1) - the 5' catalytic group I intron fragment - the 3' homology arm. Linear RNA is designed from 5' to 3' with: 5' homology arm (SEQ ID NO: 41), 3' catalytic group I intron sequence (SEQ ID NO: 46), which can be generated by 3' catalytic group I intron sequence 3' exon sequence (SEQ ID NO: 39), IRES sequence (SEQ ID NO: 53), Kozak sequence (SEQ ID NO: 37), signal peptide coding sequence (SEQ ID NO: 16 or SEQ ID NO: 17), the spike protein RBD sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or the spike protein sequence encoding the amino acid sequence shown in SEQ ID NO: 63, a stop codon, which can be catalyzed by the 5' I 5' exon sequence (SEQ ID NO: 40), 5' catalytic group I intron fragment (SEQ ID NO: 47) and 3' homology arm (SEQ ID NO: 43) recognized by the group intron fragment ). The circularized RNAs generated from this linear RNA are called circRNARBD and circRNA spike, respectively. As a control, the 3' intron sequence was mutated to a random sequence to prevent RNA circularization, and the resulting construct was called LinRNARBD.

[0454] circRNAs were generated and purified as described in Example 2. Purified circRNARBD and pre-linear RNA (LinRNARBD, in which the 3' intronic sequence was mutated to a random sequence) were resolved in agarose gel electrophoresis. Gel electrophoresis results showed that circRNARBD ran faster than LinRNA-RBD (Figure 3A), indicating that the RNA was circularized. circRNARBD is a circRNA encoding the RBD domain of the spike protein of SARS-CoV-2. The RBD domain is amino acid residues 319-542 of the full-length spike protein of SARS-CoV-2, as shown in SEQ ID NO: 2. As shown in Figure 3E, circularization of the circRNARBD was verified by reverse transcription using primers and RT-PCR analysis using specific primers (Figure 3C) (Figure 1b).

[0455] Next, the endonuclease resistance of the purified circRNA constructs was tested. Since circRNAs do not have 5' or 3' ends, circRNAs are resistant to endonucleases. The endonuclease RNase R was used to digest circRNARBD or LinRNARBD for different times, and the reaction products were analyzed by agarose gel electrophoresis. Gel electrophoresis results showed that circRNARBD was more resistant to RNase R than LinRNARBD (Fig. 3B). Example 4: Expression of SARS-CoV-2 RBD antigen in human HEK293T cells and mouse NIH3T3 cells by circRNA transfection

[0457] This example demonstrates the ability of circRNAs to express proteins (eg, the SARS-CoV-2 RBD of the S protein) in eukaryotic cells. Furthermore, this example demonstrates the surprising stability of circRNAs at room temperature for two weeks. After circRNA was incubated at room temperature for two weeks, the protein could still be expressed and secreted in cells transfected with circRNA.

[0458] After purifying circRNA (RNase R treatment and HPLC), circRNARBD was transfected into human HEK293T cells and mouse NIH3T3 cells with Lipofectamine MessengerMAX transfection reagent (Thermo LMRNA003). circRNA-EGFP and a precursor linear RNA named LinRNA-RBD were used as controls. Quantitative ELISA measurements showed that RBD protein reached ~143 ng / mL in the supernatant, which was 50-fold higher than that of the linear RNA RBD group (Fig. 3D).

[0459] After 48 h, culture supernatants of transfected cells were collected for Western blot analysis. Using the SARS-CoV-2 spike RBD antibody (ABclonal, A20135) for detection, Western blot results showed that circRNA RBD could highly express the SARS-CoV-2 RBD antigen and secrete it into the cell supernatant. Western blot results are shown in Figure 4A and Figure 4B.

[0460] circRNA is stable at room temperature of about 25°C. The purified circRNARBD was kept at room temperature about 25°C for 3, 7 or 14 days, and then transfected into human HEK293T cells. Western blot results showed that circRNARBD could efficiently express SARS-CoV-2 RBD antigen and be secreted into cell supernatant even though circRNARBD had been kept at room temperature for 14 days. The results are shown in Figure 4C.

[0461] Furthermore, in order to test the thermal stability of the circRNA-LNP preparation, the encapsulated circRNARBD-LNP was stored at 4°C or room temperature (~25°C) for 1, 3, 7, 14, 24 and 31 days before transfection . Sequentially collected circRNARBD-LNPs were transfected into cells, and the abundance of RBD antigen production was quantified by ELISA. After 7 days of storage at 4°C or at room temperature (~25°C), no reduction of RBD antigen was detected from circRNARBD-LNP (Fig. 4D). The expression levels of circRNARBD-LNP decreased to ~95% and ~75% after storage at 4°C and room temperature for 14 days, respectively (Fig. 4D). Deterioration effects do occur with extended shelf life and high temperatures.

[0462] The stability of circRNAs at room temperature has advantages for applications including vaccines and gene therapy, including storage and transportation for therapeutic circRNAs (eg, circRNA vaccines).

[0463] Example 5: The SARS-CoV-2 RBD antigen is functional and can block the infection of the SARS-CoV-2 pseudovirus.

[0464] This example demonstrates that the secreted SARS-CoV-2 RBD antigen expressed by exemplary circRNA can directly interfere with the infection of ACE2-expressing cells by SARS-CoV-2 pseudovirus.

[0465] In order to evaluate whether the secreted SARS-CoV-2 RBD antigen produced by circRNA is functional, the cell supernatant of HEK293T cells transfected with circRNA RBD or control circRNA was mixed with the SARS-CoV-2 based lentivirus encoding EGFP The pseudoviruses were incubated at 37°C for 2 h, and then the resulting SARS-CoV-2 pseudovirus / supernatant mixture was added to the medium of ACE2-overexpressing cells named HEK293-ACE2. After 48h, the cells were collected for FACS analysis, because the SARS-CoV-2 pseudovirus expressed the EGFP fluorescent marker. Cellular expression of EGFP indicated that the cells were infected by the SARS-CoV-2 pseudovirus. A commercially available neutralizing antibody to SARS-CoV-2 (ABclonal, A19215) was used as a positive control for neutralizing the SARS-CoV-2 S protein.

[0466] This pseudovirus competition experiment proves that the secreted SARS-CoV-2 RBD antigen in the supernatant produced by cells transfected with circRNA RBD can effectively block the infection of SARS-CoV-2 pseudovirus, indicating that circRNA produces The SARS-CoV-2 RBD antigen is functional at the cellular level. The secreted RBD antigen can interfere with the binding between the RBDs of the SARS-CoV-2 pseudovirus, thereby blocking the infection of cells. The results are shown in Figure 5A and Figure 5B.

[0467] Example 6: circRNA vaccines can induce SARS-CoV-2 specific immune responses and generate high levels of neutralizing antibodies

[0468] As shown in Example 5 above, the RBD antigen expressed by exemplary circRNA can directly interfere with the binding of the S protein of SARS-CoV-2 to the ACE2 receptor, thereby preventing or reducing the impact of SARS-CoV-2 pseudoviruses on cells infection. This example shows that the antigenic polypeptide expressed by circRNA (such as the RBD of coronavirus S protein) administered in vivo stimulates a specific immune response and generates a high level of neutralizing antibodies. Based on these results, the circRNAs encoding antigenic polypeptides described herein can be used as effective vaccines against viruses such as coronaviruses (eg SARS-CoV-2).

[0469] Purified circRNARBD (circRNA backbone as shown in Figure 1B, including the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, as "spike" in Figure 1B) and circRNA spike (such as The circRNA backbone shown in Figure 1B, including the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 62, as the "spike" in Figure 1B) was used to immunize BALB / c mice, respectively. The first immunization was given by intramuscular injection on day 0, and the second dose was given on day 14 to boost the immune response (Fig. 6A). On day 28, serum from the immunized mice was collected for the following assays (Fig. 6A).

[0470] First, the RBD-specific IgG titer was measured by ELISA. The ELISA results showed that the IgG titer of the circRNARBD (10 μg) group was about 32000, the IgG titer of the circRNARBD (50 μg) group was about 64000, and the placebo group had almost no RBD Specific IgG signal (Fig. 6B). Meanwhile, the neutralizing activity of immunized mouse serum was measured by an in vitro alternative neutralization assay, and the results showed that the neutralizing activity of the circRNARBD (10 μg) group was about 70%, and that of the circRNARBD (50 μg) group exceeded 95% (Fig. 6C) . Finally, neutralization activity at the cellular level was assessed using lentivirus-based pseudoviruses of SARS-CoV-2 coated with the SARS-CoV-2 Spike protein. Sera from immunized mice were incubated with SARS-CoV-2 pseudoviruses, and the incubation system was added to cultures of ACE2-overexpressing HEK293T cells. After 48h, the reporter gene-luciferase activity of the pseudovirus was measured. The results of luciferase assay showed that both circRNA RBD and circRNA spike could induce SARS-CoV-2 spike-specific neutralizing antibodies to block pseudovirus infection (Figure 6D and Figure 6E).

[0471] The above results demonstrate that circRNA vaccines can induce SARS-CoV-2-specific immune responses and generate high levels of SARS-CoV-2 spike-specific neutralizing antibodies.

[0472] Example 7: Measurement of mouse spleen weight after two doses of immunization

[0473] This example demonstrates the effect on mouse spleen weight after two doses of immunization with an exemplary circRNA (circRNARBD) encoding an antigenic polypeptide.

[0474] The circRNA administration scheme is shown in Figure 6A. Four weeks after the second dose of circRNA vaccine or placebo, mice were sacrificed and spleens of immunized mice were isolated (Fig. 7A). The body weight of each mouse was then measured, and the spleen weight from circRNARBD (10 μg) or circRNARBD (50 μg) was significantly higher than that from the placebo group (Fig. 7B).

[0475] Example 8: Expression of SARS-CoV-2 neutralizing antibodies by circRNA

[0476] This example demonstrates the use of exemplary circRNAs to express secreted virus neutralizing antibodies. Neutralizing antibodies expressed and secreted from cells transfected with the circRNA described herein can effectively block the infection of SARS-CoV-2 pseudoviruses.

[0477] circRNA can also be used to express SARS-CoV-2 neutralizing antibodies. Similar to the above-mentioned RBD antigen, the SARS-CoV-2 neutralizing antibody coding sequence was also cyclized by the above-mentioned cyclization method (Fig. 8A).

[0478] The linear RNA is designed to be circularized into a circRNA, and the linear RNA includes from 5' to 3': 5' homology arm-3' catalytic group I intron fragment-can be contained by the 3' catalytic group I 3' exon sequence recognized by subfragment (i.e. exon 2) - IRES-Kozak-SP-RBD-TAA stop codon - 5' exon sequence recognized by 5' catalytic group I intron fragment (ie Exon 1) - 5' Catalytic Group I Intron Fragment - 3' Homology Arm. Linear RNA is designed from 5' to 3' with: 5' homology arm (SEQ ID NO: 41), 3' catalytic group I intron sequence (SEQ ID NO: 46), 3' catalytic group I intron sequence 3' exon sequence (SEQ ID NO: 39), IRES sequence (SEQ ID NO: 53), Kozak sequence (SEQ ID NO: 37), signal peptide coding sequence (SEQ ID NO: 16 or SEQ ID NO: 17), encoding nAb (nAb-1 (amino acid sequence shown in SEQ ID NO: 27), nAb-2 (amino acid sequence shown in SEQ ID NO: 28), or nAb-5 (amino acid sequence shown in SEQ ID NO: 30 The amino acid sequence shown)) the nucleotide sequence, the stop codon, the 5' exon sequence (SEQ ID NO: 40) that can be recognized by the 5' catalytic group I intron fragment, the 5' catalytic group I Intron fragment (SEQ ID NO: 47) and 3' homology arm (SEQ ID NO: 43). The circular RNAs generated from these linear RNAs are called circRNAnAb-1, circRNAnAb-2 and circRNAnAB-5, respectively. As a control, the 3′ intron sequence of the linear construct designed to generate circRNAnAB-5 was mutated to a random sequence to prevent RNA circularization, and the resulting construct was named LinRNAnAB-5.

[0479] Circular RNA comprising a nucleotide sequence encoding nAb-1, nAb-2, nAb-3, nAb-4, nAb-5, nAb-6, nAb-7H or nAb-7L is generated. The amino acid sequences of the neutralizing antibodies are shown in SEQ ID NO: 26-33, respectively. Alternatively, an antibody that binds to ACE2 and blocks S protein binding, such as the amino acid sequence shown in SEQ ID NO: 34 or 35, can be used.

[0480] The exemplary circRNAs (circRNAnAb-1, circRNAnAb-2 and circRNAnAB-5) encoding nAb were transfected into HEK293T cells, and the supernatant was collected after 48h, and used for SARS-CoV-2 pseudovirus neutralization assay . A circRNA encoding luciferase (circRNALuc) and a linear precursor RNA LinRNAnAB-5 were used as negative controls, and a commercially available SARS-CoV-2 neutralizing antibody (ABclonal, A19215) was used as a positive control.

[0481] The results of the pseudovirus neutralization assay showed that, compared with the negative control, circRNAnAb-1, circRNAnAb-2 and circRNAnAB-5 could neutralize the infection of SARS-CoV-2 pseudovirus (Figure 8B). These results suggest that circRNAs can be used to express neutralizing antibodies for therapeutic purposes, such as treating coronavirus (eg, SARS-CoV-2) infections. The pseudovirus neutralization assay showed that the supernatant of HEK293T cells transfected with circRNAnAB or circRNAhACE2 bait could effectively inhibit the pseudovirus infection based on the S protein of wild SARS-CoV-2 (Figure 8C).

[0482] Next, we tested neutralizing antibodies against recently emerged SARS-CoV-2 variants, including B.1.1.7 / 501Y.V1 and B.1.351 / 501Y.V2, by pseudovirus measurements. The supernatant of circRNAnAB1-Tri and circRNAnAB3-Tri transfected cells effectively blocked B1.1.7 / 501Y.V1 and D614G pseudovirus infection (Figure 8D). However, both Nanobodies showed significantly reduced neutralizing activity against the B.1.351 / 501Y.V2 variant (Fig. 8D). The hACE2 decoy showed no inhibitory activity against the B1.1.7 / 501Y.V1 and B.1.351 / 501Y.V2 variants (Fig. 8D).

[0483] In this example, the circRNA-encoded SARS-CoV-2 nanobody exhibited strong neutralizing ability against SARS-CoV-2 natural strain D614G and B.1.1.7 / 501Y.V1 strain in vitro , but they were completely escaped by the B.1.351 / 501Y.V2 variant (Fig. 8D). In addition to viral receptors, this circRNA expression platform may also become a therapeutic drug, encoding therapeutic antibodies in vivo, such as anti-PD1 / PD-L1 antibodies. In contrast to antibody protein drugs, circRNAs can target intracellular targets, such as TP5383 and KRAS84, because they encode therapeutic antibodies in the cytoplasm, bypassing the cell membrane barrier.

[0484] Embodiment 9: circRNA encoding IDUA can restore the catalytic activity of α-1-iduronidase (IDUA) in the primary cells of the Heller syndrome mouse model.

[0485] The above examples describe exemplary circRNA backbones, generation and purification of circRNAs, use of circRNAs to produce antigenic polypeptides that can effectively generate immune responses in vivo for use as vaccines, and circRNA expression of neutralizing antibodies to treat infections (eg, SARS-CoV-2 infection). However, the circRNAs described herein can also be used to treat other diseases that may benefit from the expression of therapeutic polypeptides, such as genetic diseases associated with protein or functional protein deficiency. The results provided in this example demonstrate that circRNAs can be used to express functional proteins such as enzymes (eg IDUA). Therefore, the circRNAs provided herein can be used to generate functional therapeutic polypeptides for gene therapy applications.

[0486] Instead of the SARS-CoV-2 RBD / spike antigen, functional wild-type disease-associated proteins can also be expressed and functioned by the circRNA and methods described herein. In one example, the mouse α-l-iduronidase (IDUA) coding sequence was inserted into the circRNA backbone to generate circRNA IDUA (Fig. 9A).

[0487] Briefly, linear RNAs are designed to be circularized to generate circRNAs, and from 5' to 3', linear RNAs contain: 5' homology arm - 3' catalytic group I intron segment - accessible by 3' 3' exon sequence recognized by catalytic group I intron fragment (i.e. exon 2) - IRES-Kozak-SP-RBD-TAA stop codon - recognized by 5' catalytic group I intron fragment 5' exon sequence (ie exon 1) - 5' catalytic group I intron fragment - 3' homology arm. Linear RNA is designed from 5' to 3' with: 5' homology arm (SEQ ID NO: 41), 3' catalytic group I intron sequence (SEQ ID NO: 46), which can be generated by 3' catalytic group I intron sequence 3' exon sequence (SEQ ID NO: 39), IRES sequence (SEQ ID NO: 53), Kozak sequence (SEQ ID NO: 37), signal peptide coding sequence (SEQ ID NO: 16 or SEQ ID NO: 17), nucleotide sequence encoding IDUA (amino acid sequence shown in SEQ ID NO: 18), stop codon, 5' exon sequence that can be recognized by 5' catalytic group I intron fragment (SEQ ID NO: 40), 5' catalytic group I intron fragment (SEQ ID NO: 47) and 3' homology arm (SEQ ID NO: 43). The circular RNA generated from this linear RNA is called circRNAIDUA. As a control, the 3' intron sequence was mutated to a random sequence to prevent RNA circularization, and the resulting construct was called LinRNAIDUA.

[0488] circRNAIDUA is circularized and purified according to the method described in Example 2, and then transfected into the original protein from the Heller syndrome mouse model using cationic lipid transfection reagent Lipofectamine™ MessengerMAX transfection reagent (Thermo LRNA003). passage in MEF cells or human HEK293T / IDUA- / - cells.

[0489] After 48h, use the reported α-1-iduronidase assay (Qu et al., Nature Biotechnology, vol 37, September 2019, 1059-1069) to detect the catalysis of α-1-iduronidase active. The α-l-iduronidase assay showed that circRNAIDUA could effectively restore the catalytic activity in primary MEF cells from a mouse model of Heller's syndrome as well as in human HEK293T / IDUA- / - cells, suggesting that circRNAIDUA plays an important role in Heller's syndrome Works in primary cells of mouse origin. The results are shown in Figure 9B and Figure 9C.

[0490] Example 10: In vivo restoration of alpha-l-iduronidase catalytic activity in a mouse model of Heller syndrome.

[0491] Example 8 above demonstrates that exemplary circRNAs can express functional enzyme proteins in enzyme-deficient mouse or human cells, thereby restoring protein function in those cells. This example demonstrates that exemplary circRNAs can be used to restore protein function in vivo. In addition, circRNA-expressed proteins can restore protein function over an extended period of time (eg, at least 24 h).

[0492] Purified circRNA IDUA (30 μg per dose) was encapsulated and delivered to Heller syndrome (IDUA-deficient) mice via tail vein injection. After 4h or 24h, the Heller syndrome mice were sacrificed to separate the liver tissue, and the α-l-iduronidase activity was measured in the separated liver tissue.

[0493] α-l-iduronidase assays from livers of mice injected with circRNAIDUA or controls showed that circRNAIDUA can effectively restore α-l-iduronidase catalysis in a mouse model of Heller syndrome activity, reaching nearly 20% of the activity of wild-type mice (Figure 10). In addition, the catalytic activity increased from 4h to 24h, suggesting that circRNAIDUA has a long-lasting effect and can be used to treat genetic diseases.

[0494] Example 11: SARS-CoV-2 circRNA vaccine triggers a sustained humoral immune response through a high level of neutralizing antibodies

[0495] With its stability and immunogen encoding ability, we reasoned that circRNA could be developed into a novel vaccine. We then attempted to evaluate the immunogenicity of circRNARBD encapsulated with lipid nanoparticles in BALB / c mice (Fig. 11A). The encapsulation efficiency of circRNARBD was greater than 93%, with an average diameter of 100 nm (Fig. 11B). Animals were immunized with LNP-circRNARBD by intramuscular injection twice at a dose of 10 μg or 50 μg at two-week intervals, while blank LNP was used as placebo (Fig. 11C). 2 or 5 weeks after the LNP-circRNA RBD boost, the amount of RBD-specific IgG and pseudovirus-neutralizing activity were evaluated.

[0496] By the method previously described (Ickenstein, L. M. & Garidel, P. Lipid-based nanoparticle formulations for small molecules and RNA drugs. 890Expert Opin Drug Deliv16, 1205-1226, doi:10.1080 / 17425247.2019.16695 58 (2019), which The content is incorporated herein by reference), and the circRNA is wrapped with lipid nanoparticles (LNP). Briefly, the circRNA was diluted in 50 mM citrate buffer (pH 3.0), and the lipids were solubilized and synthesized at 50:10:38.5:1.5 (MC3-lipid:DSPC:cholesterol:PEG2000-DMG) The molar ratio is mixed in ethanol. The lipid mixture was then mixed with the circRNA solution at a volume ratio of 1:3 in a NanoAssemblr Benchtop (Precision, #NIT0046). Then, the LNP-circRNA preparation was diluted 40-fold with 1×PBS buffer (pH 7.2–7.4) and concentrated by ultrafiltration using Amicon® Ultra Centrifugal Filter Unit (Millipore). The concentration and encapsulation efficiency of circRNA were measured by Quant-iT™ RiboGreen™ RNA Measurement Kit (Invitrogen™ #R11490). The size of LNP-circRNA particles was measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS 300 (Malvern). The sample was irradiated with a red laser (l=632.8nm) and the scattered light was detected at a backscattering angle of 173 degrees. The results were analyzed using software (Zetasizer V7.13) to obtain the autocorrelation function.

[0497] circRNARBD elicited high titers of RBD-specific IgG in a dose-dependent manner, each dose and 2 weeks and 5 weeks after boosting were ~3×104 and ~1×106, indicating that circRNARBD can induce anti-SARS-CoV- 2 Long-acting antibodies to RBD (FIG. 11D).

[0498] To test the antigen-specific binding capacity of IgG from vaccinated animals, we performed a surrogate neutralization assay. Consistent with the amount of RBD-specific IgG (Fig. 11D), antibodies elicited by the circRNA RBD vaccine showed significant neutralizing capacity in a dose-dependent manner, with an NT50 of ~2 × 104 for a dose of 50 μg (Fig. 11E, Fig. 11F) .

[0499] We further demonstrated that sera from mice inoculated with circRNARBD could neutralize SARS-CoV-2 pseudoviruses (Fig. 11G) and real SARS-CoV-2 viruses (Fig. 11H). NT50 was ~5.6×103 (Fig. 11G) and NT50 was ~2.2×105 (Fig. 11H), respectively. The large amount of RBD-specific IgG, potent RBD antigen neutralization, and sustained SARS-CoV-2 neutralization capacity indicated that the circRNA RBD vaccine indeed induced a durable humoral immune response in mice.

[0500] Example 12: SARS-CoV-2 circRNA vaccine induces strong T cell immune response in spleen

[0501] B cells (the source of antibodies), CD4+T cells and CD8+T cells are the three pillars of adaptive immunity, and the effector functions they mediate are related to the control of SARS in non-hospitalized and hospitalized COVID-19 cases -CoV-2 related.

[0502] In order to detect CD4+ and CD8+ T cell immune responses in mice inoculated with circRNARBD (5 weeks after booster immunization), splenocytes were stimulated with SARS-CoV-2 spike RBD pooled peptide (Table E1 below ), and cytokine-producing T cells were quantified by intracellular cytokine staining in effector memory T cells (Tem, CD44+CD62L-). Upon stimulation with the RBD peptide library, CD4+ T cells of mice immunized with circRNARBD vaccine exhibited a Th1-biased response, producing interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), and interleukin IL-2 (FIG. 12A, FIG. 12B), but not interleukin-4 (IL-4), suggests that circRNARBD vaccines mainly induce Th1-biased rather than Th2-biased immune responses. Furthermore, multiple cytokine-producing CD8+ were detected in mice vaccinated with circRNARBD (Fig. 12C, Fig. 12D). For unknown reasons, 10 μg of circRNARBD elicited a stronger immune response in CD4+ and CD8+ effector memory T cells compared with 50 μg (Fig. 11G and Figure 11H).

[0503] Table E1: Peptide sequences of RBD antigens name sequence SEQ ID NO: S-45 GIYQTSNFRVQPTESIVR 65 S-46 RVQPTESIVRFPNITNL 66 S-47 IVRFPNITNLCPFGEVF 67 S-48 TNLCPFGEVFNATRFASV 68 S-49 VFNATRFASVYAWNRKRI 69 S-50 SVYAWNRKRISNCVADY 70 S-51 KRISNC VADYSVLYNSA 71 S-52 ADYSVLYNSASFSTFKCY 72 S-53 SASFSTFKCYGVSPTKL 73 S-54 KCYGVSPTKLNDLCFTNV 74 S-55 KLNDLCFTNVYADSFVIR 75 S-56 NVYADSFVIRGDEVRQIA 76 S-57 IRGDEVRQIAPGQTGKIA 77 S-58 IAPGQTGKIADYNYKL 78 S-59 GKIADYNYKLPDDFTGCV 79 S-60 KLPDDFTGCVIAWNSNNL 80 S-61 CVIAWNSNNLDSKVGGNY 81 S-62 NLDSKVGGNYNYLYRLFR 82 S-63 NYNYLYRLFRKSNLKPF 83 S-64 LFRKSNLKPFERDISTEI 84 S-65 PFERDISTE IYQA 85 S-66 RDISTEIYQAGSTPCNGV 86 S-67 YQAGSTPCNGVEGFNCYF 87 S-68 NGVEGFNCYFPLQSYGF 88 S-69 CYFPLQSYGFQPTNGVGY 89 S-70 GFQPTNGVGYQPYRVVVL 90 S-71 GYQPYRVVVLSFELLHA 91 S-72 VVLSFELLHAPATVCGPK 92 S-73 HAPATVCGPKKSTNLVK 93 S-74 GPKKSTNLVKNKCVNFNF 94 S-75 VKNKCVNFNFNGLTGTGV 95

[0504] Collectively, these results demonstrate that the SARS-CoV-2 circRNA RBD vaccine can induce high levels of humoral and cellular immune responses in mice. In this report, mice immunized with circRNARBD-501Y.V2 produced high titers of neutralizing antibodies. Given that the K417N-E484K-N501Y mutant in the RBD reduces its interaction with certain neutralizing antibodies (as shown in Example 8), we also demonstrated that mice immunized with circRNARBD or circRNARBD-501Y.V2 produced Neutralizing antibodies have preferential neutralizing ability to their corresponding virus strains. Recent studies have shown that 501Y.V2 does not appear to be more infectious, but has immune evasion capabilities, and multiple vaccines have been reported to be less effective against SARS-CoV-2 variants. Vaccine breakthrough infections of SARS-CoV-2 variants have also been reported. Therefore, it is important to develop and implement vaccines against emerging variants, and cricRNA vaccines are such a platform that can be rapidly tailored to specific variants. For example, a vaccine containing the E484K, N501Y, and L452R mutations in the RBD could be rapidly developed using a circRNA platform to address potential outbreaks caused by SARS-CoV-2 variants (in the recently reported B.1.617276 variant in India and in L452R was found in the B.1.429 variant of the United States).

[0505] We highlight this general strategy for designing immunogens. The coding sequence of circular RNA can be quickly adapted to deal with any emerging SARS-CoV-2 variants, as recently reported B.1.1.7 / 501Y.V1, B.1.351 / 501Y.V2, P.1 / 501Y. V3 and B.1.671 variants. In addition, circular RNAs can be rapidly and massively produced in vitro without any nucleotide modification.

[0506] Example 13: Antibodies elicited by the SARS-CoV-2 circRNA RBD-501Y.V2 vaccine showed preferential neutralization activity against the B.1.351 variant

[0507] Next, we evaluated the efficacy of a circRNA vaccine encoding RBD / K417N-E484K-N501Y derived from a B.1.351 / 501Y.V2 variant, termed circRNA RBD-501Y.V2 (Fig. 13A). BALB / c mice were immunized with intramuscular injection of circRNARBD-501Y.V2 vaccine, and then boosted every two weeks. Sera from immunized mice were collected 1 and 2 weeks after the boost. ELISA showed that RBD-501Y.V2-specific IgG titers reached 7 x 104 2 weeks after the boost (Fig. 13B). Alternative neutralization assays showed that sera from mice immunized with circRNA RBD-501Y.V2 effectively neutralized RBD antigen ( FIG. 13C ). We then proceeded to evaluate the neutralizing activity of sera from mice immunized with circRNARBD or circRNARBD-501Y.V2 vaccines against D614G, B.1.1.7 / 501Y.V1 or B.1.351 / 501Y.V2 variants. A VSV-based pseudovirus neutralization assay showed that antibodies elicited by the circRNA RBD vaccine encoding the native RBD sequence effectively neutralized all three virus strains, with the highest activity against the D614G strain (Fig. 13D). Serum from mice immunized with circRNARBD-501Y.V2 could also neutralize all three pseudoviruses, with the highest neutralizing activity against its corresponding variant 501Y.V2 (Fig. 13E).

[0508] We further tested the neutralization ability of the sera of mice immunized with circRNARBD-501Y.V2 against authentic SARS-CoV-2 strains. Consistent with the pseudovirus neutralization assay, the serum can effectively neutralize the real SARS-CoV-2 B.1.351 / 501Y.V2 strain with an NT50 of 7.1×104 (Figure 13F); and can neutralize the real SARS-CoV-2 D164G strain, the effect is poor, NT50 is 9.8×103 (Fig. 13G). Overall, antibodies elicited by circRNA vaccines exhibited the best neutralizing activity against their corresponding mutant strains. Notably, both vaccines neutralized all three strains, although with varying efficacy. Nonetheless, newer or multivalent vaccines against corresponding variants may provide better protection against natural SARS-CoV-2 strains and their looping variants.

[0509] Example 14: circRNARBD-501Y.V2 vaccine in the new mouse model for the lasting protection of real B.1.351 strain

[0510] In order to further evaluate the protective efficacy of the SARS-CoV-2 circRNA RBD-501Y.V2 vaccine in vivo, we used the B.1.351 / 501Y.V2 strain to conduct real virus challenge experiments because of its severe antibody escape ability. Consistent with a recent report, the B.1.351 / 501Y.V2 variant can infect BALB / c mice and replicate in their lungs, possibly due to mutations in the spike protein, particularly in the RBD domain, as K417N, E484K and N501Y. We then used BALB / c mice to obtain the protective efficacy of the SARS-CoV-2 circRNA RBD-501Y.V2 vaccine. BALB / c mice received two doses of 50 μg circRNARBD-501Y.V2 vaccine or placebo by intramuscular injection at two-week intervals (Fig. 14A). To evaluate the long-term protective effect of the circRNA vaccine, each immunized mouse was treated with 5×104 PFU of the authentic SARS-CoV-2 B.1.351 / 501Y.V2 strain via the intranasal (i.n.) route 7 weeks after the booster dose. Challenged, and lung tissue was collected 3 days post-challenge for detection of viral RNA (Fig. 14A). Three days before virus challenge, sera from immunized mice were collected for detection of RBD-501Y.V2-specific IgG (Fig. 14A). Nearly two months after immunization, the titer of RBD-501Y.V2-specific IgG was about 2×104 ( FIG. 14B ), and the serum showed significant neutralization ability to RBD-501Y.V2 antigen ( FIG. 14C ).

[0511] Furthermore, we found that mice in the placebo group lost increased body weight compared to vaccinated mice (FIG. 14D). Consistently, virus titers were significantly lower in the lungs of vaccinated mice compared to those mice that received placebo (Fig. 14E). These results suggest that the circRNARBD-501Y.V2 vaccine can effectively protect mice against infection with the SARS-CoV-2 B.1.351 / 501Y.V2 variant.

[0512] Exemplary sequences

[0513] SEQ ID NO: 1: The full-length S protein sequence of SARS-CoV-2

[0514] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQII...

Claims

1. A circular RNA (circRNA) comprising, in sequence, a 3' exon sequence recognizable by a 3' catalytic group I intron fragment, an IRES sequence or an m6A modified motif sequence, a Kozak sequence, a signal peptide coding sequence, a nucleic acid sequence encoding a therapeutic polypeptide, a TAA stop codon or an in-frame 2A peptide coding sequence, and a 5' exon sequence recognizable by a 5' catalytic group I intron fragment; wherein the 3' exon sequence recognizable by the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence recognizable by the 5' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 40; wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins, and target proteins.

2. The circRNA as described in claim 1 further comprises a polyAC or polyA sequence located at the 5' end of the IRES sequence.

3. The circRNA as claimed in claim 1, wherein the therapeutic polypeptide is an antigenic polypeptide.

4. The circRNA as claimed in claim 3, wherein the antigenic polypeptide comprises the spike (S) protein of a coronavirus or a fragment thereof.

5. The circRNA as claimed in claim 4, wherein the antigenic polypeptide comprises the receptor-binding domain (RBD) of the S protein.

6. The circRNA as claimed in claim 3, wherein the antigenic polypeptide further comprises a polymerizing domain.

7. The circRNA as claimed in claim 3, wherein the antigenic polypeptide comprises the S2 region of the S protein.

8. The circRNA as claimed in claim 4, wherein the antigenic polypeptide comprises amino acid residues 2-1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO:

1.

9. The circRNA of claim 5, wherein the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8-10 or SEQ ID NO: 62-63, and / or wherein the circRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11-15 and 64.

10. The circRNA as claimed in claim 1, wherein the therapeutic protein is a receptor protein.

11. The circRNA as claimed in claim 10, wherein the receptor is the ACE2 receptor.

12. The circRNA as claimed in claim 11, wherein the receptor is a high-affinity mutant ACE2 receptor.

13. The circRNA as claimed in claim 1, wherein the therapeutic protein is a target protein.

14. The circRNA as described in claim 13, wherein the target protein is an antibody.

15. A composition comprising a plurality of circRNAs as claimed in claim 1, wherein the therapeutic peptides corresponding to the plurality of circRNAs are different from each other.

16. The composition as claimed in claim 15, wherein the plurality of circRNAs target a plurality of coronavirus strains.

17. A circRNA vaccine comprising the circRNA as described in claim 1.

18. A pharmaceutical composition comprising the circRNA as described in claim 1 and a pharmaceutically acceptable carrier.

19. Use of a circRNA as described in claim 1 in the preparation of a medicament for treating or preventing an individual infection.

20. The use as described in claim 19, wherein the infection is a coronavirus infection.

21. The use as described in claim 20, wherein the infection is SARS-CoV-2 infection.

22. Use of a circRNA as claimed in claim 1 in the preparation of a medicament for treating or preventing an individual disease or condition, wherein the disease or condition is associated with insufficient protein levels and / or activity corresponding to the therapeutic protein, or wherein the disease or condition is a hereditary disease associated with one or more mutations in the protein corresponding to the therapeutic protein.

23. The use as described in claim 22, wherein: (i) The therapeutic peptide is TP53 or PTEN, and the disease or condition is cancer; (ii) The therapeutic peptide is an OTC, and the disease is ornithine transaminase deficiency; (iii) The therapeutic peptide is FAH, and the disease is tyrosinemia; (iv) The therapeutic peptide is DMD, and the disease is Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, or familial dilated cardiomyopathy; (v) The therapeutic peptide is IDUA, and the disease or condition is mucopolysaccharidosis type I (MPSI); (vi) The therapeutic peptide is COL3A1, and the disease or condition is Ehlers-Danlow syndrome; (vii) The therapeutic peptide is AHI1, and the disease or condition is Jubert syndrome; (viii) The therapeutic peptide is BMPR2, and the disease or condition is pulmonary hypertension or pulmonary venous occlusive disease; (ix) The therapeutic peptide is FANCC, and the disease or condition is Fanconi anemia; (x) the therapeutic peptide is MYBPC3, and the disease or condition is primary familial hypertrophic cardiomyopathy; or (xi) the therapeutic peptide is IL2RG, and the disease or condition is X-linked severe comprehensive immunodeficiency.

24. A linear RNA comprising, from its 5' to 3' end, the following in sequence: a 5' homologous arm, a 3' catalytic group I intron fragment, a 3' exon sequence recognizable by the 3' catalytic group I intron fragment, an IRES sequence or an m6A modified motif sequence, a Kozak sequence, a signal peptide coding sequence, a nucleic acid sequence encoding a therapeutic polypeptide, a TAA stop codon or an in-frame 2A peptide coding sequence, a 5' exon sequence recognizable by the 5' catalytic group I intron fragment, a 5' catalytic group I intron fragment, and a 3' homologous arm; wherein the 3' exon sequence recognizable by the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 39, and the 5' exon sequence recognizable by the 5' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 40; wherein the therapeutic polypeptide is selected from the group consisting of antigenic polypeptides, functional proteins, receptor proteins, and target proteins.

25. A nucleic acid construct comprising a nucleic acid sequence encoding a linear RNA as claimed in claim 24.

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