Circular RNA composition and method

Abstract

Provides a circular RNA containing an expression sequence. [Solution] In some embodiments, the circular RNA of the present invention comprises a group I intron fragment, a spacer, an IRES, a double-strand forming region, and an expression sequence. In some embodiments, the expression sequence encodes an antigen. In some embodiments, the circular RNA of the present invention has improved expression, functional stability, immunogenicity, ease of manufacture, and / or half-life compared to linear RNA. In some embodiments, the methods and constructs of the present invention result in improved cyclization efficiency, splicing efficiency, and / or purity compared to existing RNA cyclization approaches.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 63 / 027,292, filed on 19 May 2020, the contents of which are fully incorporated by reference for all purposes. [Background technology]

[0002] Traditional gene therapy involves the use of DNA to insert desired genetic information into host cells. The DNA introduced into cells is typically partially integrated into the genome of one or more transfected cells, enabling the long-term effects of the introduced genetic material within the host. While such sustained effects can offer substantial benefits, the integration of exogenous DNA into the host genome can also have many harmful effects. For example, introduced DNA may be inserted into intact genes, leading to mutations that interfere with, or even completely eliminate, the function of endogenous genes. Therefore, DNA-based gene therapy can result in potentially fatal genetic dysfunction in the treated host, such as the elimination or harmful reduction of essential enzyme production, or interference with genes critically important for regulating cell growth, potentially leading to uncontrolled or cancerous cell proliferation. In addition, traditional DNA-based gene therapy requires the inclusion of a strong promoter sequence for effective expression of the desired gene product, which can also lead to undesirable changes in the regulation of normal gene expression within cells. Furthermore, DNA-based genetic material can induce undesirable anti-DNA antibodies, potentially triggering a potentially fatal immune response. Gene therapy approaches using viral vectors can also lead to adverse immune responses. In some situations, viral vectors can even be integrated into the host genome. In addition, the production of clinical-grade viral vectors is costly and time-consuming. Targeted delivery of transgene material using viral vectors can also be difficult to control. Therefore, although DNA-based gene therapy has been evaluated for the delivery of secreted proteins using viral vectors (U.S. Patent No. 6,066,626; U.S. Publication No. 2004 / 0110709), these approaches can be limited for these various reasons.

[0003] In contrast to DNA, the use of RNA as a gene therapy agent is substantially safer because it does not carry the risk of the RNA being stably integrated into the genome of the transfected cell. Therefore, concerns that the introduced genetic material may interfere with the normal function of essential genes or cause mutations that result in harmful or carcinogenic effects are eliminated, and foreign promoter sequences are not required for the effective translation of the encoded protein, again avoiding potential harmful side effects. In addition, mRNA does not need to enter the nucleus to perform its function, whereas DNA must overcome this major barrier.

[0004] Circular RNA is useful for designing and producing RNA in stable forms. Circularization of RNA molecules offers advantages in studying RNA structure and function, especially for molecules that are easily folded in inactive conformations (Wang and Ruffner, 1998). Circular RNA is also of particular interest and may be useful for in vivo applications, particularly in the field of RNA-based regulation of gene expression and therapeutic agents, including protein replacement therapy and vaccination.

[0005] Prior to this invention, there were three main techniques for producing circularized RNA in vitro: the sprint method, the permutation-substitution intron-exon method, and the RNA ligase method. However, existing methodologies are limited by the size of the RNA that can be circularized, thus limiting their therapeutic applications. [Overview of the project]

[0006] Circular RNA is described herein along with related compositions and methods. In some embodiments, the circular RNA of the present invention comprises a group I intron fragment, a spacer, an IRES, a double-strand forming region, and an expression sequence. In some embodiments, the expression sequence encodes one or more antigens. In certain embodiments, the expression sequence is replaced with a non-coding sequence. In some embodiments, the circular RNA of the present invention has improved expression, functional stability, ease of manufacture, and / or half-life compared to linear RNA. In some embodiments, the circular RNA of the present invention has reduced immunogenicity. In some embodiments, the methods and constructs of the present invention result in improved cyclization efficiency, splicing efficiency, and / or purity compared to existing RNA cyclization approaches.

[0007] In one embodiment, a cyclic RNA polynucleotide is provided in the following order: a.3' Group I intron fragment, b. Intra-sequence ribosome entry sites (IRES), c. An expression sequence encoding one or more antigens, adjuvants, antigen-like or adjuvant-like polypeptides, or fragments thereof, and d.5' Group I intron fragment A circular RNA polynucleotide comprising the above is provided herein. In some embodiments, the 3' group I intron fragment comprises a 3' group I intron splice site dinucleotide. In some embodiments, the 5' group I intron fragment comprises a 5' group I intron splice site dinucleotide.

[0008] In one embodiment, a cyclic RNA polynucleotide is provided in the following order: a.3' Group I intron fragment, b. Intra-sequence ribosome entry sites (IRES), c. Non-coding expression sequences, and d.5' Group I intron fragment A cyclic RNA polynucleotide containing the above is provided herein.

[0009] In one embodiment, a cyclic RNA polynucleotide produced from the transcription of a vector is, in the following order: a.5' duplex forming region, b.3' Group I intron fragment, c. Intra-sequence ribosome entry sites (IRES), d. An expression sequence encoding one or more antigens, adjuvants, antigen-like or adjuvant-like polypeptides, or fragments thereof. e.5' group I intron fragment, and f.3' duplex forming region A cyclic RNA polynucleotide containing the above is provided herein.

[0010] In one embodiment, a cyclic RNA polynucleotide produced from the transcription of a vector is, in the following order: a.5' duplex forming region, b.3' Group I intron fragment, c. Intra-sequence ribosome entry sites (IRES), d. Non-coding expression sequences, e.5' group I intron fragment, and f.3' duplex forming region A cyclic RNA polynucleotide containing the above is provided herein.

[0011] In some embodiments, the vector further comprises a triphosphorylated 5' end. In some embodiments, the vector further comprises a monophosphorylated 5' end.

[0012] In some embodiments, the circular RNA polynucleotide includes a first spacer between the 5' double-stranding region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-stranding region. In some embodiments, the first and second spacers each have a length of about 10 to about 60 nucleotides. In some embodiments, the first and second double-stranding regions each have a length of about 9 to about 19 nucleotides. In some other embodiments, the first and second double-stranding regions each have a length of about 30 nucleotides.

[0013] In some embodiments, IRES includes Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated bug (Plautia stali) enterovirus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, pygmy kite P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, and Ectropis Obliqua picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human sackievirus B3, Crucifer tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic spot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human B AG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S.Cerevisiae YAP1, Tobacco HTC virus, Kabukurinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirnavirus, HCV QC64, Hitocosavirus E / D, Hitocosavirus F, Hitocosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A virus HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C The sequence of IRES contains aptamers for K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Saperovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Cissinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Disicisthovirus, Hupey Picorna-like Virus, CRPV, Sarivirus A BN5, Sarivirus A BN2, Sarivirus A 02394, Sarivirus A GUT, Sarivirus A CH, Sarivirus A SZ1, Sarivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0014] In some embodiments, the circular RNA polynucleotide consists of native nucleotides. In some embodiments, the expression sequence is codon-optimized. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one microRNA binding site present in the equivalent pre-optimization polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site present in the equivalent pre-optimization polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site present in the equivalent pre-optimization polynucleotide.

[0015] In some embodiments, the circular RNA polynucleotides are approximately 100 nucleotides to 10 kilobases in length.

[0016] In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in vivo in humans of at least about 20 hours. In some embodiments, the circular RNA polynucleotide has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in human cells that is longer than or equivalent to the duration of therapeutic effect of a comparable linear RNA polynucleotide containing the same expression sequence. In some embodiments, the circular RNA polynucleotide has a functional half-life in human cells that is longer than or equivalent to the functional half-life of a comparable linear RNA polynucleotide containing the same expression sequence. In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in vivo in humans that is longer than that of a comparable linear RNA polynucleotide having the same expression sequence. In some embodiments, the circular RNA polynucleotide has a functional half-life in vivo in humans that is longer than that of a comparable linear RNA polynucleotide having the same expression sequence.

[0017] In some embodiments, the adjuvant or adjuvant-like polypeptide is selected from the group comprising Toll-like receptor ligands, cytokines, FLt3-ligands, antibodies, chemokines, chimeric proteins, endogenous adjuvants released from dying tumors, and checkpoint inhibitor proteins. In some embodiments, the adjuvant or adjuvant-like polypeptide is selected from the group including BCSP31, MOMP, FomA, MymA, ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, rumazine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, pneumolysin, fargerin, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-α, IL-7, IL-17, IL-1β, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and andti-CD3. In some embodiments, the adjuvant or adjuvant-like polypeptide is selected from Table 10.

[0018] In one embodiment, the RNA polynucleotide is arranged in the following order: 3' intron fragment and Triphosphorylated 5' end RNA polynucleotides including the above are provided herein. In some embodiments, the RNA polynucleotide includes a 5' spacer located upstream of the 3' intron fragment and downstream of the triphosphorylated 5' end.

[0019] In one embodiment, an RNA polynucleotide comprising a 5' intron fragment and a triphosphorylated 5' terminus is provided herein. In some embodiments, the RNA polynucleotide comprises a 5' spacer located downstream of the 5' intron fragment.

[0020] In some embodiments, the RNA polynucleotide further comprises a monophosphorylated 5' end.

[0021] In one embodiment, the RNA polynucleotide is arranged in the following order: 3' intron fragment and Monophosphorylated fifth end RNA polynucleotides including the above are provided herein. In some embodiments, the RNA polynucleotide includes a 5' spacer located upstream of the 3' intron fragment and downstream of the monophosphorylated 5' end.

[0022] In one embodiment, an RNA polynucleotide comprising a 5' intron fragment and a monophosphorylated 5' terminus is provided herein. In some embodiments, the RNA polynucleotide comprises a 5' spacer located downstream of the 5' intron fragment.

[0023] In some embodiments, the RNA polynucleotide further includes a triphosphorylated 5' end.

[0024] In some embodiments, the RNA polynucleotide further comprises a poly(A) purified tag. In some embodiments, the RNA polynucleotide further comprises an initiation sequence.

[0025] In one embodiment, the RNA preparation is, a. A cyclic RNA polynucleotide according to claim 1, claim 2, or both; b. Linear RNA polynucleotides, i. A 3'-intron polynucleotide containing a monophosphorylated 5' terminus and a 3'-intron fragment, ii. 5'-intron polynucleotides containing a monophosphorylated 5' terminus and a 5'-intron fragment, iii. 3' intron polynucleotides containing a triphosphorylated 5' terminus and a 3' intron fragment, and iv. 5' intron polynucleotides containing triphosphorylated 5' terminus and 3' intron fragments A linear RNA polynucleotide containing at least one of the following: An RNA preparation is provided herein, comprising a cyclic RNA polynucleotide, wherein at least 90% of the RNA preparation is composed of the cyclic RNA polynucleotide.

[0026] In some embodiments, the 3'-intron polynucleotide or 5'-intron polynucleotide includes a spacer. In some embodiments, the 3'-intron polynucleotide or 5'-intron polynucleotide includes a polyA sequence. In some embodiments, the 3'-intron polynucleotide or 5'-intron polynucleotide includes a UTR. In some embodiments, the 3'-intron polynucleotide or 5'-intron polynucleotide includes an IRES.

[0027] In one embodiment, a pharmaceutical composition comprising a cyclic RNA polynucleotide disclosed herein, a diluent, and optionally a salt buffer is provided herein.

[0028] In one embodiment, a pharmaceutical composition comprising an RNA preparation disclosed herein, a diluent, and optionally a salt buffer is provided herein.

[0029] In one embodiment, a pharmaceutical composition comprising a cyclic RNA polynucleotide disclosed herein and a polycationic, cationic, or polymeric compound is provided herein.

[0030] In one embodiment, a pharmaceutical composition comprising an RNA preparation disclosed herein and a polycationic, cationic, or polymeric compound is provided herein.

[0031] In some embodiments, the polycationic or cationic compound is Cationic peptides or proteins, basic polypeptides, cell-permeable peptides (CPPs), Tat-derived peptides, penetratin, VP22-derived or analog peptides, pestivirus Erns, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, Antennapedia-derived peptides, pAntp, pIsl, FGF, lactoferrin, transportan, bufoly N-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, histone, cationic polysaccharide, cationic polymer, cationic lipid, dendrimer, polyimine, polyallylamine, oligofectamine, or cationic or polycationic polymer, sugar backbone polymer, silane backbone polymer, modified polyamino acid, modified acrylate, modified polybeta-amino ester (PBAE), modified amidoamine, dendrimer, block polymer consisting of a combination of one or more cationic blocks and one or more hydrophilic or hydrophobic blocks Selected from the group consisting of: In some embodiments, the polymer compound is Polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Selected from the group consisting of, Poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolal Poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(esteramide), polyamide, poly Polyalkylenes such as (ester ethers), polycarbonates, polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized cellulose (e.g., alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose), acrylic acid polymers, e.g., poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), 363 5 10 15 20 25 30 35 WO 2021 / 076805PCT / US2020 / 055844 Poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl(meth)acrylate), poly(isopropyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(octadecyl(meth)acrylate) and copolymers and mixtures thereof, polydioxanone and so Copolymers, polyhydroxyalkanoates, polypropylene fimmarates, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerols It may include. In some embodiments, the polycationic or cationic compound is Protamine, nucleolin, spermine or spermidine, poly-L-lysine (PLL), polyarginine, HIV-binding peptide, HIV-1 Tat(HIV), polyethyleneimine (PEI), DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamideglycylspermine, DIMRI: dimyristooxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-alpha-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium Lorid, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, beta-amino acid polymers or reverse polyamides, PVP (poly(N-ethyl-4-vinylpyridinium bromide)), pDMAEMA (poly(dimethylaminoethylmethyl acrylate)), pAMAM (poly(amideamine), diamine-terminated 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymer, polypropylamine dendrimer or pAMAM dendrimer, polyimine, PEI:poly(ethyleneimine), poly(propyleneimine), polyallylamine, cyclodextrin polymers, dextran polymers, chitosan, and PMOXA-PDMS copolymer It is selected from the group that includes it.

[0032] In one embodiment, a pharmaceutical composition is provided herein comprising a cyclic RNA polynucleotide disclosed herein, nanoparticles, and optionally, a targeting moiety operably attached to the nanoparticles.

[0033] In one embodiment, a pharmaceutical composition is provided herein, comprising an RNA preparation disclosed herein, nanoparticles, and optionally, a targeting portion operably attached to the nanoparticles.

[0034] In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles. In certain embodiments, the nanoparticles include one or more cationic lipids selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazole-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0035] In some embodiments, the pharmaceutical composition comprises a targeting moiety that mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue without cell isolation or purification. In some embodiments, the targeting moiety is an scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region, or a fragment thereof. In some embodiments, the cyclic RNA polynucleotide or RNA preparation is in an effective amount to treat an infection (e.g., a viral infection) in a human subject where such treatment is needed. In some embodiments, the pharmaceutical composition has an enhanced safety profile compared to a pharmaceutical composition comprising a vector containing exogenous DNA encoding an antigen. In some embodiments, less than 1% by weight of polynucleotides in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA. In some embodiments, less than 1% by weight of polynucleotides and proteins in the pharmaceutical composition is double-stranded RNA, DNA sprint, triphosphorylated RNA, phosphatase protein, protein ligase, and capping enzyme.

[0036] In one embodiment, a method for treating a subject in need is provided herein, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide disclosed herein, nanoparticles, and optionally, a targeting moiety operably attached to the nanoparticles. A method for treating a subject in need is provided herein, comprising administering a therapeutically effective amount of a composition comprising an RNA preparation disclosed herein, nanoparticles, and optionally, a targeting moiety operably attached to the nanoparticles.

[0037] In some embodiments, the subject has an infection (e.g., a viral infection). In certain embodiments, the method of treating the subject in need further includes the co-administration of an anti-inflammatory agent.

[0038] In some embodiments, the composition comprises a targeting portion that mediates receptor-mediated endocytosis to selected cells from a selected cell population without the need for cell isolation or purification. In some embodiments, the targeting portion is an scFv, nanobody, peptide, minibody, heavy chain variable region, light chain variable region, or a fragment thereof. In some embodiments, the composition comprises a targeting portion that mediates receptor-mediated endocytosis to selected cells from a selected cell population without the need for cell isolation or purification.

[0039] In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the nanoparticles comprise one or more cationic lipids, ionizable lipids, or polyβ-aminoesters. In some embodiments, the nanoparticles comprise one or more non-cationic lipids. In some embodiments, the nanoparticles comprise one or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids. In some embodiments, the nanoparticles comprise cholesterol. In some embodiments, the nanoparticles comprise arachidonic acid or oleic acid. In some embodiments, the nanoparticles encapsulate one or more cyclic RNA polynucleotides.

[0040] In one embodiment, a vector for producing a circular RNA polynucleotide is provided in the following order: 5' duplex forming region, 3' Group I intron fragment, Intra-sequence ribosome entry sites (IRES), An expression sequence encoding one or more adjuvants, antigens, or adjuvant-like or antigen-like polypeptides, or fragments thereof. 5' group I intron fragment, and 3' duplex forming region Vectors including the above are provided herein.

[0041] In one embodiment, a vector for producing a circular RNA polynucleotide is provided in the following order: 5' duplex forming region, 3' Group I intron fragment, Intra-sequence ribosome entry sites (IRES), Non-code arrays, 5' group I intron fragment, and 3' duplex forming region Vectors including the above are provided herein.

[0042] In some embodiments, the vector includes a first spacer between the 5' double-strand formation region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-strand formation region. In some embodiments, the first and second spacers each have a length of about 20 to about 60 nucleotides. In certain embodiments, the first and second spacers each include an unstructured region of at least 5 nucleotides in length. In some embodiments, the first and second spacers each include a structured region of at least 7 nucleotides in length. In some embodiments, the first and second double-strand formation regions each have a length of about 9 to 50 nucleotides. In some embodiments, the vector is codon-optimized. In certain embodiments, the vector lacks at least one microRNA binding site present in the equivalent pre-optimization polynucleotide.

[0043] In one embodiment, a prokaryotic cell containing a vector disclosed herein is provided herein. In one embodiment, a eukaryotic cell containing a cyclic RNA polynucleotide disclosed herein is provided herein. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is an antigen-presenting cell.

[0044] In one embodiment, the lipids are formulated into nanoparticles. A vaccine comprising at least one viral antigenic polypeptide, an adjuvant or adjuvant-like polypeptide, or at least one circular RNA polynucleotide having an expression sequence encoding an immunogenic fragment thereof. The following are provided herein. In some embodiments, an adjuvant or adjuvant-like polypeptide is selected from Table 10. In some embodiments, the antigenic polypeptide is adenovirus; herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus; hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HI) V); influenza virus; Guanalito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; cortivirus; banavirus; human enterovirus; hantavirus; West Nile virus; Middle East respiratory syndrome coronavirus; Japanese encephalitis virus; varicella virus; SARS-CoV-2; Eastern equine encephalitis, or any combination of two or more of the above. In some embodiments, the viral antigenic polypeptide or its immunogenic fragment is selected from or derived from any one of SEQ ID NOs. 325-336. In some embodiments, the viral antigenic polypeptide or its immunogenic fragment has an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs. 325-336, and the antigenic polypeptide or its immunogenic fragment has membrane fusion activity, attaches to cell receptors, induces fusion of the virus with the mammalian cell membrane, and / or causes the virus to bind to infected cells.

[0045] In one embodiment, the lipids are formulated into nanoparticles. A SARS-CoV-2 vaccine comprising at least one circular RNA polynucleotide having an expression sequence encoding at least one SARS-CoV-2 viral antigenic polypeptide or its immunogenic fragment. However, these are provided herein. In some embodiments, the SARS-CoV2 viral antigenic polypeptide is selected from SARS-CoV2 spike protein, Nsp1-Nsp16, ORF3a, ORF6, ORF7a, ORFb, ORF8, ORF10, SARS-CoV2 envelope protein, SARS-CoV2 membrane protein, SARS-CoV2 nucleocapsid protein, or any antigenic peptide of SARS-CoV2, or a fragment of a SARS-CoV2 peptide. In some embodiments, the SARS-CoV2 viral antigenic polypeptide is derived from SARS-CoV2 virus strains G, GR, GH, L, V, or a combination thereof.

[0046] In some embodiments, the expression sequences contained in the vaccines disclosed herein (e.g., SARS-CoV-2 vaccines) are codon-optimized. In some embodiments, the vaccines (e.g., SARS-CoV-2 vaccines) are polyvalent. In some embodiments, the vaccines (e.g., SARS-CoV-2 vaccines) are formulated in an amount effective to produce an antigen-specific immune response.

[0047] In some embodiments, the cyclic RNA polynucleotide comprises a first expression sequence encoding a first viral antigenic polypeptide and a second expression sequence encoding a second viral antigenic polypeptide.

[0048] In one embodiment, a method for inducing an immune response in a subject is provided herein, comprising administering to the subject an amount effective in producing an antigen-specific immune response in the subject. In another embodiment, a method for inducing an immune response in a subject is provided herein, comprising administering to the subject an amount effective in producing an antigen-specific immune response in the subject a SARS-CoV-2 disclosed herein.

[0049] In some embodiments, the antigen-specific immune response includes a T-cell response or a B-cell response. In some embodiments, a single dose of the vaccine is administered to the subject. In some embodiments, a booster dose of the vaccine is administered to the subject. In some embodiments, the vaccine is administered to the subject by intranasal administration, intradermal injection, or intramuscular injection. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least one logarithm compared to a predetermined threshold level. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by 1 to 3 logarithms compared to a predetermined threshold level. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least twofold compared to a predetermined threshold level. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by 2 to 10fold compared to a predetermined threshold level. In some embodiments, the predetermined threshold level is the anti-antigenic polypeptide antibody titer produced in a subject that has not been administered the vaccine containing the antigenic polypeptide. In some embodiments, a predetermined threshold level is the titer of anti-antigenic polypeptide antibodies produced in subjects administered with a live attenuated vaccine or an inactivated vaccine containing an antigenic polypeptide. In some embodiments, a predetermined threshold level is the titer of anti-antigenic polypeptide antibodies produced in subjects administered with a recombinant protein vaccine or a purified protein vaccine containing an antigenic polypeptide.

[0050] In one embodiment, a circular RNA polynucleotide having an expression sequence encoding at least one viral antigenic polypeptide, adjuvant or adjuvant-like polypeptide, or an immunogenic fragment thereof is provided herein. In one embodiment, an expression vector comprising a genetically engineered nucleic acid encoding at least one circular RNA polynucleotide disclosed herein is provided herein.

[0051] In one embodiment, a cyclic RNA polynucleotide vaccine comprising the cyclic RNA polynucleotide disclosed herein, formulated into lipid nanoparticles, is provided herein. In some embodiments, the nanoparticles have an average diameter of 50 to 200 nm. In some embodiments, the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the lipid nanoparticle carrier has a molar ratio of about 20 to 60% cationic lipids, 0.5 to 15% PEG-modified lipids, 25 to 55% sterols, and 25% non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the nanoparticles have a net neutral charge at a neutral pH.

[0052] In some embodiments of the disclosed vaccines, the cyclic RNA polynucleotide is co-formulated with an adjuvant in the same nanoparticles. In some embodiments, the adjuvant is CpG, imiquimod, aluminum, or Freund's adjuvant.

[0053] In one embodiment, a pharmaceutical composition for use in the vaccination of a target is provided herein, comprising an effective dose of at least one viral antigen, or an adjuvant or adjuvant-like polypeptide, or a cyclic RNA polynucleotide encoding an immunogenic fragment thereof, wherein the effective dose is sufficient to produce a neutralizing titer of 1,000 to 10,000 produced by a neutralizing antibody against the antigen or adjuvant or adjuvant-like polypeptide, or its immunogenic fragment, when measured in the serum of a target 1 to 72 hours after administration. In one embodiment, a pharmaceutical composition for use in the vaccination of a target is provided herein, comprising an effective dose of at least one viral antigen, or an adjuvant or adjuvant-like polypeptide, or a cyclic RNA polynucleotide encoding an immunogenic fragment thereof, wherein the effective dose is sufficient to produce a detectable level of the antigen, or adjuvant or adjuvant-like polypeptide, or its immunogenic fragment, when measured in the serum of a target 1 to 72 hours after administration. In some embodiments, the pharmaceutical composition is intended for use in a method to induce an antigen-specific immune response in a subject, the method comprising administering the vaccine or pharmaceutical composition to the subject in an amount effective in producing an antigen-specific immune response in the subject.

[0054] In one embodiment, a method for inducing, generating, or enhancing an immune response in a subject is provided herein, comprising administering to the subject an amount effective in inducing, generating, or enhancing an antigen-specific immune response in the subject. In some embodiments, the pharmaceutical composition immunizes the subject to a virus for up to two years. In some embodiments, the pharmaceutical composition immunizes the subject to a virus for more than two years. In some embodiments, the subject is exposed to a virus, is infected with the virus, or is at risk of infection by the virus. In some embodiments, the subject is immunodeficient.

[0055] In one embodiment, the use of a vaccine or pharmaceutical composition disclosed herein in the manufacture of a pharmaceutical for use in a method for inducing an antigen-specific immune response in a subject, which includes administering the subject a vaccine in an amount effective to generate an antigen-specific immune response in the subject.

[0056] In one embodiment, a method for inducing cross-reactivity to various viruses or virus strains in a mammal, comprising administering to a mammal in need of such cross-reactivity a vaccine or a pharmaceutical composition of any of the foregoing claims. In some embodiments, the method comprises administering to a mammal separately at least two cyclic RNA polynucleotides having expression sequences each encoding a consensus virus antigen. In some embodiments, the method comprises administering to a mammal simultaneously at least two cyclic RNA polynucleotides having expression sequences each encoding a consensus virus antigen. In some embodiments, the method includes: [Brief explanation of the drawing]

[0057] [Figure 1A] The images show the luminescence in the supernatant of HEK293 (Figures 1A, 1D, and 1E), HepG2 (Figure 1B), or 1C1C7 (Figure 1C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences. [Figure 1B] The images show the luminescence in the supernatant of HEK293 (Figures 1A, 1D, and 1E), HepG2 (Figure 1B), or 1C1C7 (Figure 1C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences. [Figure 1C] The images show the luminescence in the supernatant of HEK293 (Figures 1A, 1D, and 1E), HepG2 (Figure 1B), or 1C1C7 (Figure 1C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences. [Figure 1D] The images show the luminescence in the supernatant of HEK293 (Figures 1A, 1D, and 1E), HepG2 (Figure 1B), or 1C1C7 (Figure 1C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences. [Figure 1E] The images show the luminescence in the supernatant of HEK293 (Figures 1A, 1D, and 1E), HepG2 (Figure 1B), or 1C1C7 (Figure 1C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences.

[0058] [Figure 2] The images show luminescence in the supernatant of HEK293 (Figure 2A), HepG2 (Figure 2B), or 1C1C7 (Figure 2C) cells 24 hours after transfection with circular RNA containing a Gaussial alciferase expression sequence and various IRES sequences of different lengths.

[0059] [Figure 3] This shows the stability of selected IRES constructs in HepG2 (Figure 3A) or 1C1C7 (Figure 3B) cells over 3 days, as measured by luminescence.

[0060] [Figure 4] Figures 4A and 4B show protein expression from selected IRES constructs in Jurkat cells, as measured by luminescence from secreted Gaussial Alciferase in the cell supernatant.

[0061] [Figure 5] Figures 5A and 5B show the stability of selected IRES constructs in Jurkat cells over 3 days, as measured by luminescence.

[0062] [Figure 6A] This shows a comparison of 24-hour luminescence (Figure 6A) or relative luminescence over 3 days (Figure 6B) of modified linear, unpurified circular, or purified circular RNA encoding Gausial ciferase. [Figure 6B] This shows a comparison of 24-hour luminescence (Figure 6A) or relative luminescence over 3 days (Figure 6B) of modified linear, unpurified circular, or purified circular RNA encoding Gausial ciferase.

[0063] [Figure 7A] The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 7B] The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 7C] The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 7D] The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 7E] The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 7F]The transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA is shown.

[0064] [Figure 8A] This shows a comparison of the luminescence of circular RNA and modified linear RNA encoding Gaussial luciferase in human primary monocytes (Figure 8A) and macrophages (Figures 8B and 8C). [Figure 8B] This shows a comparison of the luminescence of circular RNA and modified linear RNA encoding Gaussial luciferase in human primary monocytes (Figure 8A) and macrophages (Figures 8B and 8C). [Figure 8C] This shows a comparison of the luminescence of circular RNA and modified linear RNA encoding Gaussial luciferase in human primary monocytes (Figure 8A) and macrophages (Figures 8B and 8C).

[0065] [Figure 9] Figure 9A shows relative luminescence over 3 days (Figure 9B) or 24-hour luminescence (Figure 9B) in the supernatant of primary T cells transduced using circular RNA containing Gaussial ciferase expression sequences and various IRES sequences.

[0066] [Figure 10A] Figure 10A shows 24-hour luminescence (Figure 10B) or relative luminescence over 3 days (Figure 10B) in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence, and Figure 10C shows 24-hour luminescence in PBMCs. [Figure 10B] Figure 10A shows 24-hour luminescence (Figure 10B) or relative luminescence over 3 days (Figure 10B) in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence, and Figure 10C shows 24-hour luminescence in PBMCs. [Figure 10C]Figure 10A shows 24-hour luminescence (Figure 10B) or relative luminescence over 3 days (Figure 10B) in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence, and Figure 10C shows 24-hour luminescence in PBMCs.

[0067] [Figure 11] HPLC chromatograms (Figure 11A) and cyclization efficiency (Figure 11B) of RNA constructs with different permutation sites are shown.

[0068] [Figure 12] HPLC chromatograms (Figure 12A) and cyclization efficiencies (Figure 12B) of RNA constructs with different introns and / or permutation sites are shown.

[0069] [Figure 13A] HPLC chromatograms (Figure 13A) and cyclization efficiencies (Figure 13B) of three RNA constructs with and without homology arms are shown. [Figure 13B] HPLC chromatograms (Figure 13A) and cyclization efficiencies (Figure 13B) of three RNA constructs with and without homology arms are shown.

[0070] [Figure 14] This shows the cyclization efficiency of three RNA constructs, either without homologous arms or with homologous arms of varying lengths and GC content.

[0071] [Figure 15] Figures 15A and 15B show HPLC chromatograms illustrating the contribution of strong homologous arms to improved splicing efficiency, the relationship between cyclization efficiency and nicking in selected constructs, and combinations of permutation substitution sites and homologous arms assumed to demonstrate improved cyclization efficiency.

[0072] [Figure 16]The images show fluorescence images of T cells co-cultured with Raji cells expressing GFP and firefly luciferase, either electroporated with mock electroporation (left) or electroporated with CAR-encoding circular RNA (right).

[0073] [Figure 17] Bright-field (left), fluorescence (center), and overlay (right) images of T cells co-cultured with Raji cells expressing GFP and firefly luciferase, either electroporated with mock electroporation (top) or electroporated with CAR-encoding circular RNA (bottom).

[0074] [Figure 18] This shows specific lysis of Raji target cells by T cells electroporated with mock electroporation or with circular RNA encoding a different CAR sequence.

[0075] [Figure 19] Figure 19A shows the luminescence in the supernatant of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction using linear or circular RNA containing Gaussial ciferase expression sequences and various IRES sequences, and relative luminescence over 3 days (Figure 19B).

[0076] [Figure 20A] The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 20B] The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 20C]The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 20D] The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 20E] The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown. [Figure 20F] The transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA is shown.

[0077] [Figure 21A] Figure 21A shows the specific lysis of Raji target cells by human primary CD3+ T cells electroporated with CAR-encoding circRNA, as determined by detection of firefly bioluminescence, and Figure 21B shows the induction of IFNγ transcripts 24 hours after electroporation using different amounts of circular or linear RNA encoding CAR sequences. [Figure 21B] Figure 21A shows the specific lysis of Raji target cells by human primary CD3+ T cells electroporated with CAR-encoding circRNA, as determined by detection of firefly bioluminescence, and Figure 21B shows the induction of IFNγ transcripts 24 hours after electroporation using different amounts of circular or linear RNA encoding CAR sequences.

[0078] [Figure 22] This shows the specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding CAR at different E:T ratios (Figures 22A and 22B), as determined by detection of firefly bioluminescence.

[0079] [Figure 23] This shows the specific lysis of target cells by human CD3+ T cells electroporated with CAR-encoding RNA, 1, 3, 5, and 7 days after electroporation.

[0080] [Figure 24] This demonstrates the specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding CD19 or BCMA target CARs.

[0081] [Figure 25] The total flux of organs taken from CD-1 mice administered with a circular RNA encoding FLuc, formulated with 50% lipid 10b-15, 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0082] [Figure 26] The images show highlighting the luminescence of organs isolated from CD-1 mice administered with a circular RNA encoding FLuc, formulated with 50% lipid 10b-15, 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0083] [Figure 27A] This section shows the molecular characteristics of lipids 10a-26 and 10a-27. The proton nuclear magnetic resonance (NMR) spectrum of lipid 10a-26 is also shown. [Figure 27B]This section shows the molecular characteristics of lipids 10a-26 and 10a-27. The retention time of lipid 10a-26, measured by liquid chromatography-mass spectrometry (LC-MS), is also shown. [Figure 27C] The molecular characteristics of lipids 10a-26 and 10a-27 are shown. The mass spectrum of lipid 10a-26 is shown. [Figure 27D] The molecular characteristics of lipids 10a-26 and 10a-27 are shown. The proton NMR spectrum of lipid 10a-27 is shown. [Figure 27E] This shows the molecular characteristics of lipids 10a-26 and 10a-27. The retention time of lipid 10a-27, measured by LC-MS, is also shown. [Figure 27F] The molecular characteristics of lipids 10a-26 and 10a-27 are shown. The mass spectrum of lipid 10a-27 is shown.

[0084] [Figure 28A] This document describes the molecular characteristics of lipid 22-S14 and its synthetic intermediates. The NMR spectrum of 2-(tetradecylthio)ethane-1-ol is also shown. [Figure 28B] This document describes the molecular characteristics of lipid 22-S14 and its synthetic intermediates. The NMR spectrum of 2-(tetradecylthio)ethyl acrylate is also shown. [Figure 28C] This shows the molecular characteristics of lipid 22-S14 and its synthetic intermediate. The NMR spectrum of bis(2-(tetradecylthio)ethyl)3,3'-((3-(2-methyl-1H-imidazole-1-yl)propyl)azanegyl)dipropionate (lipid 22-S14) is shown.

[0085] [Figure 29] The NMR spectrum of bis(2-(tetradecylthio)ethyl)3,3'-((3-(1H-imidazole-1-yl)propyl)azanegyl)dipropionate (lipid 93-S14) is shown.

[0086] [Figure 30A]This shows the molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-54). The proton NMR spectrum of lipid 10a-54 is shown. [Figure 30B] This shows the molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-54). The retention time of lipid 10a-54 measured by LC-MS is shown. [Figure 30C] This shows the molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-54). The mass spectrum of lipid 10a-54 is shown.

[0087] [Figure 31A] This shows the molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-53). The proton NMR spectrum of lipid 10a-53 is shown. [Figure 31B] This shows the molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-53). The retention time of lipid 10a-53 measured by LC-MS is shown. [Figure 31C] This shows the molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 10a-53). The mass spectrum of lipid 10a-53 is shown.

[0088] [Figure 32]Figure 32A shows the total flux from the spleen and liver of CD-1 mice administered with circular RNA encoding firefly luciferase (FLuc), formulated with the ionizable lipid of interest, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 32B shows the mean brightness for the in vivo distribution of protein expression.

[0089] [Figure 33] Figure 33A shows images highlighting the luminescence of organs isolated from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 33B shows whole-body IVIS images of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0090] [Figure 34] Figure 34A shows an image highlighting the luminescence of organs isolated from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 34B shows a whole-body IVIS image of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0091] [Figure 35] Figure 35A shows an image highlighting the luminescence of organs isolated from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 10a-26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 35B shows a whole-body IVIS image of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 10a-26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0092] [Figure 36A] The images show enhanced luminescence in organs isolated from administered c57BL / 6J mice, where circular RNA encapsulated in lipid nanoparticles formed with lipids 10b-15 (Figure 36A), 10a-53 (Figure 36B), or 10a-54 (Figure 36C) is encoded with FLuc. PBS was used as a control (Figure 36D). [Figure 36B] The images show enhanced luminescence in organs isolated from administered c57BL / 6J mice, where circular RNA encapsulated in lipid nanoparticles formed with lipids 10b-15 (Figure 36A), 10a-53 (Figure 36B), or 10a-54 (Figure 36C) is encoded with FLuc. PBS was used as a control (Figure 36D). [Figure 36C] The images show enhanced luminescence in organs isolated from administered c57BL / 6J mice, where circular RNA encapsulated in lipid nanoparticles formed with lipids 10b-15 (Figure 36A), 10a-53 (Figure 36B), or 10a-54 (Figure 36C) is encoded with FLuc. PBS was used as a control (Figure 36D). [Figure 36D]The images show enhanced luminescence in organs isolated from administered c57BL / 6J mice, where circular RNA encapsulated in lipid nanoparticles formed with lipids 10b-15 (Figure 36A), 10a-53 (Figure 36B), or 10a-54 (Figure 36C) is encoded with FLuc. PBS was used as a control (Figure 36D).

[0093] [Figure 37] Figures 37A and 37B show the relative luminescence in human PBMC lysates after 24-hour incubation with test lipid nanoparticles containing circular RNA encoding firefly luciferase.

[0094] [Figure 38] The expression of GFP (Figure 37A) and CD19 CAR (Figure 37B) in human PBMCs after incubation with test lipid nanoparticles containing circular RNA encoding either GFP or CD19 CAR is shown.

[0095] [Figure 39] This shows the expression of anti-mouse CD19 CAR in 1C1C7 cells lipotransfected with circular RNA containing anti-mouse CD19 CAR expression sequences and various IRES sequences.

[0096] [Figure 40] This demonstrates cytotoxicity of mouse T cells by anti-mouse CD19 CAR. The CD19 CAR is encoded and expressed in mouse T cells by electroporated circular RNA.

[0097] [Figure 41A] Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR. [Figure 41B]Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR. [Figure 41C] Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR.

[0098] [Figure 42] Figures 42A and 42B compare the expression levels of anti-human CD19 CAR expressed from circular RNA with those expressed from linear mRNA.

[0099] [Figure 43] Figures 43A and 43B compare the cytotoxic effect of anti-human CD19 CAR expressed from circular RNA with that expressed from linear mRNA.

[0100] [Figure 44] This shows cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMA CAR) expressed from a single circular RNA in T cells.

[0101] [Figure 45A] Representative FACS plots are shown, along with the frequency of tdTomato expression in various spleen immune cell subsets after treatment with LNPs formed from lipids 10a-27, 10a-26, or 10b-15. [Figure 45B] This shows the quantitative proportion of myeloid cells, B cells, and T cells expressing tdTomato, equivalent to the proportion of each cell population successfully transfected with Cre circular RNA (mean + standard deviation, n=3). [Figure 45C]This shows the percentage of additional spleen immune cell populations, including NK cells, classical monocytes, non-classical monocytes, neutrophils, and dendritic cells, expressing tdTomato after treatment with lipids 27 and 26 (mean + standard deviation, n=3).

[0102] [Figure 46A] This shows an exemplary RNA construct design with a built-in polyA sequence in the intron. [Figure 46B] This shows the chromatographic trace of unpurified circular RNA. [Figure 46C] This shows the chromatographic trace of affinity-purified circular RNA. [Figure 46D] This document describes the immunogenicity of circular RNA prepared under various IVT conditions and purification methods. (Commercial = Commercial IVT mix; Custom = Customized IVT mix; Aff = Affinity purification; Enz = Enzyme purification; GMP:GTP ratio = 8, 12.5, or 13.75).

[0103] [Figure 47] Figure 47A shows an exemplary RNA construct design with a specific binding sequence as an alternative to poly(A) for hybridization purification. Figure 47B shows the chromatographic trace of unpurified circular RNA. Figure 46C shows the chromatographic trace of affinity-purified circular RNA.

[0104] [Figure 48] Figure 48A shows the chromatographic trace of unpurified circular RNA encoding dystrophin. Figure 48B shows the chromatographic trace of enzyme-purified circular RNA encoding dystrophin.

[0105] [Figure 49]The expression (Figure 49A) and stability (Figure 49B) of purified circRNAs with different 5' spacers between the 3' intron fragment / 5' inner double-strand region and IRES in Jurkat cells are compared. (AC = only A and C were used in the spacer sequence; UC = only U and C were used in the spacer sequence).

[0106] [Figure 50] This shows the level and stability of luminescence expression in primary T cells from circular RNAs containing the original or modified IRES elements.

[0107] [Figure 51] This shows the level and stability of luminescence expression in HepG2 cells from circular RNAs containing the original or modified IRES elements shown.

[0108] [Figure 52] This shows the level and stability of luminescence expression in 1C1C7 cells from circular RNA containing the original or modified IRES element shown.

[0109] [Figure 53] This shows the luminescence expression levels and expression stability in HepG2 cells from circular RNAs containing IRES elements with inserted untranslated regions (UTRs) or hybrid IRES elements. "Scr" refers to the scramble used as a control.

[0110] [Figure 54] This study demonstrates the luminescence expression levels and expression stability in 1C1C7 cells from circular RNA containing an IRES operably ligated to a sequence encoding Gaussial ciferase and a variable stop codon cassette.

[0111] [Figure 55]This shows the luminescence expression level and expression stability in 1C1C7 cells from circular RNA containing an IRES and a variable untranslated region (UTR) inserted before the start codon of the sequence encoding Gaussial ciferase.

[0112] [Figure 56] This shows the expression level of human erythropoietin (hEPO) in Huh7 cells from a circular RNA containing two miR-122 target sites downstream of the hEPO-coding sequence.

[0113] [Figure 57] This shows the luminescence expression levels in SupT1 cells (from a human T-cell tumor line) and MV4-11 cells (from a human macrophage line) from LNPs transfected with circular RNA encoding firefly luciferase in vitro.

[0114] [Figure 58-1] This paper compares transfected primary human T cell LNPs containing cyclic RNA-dependent ApoE based on different helper lipid, PEG lipid, and ionizable lipid:phosphate ratio formulations. [Figure 58-2] This is a continuation of Figure 58-1.

[0115] [Figure 59] This paper demonstrates the uptake of LNPs containing circular RNA encoding eGFP into activated primary human T cells, with or without the use of ApoE3.

[0116] [Figure 60] This shows immune cell expression from LNPs containing circular RNA encoding Cre fluorescent protein in a Cre reporter mouse model.

[0117] [Figure 61] This shows the expression of mOX40L on immune cells in wild-type mice after intravenous injection of LNPs transfected with circular RNA encoding mOX40L.

[0118] [Figure 62A] This shows a single dose of mOX40L in LNPs transfected with circular RNA capable of expressing mOX40L. It also provides percentages of mOX40L expression in spleen T cells, CD4+ T cells, CD8+ T cells, B cells, NK cells, dendritic cells, and other myeloid cells. [Figure 62B] This shows a single dose of mOX40L in LNPs transfected with circular RNA capable of expressing mOX40L. It also provides percentages of mOX40L expression in spleen T cells, CD4+ T cells, CD8+ T cells, B cells, NK cells, dendritic cells, and other myeloid cells. [Figure 62C] This report shows a single dose of mOX40L in LNPs transfected with circular RNA capable of expressing mOX40L. It also provides changes in mouse body weight 24 hours after transfection.

[0119] [Figure 63] Figure 63A shows B cell depletion by intravenously transfected LNPs with circular RNA in mice. Figure 63B quantifies B cell depletion via B220+ B cells in living CD45+ immune cells. Figure 63B compares B cell depletion by B220+ B cells in living CD45+ immune cells with that of luciferase expressing circular RNA. Figure 63C shows the increase in B cell weight of transfected cells.

[0120] [Figure 64] Figure 64A and Figure 64B show the CAR expression levels in peripheral blood and spleen after treatment with LNPs containing circular RNA expressing anti-CD19 CARs. Anti-CD20 (aCD20) and circular RNA encoding luciferase (oLuc) were used for comparison.

[0121] [Figure 65]Figure 65A shows the overall frequency of anti-CD19 CAR expression, the frequency of anti-CD19 CAR expression on the cell surface, and the impact of IRES-specific circular RNA encoding anti-CD19 CAR on T cells on the antitumor response. Figure 65A shows the geometric mean fluorescence intensity of anti-CD19 CAR, Figure 65B shows the percentage of anti-CD19 CAR expression, and Figure 65C shows the percentage of target cell lysis performed by anti-CD19 CAR. (CK = goat gall virus; AP = apodems spicorniavirus; CK* = codon-optimized goat gall virus; PV = parabovirus; SV = Salivirus).

[0122] [Figure 66] This shows the CAR expression levels of A20 FLuc target cells after treatment with an IRES-specific circular RNA construct.

[0123] [Figure 67] This shows the luminescence expression levels of cytoplasmic (Figure 67A) and surface (Figure 67B) proteins from circular RNA in primary human T cells.

[0124] [Figure 68A] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68B] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68C]This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68D] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68E] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68F] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells. [Figure 68G] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 68A, 68B, and 68G show Gaussian luciferase expression in multiple donor cells. Figures 68C, 68D, 68E, and 68F show firefly luciferase expression in multiple donor cells.

[0125] [Figure 69]The expression of anti-CD19 CAR (Figures 69A and 69B) and anti-BCMA CAR (Figure 68B) in human T cells after treatment of K562 cells expressing firefly luciferase with lipid nanoparticles containing circular RNA encoding either anti-CD19 or anti-BCMA CAR is shown.

[0126] [Figure 70] The anti-CD19 CAR expression levels resulting from in vitro electroporation delivery of circular RNA encoding anti-CD19 CAR in a specific antigen-dependent manner are shown. Figure 70A shows Nalm6 cell lysis by anti-CD19 CAR. Figure 70B shows K562 cell lysis by anti-CD19 CAR.

[0127] [Figure 71A] This shows transfection of LNPs via the use of ApoE3 in a solution containing green fluorescent protein (GFP) expressing LNPs and circular RNA. Figure 71A shows the viability results. Figures 71B, 71C, 71D, and 71E provide expression frequencies from multiple donors. [Figure 71B] This shows transfection of LNPs via the use of ApoE3 in a solution containing green fluorescent protein (GFP) expressing LNPs and circular RNA. Figure 71A shows the viability results. Figures 71B, 71C, 71D, and 71E provide expression frequencies from multiple donors. [Figure 71C] This shows transfection of LNPs via the use of ApoE3 in a solution containing green fluorescent protein (GFP) expressing LNPs and circular RNA. Figure 71A shows the viability results. Figures 71B, 71C, 71D, and 71E provide expression frequencies from multiple donors. [Figure 71D] This shows transfection of LNPs via the use of ApoE3 in a solution containing green fluorescent protein (GFP) expressing LNPs and circular RNA. Figure 71A shows the viability results. Figures 71B, 71C, 71D, and 71E provide expression frequencies from multiple donors. [Figure 71E]This shows transfection of LNPs via the use of ApoE3 in a solution containing green fluorescent protein (GFP) expressing LNPs and circular RNA. Figure 71A shows the viability results. Figures 71B, 71C, 71D, and 71E provide expression frequencies from multiple donors.

[0128] [Figure 72A] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72B] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72C] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72D] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72E] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72F] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72G] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72H] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72I] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72J] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72K] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74. [Figure 72L] This exhibits total flux and anterior center expression of various lipid formulations. See Example 74.

[0129] [Figure 73A]Shows the cyclization efficiency of an RNA molecule encoding a stabilized (double proline mutant) SARS-CoV2 spike protein. Figure 73A shows an in vitro transcription product of a circRNA encoding the SARS-CoV2 spike of approximately 4.5 kb. Figure 73B shows a histogram of spike protein surface expression by flow cytometry after transfection of a circRNA encoding the spike into 293 cells. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody. Figure 73C shows a flow cytometry plot of spike protein surface expression in 293 cells after transfection of a circRNA encoding the spike. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody. [Figure 73B] Shows the cyclization efficiency of an RNA molecule encoding a stabilized (double proline mutant) SARS-CoV2 spike protein. Figure 73A shows an in vitro transcription product of a circRNA encoding the SARS-CoV2 spike of approximately 4.5 kb. Figure 73B shows a histogram of spike protein surface expression by flow cytometry after transfection of a circRNA encoding the spike into 293 cells. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody. Figure 73C shows a flow cytometry plot of spike protein surface expression in 293 cells after transfection of a circRNA encoding the spike. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody. [Figure 73C]Shows the cyclization efficiency of an RNA molecule encoding a stabilized (double proline mutant) SARS-CoV2 spike protein. Figure 73A shows an in vitro transcription product of a circRNA encoding the SARS-CoV2 spike of approximately 4.5 kb. Figure 73B shows a histogram of spike protein surface expression by flow cytometry after transfection of a circRNA encoding the spike into 293 cells. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody. Figure 73C shows a flow cytometry plot of spike protein surface expression in 293 cells after transfection of a circRNA encoding the spike. The transfected 293 cells were stained 24 hours after transfection with a CR3022 primary antibody and an APC-labeled secondary antibody.

[0130] [Figure 74] Provides multiple controlled adjuvant strategies. The CircRNA shown in the figure involves an in vitro unpurified sense circular RNA splicing reaction using GTP as an indicator molecule. 3p-circRNA includes purified sense circular RNA and purified antisense circular RNA containing a triphosphorylated 5' end. Figure 74A shows IFN-β induction in wild-type and MAVS knockout A549 cells in vitro, and Figure 74B shows the in vivo cytokine response to formulated circRNA generated using the indicated strategies.

[0131] [Figure 75A] Shows the intramuscular delivery of LNP containing a circular RNA construct. Provides the whole-body flux in vivo after 6 hours. [Figure 75B] Shows the intramuscular delivery of LNP containing a circular RNA construct. Provides whole-body IVIS 6 hours after a 1 μg dose of the LNP circular RNA construct. [Figure 75C] Shows the intramuscular delivery of LNP containing a circular RNA construct. Provides the ex vivo expression distribution over 24 hours.

[0132] [Figure 76] The expression of multiple circular RNAs from a single lipid formulation is shown. Figure 76A provides hEPO titers from single and mixed sets of LNPs containing circular RNA constructs. Figure 76B provides the total flux of bioluminescent expression from single or mixed sets of LNPs containing circular RNA constructs.

[0133] [Figure 77A] Figure 77A shows the frequency of spike CoV2 expression, Figure 77B shows the geometric mean fluorescence intensity (gMFI) of spike CoV2 expression, and Figure 77C compares the gMFI expression of the construct with the expression frequency. [Figure 77B] Figure 77A shows the frequency of spike CoV2 expression, Figure 77B shows the geometric mean fluorescence intensity (gMFI) of spike CoV2 expression, and Figure 77C compares the gMFI expression of the construct with the expression frequency. [Figure 77C] Figure 77A shows the frequency of spike CoV2 expression, Figure 77B shows the geometric mean fluorescence intensity (gMFI) of spike CoV2 expression, and Figure 77C compares the gMFI expression of the construct with the expression frequency. [Modes for carrying out the invention]

[0134] Detailed explanation Compositions, methods, processes, kits, and devices for the selection, design, preparation, manufacture, formulation, and / or use of circular RNA vaccines are described herein. The present invention further provides compositions, for example, pharmaceutical compositions comprising one or more circular RNA vaccines.

[0135] The circular RNA vaccine of the present invention comprises one or more circular RNA polynucleotides encoding one or more wild-type or engineered proteins, peptides, or polypeptides (e.g., adjuvants and antigens). In some embodiments, the infectious agents from which the adjuvants, adjuvant-like proteins, and antigens are derived or genetically engineered include, but are not limited to, viruses, bacteria, fungi, protozoa, and / or parasites.

[0136] In some embodiments, methods are provided for inducing, triggering, promoting, or causing an immune response in cells, tissues, or organisms, the methods comprising contacting such cells, tissues, or organisms with any of the circular RNA or linear mRNA vaccines described or taught herein.

[0137] Aspects of the present invention provide a circular RNA vaccine comprising one or more RNA polynucleotides having an expression sequence encoding a first antigenic polypeptide. In some embodiments, the circular RNA polynucleotides are formulated within a transport vehicle (e.g., lipid nanoparticles).

[0138] In some embodiments, the expression sequence is codon-optimized. In some embodiments, the first antigenic polypeptide is derived from an infectious agent. In some embodiments, the infectious agent is selected from a group consisting of viral strains and bacterial strains. In some embodiments, one or more RNA polynucleotides encode a further antigenic polypeptide. In some embodiments, the further antigenic polypeptide is encoded by an RNA polynucleotide having a codon-optimized expression sequence.

[0139] In some embodiments, one or more antigenic polypeptides are selected from the proteins or antigenic fragments listed in Table 9. In some embodiments, one or more RNA polynucleotide expression sequences and / or a second RNA polynucleotide expression sequence each independently encode an antigenic polypeptide or antigenic fragment selected from Table 9. In some embodiments, each of the one or more RNA polynucleotide expression sequences is selected from the RNA sequences or antigenic fragments listed in Table 9.

[0140] In some embodiments provided herein, the infectious agent is Adenovirus; Herpes simplex virus type 1; Herpes simplex virus type 2; Encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; Human cytomegalovirus; Human herpesvirus type 8; Human papillomavirus; BK virus; JC virus; Smallpox; Poliovirus; Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus; Norwalk virus; Coxsackievirus; Hepatitis A virus; Poliovirus; Rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; Yellow fever virus; Dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human Immunodeficiency virus (HIV); influenza virus; Gunarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; cortivirus; banavirus; human enterovirus; hantavirus; West Nile virus; Middle East Respiratory Syndrome coronavirus; Japanese encephalitis virus; varicella virus; and Eastern equine encephalitis This is a virus strain selected from a group consisting of the following:

[0141] In some embodiments, the virus is a strain of influenza A or influenza B, or a combination thereof. In some embodiments, the strain of influenza A or influenza B is associated with birds, pigs, horses, dogs, humans, or non-human primates. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. In some embodiments, the hemagglutinin protein does not contain a head domain (HA1). In some embodiments, the hemagglutinin protein contains a portion of the head domain (HA1). In some embodiments, the hemagglutinin protein does not contain a cytoplasmic domain. In some embodiments, the hemagglutinin protein contains a portion of the cytoplasmic domain. In some embodiments, the hemagglutinin protein is a cleaved hemagglutinin protein. In some embodiments, the cleaved hemagglutinin protein includes a portion of the transmembrane domain. In some embodiments, the amino acid sequence of the hemagglutinin protein or its fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the hemagglutinin amino acid sequences provided in Table 9. In some embodiments, the virus is selected from the group consisting of H1N1, H3N2, H7N9, and H10N8.

[0142] In some embodiments, the infectious agent is a bacterial strain selected from Mycobacterium tuberculosis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, and Acinetobacter baumannii. In some embodiments, the bacteria are resistant to one or more antibiotics. In some embodiments, the bacteria are Clostridium difficile. In some embodiments, C. difficile is clindamycin resistant and / or fluoroquinolone resistant. In some embodiments, the bacteria are S. aureus. In some embodiments, S. aureus is methicillin resistant and / or vancomycin resistant.

[0143] In some embodiments, the circular RNA polynucleotide comprises more than one expression sequence. In some embodiments, the expression sequence may encode more than one antigenic polypeptide. In some embodiments, the expression sequence of one or more RNA polynucleotides encodes at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic polypeptides. In some embodiments, the expression sequence of one or more RNA polynucleotides encodes at least 10, 15, 20, or 50 antigenic polypeptides. In some embodiments, the expression sequence of one or more RNA polynucleotides encodes 2 - 10, 10 - 15, 15 - 20, 20 - 50, 50 - 100, or 100 - 200 antigenic polypeptides.

[0144] In some embodiments, the circular RNA polynucleotide comprises only naturally occurring nucleic acids.

[0145] Additional embodiments provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective dose of one of the vaccines described herein to produce an antigen-specific immune response. In some embodiments, the antigen-specific immune response includes a T-cell response. In some embodiments, the antigen-specific immune response includes a B-cell response. In some embodiments, the method for producing an antigen-specific immune response involves a single dose of the vaccine. In some embodiments, the method further comprises administering one or more booster doses of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0146] The embodiments also provide any of the vaccines described herein for use in a method for inducing an antigen-specific immune response in a subject. In some embodiments, the method involves administering the vaccine to a subject in an amount effective to produce an antigen-specific immune response. In some embodiments, the circular RNA vaccine is administered in an effective dose using a dosing schedule such that at least one symptom or feature of an infectious disease is reduced in intensity, severity, or frequency, or its onset is delayed.

[0147] Another aspect provides the use of any of the vaccines described herein in the manufacture of a pharmaceutical product for use in a method of inducing an antigen-specific immune response in a subject, the method comprising administering the vaccine to the subject in an amount effective to produce an antigen-specific immune response.

[0148] In some embodiments, adjuvant polypeptides include Toll-like receptor ligands, cytokines, FLt3-ligands, antibodies, chemokines, chimeric proteins, endogenous adjuvants released from dying tumors, and checkpoint inhibitor proteins. In specific embodiments, adjuvant polypeptides are proteins that directly or indirectly stimulate T cells, B cells, NK cells, or myeloid cells. In specific embodiments, adjuvant polypeptides increase antigen peptide expression or the uptake, processing, and presentation of MHC complexes on antigen-presenting cells. In specific embodiments, adjuvant polypeptides can block MHC through downregulation.

[0149] In some embodiments, one or more adjuvant polypeptides are selected from the proteins or adjuvant fragments listed in Table 10. In some embodiments, one or more RNA polynucleotide expression sequences and / or a second RNA polynucleotide expression sequence each independently encode an adjuvant polypeptide or adjuvant fragment selected from Table 10. In some embodiments, each of the one or more RNA polynucleotide expression sequences is selected from the RNA sequences or adjuvant fragments listed in Table 10.

[0150] In certain embodiments, a vector for producing a circular RNA is provided herein, comprising a 5' double-stranding region, a 3' group I intron fragment, an optional first spacer, an intra-sequence ribosome entry site (IRES), an expression sequence, an optional second spacer, a 5' group I intron fragment, and a 3' double-stranding region. In some embodiments, these elements are arranged in the vector in the order described above. In some embodiments, the vector further comprises an internal 5' double-stranding region between the 3' group I intron fragment and the IRES, and an internal 3' double-stranding region between the expression sequence and the 5' group I intron fragment. In some embodiments, the internal double-stranding regions can form a double helix between themselves but not with an external double-stranding region. In some embodiments, the internal double-stranding regions are part of the first and second spacers. Additional embodiments include circular RNA polynucleotides comprising a circular RNA polynucleotide produced using the vector provided herein, compositions comprising such circular RNA, cells comprising such circular RNA, and methods for using and producing such vectors, circular RNA, compositions, and cells.

[0151] In some embodiments, methods are provided herein that include administering cyclic RNA polynucleotides provided herein to cells for therapeutic or useful protein production. In some embodiments, the method is advantageous in that it provides the production of a desired polypeptide in eukaryotic cells with a longer half-life than linear RNA because the cyclic RNA is resistant to ribonucleases.

[0152] Because circular RNA polynucleotides lack the free ends required for exonuclease-mediated degradation, they offer resistance to several mechanisms of RNA degradation and a longer half-life compared to equivalent linear RNA. Circularization can stabilize RNA polynucleotides, which generally have short half-lives, potentially improving the overall efficacy of exogenous mRNA in various applications. In one embodiment, the functional half-life of the circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells such as human cells), as evaluated by protein synthesis, is at least 20 hours (e.g., at least 80 hours).

[0153] definition As used herein, the terms “circRNA,” “cyclic polyribonucleotide,” or “cyclic RNA” are interchangeable and refer to polyribonucleotides that form a cyclic structure through covalent bonds.

[0154] As used herein, the term “3' group I intron fragment” refers to a sequence having 75% or greater similarity to the 3' proximal end of a natural group I intron, including a splice site dinucleotide and optionally a stretch of the natural exon sequence.

[0155] As used herein, the term “5' group I intron fragment” refers to a sequence having 75% or greater similarity to the 5' proximal end of a natural group I intron, including a splice site dinucleotide and optionally a stretch of the natural exon sequence.

[0156] As used herein, the term “permutation site” refers to a site within a group I intron where a cleavage occurs prior to the permutation of the intron. This cleavage produces 3' and 5' group I intron fragments, which are permuted onto either side of the stretch of the circularized precursor RNA.

[0157] As used herein, the term “splice site” refers to a dinucleotide that is partially or completely contained within a group I intron and in which a phosphodiester bond is cleaved during RNA cyclization.

[0158] Expression sequences in polynucleotide constructs can be separated by "cleavage site" sequences, allowing the polypeptide encoded by these sequences to be expressed in cells as distinct and separate polypeptides once translated.

[0159] A "self-cleaving peptide" refers to a peptide that is translated without a peptide bond between two adjacent amino acids, or functions to be immediately cleaved or separated into distinct first and second polypeptides without requiring any external cleavage activity (e.g., enzymatic cleavage) when a polypeptide containing a protein and a self-cleaving peptide is produced.

[0160] As used herein, the term “therapeutic protein” means any protein that, when administered directly or indirectly to a subject in the form of translated nucleic acid, has therapeutic, diagnostic, and / or prophylactic effects, and / or elicits desirable biological and / or pharmacological effects.

[0161] The α and β chains of the αβ TCR are generally considered to each have two domains or regions, namely a variable domain and a constant domain / region. The variable domain consists of a linkage of a variable region and a binding region. Therefore, in this specification and in the claims, the term “TCR alpha variable domain” refers to the linkage of the TCR alpha variable (TRAV) and TCR alpha binding (TRAJ) regions, and the term “TCR alpha constant domain” refers to the extracellular TCR alpha constant (TRAC) region or a C-terminally cleaved TRAC sequence. Similarly, the term “TCR beta variable domain” refers to the linkage of the TCR beta variable (TRBV), TCR beta diversity (TRBD), and TCR beta binding (TRBJ) regions, and the term “TCR beta constant domain” refers to the extracellular TCR beta constant (TRBC) region or a C-terminally cleaved TRBC sequence.

[0162] As used herein, the term “immunogenic” refers to an immune response to a substance. An immune response may be induced when an organism’s immune system or certain types of immune cells are exposed to an immunogenic substance. The term “non-immunogenic” refers to an immune response exceeding a detectable threshold to a substance. An immune response is undetectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic substance. In some embodiments, non-immunogenic cyclic polyribonucleotides, such as those provided herein, do not induce an immune response exceeding a predetermined threshold when measured by an immunogenic assay. In some embodiments, an innate immune response is undetectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic cyclic polyribonucleotide, such as those provided herein. In some embodiments, an adaptive immune response is undetectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic cyclic polyribonucleotide, such as those provided herein.

[0163] As used herein, the term "cyclization efficiency" refers to a measured value of the resulting cyclic polyribonucleotide compared to its linear starting material.

[0164] As used herein, the term “translation efficiency” refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of transcript encoding a protein or peptide.

[0165] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include species including purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, and pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Nucleotide analogs include nucleotides having modifications to the chemical structure of a base, sugar, and / or phosphate, including 5'-pyrimidine modifications, 8'-purine modifications, cytosine extra-ring amine modifications, and 5-bromouracil substitutions; and 2'-sugar modifications, including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH group is substituted with a group such as H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN (where R is an alkyl moiety as defined herein). Nucleotide analogs are also intended to include nucleotides accompanied by bases, such as inosine, quasine, and xanthine; sugars, such as 2'-methylribose; and unnatural phosphodiester linkages, such as methylphosphonic acid, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseuduridine, and 6-methyladenosine.

[0166] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and describe polymers composed of nucleotides, such as deoxyribonucleotides or ribonucleotides, of any length, e.g., more than about 2 nucleotides, more than about 10 nucleotides, more than about 100 nucleotides, more than about 500 nucleotides, more than 1000 nucleotides, or up to about 10,000 nucleotides or more, which can be produced enzymatically or synthetically (as described, e.g., in U.S. Patent No. 5,948,902 and the references cited herein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids, e.g., participate in Watson-Crick base-pairing interactions. Naturally occurring nucleic acids are composed of nucleotides containing guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0167] As used herein, the terms “ribonucleic acid” and “RNA” mean polymers composed of ribonucleotides.

[0168] As used herein, the terms “deoxyribonucleic acid” and “DNA” mean polymers composed of deoxyribonucleotides.

[0169] "Isolated" or "purified" generally refers to the isolation of a substance (e.g., in some embodiments, a compound, polynucleotide, protein, polypeptide, polynucleotide composition, or polypeptide composition) such that it constitutes a significant percentage (e.g., more than 1%, more than 2%, more than 5%, more than 10%, more than 20%, more than 50%, or more, typically up to about 90%–100%) of the sample in which it is present. In certain embodiments, substantially purified components constitute at least 50%, 80%–85%, and 90%–95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-dependent precipitation. Generally, a substance is purified when it is present in the sample in amounts greater than naturally occurring compared to other components of the sample.

[0170] As used herein, the terms “double-stranded,” “double-stranded,” or “hybridized” refer to nucleic acids formed by the hybridization of two single-stranded nucleic acids containing complementary sequences. In most cases, genomic DNA is double-stranded. Sequences can be fully complementary or partially complementary.

[0171] As used herein, “unstructured” with respect to RNA refers to an RNA sequence that is not predicted by RNAFold software or similar predictive tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease-protected assays.

[0172] As used herein, “structured” with respect to RNA means an RNA sequence that is predicted by RNAFold software or similar predictive tools to form a structure (e.g., a hairpin loop) with itself or with other sequences within the same RNA molecule.

[0173] As used herein, two “double-chain forming regions,” “homologous arms,” or “homologous regions” are complementary or complementary to each other if the two regions share a sufficient level of sequence identity with respect to each other’s reverse complement in order to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences have “homology” if they are identical or share sequence identity with respect to their reverse complement or “complementary” sequences. The percentage of sequence identity between a double-chain forming region and the reverse complement of the corresponding double-chain forming region can be any percentage of sequence identity that allows hybridization to occur. In some embodiments, an internal double-chain forming region of a polynucleotide of the present invention may form a double chain with another internal double-chain forming region but not with an external double-chain forming region.

[0174] Linear nucleic acid molecules are said to have a "5' end" and a "3' end" because nucleic acid phosphodiester linkages occur at the 5' and 3' carbon atoms of the sugar portion of the substituted mononucleotide. The terminal nucleotide of a polynucleotide where the new linkage is at the 5' carbon is its 5' terminal nucleotide. The terminal nucleotide of a polynucleotide where the new linkage is at the 3' carbon is its 3' terminal nucleotide. As used herein, a terminal nucleotide is a nucleotide located at the 3' or 5' end.

[0175] "Transcription" means the formation or synthesis of RNA molecules by RNA polymerase using DNA molecules as templates. The present invention is not limited to the RNA polymerase used for transcription. For example, in some embodiments, T7 type RNA polymerase can be used.

[0176] "Translation" refers to the formation of polypeptide molecules by ribosomes based on an RNA template.

[0177] It should be understood that the terms used herein are intended solely to describe and not to limit specific embodiments. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “cell” includes a combination of two or more cells, or an entire cell culture; a reference to “polynucleotide” practically includes many copies of that polynucleotide. Where used herein, the term “or” is understood to be inclusive unless specifically stated or evident from the context. Unless defined herein and in the remainder of this specification, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the invention pertains.

[0178] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerance range in the art, for example, within two standard deviations of the mean. “About” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “about.”

[0179] As used herein, the term “encode” broadly refers to any process that uses information about a polymer macromolecule to direct the creation of a second molecule distinct from a first molecule. The second molecule may have a chemical structure different from the chemical properties of the first molecule.

[0180] "Co-administration" means administering the therapeutic agent provided herein in combination with one or more additional therapeutic agents, at a time interval that is sufficiently close to the time that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa.

[0181] As used herein, the terms “treat” and “prevent,” and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, the degree of treatment or prevention that a person skilled in the art would recognize as having potential benefit or therapeutic effect varies depending on the methods disclosed herein. Treatment or prevention may include the treatment or prevention of one or more conditions or symptoms of a disease. Also, for the purposes of this specification, “prevention” may include delaying the onset of a disease, or its symptoms or conditions.

[0182] As used herein, “autoimmunity” is defined as a persistent and progressive immune response to non-infectious autoantigens, distinct from infectious non-self antigens of bacterial, viral, fungal, or parasitic origin that invade and persist within mammals and humans. Autoimmune diseases include scleroderma, Graves' disease, Crohn's disease, Sjörgen's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyglandular endocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, and thyroiditis, as well as systemic autoimmune diseases typically represented by human lupus. As used herein, “autoantigen” or “self-antigen” refers to an antigen or epitope that is specific to a mammal and is immunogenic in that mammal.

[0183] As used herein, the term “expression sequence” may refer to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide, a regulatory nucleic acid, or a non-coding nucleic acid. An exemplary expression sequence encoding a peptide or polypeptide may contain multiple nucleotide triads, each of which may encode an amino acid and be referred to as a “codon.”

[0184] As used herein, “spacer” refers to a region of a polynucleotide sequence ranging from one nucleotide to hundreds or thousands of nucleotides that separates two other elements along the polynucleotide sequence. The sequence may be defined or random. Spacers are typically non-coding. In some embodiments, the spacer includes a double-strand forming region.

[0185] As used herein, “intrasequence ribosome entry site” or “IRES” refers to an RNA sequence or structural element in the size range of 10 nt to 1000 nt or more that can initiate polypeptide translation in the absence of a typical RNA cap structure. IRESs are typically about 500 nt to 700 nt in length.

[0186] As used herein, “miRNA site” refers to a stretch of nucleotides within a polynucleotide that can form a double helix with a native miRNA sequence of at least eight nucleotides.

[0187] As used herein, “endonuclease site” refers to a stretch of nucleotides within a polynucleotide that can be recognized and cleaved by an endonuclease protein.

[0188] As used herein, “bisistronic RNA” refers to a polynucleotide containing two expression sequences that encode two different proteins. These expression sequences are often separated by a cleavable peptide such as a 2A site or an IRES sequence.

[0189] As used herein, the term “co-formulation” refers to a nanoparticle formulation comprising two or more nucleic acids or nucleic acids and other active drugs. Typically, the ratio is defined as equimolar or by a ratio measurement of two or more nucleic acids or nucleic acids and other active drugs.

[0190] As used herein, “transport vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients, etc., that are generally intended for use in connection with the administration of biologically active substances, including nucleic acids.

[0191] As used herein, the term "lipid nanoparticles" refers to a transport vehicle comprising one or more lipids (for example, in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0192] As used herein, the term "cationic lipid" refers to any of many lipid species that have a net positive charge at a selected pH, such as physiological pH.

[0193] As used herein, the term "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid.

[0194] As used herein, the term "anionic lipid" refers to any of the many lipid species that have a net negative charge at a selected pH, such as physiological pH.

[0195] As used herein, the term "ionizable lipid" refers to any of many lipid species that have a net positive charge at a selected pH, such as physiological pH 4, and a neutral charge at other pH levels, such as physiological pH 7.

[0196] The term “antibody” (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain may comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three constant domains, CH1, CH2, and CH3. Each light chain may comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL may contain three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of Ab may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, manipulated antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as “antibody complexes”), heterocomplex antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain variable fragments (scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-bonded variable fragments (sdFv), anti-idiotype (anti-id) antibodies (e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), and any of the antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a polyclonal antibody population.

[0197] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term “antibody” includes, by example, both naturally occurring and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-stranded Abs. Non-human Abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and indicated otherwise in the context, the term “antibody” also includes antigen-binding fragments or moieties of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or moieties, and single-stranded Abs.

[0198] The terms “antigen-binding molecule,” “antigen-binding portion,” or “antibody fragment” refer to any molecule containing the antigen-binding portion (e.g., CDR) of an antibody from which the molecule originates. An antigen-binding molecule may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to BCMA. In further embodiments, the antigen-binding molecule is an antibody fragment containing one or more of its complementarity-determining regions (CDRs) that specifically bind to an antigen. In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule includes or consists of an avimer.

[0199] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the 110–120 amino acids at the amino-terminus of the mature heavy chain, and approximately 90–115 amino acids in the mature light chain, which vary significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called framework regions (FRs). While we do not wish to be bound by any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily involved in antibody-antigen interaction and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework regions (FR).

[0200] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0201] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0202] Several definitions of CDR are commonly used in Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is an intermediate between the two, used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on the analysis of available composite crystal structures. The terms "Kabat numbering" and similar terms are recognized in the art and refer to a system of numbered amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding molecule. In certain embodiments, the CDR of an antibody may be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31–35, which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50–65 (CDR2), and amino acid positions 95–102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), amino acid positions 50–56 (CDR2), and amino acid positions 89–97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.In certain embodiments, the CDR of an antibody may be determined according to the Chothia numbering scheme, which points to the position of the immunoglobulin structural loop (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al, (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al, (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using Kabat numbering rules, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34, the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56, the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102, while the Chothia CDR-L1 loop is located at light chain amino acids 24-34, the Chothia CDR-L2 loop is located at light chain amino acids 50-56, and the Chothia CDR-L3 loop is located at light chain amino acids 89-97. When numbered using the Kabat numbering rules, the termination of the Chothia CDR-HI loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if 35A and 35B are not present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; and if both 35A and 35B are present, the loop terminates at 34). In certain embodiments, the CDR of the antibody described herein is determined according to the Chothia numbering scheme.

[0203] As used herein, the terms “constant region” and “constant domain” are interchangeable and have the meanings commonly understood in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chain of the antibody moiety, for example, which is not directly involved in the binding of the antibody to an antigen but can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0204] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by its dissociation constant (KD or Kd). Affinity can be measured and / or expressed by many methods known to those skilled in the art, including but not limited to the equilibrium dissociation constant (KD) and equilibrium association constant (KA or Ka). KD is calculated from the quotient of koff / kon, and KA is calculated from the quotient of kon / koff. kon refers, for example, to the association rate constant of an antibody to an antigen, and koff refers, for example, to the dissociation of an antibody to an antigen. kon and koff can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0205] As used herein, “conservative amino acid substitution” refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, etc.), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within a CDR or within a framework region of an antibody or its antigen-binding molecule may be replaced with amino acid residues having similar side chains.

[0206] As used herein, the term “heterogeneous sequence” means an exogenous sequence that is either natural or not naturally occurring in the cell or organism expressing the sequence.

[0207] As used herein, “epitope” is a term used in the art and refers to a local region of an antigen to which an antibody can specifically bind. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be composed of, for example, two or more discontinuous regions of a polypeptide (conformity, nonlinear, discontinuous, or discontinuous epitope). In some embodiments, the epitope to which the antibody binds may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenic mapping (e.g., site-directed mutagenic mapping). With regard to X-ray crystallography, crystallization can be achieved using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350, McPherson A (1990) Eur J Biochem 189:1-23, Chayen NE (1997) Structure 5:1269-1274, McPherson A (1976) J Biol Chem 251:6300-6303).Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and purified using computer software such as X-PLOR (Yale University, 1992, popularized by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., U.S. Patent Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60, Bricogne G (1997) Meth Enzymol 276A:361-423, eds Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323).

[0208] When used herein, an antigen-binding molecule, antibody, or its antigen-binding fragment "cross-competes" with a reference antibody or its reference antigen-binding fragment if the interaction between the antigen and the first binding molecule, antibody, or their antigen-binding fragment blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or their reference antigen-binding fragment to interact with the antigen. Cross-competition can be complete, for example, the binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, for example, the binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In some embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. Numerous types of competitive binding assays exist to determine whether one antigen-binding molecule competes with another. Examples include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J.Immunol.137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA with 1-125 labeling (Morel et al., 1988, Molec.Immunol.25:7-15), and solid-phase direct biotin-avidin EIA (Cheung, et al.). (Moldenhauer et al., 1990, Virology 176:546-552), and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) can be used.

[0209] As used herein, the terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are analogous terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) in such a way that such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides at a lower affinity, as determined by, for example, an immunoassay, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen binds to the antigen at a KA of at least 2log, 2.5log, 3log, 4log or more than KA when the molecule binds to another antigen.

[0210] As defined herein, the term “antigen” refers to an antigen-binding molecule, an antibody, or a molecule that binds to an antigen-binding fragment thereof. For example, an antigen can elicit an innate or adaptive immune response in a living organism. An antigen can be any immunogenic substance, particularly proteins, polypeptides, polysaccharides, nucleic acids, lipids, etc. In some embodiments, the antigen is derived from an infectious agent.

[0211] The term "self" refers to any substance originating from the same individual from which the substance is later reintroduced. For example, the manipulated autologous cell therapy (eACT®) method described herein involves the collection of lymphocytes from a patient, which are then manipulated to express, for example, a CAR construct, and then administered to the same patient.

[0212] The term "allogeneic" refers to any substance that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

[0213] As used herein, “cytokines” refers to non-antibody proteins released by one cell that can interact with a second cell and mediate a response in that second cell. As used herein, “cytokines” means proteins released by a population of cells that act on another cell as intercellular mediators. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including but not limited to macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils, and mast cells, to propagate immune responses. Cytokines can induce a variety of cellular responses. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effector cytokines, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-a, IL-lb, IL-6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effector cytokines include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), TGF-beta, IL-35, and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0214] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that are major components of the innate immune system. NK cells can induce apoptosis in tumor and virus-infected cells. They are called “natural killers” because they do not require activation to kill target cells. T cells play a major role in cell-mediated immunity (without antibody involvement). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. Helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, also known as CD8+ T cells or killer T cells), memory T cells ((i) stem memory cells (TSCMs), like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, exhibiting many functional attributes specific to memory cells), (ii) stem There are many types of T cells, including (iii) effector memory cells (TEMs), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ or CD4+FoxP3+ regulatory T cells), natural killer T cells (NKTs), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies, function as antigen-presenting cells (APCs), and, after activation by antigen interaction, can transform into memory B cells and plasma cells, both short-lived and long-lived. In mammals, immature B cells are formed in the bone marrow.

[0215] The terms “genetic manipulation” or “manipulation” refer to methods of modifying a cell’s genome, including but not limited to deleting coding or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In some embodiments, the cells to be modified are lymphocytes, such as T cells, which can be obtained from either a patient or a donor. The cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which are incorporated into the cell’s genome.

[0216] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble molecules (including alpha, cytokines, and complement) produced by these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of vertebrates of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0217] As used herein, the term “sequence identity” refers to the degree to which sequences are identical nucleotide-wise or amino acid-wise across a comparison window. Therefore, the “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, U) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exist to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The nucleotides and polypeptides described herein have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the reference sequences described herein, and typically, in the case of polypeptides, the polypeptide variant maintains the biological activity of at least one polypeptide encoded by the reference sequence.

[0218] As used herein, “adjuvant” refers to a drug or substance that modulates the immunogenicity of an antigen.

[0219] As used herein, “vaccine” means a composition, substance, or preparation that stimulates, induces, causes, or improves immunity in an organism, e.g., an animal organism, e.g., a mammalian organism (e.g., a human). In some embodiments, a vaccine provides immunity to one or more diseases or disorders in an organism, including prophylactic and / or therapeutic immunity. In some embodiments, a vaccine can be made from antigens from living, attenuated, modified, weakened, or dead forms of disease-causing microorganisms or derived therefrom, e.g., combinations of antigenic components. In some embodiments, a vaccine stimulates, induces, causes, or improves immunity in an organism, or causes or mimics an immune response in an organism, without inducing any disease or disorder. In some embodiments, a vaccine elicits an immune response after being introduced into a target tissue, extracellular space, or cell. In some embodiments, the polynucleotides of the present invention may encode an antigen, and when the polynucleotides are expressed in a cell, the expressed antigen elicits a desired immune response.

[0220] Vectors, precursor RNA, and circular RNA In certain embodiments, a circular RNA polynucleotide comprising a post-splicing 3' group I intron fragment, optionally a first spacer, an intrasequence ribosome entry site (IRES), an expression sequence, optionally a second spacer, and a post-splicing 5' group I intron fragment is provided herein. In some embodiments, these regions are in this order. In some embodiments, the circular RNA is prepared by the methods provided herein or from vectors provided herein.

[0221] In certain embodiments, transcription of a vector provided herein (e.g., comprising a 5' double-strand formation region, a 3' group I intron fragment, an optional first spacer, an intra-sequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, an optional second spacer, a 5' group I intron fragment, and a 3' double-strand formation region) results in the formation of a precursor linear RNA polynucleotide that can be cyclized. In some embodiments, this precursor linear RNA polynucleotide is cyclized when incubated in the presence of a guanosine nucleotide or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg2+).

[0222] In some embodiments, the vectors and precursor RNA polynucleotides provided herein include a first (5') double-stranding region and a second (3') double-stranding region. In certain embodiments, the first and second double-stranding regions may form a complete or incomplete double helix. Thus, in certain embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second double-stranding regions may base-pair with each other. In some embodiments, the double-stranding regions are expected to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base-pairing with unintended sequences in the RNA (e.g., non-double-stranding region sequences). In some embodiments, the double-stranding region is located at the end of the precursor RNA strand and is adjacent to or very close to a group I intron fragment, thereby bringing the group I intron fragments closer together and increasing splicing efficiency. In some embodiments, the double-stranding region is 3 to 100 nucleotides long (e.g., 3 to 75 nucleotides, 3 to 50 nucleotides, 20 to 50 nucleotides, 35 to 50 nucleotides, 5 to 25 nucleotides, 9 to 19 nucleotides). In some embodiments, the double-stranding region is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the double-stranding region has a length of about 9 to about 50 nucleotides. In one embodiment, the double-stranding region has a length of about 9 to about 19 nucleotides. In some embodiments, the double-stranding region has a length of about 20 to about 40 nucleotides. In a particular embodiment, the double-stranding region has a length of about 30 nucleotides.

[0223] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include a first (5') and / or a second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES can preserve secondary structure in those regions by preventing them from interacting and thus increasing splicing efficiency. In some embodiments, the first (between the 3' group I intron fragment and the IRES) and second (between the expression sequence and the 5' group I intron fragment) spacers include additional base-pairing regions that are not expected to base-pair with respect to the first and second double-stranding regions. In some embodiments, such spacer base-pairing brings the group I intron fragments closer together, further increasing splicing efficiency. In addition, in some embodiments, the combination of base-pairing between the first and second double-stranding regions, and separately, between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragment adjacent to the base-pairing region. A typical spacer is a contiguous sequence containing one or more of the following qualities: 1) expected to avoid interference with proximal structures, e.g., IRES, expression sequences, or introns; 2) at least 7 nt in length and no more than 100 nt; 3) located after and adjacent to a 3' intron fragment and / or before and adjacent to a 5' intron fragment; and 4) below: a) an unstructured region of at least 5 nt in length; b) a base-pairing region of at least 5 nt in length in a distal sequence containing another spacer; and c) a structured region of at least 7 nt in length, limited to the sequence of the spacer. A spacer may have several regions, including an unstructured region, a base-pairing region, a hairpin / structured region, and combinations thereof. In some embodiments, the spacer has a structured region with a high GC content. In some embodiments, a region within a spacer base pairs with another region within the same spacer. In some embodiments, a region within a spacer base pairs with a region within another spacer.In some embodiments, the spacer comprises one or more hairpin structures. In some embodiments, the spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In some embodiments, an additional spacer is present between the 3' group I intron fragment and the IRES. In some embodiments, this additional spacer prevents, or reduces to the extent that, the structuring region of the IRES interferes with the folding of the 3' group I intron fragment. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides or less long. In some embodiments, the 5' spacer sequence is 5–50, 10–50, 20–50, 20–40, and / or 25–35 nucleotides long. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In one embodiment, the 5' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a poly-AC sequence. In one embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% poly-AC content. In another embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0224] In certain embodiments, the 3' group I intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 3' proximal fragment of a native group I intron, which includes a 3' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. Typically, a 5' group I intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5' proximal fragment of a natural group I intron, which includes a 5' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. As described by Umekage et al. (2012), the outer portions of the 3' group I intron fragment and the 5' group I intron fragment are removed during circulation, so that the circular RNA provided herein contains only portions of the 3' group I intron fragment formed by any exon sequence of at least 1 nt in length and the 5' group I intron fragment formed by any exon sequence of at least 1 nt in length, provided that such sequences are present in the non-circularized precursor RNA. The portion of the 3' group I intron fragment retained by the circular RNA is referred herein to as the "post-splicing 3' group I intron fragment." The portion of the 5' group I intron fragment retained by the circular RNA is referred herein to as the "post-splicing 5' group I intron fragment."

[0225] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include intrasequence ribosome entry sites (IRESs). The inclusion of IRESs enables the translation of one or more open reading frames (e.g., open reading frames forming an expression sequence) from the circular RNA. The IRES elements attract the eukaryotic ribosome translation initiation complex, thereby facilitating translation initiation. For example, see Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298, Rees et al., BioTechniques (1996) 20:102-110, Kobayashi et al., BioTechniques (1996) 21:399-402, and Mosser et al., BioTechniques 1997 22 150-161.

[0226] A large number of IRES sequences are available, including sequences derived from a wide variety of viruses, such as leader sequences from picornaviruses like encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), polio leader sequences, hepatitis A virus leaders, hepatitis C virus IRESs, human rhinovirus type 2 IRESs (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), and giardiavirus IRESs (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397).

[0227] In some embodiments, IRES includes Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliopathy virus, human poliovirus 1, Plautia stali enteric virus, Casimir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, and Ectropis Obliqua picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic spot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, Hi BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S.Cerevisiae YAP1, Tobacco HTC virus, Kabukurinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirnavirus, HCV QC64, Hitocosavirus E / D, Hitocosavirus F, Hitocosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A virus HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C These are IRES sequences of aptamers for K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Saperovirus, Rosavirus B, Vakunsavirus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Sissinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Disicisthovirus, Hupey Picorna-like Virus, CRPV, Sarivirus A BN5, Sarivirus A BN2, Sarivirus A 02394, Sarivirus A GUT, Sarivirus A CH, Sarivirus A SZ1, Sarivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0228] In some embodiments, the polynucleotides herein include one or more expression sequences.

[0229] In certain embodiments, the vectors provided herein include a 3'UTR. In some embodiments, the 3'UTR is derived from human betaglobin, human alphaglobin, xenops betaglobin, xenops alphaglobin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFα, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chloroplast virus, woodchuck hepatitis virus, hepatitis virus post-translational regulatory element, Sindbis virus, turnip crinkle virus, tobacco etching virus, or Venezuelan encephalitis virus.

[0230] In some embodiments, the vectors provided herein include a 5'UTR. In some embodiments, the 5'UTR is derived from human betaglobin, African clawed frog betaglobin, human alphaglobin, African clawed frog alphaglobin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etching virus, cabbage virus, or adenovirus ternary leader.

[0231] In some embodiments, the vectors provided herein include a poly-A region. In some embodiments, the poly-A region is at least 12 nucleotides long, at least 30 nucleotides long, or at least 60 nucleotides long.

[0232] In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are of lengths of 300-15000, 300-14000, 300-13000, 300-12000, 300-11000, 300-10000, 400-9000, 500-8000, 600-7000, 700-6000, 800-5000, 900-5000, 1000-5000, 1100-5000, 1200-5000, 1300-5000, 1400-5000, and / or 1500-5000 nucleotides. In some embodiments, the polynucleotide is at least 300nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, 1100nt, 1200nt, 1300nt, 1400nt, 1500nt, 2000nt, 2500nt, 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, 10000nt, 11000nt, 12000nt, 13000nt, 14000nt, or 15000nt in length. In some embodiments, the polynucleotides are 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, 10000nt, 11000nt, 12000nt, 13000nt, 14000nt, 15000nt, or 16000nt or less in length. In some embodiments, the lengths of the DNA, linear RNA, and / or circular RNA polynucleotides provided herein are approximately 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10000 nt, 11000 nt, 12000 nt, 13000 nt, 14000 nt, or 15000 nt.

[0233] In some embodiments, vectors are provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' double helix formation region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) an optional second spacer sequence, i) a 5' group I intron fragment, and j) a 3' double helix formation region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' double helix formation region.

[0234] In some embodiments, vectors are provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' double-strand forming region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) an optional second spacer sequence, i) a 5' group I intron fragment, and j) a 3' double-strand forming region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' double-strand forming region.

[0235] In some embodiments, precursor RNA is provided herein. In certain embodiments, the precursor RNA is linear RNA produced by in vitro transcription of a vector provided herein. In some embodiments, the precursor RNA comprises, in the following order: a) any 5' double helix region, b) a 3' group I intron fragment, c) any first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) any second spacer sequence, i) a 5' group I intron fragment, and j) any 3' double helix region. In some embodiments, the precursor RNA comprises, in the following order: a) a 5' double helix region, b) a 3' group I intron fragment, c) any first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) any second spacer sequence, i) a 5' group I intron fragment, and j) a 3' double helix region. Precursor RNA can be unmodified, partially modified, or completely modified.

[0236] In certain embodiments, circular RNA is provided herein. In certain embodiments, circular RNA is circular RNA produced by a vector provided herein. In some embodiments, circular RNA is circular RNA produced by cyclization of a precursor RNA provided herein. In some embodiments, circular RNA comprises the following sequences: a) a first spacer sequence, b) IRES, c) a first expression sequence, d) a polynucleotide sequence encoding a cleavage site, e) a second expression sequence, and f) an optional second spacer sequence. In some embodiments, circular RNA comprises the following sequences: a) a post-splicing 3' group I intron fragment, b) a first spacer sequence, c) IRES, d) a first expression sequence, e) a polynucleotide sequence encoding a cleavage site, f) a second expression sequence, and g) a second spacer sequence, and h) a post-splicing 5' group I intron fragment. In some embodiments, the circular RNA comprises the following sequences: a) a first spacer sequence, b) a first IRES, c) a first expression sequence, d) a second IRES, e) a second expression sequence, and f) an optional second spacer sequence. In some embodiments, the circular RNA further comprises a portion of a 3' group I intron fragment that is the 3' of the 3' splice site. In some embodiments, the circular RNA further comprises a portion of a 5' group I intron fragment that is the 5' of the 5' splice site. In some embodiments, the circular RNA is of a size of at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 nucleotides. The circular RNA can be unmodified, partially modified, or fully modified.

[0237] In some embodiments, the circular RNAs provided herein have higher functional stability than mRNAs containing the same expression sequence.

[0238] In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of 5–80, 10–70, 15–60, and / or 20–50 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a longer functional half-life (e.g., at least 1.5 times longer, at least 2 times longer) than equivalent linear RNA polynucleotides encoding the same protein. In some embodiments, the functional half-life can be evaluated through detection of functional protein synthesis.

[0239] In some embodiments, the cyclic RNA polynucleotides provided herein have a half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a half-life of 5–80, 10–70, 15–60, and / or 20–50 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a longer half-life (e.g., at least 1.5 times longer, at least 2 times longer) than an equivalent linear RNA polynucleotide encoding the same protein.

[0240] In some embodiments, the circular RNAs provided herein may have a higher degree of expression than equivalent linear mRNAs, for example, a higher degree of expression 24 hours after administration of the RNA to cells. In some embodiments, the circular RNAs provided herein have a higher degree of expression than mRNAs containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail.

[0241] In some embodiments, the circular RNAs provided herein may be less immunogenic than equivalent mRNAs when exposed to the immune system of an organism or a particular type of immune cell. In some embodiments, the circular RNAs provided herein are associated with the regulation of cytokine production when exposed to the immune system of an organism or a particular type of immune cell. For example, in some embodiments, the circular RNAs provided herein are associated with reduced production of TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferons, e.g., IFN-β1, compared to mRNAs containing the same expression sequence, when exposed to the immune system of an organism or a particular type of immune cell. In some embodiments, the circular RNAs provided herein are associated with less transcription induction of TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferons, e.g., IFN-β1, compared to mRNAs containing the same expression sequence, when exposed to the immune system of an organism or a particular type of immune cell. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs containing the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail.

[0242] In some embodiments, the compositions and methods described herein provide RNA (e.g., circRNA) that has higher stability or functional stability than equivalent linear RNA without requiring nucleoside modification. In some embodiments, methods for producing nucleoside-deficient RNA produce a higher proportion of full-length transcripts than methods for producing RNA-containing nucleoside modifications resulting from a reduction in sterile transcription. In some embodiments, the compositions and methods described herein can produce large (e.g., 5kb-15kb, 6kb-15kb, 7kb-15kb, 8kb-15kb, 9kb-15kb, 10kb-15kb, 11kb-15kb, 12kb-15kb, 13kb-15kb, 14kb-15kb, 5kb-10kb, 6kb-10kb, 7kb-10kb, 8kb-10kb, 9kb-10kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, or 15kb) RNA constructs without adding sterile transcriptions related to RNA containing nucleoside modifications.

[0243] In certain embodiments, the circular RNA provided herein can be transfected directly into cells, or it can be transfected in the form of a DNA vector and transcribed within the cell. The transcription of the circular RNA from the transfected DNA vector can be mediated by an additional polymerase or a polymerase encoded by the nucleic acid transfected into the cell, or preferably by an endogenous polymerase.

[0244] In certain embodiments, the cyclic RNA polynucleotides provided herein comprise modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleosides are m 5 It is C(5-methylcytidine). In another embodiment, the modified nucleoside is m 5 In another embodiment, the modified nucleoside is m 6 A(N 6 (methyladenosine). In another embodiment, the modified nucleoside is s 2It is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2′-O-methyluridine). In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2’-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio-N 6 isopentenyladenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A (2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A (N 6 -glycylcarbamoyladenosine); t 6 A (N 6 -threonylcarbamoyladenosine); ms 2 t 6 A (2-methylthio-N 6 -threonylcarbamoyladenosine); m 6 t 6 A (N 6 -methyl-N 6 -threonylcarbamoyladenosine); hn 6 A (N 6 -hydroxynorvalylcarbamoyladenosine); ms 2 hn 6 A (2-methylthio-N 6 -hydroxynorvalylcarbamoyladenosine); Ar(p)(2’-O-ribosyladenosine(phosphate)); I (inosine); m 1 I (1-methylinosine); m 1 Im (1,2’-O-dimethylinosine); m 3C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine); ac 4 C(N 4 -acetylcytidine);f 5 C(5-formylcytidine); m 5 Cm(5,2′-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lysidine); m 1 G(1-methylguanosine); m 2 G(N 2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2′-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine); m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine);Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(unmodified hydroxywybutosine);imG(wyosine);mimG(methylwyosine);Q(keuosine);oQ(epoxykeuosine);galQ(galactosyl-keuosine);manQ(mannosyl-keuosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Alkaeosin); D (Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine); m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5U(5-Hydroxyuridine); mo 5 U(5-Methoxyuridine); cmo 5 U(Uridine 5-Oxyacetic acid); mcmo 5 U(Uridine 5-Oxyacetic acid methyl ester); chm 5 U(5-(Carboxyhydroxymethyl)uridine)); mchm 5 U(5-(Carboxyhydroxymethyl)uridine methyl ester); mcm 5 U(5-Methoxycarbonylmethyluridine); mcm 5 Um(5-Methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-Methoxycarbonylmethyl-2-thiouridine); nm 5 S 2 U(5-Aminomethyl-2-thiouridine); mnm 5 U(5-Methylaminomethyluridine); mnm 5 s 2 U(5-Methylaminomethyl-2-thiouridine); mnm 5 se 2 U(5-Methylaminomethyl-2-selenouridine); ncm 5 U(5-Carbamoylmethyluridine); ncm 5 Um(5-Carbamoylmethyl-2′-O-methyluridine); cmnm 5 U(5-Carboxymethylaminomethyluridine); cmnm 5 Um(5-Carboxymethylaminomethyl-2′-O-methyluridine); cmnm 5 s 2 U(5-Carboxymethylaminomethyl-2-thiouridine); m 6 2A(N 6 ,N 6 ,N-Dimethyladenosine); Im(2'-O-methylinosine); m 4 C(N 4 -Methylcytidine); m 4 Cm(N 4 ,2'-O-Dimethylcytidine); hm 5 C(5-Hydroxymethylcytidine); m 3 U(3-Methyluridine); cm5 U(5-carboxymethyluridine); m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine);m 2,7 G(N 2 ,7-dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine); f 5 Cm(5-formyl-2'-O-methylcytidine);m 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine); τm 5 U(5-taurinomethyluridine); τm 5 s 2 U(5-taurinomethyl-2-thiouridine)); imG-14(4-demethylyosin); imG2(isoyosin); or ac 6 A(N 6 -Acetyladenosine)

[0245] In some embodiments, the modified nucleosides include pyridine-4-onribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, and 5-taurinomethyl-2-thiouridine. , 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseuduridine, 4-methoxy-2-thio- Pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine N, Zebralin, 5-Aza-Zebralin, 5-Methyl-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopurine, 7-Deaza-Adenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-Aminopurine, 7-Deaza-8-Aza-2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-Hydroxyisopentenyl)adenosine, 2-Methylthio-N6-(cis-Hydroxyisopentenyl)adenosine, N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-Adenine, 2-Methoxy-Adenine, Inosine, 1-Methyl-Inosine, Wyosin, Wyobutosin, 7-Deaza-Guano Compounds may be selected from the group consisting of cin, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In another embodiment, the modification is independently selected from the group consisting of 5-methylcytosine, pseudouridine, and 1-methylpseudridine.

[0246] In some embodiments, the modified ribonucleoside includes 5-methylcytidine, 5-methoxyuridine, 1-methylpseudridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified nucleosides provide further stability and resistance to immunoactivation.

[0247] In certain embodiments, polynucleotides can be codon-optimized. A codon-optimized sequence may be a sequence in which the codons of a polynucleotide encoding a polypeptide are substituted to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variations in the degree of codon bias within an organism, gene, or set of genes; (iii) phylogenetic variations of codons, including context; (iv) variations in codons according to their decoded tRNAs; (v) variations in codons according to GC% at either the whole of a triplet or at one position; (vi) variations in similarity to a reference sequence, such as a naturally occurring sequence; (vii) variations in codon frequency cutoffs; (viii) structural characteristics of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of a DNA sequence based on the design of a codon substitution set; and / or (x) phylogenetic variations of the codon set of each amino acid. In some embodiments, codon-optimized polynucleotides can minimize ribozyme collisions and / or limit structural interference between the expression sequence and IRES.

[0248] In certain embodiments, the circular RNAs provided herein are produced inside a cell. In some embodiments, the precursor RNA is transcribed in the cytoplasm by bacteriophage RNA polymerase or in the nucleus by host RNA polymerase II using a DNA template (for example, using a vector provided herein in some embodiments) and then circularized.

[0249] In certain embodiments, the circular RNA provided herein is injected into an animal (e.g., a human) so that the polypeptide encoded by the circular RNA molecule is expressed inside the animal.

[0250] payload The circular RNA vaccine of the present invention comprises one or more circular RNA polynucleotides encoding one or more wild-type or engineered proteins, peptides, or polypeptides (e.g., antigens, adjuvants, or adjuvant-like proteins). In some embodiments, one or more circular RNA polynucleotides encode an antigen or adjuvant derived from an infectious agent. In some embodiments, the infectious agent from which the antigen or adjuvant is derived or genetically engineered includes, but is not limited to, viruses, bacteria, fungi, protozoa, and / or parasites. In some embodiments, the antigen is a viral antigen. In some embodiments, the antigen is a SARS-CoV-2 antigen. In some embodiments, the antigen is a SARS-CoV-2 spike protein.

[0251] In some embodiments, the circular RNA polynucleotide comprises one or more expression sequences. In some embodiments, the expression sequences may encode one or more antigenic polypeptides. In some embodiments, one or more RNA polynucleotide expression sequences encode at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic polypeptides. In some embodiments, one or more RNA polynucleotide expression sequences encode at least 10, 15, 20, or 50 antigenic polypeptides. In some embodiments, one or more RNA polynucleotide expression sequences encode 2-10, 10-15, 15-20, 20-50, 50-100, or 100-200 antigenic polypeptides.

[0252] In one embodiment, the antigens include rotavirus, foot-and-mouth disease virus, influenza A virus, influenza B virus, influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza type 2, herpes simplex virus, Epstein-Barr virus, varicella virus, porcine herpesvirus 1, cytomegalovirus, lyssavirus, anthrax, anthrax PA and derivatives, poliovirus, hepatitis A, hepatitis B, hepatitis C, hepatitis E, distemper virus, Venezuelan encephalomyelitis, and feline leukemia. Disease viruses, reoviruses, syncytial respiratory viruses, Lassa fever virus, polyomatum virus, canine parvovirus, papillomavirus, tick-borne encephalitis virus, rinderpest virus, human rhinovirus species, enterovirus species, mengovirus, paramyxovirus, avian infectious bronchitis virus, human T-cell leukemia / lymphoma virus 1, human immunodeficiency virus 1, human immunodeficiency virus 2, lymphocytic choriomeningitis virus, parvovirus B19, adenovirus, rubella virus, yellow fever virus, dengue virus, bovine syncytial respiratory virus, coronavirus, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis, Brucella abortis, Brucella melitensis, Brucella suis, Brucella ovis, Brucella species, Escherichia coli, Salmonella species, Salmonella typhi, Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella, Pseudomonas, Tuberculosis, avium, Bacille Calmette Guerin, Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella multocida, Chlamydia trachomatis, Chlamydiapsittaci, Lymphogranuloma venereum, Treponema pallidum, Haemophilus species, Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species, Borrelia burgdorferi, Regionalla pneumophila, Colstridium botulinum, Corynebacterium diphtheriae, Yersinia entercolitica, Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes, trypanosomes, Leishmania species, Filarial nematodes, trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albican, Aspergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae, malaria parasite sporozoite surface protein, malaria merozoite protein, trypanosoma surface antigen protein, pertussis, alphavirus, adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, influenza hemagglutinin, cancer antigen, tumor antigen, toxin, Clostridium perfringens Selected from or derived from the group consisting of *Perfringens* ε-toxin, lysine toxin, *Pseudomonas* exotoxin, exotoxins, neurotoxins, cytokines, cytokine receptors, monokines, monokine receptors, plant pollen, animal scales, and house dust mites.

[0253] In some embodiments, the adjuvant is selected from or derived from the group consisting of BCSP31, MOMP, FomA, MymA, ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, rumazine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, pneumolysin, fargerin, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-α, IL-7, IL-17, IL-1β, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and andti-CD3.

[0254] Immunogenic vectors and RNA preparations In some embodiments, the circular RNA vaccine of the present invention comprises one or more circular RNA polynucleotides or linear RNA polynucleotide counterparts capable of eliciting an immune response in cells. Modification or manipulation of non-immunogenic circular RNA polynucleotides can enable adjuvant-like properties (Wesselhoeft, 2019). Similarly, linear RNA polynucleotides can be manipulated to elicit an increased immune response compared to unmanipulated linear RNA polynucleotides. Examples of increased immunogenicity for linear RNA polynucleotides include various capping strategies (Pardi, 2018). Capping strategies include, but are not limited to, incorporating monophosphorylation or triphosphorylation at the terminal 5' end by adding nucleotide monophosphate to an in vivo transcription reaction. In some embodiments, altering the triphosphorylation:monophosphorylation 5' end cap ratio in the RNA preparation can be controlled based on altering the GMP:GTP ratio during in vivo transcription. In other embodiments, an enzyme (e.g., RppH) can be used to control the triphosphorylation:monophosphorylation 5' end cap ratio in the RNA preparation. The monophosphorylation:triphosphorylation ratio in any RNA preparation can be 100:1, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, or 100:1, based on the preferred level of immunogenicity. A higher triphosphorylation:monophosphorylation ratio allows for greater activation of the immune response.

[0255] In some embodiments, monophosphate or triphosphate-containing caps can be produced using synthetic methods based on providing initiator molecules during the development of RNA polynucleotides. In some embodiments, the number of triphosphates at the 5' end of RNA molecules produced by in vitro transcription can be controlled by including specific nucleotides and / or nucleosides in the in vitro transcription reaction. These nucleotides are then used with varying efficiencies as initiator nucleotides / nucleosides for new RNA chains. In the same embodiments, RNA polymerase enzymes (e.g., T7 RNA polymerase) have the ability to probabilistically select initiator nucleotides / nucleosides from available substrates. In some embodiments, by including multiple different initiator nucleosides / nucleotides (e.g., GTP and GMP) in the synthesis, several RNA molecules with 5' monophosphates and several RNA molecules with 5' triphosphates can be obtained. The ratio of initiator nucleotides / nucleosides used and the incorporation rate of specific nucleotides / nucleosides determine the proportion of RNA molecules with specific 5' end identity. In preferred embodiments for generating RNA molecules having a monophosphate 5' end, GMP is added to the T7 RNA polymerase in vitro transcription reaction at a concentration of at least 1x, most preferably 4x, of the GTP initiation concentration. In some embodiments, alternative initiator molecules, such as adenosine nucleotides / nucleosides, may be used, particularly when using alternative RNA polymerase enzymes.

[0256] In another embodiment, the monophosphate or triphosphate-containing cap method may include a splicing method. A guanosine nucleotide / nucleoside may be incorporated before the second splice site dinucleotide of the 5' splice site during group I intron and order substitution group I intron splicing. This nucleotide / nucleoside may contain zero or more phosphate groups at the 5' position. By including several different nucleosides / nucleotides (e.g., GTP and GMP), several intron products having 5' monophosphate and several intron products having 5' triphosphate can be obtained. The ratio of nucleotides / nucleosides used and the utilization rate of a particular nucleotide / nucleoside by the group I intron determine the proportion of RNA molecules having a particular 5' terminal identity. In a preferred embodiment, the ratio of nucleosides / nucleotides used is the same as that used for in vitro transcription of the precursor molecule, and splicing occurs cotranscribely. This ratio can be controlled independently by purifying the precursor RNA molecule from an in vitro transcription reaction and adding the cofactors necessary for splicing along with the desired nucleoside / nucleotide ratio. Group I introns generally accept only guanosine nucleotide / nucleoside as a cofactor, but may accept other nucleotide / nucleosides such as adenosine nucleotide / nucleoside.

[0257] In another embodiment, monophosphate or triphosphate-containing caps may be produced using enzymatic methods. The triphosphate terminus may be converted to a monophosphate or hydroxyl terminus by enzymatic treatment. Treatment of triphosphorylated RNA molecules with RNA 5' pyrophosphohydrolase (RppH) or tabacoate pyrophosphatase (TAP) converts the triphosphorylated terminus to a monophosphorylated terminus, which can then be used for ligation by a ligase enzyme such as T4 RNA ligase I without causing RIG-I. Other phosphatase enzymes, such as calf enteral phosphatase (CIP / CIAP) and shrimp alkaline phosphatase (SAP), remove terminal phosphates, thereby converting terminal monophosphates, diphosphates, or triphosphates to terminal hydroxyl groups. The terminal hydroxyl groups can then be converted to monophosphate groups using kinase enzymes such as T4 polynucleotide kinase (PNK).

[0258] In some embodiments, RNA preparations can be made more immunostimulative by using different structural or RNA polynucleotide formulations in varying proportions. In other embodiments, RNA preparations may contain both non-immunostimulative circular RNA polynucleotides and linear RNA polynucleotides containing a 5' terminal cap or immunostimulatively modified circular RNA polynucleotides. In certain embodiments, RNA preparations contain a circular RNA polynucleotide encoding an adjuvant, antigen, or adjuvant-like protein, along with a linear RNA polynucleotide or immunostimulatively modified circular RNA that helps stimulate an immune response.

[0259] Additional targets and combinations In some embodiments, methods are provided for treating or preventing microbial infections (e.g., bacterial or viral infections) and / or diseases, disorders, conditions, or symptoms associated with microbial or viral infections in a subject by administering a cyclic RNA vaccine comprising one or more polynucleotides encoding one or more peptides. The administration may be combined with antimicrobial agents described herein, such as antimicrobial agents, antimicrobial polypeptides, or small molecule antimicrobial compounds. Antimicrobial agents may include, but are not limited to, antimicrobial agents, antiviral agents, antifungal agents, antiprotozoal agents, antiparasitic agents, and antiprion agents.

[0260] Conditions related to bacterial infections Diseases, disorders, or conditions that may be treated with the circular RNA vaccine of the present invention include abscesses, actinomycosis, acute prostatitis, Aeromonas bacteria, annual ryegrass poisoning, anthrax, bacillus-purpura, bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterial pneumonia, bacterial vaginosis, bacteria-associated skin conditions, bartonellosis, BCG-oma, staphyloma, botulism, Brazilian purpura fever, Brody's abscess, brucellosis, Buruli's ulcer, Campylobacter infection, caries, Callion's disease, cat scratch disease, cellulitis, chlamydia infection, cholera, chronic bacterial prostatitis, chronic relapsing multifocal osteomyelitis, Clostridium necrotizing enterocolitis, and combined periodontic-endodontic lesions. Lesions), bovine pleuropneumonia, diphtheria, diphtheriae stomatitis, ehrlichiosis, erysipelas, epiglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spotted fever, hoof rot (infectious foot dermatitis), Garre's sclerosing osteomyelitis, gonorrhea, inguinal granuloma, human granulocytic anaplasmosis, human monocytotropic ehrlichiosis, hundred days' cough, impetigo, late congenital syphilitic eye disease, Legionnaires' disease, Lemier's syndrome, leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, lymphadenitis, meridian, meningococcal disease, meningococcal septicaemia, methicillin-resistant Staphylococcus aureus (MRSA) infection, mycobacterium Vium intracellulare (MAI), mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, cancrum oris (or gangrenous stomatitis), omphalitis, orbital cellulitis, osteomyelitis, severe post-splenectomy infection (OPSI), sheep brucellosis, pasteurellosis, periorbital cellulitis, pertussis (whooping cough), plague, pneumococcal pneumonia, Pott's disease, proctitis, pseudomonas infection, psittacosis, pyemia, suppurative myositis, Q fever, relapsing fever (typhoid fever), rheumatic fever, Rocky Mountain spotted fever (RMSF), rickettsial diseases, salmonellosis, scarlet fever, sepsis, Serratia infection, bacterial dysentery, southern tick-associated rashThis includes, but is not limited to, one or more of the following illnesses: staphylococcal scalded skin syndrome, streptococcal pharyngitis, pool granuloma, porcrine brucellosis, syphilis, syphilitic aortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever, tropical ulcer, tuberculosis, tularemia, typhoid fever, epidemic typhus, genitourinary tuberculosis, urinary tract infection, vancomycin-resistant Staphylococcus aureus infection, Waterhouse-Friderichsen syndrome, pseudotuberculosis (Yersinia), and Yersinia.

[0261] bacterial pathogen The specifications for the snowflakes are the snowflakes Among the strains of Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis、Chlamydophila psittaci、Clostridium botulinum、Clostridium difficile、Clostridium perfringens、Clostridium tetanus Staphylococcus, Corynebacterium, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, antibiotics such as Escherichia coli(ETEC), antibiotics E.coli, E.coli 0157:H7, Enter obacter sp., Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae、Legionella pneumophila、Leptospira interrogans、Listeria monocytogenes、Moraxella catarralis、Mycobacterium leprae、Mycobacterium tuberculosis、Mycoplasma pneumoniae、Neisseria gonorrhoeae、Neisseria meningitides、Preteus mirabilis、Proteus sps., Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. .

[0262] Bacterial pathogens may also include bacteria that cause drug-resistant infections, such as clindamycin-resistant Clostridium difficile, fluoroquinolone-resistant Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Enterococcus faecalis, multidrug-resistant Enterococcus faecium, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus (VRSA).

[0263] combination of antibiotics In some embodiments, the circular RNA vaccine of the present invention, for example, a circular RNA vaccine comprising a polynucleotide encoding one or more antigens of the present invention, may be administered in combination with one or more antimicrobial agents.

[0264] Antibacterial agent Antimicrobial agents include aminoglycosides (e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®), kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netylmycin (NETROMYCIN®), tobramycin (NEBCIN®), paromomycin (HUMATIN®)), ansamycin (e.g., geldanamycin, harbimycin), and carbasephalosporins (e.g., loracarbef (LORABID®)). )), carbapenems (e.g., ertapenem (INVANZ®), doripenem (DORIBAX®), imipenem / cilastatin (PRIMAXIN®), meropenem (MERREM®), cephalosporins (first generation) (e.g., cefadroxil (DURICEF®), cefazolin (ANCEF®), cephalothin or cephalothin (KEFLIN®), cephalexin (KEFLEX®), cephalosporins (second generation) (e.g., cefaclor (C) ECLOR®, MANDOL®, MEFOXIN®, CEFZIL®, Cefuroxime® (CEFTIN®, ZINNAT®), Cephalosporins (3rd generation) (e.g., SUPRAX®, OMNICEF®, CEFDIEL®, SPECTRACEF®, Cefperiol®, CEFOPERAZONE®, CEFOBID®), Cefotaxime® LAFORAN®), cefpodoxime (VANTIN®), ceftazidime (FORTAZ®), ceftibutene (CEDAX®), ceftizoxime (CEFIZOX®), ceftriaxone (ROCEPHIN®), cephalosporins (4th generation) (e.g., cefepime (MAXIPIME®)), cephalosporins (5th generation) (e.g., ceftobiprole (ZEFTERA®)), glycopeptides (e.g., teicoplanin (TARGOCID®)),Vancomycin (VANCOCIN®), Teravancin (VIBATIV®), Lincosamide (e.g., Clindamycin (CLEOCIN®), Lincomycin (LINCOCIN®)), Lipopeptides (e.g., Daptomycin (CUBICIN®)), Macrolides (e.g., Azithromycin (ZITHROMAX®, SUMAMED®, ZITROCIN®), Clarithromycin (BIAXIN®), Dylithromycin (DYN ABAC(registered trademark), erythromycin (ERYTHOCIN(registered trademark), ERYTHROPED(registered trademark)), roxithromycin, troleandomycin (TAO(registered trademark)), telithromycin (KETEK(registered trademark)), spectinomycin (TROBICIN(registered trademark))), monobactam (e.g., aztreonam(AZACTAM(registered trademark))), nitrofuran (e.g., furazolidone(FUROXONE(registered trademark)), nitrofurantoin (MACRODANTIN(registered trademark), MACROBID(registered trademark) Trademarks))), penicillin (e.g., amoxicillin (NOVAMOX®, AMOXIL®), ampicillin (PRINCIPEN®), azurocillin, carbenicillin (GEOCILLIN®), cloxacillin (TEGOPEN®), dicloxacillin (DYNAPEN®), flucloxacillin (FLOXAPEN®), mezlocillin (MEZLIN®), methicillin (STAPHCILLIN®), naphthillin (UNIPEN®) )), oxacillin (PROSTAPHLIN®), penicillin G (PENTIDS®), penicillin V (PEN-VEE-K®), piperacillin (PIPRACIL®), temocillin (NEGABAN®), ticarcillin (TICAR®), penicillin combinations (for example, amoxicillin / clavulanate (AUGMENTIN®), ampicillin / sulbactam (UNASYN®), piperacillin / tazobactam (ZOSYN®),Ticalcillin / clavranate (TIMENTIN®), polypeptides (e.g., bacitracin, colistin (COLY-MYCIN-S)®), polymyxin B, quinolones (e.g., ciprofloxacin (CIPRO®, CIPROXIN®, CIPROBAY®), enoxacin (PENETREX®), gatifloxacin (TEQUIN®), levofloxacin (LEVAQUIN®), lomefloxacin (MAXAQUIN®), moxifloxacin (AVELOX®), nalidixic acid (NEGGRAM®), norfloxacin (NOROXIN®), Ofloxacin (FLOXIN®, OCUFLOX®), trovafloxacin (TROVAN®), glepafloxacin (RAXAR®), sparfloxacin (ZAGAM®), temafloxacin (OMNIFLOX®), sulfonamides (e.g., mafenide (SULFAMYLON®), sulfonamide chrysoidine (PRONTOSIL®), sulfacetamide (SULAMYD®, BLEPH-10®), sulfadiazine (MICRO-SULFON®), silver sulfadiazine (SILVADENE®), sulfamethizol (THIOSULFIL) FORTE®), sulfamethoxazole (GANTANOL®), sulfanilimide, sulfasalazine (AZULFIDINE®), sulfisoxazole (GANTRISIN®), trimethoprim (PROLOPREVI®, TRIMPEX®), trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX) (BACTRIM®, SEPTRA®), tetracycline (e.g., demeclocycline (DECLOMYCIN®), doxycycline (VIBRAMYCIN®), minocycline (MINOCIN®), oxytetracycline (TERRAMYCIN®), tetracycline (SUMYCIN®),Drugs against mycobacteria (e.g., clofazimine (LAMPRENE (registered trademark)), dapsone (AVLOSULFON (registered trademark)), capreomycin (CAPASTAT (registered trademark)), cycloserine (SEROMYCIN (registered trademark)), ethambutol (MYAMBUTOL (registered trademark)), ethionamide (TRECATOR (registered trademark)), isoniazid (INH (registered trademark)), pyrazinamide (ALDIN) AMIDE®, rifampin (RIFADIN®, RIMACTANE®), rifabutin (MYCOBUTIN®), rifapentin (PRIFTIN®, streptomycin), and others (e.g., arsphenamine (SALVARSAN®), chloramphenicol (CHLOROMYCETIN®), fosfomycin (MONUROL®), fusidic acid (FUCIDIN®)). Examples include, but are not limited to, linezolid (ZYVOX®), metronidazole (FLAGYL®), mupirocin (BACTROBAN®), platensimycin, quinupristin / dalfopristin (SYNERCID®), rifaximin (XIFAXAN®), thianphenicol, tigecycline (TIGACYL®), and tinidazole (TINDAMAX®, FASIGYN®).

[0265] Conditions related to viral infections In some embodiments, a method is provided for treating or preventing a viral infection, and / or a disease, disorder, or condition, or symptoms thereof, in a subject by administering an antiviral polypeptide, for example, a cyclic RNA vaccine comprising one or more polynucleotides encoding the antiviral polypeptide described herein. In some embodiments, the cyclic RNA vaccine is administered in combination with an antiviral agent, for example, the antiviral polypeptide described herein or a small molecule antiviral agent.

[0266] Diseases, disorders, or conditions associated with viral infections that can be treated using the circular RNA vaccine of the present invention include acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis, infant gastroenteritis, Coxsackie infection, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes simplex and herpes simplex), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, and cellular infections. Inclusion body disease, Kaposi's sarcoma, multicentric Castleman disease, primary exudative lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, hyperplastic epithelial lesions (e.g., verruca vulgaris, verruca latum, plantar verruca, and anogenital verruca, laryngeal papilloma, epidermal verrucosa), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchitis, common cold, polio, rabies, bronchitis, pneumonia, influenza-like syndrome, severe bronchitis with pneumonia, rubella, congenital rubella, varicella, herpes zoster, and SARS-CoV-2.

[0267] Viral pathogens Examples of drugs for viral infections include infectious agents such as adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, encephalitis virus, papillomavirus, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus; This list includes, but is not limited to, hepatitis E virus; human immunodeficiency virus (HIV); influenza viruses A and B; Guanalito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; cortivirus; hantavirus; Middle East respiratory coronavirus; chikungunya fever virus; banavirus; or SARS-CoV-2. Viral pathogens may also include viruses that cause antiviral-resistant infections.

[0268] Antiviral drugs Examples of antiviral agents include abacavir (ZIAGEN®), abacavir / lamivudine / zidovudine (Trizivir®), acyclovir or acyclovir (CYCLOVIR®, HERPEX®, ACIVIR®, ACIVIRAX®, ZOVIRAX®, ZOVIR®), adefovir (Preveon®, Hepsera®), amantadine (SYMMETREL®), amprenavir (AGENERASE®), ampregen, arbidol, atazanavir (REYATAZ®), boceprevir, and cy Dofovir, Darunavir (PREZISTA®), Delavirdine (RESCRIPTOR®), Didanosine (VIDEX®), Docosanol (ABREVA®), Edoxidine, Efavirenz (SUSTINA®, STOCRIN®), Emtricitabine (EMTRIVA®), Emitricitabine / Tenofovir / Efavirenz (ATRIPLA®), Enfuvirtide (FUZEON®), Entecavir (BARACLUDE®, ENNAVIR®), Famciclovir (FAMVIR®), Homivirsen (VITRA VENE(registered trademark)), fosamprenavir (LEXIVA(registered trademark), TELZIR(registered trademark)), foscarnet (FOSCAVIR(registered trademark)), phosphonet, ganciclovir (CYTOVENE(registered trademark), CYMEVENE(registered trademark), VITRASERT(registered trademark)), GS 9137 (ELVITEGRAVIR(registered trademark)), imiquimod (ALDARA(registered trademark), ZYCLARA(registered trademark), BESELNA(registered trademark)), indinavir (CRIXIVAN(registered trademark)), inosine, inosinepranovex (IMUNOVIR(registered trademark)), interferon type I, interferon type II, interferon type III, kutapressin (NEXAVIR(registered trademark)), lamivudine (ZEFFIX(registered trademark), HEPTOVIR(registered trademark), EPIVIR(registered trademark)), lamivudine / zidovudine (COMBIVIR(registered trademark)), lopinavir,Lobilid, Maraviroc (SELZENTRY®, CELSENTRI®), Methisazone, MK-2048, Moroxidine, Nelfinavir (VIRACEPT®), Nevirapine (VIRAMUNE®), Oseltamivir (TAMIFLU®), Pegylated Interferon Alpha-2a (PEGASYS®), Penciclovir (DENAVIR®), Peramivir, Preconalil, Podophyllotoxin (CONDYLOX®), Raltegravir (ISENTRESS®), Ribavirin (COPEGUs®, REBETOL®, RIBASPHERE®, VILONA®, and VIRAZOLE®), Rimantadine (FLUMADINE®), Ritonavir (NOR Examples include, but are not limited to, VIR(registered trademark), pyramidine, saquinavir (INVIRASE(registered trademark), FORTOVASE(registered trademark)), stabudine, tea tree oil (Melaleuca oil), tenofovir (VIREAD(registered trademark)), tenofovir / emtricitabine (TRUVADA(registered trademark)), tipranavir (APTIVUS(registered trademark)), trifluridine (VIROPTIC(registered trademark)), tromantadine (VIRU-MERZ(registered trademark)), valacyclovir (VALTREX(registered trademark)), valganciclovir (VALCYTE(registered trademark)), bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (RELENZA(registered trademark)), and zidovudine (azidothymidine (AZT), RETROVIR(registered trademark), RETROVIS(registered trademark)).

[0269] Conditions related to fungal infections Diseases, disorders, or conditions associated with fungal infections that can be treated with the circular RNA vaccine of the present invention include, but are not limited to, aspergillosis, blastomycosis, candidiasis, coccidioidmycosis, cryptococcosis, histoplasmosis, misetoma, paracoccidioidomycosis, and tinea pedis. Furthermore, immunocompromised individuals are particularly susceptible to diseases caused by fungal genera such as Aspergillus, Candida, Cryptoccocus, Histoplasmosis, and Pneumocystis, which can be treated with the circular RNA vaccine of the present invention. Other fungi that can be treated with the circular RNA vaccine of the present invention include so-called cutaneous fungi and keratinophilic fungi that can attack the eyes, nails, hair, and especially the skin, causing a variety of conditions such as tinea, which is common in athlete's foot. The circular RNA vaccine of the present invention can also be used to treat fungal spores of various taxa and allergies caused by fungi.

[0270] fungal pathogen Examples of fungal pathogens include, but are not limited to, Ascomycota (e.g., Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / posadasii, Candida albicans), Basidiomycota (e.g., Filobasidiella neoformans, Trichosporon), Microsporidia (e.g., Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina (e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).

[0271] Antifungal agents Antifungal agents that can be used in combination with the circular RNA vaccine of the present invention include polyene antifungal agents (e.g., natamycin, rimocidine, philipin, nystatin, amphotericin B, candaicin, hamamycin), imidazole antifungal agents (e.g., miconazole (MICATIN®, DAKTARIN®), ketoconazole (NIZORAL®, FUNORAL®, SEBIZOLE®), clotrimazole (LOTRIMIN®, LOTRIMIN®AF, CANESTEN®), econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole (ERTACZO®), sulconazole, thioconazole), and triazole antifungal agents (e.g., albaconazole). Examples of antifungal agents include, but are not limited to, fluconazole, itraconazole, isabconazole, ravconazole, posaconazole, voriconazole, terconazole), thiazole antifungals (e.g., abafungin), allylamines (e.g., terbinafine (LAMISIL®), naftifine (NAFTIN®), butenafine (LOTRIMIN® Ultra)), echinocandins (e.g., anidurafungin, caspofungin, micafungin), and others (e.g., polygodial, benzoic acid, cyclopirox, tolnaftate (TINACTIN®, DESENEX®, AFTATE®), undecylenic acid, flucytosine, or 5-fluorocytosine, griseofulvin, haloprogin, sodium bicarbonate, allicin).

[0272] Pathological conditions related to protozoal infections Diseases, disorders, or conditions related to protozoan infections that can be treated with the circular RNA vaccine of the present invention include, but are not limited to, amoebic diseases, giardiasis, trichomoniasis, African sleeping sickness, American sleeping sickness, leishmaniasis (kala-azar), balantidiosis, toxoplasmosis, malaria, Acanthamoeba keratitis, and babesiosis.

[0273] protozoal pathogen Protozoan pathogens include, but are not limited to, Entamoeba histolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei, T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii, Plasmodium spp., and Babesia microti.

[0274] Antiparasitic drugs Exemplary antiparasitic agents include, but are not limited to, eflornithine, furazolidone (FUROXONE®, DEPEND AL-M®), melarsoprol, metronidazole (FLAGYL®), ornidazole, paromomycin sulfate (HUMATIN®), pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDAMAX®, FASIGYN®).

[0275] Conditions related to parasitic infections Parasitic infection-related diseases, disorders, or conditions that can be treated with the circular RNA vaccine of the present invention include, but are not limited to, Acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, baylisascariasis, Chagas disease, hepatotropiasis, cochlomyiasis, cryptosporidiosis, diphyllobothrium erythrocystosis, dung worm disease, echinococcosis, elephantiasis, pinworm disease, phylariasis, hirsutosis, filariasis, giardiasis, gnathostomiasis, membranoid tapeworm disease, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, metagonimus, myiasis, onchocerciasis, lice infestation, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm disease, toxocariasis, toxoplasmosis, trichocarchiosis, and whipworm disease.

[0276] parasitic pathogen Parasitic pathogens include, but are not limited to, Acanthamoeba, Anisakis, Ascaris lumbricoides, horseflies, Balantidium coli, bed bugs, Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworms, Leishmania, Linguatula serrata, liver flukes, Loa loa, Paragonimus, pinworms, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, and others.

[0277] Antiparasitic drugs Examples of antiparasitic agents include, but are not limited to, antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), antitaeniades (e.g., niclosamide, praziquantel, albendazole), antitrematodes (e.g., praziquantel), antiamoebic agents (e.g., rifampin, amphotericin B), and antiprotozoal agents (e.g., melarsopro, eflornithine, metronidazole, tinidazole).

[0278] Cutting site In some embodiments, two or more expression sequences in a polynucleotide construct can be separated by one or more cleavage site sequences. The cleavage site can be any sequence that allows two or more polypeptides to be separated. The cleavage site may be self-cleaving so that, once the polypeptides are produced, they are immediately cleaved into individual polypeptides without requiring external cleavage activity.

[0279] In some embodiments, the cleavage site may be a furin cleavage site. Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process potential precursor proteins into biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta-1 precursor, proalbumin, probeta-secretase, membrane type 1 matrix metalloproteinase, the beta subunit of pronerve growth factor, and von Willebrand factor. Furin cleaves proteins immediately downstream of a basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg) and is enriched in the Golgi apparatus.

[0280] In some embodiments, the cleavage site may encode a self-cleaving peptide.

[0281] In some embodiments, the cleavage site may operate by ribosome skipping, such as skipping the glycylpropyl bond at the C-terminus of the 2A self-cleaving peptide. In some embodiments, steric hindrance causes ribosome skipping. In some embodiments, the 2A self-cleaving peptide comprises the sequence GDVEXNPGP (SEQ ID NO: 324), where X is E or S. In some embodiments, the protein encoded upstream of the 2A self-cleaving peptide is bound to the 2A self-cleaving peptide, except for post-translational proline at the C-terminus. In some embodiments, the protein encoded downstream of the 2A self-cleaving peptide is bound to proline at the post-translational N-terminus.

[0282] In some embodiments, the self-cleaving peptide may be a 2A self-cleaving peptide derived from aftvirus or cardiovirus. Major 2A / 2B cleavage in aftviruses and cardioviruses is mediated by 2A cleavage at their C-terminus. In aptoviruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short segment of about 18 amino acids that, together with the N-terminal residue (a conserved proline residue) of protein 2B, represents an autonomous element that can mediate cleavage at its C-terminus (Donelly et al. (2001)).

[0283] 2A-like sequences have been found in picornaviruses other than aptoviruses or cardioviruses, "picornavirus-like" insect viruses, rotavirus type C, and repeat sequences within Trypanosoma and bacterial sequences (Donnelly et al. (2001)). In some embodiments, the cleavage site may include one of these 2A-like sequences, such as those listed in Table 8.

[0284] In some embodiments, the self-cleaving peptide is F2A. In some embodiments, the self-cleaving peptide is derived from foot-and-mouth disease virus. In some embodiments, the self-cleaving peptide is E2A. In some embodiments, the self-cleaving peptide is derived from equine rhinitis A virus. In some embodiments, the self-cleaving peptide is P2A. In some embodiments, the self-cleaving peptide is derived from porcine tescovirus-1. In some embodiments, the self-cleaving peptide is T2A. In some embodiments, the self-cleaving peptide is derived from Thosea asigna virus. In some embodiments, the self-cleaving peptide has the sequences listed in Table 8.

[0285] In one embodiment, the expression sequences encoding peptides separated by cleavage sites have the same level of protein expression.

[0286] In some embodiments, self-cleaving peptides are described in Liu, Z., Chen, O., Wall, JBJ et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017).

[0287] Production of polynucleotides The vectors provided herein can be prepared using standard molecular biology techniques known to those skilled in the art. For example, various elements of the vectors provided herein can be obtained using recombinant methods, for example, by screening cDNA and genomic libraries from cells, or by deriving polynucleotides from vectors known to contain polynucleotides.

[0288] The various elements of the vectors provided herein can also be generated synthetically rather than by cloning, based on known sequences. Complete sequences can be assembled from duplicate oligonucleotides prepared by standard methods. See, for example, Edge, Nature (1981) 292:756, Nambair et al., Science (1984) 223:1299, and Jay et al., J. Biol. Chem. (1984) 259:631¹.

[0289] Therefore, specific nucleotide sequences can be obtained from vectors containing the desired sequence, or synthesized entirely or partially using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques, where appropriate. One method for obtaining a nucleotide sequence encoding a desired vector element is to anneal a complementary set of duplicated synthetic oligonucleotides produced by a conventional automated polynucleotide synthesizer, followed by ligation with a suitable DNA ligase, and then amplification of the ligated nucleotide sequence via PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. In addition, oligonucleotide-specific synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-specific mutagenesis of existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of gap-filled oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0290] The precursor RNA provided herein can be generated by incubating the vector provided herein under conditions that allow transcription of the precursor RNA encoded by the vector. For example, in some embodiments, the precursor RNA is synthesized by incubating the vector provided herein, which includes an RNA polymerase promoter upstream of its 5' double-strand formation region and / or expression sequence, with a compatible RNA polymerase enzyme under conditions that allow in vitro transcription. In some embodiments, the vector is incubated intracellularly by bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II.

[0291] In certain embodiments, a method for generating precursor RNA is provided herein by performing in vitro transcription using a vector provided herein as a template (for example, a vector provided herein having an RNA polymerase promoter located upstream of the 5' double-strand formation region).

[0292] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., circular RNA polynucleotides provided herein) by incubating it in the presence of magnesium ions and guanosine nucleotides or nucleosides at a temperature at which RNA cyclization occurs (e.g., 20°C to 60°C).

[0293] Therefore, in certain embodiments, a method for producing circular RNA is provided herein. In certain embodiments, the method includes synthesizing a precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a vector comprising, in the following order, a 5' double-forming region, a 3' group I intron fragment, a first spacer, an intra-sequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, a second spacer, a 5' group I intron fragment, and a 3' double-forming region) as a template, and incubating the obtained precursor RNA in the presence of a divalent cation (e.g., a magnesium ion) and GTP to form circular RNA. In some embodiments, the precursor RNA of the present invention can be circularized in the absence of magnesium ions and GTP, and / or without the incubation step with magnesium ions and GTP. In some embodiments, transcription is carried out in the presence of excess GMP.

[0294] In some embodiments, the composition containing circular RNA is purified. The circular RNA can be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in the order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse-phase HPLC. In some embodiments, the purified composition contains less double-stranded RNA, DNA sprints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes, and / or nicked RNA than the unpurified RNA. In some embodiments, the purified composition is less immunogenic than the unpurified composition. In some embodiments, immune cells exposed to the purified composition produce less TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferon, such as IFN-β1, than immune cells exposed to the unpurified composition.

[0295] nanoparticles In certain embodiments, what is provided herein is a pharmaceutical composition comprising a circular RNA provided herein. In certain embodiments, such a pharmaceutical composition is formulated with nanoparticles to facilitate delivery.

[0296] In certain embodiments, the circular RNA provided herein may be delivered to and / or targeted to cells in a transport vehicle, such as nanoparticles or a composition containing nanoparticles. In some embodiments, the circular RNA may also be delivered to a target in a transport vehicle or a composition containing a transport vehicle. In some embodiments, the transport vehicle is nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, solid lipid nanoparticles, polymer core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the transport vehicle contains or is coated with one or more cationic lipids, non-cationic lipids, ionic lipids, PEG-modified lipids, polyglutamic acid polymers, hyaluronic acid polymers, poly-β-aminoesters, poly-beta-aminopeptides, or positively charged peptides.

[0297] In one embodiment, a transport vehicle can be selected and / or prepared to optimize the delivery of circular RNA to target cells. For example, if the target cells are antigen-presenting cells, the properties of the transport vehicle (e.g., size, charge, and / or pH) can be optimized to effectively deliver such transport vehicle to the target cells, reduce immune clearance, and / or promote retention in those target cells.

[0298] The use of a transport vehicle to facilitate the delivery of nucleic acids to target cells is intended by the present invention. Liposomes (e.g., liposomal lipid nanoparticles) are generally useful for a variety of applications in research, industry, and medicine, and are particularly useful as transport vehicles for diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol. Rev., 51:691-743, 1999), and are typically characterized as microscopic vesicles having an internal aqueous space isolated from the outer medium by one or more bilayer membranes. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer membrane can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0299] In the context of the present invention, a transport vehicle typically plays a role in transporting circular RNA to target cells. For the purposes of the present invention, the transport vehicle is prepared to contain or encapsulate a desired nucleic acid. The process of incorporating a desired entity (e.g., nucleic acid) into a liposome is often referred to as loading (Lasic, et al., FEBS Lett., 312:255-258, 1992). The nucleic acid incorporated into the liposome can be located entirely or partially within the internal space of the liposome, within the liposome bilayer, or bound to the outer surface of the liposome membrane. The purpose of incorporating circular RNA into a transport vehicle such as a liposome is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade the nucleic acid, and / or systems or receptors that cause rapid excretion of the nucleic acid. Therefore, in certain embodiments of the present invention, a selected transport vehicle can improve the stability of the circular RNA contained therein. Liposomes can enable encapsulated circRNA to reach target cells, or alternatively, restrict the delivery of such circulating RNA to other sites or cells where the presence of the administered circulating RNA may be unnecessary or undesirable. Furthermore, incorporating circulating RNA into a transport vehicle, such as a cationic liposome, can also facilitate the delivery of such circulating RNA to target cells. In some embodiments, the transport vehicles disclosed herein can, for example, facilitate the endosomal or lysosomal release of contents encapsulated within the transport vehicle (e.g., lipid nanoparticles).

[0300] Ideally, the transport vehicle is prepared to encapsulate one or more desired circular RNAs so that the composition exhibits high transfection efficiency and improved stability. While liposomes can facilitate the delivery of nucleic acids to target cells, the addition of polycations as copolymers (e.g., poly-L-lysine and protamine) can, in some cases, significantly increase the transfection efficiency of several cationic liposomes by 2 to 28 times in many cell lines, both in vitro and in vivo. (See N J. Caplen, et al., Gene Ther. 1995;2:603, S. Li, et al., Gene Ther. 1997;4,891).

[0301] In some embodiments of the present invention, the transport vehicle is formulated as lipid nanoparticles. In some embodiments, the lipid nanoparticles are formulated to deliver one or more circRNAs to one or more target cells. Examples of suitable lipids include phosphatidyl compounds (e.g., PBAEs, polyglutamic acid, polyaspartic acid, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). It is also intended that polymers be used as transport vehicles, either alone or in combination with other transport vehicles. Suitable polymers may include, for example, polyacrylates, polyhydroxyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, dendrimers, and polyethyleneimines. In some embodiments, the transport vehicle is formulated as a lipid described in U.S. Patent Application No. 16 / 065,067 (which is incorporated herein in its entirety). In some embodiments, the transport vehicle is selected based on its ability to facilitate the transfection of circRNA into target cells.

[0302] The present invention envisions the use of lipid nanoparticles as transport vehicles containing cationic lipids to load and / or encapsulate and / or enhance the delivery of circRNA to target cells that function as depots for protein production. The envisioned lipid nanoparticles may be prepared by comprising a multi-component lipid mixture in various ratios using one or more cationic lipids, non-cationic lipids, and PEG-modified lipids. Several cationic lipids are described in the literature, and many are commercially available.

[0303] Suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication 2010 / 053572 and / or U.S. Patent Application 15 / 809,680, for example, C12-200. In certain embodiments, the compositions and methods of the present invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ( Lipid nanoparticles containing ionizable cationic lipids, such as HGT5001 and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), as described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (incorporated herein by reference).

[0304] In some embodiments, the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used. (Felgner et al. Proc. Nat'l Acad. Sci. 84,7413 (1987), U.S. Patent No. 4,897,355). DOTMA may be formulated alone or in combination with neutral lipids, dioleylphosphatidylethanolamine or "DOPE" or other cationic or non-cationic lipids in a transport vehicle or lipid nanoparticles, such liposomes may be used to enhance the delivery of nucleic acids to target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycine dioctadecylamide or "DOGS", 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneaminium or "DOSPA" (Behr et al., Proc. Nat.'l Acad. Sci. 86,6982 (1989), U.S. Patent No. 5,171,678, and U.S. Patent No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP", and 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP". The cationic lipids intended are 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-Octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl1-1-(cis,cis-9',1-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleoylcarbamyl-3-dimeth This also includes diaminopropane or "DLincarbDAP", 1,2-dilinoleylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethaneamine (DLin-KC2-DMA)) (see WO2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107:276-287(2005), Morrissey, DV, et al., Nat.Biotechnol.23(8):1003-1007(2005), PCT Publication WO2005 / 121348A1). ,

[0305] The use of cholesterol-based cationic lipids is also intended by the present invention. Such cholesterol-based cationic lipids may be used alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, GL67, DC-Chol (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao, et al., Biochem. Biophys. Res. Comm. 179,280 (1991), Wolf et al., BioTechniques 23,139 (1997), U.S. Patent No. 5,744,335), or ICE.

[0306] In addition, several reagents are commercially available to enhance the effectiveness of transfection. Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, CA), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000 (Invitrogen), FUGENE (Promega, Madison, WI), TRANSFECTAM (DOGS) (Promega), and EFFECTENE (Qiagen, Valencia, CA).

[0307] Cationic lipids, such as dialkylamino, imidazole, and guanidinium lipids described in U.S. Patent No. 10,413,618, are also intended.

[0308] In other embodiments, the compositions and methods described herein relate to lipid nanoparticles comprising one or more cleavable lipids, such as compounds comprising one or more cationic lipids or cleavable disulfide (SS) functional groups (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and HGT4005), as further described in U.S. Provisional Application No. 61 / 494,745 (the entire teaching is incorporated herein by reference).

[0309] The use of polyethylene glycol (PEG)-modified phospholipids, including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide), and derivatized lipids such as derivatized ceramide (PEG-CER), is also intended by the present invention, either alone or in combination with other lipids, and together comprising a transport vehicle (e.g., lipid nanoparticles). The intended PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids with alkyl chains of C6-C20 length. The addition of such components can prevent aggregation of complexes and also provide means to extend circulating life and increase delivery of lipid-nucleic acid compositions to target cells (Klibanov et al., (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly replaced from formulations in vivo (see U.S. Patent No. 5,885,613). Particularly useful replaceable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention may comprise molar ratios of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in the transport vehicle. The PEG-terminated groups are as intended herein. In some embodiments, the PEG-terminated groups are -OH, -OCH3, acids, amines, or guanidines.

[0310] In some embodiments, RNA (e.g., circRNA) vaccines include cationic or polycationic compounds such as protamine, nucleoline, spermine, or spermidine, or other cationic peptides or proteins such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-permeable peptides (CPPs) including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived or similar peptides, pestivirus Ern, HSV, VP22 (herpes simplex virus), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, and peptides derived from Antennapedia (especially Drosophila). (from antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, histones, cationic polysaccharides, e.g., chitosan, polybrene, cationic polymers, e.g., polyethyleneimine (PEI), cationic lipids, e.g., DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DO PE: Dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamideglycylspermine, DIMRI: Dimyristooxypropyldimethylhydroxyethylammonium bromide, DOTAP: Dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-Ditetradecanoyl-N-α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,[3-Dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9:rac-[2(2,3-Dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamines, or cationic or polycationic polymers, e.g., modified polyamino acids, e.g., β-amino acid polymers or reverse polyamides, modified polyethylene, e.g., PVP (Poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, e.g., pDMAEMA (Poly(dimethylaminoethylmethyl acrylate)), modified amidoamines, e.g., pAMAM (Poly(amidoamine)), modified polybeta-aminoesters (PBAEs), e.g., dia This may be related to mine-terminal modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or pAMAM dendrimers, polyimines such as PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamines, sugar-backed polymers such as cyclodextrin polymers, dextran polymers, chitosan, silane-backed polymers such as PMOXA-PDMS copolymers, and block polymers consisting of a combination of one or more cationic blocks (selected from cationic polymers as described above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0311] The present invention also intends to use noncationic lipids, including those described in U.S. Patent Application No. 15 / 809,680. Noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine. Examples of noncationic lipids include, but are not limited to, 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearyl-2-oleoylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such noncationic lipids may be used alone or in combination with other excipients, such as cationic lipids. When used in combination with cationic lipids, the noncationic lipids may constitute 5% to about 90% or about 10% to about 70% of the total lipids present in the transport vehicle in molar ratios.

[0312] Transport vehicles (e.g., lipid nanoparticles) can be prepared by combining multiple lipid and / or polymer components. For example, transport vehicles can be prepared using C12-200, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 18:56:20:6, HGT5000, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:20:35:5, or HGT5001, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:20:35:5. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including lipid nanoparticles, and the relative molar ratios of such lipids to each other, is based on the characteristics of the selected lipids, the properties of the intended target cells, and the characteristics of the circRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusionability, and toxicity of the selected lipids. Therefore, the molar ratio can be adjusted accordingly. For example, in some embodiments, the proportion of cationic lipids in lipid nanoparticles may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The proportion of non-cationic lipids in lipid nanoparticles may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The proportion of cholesterol in lipid nanoparticles may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The proportion of PEG-modified lipids in lipid nanoparticles may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0313] Transport vehicles for use in the compositions of the present invention can be prepared by various techniques currently known in the art. Multilayered vesicles (MLVs) can be prepared using conventional techniques, for example, by depositing selected lipids on the inner wall of a suitable container or vessel, dissolving the lipids in a suitable solvent, and then evaporating or spray-drying the solvent so that a thin film remains on the inside of the container. An aqueous phase can then be added to the container using vortex motion, which results in the formation of MLVs. Monolayered vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of polyendoplasmic reticulum. In addition, ULVs can be formed by detergent removal techniques.

[0314] In certain embodiments of the present invention, the composition comprises a transport vehicle, and the circRNA is associated with both surfaces of the transport vehicle and encapsulated within the same transport vehicle. For example, during the preparation of the composition of the present invention, a cationic transport vehicle may be associated with the circRNA through electrostatic interactions.

[0315] In certain embodiments, the compositions of the present invention may be loaded with diagnostic radionuclides, fluorescent materials, or other materials detectable in both in vitro and in vivo applications. For example, suitable diagnostic materials for use in the present invention may include rhodamine-dioleoylphosphatidylethanolamine (Rh-PE), green fluorescent protein circRNA (GFP circRNA), Renilla luciferase circRNA, and firefly luciferase circRNA.

[0316] In some embodiments, the selection of an appropriate size for the transport vehicle takes into account the site of the target cell or tissue and, to some extent, the intended use for which the liposomes are produced. In some embodiments, it may be desirable to restrict the transfection of circRNA to specific cells or tissues. For example, to target hepatocytes, the transport vehicle may be sized so that its dimensions are smaller than the openings in the endothelial layer covering the hepatic sinuses of the liver. Thus, a transport vehicle of appropriate size can easily penetrate such endothelial openings and reach the target hepatocytes. Alternatively, the transport vehicle may be sized so that the dimensions of the liposomes are of a diameter sufficient to restrict or explicitly avoid distribution to certain cells or tissues. For example, the transport vehicle may be sized so that its dimensions are larger than the openings in the endothelial layer covering the hepatic sinuses of the liver, thereby restricting the distribution of the transport vehicle to hepatocytes. Generally, the size of the transport vehicle is in the range of approximately 25–250 nm. In some embodiments, the size of the transport vehicle is approximately 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or less than 10 nm.

[0317] Various alternative methods known in the art are available for dimensional determination of transport vehicle populations. One such dimensional determination method is described in U.S. Patent No. 4,737,323 (incorporated herein by reference). Sonication of liposome suspensions by either bath or probe sonication results in a gradual size reduction to small ULVs with diameters less than approximately 0.05 microns. Homogenization is another shear energy-dependent method for fragmenting larger liposomes into smaller liposomes. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until a size of selected liposomes, typically about 0.1–0.5 microns, is observed. The size of liposome vesicles can be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421–450 (1981) (incorporated herein by reference). The average liposome diameter can be reduced by sonication of the formed liposomes. To facilitate efficient liposome synthesis, intermittent sonication cycles can be performed alternately with QELS evaluation.

[0318] Furthermore, in certain embodiments, the cyclic RNA provided herein may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the cyclic RNA may be formulated in lipid nanoparticles as described in International Publication 2012 / 170930 (which is incorporated herein by reference in its entirety). In one embodiment, the lipid may be a cleavable lipid as described in International Publication 2012 / 170889 (which is incorporated herein by reference in its entirety). In one embodiment, the pharmaceutical composition of the cyclic RNA may comprise at least one of the PEGylated lipids described in International Publication 2012 / 099755 (which is incorporated herein by reference). In one embodiment, the lipid nanoparticle formulation may be prepared by the method described in International Publication 2011 / 127255 or 2008 / 103276 (each of which is incorporated herein by reference in its entirety). Lipid nanoparticles may be coated with or associated with block copolymers, such as, for example, branched polyether-polyamide block copolymers described in International Publication No. 2013 / 012476 (which is incorporated herein by reference in its entirety). Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the effectiveness of circular RNA-directed protein production, as these formulations can increase cellular transfection with circular RNA, increase the in vivo or in vitro half-life of circular RNA, and / or enable controlled release.

[0319] In other embodiments, the cyclic RNA polynucleotides provided herein can be formulated using one or more polymers. The polymers may be contained in RNA or lipid nanoparticles and / or used to encapsulate or partially encapsulate RNA or lipid nanoparticles. The polymers may be biodegradable and / or biocompatible. The polymers may be selected from, but are not limited to, the group consisting of polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-lactide-co-PPO-co-D,L-lactide), polyalkyl silciaacrylate, polyurethane, and poly-L-lysine (PLL). , hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(esteramide), polyamide, poly(ester ether), polycarbonate, polyethylene and polypropylene, polyalkylene glycol such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalate such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester such as poly(vinyl acetate), polyvinyl halide such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxane, polystyrene,Polyurethanes, derivatized celluloses (e.g., alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose), acrylic polymers, e.g., poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), 363 5 10 15 20 25 30 35 WO 2021 / 076805 PCT / US2020 / 055844 poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymer It may contain rimers, polyhydroxyalkanoates, polypropylene fimarates, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerols.

[0320] In some embodiments, polynucleotides encode proteins composed of subunits encoded by one or more genes. For example, a protein may be a heterodimer in which each chain or subunit of the protein is encoded by distinct genes. One or more circRNA molecules can be delivered by transport vehicle, with each circRNA encoding a distinct subunit of the protein. Alternatively, a single circRNA can be manipulated to encode more than one subunit (e.g., in the case of a single-stranded Fv antibody). In certain embodiments, distinct circRNA molecules encoding individual subunits may be administered by distinct transport vehicles.

[0321] The present invention also aims at the differential targeting of target cells and tissues by both passive and active targeting means. The passive targeting phenomenon utilizes the natural distribution pattern of transport vehicles in vivo without relying on the use of additional excipients or means to improve the recognition of transport vehicles by target cells. For example, transport vehicles subjected to phagocytosis by cells of the reticuloendothelial system are likely to accumulate in the liver or spleen, and therefore, means can be provided to passively guide the delivery of compositions to such target cells.

[0322] Alternatively, the present invention envisions active targeting, which involves using a targeting moiety that can bind (either covalently or noncovalently) to a transport vehicle to facilitate the localization of such transport vehicle to a particular target cell or target tissue. For example, targeting may be mediated by including one or more endogenous targeting moieties in or on the transport vehicle to facilitate distribution to target cells or tissues. Recognition of the targeting moiety by the target tissue actively facilitates the tissue distribution and cellular uptake of the transport vehicle and / or its contents in the target cells and tissues (for example, including an apolipoprotein-E targeting ligand in or on the transport vehicle facilitates the recognition and binding of the transport vehicle to an endogenous low-density lipoprotein receptor expressed by hepatocytes). As provided herein, compositions may include moieties that can improve the affinity of the composition to target cells. Targeting moieties may be ligated to the outer bilayer of lipid particles during or after formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusion polymers, such as PEAA, hemagglutinins, and other lipopeptides (see U.S. Patent Applications No. 08 / 835,281 and No. 60 / 083,294, incorporated herein by reference), as well as other properties useful for in vivo and / or intracellular delivery. In some embodiments, the compositions of the present invention demonstrate improved transfection efficiency and / or enhanced selectivity for target cells or tissues of interest. Thus, compositions comprising one or more moieties (e.g., peptides, aptamers, oligonucleotides, small molecules, vitamins, or other molecules) that can improve the affinity of the composition and its nucleic acid contents to target cells or tissues are contemplated. Preferred moieties may optionally be bound or ligated to the surface of a transport vehicle. In some embodiments, the targeting moieties may spread across the surface of the transport vehicle or be encapsulated within the transport vehicle. Preferred moieties are selected based on their physical, chemical, or biological properties (e.g., selective affinity and / or recognition of target cell surface markers or features).Cell-specific target sites and their corresponding targeting ligands can vary considerably. Suitable targeting moieties are selected to leverage the unique characteristics of target cells, thus enabling the composition to differentiate between target and non-target cells. For example, the compositions of the present invention may include surface markers (e.g., apolipoprotein B or apolipoprotein E) that selectively enhance hepatocyte recognition or affinity to hepatocytes (e.g., by receptor-mediated recognition of such surface markers and binding to such surface markers). For instance, the use of galactose as the targeting moiety may direct the composition of the present invention towards hepatocytes; alternatively, the use of mannose containing sugar residues as the targeting ligand may direct the composition of the present invention towards hepatocytes (e.g., mannose containing sugar residues that preferentially bind to asialoglycoprotein receptors present on hepatocytes). (See Hillery AM, et al., “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002), Taylor & Francis, Inc.) The presentation of such targeting moieties, compounded with a portion present on a transport vehicle (e.g., lipid nanoparticles), thus facilitates the recognition and uptake of the compositions of the present invention in target cells and tissues. Examples of suitable targeting moieties include one or more peptides, proteins, small molecules, aptamers, vitamins, and oligonucleotides.

[0323] In some embodiments, the targeting moiety selectively mediates receptor-mediated endocytosis to a specific cell population. In some embodiments, the targeting moiety can bind to hepatocyte antigens. In some embodiments, the targeting moiety is a single-strand variable fragment (scFv), nanobody, peptide, peptide-based macroring, minibody, heavy chain variable region, light chain variable region, or a fragment thereof.

[0324] In some embodiments, the circular RNA is formulated according to the process described in U.S. Patent Application No. 15 / 809,680. In some embodiments, the present invention provides a process for encapsulating circular RNA in lipid nanoparticles, comprising the steps of forming lipids within pre-formed lipid nanoparticles (i.e., formed in the absence of RNA), and then combining the pre-formed lipid nanoparticles with RNA. In some embodiments, the novel formulation process results in an RNA formulation that has higher potency (peptide or protein expression) and higher efficacy (improved biologically relevant endpoints) both in vitro and in vivo, and potentially better tolerability, compared to the same RNA formulation prepared without the step of pre-forming lipid nanoparticles (e.g., by directly combining lipids with RNA).

[0325] For specific cationic lipid nanoparticle formulations of RNA, heating of the RNA in a buffer (e.g., citrate buffer) is required to achieve high RNA encapsulation. In these processes or methods, heating after formulation (after nanoparticle formation) does not increase the efficiency of RNA encapsulation into lipid nanoparticles; therefore, heating must be performed before the formulation process (i.e., heating a separate component). In contrast, in some embodiments of the process of the present invention, the order of RNA heating does not appear to affect the RNA encapsulation percentage. In some embodiments, heating of one or more of the solution containing preformed lipid nanoparticles, the solution containing RNA, and the mixed solution containing RNA encapsulated in lipid nanoparticles does not need to occur before or after the formulation process (i.e., is maintained at ambient temperature).

[0326] RNA may be provided in a solution mixed with a lipid solution so that the RNA can be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain RNA at concentrations of approximately 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1.0 mg / ml or higher. In some embodiments, suitable RNA solutions are approximately 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml, 0. RNA may be present in concentrations ranging from 0.5 to 0.8 mg / ml, 0.05 to 0.7 mg / ml, 0.05 to 0.6 mg / ml, 0.05 to 0.5 mg / ml, 0.05 to 0.4 mg / ml, 0.05 to 0.3 mg / ml, 0.05 to 0.2 mg / ml, 0.05 to 0.1 mg / ml, 0.1 to 1.0 mg / ml, 0.2 to 0.9 mg / ml, 0.3 to 0.8 mg / ml, 0.4 to 0.7 mg / ml, or 0.5 to 0.6 mg / ml.

[0327] Typically, a suitable RNA solution may also contain buffers and / or salts. Common buffers may include Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, or sodium phosphate. In some embodiments, suitable concentrations of buffers may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 mM to 12 mM.

[0328] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, preferred concentrations of the salt in RNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.

[0329] In some embodiments, a preferred RNA solution may have a pH in the range of approximately 3.5–6.5, 3.5–6.0, 3.5–5.5, 3.5–5.0, 3.5–4.5, 4.0–5.5, 4.0–5.0, 4.0–4.9, 4.0–4.8, 4.0–4.7, 4.0–4.6, or 4.0–4.5.

[0330] Various methods can be used to prepare RNA solutions suitable for the present invention. In some embodiments, RNA can be dissolved directly in the buffer solution described herein. In some embodiments, the RNA solution can be prepared by mixing the RNA stock solution with a buffer solution before mixing it with the lipid solution for mounting. In some embodiments, the RNA solution can be prepared by mixing the RNA stock solution with a buffer solution immediately before mixing it with the lipid solution for mounting.

[0331] According to the present invention, the lipid solution contains a mixture of lipids suitable for forming lipid nanoparticles for RNA encapsulation. In some embodiments, the preferred lipid solution is ethanol-based. For example, the preferred lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, the preferred lipid solution is isopropyl alcohol-based. In yet another embodiment, the preferred lipid solution is dimethyl sulfoxide-based. In yet another embodiment, the preferred lipid solution is a mixture of preferred solvents including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[0332] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at total concentrations ranging from about 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to 80 mg / ml, 1.0 to 70 mg / ml, 1.0 to 60 mg / ml, 1.0 to 50 mg / ml, 1.0 to 40 mg / ml, 1.0 to 30 mg / ml, 1.0 to 20 mg / ml, 1.0 to 15 mg / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml, 1.0 to 8 mg / ml, 1.0 to 7 mg / ml, 1.0 to 6 mg / ml, or 1.0 to 5 mg / ml.

[0333] Any desired lipids can be mixed in any ratio suitable for encapsulating RNA. In some embodiments, a suitable lipid solution contains a mixture of desired lipids comprising cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids) and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids comprising one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and one or more PEGylated lipids.

[0334] In some embodiments, the compositions of the present invention selectively transfect or distribute target cells (i.e., do not transfect non-target cells). The compositions of the present invention may also be prepared to preferentially target a variety of target cells, including, but not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells (e.g., meninges, astrocytes, motor neurons, dorsal root ganglion cells and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial-lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, antigen-presenting cells (e.g., dendritic cells), reticulocytes, leukocytes, granulocytes, and tumor cells.

[0335] Pharmaceutical composition In certain embodiments, a composition (e.g., a pharmaceutical composition) comprising a therapeutic agent provided herein is provided herein. In certain embodiments, the therapeutic agent is an RNA polynucleotide provided herein. In some embodiments, the therapeutic agent is a cyclic RNA polynucleotide provided herein. In some embodiments, the therapeutic agent is a vector provided herein. In some embodiments, the therapeutic agent is a cell (e.g., a human cell such as a human antigen-presenting cell) comprising the RNA polynucleotide, cyclic RNA, or vector provided herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition provided herein comprises the therapeutic agent provided herein in combination with another pharmaceutically active agent or drug, e.g., an anti-inflammatory drug or antibody capable of targeting B cell antigens, e.g., an anti-CD20 antibody, e.g., rituximab.

[0336] With respect to pharmaceutical compositions, pharmaceutically acceptable carriers can be any of those conventionally used and are limited only by chemophysical considerations, such as solubility and reactivity with the active agent, as well as the route of administration. The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are generally readily available. It is preferable that pharmaceutically acceptable carriers are chemically inert to the therapeutic agent and do not have harmful side effects or toxicity under the conditions of use.

[0337] The choice of carrier is determined in part by the specific therapeutic agent, as well as the specific method used to administer the therapeutic agent. Therefore, a variety of suitable formulations of the pharmaceutical compositions provided herein exist.

[0338] In certain embodiments, the pharmaceutical composition includes a preservative. In certain embodiments, suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0339] In some embodiments, the pharmaceutical composition includes a buffer. In some embodiments, suitable buffers may include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffers may be used optionally. The buffer or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0340] In some embodiments, the concentration of the therapeutic agent in the pharmaceutical composition can vary, for example, to less than about 1% by weight, or to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% by weight or more, and can be selected mainly by fluid volume and viscosity according to a specific dosage mode selected.

[0341] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intra-arterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are merely illustrative and not limiting. More than one route may be used to administer the therapeutic agents provided herein, and in certain cases, a particular route may provide a faster and more effective response than another.

[0342] Formulations suitable for oral administration may include, or be derived from, (a) an effective amount of the therapeutic agent dissolved in a liquid solution, such as water, saline, or orange juice; (b) capsules, sachets, tablets, licks, and lozenges, each containing a predetermined amount of the active ingredient as a solid or granule; (c) powder; (d) suspension in a suitable liquid; and (e) a suitable emulsion. Liquid formulations may contain a diluent, such as water, and an alcohol, such as ethanol, benzyl alcohol, and polyethylene alcohol, with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms may be of the usual hard-shell or soft-shell gelatin type, for example, containing a surfactant, a lubricant, and an inert filler, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms may contain lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and one or more other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavorings, and other pharmacologically suitable excipients. Lozenge forms may contain therapeutic agents with flavorings, usually sucrose, acacia, or tragacanth. Lozenge may contain therapeutic agents having inert bases, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, etc., in addition to excipients known in the art.

[0343] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, antibacterial agents, and solutes to make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In some embodiments, the therapeutic agents provided herein may be administered with or without the addition of pharmaceutically acceptable surfactants, such as soaps or detergents, suspending agents, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants, in a physiologically acceptable diluent in a pharmaceutically acceptable liquid or mixture of liquids, such as water, physiological saline, aqueous dextrose and related sugar solutions, alcohols, such as ethanol or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides.

[0344] Oils can be used in parenteral formulations in some embodiments and include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0345] Soaps suitable for use in specific embodiments of parenteral formulations include fatty alkali metals, ammonium, and triethanolamine salts; suitable detergents include (a) cationic detergents, e.g., dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents, e.g., alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, e.g., fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, e.g., alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0346] In some embodiments, parenteral formulations will contain, for example, about 0.5% to about 25% by weight of the therapeutic agent in a solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain, for example, one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations will typically be in the range of, for example, about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol, sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide and propylene glycol. Parenteral formulations can be provided in sealed containers of unit or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid excipient, such as water, immediately before use for injection. Immediate injection solutions and suspensions can be prepared from the sterile powders, granules, and tablets of the types described above.

[0347] In certain embodiments, injectable formulations are provided herein. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed, pages 622-630 (1986)).

[0348] In some embodiments, topical formulations are provided herein. Topical formulations containing those useful for transdermal drug release are preferred in the circumstances of the specific embodiments provided herein for application to the skin. In some embodiments, the therapeutic agent can be made into an aerosol formulation administered by inhalation, either alone or in combination with other suitable ingredients. These aerosol formulations can be encapsulated in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, or nitrogen. They can also be formulated as pharmaceuticals for non-pressurized preparations such as nebulizers or atomizers. Such spray formulations can also be used for spraying mucous membranes.

[0349] In certain embodiments, the therapeutic agents provided herein may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes. Liposomes may help the therapeutic agent target specific tissues. Liposomes may also be used to extend the half-life of the therapeutic agent. Many methods are available for preparing liposomes, for example, as described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9,467 (1980) and U.S. Patents No. 4,235,871, No. 4,501,728, No. 4,837,028, and No. 5,019,369.

[0350] In some embodiments, the therapeutic agents provided herein are formulated in a time-release, delayed-release, or sustained-release delivery system such that delivery of the composition occurs with sufficient time to cause sensitization of the treated site before it occurs. Such systems can avoid repeated administration of the therapeutic agent, thereby increasing convenience for the subject and the physician, and may be particularly suitable for certain embodiments of the compositions provided herein. In one embodiment, the composition of the present invention is formulated to be suitable for sustained release of the circRNA contained herein. Such sustained-release composition can be conveniently administered to the subject with extended dosing intervals. For example, in one embodiment, the composition of the present invention is administered to the subject twice daily, daily, or every other day. In some embodiments, the composition of the present invention is administered to the subject twice weekly, once weekly, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, once a month, every 6 weeks, every 8 weeks, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or annually.

[0351] In some embodiments, the protein encoded by the polynucleotide of the present invention is produced by target cells over a sustained period of time. For example, the protein may be produced for longer than 1 hour, longer than 4 hours, longer than 6 hours, longer than 12 hours, longer than 24 hours, longer than 48 hours, or longer than 72 hours after administration. In some embodiments, the polypeptide is expressed at a peak level about 6 hours after administration. In some embodiments, polypeptide expression is sustained at least at a therapeutic level. In some embodiments, the polypeptide is expressed at least at a therapeutic level for longer than 1 hour, longer than 4 hours, longer than 6 hours, longer than 12 hours, longer than 24 hours, longer than 48 hours, or longer than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in the patient's tissue (e.g., liver or lung). In some embodiments, the detectable level of polypeptide is from the sustained expression from the circRNA composition over a period of longer than 1 hour, longer than 4 hours, longer than 6 hours, longer than 12 hours, longer than 24 hours, longer than 48 hours, or longer than 72 hours after administration.

[0352] In certain embodiments, the protein encoded by the polynucleotide of the present invention is produced at levels exceeding normal physiological levels. The protein level may be increased compared to a control. In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual or a population of individuals lacking the relevant protein or polypeptide. In some embodiments, the control may be the normal level of the relevant protein or polypeptide in an individual to which the composition is administered. In other embodiments, the control is the polypeptide expression level at one or more comparable time points in time, during other therapeutic interventions, for example, at direct injection of the corresponding polypeptide.

[0353] In certain embodiments, the level of the protein encoded by the polynucleotide of the present invention is detectable 3, 4, 5 days, or more than a week after administration. Increased levels of the protein may be observed in tissues (e.g., liver or lungs).

[0354] In some embodiments, the method results in a sustained cyclic half-life of the protein encoded by the polynucleotide of the present invention. For example, the protein may be detected over a longer period of time or days than the half-life observed via subcutaneous injection of the protein or the mRNA encoding the protein. In some embodiments, the half-life of the protein is 1 day, 2 days, 3 days, 4 days, 5 days, or more than 1 week.

[0355] Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems, such as poly(lactide-glycolides), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acids, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems, such as lipids including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono-di- and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused grafts; and the like. Specific examples, though not limited to these, include (a) an erosion system, where the active composition is contained in a matrix form such as those described in U.S. Patents No. 4,452,775, No. 4,667,014, No. 4,748,034, and No. 5,239,660, and (b) a diffusion system, where the active ingredient penetrates at a controlled rate from a polymer such as those described in U.S. Patents No. 3,832,253 and No. 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are suitable for implantation.

[0356] In some embodiments, the therapeutic agent can be compounded directly or indirectly into the targeting portion through a linking portion. Methods for compounding the therapeutic agent into the targeting portion are known in the art. See, for example, Wadwa et al., J, Drug Targeting 3:111 (1995) and U.S. Patent No. 5,087,616.

[0357] In some embodiments, the therapeutic agents provided herein are formulated into depot forms such that the manner in which the therapeutic agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The depot form of the therapeutic agent may be, for example, an implantable composition comprising the therapeutic agent and a porous or non-porous material such as a polymer, where the therapeutic agent is encapsulated by the material, diffused throughout the material, and / or decomposed by the non-porous material. The depot is then implanted at a desired location in the body, and the therapeutic agent is released from the implant at a predetermined rate.

[0358] Treatment method In certain embodiments, methods for treating and / or preventing pathological conditions, such as viral infections, are provided herein.

[0359] In certain embodiments, the therapeutic agents provided herein are co-administered with one or more additional therapeutic agents (for example, in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the therapeutic agents provided herein may be administered first, followed by one or more additional therapeutic agents, or vice versa. Alternatively, the therapeutic agents provided herein and one or more additional therapeutic agents may be administered simultaneously.

[0360] In some embodiments, the subject is a mammal. In some embodiments, the mammals referred to herein may be any mammal, including but not limited to rodents, such as mice and hamsters, or mammals of the order Logomorpha, such as rabbits. The mammal may be from the order Carnivora, which includes the families Felidae (cats) and Canidae (dogs). The mammal may be from the order Artiodactyla, which includes the families Bovidae (cats) and Suidae (pigs), or the order Perissodactyla, which includes the family Equidae (horses). The mammal may be from the order Primates, Ceboidi, or Simoida (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0361] array (Table 1) IRES sequence TIFF2026136198000002.tif202165TIFF2026136198000003.tif208165TIFF2026136 198000004.tif196165TIFF2026136198000005.tif172165TIFF2026136198000006.t if202165TIFF2026136198000007.tif196165TIFF2026136198000008.tif196165TIF F2026136198000009.tif208165TIFF2026136198000010.tif214165TIFF20261361980 00011.tif208165TIFF2026136198000012.tif208165TIFF2026136198000013.tif22 6165TIFF2026136198000014.tif184165TIFF2026136198000015.tif196165TIFF202 6136198000016.tif214165TIFF2026136198000017.tif196165TIFF20261361980000 18.tif208165TIFF2026136198000019.tif225165TIFF2026136198000020.tif190165

[0362] In some embodiments, the IRES of the present invention is an IRES having the sequences listed in Table 1 (SEQ ID NOs: 1 to 72). In some embodiments, the IRES is a Salivirus IRES. In some embodiments, the IRES is a Salivirus SZ1 IRES.

[0363] (Table 2) Sequence of 5' intron fragments at the anabaena permutation site TIFF2026136198000021.tif214165TIFF2026136198000022.tif220165TIFF2026136198000023.tif228165TIFF2026136198000024.tif61165

[0364] In some embodiments, the 5' intron fragment is a fragment having the sequences listed in Table 2. Typically, a construct containing the 5' intron fragments listed in Table 2 contains the corresponding 3' intron fragments listed in Table 3 (for example, both representing fragments with L9a-8 permutation sites).

[0365] (Table 3) Anabaena permutation site 3' intron fragment sequence TIFF2026136198000025.tif203165TIFF2026136198000026.tif220165TIFF2026136198000027.tif214165 TIFF2026136198000028.tif190165TIFF2026136198000029.tif208165TIFF2026136198000030.tif119165

[0366] In some embodiments, the 3' intron fragment is a fragment having the sequence listed in Table 3. In some embodiments, a construct containing the 3' intron fragments listed in Table 3 contains the corresponding 5' intron fragment as listed in Table 2 (for example, both representing fragments with L9a-8 permutation sites).

[0367] (Table 4) Sequence of 5' intron fragments at non-anabaena permutation sites TIFF2026136198000031.tif192165TIFF2026136198000032.tif48165

[0368] In some embodiments, the 5' intron fragment is a fragment having the sequence listed in Table 4. A construct containing the 5' intron fragments listed in Table 4 contains the corresponding 3' intron fragment as listed in Table 5 (for example, both representing fragments with an Azop1 intron).

[0369] (Table 5) Sequence of 3' intron fragments at non-anabaena permutation sites TIFF2026136198000033.tif191165TIFF2026136198000034.tif161165

[0370] In some embodiments, the 3' intron fragment is a fragment having the sequence listed in Table 5. A construct containing the 3' intron fragments listed in Table 5 contains the corresponding 5' intron fragment as listed in Table 4 (for example, both representing fragments with an Azop1 intron).

[0371] (Table 6) Spacer and Anabaena 5' Intron Fragment Sequences TIFF2026136198000035.tif214165TIFF2026136198000036.tif208165TIFF2026136198000037.tif220165TIFF20261361980 00038.tif214165TIFF2026136198000039.tif221165TIFF2026136198000040.tif227165TIFF2026136198000041.tif143165

[0372] In some embodiments, the spacer and 5' intron fragment are spacers and fragments having the arrangements listed in Table 6.

[0373] (Table 7) Spacer and Anabaena 3' Intron Fragment Sequences TIFF2026136198000042.tif208165TIFF2026136198000043.tif191165TIFF2026136198000044.t if185165TIFF2026136198000045.tif208165TIFF2026136198000046.tif220165TIFF2026136198 000047.tif220165TIFF2026136198000048.tif226165TIFF2026136198000049.tif214165TIFF20 26136198000050.tif220165TIFF2026136198000051.tif232165TIFF2026136198000052.tif47165

[0374] In some embodiments, the spacers and 3' intron fragments are spacers and fragments having the arrangements listed in Table 7.

[0375] (Table 8) Arrangement of sectional sites TIFF2026136198000053.tif127165

[0376] (Table 9) SARS-CoV-2 protein sequence TIFF2026136198000054.tif213165TIFF2026136198000055.tif225165TIFF20261361980 00056.tif231165TIFF2026136198000057.tif231165TIFF2026136198000058.tif231165 TIFF2026136198000059.tif142165TIFF2026136198000060.tif225165TIFF20261361980 00061.tif231165TIFF2026136198000062.tif201165TIFF2026136198000063.tif238165

[0377] In some embodiments, the antigenic polypeptide is the SARS-CoV-2 protein, a fragment of the SARS-CoV-2 protein, or derived from the SARS-CoV-2 protein or a fragment thereof. In some embodiments, the antigenic polypeptide may, but is not limited to, the SARS-CoV-2 spike protein, Nsp1-Nsp16, ORF3a, ORF6, ORF7a, ORFb, ORF8, ORF10, the SARS-CoV-2 envelope protein, the SARS-CoV-2 membrane protein, the SARS-CoV-2 nucleocapsid protein, or an immunogenic fragment of the SARS-CoV-2 spike protein.

[0378] In some embodiments, the antigen contains all or part of the sequence on Table 9. In some embodiments, the peptide contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequence on Table 9. In some embodiments, the circular RNA vaccine contains RNA encoding one or more antigens. In some embodiments, the circular RNA vaccine contains RNA encoding at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 antigens. In some embodiments, the circular RNA polynucleotide encodes one or more antigens. In some embodiments, the circular RNA polynucleotide encodes at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 antigens.

[0379] (Table 10) Adjuvant polypeptides TIFF2026136198000064.tif231165TIFF2026136198000065.tif184165TIFF2026136198000066.tif219165

[0380] In some embodiments, a polynucleotide, or a protein encoded by a polynucleotide, contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to one or more sequences disclosed herein. In some embodiments, a polynucleotide, or a protein encoded by a polynucleotide, contains a sequence identical to one or more sequences disclosed herein. In some embodiments, an expression sequence encodes a protein containing or consisting of a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequences in Table 8, or is identical to the sequences in Table 8. In some embodiments, the expression sequence encodes a protein containing or consisting of a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequences in Table 8, or is identical to the sequences in Table 8, and the IRES contains or consists of a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequences in Table 1, or is identical to the sequences in Table 1. In some embodiments, the expression sequence encodes a protein containing or consisting of a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequence in Table 8, and the 3' and 5' group I intron fragments contain or consist of corresponding sequences having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to the sequences in Tables 2 and 3, 4 and 5, or 6 and 7, or are identical to the sequences in Tables 2 and 3, 4 and 5, or 6 and 7.

[0381] Preferred embodiments are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors anticipate that such variations will be appropriately used by those skilled in the art, and they intend that the invention will be practiced in a manner different from that specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the invention unless otherwise shown herein or otherwise clearly contradicted by context. [Examples]

[0382] Wesselhoeft et al. (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In vivo. Molecular Cell. 74(3), 508-520, and Wesselhoeft et al., (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629 are incorporated herein by reference in their entirety.

[0383] The present invention will be described in more detail by reference to the following embodiments, but is not intended to be limited to these embodiments. These embodiments include all kinds of illustrative variations for the purpose of providing a complete disclosure and explanation to those skilled in the art of how to make and use the subject invention, and are not intended to limit the scope of what is considered the present invention.

[0384] Example 1 Example 1A: The outer double-strand formation region enables the cyclization of long precursor RNA using a permutation substitution intron-exon (PIE) cyclization strategy. A 1.1 kb sequence containing the full-length encephalomyocarditis virus (EMCV) IRES, a Gaussial luciferase (GLuc) expression sequence, and two short exon fragments of a permutation-substitution intron-exon (PIE) construct were inserted between the 3' and 5' introns of the permutation-substitution group I catalytic intron of the T4 phage thymidylate synthase (Td) gene. Precursor RNA was synthesized by run-off transcription. Circulation was attempted by heating the precursor RNA in the presence of magnesium ions and GTP, but no splicing product was obtained.

[0385] We designed perfectly complementary 9-nucleotide and 19-nucleotide double-strand formation regions and added them to the 5' and 3' ends of the precursor RNA. The addition of these homologous arms increased splicing efficiency from 0 to 16% in the 9-nucleotide double-strand formation region and to 48% in the 19-nucleotide double-strand formation region, as assessed by the disappearance of the precursor RNA band.

[0386] The splicing product was treated with RNase R. Sequencing across the putative splice junction of the RNase R-treated splicing reaction revealed ligated exons. Digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band, in contrast to the two bands obtained from the linear precursor digested with RNase H. This indicates that circular RNA is the primary product of the splicing reaction of precursor RNA containing an outer double-strand forming region of 9 or 19 nucleotides in length.

[0387] Example 1B: Spacers that preserve the secondary structure of the IRES and PIE splice sites increase the efficiency of annularization. A series of spacers were designed and inserted between the 3'PIE splice site and the IRES. These spacers were designed to either preserve or disrupt secondary structures within the intron sequences of the IRES, 3'PIE splice site, and / or 5' splice site. Adding spacer sequences resulted in an 87% splicing efficiency, while adding destructive spacer sequences did not result in detectable splicing.

[0388] Example 2 Example 2A: In addition to the outer double-strand formation region, the inner double-strand formation region creates a splicing bubble, enabling translation of several expression sequences. The spacers were designed to be non-homologous to unstructured, intronic, and IRES sequences and to contain a spacer-spacer double-strand formation region. These were inserted between the 5' exon and the IRES, and between the 3' exon and the expression sequence, of constructs containing an outer double-strand formation region, an EMCV IRES, and expression sequences of Gaussian luciferase (full length: 1289 nt), firefly luciferase (2384 nt), eGFP (1451 nt), human erythropoietin (1313 nt), and Cas9 endonuclease (4934 nt). Circularization was achieved for all five constructs. Circularization of constructs using T4 phage and anabena introns was nearly equivalent. Circularization efficiency was higher with shorter sequences. To measure translation, each construct was transfected into HEK293 cells. Cells transfected with Gaussia and firefly luciferase showed a strong response, as measured by luminescence; human erythropoietin was detectable in the culture medium of cells transfected with erythropoietin circRNA; and EGFP fluorescence was observed from cells transfected with EGFP circRNA. Co-transfection of cells constitutively expressing GFP with Cas9 circRNA and sgRNA for GFP resulted in the abolition of fluorescence in up to 97% of cells compared to a control with sgRNA alone.

[0389] Example 2B: Use of CVB3 IRES increases protein production. Constructs were created containing different IRESs, each containing either an internal or external double-strand formation region, as well as either a Gaussian luciferase or firefly luciferase expression sequence. Protein production was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. The coxsackievirus B3 (CVB3) IRES construct produced the highest protein in both cases.

[0390] Example 2C: The use of poly-A or poly-AC spacers increases protein production. A 30-nucleotide poly-A or poly-AC spacer was added between the IRES and the splice junction in each IRES-containing construct that produced protein in Example 2B. Gaussian alciferase activity was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. Both spacers improved expression in all constructs compared to the control construct without spacers.

[0391] Example 3 HEK293 or HeLa cells transfected with circular RNA produce more protein than cells transfected with equivalent unmodified or modified linear RNA. CircRNA encoding gausial ciferase (CVB3-GLuc-pAC), purified by HPLC, was compared with standard unmodified linear GLuc mRNA with a 5' methylguanosine cap and 3' poly(A) tail, and commercially available nucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA (Trilink). Luminescence was measured 24 hours after transfection, revealing that the circRNA produced 811.2% more protein than the unmodified linear mRNA and 54.5% more protein than the modified mRNA in HEK293 cells. Similar results were obtained in HeLa cells and in comparisons between optimized circRNA encoding human erythropoietin and linear mRNA modified with 5-methoxyuridine.

[0392] Luminescence data were collected over a 6-day period. In HEK293 cells, circRNA transfection resulted in a protein production half-life of 80 hours, compared to 43 hours for unmodified linear mRNA and 45 hours for modified linear mRNA. In HeLa cells, circRNA transfection resulted in a protein production half-life of 116 hours, compared to 44 hours for unmodified linear mRNA and 49 hours for modified linear mRNA. CircRNA produced substantially more protein over its lifetime than both unmodified and modified linear mRNA in both cell types.

[0393] Example 4 Example 4A: Purification of circRNA by RNase digestion, HPLC purification, and phosphatase treatment reduces immunogenicity. Fully purified circular RNA is significantly less immunogenic than unpurified or partially purified circular RNA. Protein expression stability and cell viability depend on cell type and circular RNA purity. Human fetal kidney 293 (HEK293) and human lung cancer A549 cells were used to create human fetal kidney 293 (HEK293) cells. a. Products of unpurified GLuc circular RNA splicing reaction, b. Products of RNase R digestion of splice reactants, c. Products obtained by RNase R digestion and HPLC purification of the splice reaction, or d. The product of the splicing reaction was transfected with RNase digestion, HPLC purification, and phosphatase treatment.

[0394] RNase R digestion of the splicing reaction was insufficient to prevent cytokine release in A549 cells compared to untransfected controls.

[0395] Although additional HPLC purification was insufficient to prevent cytokine release, interleukin-6 (IL-6) levels were significantly reduced and interferon-α1 (IFNα1) levels were significantly increased compared to the unpurified splicing reaction.

[0396] Adding phosphatase treatment after HPLC purification and before RNase R digestion dramatically reduced the expression of all upregulated cytokines evaluated in A549 cells. Secreted monocyte chemoattractant protein 1 (MCP1), IL-6, IFNα1, tumor necrosis factor α (TNFα), and IFNγ-inducible protein-10 (IP-10) were reduced to undetectable or untransfected baseline levels.

[0397] There was virtually no cytokine release in HEK293 cells. Transfecting A549 cells with higher purity circular RNA increased GLuc expression stability and cell viability. Fully purified circular RNA exhibited a stable phenotype similar to that of transfected 293 cells.

[0398] Example 4B: The circular RNA did not cause significant immunogenicity and is not a RIG-I ligand. A549 cells, a. Unpurified circular RNA, b. High molecular weight (linear and cyclic linked) RNA, c. Circular (nicked) RNA, d. Initial fraction of purified circular RNA (often overlapping with nicked RNA peaks), e. Late fraction of purified circular RNA (with less overlap with nicked RNA peaks), f. Introns excised during circulation, or g. Vehicle (i.e., untransfected control) was used for transfection.

[0399] Because it is difficult to obtain suitably pure linear precursor RNA from the splicing reaction product, the precursor RNA was separately synthesized and purified in the form of a splice site deletion mutant (DS). Cytokine release and cell viability were measured in each case.

[0400] Potent release of IL-6, RANTES, and IP-10 was observed in response to most species present in the splicing reaction product, as well as to the precursor RNA. The early circRNA fraction induced a cytokine response comparable to that of the other non-circRNA fractions. This indicates that even relatively small amounts of linear RNA contaminants can induce a substantial cellular immune response in A549 cells. The late circRNA fraction did not induced a cytokine response exceeding that of the untransfected control. A549 cell viability 36 hours after transfection was significantly higher in the late circRNA fraction compared to all other fractions.

[0401] The induction of RIG-I and IFN-β1 transcripts upon transfection of A549 cells using late circRNA HPLC fraction, precursor RNA, or unpurified splicing reaction product was analyzed. Induction of both RIG-I and IFN-β1 transcripts was weaker with the late circRNA fraction than with precursor RNA or unpurified splicing reaction product. RNase R treatment of the splicing reaction product alone was insufficient to eliminate this effect. Adding a very small amount of RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-I transcription. In HeLa cells, transfection with RNase R-digested splicing reaction product induced RIG-I and IFN-β1, but not with purified circRNA. Overall, HeLa cells were less sensitive to contaminating RNA species than A549 cells.

[0402] Time-course experiments monitoring RIG-I, IFN-β1, IL-6, and RANTES transcript induction within the first 8 hours after transfection of A549 cells with splicing reaction products or fully purified circRNA did not reveal a transient response to circRNA. Similarly, purified circRNA failed to induce pro-inflammatory transcripts in RAW264.7 mouse macrophages.

[0403] A549 cells were transfected with purified circRNA containing EMCV IRES and EGFP expression sequences. This failed to produce substantial induction of pro-inflammatory transcripts. These data demonstrate that the acyclic component of the splicing reaction is responsible for the immunogenicity observed in previous studies, and that circRNA is not a native ligand for RIG-I.

[0404] Example 5 Circular RNAs evade detection by TLRs. TLR3, 7, and 8 reporter cell lines were transfected with multiple linear or circular RNA constructs, and secreted embryonic alkaline phosphatase (SEAP) was measured.

[0405] Linear RNA was constructed by deleting introns and homologous arm sequences. The linear RNA constructs were then treated with phosphatase (or capped if capped RNA was used) and purified by HPLC.

[0406] None of the transfections attempted produced a response in TLR7 reporter cells. TLR3 and TLR8 reporter cells were activated by capped linearized RNA, polyadenylated linearized RNA, nicked circRNA HPLC fraction, and early circRNA fraction. Late circRNA fraction and m1ψ-mRNA did not induce a TLR-mediated response in any cell line.

[0407] In the second experiment, circRNA was linearized using two methods: treatment of circRNA with heat in the presence of magnesium ions, and DNA oligonucleotide-induced RNase H digestion. Both methods yielded a large amount of full-length linearized RNA and a small amount of intact circRNA. TLR3, 7, and 8 reporter cells were transfected with circular RNA, heat-degraded circular RNA, or RNase H-degraded circular RNA, and SEAP secretion was measured 36 hours after transfection. TLR8 reporter cells secreted SEAP in response to both forms of degraded circular RNA, but did not produce a greater response to circular RNA transfection than to mock transfection. Despite activation of TLR3 by linearized RNA transcribed in vitro, no activation was observed in TLR3 and TLR7 reporter cells under degradation or intact conditions.

[0408] Example 6 Unmodified circular RNA produces increased sustained in vivo protein expression compared to linear RNA. Unmodified and m1ψ-modified human erythropoietin (hEpo) linear mRNA and circRNA were injected into mice and used to transfect HEK293 cells. Equimolar transfection with m1ψ-mRNA and unmodified circRNA resulted in potent protein expression in HEK293 cells. hEpo linear mRNA and circRNA showed similar relative protein expression patterns and cell viability compared to GLuc linear mRNA and circRNA after isoweight transfection of HEK293 and A549 cells.

[0409] In mice, hEpo was detected in serum after injection of hEpo circRNA or linear mRNA into visceral fat. hEpo detected after injection of unmodified circRNA decayed more slowly than that from unmodified or m1ψ-mRNA, and was still present 42 hours after injection. Serum hEpo decreased rapidly upon injection of unpurified circRNA splicing reaction or unmodified linear mRNA. Injection of unpurified splicing reaction produced a detectable cytokine response in serum, which was not observed with other RNAs, including purified circRNA.

[0410] Example 7 Circular RNA can be effectively delivered in vivo or in vitro via lipid nanoparticles. Purified circular RNA was formulated into lipid nanoparticles (LNPs) using the ionizable lipidoid cKK-E12 (Dong et al., 2014; Kauffman et al., 2015). The particles formed a uniform multilamellar structure with an average size, polydispersity index, and encapsulation efficiency similar to particles containing commercially available control linear mRNA modified with 5 moU.

[0411] Purified hEpo circRNA, when encapsulated in LNPs and added to HEK293 cells, showed higher expression than 5moU-mRNA. Expression stability from LNP-RNA in HEK293 cells was similar to that of RNA delivered by transfection reagents, except for a slight delay in decay for both 5moU-mRNA and circRNA. Neither unmodified circRNA nor 5moU-mRNA could activate RIG-I / IFN-β1 in vitro.

[0412] In mice, LNP-RNA was delivered by local injection into visceral adipose tissue or intravenous delivery to the liver. In both cases, 6 hours after delivery, serum hEpo expression from circRNA was low but comparable to that from 5moU-mRNA. Serum hEpo detected after fat injection of unmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, and the delayed decay of expression present in serum was similar to that observed in vitro, while serum hEpo after intravenous injection of LNP-circRNA or LNP-5moU-mRNA decayed at approximately the same rate. In neither of these cases was there an increase in serum cytokines or local RIG-I, TNFα, or IL-6 transcript induction.

[0413] Example 8 Expression and functional stability of HEK293, HepG2, and 1C1C7 cells mediated by IRES. Constructs containing anabenaintron / exon regions, Gaussial alciferase expression sequences, and various IRESs were cyclized. 100 ng of each cyclized reaction product was separately transfected into 20,000 HEK293, HepG2, and 1C1C7 cells using Lipofectamine MessengerMax. Luminescence in each supernatant was evaluated after 24 hours as a measure of protein expression. In HEK293 cells, constructs containing black hivirus B, Sarivirus FHB, Aichi virus, Sarivirus HG-J1, and Enterovirus J IRESs produced the most luminescence after 24 hours (Figure 1A). In HepG2 cells, constructs containing Aichi virus, Sarivirus FHB, EMCV-Cf, and CVA3 IRESs produced high luminescence after 24 hours (Figure 1B). In 1C1C7 cells, constructs containing Sarivirus FHB, Aichivirus, Sarivirus NG-J1, and Sarivirus A SZ-1 IRES produced high luminescence within 24 hours (Figure 1C).

[0414] Larger IRESs tended to produce greater luminescence over 24 hours. Since shorter total sequence lengths tended to increase cyclization efficiency, high expression and selection of relatively short IRESs may lead to improved constructs. In HEK293 cells, constructs using blackhivirus B IRESs produced the highest luminescence, particularly compared to other IRESs of similar length (Figure 2A). Expression from IRES constructs in HepG2 and 1C1C7 cells, plotted against IRES size, is shown in Figures 2B and 2C.

[0415] The functional stability of selected IRES constructs in HepG2 and 1C1C7 cells was measured over a 3-day period. Luminescence from secreted Gaussian alciferase in the supernatant was measured every 24 hours after transfecting 20,000 cells with 100 ng of each cyclization product, followed by complete medium replacement. Sarivirus A GUT and Sarivirus FHB showed the highest functional stability in HepG2 cells, while Sarivirus N-J1 and Sarivirus FHB produced the most stable expression in 1C1C7 cells (Figures 3A and 3B).

[0416] Example 9 Expression and functional stability of IRES in Jurkat cells. Two sets of constructs containing anabenaintron / exon regions, Gaussian alciferase expression sequences, and subsets of previously tested IRESs were cyclized. 60,000 Jurkat cells were electroporated with 1 μg of each cyclized reaction. 24 hours after electroporation, luminescence from secreted Gaussian alciferase in the supernatant was measured. CVB3 IRES constructs were included in both sets for comparison between sets and with the efficacy of previously defined IRESs. CVB1 and Salivirus A SZ1 IRES constructs produced the highest expression at 24 hours. Data can be found in Figures 4A and 4B.

[0417] The functional stability of the IRES constructs in electroporated Jurkat cells was measured over a 3-day period for each round. Luminescence from secreted Gaussian alciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 μg of each cyclization reagent, followed by complete medium replacement (Figures 5A and 5B).

[0418] The Salivirus A SZ1 and Salivirus A BN2 IRES constructs exhibited higher functional stability compared to other constructs.

[0419] Example 10 Expression, functional stability, and cytokine release of circular and linear RNAs in Jurkat cells. The construct, containing the anabena intron / exon region, the Gaussian alciferase expression sequence, and the Salivirus FHB IRES, was circulated. mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified versions with 100% uridine replaced by 5-methoxyuridine (5 moU), were commercially available and purchased from Trilink. 5 moU nucleotide modification has been shown to improve mRNA stability and expression (Bioconjug Chem. 2016 Mar 16;27(3):849-53). Expression of modified mRNA in Jurkat cells, the circulation reaction product (unpurified), and circRNA purified by size exclusion HPLC (pure) were measured and compared (Figure 6A). Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 60,000 cells with 1 μg of each RNA species.

[0420] Luminescence from secreted Gaussial ciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 ug of each RNA species, followed by complete replacement of the culture medium. A comparison of functional stability data of modified mRNA and circRNA in Jurkat cells over 3 days is shown in Figure 6B.

[0421] Transcript induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) was measured 18 hours after electroporation of 60,000 Jurkat cells with 1 μg of each of the above RNA species and 3p-hpRNA (5' triphosphate hairpin RNA known as a RIG-I agonist).

[0422] Example 11 Expression of circular and linear RNAs in monocytes and macrophages. A construct containing anabena intron / exon region, a Gaussian alciferase expression sequence, and a Salivirus FHB IRES was circularized. mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified versions with 100% uridine replaced by 5-methoxyuridine (5 moU), were purchased from Trilink. Expression of the circular and modified mRNAs was measured in human primary monocytes (Figure 8A) and human primary macrophages (Figure 8B). Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 60,000 cells with 1 μg of each RNA species. Luminescence was also measured 4 days after electroporation of human primary macrophages, with the medium changed every 24 hours (Figure 8C). Differences in luminescence were statistically significant in all cases (p<0.05).

[0423] Example 12 IRES-mediated expression and functional stability in primary T cells. Constructs containing anabenaintron / exon regions, Gaussian alciferase expression sequences, and subsets of previously tested IRESs were cyclized, and the reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 1 μg of each circRNA. Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation (Figure 9A). Aichi virus and CVB3 IRES constructs were most highly expressed at 24 hours.

[0424] Luminescence was also measured every 24 hours after electroporation for 3 days to compare the functional stability of each construct (Figure 9B). The construct with Salivirus A SZ1 IRES was the most stable.

[0425] Example 13 Expression and functional stability of circular and linear RNAs in primary T cells and PBMCs. Constructs containing anabena intron / exon regions, Gaussian alciferase expression sequences, and Salivirus A SZ1 IRES or Salivirus FHB IRES were circularized. mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified versions with 100% uridine replaced by 5-methoxyuridine (5 moU), were purchased from Trilink. Expression of HPLC-purified circular and modified mRNA of Salivirus A SZ1 IRES was measured in human primary CD3+ T cells. Expression of HPLC-purified circular, unpurified, and modified mRNA of Salivirus FHB was measured in human PBMCs. Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 150,000 cells with 1 μg of each RNA species. Data for primary human T cells are shown in Figures 10A and 10B, and data for PBMCs are shown in Figure 10C. Differences in expression between purified circular RNA and unpurified circular RNA or linear RNA were significant in all cases (p<0.05).

[0426] The luminescence from secreted Gaussial ciferase in primary T cell supernatant was measured every 24 hours after electroporation for 3 days to compare the functional stability of the constructs. The data are shown in Figure 10B. The difference in relative luminescence between purified circular RNA and linear RNA from the measurement on day 1 was significant for primary T cells on both day 2 and day 3.

[0427] Example 14 Cyclization efficiency by permutation sites in anabenaintrons. RNA constructs containing CVB3 IRES, Gaussian alciferase expression sequences, anabenaintron / exon regions, spacers, internal double-strand formation regions, and homologous arms were produced. The cyclization efficiency of constructs using conventional anabenaintron permutation sites and five consecutive permutation sites at P9 was measured by HPLC. The HPLC chromatograms for the five consecutive permutation sites at P9 are shown in Figure 11A.

[0428] Circulation efficiency was measured at various permutation sites. Circulation efficiency is defined as the area under the HPLC chromatogram curve for each circRNA / (circRNA + precursor RNA). Ranked quantifications of circulation efficiency at each permutation site are shown in Figure 11B. Three permutation sites (shown in Figure 11B) were selected for further investigation.

[0429] In this example, the circular RNA was cyclized by in vitro transcription (IVT) and then purified via a spin column. The cyclization efficiency was determined for all constructs by Mg 2+ The efficiency could potentially be higher if an additional incubation step with guanosine nucleotides were included; however, the removal of this step allowed for comparison between circular RNA constructs and optimization of the circular RNA constructs. This level of optimization is particularly useful for maintaining high cyclization efficiency in large RNA constructs, such as those encoding chimeric antigen receptors.

[0430] Example 15 Efficiency of cyclic formation of alternative introns. Precursor RNAs were constructed containing permutation group 1 introns or permutation sites of various species origins, as well as several constant elements including CVB3 IRES, Gaussial ciferase expression sequences, spacers, internal double-strand formation regions, and homologous arms. Circulation data can be found in Figure 12. Figure 12A shows chromatograms of the degradation of the precursor, CircRNA, and introns. Figure 12B provides a ranked quantification of circulation efficiency based on the chromatograms shown in Figure 12A, as a function of the intron construct.

[0431] In this example, the circular RNA was cyclized by in vitro transcription (IVT) and then purified by spin column. The cyclization efficiency was determined for all constructs by Mg 2+ The efficiency may be higher if an additional incubation step with guanosine nucleotides is included; however, removing this step allows for comparison between circular RNA constructs and optimization of circular RNA constructs. This level of optimization is particularly useful for maintaining high circularization efficiency in large RNA constructs, such as those encoding chimeric antigen receptors.

[0432] Example 16 Annularization efficiency due to the presence or length of homologous arms. RNA constructs containing CVB3 IRES, Gaussian alciferase expression sequences, anabenaintron / exon regions, spacers, and internal double-strand formation regions were produced. Constructs representing three anabenaintron permutation sites were tested with 30nt, 25% GC homologous arms, or without homologous arms ("NA"). These constructs were Mg 2+ Circulation was possible without an incubation step. Circulation efficiency was measured and compared. The data can be found in Figure 13. Circulation efficiency was higher in each construct lacking homologous arms. Figure 13A provides a ranked quantification of circulation efficiency; Figure 13B provides chromatograms of precursor, circRNA, and intron degradation.

[0433] For each of the three permutation sites, constructs were prepared with arm lengths of 10 nt, 20 nt, and 30 nt, and with 25%, 50%, and 75% GC. The splicing efficiency of these constructs was measured and compared with constructs without homologous arms (Figure 14). Splicing efficiency was defined as the ratio of free introns to total RNA in the splicing reaction product.

[0434] ...

Claims

1. A circular RNA polynucleotide, in the following order: a. 3' Group I intron fragment, b. Internal ribosome entry sites (IRES), c. Expression sequences encoding one or more antigens, adjuvants, antigen-like or adjuvant-like polypeptides, or fragments thereof, and d. 5' Group I intron fragments The cyclic RNA polynucleotide comprising the above.

2. A circular RNA polynucleotide, in the following order: a. 3' Group I intron fragment, b. Internal ribosome entry sites (IRES), c. Non-coding expression sequences, and d. 5' Group I intron fragments The cyclic RNA polynucleotide comprising the above.

3. A circular RNA polynucleotide produced from the transcription of a vector, in the following order: a. 5' duplex forming region, b. 3' Group I intron fragment, c. internal ribosome entry site (IRES), d. An expression sequence encoding one or more antigens, adjuvants, antigen-like or adjuvant-like polypeptides, or fragments thereof. e. 5' group I intron fragments, and f. 3' duplex forming region The cyclic RNA polynucleotide comprising the above.

4. A cyclic RNA polynucleotide produced from the transcription of the vector, in the following order: a. 5' duplex forming region, b. 3' Group I intron fragment, c. internal ribosome entry site (IRES), d. Non-coding expression sequences, e. 5' group I intron fragments, and f. 3' duplex forming region The cyclic RNA polynucleotide comprising the above.

5. The cyclic RNA polynucleotide according to claim 3 or 4, comprising a first spacer between the 5' double-strand forming region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-strand forming region.

6. The cyclic RNA polynucleotide according to claim 5, wherein the first and second spacers each have a length of about 10 to about 60 nucleotides.

7. The cyclic RNA polynucleotide according to any one of claims 3 to 6, wherein the first and second double-strand forming regions each have a length of about 9 to about 19 nucleotides.

8. The cyclic RNA polynucleotide according to any one of claims 3 to 6, wherein the first and second double-strand forming regions each have a length of about 30 nucleotides.

9. The aforementioned IRES includes Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliopathy virus, human poliovirus 1, brown marmorated bug (Platia stalli) enteric virus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca Coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua) picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Crucifer tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic spot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n. myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, fruit fly reaper, canine Scamper, fruit fly Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, fruit fly hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco Hetch virus, cabbage crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCVQC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, human parechovirus 1, black hivirus B, Yc-3, rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, human parechovirus 5, Aichivirus, hepatitis A virus HA16, fopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, pegivirus A1220, Pacivirus A 3. Saperovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine pacivirus 1, PLV-CHN, Pacivirus A, Sisinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Houpei picorna-like virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A A circular RNA polynucleotide according to any one of claims 1 to 8, having an IRES sequence from an aptamer for SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

10. A cyclic RNA polynucleotide according to any one of claims 1 to 9, comprising natural nucleotides.

11. The circular RNA polynucleotide according to any one of claims 1 to 9, wherein the expression sequence is codon-optimized.

12. The circular RNA polynucleotide according to any one of claims 1 to 11, wherein the circular RNA polynucleotide has a length of about 100 nucleotides to about 10 kilobases.

13. A cyclic RNA polynucleotide according to any one of claims 1 to 12, having a duration of therapeutic effect in vivo in humans of at least approximately 20 hours.

14. A cyclic RNA polynucleotide according to any one of claims 1 to 13, having a functional half-life of at least about 20 hours.

15. A cyclic RNA polynucleotide according to any one of claims 1 to 14, having a duration of therapeutic effect in human cells that is longer than or equivalent to the duration of therapeutic effect of equivalent linear RNA polynucleotides containing the same expression sequence.

16. A cyclic RNA polynucleotide according to any one of claims 1 to 15, having a functional half-life in human cells that is longer than or equal to the functional half-life of an equivalent linear RNA polynucleotide containing the same expression sequence.

17. A circular RNA polynucleotide according to any one of claims 1 to 16, having an in vivo therapeutic effect duration in humans that is longer than that of equivalent linear RNA polynucleotides having the same expression sequence.

18. A circular RNA polynucleotide according to any one of claims 1 to 17, having an in vivo functional half-life in humans that is longer than that of equivalent linear RNA polynucleotides having the same expression sequence.

19. The cyclic RNA polynucleotide according to any one of claims 1, 3, and 5-18, wherein the adjuvant or adjuvant-like polypeptide is selected from the group comprising toll-like receptor ligands, cytokines, FLt3-ligands, antibodies, chemokines, chimeric proteins, endogenous adjuvants released from dying tumors, and checkpoint inhibitor proteins.

20. The adjuvant or adjuvant-like polypeptide is BCSP31, MOMP, FOMA, MymA, ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, Lumazine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, Falgellin, IFN-α, IFN-γ, I A cyclic RNA polynucleotide according to any one of claims 1, 3, and 5-19, selected from the group comprising L-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-a, IL-7, IL-17, IL-1β, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and andti-CD3.

21. The cyclic RNA polynucleotide according to any one of claims 1, 3, and 5-20, wherein the adjuvant or adjuvant-like polypeptide is selected from Table 10.

22. An RNA polynucleotide comprising a 3' intron fragment and a triphosphorylated 5' terminus in the following order:

23. The RNA polynucleotide according to claim 22, comprising a 5' spacer located upstream of the 3' intron fragment and downstream of the triphosphorylated 5' end.

24. An RNA polynucleotide comprising a 3' intron fragment and a monophosphorylated 5' terminus in the following order:

25. The RNA polynucleotide according to claim 24, comprising a 5' spacer located upstream of the 3' intron fragment and downstream of the monophosphorylated 5' end.

26. RNA polynucleotides containing a 5' intron fragment and a triphosphorylated 5' terminus.

27. The RNA polynucleotide according to claim 26, comprising a 5' spacer located downstream of the 5' intron fragment.

28. RNA polynucleotides containing a 5' intron fragment and a monophosphorylated 5' end.

29. The RNA polynucleotide according to claim 28, comprising a 5' spacer located downstream of the 5' intron fragment.

30. The RNA polynucleotide according to any one of claims 22 to 29, further comprising a poly-A purified tag.

31. The RNA polynucleotide according to any one of claims 22 to 30, further comprising an initiation sequence.

32. The cyclic RNA polynucleotide according to claim 3 or 4, wherein the vector further comprises a triphosphorylated 5' end.

33. The cyclic RNA polynucleotide according to claim 3 or 4, wherein the vector further comprises a monophosphorylated 5' end.

34. The RNA polynucleotide according to any one of claims 24-25 and 28-29, further comprising a triphosphorylated 5' end.

35. The RNA polynucleotide according to any one of claims 22-23 and 26-27, further comprising a monophosphorylated 5' end.

36. RNA preparation, a. A cyclic RNA polynucleotide according to claim 1, claim 2, or both, b. Linear RNA polynucleotides, i. 3'-intron polynucleotides containing a monophosphorylated 5' terminus and a 3'-intron fragment, ii. 5'-intron polynucleotides containing a monophosphorylated 5' terminus and a 5'-intron fragment, iii. 3'-intron polynucleotides containing a triphosphorylated 5' terminus and a 3'-intron fragment, and iv. 5' intron polynucleotides containing triphosphorylated 5' terminus and 3' intron fragments The linear RNA polynucleotide comprising at least one of the following Includes, The RNA preparation wherein the cyclic RNA polynucleotide constitutes at least 90% of the RNA preparation.

37. The RNA preparation according to claim 36, wherein the 3'-intron polynucleotide or 5'-intron polynucleotide comprises a spacer.

38. The RNA preparation according to claim 36, wherein the 3' intron polynucleotide or 5' intron polynucleotide comprises a polyA sequence.

39. The RNA preparation according to any one of claims 36 to 38, wherein the 3'-intron polynucleotide or 5'-intron polynucleotide comprises UTR.

40. The RNA preparation according to any one of claims 39, wherein the 3'-intron polynucleotide or 5'-intron polynucleotide comprises IRES.

41. A pharmaceutical composition comprising a cyclic RNA polynucleotide according to any one of claims 1 to 21, a diluent, and optionally a salt buffer.

42. A pharmaceutical composition comprising an RNA preparation according to any one of claims 36 to 40, a diluent, and optionally a salt buffer.

43. A pharmaceutical composition comprising a cyclic RNA polynucleotide according to any one of claims 1 to 21 and a polycationic, cationic, or polymeric compound.

44. A pharmaceutical composition comprising an RNA preparation according to any one of claims 36 to 40 and a polycationic, cationic, or polymeric compound.

45. The aforementioned polycationic or cationic compound Cationic peptides or proteins, basic polypeptides, cell-permeable peptides (CPPs), Tat-derived peptides, penetratin, VP22-derived or analog peptides, pestivirus Erns, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, Antennapedia-derived peptides, pAntp, pIsl, FGF, lactoferrin, transportan, bufoly N-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, histone, cationic polysaccharide, cationic polymer, cationic lipid, dendrimer, polyimine, polyallylamine, oligofectamine, or cationic or polycationic polymer, sugar backbone polymer, silane backbone polymer, modified polyamino acid, modified acrylate, modified polybeta-amino ester (PBAE), modified amidoamine, dendrimer, block polymer consisting of a combination of one or more cationic blocks and one or more hydrophilic or hydrophobic blocks A pharmaceutical composition according to claim 43 or 44, selected from the group consisting of the following.

46. The pharmaceutical composition according to claim 43 or 44, wherein the polymer compound is selected from the group consisting of polyamine, polyether, polyamide, polyester, polycarbamate, polyurea, polycarbonate, polystyrene, polyimide, polysulfone, polyurethane, polyacetylene, polyethylene, polyethyleneimine, polyisocyanate, polyacrylate, polymethacrylate, polyacrylonitrile, and polyarylate. For example, the polymer is poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly( D,L-lactide-co-caprolactone-coglycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoeste Poly(esteramide), poly(ester ether), polycarbonate, poly(ethylene terephthalate), polyalkylenes such as poly(esteramide), poly(ester ether), poly(ethylene glycol) (PEG), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl esters such as poly(vinyl acetate), polyvinyl halide such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxane, polystyrene, polyurethane, derivatized cellulose (e.g., alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose), acrylic acid polymers, e.g., poly(methyl (meth)acrylate) (PMMA),Poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), 363 5 10 15 20 25 30 35 WO 2021 / 076805 PCT / US2020 / 055844 Poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymer It may contain rimers, polyhydroxyalkanoates, polypropylene fimarates, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerols.

47. The aforementioned polycationic or cationic compound Protamine, nucleolin, spermine or spermidine, poly-L-lysine (PLL), polyarginine, HIV-binding peptide, HIV-1 Tat (HIV), polyethyleneimine (PEI), DOTMA: [1-(2,3-sioleyloxy)propyl]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamide Silspermine, DIMRI: Dimyristooxypropyldimethylhydroxyethylammonium bromide, DOTAP: Dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-Ditetradecanoyl-N-alpha-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride Lido, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, beta-amino acid polymers or reverse polyamides, PVP (poly(N-ethyl-4-vinylpyridinium bromide)), pDMAEMA (poly(dimethylaminoethylmethyl acrylate)), pAMAM (poly(amideamine)), diamine-terminated 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymer, polypropylamine dendrimer or pAMAM dendrimer, polyimine, PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, cyclodextrin polymers, dextran polymers, chitosan, and PMOXA-PDMS copolymer A pharmaceutical composition according to claim 43 or 44, selected from the group including the following.

48. A cyclic RNA polynucleotide according to any one of claims 1 to 21, a nanoparticle, and optionally a targeting portion operably connected to the nanoparticle. A pharmaceutical composition containing [the specified substance].

49. An RNA preparation according to any one of claims 36 to 40, nanoparticles, and optionally, a targeting portion operably connected to the nanoparticles. A pharmaceutical composition containing [the specified substance].

50. The pharmaceutical composition according to claim 48 or 49, wherein the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles.

51. A pharmaceutical composition according to any one of claims 41 to 50, comprising a targeting portion, wherein the targeting portion mediates receptor-mediated endocytosis or direct fusion to selected cells from a selected cell population or tissue without cell isolation or purification.

52. The pharmaceutical composition according to any one of claims 48 to 51, wherein the targeting portion is an scFv, a nanobody, a peptide, a minibody, a polynucleotide aptamer, a heavy chain variable region, a light chain variable region, or a fragment thereof.

53. The pharmaceutical composition according to any one of claims 41 to 5257 to 57, wherein the cyclic RNA polynucleotide or RNA preparation is in an amount effective for treating or preventing an infectious disease in a human subject in need.

54. The pharmaceutical composition according to any one of claims 41 to 53, having an enhanced safety profile when compared to a pharmaceutical composition comprising a vector containing exogenous DNA encoding the same antigen.

55. The pharmaceutical composition according to any one of claims 41 to 54, wherein less than 1% by weight of the polynucleotide in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA.

56. The pharmaceutical composition according to any one of claims 41 to 55, wherein less than 1% by weight of the polynucleotide and protein in the pharmaceutical composition is double-stranded RNA, DNA sprint, triphosphorylated RNA, phosphatase protein, protein ligase, and capping enzyme.

57. The pharmaceutical composition according to any one of claims 48 to 56, wherein the nanoparticles comprise one or more cationic lipids selected from the group consisting of C12-200, MC3, DLinDMA, DLinC2DMA, cKK-E12, ICE (imidazole-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLInDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

58. A method for treating a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide according to any one of claims 1 to 21, nanoparticles, and optionally, a targeting portion operably attached to the nanoparticles.

59. A method for treating a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising an RNA preparation according to any one of claims 36 to 40, nanoparticles, and optionally, a targeting portion operably connected to the nanoparticles.

60. The method according to claim 58 or 59, wherein the targeting portion is an scFv, a nanobody, a peptide, a minibody, a heavy chain variable region, a light chain variable region, or a fragment thereof.

61. The method according to any one of claims 58 to 60, wherein the nanoparticles are lipid nanoparticles, core-shell nanoparticles, or biodegradable nanoparticles.

62. The method according to any one of claims 58 to 61, wherein the nanoparticles comprise one or more cationic lipids, ionizable lipids, or polyβ-aminoesters.

63. The method according to any one of claims 58 to 62, wherein the nanoparticles comprise one or more noncationic lipids.

64. The method according to any one of claims 58 to 63, wherein the nanoparticles comprise one or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids.

65. The method according to any one of claims 58 to 64, wherein the nanoparticles contain cholesterol.

66. The method according to any one of claims 58 to 65, wherein the nanoparticles comprise arachidonic acid or oleic acid.

67. The method according to any one of claims 58 to 66, wherein the composition comprises a targeting portion, the targeting portion mediates receptor-mediated endocytosis to selected cells from a selected cell population without cell isolation or purification.

68. The method according to any one of claims 58 to 67, wherein the nanoparticles encapsulate one or more cyclic RNA polynucleotides.

69. A vector for producing circular RNA polynucleotides, in the following order: 5' duplex forming region, 3' Group I intron fragment, Intra-sequence ribosome entry sites (IRES), An expression sequence encoding one or more adjuvants, antigens, adjuvant-like or antigen-like polypeptides, or fragments thereof. 5' Group I intron fragment, and 3' duplex forming region The vector, including the vector.

70. A vector for producing circular RNA polynucleotides, comprising, in the following order: a 5' double helix formation region, a 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a non-coding sequence, a 5' group I intron fragment, and a 3' double helix formation region.

71. The vector according to claim 69 or 70, comprising a first spacer between the 5' double-strand forming region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-strand forming region.

72. The vector according to any one of claims 69 to 71, wherein the first and second spacers each have a length of about 20 to about 60 nucleotides.

73. The vector according to any one of claims 69 to 72, wherein each of the first and second spacers includes an unstructured region having a length of at least five nucleotides.

74. The vector according to any one of claims 69 to 73, wherein each of the first and second spacers includes a structuring region of at least seven nucleotides in length.

75. The vector according to any one of claims 69 to 74, wherein the first and second double-strand forming regions each have a length of about 9 to 50 nucleotides.

76. A vector according to any one of claims 69 to 75, which is codon-optimized.

77. The vector according to any one of claims 69 to 76, lacking at least one microRNA binding site present in an equivalent pre-optimization polynucleotide.

78. A prokaryotic cell comprising the vector according to any one of claims 69 to 77.

79. A eukaryotic cell comprising a cyclic RNA polynucleotide according to any one of claims 1 to 21.

80. A human cell, the eukaryotic cell according to claim 79.

81. An antigen-presenting cell, the eukaryotic cell according to claim 79 or 80.

82. It is formulated into lipid nanoparticles. A vaccine comprising at least one viral antigenic polypeptide, an adjuvant or adjuvant-like polypeptide, or at least one circular RNA polynucleotide having an expression sequence encoding an immunogenic fragment thereof.

83. The vaccine according to claim 82, wherein the adjuvant or adjuvant-like polypeptide is selected from Table 10.

84. The aforementioned antigenic polypeptide is an adenovirus; herpes simplex virus type 1; herpes simplex virus type 2; encephalitis virus, papillomavirus, varicella-zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpesvirus type 8; human papillomavirus; BK virus; JC virus; smallpox; poliovirus; hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza virus; The vaccine according to claim 82 or 83, comprising a viral polypeptide from one of the following: ganaritovirus; Junin virus; lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; orbivirus; cortivirus; vanavirus; human enterovirus; hantavirus; West Nile virus; Middle East respiratory syndrome coronavirus; Japanese encephalitis virus; varicella virus; SARS-CoV-2; Eastern equine encephalitis; or any combination of two or more of the above.

85. The vaccine according to any one of claims 82 to 84, wherein the viral antigenic polypeptide or its immunogenic fragment is selected from or derived from any one of sequence numbers 325 to 336.

86. The vaccine according to any one of claims 82 to 85, wherein the viral antigenic polypeptide or its immunogenic fragment has an amino acid sequence having at least 90% identity with any one of the amino acid sequences of SEQ ID NOs. 325 to 336, and the viral antigenic polypeptide or its immunogenic fragment has membrane fusion activity, attaches to a cell receptor, causes fusion of the virus with the mammalian cell membrane, and / or causes the virus to bind to an infected cell.

87. The vaccine according to any one of claims 82 to 86, wherein the expression sequence is codon-optimized.

88. The vaccine according to any one of claims 82 to 87, wherein the vaccine is polyvalent.

89. The vaccine according to any one of claims 82 to 88, formulated in an amount effective to produce an antigen-specific immune response.

90. The vaccine according to any one of claims 82 to 89, wherein the cyclic RNA polynucleotide comprises a first expression sequence encoding a first viral antigenic polypeptide and a second expression sequence encoding a second viral antigenic polypeptide.

91. A method for inducing an immune response in a subject, comprising administering to the subject a vaccine according to any one of claims 82 to 90 in an amount effective for producing an antigen-specific immune response in the subject.

92. The method according to claim 91, wherein the antigen-specific immune response includes a T-cell response or a B-cell response.

93. The method according to claim 91 or 92, wherein a single dose of the vaccine is administered to the subject.

94. The method according to any one of claims 91 to 93, wherein a booster dose of the vaccine is administered to the subject.

95. The method according to any one of claims 91 to 94, wherein the vaccine is administered to the subject by intranasal administration, intradermal injection, or intramuscular injection.

96. The method according to any one of claims 91 to 95, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by at least one logarithm compared to a predetermined threshold level.

97. The method according to any one of claims 91 to 96, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by 1 to 3 logarithms compared to a predetermined threshold level.

98. The method according to any one of claims 91 to 97, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by at least two times compared to a predetermined threshold level.

99. The method according to any one of claims 91 to 98, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject is increased by 2 to 10 times compared to a predetermined threshold level.

100. The method according to any one of claims 91 to 99, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject that has not been administered the vaccine containing the antigenic polypeptide.

101. The method according to any one of claims 91 to 100, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject administered with an attenuated live vaccine or an inactivated vaccine containing the antigenic polypeptide.

102. The method according to any one of claims 91 to 101, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject administered with a recombinant protein vaccine or purified protein vaccine containing the antigenic polypeptide.

103. It is formulated into lipid nanoparticles. A SARS-CoV-2 vaccine comprising at least one circular RNA polynucleotide having an expression sequence encoding at least one SARS-CoV-2 viral antigenic polypeptide or an immunogenic fragment thereof.

104. The SARS-CoV2 vaccine according to claim 102, wherein the SARS-CoV2 viral antigenic polypeptide is selected from SARS-CoV2 spike protein, Nsp1 to Nsp16, ORF3a, ORF6, ORF7a, ORFb, ORF8, ORF10, SARS-CoV2 envelope protein, SARS-CoV2 membrane protein, SARS-CoV2 nucleocapsid protein, or any antigenic peptide of SARS-CoV2, or a fragment of a SARS-CoV2 peptide.

105. The SARS-CoV2 vaccine according to claim 102103 or 104, wherein the SARS-CoV2 virus antigenic polypeptide is derived from SARS-CoV2 virus strain G, strain GR, strain GH, strain L, strain V, or a combination thereof.

106. The SARS-CoV2 vaccine according to any one of claims 103 to 105, wherein the expression sequence is codon-optimized.

107. The SARS-CoV-2 vaccine according to any one of claims 103 to 106, wherein the vaccine is polyvalent.

108. A SARS-CoV-2 vaccine according to any one of claims 103 to 107, formulated in an amount effective to produce an antigen-specific immune response.

109. A method for inducing an immune response in a subject, comprising administering to the subject a SARS-CoV-2 vaccine according to any one of claims 103 to 108 in an amount effective for producing an antigen-specific immune response in the subject.

110. The method according to claim 109, wherein the antigen-specific immune response includes a T-cell response or a B-cell response.

111. The method according to claim 109 or 110, wherein a single dose of the vaccine is administered to the subject.

112. The method according to any one of claims 109 to 111, wherein a booster dose of the vaccine is administered to the subject.

113. The method according to any one of claims 109 to 112, wherein the vaccine is administered to the subject by intranasal administration, intradermal injection, or intramuscular injection.

114. The method according to any one of claims 109 to 113, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by at least one logarithm compared to a predetermined threshold level.

115. The method according to any one of claims 109 to 114, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by 1 to 3 logarithms compared to a predetermined threshold level.

116. The method according to any one of claims 109 to 115, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by at least two times compared to a predetermined threshold level.

117. The method according to any one of claims 109 to 116, wherein the titer of the anti-antigenic polypeptide antibody produced in the subject increases by 2 to 10 times compared to a predetermined threshold level.

118. The method according to any one of claims 109 to 117, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject that has not been administered the vaccine containing the antigenic polypeptide.

119. The method according to any one of claims 109 to 118, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject administered with an attenuated live vaccine or an inactivated vaccine containing the antigenic polypeptide.

120. The method according to any one of claims 109 to 119, wherein the predetermined threshold level is the titer of an anti-antigenic polypeptide antibody produced in a subject administered with a recombinant protein vaccine or purified protein vaccine containing the antigenic polypeptide.

121. A circular RNA polynucleotide having an expression sequence encoding at least one viral antigenic polypeptide, adjuvant, or adjuvant-like polypeptide, or an immunogenic fragment thereof.

122. An expression vector comprising a genetically engineered nucleic acid encoding at least one circular RNA polynucleotide as described in any one of claims 1 to 21.

123. A cyclic RNA polynucleotide vaccine comprising the cyclic RNA polynucleotide described in claim 121, formulated into lipid nanoparticles.

124. The cyclic RNA polynucleotide vaccine according to claim 123, wherein the nanoparticles have an average diameter of 50 to 200 nm.

125. The cyclic RNA polynucleotide vaccine according to claim 123 or 124, wherein the lipid nanoparticles include a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

126. The cyclic RNA polynucleotide vaccine according to any one of claims 123 to 125, wherein the lipid nanoparticle carrier comprises a molar ratio of approximately 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid.

127. The cyclic RNA polynucleotide vaccine according to any one of claims 123 to 126, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol.

128. The cyclic RNA polynucleotide vaccine according to any one of claims 123 to 127, wherein the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

129. The cyclic RNA polynucleotide vaccine according to any one of claims 123 to 128, wherein the nanoparticles have a polydispersity value of less than 0.

4.

130. The cyclic RNA polynucleotide vaccine according to any one of claims 123 to 129, wherein the nanoparticles have a net neutral charge at a neutralized pH value.

131. A pharmaceutical composition for use in the vaccination of a target, comprising an effective dose of a cyclic RNA polynucleotide encoding at least one viral antigen, or an adjuvant or adjuvant-like polypeptide, or an immunogenic fragment thereof, wherein the effective dose is sufficient to produce a neutralizing titer of 1,000 to 10,000 produced by a neutralizing antibody against the antigen, or the adjuvant or adjuvant-like polypeptide, or an immunogenic fragment thereof, when measured in the serum of the target 1 to 72 hours after administration.

132. A pharmaceutical composition for use in the vaccination of a target, comprising an effective dose of a cyclic mRNA polynucleotide encoding at least one viral antigen, or an adjuvant or adjuvant-like polypeptide, or an immunogenic fragment thereof, wherein the effective dose is sufficient to produce a detectable level of the antigen, or an adjuvant or adjuvant-like polypeptide, or an immunogenic fragment thereof, when measured in the serum of the target 1 to 72 hours after administration.

133. A method for inducing, generating, or enhancing an immune response in a subject, comprising administering to the subject a pharmaceutical composition according to claim 131 or 132 in an amount effective for inducing, generating, or enhancing an antigen-specific immune response in the subject.

134. The method according to claim 133, wherein the pharmaceutical composition immunizes the subject to the virus for up to two years.

135. The method according to claim 133 or 134, wherein the pharmaceutical composition immunizes the subject to the virus for more than two years.

136. The method according to any one of claims 133 to 135, wherein the subject is exposed to the virus, the subject is infected with the virus, or the subject is at risk of infection by the virus.

137. The method according to any one of claims 133 to 136, wherein the subject is in an immune-deficient state.

138. A method for inducing an antigen-specific immune response in a subject, comprising administering the vaccine or the pharmaceutical composition to the subject in an amount effective to generate an antigen-specific immune response in the subject. A vaccine according to any one of claims 82-90, 103-108, and 123-130, or a pharmaceutical composition according to claim 131 or 132, for use in [the specified field].

139. A method for inducing an antigen-specific immune response in a subject, comprising administering the vaccine to the subject in an amount effective in generating an antigen-specific immune response in the subject. Use of a vaccine according to any one of claims 82-90, 103-108, and 123-130, or a pharmaceutical composition according to claim 131 or 132, in the manufacture of a pharmaceutical for use in [the specified area].

140. A method for inducing cross-reactivity to various viruses or viral strains in a mammal, comprising administering to the mammal in need of such cross-reactivity a vaccine or a pharmaceutical composition according to any of the prior claims.

141. The method according to claim 140, wherein at least two circular RNA polynucleotides, each having an expression sequence encoding a consensus virus antigen, are administered separately to the mammal.

142. The method according to claim 140 or 141, wherein at least two circular RNA polynucleotides, each having an expression sequence encoding a consensus virus antigen, are administered simultaneously to the mammal.

143. The vaccine according to any one of claims 82-90, 103-108, and 123-130, wherein the cyclic RNA polynucleotide is co-formulated with an adjuvant in the same nanoparticle.

144. The vaccine according to any one of claims 82-90, 103-108, 123-130, and 143, wherein the adjuvant is CpG, imiquimod, aluminum, or Freund's adjuvant.

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