Immunogenic compositions and their use

Abstract

To provide compositions, pharmaceutical preparations, and uses of polyribonucleotides encoding one or more immunogenic polypeptides. [Solution] In particular, this disclosure features cyclic polyribonucleotides encoding one or more immunogenic polypeptides. This disclosure provides compositions, pharmaceutical preparations, and uses of polyribonucleotides (e.g., cyclic or linear polyribonucleotides) encoding one or more immunogens. In particular, this disclosure provides cyclic polyribonucleotides encoding multiple immunogens, and compositions comprising multiple cyclic polyribonucleotides. This disclosure further relates to methods of using cyclic polyribonucleotides encoding one or more polypeptide immunogens. The compositions and pharmaceutical preparations of cyclic polyribonucleotides described herein may induce an immune response in a subject upon administration.

Description

[Technical Field]

[0001] Sequence List This application was filed electronically in ASCII format and is incorporated herein by reference in its entirety. Includes a sequence listing. The aforementioned ASCII copy, created on May 18, 2021, contains 5150 9-024WO3_Sequence_Listing_05.18.21_ST25 and It has been named and is 41,145 bytes in size. [Background technology]

[0002] Vaccination has made a significant contribution to the health of both humans and animals. Since the invention of the first vaccine, vaccines have been used to evoke an immune response in the target population. Therefore, it has come to be considered the most successful method for preventing many infectious diseases. According to the World Health Organization, immunization is currently It prevents 2 to 3 million deaths every year across all age groups. Today, vaccines The virus includes more than 20 diseases, including diphtheria, tetanus, pertussis, influenza, and measles. Developed to prevent and control the spread of infectious diseases, it led to the complete eradication of smallpox. Furthermore, there remains a need to develop improved immunogenic compositions and their uses. [Overview of the project] [Means for solving the problem]

[0003] This disclosure relates to polyribonucleotides (e.g., cyclic or) that encode one or more immunogens. This invention provides compositions, pharmaceutical preparations, and uses of linear polyribonucleotides. The diagram shows a cyclic polyribonucleotide encoding multiple immunogens, and multiple cyclic polyribonucleotides. Provided is a composition comprising a nucleotide. The present disclosure further relates to a method of using a circular polynucleotide encoding one or more polypeptide immunogens. The circular polynucleotide compositions and pharmaceutical preparations described herein can elicit an immune response in a subject upon administration. The circular polynucleotide compositions and pharmaceutical preparations described herein can be used to treat or prevent a disease, disorder, or condition in a subject. In one aspect, the present disclosure provides a circular polynucleotide comprising a plurality of sequences, each sequence encoding a polypeptide immunogen, and at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine) of the polypeptide immunogens identifying different proteins, each of the different proteins identifying the same target. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin.

[0004] In one aspect, the present disclosure provides a circular polynucleotide comprising a plurality of sequences, each sequence encoding a polypeptide immunogen, and at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine) of the polypeptide immunogens identifying different proteins, each of the different proteins identifying the same target. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin.

[0005] In some embodiments, each of the polypeptide immunogens identifies a different protein. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the target is a pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the pathogen is a virus, bacterium, fungus, or parasite. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the pathogen is a virus and each of the different proteins is a viral protein associated with the virus. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the pathogen is a bacterium and each of the different proteins is a bacterial protein associated with the bacterium. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the target is a cancer cell. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, each of the different proteins is a different tumor antigen associated with the cancer cell. In some embodiments, the target is an allergen or toxin. In some embodiments, the target is an allergen or toxin.

[0006] In another aspect, the disclosure provides a cyclic polyribonucleotide comprising multiple sequences, each of which The column codes for polypeptide immunogens and at least two polypeptide immunogens (e.g., At least 3, at least 4, at least 5, at least 6, at least 7, Identifying at least eight, or at least nine, different targets. In some embodiments, Each polypeptide immunogen identifies a different target. In some embodiments, Each target is a different pathogen. In some embodiments, each target is independently a cancer cell. These are cells, viruses, bacteria, fungi, or parasites. In some embodiments, each target is different It is a virus. In some embodiments, each target is a different bacterium. In this embodiment, the targets include viruses and bacteria.

[0007] In some embodiments, multiple immune responses are encoded by cyclic polyribonucleotides. Each of the originals shares less than 90% sequence identity.

[0008] In some embodiments of any one of the cyclic polyribonucleotides described herein Cyclic polyribonucleotides consist of 500 to 20,000 molecules (for example, 500 to 10,000 molecules). 0 pieces, 500~9,000 pieces, 500~8,000 pieces, 500~5,000 pieces, 500~ 4,000 pieces, 500~3,000 pieces, 1,000~10,000 pieces, 1,000~8, 000 pieces, 1,000~5,000 pieces, 3,000~8,000 pieces, 4,000~9,0 Contains 00 or 10,000-20,000 ribonucleotides. In the application form, the cyclic polyribonucleotide contains 500 to 5,000 units. In the application form, cyclic polyribonucleotides consist of 1,000 to 5,000 ribonucleonucleotides. It contains. In some embodiments, the cyclic polyribonucleotide is 5,000 to 10, Contains 000 ribonucleotides. In some embodiments, cyclic polyribonucleotides D has at least 500 ribonucleotides (for example, at least 600, and at least... Also 1000, at least 1500, at least 2000, at least 2500, At least 3000, at least 3500, at least 4000, at least 45 00, at least 5000, at least 5500, at least 6000, fewer 6500 each, at least 7000, at least 7500, at least 8000 , at least 8500, at least 9000, or at least 9500 ribonuk Contains leotide.

[0009] In some embodiments, the cyclic polyribonucleotide comprises at least three, at least 4, at least 5, at least 6, at least 7, at least 8, or fewer Each contains nine sequences, each encoding a polypeptide immunogen. Several implementations In this state, cyclic polyribonucleotides are arranged in groups of two or three, or 2 to 5 (for example, 2, 3, or 4). ), or including an array of 5 to 10 (for example, an array of 5, 6, 7, 8, or 9), Each sequence encodes a polypeptide immunogen.

[0010] In some embodiments, at least one sequence encoding a polypeptide immunogen is Further encoding of the signal sequence. In some embodiments, polypeptide immunogen coding Each sequence further codes for a signal sequence. In some embodiments, polyp Each of the sequences encoding each of the cytoimmunogens is an internal ribosome entry site (IRE). It is operably linked to S). It includes a single IRES. In some embodiments, each of the polypeptide immunogens is , by a single open reading frame operably linked to a single IRES The expression of each polypeptide immunogen is encoded and open reading frame by amino acids. Generates a polypeptide containing an array.

[0011] In some embodiments, the polypeptide immunogen is controlled by a polypeptide linker. They are separated. In some embodiments, polypeptide immunogens are each cleaved Separated by the main. In some embodiments, each stagger element (stagg The er element is a 2A self-cleaving peptide. In some embodiments, the ring The polyribonucleotide comprises multiple IRESs. In some embodiments, each IRES S is activated in an open reading frame containing a sequence encoding a polypeptide immunogen. They are connected in a way that allows them to be linked.

[0012] In some embodiments, at least one sequence encoding an immunogen is a signal sequence. Further encoding. In some embodiments, each sequence encoding an immunogen is a signal Further encoding the sequence. In some embodiments, at least one encoding an immunogen The sequence does not encode a signal sequence. In some embodiments, it encodes an immunogen. None of the arrays further encode the signal arrays.

[0013] In some embodiments of any one of the cyclic polyribonucleotides described herein A cyclic polyribonucleotide is a first polyribonucleotide having a 5' end and a 3' end. It contains tide, and the 5' and 3' ends are hybridized to the second polynucleotide, respectively. This process links the 5' and 3' ends of the first polyribonucleotide, forming a ring. Forms polyribonucleotides. In some embodiments, cyclic polyribonucleotides This is generated by sprint ligation. In some embodiments, cyclic poly The ribonucleotide provides linear polyribonucleotides having 3' and 5' ends. It is generated by such means that the 3' and 5' ends each contain a portion of the intron. The 3' and 5' terminal introns catalyze the splicing reaction. This then covalently bonds the 5' and 3' ends to produce a cyclic polyribonucleotide. To accomplish. In some embodiments, the intron is a Group I self-splicing It is a trontron.

[0014] In another aspect, the disclosure includes multiple sequences, each comprising a sequence encoding a polypeptide immunogen. The present invention provides a composition comprising several cyclic polyribonucleotides. In some embodiments, multiple Each of the cyclic polyribonucleotides is a cyclic polyribonucleotide as described herein. In some embodiments, each polypeptide immunogen identifies a target. It comprises one or more epitopes. In some embodiments, the composition comprises a first polypeptide A first cyclic polyribonucleotide containing a sequence encoding an immunogen, and a second A sequence containing at least two cyclic polyribonucleotides encoding polypeptide immunogens The first and second polypeptide immunogens contain different proteins, and each different type The proteins identify the same target. In some embodiments, the composition is a first polypeptide A first cyclic polyribonucleotide containing a sequence encoding an immunogen, and a second A sequence containing at least two cyclic polyribonucleotides encoding polypeptide immunogens The first polypeptide immunogen identifies the first target, and the second polypeptide immunogen... This identifies a second target. In some embodiments, each target is independently identified as cancer cells, etc. These are bacteria, fungi, parasites, toxins, or allergens. In some embodiments, the target The target is a pathogen. In some embodiments, the pathogen is a virus, bacteria, fungus, or parasite. It is a living parasite. In some embodiments, the target is a cancer cell, an allergen, or a toxin. In some embodiments, each polypeptide immunogen is operably linked to an IRES. ru.

[0015] In another aspect, this disclosure relates to the cyclic polyribonucleotides, compositions, or described herein. The present invention provides a pharmaceutical composition comprising one of the pharmaceutical preparations and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a cyclic polyribonucleotide as described herein. , one of the following: composition, or pharmaceutical preparation, and protamine or protamine salt (for example, Contains protamine sulfate. In some embodiments, the pharmaceutical composition further contains adjuvants. It includes. In some embodiments, the adjuvant is an inorganic adjuvant, a small molecule adjuvant. Peptides, and oil-in-water emulsions, lipids or polymers, peptides or peptidoglycans, Carbohydrates or polysaccharides, saponins, RNA-based adjuvants, DNA-based adjuvants Related to viruses, bacterial adjuvants, hybrid molecules, fungi, or oocyte microorganisms. These are molecular patterns (MAMPs), inorganic nanoparticles, or multi-component adjuvants. In some embodiments, the adjuvant is an inorganic adjuvant. The adjuvant is an aluminum salt or a calcium salt. In some embodiments, The adjuvant is a small molecule. In some embodiments, the small molecule is imiquimod, resicci. It is a mod or garziki mod. In some embodiments, the adjuvant is an oil-in-water type. It is a emulsion. In some embodiments, the oil-in-water emulsion is squalene, M F59, AS03, Montanide preparations, and Montanide IS (optional selection) Incomplete flow of A51 or Montanide ISA720 or oil-in-water emulsion. It is an adjuvant. In some embodiments, the adjuvant is a lipid or polymer. —In some embodiments, the lipid or polymer is polymer nanoparticles, optionally By choice, PLGA, PLG, PLA, PGA, or PHB, liposomes, and by choice, Losomes or CAF01, lipid nanoparticles or their components, lipopolysaccharide (LPS), optional Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), Popeptide, optionally selected as Pam2 (Pam2CSK4) or Pam3 (Pam3CS K4), or glycolipid, or optionally trehalose dimycolate. Several implementations In terms of form, the adjuvant is a peptide or peptidoglycan. Several embodiments So, peptides or peptidoglycans are synthesized or purified from Gram-negative bacteria or Gram-positive bacteria. By selecting the bacteria, N-acetyl-muramyl-L-alanyl-D-isoglutamine (M DP), flagellin fusion protein, mannose-binding lectin (MBL), cytokinase It matches all or part of a chemokine. In some embodiments, it is an adjuvant. The first element is a carbohydrate or polysaccharide. In some embodiments, the carbohydrate or polysaccharide is dextrose. Tran (branched microbial polysaccharide), dextran sulfate, lentinan, zymosan, β-glucan, Deltin, mannan, or chitin. In some embodiments, the adjuvant is It is a saponin. In some embodiments, the saponin is bound to one or more sugar side chains. A glycoside or polycyclic aglycone, optionally ISCOMS, ISCOMS matrix It is either xx or QS-21. In some embodiments, the adjuvant is RNA-based. It is an adjuvant. In some embodiments, the RNA-based adjuvant is a polyadjuvant. Li-IC, poly-IC:LC, hairpin RNA, 5'PPP-containing sequence by optional selection, virus S sequences, polyU-containing sequences, dsRNA, natural or synthetic immunostimulatory RNA sequences, nucleic acid-like sequences The body may optionally contain cyclic dinucleotides such as cyclic GMP-AMP or cyclic diGMP, These are immunostimulatory base analogs, optionally C8 substituted or N7,C8 disubstituted guanine ribonucleates. It is a rheotide. In some embodiments, the adjuvant is a DNA-based adjuvant. In some embodiments, the DNA-based adjuvant is CpG, dsDN. A, or a natural or synthetic immunostimulatory DNA sequence. In some embodiments, A The juvant is a virus particle. In some embodiments, the virus particle is a virus The chromosome is, optionally, a phospholipid cell membrane bilayer. In some embodiments, adjuv The adjuvant is a bacterial adjuvant. In some embodiments, the bacterial adjuvant is a bacterium. Rajerin, LPS, or bacterial toxins, optionally enterotoxins, thermolabic toxins, or It is cholera toxin. In some embodiments, the adjuvant is a hybrid molecule. In some embodiments, the adjuvant is CpG conjugated to imiquimod. In some embodiments, the adjuvant is a fungal or oocyte microorganism-related molecule. It is a turn (MAMP). In some embodiments, the fungal or oocyte MAMP is a turn. It is tin or β-glucan. In some embodiments, the adjuvant is inorganic nanoparticles. In some embodiments, inorganic nanoparticles are gold nanorods, silica-based nanoparticles. The child is optionally mesoporous silica nanoparticles (MSNs). Several embodiments In this case, the adjuvant is a multi-component adjuvant. In some embodiments, a multi-component adjuvant The juvant is AS01, AS03, AS04, full Freund adjuvant, or CA It is F01.

[0016] In another aspect, the Disclosure provides a method for treating or preventing a disease, disorder, or symptom in question. Furthermore, this method applies to cyclic polyribonucleotides, compositions, pharmaceutical preparations, and other materials described herein. This includes administering one of the pharmaceutical compositions to a target. In some embodiments, the disease The illness, disorder, or symptoms are caused by a viral, bacterial, or fungal infection. Several implementations In terms of form, the disease, disorder, or symptom is cancer. In some embodiments, the disease, disorder, or The symptoms are related to exposure to allergens. In some embodiments, diseases, disorders, Alternatively, the symptoms may be related to exposure to toxins.

[0017] In another aspect, the present disclosure provides a method for inducing an immune response in a subject, the method being , the cyclic polyribonucleotides, compositions, pharmaceutical preparations, or pharmaceutical compositions described herein This includes administering one of the following to a target. In some embodiments, the target is a mammal. Yes. In some embodiments, the subject is human. In some embodiments, the method is They are non-human mammals. In some embodiments, non-human mammals include cattle, sheep, and yaks. The subject is a ghee, pig, dog, horse, or cat. In some embodiments, the subject is a bird. In some embodiments, the bird is a hen, a rooster, a turkey, or a parrot. In some embodiments, the method further includes administering an adjuvant to the target. In terms of application methods, the adjuvant is cyclic polyribonucleotide, linear polyribonucleotide, or a molecular entity separate from the preparation or composition thereof. In some embodiments, A Adjuvants include inorganic adjuvants, small molecule adjuvants, and oil-in-water emulsions and lipids. Substances or polymers, peptides or peptidoglycans, carbohydrates or polysaccharides, saponins, RNA Base adjuvants, DNA-based adjuvants, virus particles, bacterial adjuvants Hybrid molecules, fungal or oocyte microorganism-related molecular patterns (MAMPs), inorganic nano It is a particle or a multi-component adjuvant. In some embodiments, the adjuvant is poly It is a peptide adjuvant. In some embodiments, the polypeptide adjuvant is Cytokines, chemokines, co-stimulatory molecules, innate immune system stimulants, signaling molecules, transcriptional activity These are sexualization factors, cytokine receptors, bacterial components, or components of the innate immune system. In terms of application, the adjuvant is an innate immune system stimulant. In some embodiments, the adjuvant is an innate immune system stimulant. Immune system stimulants include GU-rich motifs, AU-rich motifs, and structural regions containing dsRNA. or selected from RNA containing an aptamer.

[0018] In some embodiments, the cyclic polyribonucleotides, compositions, and pharmaceuticals described herein are used. One of the preparations or pharmaceutical compositions is administered to the subject as a single dose. In the embodiments described herein, the cyclic polyribonucleotide, composition, pharmaceutical preparation, or This refers to a situation where one of the pharmaceutical compositions is administered to a target two or more times, three or more times, four or more times, or five or more times. In some embodiments, the cyclic polyribonucleotide described herein, composition The administration of one of the substances, pharmaceutical preparations, or pharmaceutical compositions is approximately every week, or approximately every two weeks. Approximately every 3 weeks, approximately every 1 month, approximately every 2 months, approximately every 3 months, approximately every 4 months, approximately every 5 months Every, approximately every 6 months, approximately every 1 year, approximately every 2 years, approximately every 3 years, approximately every 4 years, approximately every 5 years, Alternatively, it is performed approximately every 10 years.

[0019] In some embodiments, the method involves targeting polypeptide immunogens (e.g., polypeptides). Further includes administering protein subunits (including immunogens). Several implementations In this state, polypeptide immunogens are encoded by a sequence of cyclic polyribonucleotides. Matching polypeptide immunogens (e.g., 90%, 95%, 96%, 97%, 98%, or (They share 100% amino acid sequence identity). In some embodiments, polypeptides The immunogen is a cyclic polyribonucleotide, immunogenic composition, pharmaceutical preparation, as described herein. Alternatively, it is administered to the subject after administering one of the pharmaceutical compositions. In some embodiments, Polypeptide immunogens maintain the immune response to polypeptide immunogens in the target. It will be strengthened.

[0020] In another aspect, the Disclosure provides a method for maintaining or enhancing an immune response in a subject. The method involves (i) targeting cyclic polyribonucleotides encoding polypeptide immunogens. (ii) a step of administering a polypeptide immunogen to the target, Step (ii) takes place after Step (i) and lasts from one week to six months (for example, from one month to five months). (This is done over a period of 2 to 3 months, 2 weeks to 3 months, or 3 to 6 months) and polypeptide Immunogen administration is performed when the polypeptide immunogen in step (ii) is administered to the target. Maintain or enhance the immune response to butyl immunogens. In some embodiments, polypeptide A cytoimmunogen comprises one or more epitopes for target identification. In some embodiments, The target is a pathogen. In some embodiments, the target is cancer cells, allergens, or It is a toxin.

[0021] Numbered embodiments: [1] A cyclic polyribonucleotide comprising multiple sequences, each sequence being a polypeptide molecule The epidemiogen codes for at least two polypeptide immunogens, which identify different targets, and are circular. Polyribonucleotide. [2] Each polypeptide immunogen identifies a different target, the cyclic polylith of item [1] Bonucleotide. [3] Cyclic polyribonucleotides of item [1] or [2], each target being a different pathogen. [4] Each target is independently a virus, bacterium, fungus, or parasite, as in the annular [3] of item [3]. Polyribonucleotide. [5] Each target is a different virus, a cyclic polyribonucleotide of item [4]. [6] Each target is a different bacterium, the cyclic polyribonucleotide of item [4]. [7] Cyclic polyribonucleotides of item [4], including viruses and bacteria, which are targets. [8] A cyclic polyribonucleotide comprising multiple sequences, each sequence being a polypeptide immunonucleotide Identify a peptide immunogen that encodes an epidemiogen and has at least two different proteins. Each of these proteins is a cyclic polyribonucleotide that identifies the same target. [9] Each polypeptide immunogen identifies a different protein, the circular [8] Polyribonucleotide.

[10] A cyclic polyribonucleotide of item [8] or [9], the target being a pathogen.

[11] The cyclic polylith of item

[10] , in which the pathogen is a virus, bacterium, fungus, or parasite. Bonucleotide.

[12] The pathogen is a virus, and each of the different proteins is associated with the virus. A viral protein, the cyclic polyribonucleotide of item

[11] .

[13] The pathogen is a bacterium, and each of the different proteins is associated with the bacterium. A cyclic polyribonucleotide, which is an protein

[11] .

[14] A cyclic polyribonucleotide of item [8] or [9] whose target is cancer cells.

[15] A cyclic polyribonucleotide of section

[0014] , each of which is a different tumor antigen associated with cancer cells.

[16] The target is an allergen or toxin of the cyclic polyribonucleotide of item [8] or [9]. Ochido.

[17] containing 500 to 20,000 ribonucleotides, of items [1] to

[16] Any one of the cyclic polyribonucleotides.

[18] A cyclic polyribonucleotide of item

[17] containing 500 to 10,000 ribonucleotides. nucleotide.

[19] A cyclic polyribonucleotide of item

[18] containing 500 to 5,000 ribonucleotides. Cleotide.

[20] containing at least 500 ribonucleotides, any of items [1] to

[16] A single cyclic polyribonucleotide.

[21] A cyclic polyribonucleotide of item

[20] containing at least 1,000 ribonucleotides. nucleotide.

[22] A cyclic polyribonucleotide of item

[21] containing at least 5,000 ribonucleotides. nucleotide.

[23] at least three, at least four, at least five, at least six, at least It also contains seven, at least eight, or at least nine sequences, each sequence being an immunogenic polyp A cyclic polyribonucleotide of any one of the terms [1] to

[22] that codes for a plutido.

[24] Consists of sequences 2-3, 2-5, or 5-10, each sequence being a polypeptide immunogen A cyclic polyribonucleotide that codes for one of the terms [1] to

[22] .

[25] At least one sequence encoding a polypeptide immunogen further signals the sequence A cyclic polyribonucleotide that codes for one of the terms [1] to

[22] .

[26] Each sequence encoding a polypeptide immunogen further encodes a signal sequence. A cyclic polyribonucleotide of any one of the terms [1] to

[22] .

[27] Each of the sequences encoding each polypeptide immunogen is an internal ribosome One of items [1] to

[26] is operably connected to the entry area (IRES). A cyclic polyribonucleotide.

[28] A cyclic polyribonucleotide of item

[27] containing a single IRES.

[29] Each polypeptide immunogen is operably linked to a single IRES Coded by the OpenReadingFrame, and the OpenReadingFrame The expression of the cyclic polyribonucleotide of section

[0028] generates polypeptides containing the amino acid sequence of each polypeptide immunogen.

[30] The polypeptide immunogens are separated by polypeptide linkers. , the cyclic polyribonucleotide of item

[29] .

[31] Cyclic polyribonucleotides of item

[0029] , wherein each polypeptide immunogen is separated by a cleavage domain.

[32] Each cleavage domain is a 2A self-cleaving peptide, the cyclic polyribonu of item

[31] Cleotide.

[33] A cyclic polyribonucleotide of item

[27] containing multiple IRESs.

[34] Each IRES contains an open reading that includes a sequence encoding a polypeptide immunogen. A cyclic polyribonucleotide of item

[33] operably linked to a frame.

[35] comprising a first polyribonucleotide having a 5' end and a 3' end, the 5' end and The 3' ends each hybridize to a second polynucleotide, thereby creating the first The 5' and 3' ends of a polyribonucleotide are linked together to form a cyclic polyribonucleotide. A cyclic polyribonucleotide comprising one of the terms [1] to

[34] .

[36] The cyclic polyribonucle of term

[35] generated by sprint ligation. Leotid.

[37] By providing linear polyribonucleotides having a 3' end and a 5' end It is generated, and each of the 3' and 5' ends contains part of an intron, and the 3' end The intron portion and the 5'-terminus intron portion catalyze the splicing reaction, and thereafter This involves covalently bonding the 5' and 3' ends to produce a cyclic polyribonucleotide, item [ A cyclic polyribonucleotide of any one of the following terms: [1] to

[34] .

[38] If an intron is a Group I self-pricing intron, then

[37] A cyclic polyribonucleotide.

[39] Multiple cyclic polyribonu sequences, each containing a sequence encoding a polypeptide immunogen. An immunogenic composition containing a creotide.

[40] Each of the multiple cyclic polyribonucleotides is one of the following [1] to

[38] An immunogenic composition of item

[39] , wherein the cyclic polyribonucleotide is item one.

[41] (a) at least a first circular matrix comprising a sequence encoding a first polypeptide immunogen (b) A small number of sequences containing polyribonucleotides and a second polypeptide immunogen. At the very least, it contains a second cyclic polyribonucleotide, and the first and second polypeptide immunogens The process involves identifying different proteins, and each of these different proteins identifies the same target.

[39] Immunogenic composition.

[42] An immunogenic composition of item

[41] , wherein the target is a pathogen.

[43] The immunogenicity group of item

[42] , in which the pathogen is a virus, bacterium, fungus, or parasite. Finished product.

[44] Immunogenic compositions of item

[43] in which the pathogen is a cancer cell, an allergen, or a toxin. .

[45] (a) at least a first circular matrix comprising a sequence encoding a first polypeptide immunogen (b) A small number of sequences containing polyribonucleotides and a second polypeptide immunogen. At the very least, it contains a second cyclic polyribonucleotide, and the first polypeptide immunogen is the first Identify the target, and a second polypeptide immunogen identifies the second target, as described in section

[39] . genic composition.

[46] The immunogenic composition of item

[45] , wherein each target is a pathogen.

[47] Each target independently targets cancer cells, viruses, bacteria, fungi, parasites, toxins, or allergens. An immunogenic composition of item

[45] or

[46] , which is a rugen.

[48] ​​Each polypeptide immunogen is operably linked to an IRES, section

[39] ~ An immunogenic composition according to any one of the items in

[47] .

[49] A cyclic polyribonucleotide from any one of the items [1] to

[38] or item

[39] A pharmaceutical compound comprising an immunogenic composition of any one of the items in

[48] and a pharmaceutically acceptable excipient. Finished product.

[50] The pharmaceutical composition of item

[49] , further comprising an adjuvant.

[51] Adjuvants include inorganic adjuvants, small molecule adjuvants, and oil-in-water emulsions. John, lipids or polymers, peptides or peptidoglycans, carbohydrates or polysaccharides, saponins RNA-based adjuvants, DNA-based adjuvants, virus particles, bacterial adjuvants Juvant, hybrid molecule, fungal or oocyte microorganism-associated molecular pattern (MAMP) A pharmaceutical composition according to item

[50] , which is inorganic nanoparticles or a multi-component adjuvant.

[52] The pharmaceutical composition of item

[51] , wherein the adjuvant is an inorganic adjuvant.

[53] The inorganic adjuvant is an aluminum salt or a calcium salt, as in item

[52] . A pharmaceutical composition.

[54] The pharmaceutical composition of item

[51] , wherein the adjuvant is a small molecule.

[55] The small molecule is imiquimod, reximod, or gardiquimod, as described in paragraph

[51] . A pharmaceutical composition.

[56] The pharmaceutical composition of item

[51] , wherein the adjuvant is an oil-in-water emulsion.

[57] Oil-in-water emulsions include squalene, MF59, AS03, and Montanid e-formulation, optional selection of Montanide ISA51 or Montanide A pharmaceutical composition according to item

[0056] , which is ISA720 or an incomplete Freund's adjuvant of an oil-in-water emulsion.

[58] The pharmaceutical composition of item

[51] , wherein the adjuvant is a lipid or polymer.

[59] Lipids or polymers, polymer nanoparticles, optionally PLGA, PLG, P LA, PGA, or PHB, liposomes, optionally visomes or CAF01, lipids High-quality nanoparticles or their components, lipopolysaccharide (LPS), and optionally monophosphoryl lipid A (MPLA) or glucopyranosyllipid A (GLA), lipopeptides, optionally Pam2 (Pam2CSK4) or Pam3 (Pam3CSK4), or glycolipids, optional selection. A pharmaceutical composition according to item

[58] , wherein the selection is trehalose dimicholate.

[60] The pharmaceutical composition of item

[51] in which the adjuvant is a peptide or peptidoglycan. thing.

[61] Peptidoglycan peptides are synthesized or purified Gram-negative bacteria or Gram-positive bacteria. By selecting the bacteria, N-acetyl-muramyl-L-alanyl-D-isoglutamine (M DP), flagellin fusion protein, mannose-binding lectin (MBL), cytokinase A pharmaceutical composition of item

[60] , which corresponds to all or part of a chemokine.

[62] The pharmaceutical composition of item

[51] , wherein the adjuvant is a carbohydrate or polysaccharide.

[63] Carbohydrates or polysaccharides, dextran (branched microbial polysaccharide), dextran sulfate These are lentinan, zymosan, β-glucan, deltin, mannan, or chitin. The pharmaceutical composition of item

[62] .

[64] The pharmaceutical composition of item

[51] , wherein the adjuvant is a saponin.

[65] Saponins, one or more sugar side chains, optionally ISCOMS, ISCOM It is an S matrix, or a glycoside or polycyclic aglycone bound to QS-21. , the pharmaceutical composition of item

[64] .

[66] The pharmaceutical composition of item

[51] wherein the adjuvant is an RNA-based adjuvant. .

[67] RNA-based adjuvants include poly-IC, poly-IC:LC, hairpin RNA, Optional selections include 5'PPP-containing sequences, viral sequences, polyU-containing sequences, dsRNA, and natural sequences. Alternatively, synthetic immunostimulatory RNA sequences, nucleic acid analogs, or optionally cyclic GMP-AMP or Cyclic dinucleotides such as cyclic diGMP, or immunostimulatory base analogs, optionally C The pharmaceutical composition of item

[66] , which is an 8-substituted or N7,C8 disubstituted guanine ribonucleotide. .

[68] The pharmaceutical composition of item

[51] wherein the adjuvant is a DNA-based adjuvant. .

[69] DNA-based adjuvants are CpG, dsDNA, or natural or synthetic adjuvants. A pharmaceutical composition of item

[68] , wherein the immunostimulatory DNA sequence is present.

[70] The pharmaceutical composition of item

[51] , wherein the adjuvant is a virus particle.

[71] Viral particles are visomes, which are selected to be phospholipid cell membrane bilayers.

[70] Pharmaceutical composition.

[72] The pharmaceutical composition of item

[51] , wherein the adjuvant is a bacterial adjuvant.

[73] Bacterial adjuvants, flagellin, LPS, or bacterial toxins, can be selected to be effective. A pharmaceutical composition of item

[72] , which is a cholerotoxin, a heat-labile toxin, or a cholera toxin.

[74] A pharmaceutical composition of item

[51] wherein the adjuvant is a hybrid molecule.

[75] The adjuvant is a CpG conjugated to imiquimod, as described in paragraph

[74] . Pharmaceutical composition.

[76] The adjuvant is a fungal or oocyte microorganism-associated molecular pattern (MAMP). , the pharmaceutical composition of item

[51] .

[77] The pharmaceuticals of item

[76] in which fungal or oocyte MAMP is chitin or β-glucan. composition.

[78] The pharmaceutical composition of item

[51] , wherein the adjuvant is an inorganic nanoparticle.

[79] Inorganic nanoparticles are gold nanorods, silica-based nanoparticles, and optionally meso A pharmaceutical composition of item

[78] , wherein porous silica nanoparticles (MSNs).

[80] The pharmaceutical composition of item

[51] , wherein the adjuvant is a multi-component adjuvant.

[81] Multicomponent adjuvants include AS01, AS03, AS04, and complete Freund's adjuvants. A pharmaceutical composition of item

[80] , which is a bant or CAF01.

[82] A cyclic polyribonucleotide from any one of the items [1] to

[38] or item

[39] Lipid nanoparticles (LNPs) comprising any one of the immunogenic compositions in item

[48] .

[83] LNPs of item

[82] , including ionic lipids.

[84] LNPs of item

[82] , including cationic lipids.

[85] Cationic lipids: [ka] An LNP of item

[84] having the structure of .

[86] One or more neutral lipids, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, steroids, e.g., cholesterol, and / or one or Multiple polymer complex lipids, for example, PEG-DAG, PEG-PE PEG-S-DAG, PEG-cer, or PEG-dialkyloxypropylcarbamate The item further comprises (dialkyoxypropylcarbamate)

[82] The LNP of any one of the terms in ~

[85] .

[87] A method for treating or preventing a disease, disorder, or symptom of the subject, comprising a cyclic polyribonucleotide of any one of items [1] to

[0038] , or any one of items

[39] to

[48] An immunogenic composition of any one of the items

[49] to

[81] , or item

[82] A method comprising administering to any one of the LNPs in ~

[86] .

[88] The method of paragraph

[0087] , wherein the disease, disorder, or symptom is a viral infection, bacterial infection, or fungal infection.

[89] The method of paragraph

[87] , in which the disease, disorder or symptom is cancer.

[90] The method of paragraph

[87] in which a disease, disorder or symptom is related to exposure to an allergen.

[91] The method of paragraph

[87] in which a disease, disorder or symptom is related to exposure to a toxin.

[92] A method for inducing an immune response in a subject, any of items [1] to

[38] One cyclic polyribonucleotide, one immunogenic composition of any one of the items

[39] -

[48] A substance, a pharmaceutical composition of any one of items

[49] to

[81] , or an item of items

[82] to

[86] A method comprising administering to any one of the LNPs.

[93] A method according to any one of the items

[87] to

[92] , wherein the subject is a mammal.

[94] The method of item

[93] , wherein the subject is human.

[95] The method of item

[93] , wherein the method is a non-human mammal.

[96] Non-human mammals are cattle, sheep, goats, pigs, dogs, horses, or cats. The method of item

[93] .

[97] A method according to any one of the terms

[87] to

[96] , wherein the subject is a bird.

[98] The method of paragraph

[97] , wherein the bird is a hen, a rooster, a turkey, or a parrot.

[99] Any of paragraphs

[87] -

[98] , which further include administering an adjuvant to the subject. Any one of the methods.

[0100] Adjuvants include inorganic adjuvants, small molecule adjuvants, and oil-in-water emulsions. Lubricant, lipids or polymers, peptides or peptidoglycans, carbohydrates or polysaccharides, suppositories Nin, RNA-based adjuvants, DNA-based adjuvants, virus particles, bacteria Adjuvants, hybrid molecules, fungal or oocyte microorganism-related molecular patterns (MAMPs) The method of item

[99] , wherein the material is inorganic nanoparticles or a multi-component adjuvant.

[0101] The method of section

[99] , wherein the adjuvant is a polypeptide adjuvant.

[0102] Polypeptide adjuvants are cytokines, chemokines, co-stimulatory molecules, and natural Immune system stimulants, signaling molecules, transcription activators, cytokine receptors, bacterial components, and The method described in section

[0101] involves a component of the innate immune system.

[0103] The method of section

[99] , wherein the adjuvant is an innate immune system stimulant.

[0104] Innate immune system stimulants include GU-rich motif, AU-rich motif, and dsRN. The method of item

[0103] , selected from a structural region containing A or RNA containing an aptamer.

[0105] One cyclic polyribonucleotide from any of items [1] to

[38] , item

[39] An immunogenic composition according to any one of items

[48] , or a medical composition according to any one of items

[49] to

[81] A drug composition, or an LNP of any one of the items

[82] to

[86] , is targeted as a single dose. The method of administration described in any one of the items

[87] to

[0104] .

[0106] One cyclic polyribonucleotide from any of items [1] to

[38] , item

[39] An immunogenic composition according to any one of items

[48] , or a medical composition according to any one of items

[49] to

[81] A drug composition, or an LNP of any one of items

[82] to

[86] , administered to a subject two or more times, three times The method described in any one of the items

[86] to

[99] above, administered four or more times, or five or more times. .

[0107] One cyclic polyribonucleotide from any of items [1] to

[38] , item

[39] An immunogenic composition according to any one of items

[48] , or a medical composition according to any one of items

[49] to

[81] The drug composition, or LNP of any one of items

[82] to

[86] , is administered approximately every week. Approximately every two weeks, every three weeks, every one month, every two months, every three months, every four months And, approximately every 5 months, approximately every 6 months, approximately every 1 year, approximately every 2 years, approximately every 3 years, approximately every 4 years, The method described in item

[0106] , which is performed approximately every 5 years or approximately every 10 years.

[0108] Polypeptide immunogens (e.g., protein polypeptide immunogens containing polypeptide immunogens) The person who administers the drug to the target of any one of the items

[87] to

[0104] , which further includes administering the drug to the target of [the drug]. Law.

[0109] Polypeptide immunogens are encoded by sequences of cyclic polyribonucleotides. It matches polypeptide immunogens (e.g., 90%, 95%, 96%, 97%, 98%) The method of item

[0108] (or sharing 100% amino acid sequence identity).

[0110] The polypeptide immunogen is a cyclic polyribonuclear compound found in any one of the items [1] to

[38] . A creotide, an immunogenic composition according to any one of items

[39] to

[48] , a pharmaceutical composition according to any one of items

[49] to

[0081] , or an LNP according to any one of items

[82] to

[86] The method of item

[0108] , which is administered to the subject after the administration of the other method.

[0111] Polypeptide immunogens induce an immune response to polypeptide immunogens in a target. A method of maintaining or enhancing according to any one of the items

[0108] to

[0110] .

[0112] A method for maintaining or enhancing the immune response in a subject, comprising (i) polypeptide (ii) A step of administering a cyclic polyribonucleotide encoding an immunogen to the target, and A method comprising the step of administering a polypeptide immunogen to a target, wherein step (ii) Step (i) is performed between 1 week and 6 months after step (ii), and the polypeptide immunity in step (ii) The administration of the epidemic agent maintains or enhances the immune response to polypeptide immunogens in the subject. method.

[0113] A polypeptide immunogen contains one or more epitopes that identify a target. Method

[0112] .

[0114] The method of item

[0113] , wherein the target is a pathogen.

[0115] The method of item

[0113] , wherein the target is cancer cells, allergens, or toxins.

[0022] definition This disclosure is described in relation to specific embodiments and with reference to specific figures, but this disclosure is not limited to, The following uses are not limited to those, but are limited only by the claims. Words should generally be understood in their common sense unless otherwise specified.

[0023] As used herein, the term “adaptive immune response” refers to humoral or cellular immune response. This means one of the answers. For the purposes of this disclosure, “humoral immune response” is defined by antibody molecules. This refers to an immune response mediated by T lymphocytes and / or other cells, while "cellular immune response" refers to an immune response mediated by T lymphocytes and / or other cells. It is mediated by white blood cells.

[0024] As used herein, the term “adjuvant” means an agent that enhances the immune response, Otherwise, it refers to compounds that are altered or modified. Modification of the immune response includes antibodies and cellular antibodies. This includes enhancing or expanding the specificity of one or both of the disease response. Modification of the immune response is specific. This may also mean reducing or suppressing the immunogen-specific immune response.

[0025] As used herein, the term “related” to a disease, disorder, or symptom refers to an entity and a symptom. Any cause or correlation between the onset or severity of disease, disability, or symptoms in the subject It refers to a relationship. For example, if the target is related to a disease, disorder, or symptom, then the target is the disease. It can be the causative pathogen of a disorder or symptom. For example, a virus is the causative pathogen of a viral infection. Bacteria can be pathogens that cause bacterial infections, and fungi can be pathogens that cause fungal infections. Parasites can be the causative pathogens of parasitic infections, and cancer cells can be the causative pathogens of cancer. Toxins can be pathogens that cause toxicity, or allergens can be the source of allergic reactions. It may be the causative pathogen. Targets associated with disease, disorder, or symptoms may also be disease It may correlate with an increased likelihood or severity of developing illness, disability, or symptoms.

[0026] As used herein, the term "carrier" refers to a composition applied to a subject, tissue, or cell. Compounds and combinations that facilitate the transport or delivery of (for example, linear or cyclic polyribonucleotides). It means a substance, reagent, or molecule. Non-limiting examples of carriers include carbohydrate carriers (e.g., without Water-modified phytoglycogen or glycogen-type substance), nanoparticles (e.g., cyclic or linear) Nanoparticles encapsulated or covalently bonded to polyribonucleotides, liposomes, fuso Somes, reticulocytes differentiated in vitro, exosomes, protein carriers (e.g., polymethyl A protein covalently bonded to a bonucleotide, or a cationic carrier (e.g., cationic It contains lipopolymer or transfection reagents.

[0027] As used herein, the term “cell permeable agent” means that, upon contact with cells, it penetrates the cells. This refers to a substance that promotes the entry of cells. In some cases, cell permeabilizers are, for example, substances that promote the entry of cells into the cell membrane. Direct electrostatic interaction with negatively charged phospholipids, or membrane proteins or phospholipids. Direct permeability of the cell membrane is promoted through temporary pore formation by inducing structural changes in the bilayer. It progresses. In some cases, cell permeabilisers promote cell migration via endocytosis. To advance. For example, under certain circumstances, cell permeability agents stimulate cells to undergo endocytosis. The process can be undertaken, thereby allowing the cell membrane to fold inward into the cell. Specific implementation Morphologically, cell permeabilizers help form temporary structures that can permeate and transport substances across the cell membrane. While we do not wish to be bound by this theory, the cell permeabilizing agents provided herein are fine It can increase the permeability of the cell membrane or increase the internalization of molecules into the cell, and as a result In addition, compared to delivery which is identical except for the absence of a cell permeability agent, cells simultaneously receive the cell permeability agent. When in contact, delivery to cells can be more efficient.

[0028] As used herein, "circRNA" or "cyclic polyribonucleotide" The terms "cyclic RNA" and "cyclic polyribonucleotide molecule" are used interchangeably. Polypropylene has a structure that does not have free ends (i.e., free 3' and / or 5' ends). Bonucleotide molecules, for example, cyclic or terminal-less structures via covalent or non-covalent bonds. This refers to polyribonucleotide molecules that form a structure.

[0029] As used herein, the term "cyclization efficiency" refers to the obtained cyclic polyribonucle This is a measurement of rheotide relative to its acyclic starting material.

[0030] When used herein, the term "circRNA preparation" or "cyclic polyribonucleoprotein" is used. The terms "circular RNA preparation" or "circular RNA preparation" are used interchangeably. A composition comprising a molecule, a diluent, a carrier, a first adjuvant, or a combination thereof. It tastes good.

[0031] The phrase "compounds, compositions, products, etc. for treatment or adjustment" refers to compounds, compositions, etc. It should be understood as referring to finished products, etc., and that these products themselves are the subject of instructions for treatment or adjustment. It should be understood that this refers to compounds, compositions, products, etc., that are suitable for a particular purpose. The phrase "compounds, compositions, products, etc. for the purpose of" means that, as a preferred embodiment, Further disclosures are made regarding the use of compounds, compositions, products, etc., for treatment and adjustment.

[0032] "Compounds, compositions, products, etc. for use in ~" or "Pharmaceuticals, pharmaceutical compositions for ~" "Use of compounds, compositions, products, etc. in the manufacture of substances, veterinary compositions, diagnostic compositions, etc." The phrase "such compounds, compositions, products, etc. may be applied to the body of a human or animal" means that such compounds, compositions, products, etc. may be applied to the body of a human or animal. This indicates that it is used in treatment. These include embodiments and procedures related to treatment methods, etc. The embodiments or claims are deemed equivalent disclosures of the claims. Therefore, if an embodiment or claim is "suffering from a disease", When referring to "compounds for use in the treatment of humans or animals suspected of having," This refers to the manufacture of pharmaceuticals for treating humans or animals suspected of having a disease. "Use of compounds in" or "Administration of compounds to humans or animals suspected of having a disease" This can also be seen as disclosing "methods of treatment by taking action."

[0033] The term "diluent" refers to the compositions described herein (e.g., cyclic or linear polyribonu This refers to a vehicle containing an inert solvent capable of diluting or dissolving a composition containing creotide. The diluent may be an RNA solubilizer, a buffer, an isotonic agent, or a mixture thereof. It may be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents. A medium, solubilizer, and emulsifier, such as ethyl alcohol, isopropyl alcohol, or carbonic acid. Ethyl, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, etc.) (Sorghum oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrochloride Fatty acid esters of drofluryl alcohol, polyethylene glycol, and sorbitan , and 1,3-butanediol are included. Non-limiting examples of solid diluents include calcium carbonate. Um, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, phosphorus Calcium hydrogen oxyphosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose Lullose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry Contains dried starch, cornstarch, or powdered sugar.

[0034] As used herein, the terms “disease,” “disorder,” and “symptom” are used in relation to each other. , an abnormal state of health, for example, one that is normally diagnosed or treated by a medical professional, Alternatively, it refers to a condition that will be diagnosed or treated.

[0035] As used herein, the term “epitope” refers to an antibody or T cell receptor. This refers to a part or all of an immunogen that is recognized, targeted, or bound to. An epitope is a linear epitope. A pitope can be, for example, a continuous sequence of nucleic acids or amino acids. An epitope is a three-dimensional structure. Epitope formation, for example, the formation of epitopes in the folded three-dimensional structure of proteins. It may be an epitope containing an acid. The three-dimensional epitope is a discrepancy from the primary amino acid sequence. It may contain subsequent amino acids. As another example, a three-dimensional epitope may contain its secondary or three-dimensional structure. Based on the following structure, nucleic acids that form epitopes in the folded three-dimensional structure of immunogenic sequences It is included.

[0036] As used herein, the term "encryptogen" is used. The term helps to reduce, evade, and / or avoid detection by immune cells, and A ring that reduces the induction of an immune response to a ring or linear polyribonucleotide. This refers to the nucleic acid sequence or structure of a polyribonucleotide.

[0037] As used herein, the term “expression sequence” refers to the product, for example, a peptide or pylori product. A nucleic acid sequence or regulatory nucleic acid that encodes a peptide or polypeptide. An exemplary expression sequence may contain multiple triplicates, each of which is an amide. It can code for no acids and is called a "codon".

[0038] As used herein, "identify" or "identify (id)" The term "entifies" means to indicate, establish, or recognize the identity of an entity. This refers to the target. For example, an immunogen or its epitope can identify a target, that is, the target However, it contains an immunogen or its epitope, and the immunogen or its epitope is derived from the target. to, and / or the immunogen or its epitope exhibits a high degree of similarity to part or all of the target. It means having; the recognition of the binding of an antibody or T cell receptor to an immunogen or its epitope. Through recognition, the target can be identified. The immunogen or its epitope identifies the target. A combination, immunogen, or its epitope distinguishes a target from one or more other targets. Polypeptide immunogens can identify proteins. In other words, Lipeptide immunogens are proteins or parts of proteins, particularly protein epitopes. It is a component, part of it, derived from it, or shares a high degree of similarity with it. .

[0039] As used herein, the term “impurity” means a composition, for example, as described herein. Undesirable substances present in the listed pharmaceutical composition. In some embodiments, impurities These are process-related impurities. In some embodiments, the impurities are present in the final composition. Other than the desired product, for example, active drug components, for example, cyclic or linear polyribonuclear compounds as described herein. These are product-related substances other than creotides. When used herein, they are referred to as "process-related impurities." The term "product" refers to the final composition other than the linear polyribonucleotide described herein, preparation Undesirable substances in a material or product, used in the manufacture of a composition, preparation, or product, are present. It is a substance that is produced or generated. In some embodiments, process-related impurities are poly An enzyme used for the synthesis or cyclization of ribonucleotides. The term “product-related substance” refers to a composition, preparation, or product, or any intermediate thereof. It is a substance or by-product generated during the synthesis of a body. In some embodiments, it is a product-related substance. is a deoxyribonucleotide fragment. In some embodiments, the product-related substance is de It is an oxyribonucleotide monomer. In some embodiments, the product-related substance is 1 One or more derivatives or fragments of polyribonucleotides described herein, for example, 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or It is a fragment of triribonucleic acid.

[0040] As used herein, the term “immunogen” means an antibody or T cell receptor Any molecule containing one or more epitopes that are recognized, targeted, or bound. Alternatively, it refers to a molecular structure. In particular, an immunogen is something that induces an immune response in a target (for example, Honmei (Immunogenicity as defined in the detailed document). An immunogen is a substance that can induce an immune response in a target. An immune response is a series of molecules, cells, and reactions that are triggered when the immune system encounters an immunogen. It refers to biological phenomena. Immune responses can be humoral and / or cellular immune responses. This may include antibody production and proliferation of B and T cells. Determining whether an immune response has occurred is crucial. To determine the course of the disease and observe its progression, the immunized target is subjected to an immune response to a specific immunogen. The appearance of substances can be monitored. The immune response to most immunogens is specific. It induces the production of both antibodies and specific effector T cells. In some embodiments, Immunogens are exogenous to the host. In some embodiments, immunogens are exogenous to the host. It is not exogenous. Immunogens are polypeptides, polysaccharides, polynucleotides, or lipids. It may contain all or part of the immunogen, as well as polypeptides, polysaccharides, polynucleotides, and / or it may be a mixture of lipids. For example, the immunogen may be a translationally modified polypeptide. It's possible. "Polypeptide immunogen" refers to an immunogen that contains polypeptides. An immunogen may contain one or more posttranslational modifications and / or one or more additional It can form complexes with the molecule and / or select a tertiary or quaternary structure. This allows for the determination of the immunogenicity of polypeptides, or the polypeptide It can affect the immunogenicity of the drug.

[0041] As used herein, the term "immunogenic" refers to a specific immune response assay. It is the potential ability to induce a response exceeding a predetermined threshold in a substance. Assays are examples. For example, the expression of specific inflammatory markers, the production of antibodies, or immunogenicity as described herein. It may be that the immune system of an organism or a particular type of immune cells is immune. Exposure to the substance can trigger an immune response.

[0042] Immunogenic responses include whole antibody assays, confirmatory tests, antibody titer measurement, and isotyping. Furthermore, by evaluating antibodies in the target plasma or serum using neutralizing antibody assessment, evaluation can be performed. The total antibody assay can be performed on the serum or plasma of the subject to which the immunogen was administered. It measures all antibodies produced as part of the immune response. The commonly used test is ELISA (Enzyme-linked immunosorbent assay), and ELISA A binds to the target antibody, including IgM, IgD, IgG, IgA, and IgE, in the test. Antibodies in the serum are detected. The immunogenic response is further evaluated by a confirmatory assay. This can be done. Following the overall antibody evaluation, a confirmatory assay is used to obtain the results of the overall antibody assay. This can be confirmed. A competitive assay can be used to confirm that the antibody is specifically binding to the target. And, positive findings in the screening assay, the test serum or detection reagent and the assay This confirms that it is not the result of nonspecific interactions with other substances.

[0043] Immunogenic responses can be evaluated by isotyping and titer measurement. Sotyping assays can be used to evaluate only the relevant antibody isotypes. For example, the expected isotypes could be IgM and IgG. The total number of antibodies present was specifically detected and quantified by isotyping and titer measurement. It can be compared to that.

[0044] The immunogenic response can be evaluated by a neutralizing antibody assay (nAb). Using an in vivo assay (nAb), antibodies produced in response to an immunogen neutralize the immunogen. This inhibits the immunogen from affecting the target, leading to abnormal pharmacokinetic behavior. It is possible to determine whether or not to rub. In many cases, nAb assays use antibodies to target cells. This is a cell-based assay that involves incubation together with [the specified substance]. However, it is not limited to [the specified substance]. Cell proliferation, viability, antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), Inhibition of cytopathic effects (CPE), apoptosis, ligand-stimulated cell signaling (Liga (and Stimulated Cell Signaling), enzyme activity, reporter Includes gene assays, protein secretion, metabolic activity, stress, and mitochondrial function. Various cell-based nAb assays can be used. Absorbance is used for readout of the detection results. This includes fluorescence, luminescence, chemiluminescence, or flow cytometry. Ligand binding assembly Using method (i), the binding affinity between the immunogen and the antibody is measured in vitro, and the neutralization effect is evaluated. It can also be done this way.

[0045] Furthermore, the induction of cellular immune responses is performed using cellular markers obtained from T cells of the target. This can be evaluated by measuring T cell activation in the subject. Pulls, lymph node biopsies, or tissue samples are collected from the subjects, and one T cell is selected from the sample. Or multiple (e.g., two, three, four, or more) activation markers: CD25, C D71, CD26, CD27, CD28, CD30, CD154, CD40L, CD13 4. T cell activation can be evaluated for CD69, CD62L, or CD44. This can also be evaluated using the same method in an in vivo animal model. This assay is Immunogens can be injected into T cells in vitro (e.g., subjects, animal models, storage facilities, or commercial sources). By adding the markers mentioned above to the T cells obtained and measuring them, T cell activation can be evaluated. This can also be done using a similar approach to other immune cells, such as eosinophils. Markers: CD35, CD11b, CD66, CD69, and CD81), dendritic cells ( (Manufacturer: IL-8, MHC Class II, CD40, CD80, CD83, and CD86) basophils (CD63, CD13, CD4, and CD203c), and neutrophils (CD11 The effect on the activation of b, CD35, CD66b, and CD63 can be evaluated. These markers can be obtained using flow cytometry, immunohistochemistry, and in situ hive biopsy. Evaluation is performed using other assays that enable rediization and measurement of cell markers. It is possible to determine the effect by comparing the results before and after administration of the immunogen. can.

[0046] As used herein, the term “induces an immune response” means an immune response by an object. This refers to initiating, amplifying, or maintaining an immune response. Inducing an immune response is an adaptive immune response. This may refer to an epidemic response or an innate immune response. The induction of an immune response can be measured as described above. Cut.

[0047] As used herein, the term “linear counterpart” refers to a cyclic polyribonucleotide. Nucleotide sequences that are the same as or similar to (e.g., 100%, 95%, 90%, 85%, 80%) Having sequence identity of %, 75%, or any percentage in between, and two Poly These are ribonucleotide molecules (and their fragments). In some embodiments, linear counterparts ( For example, the pre-cyclic form has the same or similar nucleotide configuration as a cyclic polyribonucleotide. Columns (for example, 100%, 95%, 90%, 85%, 80%, 75%, or any percentage in between). (Percentage of sequence identity) and having the same or similar nucleic acid modifications, and two free Polyribonucleotides having terminals (i.e., acyclic forms (and fragments thereof) of cyclic polyribonucleotides These are nucleotide molecules (and their fragments). In some embodiments, the linear counterpart is a ring. Nucleotide sequences that are the same as or similar to polyribonucleotides (e.g., 100%, 95%) , 90%, 85%, 80%, 75%, or any percentage in between, identical arrays (Sex) and having different nucleic acid modifications, or having the same or similar nucleic acids as cyclic polyribonucleotides Cleotide sequences (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or so They have any percentage of sequence identity between them but do not have nucleic acid modifications, and It has two free ends (i.e., the acyclic form (and fragments thereof) of a cyclic polyribonucleotide). These are polyribonucleotide molecules (and fragments thereof). In some embodiments, they are linear The fragment of the corresponding polyribonucleotide molecule is the linear corresponding polyribonucleotide molecule. Shorter linear counterparts are any part of a polyribonucleotide molecule. Several implementations In this configuration, the linear counterpart further includes a 5' cap. In some embodiments, the linear counterpart The material further comprises a polyadenosine tail. In some embodiments, the linear counterpart is 3' Further includes UTR. In some embodiments, the linear counterpart further includes 5'UTR. .

[0048] As used herein, "linear RNA" or "linear polyribonucleotide" or " The term "linear polyribonucleotide molecule" is used interchangeably, with the 5' and 3' ends being... This refers to a polyribonucleotide molecule. Either the 5' end and / or the 3' end are: The ends may be free or they may be bound to another part. Linear RNA is circularized. contains RNA that has not been (e.g., not pre-circularized), e.g., a sprint ligation, or cyclization by chemical, enzymatic, ribozyme or splicing catalysts can be used as a starting material for cyclization by the method.

[0049] As used herein, the term "mixture" means a material consisting of two or more different substances that are mixed. In some cases, the mixtures described herein can be homogeneous mixtures of two or more different substances. For example, the mixture can be throughout any given sample of the mixture, and its components (e.g., two or more substances) can have the same ratio. In some cases, the mixtures provided herein can be heterogeneous mixtures of two or more different substances. For example, the ratio of the components of the mixture (e.g., two or more substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. substances. In some cases, the mixtures described herein can be homogeneous mixtures of two or more different substances. For example, the mixture can be throughout any given sample of the mixture, and its components (e.g., two or more substances) can have the same ratio. In some cases, the mixtures provided herein can be heterogeneous mixtures of two or more different substances. For example, the ratio of the components of the mixture (e.g., two or more substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. substances. In some cases, the mixtures described herein can be homogeneous mixtures of two or more different substances. For example, the mixture can be throughout any given sample of the mixture, and its components (e.g., two or more substances) can have the same ratio. In some cases, the mixtures provided herein can be heterogeneous mixtures of two or more different substances. For example, the ratio of the components of the mixture (e.g., two or more substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. body, and its components (e.g., two or more substances) can have the same ratio. In some cases, the mixtures provided herein can be heterogeneous mixtures of two or more different substances. For example, the ratio of the components of the mixture (e.g., two or more substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. substances. In some cases, the mixtures provided herein can be heterogeneous mixtures of two or more different substances. For example, the ratio of the components of the mixture (e.g., two or more substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. substances) can vary throughout the mixture. In some cases, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. Thus, the mixture is a solution. For example, the mixture is present in the liquid phase. In some cases, the liquid solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. body solution can be regarded as containing a liquid solvent and a solute. Mixing a solute into a liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. and the solute into the liquid solvent can be called the "dissolving" process. In some cases, the liquid solution is a solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. solution containing a liquid in a liquid (e.g., a liquid solute dissolved in a liquid solvent), a solution containing a solid in a liquid (e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. e.g., a solid solute dissolved in a liquid solvent), or a solution containing a gas in a liquid (e.g., a solid solute dissolved in a liquid solvent). In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. In some cases, there are multiple solvents and / or multiple solutes. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. In some cases, the mixture is a colloid, a liquid suspension, or an emulsion. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase. In some cases, the mixture is a solid mixture. For example, the mixture is present in the solid phase.

[0050] As used herein, the term "modified ribonucleotide" refers to a sugar, a nucleobase, means a nucleotide having at least one modification to the internucleoside linkage .

[0051] As used herein, the term "naked delivery" means a formulation for delivery to cells without the aid of a carrier and without using covalent modifications to moieties that assist in the delivery to cells . Naked delivery formulations do not include any transfection reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein carriers. For example, a naked delivery formulation of a cyclic or linear polyribonucleotide contains the cyclic or linear polyribonucleotide without covalent modification and is a formulation without a carrier . . .

[0052] As used herein, the terms "nick RNA" or "nick linear polyribonucleotide" or "nick linear polyribonucleotide molecule" are used interchangeably and mean a polyribonucleotide molecule having 5' and 3' ends resulting from the cleavage or degradation of circular RNA . . .

[0053] As used herein, the term "non-circular RNA" means all nick RNAs and linear RNAs .

[0054] Terms such as "obtainable by" or "manufacturable by" are used to indicate that a claim or embodiment refers to a compound, composition, product, etc. itself, i.e., the compound, composition, product, etc. can be obtained or manufactured by the method described for the manufacture of the compound, composition, product, etc., but the compound, composition, product, etc. can also be obtained or manufactured by methods other than the described method . . . "Obtained by" or "manufactured by" are used to indicate that a compound, composition, product, etc. can be obtained or manufactured by the method described for the manufacture of the compound, composition, product, etc., but the compound, composition, product, etc. can also be obtained or manufactured by methods other than the described method Terms such as "obtained" refer to compounds, compositions, or products obtained by the listed specific methods. It indicates that it is either available by ~ or can be manufactured by ~. Which terms also include terms such as "available by ~" and "manufacturable by ~" Preferred embodiments are disclosed, such as "available by ~" and "manufacturable by ~". I want you to understand that.

[0055] As used herein, the term "pathogen" means, for example, a substance that directly infects a target. By producing pathogens that cause disease or symptoms of disease in the target, and To induce disease or signs of disease in a target by eliciting an immune response from that target. This refers to infectious pathogens. As used herein, pathogens are not limited to... Moss, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids, and viruses, or so Any combination of these is included, and each pathogen can be targeted alone or in conjunction with another pathogen. It can induce disease or symptoms.

[0056] As used herein, the term “payload” means the poly(poly(poly(poly(poly(poly(poly)) disclosed herein))” This refers to any molecule delivered by ribonucleotides. In some cases, paylow The term "do" refers to nucleic acids, proteins, chemicals, ribonucleoproteins, or any combination thereof. In some cases, the payload is within the polyribonucleotides disclosed herein. It is a nucleic acid sequence directly contained in the payload. In some cases, the payload may be, for example, a complementary hive. Through redylation or through protein-nucleic acid interactions, as disclosed herein It binds to or associates with polyribonucleotides. In certain cases, the payload is polyri a protein encoded by a nucleic acid sequence contained within, bound to, or associated with a polynucleotide. In some cases, "bound" means a covalent bond or non-covalent interaction between two molecules. In some cases, "associated" when used in the context of an interaction between a payload and a polynucleotide means that the payload is directly linked to the polynucleotide through one or more other molecules between them. In some cases, the binding or association can be transient. In some cases, the payload binds or associates with the polynucleotide under certain conditions but does not bind or associate under other conditions, e.g., depending on the surrounding pH conditions or the presence or absence of a stimulus or binding partner.

[0057] The term "pharmaceutical composition" is also intended to disclose that the cyclic or linear polynucleotide contained within the pharmaceutical composition can be used for the treatment of the human or animal body by therapy. Thus, the pharmaceutical composition means being equivalent to "cyclic or linear polynucleotide for use in therapy".

[0058] As used herein, the term "polynucleotide" means a molecule containing one or more nucleic acid subunits or nucleotides and can be used interchangeably with "nucleic acid" or "oligonucleotide". The polynucleotide can contain one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. The nucleotide is a nucleoside and At least one, two, three, four, five, six, seven, eight, nine, ten, or more The above may contain a phosphate (PO3) group. Nucleotides are nucleic acid bases, pentoses (ribose or It may contain any of the deoxyribose compounds and one or more phosphate groups. Ribonucleo A nucleotide is a nucleotide whose sugar is ribose. It is also called a polyribonucleotide or ribonucleic acid. Alternatively, RNA may contain multiple ribonucleotides polymerized via phosphodiester bonds. It can refer to a polymer. A deoxyribonucleotide is a nucleonucleotide whose sugar is deoxyribose. It's Chido.

[0059] Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, are phosphodiester. This refers to a polymer containing multiple deoxyribonucleotides polymerized via nucleotide bonds. Cleotide may be nucleoside monophosphate or nucleoside polyphosphate. Chido includes detectable tags such as luminescent tags and markers (e.g., fluorophores), For example, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCT) P), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and Deoxyribo can be selected from deoxythymidine triphosphate (dTTP) dNTP. This refers to deoxyribonucleoside polyphosphates such as nucleoside triphosphates (dNTPs). Nucleotides contain any subunits that can be incorporated into a growing nucleic acid chain. Obtain. Such subunits are A, C, G, T, or U, or one or more phases. It is specific to the complementary A, C, G, T, or U, or purines (i.e., A or G, or (Its variant) or complementary to pyrimidine (i.e., C, T, or U, or its variant) It can be any other subunit. In some examples, polynucleotides are deoxygenated. These are siribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, polynucleotides are, to give a few examples, small interfering RNAs (s iRNA, microRNA (miRNA), plasmid DNA (pDNA), small molecule Apin RNA (shRNA), nuclear small RNA (snRNA), messenger RNA (mRNA), mRNA precursor (pre-mRNA), antisense RNA (asRNA) ) are nucleotide sequences, and are single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. It encompasses both of any structural embodiments of the polynucleotide molecule. In some cases, the polynucleotide molecule is ring It is in this state. Polynucleotides can have various lengths. Nucleic acid molecules are at least about 10 Bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases , 400 bases, 500 bases, 1 kilobase (kb), 2kb, 3kb, 4kb, 5kb, 1 Polynucleotides can have lengths of 0kb, 50kb, or more. It can be isolated from. As embodied herein, the polynucleotide sequence is Isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA molecules It may include NA analogs.

[0060] Polynucleotides, for example, polyribonucleotides or polydeoxyribonucleotides This includes non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. It may contain one or more nucleotide variants that include a creotide. Examples of modified nucleotides. This includes, but is not limited to, diaminopurine, 5-fluorouracil, 5-bromo Uracil, 5-Chlorouracil, 5-Iodouracil, Hypoxanthine, Xanthine, 4 -Acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxy Methylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, Dihydrouracil, β-D-galactosylkeosin, inosine, N6-isopentenyl Denine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl Chiladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino Methyl-2-thiouracil, β-D-mannosylkeosin, 5'-methoxycarboxymethyl Chiluracil, 5-Methoxyuracil, 2-Methylthio-D46-Isopentenyladeno Uracil-5-oxyacetic acid (v), Wibutoxosin, Pseudouracil, Keosin , 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil Cyl, 5-methyluracil, uracil-5-oxyacetate methyl ester, uracil-5- Oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-k It contains (ruboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, etc. In some cases, nucleotides include modifications to their triphosphate moieties. This may include modifications to parts of the chain. Non-limiting examples of such modifications include longer phosphate chains (e.g., For example, having 4, 5, 6, 7, 8, 9, 10, or more phosphate groups. Modification of the phosphate chain and thiol moiety (e.g., α-thiotriphosphate and β-thiotriphosphate) Nucleic acid molecules also consist of a base moiety, a sugar moiety, or a phosphate backbone (for example, typical It contains one or more atoms that can be used to form hydrogen bonds with complementary nucleotides. and / or typically one or more nucleotides that cannot form hydrogen bonds with complementary nucleotides. Nucleic acid molecules can also be modified with N-hydroxysuccinimide (NH₃). To enable covalent bonding of amine-reactive moieties such as S, aminoallyl (amino ally 1)-dUTP(aa-dUTP) and aminohexylacrylamide-d The oligonucleotides disclosed herein may contain amine-modified groups such as CTP (aha-dCTP). Standard DNA base pair or RNA base pair substitutes in the 3D are high per cubic mm. Bit density, higher safety (resistance to accidental or intentional synthesis of natural toxins), light This may provide easier identification or lower secondary structure in programmed polymerases. Such as compatible with natural and mutant polymerases for de novo and / or amplification synthesis. Alternative base pairs are incorporated herein by reference for all purposes, Betz K ,Malyshev DA,Lavergne T,Welte W,Diederic hs K, Dwyer TJ, Ordoukhanian P, Romesberg F E,Marx A.Nat.Chem.Biol.2012 Jul;8(7):612 It is described in -4.

[0061] As used herein, "polypeptide" is most often linked by peptide bonds. This term refers to a polymer of linked amino acid residues (natural or unnatural). When used herein, proteins, polypeptides of any size, structure, or function Polypeptides refer to genes and peptides. Polypeptides include gene products and naturally occurring polypeptides. synthetic polypeptides, homologs, orthologs, paralogs, their fragments and other equivalents, modified Allomorphs and analogs may be included. A polypeptide may be a single molecule or a dimorph. Polypeptides may also be multimolecular complexes such as wholes, trimers, or tetramers. It may contain single-chain or multi-chain polypeptides such as antibodies or insulin, and may be associated or linked. It is possible. The most common disulfide bond is found in multi-chain polypeptides. The term "artificial" refers to one or more amino acid residues corresponding to naturally occurring amino acids. It can also be applied to amino acid polymers, which are chemical analogs.

[0062] As used herein, the term “prevent” means that a disease, disorder, or symptom may not occur. To reduce the likelihood of or to mitigate the severity of any subsequent illness or disability. This means that a therapeutic agent prevents the onset of a disease or symptom, or reduces its severity. Therefore, it is administered to individuals who are at higher risk of developing disease or disability compared to members of the general population. It is possible. Therapeutic agents can be used as a preventative measure, for example, to prevent the onset of signs or manifestations of disease or disorder. It can be administered before [the relevant procedure].

[0063] As used herein, the term "pseudo-helical structure" refers to a cyclic polyribonucleotide. This is the higher-order structure of ocide, in which at least a portion of the cyclic polyribonucleotide is folded into a helical structure. It's folded up.

[0064] As used herein, the term "pseudo-double-stranded secondary structure" refers to a cyclic polyribonucle. This is the higher-order structure of rheotide, in which at least a portion of the cyclic polyribonucleotide forms an inner double helix. It is forming.

[0065] As used herein, the term “regulatory element” refers to a cyclic or linear polyribonucleotide. This refers to the part of the nucleic acid sequence, etc., that modifies the expression of the expression sequence within the ocide.

[0066] As used herein, the term "repetitive nucleotide sequence" refers to DNA or These are repeating nucleic acid sequences within an RNA stretch or throughout the entire genome. In some embodiments, The repeating nucleotide sequence contains poly-CA or poly-TG(UG) sequences. Several implementations Morphologically, repetitive nucleotide sequences include the Alu family of introns.

[0067] As used herein, the term “replica element” means an element useful for reproduction or a ring It is a sequence and / or motif that initiates the transcription of polyribonucleotides.

[0068] As used herein, the term “surface area” of the body in question refers to the body in question being exposed to the external environment. It means any area of ​​an object that is exposed to or may be exposed to the substance. The body of the object, for example. The surface area of ​​a mammal's body, for example, the human body, includes the skin, oral cavity, nasal cavity, ear cavity, gastrointestinal tract, respiratory tract, This may include the vagina, cervix, uterus, urinary tract, and the surface area of ​​the eye. In some cases, the body of the subject may also be included. Surface area often refers to the outer region where epithelial cells are arranged. For example, skin, It may be a type of surface area discussed herein, and may consist of the epidermis and dermis. The epidermis is It forms the outermost layer of the skin and may contain organic aggregates of epithelial cells, among many other types of cells.

[0069] As used in this specification, the terms "stagger element" and The term refers to a segment, such as a nucleotide sequence, that induces ribosome rest during translation. In some embodiments, the staggered element has a strong alpha-helix tendency followed by a consensus The sequence -D(V / I)ExNPG is a non-conserved sequence of amino acids followed by P, where x is any amino acid. It is an acid. In some embodiments, the stagger element is a chemical part such as glycerol. This may include non-nucleic acid linkages, chemical modifications, modified nucleic acids, or any combination thereof.

[0070] As used herein, the term “substantially does not contain” means “substantially does not contain” biological, chemical, Compositions and preparations that are below the level required to induce physical and / or pharmacological effects. This refers to the level of a component in a substance, product, or any intermediate thereof. Several embodiments So, if the composition, preparation, or product is detectable only in trace amounts, Related detection techniques (e.g., chromatography (using columns, using paper, etc.) Using HPLC, using UHPLC, etc., or by IC, S (By EC, by reverse phase, by anion exchange, by mixed mode, etc.) or electric Gas electrophoresis (UREA PAGE, tip-based, polyacrylamide gel, RNA, capillaries) (Larry, c-IEF, etc.) (The level of that is mass spectrometry, ultraviolet-visible light, fluorescence, light scattering, refractive index) Detection techniques based on or detection techniques that use silver, dye staining, or radioactive decay for detection. Detectable by using or not using a derivatization method before or after separation. If the level is below the effective level, the component is substantially absent. Alternatively, a composition, preparation, or Whether a product substantially contains an ingredient can be determined by mass spectrometry, microscopy, or circularly polarized light analysis. Fluorescence analysis by UV or UV-vis spectrophotometric chromatography (CD) (example) For example, surface plasmon resonance (SPR) by RNase H analysis using Qubit. Separation techniques that utilize silver, dye staining, or radioactive decay for detection. It is possible to make a decision without using it.

[0071] As used herein, the term “substantially resistant” means, compared to the references, For the effect pedal, at least 50%, 55%, 60%, 65%, 70%, 75%, 8 Resistance to 0%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% It is a sexual thing.

[0072] As used herein, the term "sterilizer" means bacteriostatic, bactericidal, and / Or any method that actively kills microorganisms, inactivates microorganisms, or prevents the growth of microorganisms This refers to the active substance. A sterilizing agent that kills microorganisms may be an antibacterial agent and / or a preservative. In one embodiment, the sterilizing agent is a liquid such as alcohol, iodine, or hydrogen peroxide. In some embodiments, the sterilizer is UV light or laser light. In this state, the sterilizing agent is delivered electrically or by other means (e.g., steam, contact). It is heat.

[0073] As used herein, the term “stoichiometric translation” refers to cyclic or linear polyribo This is the production of substantially equivalent expression products translated from nucleotides. For example, two expression In the case of cyclic or linear polyribonucleotides having a sequence, cyclic or linear polyribonucleo Stoichiometric translation of tide occurs when the expression products of two expression sequences are substantially equal in quantity, for example, The difference in amount (e.g., molar molecular weight) between the two expression sequences is approximately 0, less than 1%, or less than 2%. Less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10% It may be less than 15%, less than 20%, or any percentage in between. It means that.

[0074] As used herein, the terms "systemic delivery" or "systemic administration" refer to the circulatory system (e.g., For example, it refers to the route of administration of a pharmaceutical composition or other substance to the blood system or lymphatic system. Administration methods include oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or Any combination of these may be included. When used herein, "non-whole-body delivery" or " The term "non-systemic administration" refers to any other method of administration other than systemic delivery of a pharmaceutical composition or other substance. It can refer to a pathway, for example, the substance being delivered to the target body's circulatory system (e.g., the blood system and It does not belong to the lymphatic system.

[0075] As used herein, the term “sequence identity” refers to global or local identity. Using the alignment algorithm, two peptide sequences or two nucleotide sequences It is determined by the alignment. Therefore, the arrays are determined by (default parameters) Using data, for example, optimally aligned by the program GAP or BESTFIT (In cases where) they share at least a certain minimum percentage of sequence identity, they are "substantially identical" It can be called "one" or "essentially similar." GAP is Needleman and Wu Using the NSC global alignment algorithm, the two arrays are aligned to their full length. The data is aligned, the number of matches is maximized, and the number of gaps is minimized. In general, gap creation... Growth penalty = 50 (nucleotides) / 8 (proteins) and gap expansion penalty = The default GAP parameter of 3 (nucleotides) / 2 (proteins) is used. For nucleotides, the default score matrix used is nwsgapdna. For proteins, the default score matrix is ​​Blosum62 (Henik off & Henikoff, 1992, PNAS 89, 915-919). Sequence aligner. The performance and sequence identity percentage scores were determined by Accelrys Inc., 96 85 Scranton Road, San Diego, CA 92121-3752 GCG Wisconsin Package, Version available from the USA 10.3 or EmbossWin version 2.10.0 (Program "Needle") This can be determined using computer programs such as (using). Alternatively, this In addition, percent identity is performed using algorithms such as FASTA and BLAST. This can be determined by searching the database. Sequence identity is determined by the entire sequence. This refers to sequence identity over a long distance.

[0076] A "signal sequence" is a newly synthesized protein that targets polypeptide sequences to the secretory pathway. A polypeptide sequence located at the N-terminus of a high-quality polypeptide sequence, for example, a polypeptide with a length of 10 to 30 amino acids. This refers to the CHIDO sequence.

[0077] As used herein, the term “target” includes one or more epitopes. It refers to any entity. For example, a target could be a chemical part, a part of a molecule, or a molecule (e.g., an allergen). (or toxins), polymers (e.g., polypeptides, nucleic acids, or carbohydrates), polymers Post-translational correction of polymers (e.g., phosphorylation, glycosylation, acylation, alkylation, etc.) Decorative states, higher-order polymer structures (e.g., complexes of two or more polypeptides), cells (e.g., Cancer cells), parts of cells (e.g., tumor antigens), receptors on the cell surface, pathogens (e.g., viruses) Rus or part of a virus; bacteria or part of bacteria; fungi or part of fungi; or parasites It may be a part of an insect or parasite, or a tissue form.

[0078] As used herein, the terms “to treat” or “to treat” refer to the subject matter. This refers to the therapeutic treatment of a disease or disorder (e.g., infection, cancer, toxicity, or allergic reaction). The effects of the treatment include the reversal of one or more signs or manifestations of the disease or disease or disorder. Reversal, reduction, reduction of severity, cure, inhibition of progression, reduction of the likelihood of recurrence, state of disease or disability Stabilization (i.e., no worsening) of the disease or disorder in the absence of therapeutic intervention. This may include preventing the spread of disease or disorder compared to the condition and / or symptoms.

[0079] As used herein, the term “terminating element” refers to a cyclic or linear polyribonucleotide. This refers to the nucleic acid sequence and other parts that terminate the translation of the expression sequence in ocid.

[0080] As used herein, the term "total ribonucleotide molecule" refers to a ribonucleotide molecule. When measured by the total amount of rheotide molecules, linear polyribonucleotide molecules, cyclic polyribonucleotides, etc. Bonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, and their This refers to the total amount of any ribonucleotide molecule, including fragments and their modified forms. ru.

[0081] As used herein, the term "translation efficiency" refers to the production of ribonucleotide transcripts. This refers to the rate or amount of protein or peptide production from a substance. In one embodiment, translation Efficiency is measured, for example, in an in vitro translation of a given translation system, such as rabbit reticulocyte lysate. In translation systems, or in in vivo translation systems such as eukaryotic or prokaryotic cells, for example In a given period, produced per given amount of transcript encoding a protein or peptide. It can be expressed as the amount of protein or peptide being processed.

[0082] As used herein, the term “translation initiation sequence” refers to a cyclic or linear polyribonu This is the nucleic acid sequence that initiates translation of the expression sequence in creotide. In embodiments of the present invention, for example, the following items are provided. (Item 1) A cyclic polyribonucleotide comprising multiple sequences, each sequence encoding a polypeptide immunogen, wherein at least two of the polypeptide immunogens identify different targets. (Item 2) Each of the polypeptide immunogens identifies a different target, as described in item 1, the cyclic polyribonucleotide. (Item 3) A cyclic polyribonucleotide as described in item 1 or 2, wherein each target is a different pathogen. (Item 4) Each target is independently a virus, bacterium, fungus, or parasite, as described in item 3, a cyclic polyribonucleotide. (Item 5) Each target is a different virus, as described in item 4, a cyclic polyribonucleotide. (Item 6) Each target is a different bacterium, as described in item 4, and is a cyclic polyribonucleotide. (Item 7) The aforementioned target is a cyclic polyribonucleotide as described in item 4, including viruses and bacteria. (Item 8) A cyclic polyribonucleotide comprising multiple sequences, each sequence being a cyclic polyribonucleotide encoding a polypeptide immunogen, wherein at least two of the polypeptide immunogens identify different proteins, and each of the different proteins identifies the same target. (Item 9) Each of the polypeptide immunogens identifies a different protein, as described in item 8, a cyclic polyribonucleotide. (Item 10) The cyclic polyribonucleotide described in item 8 or 9, wherein the target is a pathogen. (Item 11) The cyclic polyribonucleotide described in item 10, wherein the pathogen is a virus, bacterium, fungus, or parasite. (Item 12) The cyclic polyribonucleotide according to item 11, wherein the pathogen is a virus, and each of the different proteins is a viral protein associated with the virus. (Item 13) The cyclic polyribonucleotide described in item 11, wherein the pathogen is a bacterium, and each of the different proteins is a bacterial protein associated with the bacterium. (Item 14) The cyclic polyribonucleotide described in item 8 or 9, wherein the target is cancer cells. (Item 15) A cyclic polyribonucleotide as described in item 14, wherein each of the aforementioned different proteins is a different tumor antigen associated with the cancer cells. (Item 16) The cyclic polyribonucleotide described in item 8 or 9, wherein the target is an allergen or toxin. (Item 17) A cyclic polyribonucleotide, as described in any one of items 1 to 16, containing 500 to 20,000 ribonucleotides. (Item 18) A cyclic polyribonucleotide as described in any one of items 1 to 16, containing at least 1,000 ribonucleotides. (Item 19) A cyclic polyribonucleotide as described in any one of items 1 to 18, comprising at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine sequences, each sequence encoding a polypeptide immunogen. (Item 20) A cyclic polyribonucleotide as described in any one of items 1 to 18, comprising sequences 2-3, 2-5, or 5-10, where each sequence encodes a polypeptide immunogen. (Item 21) A cyclic polyribonucleotide as described in any one of items 1 to 20, wherein at least one sequence encoding a polypeptide immunogen further encodes a signal sequence. (Item 22) A cyclic polyribonucleotide according to any one of items 1 to 21, wherein each of the sequences encoding each of the polypeptide immunogens is operably linked to an internal ribosome entry site (IRES). (Item 23) A cyclic polyribonucleotide as described in item 22, containing a single IRES. (Item 24) The cyclic polyribonucleotide according to item 23, wherein each of the polypeptide immunogens is encoded by a single open reading frame operably linked to a single IRES, and the expression of the open reading frame generates a polypeptide comprising the respective amino acid sequences of the polypeptide immunogens. (Item 25) The cyclic polyribonucleotide described in item 24, wherein each polypeptide immunogen is separated by a polypeptide linker. (Item 26) The cyclic polyribonucleotide described in item 24, wherein each polypeptide immunogen is separated by a cleavage domain. (Item 27) A cyclic polyribonucleotide as described in item 26, wherein each cleavage domain is a 2A self-cleaving peptide. (Item 28) A cyclic polyribonucleotide as described in item 22, containing multiple IRESs. (Item 29) A cyclic polyribonucleotide as described in item 28, wherein each IRES is operably linked to an open reading frame containing a sequence encoding a polypeptide immunogen. (Item 30) An immunogenic composition comprising multiple cyclic polyribonucleotides, each containing a sequence encoding a polypeptide immunogen. (Item 31) The immunogenic composition according to item 30, wherein each of the plurality of cyclic polyribonucleotides is a cyclic polyribonucleotide according to any one of items 1 to 29. (Item 32) The immunogenic composition according to item 31, comprising (a) at least a first cyclic polyribonucleotide comprising a sequence encoding a first polypeptide immunogen, and (b) at least a second cyclic polyribonucleotide comprising a sequence encoding a second polypeptide immunogen, wherein the first and second polypeptide immunogens identify different proteins, and each of the different proteins identifies the same target. (Item 33) The immunogenic composition according to item 32, wherein the target is a pathogen. (Item 34) The immunogenic composition according to item 33, wherein the pathogen is a virus, bacteria, fungus, or parasite. (Item 35) The immunogenic composition according to item 32, wherein the target is a cancer cell, an allergen, or a toxin. (Item 36) (a) at least a first cyclic polyribonucleotide comprising a sequence encoding a first polypeptide immunogen, and (b) at least a sequence comprising a sequence encoding a second polypeptide immunogen. The immunogenic composition according to item 30, wherein the immunogenic composition also comprises a second cyclic polyribonucleotide, the first polypeptide immunogen identifies a first target, and the second polypeptide immunogen identifies a second target. (Item 37) An immunogenic composition as described in item 36, wherein each target is a pathogen. (Item 38) An immunogenic composition according to item 36 or 37, wherein each target is independently a cancer cell, virus, bacterium, fungus, parasite, toxin, or allergen. (Item 39) An immunogenic composition according to any one of items 30 to 38, wherein each polypeptide immunogen is operably linked to an IRES. (Item 40) A pharmaceutical composition comprising a cyclic polyribonucleotide as described in any one of items 1 to 29 or an immunogenic composition as described in any one of items 30 to 39, and a pharmaceutically acceptable excipient. (Item 41) The pharmaceutical composition described in item 40, further comprising an adjuvant. (Item 42) The pharmaceutical composition according to item 41, wherein the adjuvant is an inorganic adjuvant, a small molecule adjuvant, and an oil-in-water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a virus particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte microorganism-associated molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component adjuvant. (Item 43) A method for treating or preventing a target disease, disorder, or symptom, comprising administering to the target a cyclic polyribonucleotide described in any one of items 1 to 29, an immunogenic composition described in any one of items 30 to 39, or a pharmaceutical composition described in any one of items 40 to 42. (Item 44) The method according to item 43, wherein the disease, disorder, or symptom is a viral infection, bacterial infection, or fungal infection. (Item 45) The method according to item 43, wherein the disease, disorder, or symptom is cancer. (Item 46) The method described in item 43, wherein the disease, disorder, or symptom is related to exposure to an allergen. (Item 47) The method described in item 43, wherein the disease, disorder, or symptom is related to exposure to a toxin. (Item 48) A method for inducing an immune response in a subject, as described in any one of items 1 to 29. A method comprising administering to the subject a cyclic polyribonucleotide, an immunogenic composition according to any one of items 30 to 39, or a pharmaceutical composition according to any one of items 40 to 42. (Item 49) The method according to any one of items 43 to 48, further comprising administering an adjuvant to the subject. (Item 50) The adjuvant may be an inorganic adjuvant, a small molecule adjuvant, an oil-in-water emulsion, a lipid or polymer, a peptide or peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a virus particle, or a bacterial adjuvant. The method according to item 49, which is an adjuvant, hybrid molecule, fungal or oocyte microorganism-associated molecular pattern (MAMP), inorganic nanoparticles, or multicomponent adjuvant. (Item 51) The method according to any one of items 43 to 50, wherein a cyclic polyribonucleotide described in any one of items 1 to 29, an immunogenic composition described in any one of items 30 to 39, or a pharmaceutical composition described in any one of items 40 to 42 is administered to the subject as a single dose. (Item 52) The method according to any one of items 43 to 50, wherein a cyclic polyribonucleotide described in any one of items 1 to 29, an immunogenic composition described in any one of items 30 to 39, or a pharmaceutical composition described in any one of items 40 to 42 is administered to the subject two or more times, three or more times, four or more times, or five or more times. (Item 53) The method according to item 52, wherein the administration of a cyclic polyribonucleotide described in any one of items 1 to 29, an immunogenic composition described in any one of items 30 to 39, or a pharmaceutical composition described in any one of items 40 to 42 is performed approximately every week, every two weeks, every three weeks, every month, every two months, every three months, every four months, every five months, every six months, every year, every year, every two years, every three years, every four years, every five years, or every ten years. (Item 54) The method according to any one of items 43 to 53, further comprising administering a polypeptide immunogen to the subject. (Item 55) The method according to item 54, wherein the polypeptide immunogen is administered to the subject after administering a cyclic polyribonucleotide according to any one of items 1 to 29, an immunogenic composition according to any one of items 30 to 39, or a pharmaceutical composition according to any one of items 40 to 42. (Item 56) The method according to item 54 or item 55, wherein the polypeptide immunogen maintains or enhances the immune response to the polypeptide immunogen in the subject. (Item 57) A method for maintaining or enhancing the immune response in a subject, (i) A step of administering a cyclic polyribonucleotide encoding a polypeptide immunogen to the subject; (ii) The step of administering the polypeptide immunogen to the subject. Includes, A method wherein step (ii) is performed one week to six months after step (i), and the administration of the polypeptide immunogen in step (ii) maintains or enhances the immune response to the polypeptide immunogen in the subject. [Brief explanation of the drawing]

[0083] [Figure 1]Figure 1 is a schematic diagram of an exemplary circular RNA containing two expression sequences, each encoding an immunogen. The circular RNA contains two open reading frames (ORFs), each encoding an expression sequence, and each ORF is operably ligated to an IRES. [Figure 2] Figure 2 is a schematic diagram of an exemplary circular RNA containing two expression sequences, each of which is an immunogen. The circular RNA contains two expression sequences separated by a 2A sequence, all of which are operably ligated to an IRES. [Figure 3] Figure 3 shows a schematic diagram of multiple polyribonucleotides, each containing an ORF that encodes an immunogen. [Figure 4] Figure 4 shows RBD immunogens encoded by circular RNAs that are detected in BJ fibroblasts and HeLa cells, but not in BJ fibroblasts and HeLa cells, including vehicle controls. [Figure 5] This study demonstrates that a sustained anti-RBD antibody response was obtained in a mouse model after administration of a circular RNA encoding a SARS-CoV-2 RBD immunogen, combined with a cationic polymer (e.g., protamine). [Figure 6] This study demonstrates that an anti-spike response was obtained in a mouse model after administration of circular RNA encoding a SARS-CoV-2 RBD immunogen, combined with a cationic polymer (e.g., protamine). [Figure 7] The images show the anti-RBD IgG2a and IgG1 isotype levels obtained after administration of a circular RNA encoding the SARS-CoV-2 RBD antigen, combined with a cationic polymer (e.g., protamine), in a mouse model. [Figure 8] This study shows long-term in vivo protein expression from circular RNA after intramuscular injection of circular RNA preparations (containing Trans-IT, containing protamine, and without), protamine vehicle alone, and in non-injection control mice. [Figure 9]The graph shows long-term in vivo protein expression from circular RNA after co-intramuscular delivery of Addavax® adjuvants with (i) a non-combined circular RNA preparation (left graph), (ii) circular RNA combined with TransIT (middle graph), and (iii) circular RNA combined with protamine (right graph). In each case, the Addavax® adjuvant was delivered as individual injections at 0 and 24 hours. [Figure 10] (i) circular RNA combined with protamine, (ii) circular RNA combined with protamine after injection of Addavax® adjuvant at 24 hours, (iii) protein expression from circular RNA after intradermal delivery of protamine vehicle alone, and (iv) long-term in vivo protein expression from circular RNA in non-injected control mice. [Figure 11] Figure 11 shows the binding of the probe to circular and linear RNA, and subsequent RNA degradation by RNase H. Circular RNA is detected as a single cleaved linear band, compared to linear and chain RNA, which are detected as multiple bands. Degradation was detected by electrophoresis of the sample on a denatured polyacrylamide gel and comparing the degradation bands with and without RNase H. [Figure 12] Figure 12 is an image showing a protein blot of expression products from circular RNA or linear RNA containing stagger elements. [Figure 13] Figure 13 shows an exemplary circular RNA generation by self-splicing. [Figure 14] Figure 14 is an image showing a blot of the protein expression product from circular RNA or linear RNA. [Figure 15] Figure 15 shows experimental data demonstrating increased persistence of Gaussial ciferase expression in mice after re-administration of cyclic polyribonucleotides ("endless") compared to linear polyribonucleotide counterparts ("linear"). [Figure 16]Figure 16 shows experimental data demonstrating the increased persistence of Gaussial luciferase expression in mice after staggered administration of cyclic polyribonucleotides ("three doses without ends") compared to staggered administration of linear polyribonucleotides ("three doses without ends"), a single dose of cyclic polyribonucleotides ("endless"), or a single dose of linear polyribonucleotides ("linear"). [Figure 17] Figure 17 shows experimental data demonstrating the increased persistence of Gaussial luciferase expression in mice after a single dose of cyclic polyribonucleotide ("endless RNA") compared to a single dose of the linear polyribonucleotide counterpart ("linear RNA"), after staggered administration of the linear polyribonucleotide counterpart ("three doses of linear RNA") compared to a single dose ("linear RNA"), or after staggered administration of cyclic polyribonucleotide ("three doses of endless RNA") compared to a single dose ("endless RNA"). [Figure 18] Figure 18 shows the long-term in vivo protein expression of cyclic polyribonucleotides administered intramuscularly without a carrier, and the level of protein activity in plasma several days after injection. [Figure 19] Figure 19 shows that intravenously administered cyclic polyribonucleotides expressed protein in vivo for an extended period, demonstrating the level of protein activity in plasma several days after injection, and that at least five re-administrations were possible. [Figure 20A] Multiple immunogen expressions from cyclic polyribonucleotides were observed. RD immunogen expression was detected from cyclic RNA encoding SARSs-CoV-2 RBD immunogen and GLuc polypeptide. [Figure 20B] Multiple immunogen expressions from cyclic polyribonucleotides were observed. GLuc activity was detected from the SARSs-CoV-2 RBD immunogen and the cyclic RNA encoding the GLuc polypeptide. [Figure 21A]This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 RBD immunogen and a second circular RNA encoding the GLuc polypeptide. Anti-RBD antibodies were obtained 17 days after injection. [Figure 21B] This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 RBD immunogen and a second circular RNA encoding the GLuc polypeptide. GLuc activity was detected two days after injection. [Figure 22A] This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 RBD immunogen and a second circular RNA encoding the influenza hemagglutinin (HA) immunogen. Anti-RBD antibodies were obtained 17 days after injection. [Figure 22B] This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 RBD immunogen and a second circular RNA encoding the influenza hemagglutinin (HA) immunogen. Anti-HA antibodies were obtained 17 days after injection. [Figure 23A] This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 spike immunogen and a second circular RNA encoding the influenza hemagglutinin (HA) immunogen. Anti-RBD (spike domain) antibodies were obtained 17 days after injection. [Figure 23B] This study demonstrates the immunogenicity of multiple immunogens derived from circular RNAs in a mouse model. Mice were inoculated with a first circular RNA encoding the SARS-CoV-2 spike immunogen and a second circular RNA encoding the influenza hemagglutinin (HA) immunogen. Anti-HA antibodies were obtained 17 days after injection. [Figure 24]This study demonstrates the anti-HA antibody response in mice administered with circular RNAs encoding multiple immunogens. Mice were administered with circular RNAs encoding SARS-CoV-2 RBD immunogen, SARS-CoV-2 spike immunogen, influenza HA immunogen, SARS-CoV-2 RBD immunogen and influenza HA immunogen, SARS-CoV-2 RBD immunogen and GLuc polypeptide, or SARS-CoV-2 RBD immunogen and SARS-CoV-2 spike immunogen. Anti-influenza HA antibodies were measured using hemagglutination inhibition assays (HAIs). Figure 24 shows the HAI titers in samples administered with circular RNA preparations encoding influenza HA immunogen, either alone or in combination with SARS-CoV-2 immunogens, such as RBD or spike. [Modes for carrying out the invention]

[0084] This disclosure relates to cyclic or linear polyribonucleonucleotides encoding one or more polypeptide immunogens. This disclosure provides compositions and pharmaceutical preparations of rheotides, as well as their uses. In particular, this disclosure provides multiple A number of cyclic or linear polyribonucleotides encoding immunogens, and a plurality of cyclic or linear poly This disclosure provides an immunogenic composition containing lilibonucleotides. A pharmaceutical composition comprising one or more cyclic or linear polyribonucleotides encoding a medical The pharmaceutical preparation is further characterized by the set of cyclic or linear polyribonucleotides described herein. The compounds and pharmaceutical preparations described herein may induce an immune response in subjects upon administration. Compositions and pharmaceutical preparations of cyclic or linear polyribonucleotides are intended for use in treating diseases and disorders in the target area. It can be used to treat or prevent symptoms.

[0085] Polyribonucleotides Polyribonucleotides, in addition to one or more immunogens described herein, are as follows: The elements listed are also included. In certain embodiments, the polyribonucleotide is a cyclic polyribonucleo It's Chido.

[0086] In some embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) are used. ) consists of at least approximately 20 nucleotides, at least approximately 30 nucleotides, and at least approximately 4 0 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, less At least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides Cleotide, at least about 400 nucleotides, at least about 500 nucleotides, less At least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 0 nucleotides, at least approximately 8,000 nucleotides, at least approximately 9,000 nucleotides Otid, at least approximately 10,000 nucleotides, at least approximately 12,000 nucleotides D, at least approximately 14,000 nucleotides, at least approximately 15,000 nucleotides, At least approximately 16,000 nucleotides, at least approximately 17,000 nucleotides, at least At least approximately 18,000 nucleotides, at least approximately 19,000 nucleotides, or less It is at least about 20,000 nucleotides.

[0087] In some embodiments, polyribonucleotides (e.g., cyclic polyribonucleotides) are used. ) may be large enough to accommodate the ribosome binding site. Several implementations In this state, the maximum size of a cyclic polyribonucleotide is determined by the generation of the cyclic polyribonucleotide. and / or within the size limits of the technical constraints of using cyclic polyribonucleotides. To obtain. I don't want to be bound by any particular theory, but multiple segments of RNA "Strings" of RNA are generated from DNA, and their 5' and 3' free ends are annealed to form RNA strings. It is possible to generate " which is ultimately circularized, with one 5 free ends and one Only the 3' free end may remain. In some embodiments, the cyclic polyribonucleotide The maximum size may be limited by the ability to package RNA and deliver it to its target. In some embodiments, the size of the cyclic polyribonucleotide is such that the immunogen or so It is long enough to encode useful polypeptides such as epitopes, and therefore , at least 20,000 nucleotides, at least 15,000 nucleotides, less Each has 10,000 nucleotides, at least 7,500 nucleotides, or at least 5 ,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides Rheotide, at least 2,000 nucleotides, at least 1,000 nucleotides, small At least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides Otide, at least 200 nucleotides, at least 100 nucleotides, or at least A length of 70 nucleotides may also be useful. In some embodiments, cyclic polyribonu The maximum size of creotides is one or more (for example, two or more, three or more, four or more, and It is long enough to encode five or more immunogens. In some embodiments, a ring The maximum size of a polyribonucleotide is 2 to 5 (e.g., 3, 4, and 5). It is long enough to code the source of the epidemic.

[0088] Cyclic polyribonucleotide elements In some embodiments, the cyclic polyribonucleotide includes a sequence encoding an immunogen. In addition to the above, it includes one or more of the elements described herein. In some embodiments, Cyclic polyribonucleotides either lack a poly(A) sequence or a free 3' end. Alternatively, it may lack the RNA polymerase recognition motif, or any combination thereof. It is missing. In some embodiments, the cyclic polyribonucleotide is, by reference, its entire The body is disclosed in International Publication No. 2019 / 118919, which is incorporated herein. Includes any feature or any combination of features.

[0089] immunogen The cyclic or linear polyribonucleotides described herein encode at least one immunogen. It also contains one sequence. The immunogen is recognized by a given antibody or T cell receptor and is targeted. It includes one or more epitopes that are transformed or combined. Epitopes are linear epitopes. A tope can be, for example, a continuous sequence of nucleic acids or amino acids. An epitope is a three-dimensional structure. Epitopes, for example, amino acids that form epitopes in the folded three-dimensional structure of proteins. It may be an acid-containing epitope. The three-dimensional epitope is a discontinuity from the primary amino acid sequence. It may contain amino acids. As another example, the three-dimensional epitope may have its secondary or tertiary structure. Based on its structure, the immunogenic sequence contains nucleic acids that form epitopes in the folded three-dimensional structure. Born.

[0090] In some embodiments, the immunogen is a protein, peptide, glycoprotein, or lipoprotein. Proteins, phosphoproteins, ribonucleoproteins, carbohydrates (e.g., polysaccharides), lipids (e.g., (phospholipids or triglycerides), or all or one of nucleic acids (e.g., DNA, RNA) Includes the section.

[0091] In other embodiments, the immunogen may be a protein immunogen or an epitope (e.g., protein Peptides derived from proteins, glycoproteins, lipoproteins, phosphoproteins, or ribonucleoproteins. It contains (hydroimmunogens or peptide epitopes). Immunogens are amino acids, sugars, lipids, phosphorus It may contain a folyl group, a sulfonyl group, or a combination thereof.

[0092] In certain embodiments, the immunogen is a polypeptide immunogen.

[0093] Polypeptide immunogens undergo post-translational modifications, such as glycosylation, ubiquitination, and phosphorylation. Nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipid It may include transformation.

[0094] In one embodiment, the immunogen is at least 4, at least 5, at least 6, and at least at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or the It contains an epitope comprising the above amino acids. In one embodiment, the epitope is 4 or less. , 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less , 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less , 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less or containing 30 or fewer amino acids, or fewer amino acids. In one embodiment, The epitopes are 1, 2, 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, It contains or comprises 28, 29, or 30 amino acids. In one embodiment, epi The epitope contains 5 amino acids. In one embodiment, the epitope contains 6 amino acids. It contains amino acids. In one embodiment, the epitope contains seven amino acids. In one embodiment, the epitope contains eight amino acids. Therefore, the epitope can consist of approximately 8 to 11 amino acids. In one embodiment, the epi A tope can consist of approximately 9 to 22 amino acids.

[0095] Immunogens are immunogens recognized by B cells, immunogens recognized by T cells, or This may include combinations thereof. In one embodiment, the immunogen is recognized by B cells. It contains an immunogen. In one embodiment, the immunogen is an immunogen recognized by B cells. In one embodiment, the immunogen includes an immunogen recognized by T cells. In the context of administration, immunogens are immunogens that are recognized by T cells.

[0096] Epitopes are those recognized by B cells, immunogens recognized by T cells, and This may include combinations thereof. In one embodiment, the epitope is recognized by B cells. It includes an epitope that is recognized. In one embodiment, the epitope is recognized by B cells. It is an epitope that is recognized by T cells. It contains an epitope that is recognized by T cells. It is an epitope.

[0097] Techniques for identifying immunogens and epitopes within a computer include, for example, the following: The entirety of which is incorporated herein by reference, Sanchez-Trincado JL ,et al.(Fundamentals and methods for Ta nd B-cell epitope prediction,J.Immunol.R es.,2017:2680160.doi:10.1155 / 2017 / 268016 0(2017));Grifoni,A,et al.(A Sequence Hom ology and Bioinformatic Approach Can Pre dict Candidate Targets for Immune Respon ses to SARS-CoV-2,Cell Host Microbe,27(4 ):671-680(2020));Russi RC et al.(In sili co prediction of epitopes recognized by T cells and B cells in PmpD:First step t awards to the design of a Chlamydia trac homatis vaccine,Biomedical J.,41(2):109- 117(2018));Baruah V,et al.(Immunoinforma tics‐aided identification of T cells and B cell epitopes in the surface glycoprot ein of 2019‐nCoV, J. Med. Virol., 92(5), doi: It is disclosed in 10.1002 / jmv.25698(2020)).

[0098] In some embodiments, the immunogen comprises polynucleotides. In some embodiments, the immunogen is a polynucleotide. In some embodiments, the immunogen is RNA. In some embodiments, the immunogen is It contains DNA. In some embodiments, the immunogen is DNA. Polynucleotides are encoded as cyclic or linear polyribonucleotides.

[0099] The cyclic or linear polylibonucleotides of this disclosure include or encode various immunogens. . The cyclic or linear polyribonucleotides are at least 1, at least 2, at least 3, At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 3 0, at least 40, at least 50, at least 60, at least 70, at least 8 0, at least 90, at least 100, at least 120, at least 140, less 160, at least 180, at least 200, at least 250, at least 30 0, at least 350, at least 400, at least 450, at least 500, or Contains or codes for more immunogens.

[0100] In one embodiment, the cyclic or linear polyribonucleotide is, for example, 1 or less, 2 or more. Lower, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less , 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or so This includes or codes for the following immunogens:

[0101] In one embodiment, the cyclic or linear polyribonucleotides are approximately 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 9 0, 100, 120, 140, 160, 180, 200, 250, 300, 350, 40 Contains or codes for 0, 450, or 500 immunogens.

[0102] In some embodiments, cyclic or linear polyribonucleotides are used to coat multiple immunogens. In some embodiments, the cyclic or linear polyribonucleotide is 1 to 100 Contains or encodes an immunogen. In some embodiments, cyclic or linear polyribonucleotides. Otide contains or encodes 1 to 50 immunogens. In some embodiments, it is cyclic or Linear polyribonucleotides contain or encode 1 to 10 immunogens; for example, ring Polyribonucleotides can be 1, 2, 3, 4, 5, 6, 7, or 8 in number. , 9 or 10 immunogens are encoded. In some embodiments, cyclic or linear polyri A bonucleotide contains or encodes two immunogens. In some embodiments, a ring Polyribonucleotides, either linear or morphological, contain or encode three immunogens. In the embodiment, the cyclic or linear polylibonucleotide contains or codes for four immunogens. In some embodiments, cyclic or linear polyribonucleotides are used to produce five immunogens. To include or code.

[0103] In some embodiments, multiple immunogens each identify the same target. If so, a single target may include each of multiple immunogens, and each of multiple immunogens They may originate from the same target, and / or each of the multiple immunogens may be high in part or all of the target. They can share a degree of similarity. For example, the target may be a cell, and each immunogen may have its own characteristics. It can match the proteins of cells. For example, the target could be a specific cancer cell, and each immunogen This may match the tumor antigen associated with the cancer. Therefore, in some embodiments, Each of the multiple immunogens originates from a different protein targeting the same subject.

[0104] In some embodiments, multiple immunogens are derived from different targets. In this state, multiple immunogens can originate from various capsid proteins of a given virus. For example, one immunogen originates from orthopoxvirus. Another immunogen may be derived from hepadnavirus, and the third The immunogen may originate from a flavivirus. For example, polyribo A nucleotide can encode multiple immunogens, each immunogen being, for example, yellow fever virus, chikungun virus. Due to the Chikungunya virus, Zika, Hepatitis A, and Hepatitis B Polyribonucleotides are involved in yellow fever virus and Chikungunya fever. The following are immunodeficiencies derived from the nya virus, Zika, hepatitis A, or hepatitis B. It can encode epidemics. Polyribonucleotides can encode Japanese encephalitis, Chikungunya fever (Chiku The virus originates from Zika, hepatitis A, and hepatitis B, respectively. Polyribonucleotides can encode multiple immunogens, each immunogen. The source of the disease is SARS-CoV-2, poxvirus, respiratory syncytial virus, or human papillomavirus (HPV). It originates from the Romavirus. Polyribonucleotides are found in SARS-CoV-2 and poxu. Immunity derived from viruses, respiratory syncytial viruses, and human papillomavirus It can encode immunogens. A polyribonucleotide can encode multiple immunogens, and each immunogen can encode multiple immunogens. It originates from herpesviruses (CMV, EBV, or VZV). Polyribonucleotides are , encoding immunogens derived from herpesviruses: CMV, EBV, or VZV, respectively. It is possible. A polyribonucleotide may encode multiple immunogens, and each immunogen may... It originates from shingles (Singles) or West Nile virus. Polyribonucleotides immunogens derived from herpes zoster (shingles) and West Nile virus, respectively, It is possible.

[0105] In some embodiments, multiple immune responses are encoded by cyclic polyribonucleotides. Each of the originals shares less than 90% sequence identity.

[0106] Immunogens include, for example, viral surface proteins, viral membrane proteins, and viral envelope proteins. Rope protein, viral capsid protein, viral nucleocapsid protein , viral spike protein, viral entry protein, viral membrane fusion protein, Viral structural proteins, viral non-structural proteins, viral regulatory proteins, viruses Slip proteins, secreted viral proteins, viral polymerase proteins , viral DNA polymerase, viral RNA polymerase, viral protease, Viral glycoprotein, viral fusogen, viral helical capsid protein, virus Icosahedral capsid protein, viral matrix protein, viral replica These are derived from viruses, such as viral enzymes, viral transcription factors, or viral enzymes.

[0107] In some embodiments, the immunogen is derived from one of these viruses:

[0108] Orthomyxovirus: Useful immunogens include hemagglutinin, neuraminidase, or ma It may be derived from influenza A, B, or C viruses, such as the Trix M2 protein. If the immunogen is influenza A virus hemagglutinin, this immunogen is any Subtypes, for example, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10 It may derive from H11, H12, H13, H14, H15, or H16.

[0109] Viruses of the Paramyxoviridae family: Viral immunogens This includes, but is not limited to, pneumoviruses (for example, respiratory syncytial virus ( RSV), rubravirus (for example, mumps virus), paramyxovirus (for example) (e.g., parainfluenza virus), metapneumovirus, and morbillivirus (example) For example, measles virus, henipavirus (for example, Nipah virus) It includes elements derived from s).

[0110] Poxviridae family: Limited to viral immunogens. However, this includes, but is not limited to, smallpox viruses such as varicella and varicella. It contains substances derived from the sopoxvirus.

[0111] Picornavirus: While not limited to viral immunogens, enterovirus S, rhinovirus, heparnavirus, cardiovirus, and aftvirus, etc. This includes those derived from picornaviruses. In one embodiment, enteroviruses are Poliovirus, for example, type 1, type 2 and / or type 3 poliovirus. Another embodiment In this case, the enterovirus is the EV71 enterovirus. In another embodiment, The terrorist virus is either a Coxsackie A or B virus.

[0112] Bunyavirus: While not limited to viral immunogens, California brain Orthobunyaviruses such as inflammatory viruses, and phleboviruses such as Rift Valley fever virus. or include those derived from nairoviruses such as Crimean-Congo hemorrhagic fever virus. .

[0113] Heparnavirus: While not limited to viral immunogens, hepatitis A virus This includes substances derived from heparnaviruses such as HAV.

[0114] Filoviruses: While not limited to viral immunogens, Ebola virus ( (Including Ebola virus from Zaire, Côte d'Ivoire, Reston, or Sudan) or Mar This includes those derived from filoviruses such as Rubrug virus.

[0115] Togavirus: Viral immunogens include, but are not limited to, rubivirus, al This includes viruses derived from Togaviruses such as Favirus or Alterivirus. This contains rubella virus.

[0116] Flaviviruses: While not limited to viral immunogens, they are also responsible for tick-borne encephalitis. TBE virus, dengue (type 1, 2, 3, or 4) virus, yellow fever virus, Japanese This encephalitis virus, Kyasanur forest virus, West Nile encephalitis virus, St. Louis encephalitis This includes viruses such as Russian spring-summer encephalitis virus, Poissant encephalitis virus, and Zika virus. .

[0117] Pestivirus: While not limited to viral immunogens, bovine viruses Pestiviruses such as diarrhea (BVDV), classical swine fever (CSFV), or border disease (BDV) It includes things derived from S.

[0118] Hepadnavirus: While not limited to viral immunogens, hepatitis B virus This includes those derived from hepadnavirus, such as s. Hepatitis B virus immunogens are type B. It may be a hepatitis virus surface immunogen (HBsAg).

[0119] Other hepatitis viruses: Viral immunogens are not limited to, but include hepatitis C virus Those derived from hepatitis delta virus, hepatitis E virus, or hepatitis G virus. It is included.

[0120] Rhabdovirus: While not limited to viral immunogens, lyssavirus ( For example, rabies virus and rhabdoviruses such as becyclovirus (VSV) It includes things like that.

[0121] Caliciviridae family: Limited to viral immunogens. It is not a thing, but caliciviruses such as Norwalk virus (norovirus) (Cal The family Civiciridae, and other viruses such as Hawaiian virus and Snow Mountain virus. —Includes substances derived from walk-like viruses.

[0122] Retroviruses: While not limited to viral immunogens, they include tumor viruses and retroviruses. derived from HIV (e.g., HIV-1 or HIV-2) or Supmavirus It includes.

[0123] Reoviruses: While not limited to viral immunogens, orthoreoviruses are also included. This includes those derived from rotavirus, orbivirus, or cortivirus.

[0124] Parvovirus: While not limited to viral immunogens, parvovirus B It includes elements derived from 19.

[0125] Bocavirus: While not limited to viral immunogens, some originate from the Bocavirus. This includes things that...

[0126] Herpesvirus: While not limited to viral immunogens, human herpesvirus Virus-derived substances, for example, herpes simplex virus (HSV) (e.g., HSV type 1 and Type 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Itomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus This includes Virus 7 (HHV7) and Human Herpesvirus 8 (HHV8).

[0127] Papovavirus: While not limited to viral immunogens, papillomavirus This includes those derived from septic and polyomaviruses. (Human) papillomavirus is, Serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 4 1, 42, 47, 51, 57, 58, 63, or 65, for example, serotypes 6, 11, 16 and It could be one or more of the following:

[0128] Orthohantavirus: While not limited to viral immunogens, hantavirus It includes elements originating from Rus.

[0129] Arenavirus: While not limited to viral immunogens, guanalituvirus S, Junin virus, Lassa virus, Lujo virus, Machupo virus, Sabia virus It may contain substances derived from Rus or Whitewater Arroyo virus.

[0130] Adenovirus: The viral immunogen is adenovirus serotype 36 (Ad-36). It includes things that come.

[0131] Community-acquired respiratory virus (Tory virus): Viral immunogens include those derived from community-acquired respiratory viruses. This includes [something].

[0132] Coronavirus: While not limited to viral immunogens, SARS coronavirus Viruses (e.g., SARS-CoV-1 and SARS-CoV-2), MERS coronavirus Viruses, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and swine infectious It contains substances derived from the gastroenteritis virus (TGEV). Coronavirus immunogens are spa It may be a receptor-binding domain (RBD) of an ecpolypeptide or spike protein. Coronavirus immunogens are also known as envelope polypeptides, membrane polypeptides, or nuclei It may be an ocapsid polypeptide.

[0133] In some embodiments, the immunogen is derived from a virus that infects fish. In this embodiment, the immunogen induces an immune response to a virus that infects fish. For example, viruses that infect fish include infectious salmon anemia virus (ISAV) and salmon pancreatic disease. Virus (SPDV), Infectious Pancreatic Necrotic Virus (IPNV), Channel Catfish Virus (CCV), Fish Lymphocystis Disease Virus (FLDV), Infectious Hematopoietic Necrosis Virus (I HNV), Koi Herpesvirus, Salmon Picorna-like Virus (salmon picor (NA-like virus) (As the picoruna-like virus of Atlantic salmon) Also known as), landlocked salmon virus (LSV), Atlantic salmon rotavirus (ASR), Mass strawberry disease Virus (TSD), coccyx tumor virus (CSTV), or viral hemorrhagic sepsis Select from VHSV.

[0134] In some embodiments, the immunogen is derived from the host cell of the target. For example, a virus Antibodies that block invasion are immunogens derived from components of host cells that the virus uses as invasion factors. Alternatively, it can be generated by using an epitope.

[0135] Immunogens include, for example, bacterial surface proteins, bacterial membrane proteins, and bacterial envelope proteins. Proteins, bacterial inner membrane proteins, bacterial outer membrane proteins, bacterial periplasmic proteins, bacterial invasion Enteric proteins, bacterial membrane fusion proteins, bacterial structural proteins, bacterial non-structural proteins, bacterial proteins, bacterial polymerase proteins, bacterial DNA polymerase, bacterial RNA Polymerase, bacterial protease, bacterial glycoprotein, bacterial transcription factor, bacterial enzyme, or It originates from bacteria, such as mycotoxins.

[0136] In some embodiments, the immunogen induces an immune response from one of these bacteria: Treptococcus agalactiae )(Group B streptococcus or GB) (Also known as S) Streptococcus pyogene s) (Also called Group A Streptococcus (GAS)) To be found out; Staphylococcus aureus; Methicillin Methicillin-resistant Staphylococcus aureus (ococcus aureus) (MRSA); Staphylococcus epidermidis (Staphylococcus) *Treponema ccus epidermis* (syphilis treponema) dum); Francisella tularensis; Rickettsia ( Rickettsia genus; Yersinia pestis (plague bacterium); Neisseria meningitidis ( Neisseria meningitidis): The immune source is not limited to... But, adhesion factors, autotransporters, toxins, iron acquisition proteins, H factor binding proteins It contains membrane proteins such as chlorine; Streptococcus pneumoniae umoniae); Moraxella catarrha lis); Bordetella pertussis: Limited as an immune source. Although it is not done, pertussis toxin or toxoid (PT), fibrous hemagglutinin (F It contains HA, partactin, agglutinogens 2 and 3; Clostridium tetanus ( tetani): The typical immunogenicity is tetanus toxoid; Corynebacterium ziph Terrier (Cornynebacterium diphtheriae): Typical immune The source is diphtheria toxoid; Haemophilus influenzae (Haemophi Pseudomonas aerugino; Pseudomonas aerugino sa); Chlamydia trachomatis; Chlamydia pneumoniae; Helicobacter pylori Helicobacter pylori; Escherichia c oli)(Immunogens are not limited to, but include enterotoxigenic Escherichia coli (E. coli)) Enteroaggregative Escherichia coli (E. coli) (EAggEC), diffuse adhesive colon Bacteria (E.coli) (DAEC), Pathogenic Escherichia coli (E.coli) (EPEC), Extraintestinal Pathogenic Escherichia coli (E.coli) (ExPEC) and / or Enterohemorrhagic Escherichia coli (E.coli) (Contains immunosources derived from li)(EHEC). ExPEC strains include urinary tract pathogenic colon. E. coli (UPEC) and E. coli (MN) associated with meningitis / holemia This includes EC. Also, Bacillus anthracis; Welsh Clostridium perfringens or Clostridium botulinum Tridium botulinums; Legionella mophylla a pneumophila; Coxiella burnetii netiid; genus Brucella, e.g., B. abotus (B. abotus). B. ortus, B. canis, B. melitensis sis), B. neotomae, B. ovis, B . Switzerland (B. suis), and B. pinnipediae (B. pinnipediae); The genus Francisella, for example, F. novici da), F. philomiragia, and F. tularensis (F.tularensis); Neisseria gonorrhoeae Haemophilus ducreyi; Enterococcus faecalis Squirrel (Enterococcus faecalis) or Enterococcus faecalis Enterococcus faecium; Staphylococcus saprophyticus Staphylococcus saprophyticus; Yersinia Enterocolitica (Yersinia enterocolitica); human-type tuberculosis bacterium (Mycobacterium tuberculosis); Listeria monosite Listeria monocytogenes; Vibrio cholerae cholerae); Salmonella typhi; Borrelia bullosa Borrelia burgdorferi; Porphyromonas gin Porphyromonas gingivalis; and Klebsiella ( This includes the genus Klebsiella.

[0137] Immunogens include, for example, fungal surface proteins, fungal membrane proteins, and fungal envelope proteins. Calcium, fungal inner membrane protein, fungal outer membrane protein, fungal periplasm protein, fungal invasion Enteric proteins, fungal membrane fusion proteins, fungal structural proteins, fungal non-structural proteins, Fungal proteins, fungal polymerase proteins, fungal DNA polymerase, fungal RNA polymerase, fungal protease, fungal glycoprotein, fungal transcription factor, fungal enzyme, or fungal It originates from fungi, such as mycotoxins.

[0138] In some embodiments, the fungal immunogen is derived from dermatophytes including: Epidel Epidermophyton floccusum, Miku Microsporum audouini, Microsporum Microsporum canis, Microsporum distortum (Microsporum distortum), Microsporum equinum (Mi Crosporum equinum, Microsporum gypsum (Microspo rum gypsum), Microsporum nanum m) Trichophyton concentrium Trichophyton equinus m), Trichophyton gallinae, Trichophyton gypseum, Trichophyton Trichophyton megnini, Trichophyton Men Trichophyton mentagrophytes, Trichophyton Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton · Trichophyton schoenleini, Trichophyton Trichophyton tonsurans, Trichophyton Trichophyton verrucosum, T. verruco T. verrucosum var. album, T. discoides variety Species (var. discoides), ochraceum variety (var. ochraceum) Trichophyton violaceum, and / or Trichophyton faviforme me); or Aspergillus fumigatus Aspergillus flavus, Aspergillus (us), Aspergillus flavus, Aspergillus Aspergillus niger, Aspergillus nidula Aspergillus nidulans, Aspergillus teleus (As Aspergillus terreus, Aspergillus sidoui Aspergillus flavatus (Aspergillus flavatus) Aspergillus glaucus (avatus), Aspergillus glaucus s), Blastoschizomyces capitatus itatus), Candida albicans, Can Candida enolase, Candida tropicalis (Ca Candida tropicalis, Candida glabrata (Candida gl abrata), Candida krusei, Candida pa Candida parapsilosis (Candida stellatoi) Candida stellatoidea, Candida sylvestre Candida parakwsei, Candida parakwsei Candida lusitaniae, Candida pseudotoro Candida pseudotropicalis, Candida gilliel Candida guilliermondi, Cladosporium kallio Cladosporium carrionii, Coccidioides imitis (Coccidioides immitis), Blastomyces dermatides ( Blastomyces dermatidis, Cryptococcus neoforman Cryptococcus neoformans, Geotrichum crebatum Geotrichum clavatum, Histoplasma capsulatum (Hist Klebsiella oplasma capsulatum, Klebsiella pneumoniae (Ella pneumoniae), Microsporidia Encephalitozoon spp., Septata Septa intestinalis and enterocit Derived from Enterocytozoon bieneusi; Although not very common, the genus Brachiola (spp.) and Microspor * Microsporidium spp., Nosema sp. p.), Pleistophora spp., Trachyplas Tophora genus (Trachipleistophora spp.), Vittaforma genus (V ittaforma spp), Paracosidioides brasiliensis (Paraco ccidioides brasiliensis), Pneumocystis carinii (Pn Eumocystis carinii, Pythium insidiosum mn insidiosum, Pityrosporum ovale ovale), Saccharomyces cerevisiae Saccharomyces boular dii), Saccharomyces pombe, cedar Scedosporium apiosperum, Sporothrix schenckii, Tricos Trichosporon beigelii, Toxoplasma Toxoplasma gondii, Penicillium marnefei (P Enicillium marneffei, Malassezia genus spp.), Fonsecaea spp., Wangiera genus Giella spp.), Sporothrix genus, Basidiobolus spp., Conidiobolus genus (Co Rhizopus spp. (Nidiobolus spp.), Rhizopus genus, Mold genus (Mucor spp.), Uniced fungus genus (Absidia spp.), Kuwareka Mortierella spp., Cunninghamel Saxenaea spp., Alternaria genus (A (Iternaria spp), Curvularia genus, He Helminthosporium spp., Fusarium genus Fusarium spp), Aspergillus genus, Penicillium spp., Monolini a spp), Rhizoctonia spp, Pesylomyces genus Paecilomyces spp), Pithomyces spp , and derived from the genus Cladosporium spp.

[0139] Immunogens include, for example, eukaryotic parasite surface proteins, eukaryotic parasite membrane proteins, and eukaryotic parasites. Envelope proteins, eukaryotic parasite invasion proteins, eukaryotic parasite membrane fusion proteins, Nuclear parasite structural proteins, eukaryotic parasite non-structural proteins, secretory eukaryotic parasite proteins Quality, eukaryotic parasite polymerase protein, eukaryotic parasite DNA polymerase, eukaryotic Biological parasite RNA polymerase, eukaryotic parasite protease, eukaryotic parasite glycoprotein Derived from cereals, eukaryotic parasite transcription factors, eukaryotic parasite enzymes, or eukaryotic parasite toxins. ru.

[0140] In some embodiments, the immunogen is Plasmodium falciparum (Platypleura falciparum). Plasmodium vivax (P. vivax), Plasmodium malariae (P. malariae) e) Plasmodium such as Plasmo ovale (P. ovale), or Plasmodium It induces an immune response against parasites of the genus *Dium*. In some embodiments, the immune response is Originally, it belonged to the family Caligidae, particularly Lepeophthys. Parasites of the genera *therus* and *Caligus*, for example, *Repeoftei* Lepeophtheirus salmonis or Caligus Fish lice such as Caligus rogercresseyi To induce an immune response against which parasite. In some embodiments, the immunogen is used to induce an immune response against the parasite. Induces an immune response against Toxoplasma gondii. To emit.

[0141] In some embodiments, the immunogen is a cancer immunogen (such as a neoepitope). For example , the immunogen is used to treat acute leukemia, astrocytoma, biliary cancer (biliary cancer). Cancer (cholangiocarcinoma), bone cancer, breast cancer, brainstem glioma, tracheal cancer Broochalveolar cell lung cancer, adrenal gland cancer, anal cancer, bladder cancer, endocrine cancer, esophageal cancer, head and neck cancer, kidney cancer, Parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureteral cancer, urethral cancer, children Cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, Colorectal cancer, cutaneous melanoma, ependymoma, epidermoid, Ewing's sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme Glioma, hematological malignancies, hepatocellular carcinoma (liver carcinoma), liver cancer, Hodgkin's disease, intraocular melanoma, Kaposi's disease Sarcoma, lung cancer, lymphoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, central nervous system Neoplasms of the nervous system (CNS), neuronal carcinoma, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, Pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma (Schwanoma), skin cancer, spinal axial tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, Tumors including uterine cancer, or any refractory form of any of the above cancers, and their metastases, or any of them These are novel antigens and / or neoepitopes related to the combination.

[0142] In some embodiments, the immunogen is a tumor antigen selected from: (a) NY -Cancer testicular antigens such as ESO-1, SSX2, and SCP1, as well as RAGE, BAGE, and GA GE and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, M AGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 , and MAGE-12 (e.g., melanoma, lung, head and neck, NSCLC, breast, gastrointestinal tract, and bladder) (b) Mutant antigens, e.g., p53( For example, p21 / Ra (associated with various solid tumors such as colorectal cancer, lung cancer, and head and neck cancer) s (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CDK4 (e.g., melanoma) (related to), MUMl (e.g., related to melanoma), caspase-8 (e.g., head and neck (associated with local cancer), CIA0205 (e.g., associated with bladder cancer), HLA-A2-R1 701, β-catenin (e.g., associated with melanoma), TCR (e.g., T cell non-Hodgkin (associated with lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), Tri Ous phosphate isomerase, KIA0205, CDC-27, and LDLR-FUT; c) Overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (For example, related to Hodgkin's disease), proteinase 3 (for example, related to chronic myeloid leukemia) (related), WT1 (for example, associated with various leukemias), carbonic anhydrase (for example, related to renal cancer) (related), aldolase A (e.g., related to lung cancer), PRAME (e.g., melanoma) (related to), HER-2 / neu (for example, related to breast cancer, colon cancer, lung cancer, and ovarian cancer) (e.g., mammoglobin, alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g.) For example, (associated with colorectal cancer), gastrin (associated with pancreatic and gastric cancer), telomere Lase catalytic protein, MUC-1 (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast cancer, colon cancer), and cancer Fetal antigens (e.g., associated with cancers of the gastrointestinal tract such as breast cancer, lung cancer, and colorectal cancer); (d) co Antigens, e.g., MART-1 / Melan A, gplOO, MC1R, melanocytes Stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and Tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma) - Melanocyte differentiation antigens; (e) PAP, PSA, PSMA, etc., associated with prostate cancer. (f) Immunoglobulins (g) Lymphoidiotype (e.g., associated with myeloma or B-cell lymphoma); (g) Novel antigen. In certain embodiments, the tumor immunogenicity may include, but is not limited to, pi5, Hom / M el-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-R Human papillomavirus containing AR, Epstein-Barr virus antigen, EBNA, E6 and E7 Virus (HPV) antigen, hepatitis B and C virus antigen, human T-cell lymphotropic virus antigen, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn -23HI, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuM a, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791T gp72, β-HCG, BCA225, BTAA, CA125, CA15-3(CA27 .29YBCAA), CA195, CA242, CA-50, CAM43, CD68\K P1, CO-029, FGF-5, Ga733(EpCAM), HTgp-175, M3 44, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCA S1, SDCCAG16, TA-90 (Mac-2 binding protein cyclophyllin C-binding protein) This includes linked proteins, TAAL6, TAG72, TLP, and TPS.

[0143] In some embodiments, the immunogen induces an immune response to: pollen allergens (Allergens from trees, herbs, weeds, and grass pollen); allergens from insects or spiders (insects) Containers, saliva, and toxic allergens, such as dust mite allergens, cockroach allergens, and small insect allergens. Hymenopthera poisonous allergens; animal hair and dander allergens (For example, from dogs, cats, horses, mice, etc.); and food allergens (for example) (For example, gliadin). Important pollen allergens from trees, grasses, and herbs are limited to... However, birch (Betula), alder (Alnus), hazel (Cor) ylus), hornbeam (Carpinus) and olive (Olea), cedar (Crypto) Taxonomy including meria and Juniperus, and plane trees (Platanus) The above are the Fagales, Oleales, and Pinales orders. s), and the family Platanaceae, genus Lolium, Phleum, Poa, Cynodon, Dak Dactylis, Holcus, Phalaris Grasses of the genera *Laris*, *Secale*, and *Sorghum* Includes the order Poales, genus Ambrosia, genus Artemisia. Asterales (As) including herbs of the genus Parieta (emisia), and Parieta Derived from terales and Urticales. Other important absorbents The allergens present are house dust mites (Dermatophagoides) and Eurogly. Derived from house dust mites of the genus Euroglyphus, storage mites, for example, Lepido Genus Lepidoglyphys, Genus Glycyphagus (us), and things of the genus Tyrophagus, cockroaches, small insects , and fleas, for example, the German cockroach (Blatella), the American cockroach (P eriplaneta), the genus Chironomidae (Chironomus), and the genus Ct Derived from enocepphalides, found in mammals such as cats, dogs, and horses. Sources include honeybees (Apidae), wasps (Vespidea), and ants. The taxonomic order Hymenoptera, which includes Formicoidae Contains toxic allergens, including those derived from insects that sting or bite. .

[0144] In some embodiments, the immunogen is, for example, snakes (e.g., most species of rattles) Snakes (for example, the Eastern Diamondback Rattlesnake), brown snake species (for example, the King Brown Rattlesnake) Snakes and Eastern brown snakes), Russell's vipers, cobras (for example, in (Docobra, King Cobra), certain types of kraits (for example, the common krait) ), Mamba (e.g., Black Mamba), Carpet Viper, Boomslang, Dubo The spiny sea snake (Dubois sea snake), taipan species (for example, coastal taipan) Coastal Taipan and Inland Taipan n) Snakes, lancehead species (e.g., feldlance and tel) Siopero, Bushmaster, Copperhead, Cottonmouth, Coral Snake, Death Snake Dar, Belcher's sea snake, Tiger snake, Australian black snake (Au (Stralalian black snake), spiders (for example, black recluse spiders, black spiders) Widow spider, black wandering spider (Brazilian wandering spider) Funnel-web spider, button spider (on spider), Australian redback spider, Katipo, Ni false black widow, Chilea (n recluse spider), mouse spider, jo Macrothele species, Sicarius species, Hexa Hepthalma species (specific species of tarantulas), scorpions and other arachnids (examples) For example, the fat-tailed scorpion, Death Deathstalker scorpion, India Indian red scorpion, Centriloides Brazilian yellow scorpion (Brazilian ye) Tityus species (such as the small scorpion), insects (for example) honeybee species, wasp species, certain ants such as fire ants, some species of lepidopteran caterpillars, centipedes A specific species of this species, Remipedo xibarbanus turmensis. albanus tulumensis), fish (for example, a specific species of catfish (for example) , striped eel catfish and other members of the Eel family (ee (Little tail catfish), certain species of stingrays (e.g., blue spotted stingray), minnow Scorpionfish, stonefish, scorpionfish, pufferfish, rabbitfish, goblinfish, blacktip reef fish Stonefish, striped blenny, yellowfin seabream rgazer), Chimera, Weever, Tsunoza (dogfish shark), cnidarians (for example, certain species of jellyfish (for example, Irukandji jellyfish and box jellyfish), hydrozoans (for example, Portuguese man-of-war (Portu Guess Man o' War), sea anemones, certain species of coral, lizards ( For example, the American beaded lizard (gila monster), the Mexican bearded dragon Ge (Mexican bearded lizard), Varanus Certain species (e.g., Komodo dragon), Perentie monitor lizard , and lace monitor), mammals (e.g., Minamipla Southern short-tailed shrew, duck Leaf shrike, European mole, water shrew (ew), Mediterranean water shrew w), Planari shrew (Northern short-tailed shrew) w), Elliot's short-tailed shrew Shrew), specific species of solenodon (e.g., Cuban solenodon, Haitian solenodon) ), slow loris), mollusks (e.g., certain species of cone snails), cephalopods (e.g., octopuses) Certain species (for example, the blue-ringed octopus, i (Cucumbers and cuttlefish), amphibians (e.g., poison dart frogs, blue-spotted frogs, and squirrels) Frogs such as Greening's frog, salamanders ( For example, the fire salamander and the Iberian thorn. The toxins in the venomous fluids of the Iberian ribbed newt, etc. To come.

[0145] In some embodiments, the toxin is derived from a plant or fungus (e.g., a mushroom).

[0146] In some embodiments, the toxin immunogen is cyanotoxin, dinotoxin (dinot oxins, myotoxins, cytotoxins (e.g., lysine, apitoxins, mycotoxins) (For example, aflatoxin), ochratoxin, citrinin, ergot alkaloids, patricide (Fusarium, fumonisin, trichothecene, cardiotoxin), tetrodotoxin batrachotoxin, botulinum toxin A, tetanus toxin A, diphtheria toxin, dioxin muscarine, buphotoxin, sarin, hematotoxins, phototoxins, necrotizing toxins, nephrotoxins, and neurotoxins (e.g., calciceptin, cobrotoxin, calcicurdin, fasciculin) -I (derived from toxins such as cariotoxin)

[0147] Any number of immunogens derived from microorganisms or cancer cells can be used in the form of cyclic or linear polyribonucleotides. It is possible. In some cases, the immunogen is related to one of the microorganisms disclosed above. or expressed thereby. In some embodiments, the immunogen is disclosed above. It is associated with or expressed by two or more microorganisms. In some cases, it is immunosuppressant. The epidemic is associated with or expressed by one of the cancers disclosed above. In that embodiment, the immunogen is associated with or related to two or more of the cancers disclosed above. It is expressed by. In some embodiments, the immunogen is derived from the toxin disclosed above. In some embodiments, the immunogen is derived from two or more toxins disclosed above. .

[0148] Two or more microorganisms may or may not be related. In some cases, two or more. The microorganisms are phylogenetically related. For example, the cyclic or linear polyribonucleotides of the present disclosure Ochido is two or more viruses, two or more members of a virus family, virus k Two or more members of the LASS family, two or more members of the Virus family, two or more members of the Virus genus Members of a virus species, two or more members of a virus species, and immunogens derived from two or more bacterial pathogens Includes or codes for these. In some embodiments, two or more microorganisms are phylogenetic. They are not directly related.

[0149] In some cases, two or more microorganisms are phenotypically related. For example, the ring of the present disclosure Polyribonucleotides in a linear or linear form contain two or more respiratory pathogens, two or more selective agents, and heavy Two or more microorganisms associated with the disease, or two or more associated with adverse outcomes in immunocompromised subjects. Microorganisms, two or more microorganisms associated with adverse outcomes related to pregnancy, two associated with hemorrhagic fever This includes or encodes immunogens derived from the above microorganisms.

[0150] The immunogens in this disclosure may include wild-type sequences. The term "wild-type" is used when referring to immunogens. These are natural and sequences encoded by genomes (e.g., viral genomes) (e.g., This refers to nucleic acid sequences or amino acid sequences. A certain species (for example, a microbial species) has one wild-type variant. A column, or two or more wild-type sequences (e.g., one standard wild-type sequence present in a reference microbial genome) It may have a type sequence and a wild-type sequence of further mutants arising from mutations. ru.

[0151] When referring to an immunogen, the terms "derivative" and "derived from" refer to one or more nucleic acids. Alternatively, the amino acids may differ from the wild-type sequence, for example, by one or more amino acid insertions compared to the wild-type sequence. This refers to sequences (e.g., nucleic acid sequences or amino acid sequences) that contain additions, deletions, and / or substitutions. .

[0152] The immunogen derivative sequence is a wild-type sequence, for example, wild-type nucleic acid, protein, immunogen, or E For the pitope sequence, at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any of the above. The sequences described above possess sequence identity.

[0153] In one embodiment, an immunogen is one that affects the structure of the encoded protein. The above amino acid insertions, deletions, substitutions, or combinations thereof are included. Therefore, an immunogen is a one or more amino acid insertion that affects the function of the encoded protein. It contains deletions, substitutions, or combinations thereof. In one embodiment, the immunogen is used in cells. Therefore, one or more amino acids that affect the expression or processing of the encoded protein. It contains acid insertion, deletion, substitution, or a combination thereof.

[0154] In some embodiments, the immunogen affects the structure of the encoded immunogenic nucleic acid. This includes insertions, deletions, substitutions, or combinations thereof of one or more nucleic acids.

[0155] Amino acid insertions, deletions, substitutions, or combinations thereof introduce sites for post-translational modification. Obtain (e.g., glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetate) Chillation, amidation, hydroxylation, sulfation, or lipidation sites, or targeted for cleavage (Introducing a sequence). In one embodiment, amino acid insertions, deletions, substitutions, or the same The combination removes sites for post-translational modification (e.g., glycosylation, ubiquitination, Phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipid (Removes the target site or sequence to be cut). In one embodiment, Acid insertions, deletions, substitutions, or combinations thereof modify sites for post-translational modification (e.g.) For example, glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, Modify the amidation, hydroxylation, sulfation, or lipidation sites, or the efficiency or properties of cleavage. (Modifies the sea urchin part).

[0156] Amino acid substitutions can be conservative or non-conservative. Conservative amino acid substitutions are similar to One amino acid substitute for another amino acid with biochemical properties (e.g., charge, size, and / or hydrophobicity) It can be a substitution of one amino acid. Non-conservative amino acid substitutions result in different biochemical properties (e.g., One amino acid in place of another amino acid having charge, size, and / or hydrophobicity Substitution is possible. Conservative amino acid changes, for example, affect the secondary or tertiary structure of a polypeptide. This can be a substitution with minimal impact. A conservative amino acid change is one hydrophilic amino acid. This could be an amino acid change from one to another hydrophilic amino acid. Hydrophilic amino acids are Thr(T) , Ser(S), His(H), Glu(E), Asn(N), Gln(Q), Asp( D), may contain Lys(K) and Arg(R). Conservative amino acid changes include one hydrophobic component. This can be an amino acid change from one amino acid to another hydrophilic amino acid. Hydrophobic amino acids are Il e(I), Phe(F), Val(V), Leu(L), Trp(W), Met(M), May contain Ala(A), Gly(G), Tyr(Y), and Pro(P). Conservative Amino Acids An acid change can be an amino acid change from one acidic amino acid to another. Sexual amino acids may include Glu(E) and Asp(D). There is one conservative amino acid change. This can be an amino acid change from one basic amino acid to another. Basic amino acids It may contain His(H), Arg(R), and Lys(K). Conservative amino acid changes are, This can be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids are It may include Asn(N), Gln(Q), Ser(S), and Thr(T). Conservative amine A noacid change can be an amino acid change from one nonpolar amino acid to another. Nonpolar amino acids include Leu(L), Val(V), Ile(I), Met(M), and Gl. It may contain y(G) and Ala(A). Conservative amino acid changes are one aromatic amino acid or This could be an amino acid change to another aromatic amino acid. Aromatic amino acids include Phe(F), It may contain Tyr(Y) and Trp(W). The conservative amino acid change is one aliphatic amino acid. This can be an amino acid change from an acid to another aliphatic amino acid. Aliphatic amino acids include Ala(A ), may include Val(V), Leu(L), and Ile(I). In one embodiment, Conservative amino acid substitutions belong to the following groups: Group I: ala, pro, gly, gln , asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; group IV: lys, a rg, his; Group V: phe, tyr, trp, his; and Group VI: as In p and glu, one of the amino acids is changed from one amino acid to another. ru.

[0157] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), at least 1, at least 2, and at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, less At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, less 40, at least 45, at least 50, at least 60, at least 70, less Each contains 80, at least 90, or at least 100 amino acid deletions.

[0158] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), at least 1, at least 2, and at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, less At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, less Each contains 40, at least 45, or at least 50 amino acid substitutions.

[0159] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), values ​​of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less Lower, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 Below, 15 or below, 16 or below, 17 or below, 18 or below, 19 or below, 20 or below, 25 or below, 30 The following include amino acid substitutions of 35 or less, 40 or less, 45 or less, or 50 or less.

[0160] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), 1-2, 1-3, 1-4, 1-5, 1- 6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2 ~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 2~15, 2~2 0, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9 , 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or It contains 20 to 25 amino acid substitutions.

[0161] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids Includes substitution.

[0162] One or more amino acid substitutions are at the N-terminus or C-terminus of an amino acid sequence, or a combination thereof. Yes, it's possible. Amino acid substitutions can be continuous, discontinuous, or a combination of both.

[0163] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is described herein. Compared to the given sequence (e.g., wild-type sequence), values ​​of 1 or less, 2 or less, 3 or less, 4 or less, 5 or less Lower, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 Below, 15 or below, 16 or below, 17 or below, 18 or below, 19 or below, 20 or below, 25 or below, 30 Below, 35 or below, 40 or below, 45 or below, 50 or below, 60 or below, 70 or below, 80 or below, 90 Below: 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, or 200 or less. It includes amino acid deficiencies.

[0164] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is a wild-type sequence and Compared to 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 , 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7 , 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3- 4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-3 0, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5- 30, 5-40, 10-15, 15-20, 20-25, 20-30, 30-50, 50 Contains approximately 100, or 100-200, amino acid deletions.

[0165] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is a wild-type sequence and Compared to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Contains 16, 17, 18, 19, or 20 amino acid deletions.

[0166] One or more amino acid deletions occur at the N-terminus, C-terminus, or a combination thereof within an amino acid sequence. Yes, it's possible. Amino acid deletions can be continuous, discontinuous, or a combination of both.

[0167] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is a wild-type sequence and Compared to, at least 1, at least 2, at least 3, at least 4, at least 5, less At least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or fewer Both contain 50 amino acid insertions.

[0168] In one embodiment, the immunogen derivative or epitope derivative of the present disclosure is a wild-type sequence and Compared to 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 25 or less, 30 or less, 35 or less, 40 or less, 45 or less, or including insertion of 50 or fewer amino acids.

[0169] In one embodiment, the antigen derivative or epitope derivative of the Disclosure is compared to the wild-type sequence. All, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4 , 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-3 Contains 0, 5-40, 10-15, 15-20, or 20-25 amino acid insertions.

[0170] In one embodiment, the antigen derivative or epitope derivative of the Disclosure is compared to the wild-type sequence. All, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 Includes 6, 17, 18, 19, or 20 amino acid insertions.

[0171] One or more amino acid insertions occur at the N-terminus, C-terminus, or a combination thereof within an amino acid sequence. Yes, it's possible. Amino acid insertions can be continuous, discontinuous, or a combination of both.

[0172] In some embodiments, the immunogen is expressed by cyclic or linear polyribonucleotides. In some embodiments, the immunogen is a row of cyclic or linear polyribonucleotides. It is a product of ring-circle amplification.

[0173] Immunogens can be produced in considerable quantities. Therefore, immunogens can be produced It can be any protein molecule that can do this. The immunogen is a polypeptide that can be secreted from cells. It may be a polypeptide that can be localized in the cytoplasm, nucleus, or membrane fraction of a cell. In some embodiments, the cyclic or linear polyribonucleotides of the Disclosure encode the The polypeptide comprises a fusion protein containing two or more immunogens disclosed herein. In some embodiments, the cyclic or linear polyribonucleotides of the present disclosure are used to coat The polypeptide being synthesized includes an epitope. In some embodiments, the cyclic or Polypeptides encoded by linear polyribonucleotides are disclosed herein. A fusion protein comprising two or more epitopes, for example, one or more microorganisms of the present disclosure. Contains an artificial peptide sequence that includes multiple predicted epitopes from [source].

[0174] In some embodiments, immunogens can be expressed from cyclic or linear polyribonucleotides. These are membrane proteins, and for example, polypeptides commonly found as membrane proteins. Includes sequences, or polypeptide sequences modified to become membrane proteins. In terms of application, it can be expressed from cyclic or linear polyribonucleotides as disclosed herein. Exemplary immunogens include intracellular immunogens or cytoplasmic immunogens.

[0175] In some embodiments, the immunogen has an amino acid length of less than approximately 40,000, and approximately 35,000 Amino acid length less than 0, amino acid length less than approximately 30,000, amino acids less than approximately 25,000 Length, amino acid length less than approximately 20,000, amino acid length less than approximately 15,000, approximately 10,000 Amino acid length less than 0, amino acid length less than approximately 9,000, amino acid length less than approximately 8,000, Amino acid length less than approximately 7,000, amino acid length less than approximately 6,000, and less than approximately 5,000 amino acid length, amino acid length less than approximately 4,000, amino acid length less than approximately 3,000, approximately 2,50 Amino acid length less than 0, amino acid length less than approximately 2,000, amino acid length less than approximately 1,500, Amino acid length less than approximately 1,000, amino acid length less than approximately 900, amino acid length less than approximately 800 , amino acid length less than approximately 700, amino acid length less than approximately 600, amino acid length less than approximately 500, Amino acid length less than approximately 400, amino acid length less than approximately 300, amino acid length less than approximately 250, approximately Amino acid length less than 200, amino acid length less than approximately 150, amino acid length less than approximately 140, approximately 1 Amino acid length less than 30, amino acid length less than approximately 120, amino acid length less than approximately 110, approximately 10 Amino acid length less than 0, amino acid length less than approximately 90, amino acid length less than approximately 80, less than approximately 70 amino acid length, amino acid length less than approximately 60, amino acid length less than approximately 50, amino acid length less than approximately 40 Length, amino acid length less than approximately 30, amino acid length less than approximately 25, amino acid length less than approximately 20, approximately 1 Having an amino acid length of less than 5, an amino acid length of less than approximately 10, and an amino acid length of less than approximately 5, Any amino acid length between these two amino acids, or less, may be useful.

[0176] In some embodiments, cyclic or linear polyribonucleotides are used to make one or more immune It contains the original sequence and is configured for sustained expression in target cells in vivo. In some embodiments, cyclic or linear polyribonucleotides are used in cells at a later point in time. The expression of one or more expression sequences within the molecule is configured to be equal to or higher than that at the previous time point. In such embodiments, the expression of one or more immunogen sequences is made relatively stable. The expression of immunogen sequences can be maintained at a certain level or increased over time. It can be made relatively stable over a period of time. The expression of the immunogen sequence is transient or limited For the duration specified, for example, at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, It can be kept relatively stable for 7, 8, 9, or 10 days.

[0177] In some embodiments, cyclic or linear polyribonucleotides are, for example, transient or long Periodically, one or more immunogens are expressed in the target. In a particular embodiment, the immunogen The onset of symptoms can range from at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, and 12 hours. Intervals: 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 1 7 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 2 5 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer It can also last for a long time, any duration between them. In certain embodiments, immunogen expression lasts for about 3 From 0 minutes to approximately 7 days, or within approximately 1 hour, 2 hours, 3 hours, or 4 hours. Within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, 11 hours Within hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, 1 Within 7 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours Within 24 hours, within 36 hours, within 48 hours, within 60 hours, within 72 hours, within 4 days Within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, 1 Within 1 day, within 12 days, within 13 days, within 14 days, within 15 days, within 16 days Within 17 days, within 18 days, within 19 days, within 20 days, within 21 days, within 22 days Within 23 days, within 24 days, within 25 days, within 26 days, within 27 days, 28 Within a certain number of days, within 29 days, within 30 days, within 60 days, or any time in between. To be continued.

[0178] The expression of immunogens is at least the cyclic or linear polyribonucleotides provided herein. This includes translating regions of a cyclic or linear polyribonucleotide. Translate the target to produce a polypeptide containing one or more immunogens, and This stimulates the production of an adaptive immune response (e.g., antibody response and / or T cell response) in the target. It can be stimulated. In some embodiments, the cyclic or linear polyribonucleotides of the present disclosure Translate the cydo to produce one or more immunogens in human or animal subjects, and This leads to adaptive immune responses in humans or animals (e.g., antibody responses and / or T-cell responses). It stimulates the production of ).

[0179] In some embodiments, the method for immunogenic expression is a cyclic or linear polyribonucleo At least 10%, at least 20%, at least 30%, and at least 40% of the total length of the Chido %, at least 50%, at least 60%, at least 70%, at least 80%, less Includes translation into polypeptides of at least 90%, or at least 95%. Several implementations In this state, the method for immunogenic expression is to use cyclic or linear polyribonucleotides, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids Acid, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, At least 200 amino acids, at least 250 amino acids, at least 300 amino acids, small At least 400 amino acids, at least 500 amino acids, at least 600 amino acids, less At least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or a small amount This includes translation into a polypeptide of at least 1000 amino acids. In some embodiments, The method for protein expression involves cyclic or linear polyribonucleotides, approximately 5 amino acids, approximately 10 amino acids, approximately 15 amino acids, approximately 20 amino acids, approximately 50 amino acids, approximately 100 amino acids, Approximately 150 amino acids, approximately 200 amino acids, approximately 250 amino acids, approximately 300 amino acids, approximately 400 Amino acids, approximately 500 amino acids, approximately 600 amino acids, approximately 700 amino acids, approximately 800 amino acids This includes translation into polypeptides of approximately 900 amino acids or approximately 1000 amino acids. In this embodiment, the method provides for cyclic or linear polyribonucleotides as provided herein. A continuous polypeptide, separate polypeptides as provided herein, or both Includes translation to.

[0180] In some embodiments, methods for immunogenic expression involve modification of the translation product, Foldy This includes ing or other post-translational modifications. In some embodiments, the method for immunogenic expression is This includes, for example, post-translational modifications mediated by cellular mechanisms in vivo.

[0181] signal sequence In some embodiments, the cyclic or linear polyribonucleotides disclosed herein are Exemplary immunogens that can be expressed include secreted proteins, for example, those with naturally occurring signal sequences. Proteins containing (e.g., immunogens), or those that do not normally encode a signal sequence, but one Examples of those modified to include include annular or linear polyri Immunogens encoded by bonucleotides include secretory signals. For example, secretory signals Naru may be a naturally encoded secretory signal in secretory proteins. In this example, the secretory signal may be a modified secretory signal in a secretory protein. i. In other embodiments, immunogens encoded by cyclic or linear polyribonucleotides It does not include secretory signals.

[0182] In some embodiments, cyclic or linear polyribonucleotides are multiple immunonucleotides of the same immunogen. Copies of the same immunogen (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9) It encodes copies (10, 10, or more). In some embodiments, a small number of immunogens At least one copy contains the signal sequence, and at least one copy of the immunogen contains the signal sequence. It does not contain a gnall sequence. In some embodiments, cyclic or linear polyribonucleotides are , multiple immunogens (e.g., multiple different immunogens or multiple immunogens with less than 100% sequence identity) It codes for a number of immunogens, where at least one of the multiple immunogens contains a signal sequence. At least one copy of the multiple immunogens does not contain the signal sequence.

[0183] In some embodiments, the signal sequence, for example, when endogenously expressed, is wild-type. These are signal sequences, located at the N-terminus of the corresponding wild-type immunogen. In this embodiment, the signal sequence is heterogeneous to the immunogen, for example, the wild-type immunogen When expressed endogenously, it is absent. The polyribonucleotide sequence encoding the immunogen is , remove the nucleotide sequence encoding the wild-type signal sequence and / or the heterogeneous signal It may be modified to include an array that encodes the array.

[0184] Immunogens encoded by polyribonucleotides are signals that guide them into the secretory pathway. The signal sequence may also be included. In some embodiments, the signal sequence is used to target immunogens to specific cells. It may be guided to reside in organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes). In some embodiments, the signal sequence guides the immunogen to be secreted from the cell. In the case of secreted proteins, the signal sequence is cleaved after secretion, resulting in the maturation of the protein. In other embodiments, the signal sequence may be embedded within the membrane of a cell or a specific organelle. Transmembrane segmentation allows proteins to be fixed to the membranes of cells, endoplasmic reticulum, or Golgi apparatus. A ment can be created. In certain embodiments, the signal sequence of a transmembrane protein is a polyp This is a short sequence at the N-terminus of the plutide. In other embodiments, the first transmembrane domain is a protein It acts as the first signal sequence, targeting the crystalline structure to the membrane.

[0185] In some embodiments, the immunogen encoded by polyribonucleotides is secreted. Includes a signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence. It is one of them.

[0186] Adjustment element Cyclic polyribonucleotides or linear polyribonucleotides are regulatory elements, for example, cyclic polyribonucleotides. or includes sequences that regulate the expression of expression sequences within linear polyribonucleotides. The regulatory elements are It may include sequences located adjacent to the expression sequence encoding the expression product. The regulatory elements are adjacent to It can be operablely linked to the sequence. The regulatory element is expressed when the regulatory element is not present. The amount of the product expressed can be increased compared to the amount of the product. Using a certain regulatory element, cyclic Alternatively, increase the expression of one or more immunogens encoded by linear polyribonucleotides. It can be done. Similarly, using a certain regulatory element, cyclic or linear polyribonucleotides can be made to The expression of one or more immunogens encoded by can be reduced. In that embodiment, the expression of an immunogen can be increased using a certain regulatory element, and Using a different regulatory element, the development of a different immunogen against the same cyclic or linear polyribonucleotide. The expression can be reduced. Furthermore, one regulatory element can be linked to multiple expressions in parallel. It may increase the amount of the product expressed for the sequence (e.g., immunogen). Therefore, one The regulatory elements can promote the expression of one or more expression sequences (e.g., immunogens). Multiple regulatory elements can also be used, for example, to regulate the expression of different expression sequences in different ways. Obtain. In one embodiment, the modulatory element provided herein is a selective translation sequence. This may include. As used herein, the term "selective translation sequence" means a ring or These are linear polyribonucleotides, for example, expression sequences in specific riboswitch aptazymes. This refers to nucleic acid sequences that selectively initiate or activate translation. Regulatory elements are selective decomposition and distribution. It may also include columns. As used herein, the term “selective decomposition sequence” means a ring. Alternatively, the expression of linear polyribonucleotides, or cyclic or linear polyribonucleotides. This refers to a nucleic acid sequence that initiates the degradation of the product. In one embodiment, the regulatory element is a translation module. It is a translation modulator. A translation modulator translates the expression sequence in a cyclic polyribonucleotide. It can be adjusted. A translation modulator can be a translation enhancer or suppressor. In some embodiments, the translation initiation sequence may function as a modifier. Further examples of modifiers However, the entirety of International Publication No. 2019 / 118919 is incorporated herein by reference. This is described in Lett's paragraphs

[0154] to

[0161] .

[0187] A codon that initiates translation, for example, but not limited to, a start codon or an alternative start codon Nucleotides adjacent to a donor affect the translation efficiency and length of cyclic or linear polyribonucleotides. It is known that this affects the structure (e.g., Matsuda and See Mauro PLoS ONE, 2010 5:11; the full content is as follows: (The body is incorporated herein by reference). Nucleo adjacent to the codon that initiates translation. By masking one of the nucleotides, translational development of cyclic or linear polyribonucleotides can be performed. The starting position, translation efficiency, length, and / or structure may be changed.

[0188] In one embodiment, the codon is masked or hidden, and the masked start codon or an alternative is used. To reduce the probability of translation initiation at a start codon, a masking agent is used at the start codon or an alternative. It may be used near the start codon. In another embodiment, translation is opened with an alternative start codon. To increase the likelihood of initiation, masking agents are used to process cyclic or linear polyribonucleotides. You may mask the start codon.

[0189] Translation start sequence In some embodiments, cyclic or linear polyribonucleotides encode immunogens. A translation initiation sequence, for example, including a start codon. In some embodiments, the translation initiation sequence is The sequence includes a Kossack or Shine-Dalgarno sequence. In some embodiments, the translation initiation sequence is , including a Kosack sequence. In one embodiment, the translation initiation sequence is Kosack or Shine • Contains a Dalgan sequence. In one embodiment, a cyclic or linear polyribonucleotide is The expression sequence includes a translation initiation sequence adjacent to it, such as a Kozak sequence. In one embodiment, The translation start sequence is a non-coding start codon. In one embodiment, the translation start sequence, For example, the Kozak sequence is present on one or both sides of each expression sequence to separate the expression product. In one embodiment, a cyclic or linear polyribonucleotide is placed adjacent to the expression sequence. It includes at least one adjacent translation initiation sequence. In one embodiment, the translation initiation sequence is To impart conformational flexibility to cyclic or linear polyribonucleotides. In one embodiment, The translation initiation sequence is located within a substantially single-stranded region of a cyclic polyribonucleotide. Further examples of columns are provided in International Publication No. 2019 / 118, which is incorporated herein by reference in its entirety. It is described in paragraphs

[0163] to

[0165] of pamphlet No. 919.

[0190] Cyclic or linear polyribonucleotides, but not limited to, at least 2, at 3, at least 4, at least 5, at least 6, at least 7, at least 8, less 9, at least 10, at least 11, at least 12, at least 13, and at least 14, at least 15, at least 16, at least 17, at least 18, and at least 19, at least 20, at least 25, at least 30, at least 35, and at least Two or more start codons, such as 40, at least 50, at least 60, or more than 60 It may include a start codon. The translation may start with the first start codon, or the first start codon It can start downstream of the codon.

[0191] In some embodiments, a cyclic or linear polyribonucleotide is a first start codon, For example, it may start with a codon other than AUG. Translation of cyclic or linear polyribonucleotides , International Patent Publication No. 2019 / 118919A1 pamphlet (the entire pamphlet is available for reference) Alternative translation initiation sequences as described in

[0164] (as incorporated herein by reference) It can start with this.

[0192] In some embodiments, translation involves eukaryotic initiation factor 4A (eIF) with locagreate. The process is initiated by 4A) (translation is interrupted by 43S scanning and RocA-e Immature upstream translation initiation and decreased protein expression from transcripts containing the IF4A target sequence. It is suppressed by causing, for example, www.nature.com / art (See icles / nature17978).

[0193] IRES In some embodiments, the cyclic or linear polyribonucleotides described herein are internal It includes a ribosome entry site (IRES) element. In some embodiments, as described herein The cyclic or linear polyribonucleotides listed are two or more (for example, 2, 3, 4, and 5) It includes an internal ribosome entry site (IRES) element. In some embodiments, it is circular or Linear polyribonucleotides have one or more IRES sequences on one or both sides of each expression sequence. The inclusion of several causes separation of the resulting peptides and / or polypeptides. In this embodiment, IRES is at least one (e.g., two, three, four, five or more) It is adjacent to both sides of the expression sequence of ). Suitable for inclusion within circular or linear polyribonucleotides. These IRES elements may be RNA sequences capable of associating with eukaryotic ribosomes. In some embodiments, IRES is encephalomyelitis virus (encephalomyoc It is arditis virus (EMCV)IRES. In some embodiments, IRES stands for Coxsackievirus (CVB3) IR This is ES. A further example of IRES is International Patent Publication No. 2019 / 11891. This is described in paragraphs

[0166] to

[0168] of Pamphlet No. 9 (the entirety of which is incorporated herein by reference).

[0194] Disconnected domain The cyclic or linear polyribonucleotides of this disclosure have cleavage domains (e.g., stagger elements). It may include (or a cleaved sequence).

[0195] The term "stagger element" refers to a nucleotide that induces ribosome pause during translation. This refers to parts such as arrays. In one embodiment, staggered elements have a strong alpha helix tendency. The non-conserved amino acid sequence, followed by the consensus sequence -D(V / I)ExNPGP(here) So x = any amino acid) (SEQ ID NO: 7). In one embodiment, staga - The elements are glycerol, non-nucleic acid linkage moieties, chemical modifications, modified nucleic acids, or any combination thereof. It may include chemical components such as compounding.

[0196] In one embodiment, a cyclic or linear polyribonucleotide is adjacent to the expression sequence. It contains at least one staggered element. In one embodiment, a cyclic or linear polyribonucle The rheotide contains stagger elements adjacent to each expression sequence. In one embodiment, stagger - The element is present on one or both sides of each expression sequence, and is used to isolate the expression product, e.g., immunogen. This results in the following: In one embodiment, the stagger element is part of one or more expression sequences. In one embodiment, a cyclic or linear polyribonucleotide is expressed in one or more expression sequences ( For example, an immunogen containing one or more expression sequences, each of which is a cyclic or linear polyribonucleated The stagger element in rheotide separates it from subsequent expression sequences (e.g., immunogen). In one embodiment, the stagger element is derived from (a) two translations of a single expression sequence. or (b) prevent the generation of a single polypeptide from one or more translations of two or more expression sequences. In one embodiment, the stagger element is a compound separated from one or more expression sequences. It is a column. In one embodiment, the stagger element is an expression sequence among one or more expression sequences. Includes part of the column.

[0197] An example of a stagger element is cited herein by reference in its entirety in International Publication No. 2019 / 1 It is described in paragraphs

[0172] to

[0175] of pamphlet No. 18919.

[0198] In some embodiments, multiple encodings by cyclic or linear ribonucleotides Immunogens may be separated by IRES between each immunogen. IRES is used to separate all immunogens The same IRES may be used between different immunogens. The IRES may differ between different immunogens. In this embodiment, multiple immunogens may be separated by 2A autocleaved peptides. Furthermore, multiple immunogens encoded by cyclic or linear ribonucleotides are IRES and The 2A sequence may be separated by both the first immunogen and the second immunogen. For example, IRES may be separated by the first immunogen and the second immunogen. While the 2A peptide may be present between the second and third immunogens, It may be present in between. Using a selection of a specific IRES or 2A self-cleaving peptide, IRE The expression level of the immunogen may be controlled under the control of the S or 2A sequence. For example, the selected I Even if expression in polypeptides is enhanced or reduced depending on RES and / or 2A peptide good.

[0199] Maintaining rolling circle translation while avoiding the production of continuous expression products, such as immunogens. To achieve this, stagger elements may be included to induce ribosome arrest during translation. In some embodiments, the stagger element is at least one of one or more expression sequences It is located at the 3' end. The stagger element is a rolling element of a cyclic or linear polyribonucleotide. It can be designed to halt ribosomes during translation. The stagger element is not limited to However, there is no 2A-like or CHYSEL (SEQ ID NO: 8) (cis-acting hydrolase element). ) may include an array. In some embodiments, the staggered elements are X1X2X3EX5 NPGP (wherein X1 is absent or G or H, and X2 is absent or D or G) X3 is D, V, I, S, or M, and X5 is any amino acid (sequence number) It encodes a sequence having a C-terminal consensus sequence which is number 9). In the application form, this sequence consists of a non-conserved amino acid sequence with strong α-helical properties, followed by... Consensus sequence -D(V / I)ExNPGP(where x = any amino acid (SEQ ID NO)) 7) includes ). Some non-restrictive examples of stagger elements include GDVESNPGP( SEQ ID NO: 10), GDIEENPGP (SEQ ID NO: 11), VEPNPGP (SEQ ID NO: 12) ), IETNPGP (SEQ ID NO: 13), GDIESNPGP (SEQ ID NO: 14), GDVE LNPGP (SEQ ID NO: 15), GDIETNPGP (SEQ ID NO: 16), GDVENPGP (Sequence number 17), GDVEENPGP (Sequence number 18), GDVEQNPGP (Sequence number 18) No. 19), IESNPGP (Sequence ID 20), GDIELNPGP (Sequence ID 21), H DIETNPGP (SEQ ID NO: 22), HDVETNPGP (SEQ ID NO: 23), HDVEM NPGP (SEQ ID NO: 24), GDMESNPGP (SEQ ID NO: 25), GDVETNPGP (Sequence ID 26), GDIEQNPGP (Sequence ID 27), and DSEFNPGP (Sequence ID 26), Number 28) is cited.

[0200] In some embodiments, the stagger element described herein is a concept described herein. The expression product is cleaved between the G and P in the nucleotide sequence, for example. As one non-limiting example, a circular or Linear polyribonucleotides require at least one stagger to cleave the expression product. Contains elements. In some embodiments, cyclic or linear polyribonucleotides are at least It contains staggered elements adjacent to a single expression sequence. In some embodiments, it is circular or linear. Polyribonucleotides contain a stagger element after each expression sequence. In some embodiments, Circular or linear polyribonucleotides are staggered, present on one or both sides of each expression sequence. - By including this element, the translation of individual peptides and / or polypeptides from each expression sequence is extracted. To wake up.

[0201] In some embodiments, the stagger element induces ribosome arrest during translation. or contains multiple modified nucleotides or non-natural nucleotides. Non-natural nucleotides are, Butyrate nucleic acids (PNA), morpholino and locked nucleic acids (LNA), and glycol nucleic acids It may also contain (GNA) and threose nucleic acid (TNA). Examples of these include naturally occurring They are distinguished by the presence of DNA or RNA and by changes in the molecular backbone. Exemplary modifications These include sugars, nucleic acid bases, and nucleoside bonds that can induce ribosome arrest during translation (e.g., Any modification to the linked phosphate / phosphate diester bond / phosphate diester skeleton, and any combination thereof. Some of the exemplary modifications provided herein This is described elsewhere in this specification.

[0202] In some embodiments, the stagger element is located in a cyclic or linear polyribonucleotide. It exists in the form of a cyclic or linear polyribonucleotide. For example, in some exemplary cyclic or linear polyribonucleotides The stagger element is the termination sequence of the first expression sequence in a cyclic or linear polyribonucleotide. and a nucleus that separates the termination sequence from the first translation start sequence of expression that is consecutive to the first expression sequence. Includes an ocid spacer sequence. In some examples, the first stagger of the first expression sequence The element is the first expression sequence in a cyclic or linear polyribonucleotide that is followed by the first expression It is located upstream of the translation initiation sequence (5' side of it). In some cases, the first expression sequence The expression sequences in the column and the first expression sequence are 2 cyclic or linear polyribonucleotides. These are two separate expression sequences. The distance between the first stagger element and the first translation initiation sequence is: This may enable sequential translation of the first expression sequence and subsequent expression sequences. Morphologically, the first stagger element contains a termination sequence and the expression product of the first expression sequence is linked to it. The expression product is separated from the expression product of the subsequent expression sequence, thereby producing the separated expression product. In that case, the first translation initiation sequence of a sequence in a circular or linear polyribonucleotide A cyclic or linear polyribonucleotide containing a first stagger element upstream is continuously translated. On the other hand, it is located upstream of the second translation initiation sequence of the expression sequence that is continuous with the second expression sequence. The corresponding cyclic or linear polyribonucleotide containing the stagger element of the second expression sequence is successive It is not subsequently translated. In some cases, the expression sequence is in a cyclic or linear polyribonucleotide. There is only one such expression sequence, and the first expression sequence and its subsequent expression sequences are the same expression sequence. In some exemplary cyclic or linear polyribonucleotides, the stagger element is cyclic or The first termination sequence of the first expression sequence in a linear polyribonucleotide, and the downstream translation initiation sequence Includes nucleotide spacer sequences that separate the termination sequence from the column. Some such examples So, the first stagger element is the first expression sequence in a cyclic or linear polyribonucleotide. It is located upstream (5' side) of the first translation start sequence. In some cases, the first star The distance between the Gar element and the first translation initiation sequence is the distance between the first expression sequence and any consecutive expression sequences. Enables continuous translation of the sequence. In some embodiments, the first stagger element is the first The first expression product of the expression sequence is separated from the next expression product of the first expression sequence, and The isolated expression product is produced. In some cases, in cyclic or linear polyribonucleotides The first expression sequence contains a first stagger element upstream of the first translation initiation sequence, in a circular or linear manner. Polyribonucleotides are continuously translated, while the corresponding cyclic or linear polyribonucleotides The second expression sequence in the creotide contains a corresponding stagger element upstream of the second translation initiation sequence. The cyclic or linear polyribonucleotides are not continuously translated. In some cases, the second The distance between the stagger element and the second translation start sequence is the distance between the corresponding circular or linear polyribonu In creotides, the first stagger element in a cyclic or linear polyribonucleotide and At least 2, 3, 4, 5, 6, or 7 times the distance between the first translation start sequence and the next sequence. 8 times, 9 times, or 10 times larger. In some cases, the first stagger element and the first translation opening The distance from the starting sequence is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16 nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40 nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or It is more than that. In some embodiments, a second stagger element and a second translation start sequence and The distance between them is less than the distance between the first stagger element and the first translation start sequence. Also 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11 nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19 nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55 nt, 60nt, 65nt, 70nt, 75nt, or greater. Several implementations Morphologically, cyclic or linear polyribonucleotides contain two or more expression sequences.

[0203] In some embodiments, the cyclic or linear polyribonucleotide is at least one cross It includes a cleavage sequence. In some embodiments, the cleavage sequence is adjacent to the expression sequence. In some embodiments, the cleavage sequence is located between the two expression sequences. In some embodiments, The cleavage sequence is contained within the expression sequence. In some embodiments, a cyclic or linear polyribonu The creotide contains a cleavage sequence between 2 and 10. In some embodiments, it is cyclic or linear. Polyribonucleotides contain cleavage sequences between 2 and 5. In some embodiments, multiple The cleavage sequences are located between multiple expression sequences; for example, cyclic or linear polyribonucleotides. It contains three expression sequences and two cleavage sequences, such that cleavage sequences exist between each expression sequence. That is also acceptable. In some embodiments, cyclic or linear polyribonucleotides are, for example, Sacrificial circRNA (immolating circRNA) or cleavable cir The cRNA or self-cleaved circRNA contains a cleavage sequence. In some embodiments, a ring Ricosal or linear polyribonucleotides are cyclic or linear polyribonucleotides, for example, mi To multiple products such as RNA, linear RNA, and smaller circular or linear polyribonucleotides. It contains two or more cleavage sequences that cause separation.

[0204] In some embodiments, the cleavage sequence includes a ribozyme RNA sequence. NA enzymes (also called catalytic RNA, derived from ribonucleic acid enzymes) are R enzymes that catalyze chemical reactions. It is an NA molecule. Many natural ribozymes have one of their own phosphodiester bonds. It catalyzes either hydrolysis or hydrolysis of binding to other RNAs, but ribosomes It has also been found to catalyze the aminotransferase activity of . The catalytic RNA is It can be "evolved" through in vitro methods. Similar to the riboswitch activity discussed above, riboswitches Immunopropyl alcohol and their reaction products can regulate gene expression. In some embodiments, catalysts are used. RNA or ribozymes are used to chemically transform molecules from bulk volume. They can be located within larger uncoding RNAs, such as those present in multiple copies within a cell. In some embodiments, both the aptamer and ribozyme are present in the same uncoding RNA. It can be coded as follows.

[0205] In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, polyribonucleotides are, for example, two or more immunogens in a single open reed. When encoded by an ORF (Original Image Frame), it may encode two or more immunogens. For example, two or more immunogens are coded by a single open reading frame. It may be done, and its expression is controlled by IRES. In some embodiments, OR F further encodes a polypeptide linker; for example, the expression product of ORF is It consists of two or more immunogens, each containing a polypeptide linker (e.g., 5-200, 5- Linkers of 100, 5-50, 5-20, 50-100, or 50-200 amino acids It codes what is separated by the sequence it codes. The polypeptide linker is the cleavage part For example, proteases (for example, polyribonucleotides) in the target after administration to the target. It may also include a cleavage site that is recognized and cleaved by an endogenous protease. In one embodiment, a single expression product containing the amino acid sequences of two or more immunogens is cleaved during expression. This process allows two or more immunogens to be separated after expression. Exemplary protease cleavage region. For example, metalloproteinase (for example, matrix metalloproteinase (MM P), any one or more of MMP1-28, disintegrins and metallopro Teinases (ADAM, ADAM2, 7-12, 15, 17-23, 28-30 and 33) Any one or more of the following: serine proteases, urokinase-type plasminogens Activator, matryptase, cysteine ​​protease, aspartate protein It acts as a protease cleavage site recognized by cathepsin proteases or cathepsin proteases. The amino acid sequence is known to those skilled in the art. In some embodiments, the protease is M It is MP9 or MMP2. In some embodiments, the protease is matryptase. That is the case.

[0206] In some embodiments, the cyclic or linear polyribonucleotides described herein are sacrificed. Continental cyclic or linear polyribonucleotides, cleavable cyclic or linear polyribonucleotides Cleotides, or self-cleaving cyclic or linear polyribonucleotides. Cyclic or Linear polyribonucleotides include, for example, RNA, lncRNA, lincRNA, and miR NA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA It can deliver cellular components including cyclic or linear polyribonucleotides. D is (i) self-cleaving element; (ii) cleaving mobilization site; (iii) decomposable linker; (iv) ) Chemical linker; and / or (v) containing miRNA separated by spacer sequence In some embodiments, circRNA is (i) a self-cleaving sequence; (ii) a cleaving sequence. (iii) Member parts (e.g., ADAR); (iii) Degradable linker (e.g., glycerol); ( iv) Chemical linker; and / or (v) siRNA isolated by spacer sequence This includes, as non-limiting examples of self-cleaving sequences, hammerheads, splicing elements, Hairpin, hepatitis delta virus (HDV) Examples include Varkud Satellite (VS) and glmS Ribozyme.

[0207] Ratio of regulatory elements and expression products In one embodiment, a cyclic or linear polyribonucleotide is used as part of a regulatory nucleic acid combination. Includes columns or regulatory nucleic acids, such as those that regulate the expression of endogenous and / or exogenous genes. It comprises one or more expression sequences encoding nucleic acids. In one embodiment, according to this specification The cyclic or linear polyribonucleotide expression sequences provided are, but are not limited to, tR NA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA , piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and Antisense for regulatory nucleic acids such as non-coding RNAs like hnRNA It may contain a certain sequence.

[0208] An exemplary regulatory nucleic acid is cited herein by reference in its entirety in International Publication No. 2019 / 1 It is described in paragraphs

[0177] to

[0194] of pamphlet No. 18919.

[0209] In one embodiment, translation of the cyclic polyribonucleotide provided herein Efficiency is referenced, for example, linear equivalents, linear expression sequences, or linear polyribonu for cyclization. It is more expensive than creotide. In one embodiment, the cyclic polyribo provided herein Nucleotides have translation efficiencies of at least 5%, 10%, 15%, 20%, and 25% compared to the reference. %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 20 0%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 70 %, 800%, 900%, 1000%, 2000%, 5000%, 10000%, 100 It has a translation efficiency of 000% or higher. In one embodiment, cyclic polyribonu Cleotide has a 10% higher translation efficiency than its linear equivalent. In one embodiment, Furthermore, cyclic polyribonucleotides have a 300% higher translation efficiency than their linear counterparts. .

[0210] In one embodiment, cyclic or linear polyribonucleotides are expressed in stoichiometric ratios. It generates substances. Rolling circle translation generates expression products sequentially at substantially equal rates. To generate. In one embodiment, cyclic or linear polyribonucleotides are expressed as actual products. It has a stoichiometric translation efficiency such that it produces qualitatively equivalent ratios. And, cyclic or linear polyribonucleotides can express multiple products, for example, 2, 3, 4, 5, Stoichiometric translation of products from expression sequences 6, 7, 8, 9, 10, 11, 12, or more. It has translation efficiency. In some embodiments, cyclic or linear polyribonucleotides are expressed This results in substantially different ratios of products. For example, the translation efficiency of multiple expression products is 1:10, 000;1:7000, 1:5000, 1:1000, 1:700, 1:500, 1:1 The ratios can be 00, 1:50, 1:10, 1:5, 1:4, 1:3, or 1:2. How many? In that embodiment, the ratio of multiple expression products may be modified using a regulatory element.

[0211] translation In one embodiment, once translation of cyclic polyribonucleotides is initiated, the cyclic polyribonucleotide Ribosomes bound to bonucleotides are at least once connected to cyclic polyribonucleotides. The cyclic polyribonucleotide is not detached before the translation is completed. In one embodiment, The cyclic polyribonucleotides described herein possess the ability to perform rolling circle translation. In one embodiment, during rolling circle type translation, cyclic polyribonucleotides Once translation of the cyclic polyribonucleotide begins, the ribosome bound to the cyclic polyribonucleotide will Lilibonucleotides at least 2 times, at least 3 times, at least 4 times, at least 5 times Moves, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 1 0 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, few At least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, At least 100 times, at least 150 times, at least 200 times, at least 250 times, At least 500 times, at least 1000 times, at least 1500 times, at least 200 0 times, at least 5000 times, at least 10000 times, at least 105 times, or fewer It does not detach from the cyclic polyribonucleotide before completing at least 106 translations.

[0212] In one embodiment, rolling circle translation of cyclic polyribonucleotides is performed in a ring polypeptide products translated from two or more translations of a polyribonucleotide ("sequential") This results in the production of the expression product. In one embodiment, the cyclic polyribonucleotide is Rolling circle translation of cyclic polyribonucleotides containing staggered elements is performed by cyclic poly polypeptide products generated from one or fewer translations of riribonucleotides ( This results in the generation of a “separate” expression product. In one embodiment, a cyclic polyribonucleo Tide is produced during the rolling-circle translation of cyclic polyribonucleotides. Petit (moles / moles) at least 10%, 20%, 30%, 40%, 50%, and less 60%, at least 70%, at least 80%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, or 1 It is configured such that 00% is a separate polypeptide. In some embodiments, a cyclic polypeptide Lilibonucleotides are all polypeptides in which at least 99% are separate polypeptides. Designed to do so. In one embodiment, the amount ratio of distinct products across the entire polypeptide. This is tested in an in vitro translation system. In one embodiment, it is used to test the quantity ratio. The in vitro translation system contains rabbit reticulocyte lysate. In one embodiment, the quantity ratio The rate is determined in the in vivo translation system of eukaryotic or prokaryotic cells, cultured cells, or cells within organisms. They will be tested.

[0213] Non-translated areas In one embodiment, the cyclic polyribonucleotide includes an untranslated region (UTR). The UTR of a genomic region containing a gene can be transcribed but not translated. In one embodiment In this context, the UTR may be located upstream of the translation initiation sequence of the expression sequence described herein. In one embodiment, the UTR may be included downstream of the expression sequence described herein. In some cases, one UTR for the first expression sequence is used for the second expression sequence. It is the same as, consecutive with, or overlaps with another UTR. Morphologically, the intron is a human intron. In one embodiment, the intron The intron is a full-length human intron, for example, ZKSCAN1.

[0214] The exemplary untranslated area is incorporated herein by reference in its entirety by International Publication No. 2019 / It is described in paragraphs

[0197] to

[0201] of pamphlet No. 118919.

[0215] In one embodiment, the cyclic polyribonucleotide contains a polyA sequence. The sequence A is incorporated herein by reference in its entirety by International Publication No. 2019 / 118919. It is described in paragraphs

[0202] to

[0205] of the pamphlet. In one embodiment... Furthermore, cyclic polyribonucleotides lack a poly(A) sequence.

[0216] In some embodiments, the cyclic polyribonucleotide is composed of adenosine and uridine. Includes UTR containing one or more stretches. These AU Rich signatures This can increase the turnover rate of the expression product.

[0217] The introduction, removal, or modification of the AU-rich element (ARE) of UTR is a cyclic polyribonuclear Cleotide stability, or immunogenicity (e.g., one or more marks of immune or inflammatory responses) It may be useful for regulating the level of car. Modifying certain cyclic polyribonucleotides. In this process, one or more copies of ARE may be introduced into the cyclic polyribonucleotide. Copies of ARE can regulate the translation and / or production of the expression product. Similarly, copies of ARE can regulate the translation and / or production of the expression product. By fixing and removing or modifying them into cyclic polyribonucleotides, intracellular stability can be regulated. This can, in turn, affect the translation and production of the resulting protein.

[0218] Any UTR from any gene is incorporated into each adjacent region of a circular polyribonucleotide. It should be understood that this is acceptable.

[0219] In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR, and It has the ability to express proteins from one or more expression sequences. In some embodiments, Cyclic polyribonucleotides lack a 3'UTR and have one or more expression sequences. It has the ability to express proteins from. In some embodiments, cyclic polyribonucleotides The protein lacks a poly(A) sequence, and the ability to express protein from one or more of its expression sequences is limited. It has power. In some embodiments, cyclic polyribonucleotides lack a termination sequence. It has the ability to express proteins from one or more of these expression sequences. In this state, cyclic polyribonucleotides lack an internal ribosome entry site, and one of them Alternatively, it has the ability to express proteins from multiple expression sequences. In some embodiments, it is circular. Polyribonucleotides lack a cap, and one or more of their expression sequences are detached. It has the ability to express proteins. In some embodiments, the cyclic polyribonucleotide is 5 Lacking 'UTR, 3'UTR, and IRES, and derived from one or more expression sequences It has the ability to express proteins. In some embodiments, cyclic polyribonucleotides are The following sequences: sequences encoding one or more miRNAs, one or more replication proteins Sequences encoding quality, sequences encoding exogenous genes, therapeutics Arrays that code for translation, modifiers (e.g., translation modulators, translation enhancers or (is a suppressor), translation initiation sequence, one or more regulatory nucleic acids targeting endogenous genes ( For example, siRNA, lncRNA, shRNA, and therapeutic mRNA or protein. Includes one or more arrays that code for .

[0220] In some embodiments, cyclic polyribonucleotides lack a 5'UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR. In one embodiment, the cyclic polyribonucleotide lacks a poly(A) sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence. In this state, cyclic polyribonucleotides lack an internal ribosome entry site. In this embodiment, the cyclic polyribonucleotide is made susceptible to degradation by exonucleases. Lacking. In some embodiments, cyclic polyribonucleotides lack degradation sensitivity. The fact that cyclic polyribonucleotides are not degraded by exonucleases or, in the presence of an exonuclease, it is broken down to a limited extent, for example, This may mean that they are equivalent or similar in the absence of sonuclease. Several embodiments Therefore, cyclic polyribonucleotides are not degraded by exonucleases. In that embodiment, when a cyclic polyribonucleotide is exposed to an exonuclease... , degradation is reduced. In some embodiments, cyclic polyribonucleotides are caps It lacks binding to protein-binding proteins. In some embodiments, cyclic polyribonucleotides Ochido is missing a 5' cap.

[0221] Termination sequence Cyclic polyribonucleotides encode one or more expression sequences (e.g., immunogens). ) may include, and each expression sequence may or may not have a termination sequence. A further example is the International Patent Publication pamphlet No. 2019 / 118919 (that Paragraphs

[0169] to

[0170] (the entire text is incorporated herein by reference) are described below. It is listed.

[0222] In some embodiments, the cyclic polyribonucleotide contains a polyA sequence. In one embodiment, the length of the poly(A) sequence exceeds 10 nucleotides. PolyA sequences are longer than 15 nucleotides (for example, at least about 10, about 15, about 2) 0, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 70, approximately 8 0, approximately 90, approximately 100, approximately 120, approximately 140, approximately 160, approximately 180, approximately 200, approximately 250 , approximately 300, approximately 350, approximately 400, approximately 450, approximately 500, approximately 600, approximately 700, approximately 800 Approximately 900, 1,000, 1,100, 1,200, 1,300, 1,400 Approximately 1,500, 1,600, 1,700, 1,800, 1,900, 2,0 (00, approximately 2,500, and approximately 3,000 nucleotides or more). In one embodiment, the poly-A sequence is as described in International Patent Publication No. 2019 / 118919

[0202] ~ of Pamphlet A1 (the entirety of which is incorporated herein by reference) Designed according to the description of the poly-A sequence within

[0204] .

[0223] In some embodiments, the cyclic polyribonucleotide contains or lacks polyA. or for modulating the properties of one or more cyclic polyribonucleotides It has modified poly-A. In some embodiments, it lacks poly-A or has modified poly-A. The cyclic polyribonucleotides possess one or more functional properties, for example, immunogenicity ( For example, levels of one or more markers of an immune or inflammatory response, half-life, expression efficiency, etc. It will improve.

[0224] regulatory nucleic acids In some embodiments, cyclic polyribonucleotides are, for example, endogenous genes and / Or, it includes one or more expression sequences encoding regulatory nucleic acids that modify the expression of exogenous genes. In some embodiments, cyclic polyribonucleotides such as those provided herein The expression sequences are not limited to tRNA, lncRNA, miRNA, rRNA, and sn RNA, microRNA, siRNA, piRNA, snoRNA, snRNA, exR Regulatory nucleic acids similar to non-coding RNAs such as NA, scaRNA, Y RNA, and hnRNA. It may contain an array that is antisense to it.

[0225] In one embodiment, the regulatory nucleic acid targets genes such as host genes. The regulatory nucleic acid is a national International Patent Publication Brochure No. 2019 / 118919A1 (the entire brochure is available for reference) The following are described in paragraphs

[0177] and

[0181] to

[0189] of the (more incorporated herein) It may contain any of the regulatory nucleic acids.

[0226] In some embodiments, the expression sequence is one or more of the features described herein, for example , a sequence encoding one or more peptides or proteins, one or more regulatory elements, One or more regulatory nucleic acids, for example, one or more uncoding RNAs, other expression sequences, and so This includes any combination of these.

[0227] In one embodiment, the cyclic polyribonucleotide contains one or more RNA-binding sites. MicroRNAs (or miRNAs) bind to the 3'UTR of nucleic acid molecules, thus stabilizing the nucleic acid molecule. Short-chain non-transfers that downregulate gene expression by reducing qualitative properties or inhibiting translation. It is a coding RNA. A circular polyribonucleotide is one or more microRNA labels. This may include target sequences, microRNA sequences, or microRNA seeds. Such sequences may include The entire content of U.S. Patent Application Publication 2005 / 02612 is incorporated herein by reference. As taught in Specification No. 18 and U.S. Patent Application Publication No. 2005 / 0059005 It can correspond to any known microRNA such as those found in [the text]. The microRNA sequence is "C The "code" region, that is, the sequence within the region from position 2 to 8 of a mature microRNA, is a miRNA. MicroRNA containing sequences that exhibit complete Watson-Crick complementation to the target sequence. The seed may contain positions 2-8 or 2-7 of the mature microRNA. Several implementations Morphologically, microRNA seeds have 7 nucleotides (for example, the nucleotides of a mature microRNA). It may also contain creotides 2-8), where seed complementation in the corresponding miRNA target. The target site is adjacent to the adenine (A) at position 1 of the microRNA. In one embodiment, the microRNA seed is a 6-nucleotide (for example, a mature microRNA) It may also contain nucleotides 2-7 of NA, where the corresponding miRNA target The complementary site is adjacent to the adenine (A) at position 1 of the microRNA. For example, Grimson et al; Mol Cell. 2007 6;27:91 See -105 (the entire section of which is incorporated herein by reference).

[0228] protein binding In some embodiments, cyclic polyribonucleotides are proteins, for example, ribonucleotides. One or more proteins that enable the `me` to bind to an internal site within the RNA sequence. Includes a binding site. Protein binding sites, for example, ribosome binding sites, are cyclic polyribonucleated. By designing within a rheotide, cyclic polyribonucleotides become cyclic polyribonucleos. By masking the cydops from components of the host's immune system, they evade detection by the host's immune system. It may be possible or reducing, regulating decomposition, or regulating translation.

[0229] In some embodiments, a cyclic polyribonucleotide is a combination of at least one immunoprotein. By including a chromosome binding site, for example, immune responses, such as CTLs (cytotoxic T lymphocytes) ) Avoids the response. In some embodiments, the immunoprotein binding site is an immunoprotein Nucleo that binds to the substance and assists in masking exogenous cyclic polyribonucleotides. It is a cyd sequence. In some embodiments, the immunoprotein binding site is an immunoprotein Nuclei that bind to and assist in the concealment of cyclic polyribonucleotides as exogenous or foreign. This is an Otid sequence.

[0230] The traditional mechanism of ribosome association to linear RNA involves the capped 5' end of the RNA. It includes ribosome binding. As soon as the ribosome moves from the 5' end to the start codon, A peptide bond is formed. According to this disclosure, within the translation of a cyclic polyribonucleotide Partial initiation (i.e., cap-independent) does not require a free end or a capped end. Furthermore, ribosomes bind to the uncapped internal region, thereby allowing the ribosome to connect with the polypeptide. The cytoplasmic extension is initiated at the start codon. In some embodiments, the cyclic polyribonucleotide The dot contains one or more RNA sequences, including a ribosome binding site, such as a start codon. nothing.

[0231] The natural 5'UTR has features that play a role in translation initiation. It is a ribosome. The Kozak gene is generally known to be involved in the process of initiating the translation of numerous genes. It contains a signature like a column. The Kozak sequence is a consensus CCR(A / G)C. CAUGG (SEQ ID NO: 29) (where R is the purine 3 bases upstream of the start codon (AUG)) It has (adenine or guanine) followed by another "G". 5'U TR is also known to form secondary structures involved in elongation factor binding.

[0232] In some embodiments, cyclic polyribonucleotides bind to proteins. It encodes a protein-binding sequence. In some embodiments, the protein-binding sequence is a cyclic poly Ribonucleotides are targeted or localized to specific targets. In some embodiments, The protein-binding sequence specifically binds to the arginine-rich region of the protein.

[0233] In some embodiments, the protein binding site is, but is not limited to, ACIN1 , AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, C APRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7, CST F2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DG CR8, EIF3A, EIF4A3, EIF4G2, ELAVL1, ELAVL3, FA M120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1, FXR 2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, H NRNPK, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, IGF2 BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, L IN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1 , MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2 , POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22, R BM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1, TNRC 6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF 1, WDR33, XRN2, YBX1, YTHDC1, YTHDF1, YTHDF2, Y WHAG, ZC3H7B, PDK1, AKT1, and any other proteins that bind to RNA Examples include binding sites to proteins such as cereals.

[0234] Encryptogen As described herein, cyclic polyribonucleotides reduce the innate immune response of cells, escaping Contains an encryptogen for avoidance or evasion. In one embodiment, when delivered to a cell, Reference compounds, e.g., linear polynucleotides corresponding to the described cyclic polyribonucleotides. Responds induced by cyclic polyribonucleotides lacking tide or cryptogen In comparison to the answer, cyclic polyribonucleotides result in a reduced immune response from the host. This is provided herein. In some embodiments, cyclic polyribonucleotides are used Reduced immunogenicity compared to cryptogenic counterparts (e.g., reduced levels of It has one or more markers of an immune or inflammatory response.

[0235] In some embodiments, the cryptogen enhances stability. (Stability of nucleic acid molecules) And regarding the regulatory role played by UTR from a translation perspective, a growing body of evidence is emerging. The regulatory features of UTR enhance the stability of cyclic polyribonucleotides, thus enabling It may be included in cryptogen.

[0236] In some embodiments, the 5' or 3'UTR is an endoplasmic polyribonucleotide. It can constitute a cryptogen. For example, the modification of the AU-rich element (ARE) of UTR Removal modulates the stability or immunogenicity of cyclic polyribonucleotides (e.g., immune or It may be useful in regulating the levels of one or more markers of the inflammatory response.

[0237] In some embodiments, AU-rich elements (AREs) within the expression sequence, for example, translatable Removal of modifications to the translatable region modulates the stability or immunogenicity of cyclic polyribonucleotides. (For example, useful for regulating the levels of one or more markers of an immune or inflammatory response) It is possible.

[0238] In some embodiments, the encryptogen is a miRNA binding site or any other non- Includes a binding site for the translated RNA. For example, the ring described herein for the miR-142 site. Incorporation into ring polyribonucleotides not only regulates expression within hematopoietic cells, but also ring Reduces or eliminates the immune response to proteins encoded by polyribonucleotides. It can be made to disappear.

[0239] In some embodiments, the encryptogen is a protein, such as an immunoprotein. It includes one or more protein binding sites that enable it to bind to an RNA sequence. By designing the protein binding site within a cyclic polyribonucleotide, Nucleotides mask cyclic polyribonucleotides from components of the host immune system. Therefore, it can either evade or reduce detection by the host's immune system, or regulate its degradation. They are either dolphins or regulating translation. In some embodiments, cyclic polyribonucleotides It includes at least one immune protein binding site, for example, an immune response, for example , evading the CTL response. In some embodiments, the immunoprotein binding site is located in the immunota It binds to proteins and assists in masking exogenous cyclic polyribonucleotides. It is a creotide sequence.

[0240] In some embodiments, the encryptogen comprises one or more modified nucleotides. Exemplary modifications may block or reduce the immune response to cyclic polyribonucleotides. , sugars, nucleic acid bases, nucleoside bonds (e.g., linked phosphate / phosphate diester bonds / This may include any modification of the phosphate diester skeleton and any combination thereof. Some exemplary modifications are provided herein.

[0241] In some embodiments, cyclic polyribonucleotides are used to stimulate an immune response from the host, The response induced by a compound, for example, a cyclic polyribonucleotide lacking modification, and Includes one or more modifications, as described elsewhere in this specification, for comparison and reduction. In particular, the addition of one or more inosine molecules makes the RNA endogenous to the viral. It has been shown that they can be distinguished. For example, Yu, Z. et al. (2015) RNA e diting by ADAR1 marks dsRNA as “self”.Cel See l Res.25,1283–1284 (the entire section is referenced by reference). sea ​​bream.

[0242] In some embodiments, cyclic polyribonucleotides are converted into shRNA or siRNA. It comprises one or more expression sequences for a processingable RNA sequence, and shRNA or siRNA targets RIG-I and reduces RIG-I expression. RIG-I is exogenous It can sense native circular RNA and induce the degradation of foreign circular RNA. Therefore, The sequence in shRNA, siRNA, or any other regulatory nucleic acid that targets RIG-1 The encapsulated cyclic polynucleotide provides immunity to cyclic polyribonucleotides, for example, It may reduce chief cell immunity.

[0243] In some embodiments, the cyclic polyribonucleotide is Sequences, elements, or It lacks structure. In some such embodiments, cyclic polyribonucleotides are Poly(A) sequence, 5' end, 3' end, phosphate group, hydroxyl group, or any combination thereof. It is acceptable to omit the matching parts.

[0244] Nucleotide spacer sequence In some embodiments, the cyclic polyribonucleotide includes a spacer sequence. In several embodiments, the cyclic polyribonucleotide comprises at least one spacer sequence. Includes. In some embodiments, the cyclic polyribonucleotides are 1, 2, 3, 4, 5, 6 , including 7 or more spacer arrays.

[0245] In some embodiments, the cyclic polyribonucleotide includes a spacer sequence. In the embodiment, the elements of the polyribonucleotide are arranged by a spacer sequence or a linker. They can be separated from each other. An example of a spacer arrangement is incorporated herein by reference in whole. Paragraphs

[0293] to

[0302] of the International Publication No. 2019 / 118919 pamphlet It is stated.

[0246] Non-nucleic acid linker The cyclic polyribonucleotides described herein may include non-nucleic acid linkers. In several embodiments, the cyclic polyribonucleotide is a sequence or element described herein. One or more of the links have a non-nucleic acid linker between them. In one embodiment, the 1 described herein One or more sequences or elements are linked with a linker. Non-nucleic acid linkers are chemically linked. The union may be, for example, one or more covalent or non-covalent bonds. Several implementations In this context, non-nucleic acid linkers are peptide or protein linkers. - may be between 2 and 30 or more amino acids. The cyclic poly(P) described herein Lilibonucleotides may also contain non-nucleic acid linkers. An example of a non-nucleic acid linker is: , International Patent Publication No. 2019 / 118919 pamphlet (the entire pamphlet is referenced) As described in paragraphs

[0303] to

[0307] of (more incorporated herein) .

[0247] In one embodiment, the cyclic polyribonucleotide further comprises another nucleic acid sequence. In one embodiment, the cyclic polyribonucleotide includes DNA, RNA, or artificial nucleic acids. It may contain other sequences. These other sequences may include, but are not limited to, genomic DNA and cDNA. , or tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or others Examples include sequences encoding RNAi molecules. In one embodiment, cyclic polyribonu Cleotides target different gene loci of the same gene expression product as cyclic polyribonucleotides. It contains siRNA to achieve this. In one embodiment, a cyclic polyribonucleotide is Targeting gene expression products different from those present in cyclic polyribonucleotides It includes siRNA for this purpose.

[0248] Stability and half-life In some embodiments, cyclic polyribonucleotides have specific sequence characteristics. For example The cyclic polyribonucleotide may contain certain nucleotide compositions. In such embodiments, the cyclic polyribonucleotide is one or more purines (ade It may also include a nin and / or guanosine-rich region. Some such embodiments Therefore, a cyclic polyribonucleotide may contain one or more purine-deficient regions. In some embodiments, the cyclic polyribonucleotide has one or more AU-rich regions or It may include elements (ARE). In some embodiments, cyclic polyribonucleotides Otide may contain one or more adenine-rich regions.

[0249] In some embodiments, cyclic polyribonucleotides are described elsewhere in this specification. It may include one or more repeating elements. In some embodiments, a ring-shaped Lilibonucleotides include one or more modifications as described elsewhere in this specification.

[0250] Cyclic polyribonucleotides are subjected to one or more substitutions, insertions and / or This may include addition, deletion, and covalent modification of the parent polyribonucleotide. cyclic polyribochemicals having one or more insertions, additions, deletions, and / or covalent modifications. Nucleotides are included within the scope of this disclosure. Exemplary modifications are included in the international publication of the international patent publication. Brochure No. 2019 / 118919 (the entire brochure is incorporated herein by reference) It is described in paragraphs

[0310] to

[0325] of the book.

[0251] In some embodiments, the cyclic polyribonucleotide has a higher-order structure, for example, a secondary or It includes a tertiary structure. In some embodiments, complementary segments of cyclic polyribonucleotides are included. The two-stranded segment is maintained together by hydrogen bonds between pairs, for example, between AU and CG. It folds itself into the base. In some embodiments, it is also known as a stem. A clasp is formed intramolecularly, and it has a double-stranded segment linked to an end-loop. In some embodiments, the cyclic polyribonucleotide has a pseudo-double-stranded secondary structure. It has at least one segment.

[0252] In some embodiments, one or more sequences of cyclic polyribonucleotides are substantially It includes single-stranded and double-stranded regions. In some embodiments, the ratio of single-stranded to double-stranded regions This may affect the functionality of cyclic polyribonucleotides.

[0253] In some embodiments, one or more sequences of cyclic polyribonucleotides are substantially It is single-stranded. In some embodiments, it is substantially single-stranded cyclic polyribonucleo One or more sequences of the chido may contain protein or RNA binding sites. In this embodiment, the substantially single-stranded cyclic polyribonucleotide sequence is stereostructured Flexibility can enable enhanced interactions. In some embodiments, cyclic poly The ribonucleotide sequence is such that it binds to or enhances protein or nucleic acid binding. It is modified with the intention of including the following structure.

[0254] In some embodiments, cyclic polyribonucleotides are substantially double-stranded. In some embodiments, one or more cyclic polyribonucleotides that are substantially double-stranded are used. The sequence may include a three-dimensional recognition site, such as a riboswitch or aptazyme. In some embodiments, a substantially double-stranded cyclic polyribonucleotide sequence is... It may be structurally robust. In some such examples, the arrangement is structurally robust. This is because cyclic polyribonucleotides sterically hinder their binding to proteins or nucleic acids. This is possible. In some embodiments, the sequence of cyclic polyribonucleotides is a protein It is modified with the intention of including secondary structures that avoid or reduce qualitative or nucleic acid binding.

[0255] There are 16 possible base pairs, but of these, six (AU, GU, GC, UA, UG, C) are considered. G) can form an actual base pair. The rest are called mismatches and are very low in the helix. It occurs with high frequency. In some embodiments, the structure of a cyclic polyribonucleotide is its mechanism It cannot be easily destroyed without affecting the ability and lethal consequences, and it is a secondary structure It provides options for maintaining the primary structure of the stem (i.e.) (Their nucleotide sequences) can still change, but they still maintain the helical region. The properties of a base are derived from its higher-order structure, and substitution is possible as long as the secondary structure is preserved. In some embodiments, the cyclic polyribonucleotide has a pseudo-helical structure. In some embodiments, the cyclic polyribonucleotide has a pseudo-helical structure. It has at least one segment. In some embodiments, it has a cyclic polyribonucleotide. Otid contains at least one of U-rich or A-rich sequences or a combination thereof. In some embodiments, the U-rich and / or A-rich arrays have a triple pseudo-helical structure. They are arranged in a manner that produces a structure. In some embodiments, cyclic polyribonucleo The cydo has a double pseudo-helical structure. In some embodiments, a cyclic polyribonucleotide Otido has one or more segments (e.g., 2, 3, It has 4, 5, 6, or more. In some embodiments, it has a cyclic polyribonucleotide. The cyd comprises at least one C-rich and / or G-rich sequence. Several embodiments So, C-rich and / or G-rich sequences are designed to produce a triple pseudo-helical structure. They are arranged in this way. In some embodiments, cyclic polyribonucleotides are used to aid in stabilization. It has an intramolecular triple pseudo-helical structure.

[0256] In some embodiments, cyclic polyribonucleotides are (e.g., diester phosphates) It has two pseudo-helical structures (separated by bonding), and their terminal base pairs These layers are stacked, and the pseudo-helical structure becomes collinear, resulting in a "coaxially stacked" substructure. vinegar.

[0257] In some embodiments, the cyclic polyribonucleotide has one or more motifs, e.g. For example, a tertiary structure having pseudoknots, g-quadruplicates, helices, and coaxial stacking. .

[0258] Further examples of cyclic polyribonucleotide structures such as those disclosed herein are internationally recognized. International Publication No. 2019 / 118919 of the Public Publication of the Public (the entire pamphlet is available by reference to the Public Publication of the Public Publication) It is described in paragraphs

[0326] to

[0333] (which are referenced in the detailed text).

[0259] As a result of this cyclization, cyclic polyribonucleotides distinguish them from linear RNA. It may contain several characteristics. For example, cyclic polyribonucleotides, compared to linear RNA, Therefore, it is less susceptible to degradation by exonucleases. Bonucleotides, in particular, when incubated in the presence of exonucleases, It is more stable than linear RNA. Compared to linear RNA, it has an increased amount of cyclic polyribonucleotides. Due to its stability, the cyclic polyribonucleotide is a cell transformation reagent that generates polypeptides. (For example, it becomes more useful as an immunogen.) Circular polyribonucleotides compared to linear RNA. Due to the increased stability of ocide, cyclic polyribonucleotides can be transmitted more easily than linear RNA. It can be stored for a longer period. Cyclic polyribonucleotides treated with exonuclease. The stability of the RNA degradation is a standard in the art that determines whether RNA degradation has occurred. It can be tested using methods (for example, by gel electrophoresis).

[0260] Furthermore, unlike linear RNA, cyclic polyribonucleotides are cyclic polyribonucleotides. When the drug is incubated with phosphatases such as calf intestinal phosphatase, They are less sensitive to dephosphorylation.

[0261] In one embodiment, preparation of cyclic polyribonucleotides provided herein For example, a linear polyribonucleotide that has the same nucleotide sequence but is not circular. It has a longer half-life than ocide (e.g., a linear equivalent). In one embodiment, ring Polyribonucleotides are resistant to degradation, such as by exonucleases. In one embodiment, cyclic polyribonucleotides are resistant to autodegradation. In one embodiment, the cyclic polyribonucleotide is cleaved at an enzymatic cleavage site, for example, a dicer cleavage site. It lacks a cleavage site. In some embodiments, cyclic polyribonucleotides are, for example, reference. For example, compared to the linear counterpart, at least about 5%, at least about 10%, at least about 20% , at least about 30%, at least about 40%, at least about 50%, at least about 60% , at least about 70%, at least about 80%, at least about 90%, at least about 100 %, at least about 120%, at least about 140%, at least about 150%, at least Approximately 160%, at least approximately 180%, at least approximately 200%, at least approximately 300%, less At least approximately 400%, at least approximately 500%, at least approximately 600%, at least approximately 70 0% at least about 800%, at least about 900%, at least about 1000%, or less Both have half-lives that are approximately 10,000% longer.

[0262] In some embodiments, cyclic polyribonucleotides persist within the cell during cell division. In some embodiments, cyclic polyribonucleotides exist in daughter cells after mitosis. To be continued. In some embodiments, cyclic polyribonucleotides are replicated within the cell, and daughters It is passed through cells. In some embodiments, cyclic polyribonucleotides are cyclic polyri It includes a replication element that mediates the self-replication of a bonucleotide. In some embodiments, The replication element is complementary to the cyclic polyribonucleotide of the cyclic polyribonucleotide. It mediates the transcription to linear polyribonucleotides that are linearly complementary. Several embodiments So, complementary linear polyribonucleotides are found in cells, in vivo, and complementary cyclic polyribonucleotides are found in cells. It can be cyclized into bonucleotides. In some embodiments, complementary polyribonucleo The nucleotide has the same or similar nucleotide sequence as the initial cyclic polyribonucleotide. It can further self-replicate into another cyclic polyribonucleotide. One exemplary self-replicating element The nt includes HDV replication domains (Beeharry et al, Virol, 20 (As described in 14,450-451:165-173). In some embodiments... The cells contain at least one cyclic polyribonucleotide, at least 25%, 50%, Passaging to daughter cells with an efficiency of 60%, 70%, 80%, 85%, 90%, 95%, or 99% In some embodiments, cells undergoing meiosis have cyclic polyribonucleotides. to at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and It passes to daughter cells with 99% efficiency. In some embodiments, the cells that have undergone mitosis The cells contain cyclic polyribonucleotides at least 25%, 50%, 60%, 70%, and 80%. The cells are passed to daughter cells with an efficiency of %, 85%, 90%, 95%, or 99%.

[0263] Further improvements in the stability and half-life of cyclic polyribonucleotides as disclosed herein. An example is the brochure for International Patent Publication No. 2019 / 118919 (the entire brochure is available for reference). As described in paragraphs

[0308] to

[0309] of (as incorporated herein by reference) Yes, they are.

[0264] qualification A cyclic polyribonucleotide is a reference sequence, in particular, a parent polyribonucleotide, with respect to 1 This may include one or more substitutions, insertions and / or additions, deletions, and covalent modifications. , included within the scope of this disclosure.

[0265] In some embodiments, the cyclic polyribonucleotide has one or more post-transcriptional modifications ( For example, capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation , acylation, phosphorylation, methylation and acetylation of lysine and arginine residues, and thio Includes R groups and nitrosylation of tyrosine residues. One or more post-transcriptional modifications are R Any of the more than 100 different nucleoside modifications identified in NA It can be a post-transcription modification of meaning (Rozenski, J., Crain, P., and McCl). oskey, J. (1999).The RNA Modification Data base:1999 update.Nucl Acids Res 27:196-1 97). In some embodiments, the first isolated nucleic acid is messenger RNA (mRNA). ) includes. In some embodiments, mRNA is included in International Patent Publication No. 2019 / The invention comprises at least one nucleoside selected from the group described in

[0311] of Pamphlet No. 118919A1 (the entirety of which is incorporated herein by reference). .

[0266] Cyclic polyribonucleotides are, for example, linked to sugars, nucleic acid bases, or nucleoside bonds (for example) Any useful Modifications may be included. One or more atoms of the pyrimidine nucleic acid base may be optionally substituted. Amino compounds, optionally substituted thiols, optionally substituted alkyl groups (e.g.) For example, substituted with methyl or ethyl, or halo (for example, chloro or fluoro) (repl It may be aced or substituted. In certain embodiments, substituted The modifications (e.g., one or more modifications) are present in both the sugar and nucleoside bonds. It exists. The modifications are of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and threose nucleic acid. (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LN Modifications may be made to A) or hybrids thereof. Further modifications are described herein. It is described there.

[0267] In some embodiments, cyclic polyribonucleotides are used to increase translation efficiency. Includes at least one N(6)methyladenosine (m6A) modification. Several embodiments So, is N(6) methyladenosine (m6A) modification an immunogen for cyclic polyribonucleotides? It reduces the ability to reduce the level of one or more markers of an immune or inflammatory response. obtain.

[0268] In some embodiments, the modifications may include chemical or cell-induced modifications. For example, Some non-limiting examples of intracellular RNA modification are found in Nat Reviews Mol Cell. Lewis and Pan, “RN” from Biol, 2017, 18:202-210 A modifications and structures cooperate Described in "to guide RNA-protein interactions" It is listed.

[0269] In some embodiments, the chemical relationship between cyclic polyribonucleotides and ribonucleotides Modification may enhance immune evasion. Cyclic polyribonucleotides are well established in the art. Established methods, for example, “Current protocols in nucleic acid chemistry,”Beaucage,S Let al.(Eds .), John Wiley & Sons, Inc., New York, NY, US Synthesized and / or by means of A (as incorporated herein by reference) It may be modified. Modifications include, for example, terminal modifications, for example, 5' terminal modifications (phosphorylation (mo (North, G, and Tri), conjugation, reverse bond, etc.), 3'-terminus modification (conjugate) (e.g., nucleotides, reverse bonds), base modifications (e.g., stabilizing bases, ankylosis) (Substitution of a stenosing base, or a base that forms a base pair with an expanded repertoire of partners.) Includes base removal (debasement nucleotide) or conjugate base. Modified ribonucle Otido bases may also contain 5-methylcytidine and pseudouridine. In this embodiment, the base modification has several functional effects, including the cyclic polyribonucleo It can regulate the expression, immune response, stability, and intracellular localization of tides. In some embodiments, The modifications include biorthogonal nucleotides, such as non-native bases. For example, Kimoto et al,Chem Commun(Camb),2017,53:12309,D See OI:10.1039 / c7cc06661a (referred to here by reference). I want to.

[0270] In some embodiments, one or more ribonucleonucleotides of a cyclic polyribonucleotide Sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, as well as skeletal modifications, are related to phosphate dietary modifications. This may include modifications or substitutions of stealth bonds. Specific examples of cyclic polyribonucleotides include, However, this does not apply to modified skeletons or non-natural nuclei, including modification or substitution of phosphate diester bonds. Examples include cyclic polyribonucleotides that include osidic bonds, such as nucleoside modifications. A cyclic polyribonucleotide having a modified skeleton is, in particular, one that does not have a phosphorus atom in the skeleton. Including the following. In the intent of this application, and as sometimes referred to in the art, nucleo Modified RNA that does not have a phosphorus atom in the intersidal skeleton can also be considered an oligonucleoside. This is possible. In certain embodiments, cyclic polyribonucleotides are located within the nucleoside interbackframe. This will result in the inclusion of ribonucleotides containing a phosphorus atom.

[0271] Modified cyclic polyribonucleotide skeletons include, for example, phosphorothioates and chiral nucleotides. Sulfrothioate, dithiophosphate, phosphotriester, aminoalkylphosphotriester Tel, methyl and other alkylphosphonates, for example, 3'-alkylenephosphonates and Chiral phosphonates, phosphinates, phosphoramidates, for example, 3'-aminophosphates Sulfoamides and aminoalkylphosphoramides, thionophosphoramides, Thionoalkylphosphonates, thionoalkylphosphotriesters, and normal 3'-5 Boranophosphates having a bond, their 2'-5' bond analogues, and nucleoside units Inverted poles where adjacent pairs are joined at 3'-5' and 5'-3' or 2'-5' and 5'-2'. It may include substances that have properties. Various salts, mixed salts, and free acid forms are also included. In this embodiment, the cyclic polyribonucleotide can be negatively or positively charged.

[0272] Modified nucleotides that can be incorporated into cyclic polyribonucleotides are those that form nucleoside bonds. It can be modified (for example, the phosphate skeleton). In this specification, the polynucleotide skeleton In relation to this, the terms "phosphate" and "diester phosphate" are used interchangeably. The phosphate group of the skeleton is modified by substituting one or more oxygen atoms with different substituents. It can be decorated. Furthermore, modified nucleosides and nucleotides have an unmodified phosphate moiety, which is a natural characteristic. This may include large-scale substitutions involving other nucleoside bonds, as described in the detailed document. Modified phosphate Examples of the base material, though not limited to them, include phosphorothioates, phosphoroselenates, and boranol. Hydrogen acid, boranophosphate ester, hydrogen phosphonate, phosphoramidate, phosphorodiamidate Examples include dithiophosphates, alkyl or aryl phosphonates, and phosphotriesters. Ophosphate has both unbonded oxygen atoms substituted with sulfur. Also, phosphate linker It consists of nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged Modification can be made by substitution of bound oxygen with bridged methylene phosphonates.

[0273] The α-thio-substituted phosphate moiety is a non-natural phosphorothioate bone in RNA and DNA polymers. Provided to provide stability via dendritic bonding. Phosphothioate DNA and RNA In the cellular environment, it exhibits enhanced nuclease resistance followed by an extended half-life. Phosphothioates linked to cyclic polyribonucleotides are part of the cell's innate immune system. This is expected to reduce the innate immune response through weaker binding / activation.

[0274] In specific embodiments, the modified nucleoside is an α-thionucleoside (e.g., 5'- O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine(a- Thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thio Contains (phosphate)-uridine or 5'-O-(1-thiophosphate)-psoidouridine.

[0275] This disclosure may include nucleoside interbonds that do not contain a phosphite atom, and may be used in accordance with this disclosure. Other nucleoside-to-nucleoside bonds are described herein.

[0276] In some embodiments, cyclic polyribonucleotides are one or more cytotoxic nucleotides. It may contain a cleoside. For example, cytotoxic nucleosides are rings such as those with bifunctional modifications. It may be incorporated into polyribonucleotides. Cytotoxic nucleosides are not limited to However, adenosine arabinoside, 5-azacitidine, 4'-thio-azacitidine, citric acid Lopentenylcytosine, cladribine, clopharabine, cytarabine, cytosine arabine Sid, l-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)- Cytosine, decitabine, 5-fluorouracil, fludarabine, phloxuridine, gem A combination of cytabine, tegafur, and uracil, tegafur ((RS)-5-fluoro -l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione) Troxacitabine, tezacitabine, 2'-deoxy-2'-methylidencytidine (DM It may also contain DC, and 6-mercaptopurine. As a further example, fludara phosphate Bin, N4-behenoyl-l-β-D-arabinofuranosilcytosine, N4-octadessy Ru-1-β-D-arabinofuranosilcytosine, N4-palmitoyl-l-(2-C-cytosine Ano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-405 5 (cytarabine 5'-elaidic acid ester) is one example.

[0277] Cyclic polyribonucleotides can be uniformly modified along the entire length of the molecule or not. i. For example, one or more or all types of nucleotides (for example, naturally occurring nucleotides) Cleotide, purine or pyrimidine, or any one of A, G, U, C, I, pU (or multiple or all of them) are cyclic polyribonucleotides, or a given predetermined sequence region thereof. The region may or may not be uniformly modified. In some embodiments, cyclic poly The bonucleotide contains pseudouridine. In some embodiments, cyclic polyribonu Cleotides contain inosine, which is a cyclic polyribonucleotide that combines endogenous RNA with other substances. It may contribute to the immune system, characterized as a Rustic RNA. Inosine incorporation is also RN A may mediate improved stability / reduced degradation. For example, Yu, Z. et al. (2015) )RNA editing by ADAR1 marks dsRNA as“sel f.Cell Res. 25, 1283-1284 (the entire article is referenced by reference) Please refer to ).

[0278] In some embodiments, a cyclic polyribonucleotide (or a given sequence region thereof) All nucleotides are modified. In some embodiments, the modification enhances expression. m6A that can reduce the immune response; inosine that can reduce RNA stability or translational readthrough ( Pseudouridine, which can enhance stagger elements; m5C, which can enhance stability; and intracellular It may also contain 2,2,7-trimethylguanosine, which assists in rearrangement (e.g., nuclear localization). .

[0279] Different sugar modifications, nucleotide modifications, and / or internucleoside bonds (e.g., skeletal structures) ) may be located at various positions within the cyclic polyribonucleotide. Those skilled in the art will know that the nucleo The fact that cytonucleotide analogs or other modifications can be located at any position on the cyclic polyribonucleotide means that You will understand that the function of cyclic polyribonucleotides is not substantially reduced by the modification. Furthermore, non-coding region modifications are also acceptable. Cyclic polyribonucleotides are (total nucleotides) The content, or one or more types of nucleotides, namely A, G, U, or C. (For any one or more of them) approximately 1% to approximately 100%, or any percentage in between. Rates (for example, 1%~20%, 1%~25%, 1%~50%, 1%~60%, 1%~70%) %, 1%~80%, 1%~90%, 1%~95%, 10%~20%, 10%~25%, 1 0%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 1 0%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100% 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95% 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~10 0%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~ It may contain 100% and 95%-100% modified nucleotides.

[0280] structure In some embodiments, the cyclic polyribonucleotide has a higher-order structure, for example, a secondary or It includes a tertiary structure. In some embodiments, complementary segments of cyclic polyribonucleotides are included. The two-stranded segment is maintained together by hydrogen bonds between pairs, for example, between AU and CG. It folds itself into the base. In some embodiments, it is also known as a stem. A clasp is formed intramolecularly, and it has a double-stranded segment linked to an end-loop. In some embodiments, the cyclic polyribonucleotide has a pseudo-double-stranded secondary structure. It has at least one segment. In some embodiments, it has a pseudo-double-stranded secondary structure. The segment having at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, It has 80, 85, 90, 95, 100, or more paired nucleotides. In this embodiment, the cyclic polyribonucleotide has one or multiple pseudo-double-stranded secondary structures. It has a number segment (e.g., 2, 3, 4, 5, 6, or more). In terms of application patterns, the segments are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, Only 85, 90, 95, 100, or more nucleotides are isolated.

[0281] In some embodiments, one or more sequences of cyclic polyribonucleotides are substantially It includes single-stranded and double-stranded regions. In some embodiments, the ratio of single-stranded to double-stranded regions This may affect the functionality of cyclic polyribonucleotides.

[0282] In some embodiments, one or more sequences of cyclic polyribonucleotides are substantially It is single-stranded. In some embodiments, it is substantially single-stranded cyclic polyribonucleo One or more sequences of the chido may contain protein or RNA binding sites. In this embodiment, the substantially single-stranded cyclic polyribonucleotide sequence is stereostructured Flexibility can enable enhanced interactions. In some embodiments, cyclic poly The ribonucleotide sequence is such that it binds to or enhances protein or nucleic acid binding. It is modified with the intention of including the following structure.

[0283] In some embodiments, the cyclic polyribonucleotide has at least one binding site, For example, at least one protein binding site, at least one miRNA binding site, At least one lncRNA binding site, at least one tRNA binding site, and at least one rRNA binding site, at least one snRNA binding site, at least one s iRNA binding site, at least one piRNA binding site, at least one snoRN A binding site, at least one snRNA binding site, at least one exRNA binding site Position, at least one scaRNA binding site, at least one Y RNA binding site, few It has at least one hnRNA binding site and / or at least one tRNA motif. do.

[0284] In some embodiments, cyclic polyribonucleotides have higher-order structures, for example, international patents Publicly available International Publication No. 2019 / 118919A1 (the entire pamphlet is available by reference) Designed to include those described in (as referenced in the specification).

[0285] Production method In one embodiment, cyclic polyribonucleotides are non-natural and are synthesized using recombinant technology. For example, obtained in vitro using DNA plasmids, chemically synthesized, or combinations thereof. It includes a deoxyribonucleic acid sequence that can be generated using the combination.

[0286] The DNA molecules used to generate the RNA ring are the DNA sequences of the original, natural nucleic acid sequence. The modified form, or synthetic polypeptides not normally found in nature (e.g., chimeric molecules or DNA sequences encoding fusion proteins (e.g., fusion proteins containing multiple immunogens) This disclosure may include, but is not limited to, DNA and RNA molecules. Site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, nucleic acid fluctuating Restriction enzyme cleavage of ligants, ligation of nucleic acid fragments, polymerase chain reaction ( PCR amplification and / or mutagenesis of selected regions of nucleic acid sequences, oligonucleotides Synthesis of mixtures and ligation of mixture groups for "constructing" mixtures of nucleic acid molecules This includes various techniques such as combinations of these, including classical mutagenesis and recombination techniques. It can be used to modify.

[0287] Cyclic polyribonucleotides are, but are not limited to, any combination of chemical and enzymatic synthesis. It can be prepared according to the available technology. In one embodiment, a linear primary structure or line The mRNA is cyclic or concatemerized to form the cyclic polyribonucleonucleotide described herein. Cleotides may be formed. The mechanism of cyclization or concatemerization is not limited to chemical This is carried out by methods such as enzymatic, sprint ligation, or ribozyme-catalyzed methods. It is possible. The newly formed 5'- / 3'- bond can be an intramolecular bond or an intermolecular bond. ru.

[0288] The method for producing cyclic polyribonucleotides described herein is, for example, Khud Yakov & Fields,Artificial DNA:Methods an d Applications,CRC Press(2002);in Zhao,S Synthetic Biology:Tools and Applications, (First Edition), Academic Press (2013); and E gli & Herdewijn,Chemistry and Biology of Artificial Nucleic Acids,(First Edition ), described in Wiley-VCH (2012).

[0289] Various methods for synthesizing cyclic polyribonucleotides have also been described in the art. (For example, the entirety of each of the following is incorporated herein by reference: U.S. 6 U.S. Patent No. 210931, U.S. Patent No. 5773244, U.S. Patent No. 5766903 U.S. Patent No. 5712128, U.S. Patent No. 5426180, U.S. National Patent Application Publication No. 20100137407, International Publication No. 1992001813 Please refer to the brochure and International Publication No. 2010 / 084371.

[0290] Ring formation In one embodiment, linear polyribonucleotides for cyclization are cyclized or con It can be catemerized. In one embodiment, a linear polyribonucleotide for cyclization. The compound may be cyclized in vitro before formulation and / or delivery. In one embodiment, the ring Linear polyribonucleotides for morphogenesis can be cyclically formed within cells.

[0291] a. Extracellular circulation In one embodiment, linear polyribonucleotides for cyclization are obtained by a chemical method They are then cyclically formed or concatemerized to form cyclic polyribonucleotides. In this method, the 5' end of nucleic acids (e.g., linear polyribonucleotides for cyclization) When the 5' and 3' ends are brought close together, a new covalent bond is formed between the 5' and 3' ends of the molecule. It contains a chemically reactive group that can form a [component]. The 5' end contains an NHS ester reactive group. The 3' end may contain a 3'-amino-terminal nucleotide, and in organic solvents, linear The 3'-amino-terminal nucleotide at the 3' end of the RNA molecule is 5'-NHS-ester. A nucleophilic attack occurs on the amide portion, forming a new 5'- / 3'-amide bond. It is.

[0292] In one embodiment, DNA or RNA ligase is used to process 5'-phosphorylated nucleic acid molecules. (For example, linear polyribonucleotides for cyclization) of nucleic acids (for example, linear nucleic acids) It enzymatically links to the hydroxyl group to form a new phosphorodiester bond. In the reaction, linear polyribonucleotides for cyclization are used according to the manufacturer's protocol. Therefore, incubate with 1-10 units of T4 RNA ligase at 37°C for 1 hour. It is published by New England Biolabs, Ipswich, MA. The ligation reaction involves 5'- and 3'- ligation reactions aligning to assist the enzymatic ligation reaction. This can occur in the presence of linear nucleic acids capable of base pairing with both regions. In one embodiment, In this context, ligation is sprint ligation. For example, SplintR (Registered Trademark) Splint ligases, similar to ligase, are used in splint ligation. It is possible. In sprint ligation, single-stranded polynucleotides such as single-stranded RNA are used. (Sprint) is formed when the two ends are juxtaposed with a single-strand sprint during hub reduction. Designed to hybridize with both ends of a linear polyribonucleotide so that it can be hybridized. Therefore, sprintrigase is a juxtaposition of linear cyclic polyribonucleotides. The ligation of the two terminals can be catalyzed to produce a cyclic polyribonucleotide.

[0293] In one embodiment, a DNA or RNA ligase is used in the synthesis of cyclic polynucleotides. Used. In one embodiment, the 5' end of a linear polyribonucleotide for cyclization. Either the end or the 3' end is a linear polyribo for cyclization during in vitro transcription. The nucleotide is used to circulate the 5' end of the linear polyribonucleotide for cyclization. Active ribozyme sequences that can be ligated to the 3' end of linear polyribonucleotides The ligase ribozyme sequence may be encoded to include the following: Ligase ribozymes are glue Possibly derived from p-I introns, hepatitis delta virus, hairpin ribozymes, or SE It can be selected by LEX (systematic evolution of ligands through exponential enrichment). The immunoglycase reaction can be carried out at a temperature of 0–37°C for 1–24 hours.

[0294] In one embodiment, at least one linear polyribonucleotide is used for cyclization. The non-nucleic acid portion is used to form a cyclic or concatemer. In one embodiment, At least one non-nucleic acid moiety is used to circulate a linear polyribonucleotide for cyclization or To concatemerize, the vicinity of the 5' end of the linear polyribonucleotide for cyclization. and / or may react with a region or feature near the 3' end. In another embodiment, at One non-nucleic acid moiety is a linear polyribonucleotide 5' end and / or 3' end for cyclization. Possible non-nucleic acid portions are: They may be of the same or different species. As a non-limiting example, the non-nucleic acid portion may be hydrophobic or ionic. These bonds may include biodegradable bonds and / or cleavable bonds. Another non-limiting example is... The non-nucleic acid portion is the ligation portion. As yet another non-limiting example, the non-nucleic acid portion is the ligation portion. The portion is an oligonucleotide such as an aptamer or non-nucleic acid linker as described herein. Alternatively, it may be a peptide portion.

[0295] In one embodiment, linear polyribonucleotides for cyclization are used for cyclization. At the 5' and 3' ends of linear polyribonucleotides, in their vicinity, or near them Non-nucleic acid moieties attached to the 5' and 3' ends that cause interatomic and intermolecular surface attraction. Depending on the fraction, it is cyclic or concatemerized. As a non-limiting example, one or more cyclics Linear polyribonucleotides for this purpose are cyclized or concatenated by intermolecular or intramolecular forces. It can be merized. Non-restrictive examples of intermolecular forces include dipole-dipole forces and dipole-induced dipole forces. Polar forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces are examples. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, and resonance bonds. Conjugation, agnostic bond, dipole bond, conjugation, hyperconjugation And antibonding are examples.

[0296] In one embodiment, the linear polyribonucleotide for cyclization is located near the 5' end. and may contain a ribozyme RNA sequence near the 3' end. The ribozyme RNA sequence is a sequence When exposed to the rest of the ribozyme, it can covalently bond to the peptide. In one embodiment Peptides covalently bound to ribozyme RNA sequences near the 5' and 3' ends are mutually It binds to and cyclically or concatemerizes linear polyribonucleotides for cyclization. In another embodiment, it can covalently bind to ribozyme RNA near the 5' and 3' ends. The peptides used are, but are not limited to, those related to the technology of protein ligation. In ligation using various known methods, a linear primary construct or linear mRNA is supplied. After that, they can be made cyclic or concatenated. Linear primary structures or Non-limiting examples of ribozymes or peptides incorporated for use with linear RNA and / or This is a non-exclusive list of methods for covalent bonding, U.S. Patent Application Publication No. 2003008276 This is described in Specification No. 8, and its entire contents are incorporated herein by reference.

[0297] In one embodiment, the linear polyribonucleotide for cyclization is, for example, 5'- Rephosphate is converted to RNA 5' pyrophosphohydrolase (RppH) or ATP diphospho By contact with hydrolase (apirase), it is converted to 5' monophosphate. It may contain the 5' triphosphate of nucleic acids. Alternatively, linear polyribonucleic acid for cyclization. The conversion of the 5'-triphosphate of rheotide to the 5'-monophosphate is (a) cyclic For the purpose of conversion, the 5' nucleotide of linear polyribonucleotides is converted by phosphatase (for example, A Antarctic phosphatase, alkaline phosphatase derived from shrimp Remove all three phosphates by contacting them with a phosphatase (or phosphatase derived from calf small intestine). (b) the 5' nucleotide after step (a) is subjected to the following steps: A two-step reaction including contact with a kinase (e.g., polynucleotide kinase) Therefore, it can be done.

[0298] In one embodiment, the cyclization efficiency of the cyclization method provided herein is low. At least about 10%, at least about 15%, at least about 20%, at least about 25%, and less At least about 30%, at least about 35%, at least about 40%, at least about 45%, and less At least 50%, at least 60%, at least 70%, at least 80%, and less At most about 90%, and at least about 95% or 100%. In one embodiment, the present invention The cyclic efficiency of the cyclic method provided in the details is at least about 40%. In terms of application methods, the provided annularization method has an annularization efficiency of approximately 10% to approximately 100%; For example, the cyclic formation efficiencies are approximately 15%, 20%, 25%, 30%, 35%, and 40%. Approximately 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 80% It may be about 85%, about 90%, about 95%, and about 99%. In one embodiment, the ring The cyclization efficiency is approximately 20% to 80%. In one embodiment, the cyclization efficiency is approximately 30%. The cyclization efficiency is approximately 60%. In one embodiment, the cyclization efficiency is approximately 40%.

[0299] b. Splicing elements In one embodiment, the cyclic polyribonucleotide is at least one splicin Includes splice elements. Exemplary splice elements are incorporated herein by reference in their entirety. Paragraph

[0270] of the brochure, International Patent Publication Number 2019 / 118919 It is described in

[0275] .

[0300] In one embodiment, the cyclic polyribonucleotide is at least one splicin Contains a spra element. In the cyclic polyribonucleotide provided herein, The splicing element can mediate the splicing of cyclic polyribonucleotides, resulting in complete splicing. It can be an ising element. Alternatively, a splicing element can also be a completed splicing It may be a residual splicing element from an event. For example, in some cases, linear polyribonu The splicing element of creotide results in the cyclization of linear polyribonucleotides. It can mediate the rising event, thereby the resulting cyclic polyribonucleotide is this This includes residual splicing elements from splicing-mediated cyclic events, in some cases. Therefore, residual splicing elements cannot mediate any splicing. In this case, residual splicing elements still mediate splicing under certain circumstances. It is possible. In one embodiment, the splicing element has at least one expression It is adjacent to the sequence. In one embodiment, the cyclic polyribonucleotide is adjacent to each expression sequence Includes adjacent splicing elements. In one embodiment, the splicing elements are Located on one or both sides of each expression sequence, the expression product, e.g., peptides and / or polypeptides This brings about the separation of the do.

[0301] In one embodiment, when a cyclic polyribonucleotide is replicated, it is spliced. It includes an internal splice element where the ends are joined together. Some examples are splices Site sequences and short reverse repeats (30-40 nt), e.g., AluSq2, AluJr, and AluSz, reverse array in adjacent introns, Alu elements in adjacent introns , and observed in cis-array elements adjacent to backsplice events A suptable motif (a suptable motif for enrichment), for example, adjacent Located 200 bp before (upstream of) or after the back splice site containing the exon. It may include small introns (<100nt) having (downstream) sequences. Furthermore, cyclic polyribonucleotides are used as internal splicing elements in this specification. This includes at least one repeating nucleotide sequence as described in the section. Such embodiments In this context, the repeating nucleotide sequence includes repeating sequences from the Alu family of introns. It is possible. In one embodiment, the splicing repeat ribosome-binding protein is circular. Polyribonucleotide biosynthesis (e.g., Muscleblind and Quaking (Q...

Claims

[Claim 1] The invention described herein.

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