Circular RNA composition and method

Abstract

Circular RNAs are described herein, along with related compositions and methods. In some embodiments, the circular RNAs of the present invention comprise a post-spliced ​​group I intron fragment, a spacer, an IRES, an optional duplex-forming region, and more than one expression sequence. In some embodiments, the expression sequences are separated by one or more polynucleotide sequences encoding cleavage sites. In some embodiments, the circular RNAs of the present invention have improved expression, functional stability, immunogenicity, ease of manufacture, and / or half-life compared to linear RNA. In some embodiments, the methods and constructs of the present invention result in improved circularization efficiency, splicing efficiency, and / or purity compared to existing RNA circularization approaches.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 62 / 992,518, filed on March 20, 2020, the contents of which are fully incorporated by reference for all purposes. [Background technology]

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

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

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

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

[0006] overview Circular RNA is described herein along with related compositions and methods. In some embodiments, the circular RNA of the present invention comprises a post-splicing group I intron fragment, a spacer, an IRES, an optional double-strand forming region, and one or more expression sequences. In some embodiments, the circular RNA of the present invention comprises a double-strand forming region. In some embodiments, the expression sequences are separated by one or more polynucleotide sequences encoding a cleavage site. In some embodiments, the cleavage site is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide. In some embodiments, the first and second expression sequences are separated by a ribosome skipping element. In some embodiments, each expression sequence encodes a therapeutic protein. In some embodiments, the first expression sequence encodes a cytokine or a functional fragment thereof. In some embodiments, the first expression sequence encodes a transcription factor. In some embodiments, the first expression encodes an immune checkpoint inhibitor. In some embodiments, the first expression sequence encodes a chimeric antigen receptor. In some embodiments, the first expression sequence encodes a first T cell receptor (TCR) chain, and the second expression encodes a second TCR chain. In some embodiments, the circular RNA of the present invention has improved expression, functional stability, ease of manufacture, and / or half-life compared to linear RNA. In some embodiments, the circular RNA of the present invention has reduced immunogenicity. In some embodiments, the methods and constructs of the present invention result in improved cyclization efficiency, splicing efficiency, and / or purity compared to existing RNA cyclization approaches.

[0007] In some embodiments, the circular RNA polynucleotide contains one or more microRNA binding sites. The microRNA binding sites are recognized by microRNA expressed in the liver. In some embodiments, the microRNA binding sites are recognized by miR-122.

[0008] One aspect of this application provides a circular RNA polynucleotide comprising, in the following order: a post-splicing 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, and a post-splicing 5' group I intron fragment.

[0009] In some embodiments, the circular RNA polynucleotide comprises a polynucleotide sequence encoding a cleavage site between a first expression sequence and a second expression sequence. In some embodiments, the cleavage site is a self-cleavage spacer. In some embodiments, the self-cleavage spacer is a 2A self-cleavage peptide.

[0010] In some embodiments, the circular RNA polynucleotide includes a second IRES between a first expression sequence and a second expression sequence. In some embodiments, the first IRES consists of or includes any sequence from SEQ ID NOs: 1 to 72. In some embodiments, the second IRES consists of or includes any sequence from SEQ ID NOs: 1 to 72.

[0011] In some embodiments, the first expression sequence encodes a first therapeutic protein, and the second expression sequence encodes a second therapeutic protein. In some embodiments, the first or second expression sequence encodes an antibody. In some embodiments, the first or second expression sequence encodes a chimeric antigen receptor. In some embodiments, the first or second expression sequence encodes a transcription factor. In some embodiments, the first or second expression sequence encodes a cytokine. In some embodiments, the first or second expression sequence encodes an immunosuppressant molecule. In some embodiments, the first or second expression sequence encodes a costimulatory molecule agonist. In some embodiments, the first or second expression sequence encodes an inhibitor of an immune checkpoint molecule. In some embodiments, the first expression sequence encodes the alpha chain of the T cell receptor (TCR), and the second expression sequence encodes the beta chain of the T cell receptor (TCR).

[0012] In some embodiments, the first expression sequence encodes the beta chain of the T cell receptor (TCR), and the second expression sequence encodes the alpha chain of the T cell receptor (TCR). In some embodiments, the first expression sequence encodes the gamma chain of the T cell receptor (TCR), and the second expression sequence encodes the delta chain of the T cell receptor (TCR). In some embodiments, the first expression sequence encodes the delta chain of the T cell receptor (TCR), and the second expression sequence encodes the gamma chain of the T cell receptor (TCR). In some embodiments, the first expression sequence encodes the T cell receptor (TCR), and the second expression sequence encodes a chemokine. In some embodiments, the first expression sequence encodes a chemokine, and the second expression sequence encodes the T cell receptor (TCR). In some embodiments, the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a PD1 or PDL1 antagonist. In some embodiments, the first expression sequence encodes a PD1 or PDL1 antagonist, and the second expression sequence encodes a chimeric antigen receptor (CAR). In some embodiments, the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a chemokine. In some embodiments, the first expression sequence encodes a chemokine, and the second expression sequence encodes a chimeric antigen receptor (CAR). In some embodiments, the first expression sequence encodes a transcription factor, and the second expression sequence encodes a cytokine.

[0013] In some embodiments, the first expression sequence encodes a T cell receptor (TCR), and the second expression sequence encodes a cytokine. In some embodiments, the cytokine is selected from IL-2, IL-7, IL-12, and IL-15.

[0014] In some embodiments, the first expression sequence encodes a T cell receptor (TCR), and the second expression sequence encodes a transcription factor. In some embodiments, the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25.

[0015] In some embodiments, the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a cytokine. In some embodiments, the cytokine is selected from IL-2, IL-7, IL-12, and IL-15.

[0016] In some embodiments, the first expression sequence encodes a cytokine, and the second expression sequence encodes a transcription factor. In some embodiments, the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. In some embodiments, the cytokine is selected from IL-10, IL-12, and TGFβ.

[0017] In some embodiments, the first expression sequence encodes a transcription factor, and the second expression sequence encodes a chemokine. In some embodiments, the first expression sequence encodes a chemokine, and the second expression sequence encodes a transcription factor. In some embodiments, the transcription factor is selected from FOXP3, STAT5B, and HELIOS. In some embodiments, the chemokine is a CC chemokine, a CXC chemokine, a C chemokine, or a CX3C chemokine. In some embodiments, the chemokine is selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.

[0018] In some embodiments, the first expression sequence encodes a tumor antigen, and the second expression sequence encodes a cytokine. In some embodiments, the first expression sequence encodes a cytokine, and the second expression sequence encodes a tumor antigen. In some embodiments, the antigen is a nascent antigen. In some embodiments, the cytokine is IFNγ.

[0019] In some embodiments, a first expression sequence encodes a CAR, and a second expression sequence encodes a CAR.

[0020] In some embodiments, a first expression sequence encodes a cytokine, and a second expression sequence encodes a cytokine. In some embodiments, the first or second expression sequence encodes a cytokine selected from IL-10, TGFβ, and IL-35. In some embodiments, the first or second expression sequence encodes a cytokine selected from IFNγ, IL-2, IL-7, IL-15, and IL-18.

[0021] In some embodiments, the first expression sequence encodes a T cell receptor (TCR), and the second expression sequence encodes a T cell receptor (TCR). In some embodiments, the first expression sequence encodes a chemokine, and the second expression sequence encodes a chemokine. In some embodiments, the first or second expression sequence encodes an immunosuppressive enzyme. In some embodiments, the first expression sequence encodes a rate-limiting enzyme, and the second expression sequence encodes a flux-restriction enzyme. In some embodiments, the first expression sequence encodes a flux-restriction enzyme, and the second expression sequence encodes a rate-limiting enzyme.

[0022] In some embodiments, the first expression sequence encodes a transcription factor, and the second expression sequence encodes a survival factor. In some embodiments, the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. In some embodiments, the survival factor is selected from BCL-XL.

[0023] In some embodiments, the first or second expression sequence encodes a chaperone protein or complex. In some embodiments, the first expression sequence encodes a transcription factor, and the second expression sequence encodes a chaperone protein or complex. In some embodiments, the first expression sequence encodes a chaperone protein or complex, and the second expression sequence encodes a transcription factor. In some embodiments, the chaperone protein or complex is selected from Skp, Spy, FkpA, SurA, Hsp60, Hsp70, GroEL, GroES, Hsp90, HtpG, Hsp100, ClpA, ClpX, ClpP, and Hsp104. In some embodiments, the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25.

[0024] In some embodiments, one or both of the expression sequences encode a signaling protein.

[0025] In some embodiments, a first expression sequence encodes an enzyme, and a second expression sequence encodes a negative regulatory inhibitor of the first expression sequence. In some embodiments, the first expression sequence encodes a negative regulatory inhibitor protein of the enzyme encoded by the second expression sequence. In some embodiments, the negative regulatory inhibitor is selected from p57kip2, a BAX inhibitor, and TIPE2.

[0026] In some embodiments, the first expression sequence encodes a dominant-negative protein, and the second expression sequence encodes an immune protein. In some embodiments, the first expression sequence encodes an immune protein, and the second expression sequence encodes a dominant-negative protein. In some embodiments, the first or second expression sequence encodes an anti-inflammatory protein.

[0027] In some embodiments, the first expression sequence encodes a transcription factor, and the second expression sequence can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). In some embodiments, the first expression sequence can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and the second expression sequence is a transcription factor. In some embodiments, the expression sequence that can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) is a cytosine deaminase.

[0028] In some embodiments, the circular RNA polynucleotide includes a first spacer between the 5' double-strand formation region and the post-splicing 3' group I intron fragment, and a second spacer between the post-splicing 5' group I intron fragment and the 3' double-strand formation region. In some embodiments, the first and second spacers each have a length of about 10 to about 60 nucleotides. In some embodiments, the first and second double-strand formation regions each have a length of about 9 to about 19 nucleotides. In some embodiments, the first and second double-strand formation regions each have a length of about 30 nucleotides. In some embodiments, IRES includes Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated bug (Plautia stali) enterovirus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, pygmy kite P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, and Ectropis obliqua) picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast (S. cerevisiae) TFIID, budding yeast YAP1, tobacco etch virus, cabbage virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A1220, Pacivirus A 3. IRES sequences from aptamers for saperovirus, rosavirus B, Bakunsa virus, tremovirus A, swine pacivirus 1, PLV-CHN, pacivirus A, sisinivirus, hepacivirus K, hepacivirus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Hupey picorna-like virus, CRPV, sarivirus A BN5, sarivirus A BN2, sarivirus A 02394, sarivirus A GUT, sarivirus A CH, sarivirus A SZ1, sarivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0029] In some embodiments, the circular RNA polynucleotide contains native nucleotides. In some embodiments, the circular RNA polynucleotide consists of native nucleotides. In some embodiments, the expression sequence is codon-optimized.

[0030] In some embodiments, the circular RNA polynucleotide is optimized to lack at least one microRNA binding site present in the equivalent pre-optimization polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site present in the equivalent pre-optimization polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site present in the equivalent pre-optimization polynucleotide.

[0031] In some embodiments, the circular RNA polynucleotide is the circular RNA polynucleotide according to any one of the prior claims, wherein the circular RNA polynucleotide is about 100 nucleotides to about 15 kilobases in length.

[0032] In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in vivo of at least about 20 hours in humans. In some embodiments, the circular RNA polynucleotide has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA polynucleotide has a longer duration of therapeutic effect in human cells than or equivalent to that of equivalent linear RNA polynucleotides containing the same expression sequence. In some embodiments, the circular RNA polynucleotide has a longer duration of therapeutic effect in human cells than that of equivalent linear RNA polynucleotides containing the same expression sequence. In some embodiments, the circular RNA polynucleotide has a longer duration of therapeutic effect in vivo in humans than that of equivalent linear RNA polynucleotides having the same expression sequence. In some embodiments, the circular RNA polynucleotide has a longer in vivo functional half-life in humans than that of equivalent linear RNA polynucleotides having the same expression sequence.

[0033] In another embodiment, this application provides a pharmaceutical composition comprising a cyclic RNA polynucleotide, nanoparticles, and optionally a targeting moiety operably connected to the nanoparticles as described herein. In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles.

[0034] In some embodiments, the pharmaceutical composition comprises a targeting moiety that mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue without cell isolation or purification. In some embodiments, the pharmaceutical composition comprises a targeting moiety operably connected to nanoparticles. In some embodiments, the targeting moiety is an scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or a fragment thereof.

[0035] In some embodiments, the pharmaceutical composition contains less than 1% by weight of polynucleotides, which are double-stranded RNA, DNA sprints, or triphosphorylated RNA in the composition. In some embodiments, the pharmaceutical composition contains less than 1% by weight of polynucleotides and proteins, which are double-stranded RNA, DNA sprints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes in the pharmaceutical composition.

[0036] In another aspect, the Disclosure provides a method for treating a subject requiring treatment, comprising administering a therapeutically effective amount of a composition comprising the cyclic RNA polynucleotides, nanoparticles, and optionally, a targeting moiety operably attached to the nanoparticles as described herein.

[0037] In some embodiments, the composition comprises a targeting moiety that selectively mediates receptor-mediated endocytosis to cells in a selected cell population without cell selection or purification. In some embodiments, the targeting moiety is an scFv, nanobody, peptide, minibody, heavy chain variable region, light chain variable region, or a fragment thereof. In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, or biodegradable nanoparticles.

[0038] In some embodiments, the nanoparticles comprise one or more cationic lipids, ionizable lipids, or polyβ-aminoesters. In some embodiments, the nanoparticles comprise one or more non-cationic lipids. In some embodiments, the nanoparticles comprise one or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids. In some embodiments, the nanoparticles comprise cholesterol. In some embodiments, the nanoparticles comprise arachidonic acid or oleic acid. In some embodiments, the provided nanoparticles comprise one or more cyclic RNA polynucleotides.

[0039] In some embodiments, the target is acute lymphoblastic leukemia; acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B-cell malignancies; bladder cancer (e.g., bladder carcinoma); bone cancer; brain cancer (e.g., medulloblastoma); breast cancer; cancer of the anus, anal canal, or anorectum; eye cancer; intrahepatic bile duct cancer; joint cancer; neck cancer; gallbladder cancer; pleural cancer; cancer of the nose, nasal cavity, or middle ear; oral cancer; vulvar cancer; chronic lymphoblastic leukemia; chronic myeloid carcinoma; colon cancer; esophageal cancer; cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumors; head and neck cancer (e.g., squamous cell carcinoma of the head and neck); Hodgkin's lymphoma Having a cancer selected from the group consisting of: hypopharyngeal cancer; kidney cancer; laryngeal cancer; leukemia; humoral neoplasm; liver cancer; lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma); lymphoma; mesothelioma; mast cell tumor; melanoma; multiple myeloma; nasopharyngeal cancer; non-Hodgkin lymphoma; chronic lymphocytic leukemia B; hairy cell leukemia; acute lymphoblastic leukemia (ALL); Burkitt lymphoma; ovarian cancer; pancreatic cancer; peritoneal cancer; omental cancer; mesenteric cancer; pharyngeal cancer; prostate cancer; rectal cancer; kidney cancer; skin cancer; small intestine cancer; soft tissue cancer; solid tumor; synovial sarcoma; gastric cancer; testicular cancer; thyroid cancer; and ureteral cancer. In some embodiments, the subject has an autoimmune disorder selected from systemic autoimmune diseases typically represented by scleroderma, Graves' disease, Crohn's disease, Sjögren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyglandular endocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and human lupus.

[0040] In another aspect, the present application provides a vector for producing a circular RNA polynucleotide, comprising, in the following order: a 5' double-strand formation region, a 3' group I intron fragment, an intra-sequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, a 5' group I intron fragment, and a 3' double-strand formation region.

[0041] In some embodiments, the vector includes a polynucleotide sequence encoding a cleavage site between a first expression sequence and a second expression sequence. In some embodiments, the cleavage site is a self-cleaving spacer. In some embodiments, the self-cleaving spacer is a 2A self-cleaving peptide.

[0042] In some embodiments, the vector includes a first spacer between the 5' double-strand forming region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-strand forming region. In some embodiments, the first and second spacers each have a length of about 5 to about 60 nucleotides. In some embodiments, the first and second spacers each include an unstructured region of at least 5 nucleotides in length. In some embodiments, the first and second spacers each include a structured region of at least 7 nucleotides in length. In some embodiments, the first and second double-strand forming regions each have a length of about 9 to 50 nucleotides.

[0043] In some embodiments, the vector is codon-optimized. In some embodiments, the vector lacks at least one microRNA binding site that is present in the equivalent pre-optimization polynucleotide.

[0044] In another embodiment, this application provides eukaryotic cells comprising the cyclic RNA polynucleotide described herein. In some embodiments, the eukaryotic cells are human cells. In some embodiments, the eukaryotic cells are immune cells. In some embodiments, the eukaryotic cells are T cells.

[0045] In one embodiment, the circular RNA polynucleotide provided herein comprises, in the following order: a post-splicing 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, and a post-splicing 5' group I intron fragment. In one embodiment, the circular RNA polynucleotide provided herein comprises, in the following order: a post-splicing 3' group I intron fragment, a first intrasequence ribosome entry site (IRES), a first expression sequence, a second IRES, a second expression sequence, and a post-splicing 5' group I intron fragment. In some embodiments, the first and second expression sequences encode different therapeutic proteins. In some embodiments, the first and second expression sequences encode the same therapeutic protein.

[0046] In one embodiment, the circular RNA polynucleotide provided herein is produced from the transcription of a vector and comprises, in the following order: an optional 5' double-stranding region, a post-splicing 3' group I intron fragment, an intra-sequence ribosome entry site (IRES), an expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, a 5' group I intron fragment, and an optional 3' double-stranding region. In one embodiment, the circular RNA polynucleotide provided herein is produced from the transcription of a vector and comprises, in the following order: an optional 5' double-stranding region, a 3' group I intron fragment, a first intra-sequence ribosome entry site (IRES), an expression sequence, a second IRES, a second expression sequence, a 5' group I intron fragment, and an optional 3' double-stranding region. In some embodiments, the circular RNA polynucleotide or vector provided herein comprises 3' and 5' double-stranding regions.

[0047] In some embodiments, the circular RNA polynucleotide includes a first spacer between the 5' double-stranding region and the post-splicing 3' group I intron fragment, and a second spacer between the post-splicing 5' group I intron fragment and the 3' double-stranding region. In some embodiments, the first and second spacers each have a length of about 10 to about 60 nucleotides. In certain embodiments, the first and second double-stranding regions each have a length of about 9 to about 19 nucleotides. In certain other embodiments, the first and second double-stranding regions each have a length of about 30 nucleotides.

[0048] In certain embodiments, IRES is selected from Table 17 having the sequence of IRES, or is a functional fragment or variant thereof. In some embodiments, IRES is Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated stink bug enterovirus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulatavirus-1, human immunodeficiency virus type 1, Homalodisca coagulatavirus-1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, white-winged oak picorna-like virus, brain Myocarditis virus, Drosophila C virus, Human coxsackievirus B3, Brassicaceae tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen bee brood virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute honeybee paralysis virus, Hibiscus chlorotic ring spot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila Antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast TFIID, budding yeast YAP1, tobacco etch virus, cabbage claw virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sarivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A1220, Pacivirus A 3. IRES sequences from aptamers for saperovirus, rosavirus B, Bakunsa virus, tremovirus A, swine pacivirus 1, PLV-CHN, pacivirus A, sisinivirus, hepacivirus K, hepacivirus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Hupey picorna-like virus, CRPV, sarivirus A BN5, sarivirus A BN2, sarivirus A 02394, sarivirus A GUT, sarivirus A CH, sarivirus A SZ1, sarivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0049] In some embodiments, the first and second poly-A sequences each have a length of 15 to 50 nt. In some embodiments, the first and second poly-A sequences each have a length of about 20 to 25 nt.

[0050] In certain embodiments, the circular RNA polynucleotide consists of naturally occurring nucleotides. In some embodiments, the circular RNA contains at least about 80%, at least 90%, at least about 95%, or at least about 99% naturally occurring nucleotides. In certain embodiments, the expression sequence is codon-optimized. In certain embodiments, the circular RNA polynucleotide is optimized to lack at least one microRNA binding site present in the equivalent pre-optimization polynucleotide. In certain embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site present in the equivalent pre-optimization polynucleotide. In certain embodiments, the circular RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site present in the equivalent pre-optimization polynucleotide.

[0051] In some embodiments, the circular RNA polynucleotide is about 100 nucleotides to about 15 kilobases in length. In certain embodiments, the circular RNA polynucleotide of this disclosure has a duration of therapeutic effect in vivo of at least about 20 hours in humans. In certain embodiments, the circular RNA polynucleotide has a functional half-life of at least about 20 hours. In certain embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in human cells that is longer or equivalent to that of a comparable linear RNA polynucleotide containing the same expression sequence. In certain embodiments, the circular RNA polynucleotide has a functional half-life in human cells that is longer than that of a comparable linear RNA polynucleotide containing the same expression sequence. In certain embodiments, the circular RNA polynucleotide has a functional half-life in human cells that is longer than that of a comparable linear RNA polynucleotide having the same expression sequence. In some embodiments, the reference linear RNA polynucleotide is a linear, unmodified or nucleoside-modified, fully processed mRNA containing a cap1 structure and a polyA tail of at least 80 nt in length.

[0052] In some embodiments, the pharmaceutical composition has a functional half-life in human cells that is longer than or equivalent to that of a predetermined threshold. In some embodiments, the pharmaceutical composition has a functional half-life in vivo in human cells that is longer than that of a predetermined threshold. In some embodiments, the functional protein assay is an in vitro luciferase assay. In some embodiments, the functional protein assay involves measuring the level of a protein encoded by the expression sequence of a circular RNA polynucleotide in patient serum or tissue samples. In some embodiments, the predetermined threshold is the functional half-life of a reference linear RNA polynucleotide containing the same expression sequence as the circular RNA polynucleotide. In some embodiments, the pharmaceutical composition has a functional half-life of at least about 20 hours.

[0053] In one embodiment, a pharmaceutical composition comprising a cyclic RNA polynucleotide, nanoparticles, and optionally a targeting moiety operably connected to the nanoparticles is provided herein. In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles. In certain embodiments, the nanoparticles comprise one or more cationic lipids selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (imidazole-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0054] In certain embodiments, the pharmaceutical composition comprises a targeting moiety that mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue without cell isolation or purification. In some embodiments, the targeting moiety is an scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof. In certain embodiments, the cyclic RNA polynucleotide is present in an effective amount to treat an autoimmune disorder or cancer in a human subject requiring it. In certain embodiments, the pharmaceutical composition has an enhanced safety profile compared to a pharmaceutical composition comprising a vector containing exogenous DNA encoding the same expression sequence.

[0055] In certain embodiments, less than 1% by weight of polynucleotides in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA. In certain embodiments, less than 1% by weight of polynucleotides and proteins in the pharmaceutical composition is double-stranded RNA, DNA sprint, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes.

[0056] A method for treating a subject requiring treatment is provided herein, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide, nanoparticles, and optionally, a targeting moiety operably connected to the nanoparticles. In some embodiments, the subject has an autoimmune disorder or cancer.

[0057] In some embodiments, the targeting portion is an scFv, nanobody, peptide, minibody, heavy chain variable region, light chain variable region, or a fragment thereof. In some embodiments, the composition comprises a targeting portion that mediates receptor-mediated endocytosis to selected cells from a selected cell population without the need for cell isolation or purification.

[0058] In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the nanoparticles contain one or more cationic lipids, ionizable lipids, or polyβ-aminoesters. In some embodiments, the nanoparticles contain one or more non-cationic lipids. In some embodiments, the nanoparticles contain one or more PEG-modified lipids, polyglutamate lipids, structural lipids, or hyaluronic acid lipids. In certain embodiments, the nanoparticles contain cholesterol. In some embodiments, the structural lipid is beta-sitosterol. In some embodiments, the structural lipid is not beta-sitosterol. In some embodiments, the nanoparticles contain arachidonic acid or oleic acid. In some embodiments, the nanoparticles contain one or more cyclic RNA polynucleotides.

[0059] In some embodiments, the structural lipids bind to C1q and / or promote the binding of transport vehicles containing the structural lipids to C1q compared to control transport vehicles lacking the structural lipids, and / or increase the uptake of transport vehicles with bound C1q into immune cells compared to control transport vehicles lacking the structural lipids.

[0060] In some embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, or PEG-1. In some embodiments, the PEG-modified lipid is DSPE-PEG(2000).

[0061] In some embodiments, the pharmaceutical composition further comprises a helper lipid. In some embodiments, the helper lipid is DSPC or DOPE.

[0062] In some embodiments, the pharmaceutical composition comprises DOPE, cholesterol, and DSPE-PEG.

[0063] In some embodiments, the transport vehicle contains about 0.5% to about 4% PEG-modified lipids in molar ratio. In some embodiments, the transport vehicle contains about 1% to about 2% PEG-modified lipids in molar ratio.

[0064] In some embodiments, the molar ratio of ionizable lipids:DSPC:cholesterol:DSPE-PEG(2000) is 62:4:33:1.

[0065] In some embodiments, the transport vehicle includes ionic lipids, DOPE, cholesterol, and DSPE-PEG(2000).

[0066] In some embodiments, the molar ratio of ionizable lipids:DSPC:cholesterol:DSPE-PEG(2000) is 50:10:38.5:1.5.

[0067] In some embodiments, the transport vehicle has a nitrogen:phosphate (N:P) ratio of about 3 to about 6.

[0068] In some embodiments, the transport vehicle is formulated for the endosomal release of cyclic RNA polynucleotides.

[0069] In some embodiments, the transport vehicle is capable of binding to APOE. In some embodiments, the transport vehicle interacts with apolipoprotein E (APOE) less than an equivalent transport vehicle loaded with a reference linear RNA having the same expression sequence as a circular RNA polynucleotide. In some embodiments, the outer surface of the transport vehicle is substantially devoid of APOE binding sites.

[0070] In some embodiments, the transport vehicle has a diameter of less than approximately 120 nm. In some embodiments, the transport vehicle does not form aggregates having a diameter greater than 300 nm.

[0071] In some embodiments, the transport vehicle has a diameter of less than approximately 120 nm. In some embodiments, the transport vehicle does not form aggregates having a diameter greater than 300 nm.

[0072] In some embodiments, the transport vehicle has an in vivo half-life of less than approximately 30 hours.

[0073] In some embodiments, the transport vehicle is capable of LDLR-dependent uptake into cells.

[0074] In some embodiments, the pharmaceutical composition is substantially free of linear RNA.

[0075] In some embodiments, the pharmaceutical composition further includes a targeting portion functionally connected to a transport vehicle. In some embodiments, the targeting portion specifically binds to or indirectly binds to an immune cell antigen. In some embodiments, the immune cell antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of CD2, CD3, CD5, CD7, CD8, CD4, beta-7 integrin, beta-2 integrin, and C1q.

[0076] In some embodiments, the pharmaceutical composition further comprises an adapter molecule including a transport vehicle binding site and a cell binding site, wherein the targeting portion specifically binds to the transport vehicle binding site and the cell binding site specifically binds to a target cell antigen. In some embodiments, the target cell antigen is an immune cell antigen. In some embodiments, the immune cell antigen is a T cell antigen, NK cell, NKT cell, macrophage, or neutrophil. In some embodiments, the T cell antigen is selected from the group consisting of CD2, CD3, CD5, CD7, CD8, CD4, beta-7 integrin, beta-2 integrin, CD25, CD39, CD73, A2a receptor, A2b receptor, and C1q. In some embodiments, the immune cell antigen is a macrophage antigen. In some embodiments, the macrophage antigen is selected from the group consisting of mannose receptor, CD206, and C1q.

[0077] In some embodiments, the targeting portion is a small molecule. In some embodiments, the small molecule binds to an exogenous enzyme on an immune cell, the exogenous enzyme being selected from the group consisting of CD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. In some embodiments, the small molecule is mannose, lectin, asibicin, biotin, or digoxigenin.

[0078] In some embodiments, the targeting portion is a single-chain Fv(scFv) fragment, a nanobody, a peptide, a peptide-based macrocycle, a minibody, a small molecule ligand such as folic acid, arginylglycylaspartate (RGD), or phenol-soluble modulin alpha-1 peptide (PSMA1), a heavy chain variable region, a light chain variable region, or a fragment thereof.

[0079] In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than approximately 2 weeks. In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than approximately 1 week. In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than approximately 30 hours. In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than the functional half-life of a cyclic RNA polynucleotide.

[0080] In another embodiment, the application provides a method for treating or preventing a disease, disorder, or pathological condition, comprising administering an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the disease, disorder, or pathological condition is associated with the abnormal expression, activity, or localization of a polypeptide selected from Table 27 or 28. In some embodiments, a cyclic RNA polynucleotide encodes a therapeutic protein. In some embodiments, therapeutic protein expression in the spleen is greater than therapeutic protein expression in the liver. In some embodiments, therapeutic protein expression in the spleen is at least about 2.9 times that in the liver. In some embodiments, the therapeutic protein is not expressed at a functional level in the liver. In some embodiments, the therapeutic protein is not expressed at a detectable level in the liver. In some embodiments, therapeutic protein expression in the spleen is at least about 63% of total therapeutic protein expression.

[0081] In some embodiments, the linear RNA polynucleotide includes a 3' anabaena group I intron fragment and a 5' anabaena group I intron fragment. In some embodiments, the reference RNA polynucleotide includes a reference 3' anabaena group I intron fragment and a reference 5' anabaena group I intron fragment. In some embodiments, the reference 3' anabaena group I intron fragment and the reference 5' anabaena group I intron fragment were generated using an L6-5 permutation substitution site. In some embodiments, the 3' anabaena group I intron fragment and the 5' anabaena group I intron fragment were not generated using an L6-5 permutation substitution site. In some embodiments, the 3' anabaena group I intron fragment includes or consists of a sequence selected from SEQ ID NOs. 112-123 and 125-150. In some embodiments, the 5' anabaena group I intron fragment includes a corresponding sequence selected from SEQ ID NOs. 73-84 and 86-111. In some embodiments, the 5' anabaena group I intron fragment includes or consists of a sequence selected from SEQ ID NOs. 73-84 and 86-111. In some embodiments, the 3' anabaena group I intron fragment includes or consists of a corresponding sequence selected from SEQ ID NOs. 112-124 and 125-150.

[0082] In some embodiments, IRES includes a nucleotide sequence selected from SEQ ID NOs: 348-351. In some embodiments, the reference IRES is CVB3. In some embodiments, IRES is not CVB3. In some embodiments, IRES includes a sequence selected from SEQ ID NOs: 1-64 and 66-72.

[0083] In another aspect, this application discloses cyclic RNA polynucleotides produced from linear RNA disclosed herein.

[0084] In another aspect, the application discloses a circular RNA comprising a 5' to 3' group I intron fragment, an IRES, an expression sequence, and a 5' group I intron fragment, wherein the IRES comprises a nucleotide sequence selected from SEQ ID NOs. 348 to 351.

[0085] In some embodiments, the circular RNA polynucleotide further includes a spacer between the 3' group I intron fragment and the IRES.

[0086] In some embodiments, the cyclic RNA polynucleotide further includes first and second double-stranding regions capable of forming a double helix. In some embodiments, the first and second double-stranding regions each have a length of about 9 to 19 nucleotides. In some embodiments, the first and second double-stranding regions each have a length of about 30 nucleotides.

[0087] In one embodiment, the vector provided herein for producing a circular RNA polynucleotide comprises, in the following order: an optional 5' double-stranding region, a 3' group I intron fragment, an intra-sequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, a 5' group I intron fragment, and an optional 3' double-stranding region. In one embodiment, the vector provided herein for producing a circular RNA polynucleotide comprises, in the following order: an optional 5' double-stranding region, a 3' group I intron fragment, a first intra-sequence ribosome entry site (IRES), a first expression sequence, a second IRES, a second expression sequence, a 5' group I intron fragment, and an optional 3' double-stranding region. In some embodiments, the polynucleotide comprises a 3' double-stranding region and a 5' double-stranding region. In certain embodiments, the vector includes a first spacer between the 5' double-stranding region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-stranding region. In certain embodiments, the first and second spacers each have a length of about 5 to about 60 nucleotides. In certain embodiments, the first and second spacers each have a length of about 8 to about 60 nucleotides. In certain embodiments, the first and second spacers each include an unstructured region of at least 5 nucleotides in length. In certain embodiments, the first and second spacers each include a structured region of at least 7 nucleotides in length. In certain embodiments, the first and second double-stranding regions each have a length of about 9 to 50 nucleotides. In certain embodiments, the vector is codon-optimized. In certain embodiments, the vector lacks at least one microRNA binding site present in an equivalent pre-optimization polynucleotide.

[0088] In one embodiment, the present invention provides a prokaryotic cell comprising a vector for producing a circular RNA polynucleotide, comprising, in the following order: an optional 5' double-stranding region, a 3' group I intron fragment, an intra-sequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, a 5' group I intron fragment, and an optional 3' double-stranding region. In some embodiments, the polynucleotide comprises a 3' double-stranding region and a 5' double-stranding region.

[0089] In one embodiment, eukaryotic cells comprising the cyclic RNA polynucleotide of the present disclosure are provided herein. In some embodiments, the eukaryotic cells are human cells. In some embodiments, the eukaryotic cells are immune cells. In some embodiments, the eukaryotic cells are T cells, NK cells, NKT cells, macrophages, or neutrophils. [Invention 1001] A circular RNA polynucleotide comprising, in the following order: a post-splicing 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, and a post-splicing 5' group I intron fragment. [Invention 1002] A circular RNA polynucleotide of the present invention 1001, comprising a polynucleotide sequence encoding a cleavage site between the first expression sequence and the second expression sequence. [Invention 1003] The cyclic RNA polynucleotide of the present invention 1002, wherein the cleavage site is a self-cleavage spacer. [Invention 1004] The cyclic RNA polynucleotide of the present invention 1003, wherein the self-cleaving spacer is a 2A self-cleaving peptide. [Invention 1005] A circular RNA polynucleotide of the present invention 1001, comprising a second IRES between the first expression sequence and the second expression sequence. [Invention 1006] The circular RNA of the present invention 1005, wherein the first IRES consists of or contains any sequence of sequence numbers 1 to 72. [Invention 1007] The circular RNA of the present invention 1005 or 1006, wherein the second IRES consists of or contains any sequence of sequence numbers 1 to 72. [Invention 1008] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence encodes a first therapeutic protein, and the second expression sequence encodes a second therapeutic protein. [Invention 1009] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes an antibody. [Invention 1010] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes a chimeric antigen receptor. [Invention 1011] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes a transcription factor. [Invention 1012] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes a cytokine. [Invention 1013] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes an immunoinhibitory molecule. [Invention 1014] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes an agonist of a co-stimulatory molecule. [Invention 1015] A cyclic RNA polynucleotide according to any of the prior art inventions, wherein the first expression sequence or the second expression sequence encodes an inhibitor of an immune checkpoint molecule. [Invention 1016] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes the alpha chain of the T cell receptor (TCR), and the second expression sequence encodes the beta chain of the T cell receptor (TCR). [Invention 1017] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes the beta chain of the T cell receptor (TCR), and the second expression sequence encodes the alpha chain of the T cell receptor (TCR). [Invention 1018] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes the gamma chain of the T cell receptor (TCR), and the second expression sequence encodes the delta chain of the T cell receptor (TCR). [Invention 1019] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes the delta chain of the T cell receptor (TCR), and the second expression sequence encodes the gamma chain of the T cell receptor (TCR). [Invention 1020] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a T cell receptor (TCR) and the second expression sequence encodes a cytokine. [Invention 1021] A circular RNA polynucleotide according to any of the invention 1001 to 1008, wherein the first expression sequence encodes a cytokine and the second expression sequence encodes a T cell receptor (TCR). [Invention 1022] The cytokine is selected from IL-2, IL-7, IL-12, and IL-15, and is a cyclic RNA polynucleotide according to any of the inventions 1020 to 1021. [Invention 1023] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a T cell receptor (TCR) and the second expression sequence encodes a chemokine. [Invention 1024] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chemokine and the second expression sequence encodes a T cell receptor (TCR). [Invention 1025] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a T cell receptor (TCR) and the second expression sequence encodes a transcription factor. [Invention 1026] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor and the second expression sequence encodes a T cell receptor (TCR). [Invention 1027] A cyclic RNA polynucleotide according to any of the present invention 1025-1026, wherein the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. [Invention 1028] A circular RNA polynucleotide according to any of the invention 1001 to 1008, wherein the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a PD1 or PDL1 antagonist. [Invention 1029] A circular RNA polynucleotide according to any of the invention 1001 to 1008, wherein the first expression sequence encodes a PD1 or PDL1 antagonist, and the second expression sequence encodes a chimeric antigen receptor (CAR). [Invention 1030] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a cytokine. [Invention 1031] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a cytokine and the second expression sequence encodes a chimeric antigen receptor (CAR). [Invention 1032] The cytokine is selected from IL-2, IL-7, IL-12, and IL-15, and is a cyclic RNA polynucleotide according to any of the invention 1030 to 1031. [Invention 1033] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chimeric antigen receptor (CAR), and the second expression sequence encodes a chemokine. [Invention 1034] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chemokine and the second expression sequence encodes a chimeric antigen receptor (CAR). [Invention 1035] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor and the second expression sequence encodes a cytokine. [Invention 1036] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a cytokine and the second expression sequence encodes a transcription factor. [Invention 1037] The cyclic RNA polynucleotide according to any of the present invention 1035-1036, wherein the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. [Invention 1038] The cytokine is selected from IL-10, IL-12, and TGFβ, and is a cyclic RNA polynucleotide according to any of the present invention 1035 to 1037. [Invention 1039] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor and the second expression sequence encodes a chemokine. [Invention 1040] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chemokine and the second expression sequence encodes a transcription factor. [Invention 1041] The aforementioned transcription factor is selected from FOXP3, STAT5B, and HELIOS, and is a circular RNA according to any of the present invention 1039-1040. [Invention 1042] A cyclic RNA polynucleotide according to any of the invention items 1039 to 1041, wherein the chemokine is a CC chemokine, a CXC chemokine, a C chemokine, or a CX3C chemokine. [Invention 1043] The chemokines are CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, A cyclic RNA polynucleotide of any of the present invention 1039-1042, selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1. [Invention 1044] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a tumor antigen and the second expression sequence encodes a cytokine. [Invention 1045] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a cytokine and the second expression sequence encodes a tumor antigen. [Invention 1046] The aforementioned antigen is a nascent antigen, a cyclic RNA polynucleotide according to any of the present invention 1044 to 1045. [Invention 1047] The cytokine is IFNγ, and the cyclic RNA polynucleotide is any of the 1044-1046 of the present invention. [Invention 1048] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a CAR, and the second expression sequence encodes a CAR. [Invention 1049] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a cytokine, and the second expression sequence encodes a cytokine. [Invention 1050] The cyclic RNA polynucleotide of the present invention 1049, wherein the first or second expression sequence encodes a cytokine selected from IL-10, TGFβ, and IL-35. [Invention 1051] A cyclic RNA polynucleotide according to any of the invention 1049-1050, wherein the first or second expression sequence encodes a cytokine selected from IFNγ, IL-2, IL-7, IL-15, and IL-18. [Invention 1052] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a T cell receptor (TCR), and the second expression sequence encodes a T cell receptor (TCR). [Invention 1053] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chemokine, and the second expression sequence encodes a chemokine. [Invention 1054] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first or second expression sequence encodes an immunosuppressive enzyme. [Invention 1055] A circular RNA polynucleotide according to any of the invention 1001 to 1008, wherein the first expression sequence encodes a rate-limiting enzyme and the second expression sequence encodes a flux restriction enzyme. [Invention 1056] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a flux restriction enzyme and the second expression sequence encodes a rate-limiting enzyme. [Invention 1057] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor and the second expression sequence encodes a survival factor. [Invention 1058] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a survival factor and the second expression sequence encodes a transcription factor. [Invention 1059] The cyclic RNA polynucleotide according to any of the inventions 1057 to 1058, wherein the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. [Invention 1060] The survival factor is a circular RNA polynucleotide selected from BCL-XL, one of the 1057-1059 of the present invention. [Invention 1061] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first or second expression sequence encodes a chaperone protein or complex. [Invention 1062] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor and the second expression sequence encodes a chaperone protein or complex. [Invention 1063] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a chaperone protein or complex, and the second expression sequence encodes a transcription factor. [Invention 1064] The chaperone protein or complex is selected from Skp, Spy, FkpA, SurA, Hsp60, Hsp70, GroEL, GroES, Hsp90, HtpG, Hsp100, ClpA, ClpX, ClpP, and Hsp104, and is a cyclic RNA polynucleotide according to any of the inventions 1061 to 1063. [Invention 1065] The cyclic RNA polynucleotide according to any of the present invention 1061 to 1064, wherein the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. [Invention 1066] A circular RNA polynucleotide according to any of the present invention 1001-1008, wherein one or both expression sequences encode a signaling protein. [Invention 1067] A cyclic RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes an enzyme, and the second expression sequence encodes a negative regulatory inhibitor of the first expression sequence. [Invention 1068] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a negative regulatory inhibitor protein of the enzyme encoded by the second expression sequence. [Invention 1069] The negative regulatory inhibitor is selected from p57kip2, a BAX inhibitor, and TIPE2, wherein the cyclic RNA polynucleotide is one of the 1067-1068 inventions. [Invention 1070] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes a dominant-negative protein and the second expression sequence encodes an immunoprotein. [Invention 1071] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first expression sequence encodes an immunoprotein and the second expression sequence encodes a dominant-negative protein. [Invention 1072] A circular RNA polynucleotide according to any of the present invention 1001 to 1008, wherein the first or second expression sequence encodes an anti-inflammatory protein. [Invention 1073] A cyclic RNA polynucleotide according to any of the invention 1001 to 1008, wherein the first expression sequence encodes a transcription factor, and the second expression sequence can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). [Invention 1074] The first expression sequence is capable of converting 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and the second expression sequence is a transcription factor, wherein each of the cyclic RNA polynucleotides of the present invention 1001 to 1008 is described above. [Invention 1075] A cyclic RNA polynucleotide according to any of the present invention 1073 to 1074, wherein the expression sequence capable of converting 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) is cytosine deaminase. [Invention 1076] A cyclic RNA polynucleotide according to any prior art invention, comprising a first spacer between the 5' double-strand formation region and the post-splicing 3' group I intron fragment, and a second spacer between the post-splicing 5' group I intron fragment and the 3' double-strand formation region. [Invention 1077] The first and second spacers each have a length of approximately 10 to approximately 60 nucleotides, wherein each is a circular RNA polynucleotide according to the present invention 1076. [Invention 1078] The first and second double-stranding regions each have a length of approximately 9 to approximately 19 nucleotides, wherein each is a cyclic RNA polynucleotide according to any of the prior art inventions. [Invention 1079] A cyclic RNA polynucleotide according to any of the invention 1077 to 1078, wherein the first and second double-strand forming regions each have a length of approximately 30 nucleotides. [Invention 1080] The aforementioned IRESs include Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, red imported fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated stink bug (Plautia stali) enterovirus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, small kite P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, and Ectropis obliqua) picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila Reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila Hairless, Budding Yeast (S.Cerevisiae TFIID, budding yeast YAP1, tobacco Etch virus, cabbage crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, human parechovirus 1, black hivirus B, Yc-3, rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, human parechovirus 5, Aichivirus, hepatitis A virus HA16, fopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Saperovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Sisinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Disicisthovirus, Houpei Picorna-like Virus, CRPV, Sarivirus A BN5, Sarivirus A BN2, Sarivirus A 02394, Sarivirus A GUT, Sarivirus A CH, Sarivirus A A cyclic RNA polynucleotide having an IRES sequence from an aptamer for SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G, according to any of the prior art inventions. [Invention 1081] A cyclic RNA polynucleotide consisting of natural nucleotides, according to any of the prior inventions. [Invention 1082] The expression sequence is a cyclic RNA polynucleotide from any of the prior art inventions, wherein the expression sequence is codon-optimized. [Invention 1083] A cyclic RNA polynucleotide from any of the prior inventions, which is optimized to lack at least one microRNA binding site present in an equivalent pre-optimization polynucleotide. [Invention 1084] A cyclic RNA polynucleotide from any of the prior inventions, which is optimized to lack at least one endonuclease-sensitive site present in an equivalent pre-optimization polynucleotide. [Invention 1085] A cyclic RNA polynucleotide from any of the prior inventions, which is optimized to lack at least one RNA editing sensitive site present in an equivalent pre-optimization polynucleotide. [Invention 1086] A circular RNA polynucleotide, either prior to or related to the present invention, having a length of approximately 100 nucleotides to approximately 15 kilobases. [Invention 1087] A cyclic RNA polynucleotide from any of the prior art inventions having a duration of therapeutic effect in vivo in humans of at least approximately 20 hours. [Invention 1088] A cyclic RNA polynucleotide having a functional half-life of at least approximately 20 hours, as described in any of the prior art inventions. [Invention 1089] A cyclic RNA polynucleotide having a duration of therapeutic effect in human cells that is longer than or equal to that of an equivalent linear RNA polynucleotide containing the same expression sequence, as described in any of the prior art inventions. [Invention 1090] A cyclic RNA polynucleotide from any of the prior art inventions having a functional half-life in human cells that is longer than or equal to that of an equivalent linear RNA polynucleotide containing the same expression sequence. [Invention 1091] A cyclic RNA polynucleotide, either of the prior art inventions, having a longer duration of in vivo therapeutic effect in humans than equivalent linear RNA polynucleotides having the same expression sequence. [Invention 1092] A cyclic RNA polynucleotide from the prior art invention having a longer in vivo functional half-life in humans than equivalent linear RNA polynucleotides having the same expression sequence. [Invention 1093] A pharmaceutical composition comprising any of the prior art cyclic RNA polynucleotides, nanoparticles, and optionally a targeting moiety operably connected to the nanoparticles. [Invention 1094] The pharmaceutical composition of the present invention 1093, wherein the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles. [Invention 1095] A pharmaceutical composition according to the present invention 1093 or 1094, comprising a targeting portion, wherein the targeting portion mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue without cell isolation or purification. [Invention 1096] A pharmaceutical composition according to any one of the present invention 1093 to 1095, comprising a targeting portion operably connected to the nanoparticles. [Invention 1097] A pharmaceutical composition according to any one of the invention 1093 to 1096, wherein the targeting portion is an scFv, a nanobody, a peptide, a minibody, a polynucleotide aptamer, a heavy chain variable region, a light chain variable region, or a fragment thereof. [Invention 1098] A pharmaceutical composition according to any one of the invention 1093 to 1097, wherein less than 1% by weight of the polynucleotide in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA. [Invention 1099] A pharmaceutical composition according to any one of the invention 1093 to 1098, wherein less than 1% by weight of the polynucleotide and protein in the pharmaceutical composition is double-stranded RNA, DNA sprint, triphosphorylated RNA, phosphatase protein, protein ligase, and capping enzyme. [Invention 1100] A method for treating a subject requiring treatment, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide, nanoparticles, and optionally a targeting moiety operably connected to the nanoparticles, according to any of invention 1001 to 1092. [Invention 1101] The method of the present invention 1100, wherein the targeting portion is an scFv, a nanobody, a peptide, a minibody, a heavy chain variable region, a light chain variable region, or a fragment thereof. [Invention 1102] The method according to any one of the present invention 1100 to 1101, wherein the nanoparticles are lipid nanoparticles, core-shell nanoparticles, or biodegradable nanoparticles. [Invention 1103] The method according to any one of the present invention 1100 to 1102, wherein the nanoparticles comprise one or more cationic lipids, ionizable lipids, or polyβ-aminoesters. [Invention 1104] The method according to any one of the present invention 1100 to 1103, wherein the nanoparticles contain one or more noncationic lipids. [Invention 1105] The method according to any one of the present invention 1100 to 1104, wherein the nanoparticles comprise one or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids. [Invention 1106] The method according to any one of the present invention 1100 to 1105, wherein the nanoparticles contain cholesterol. [Invention 1107] The method according to any one of the present invention 1100 to 1106, wherein the nanoparticles contain arachidonic acid or oleic acid. [Invention 1108] The method according to any one of the present invention 1100 to 1107, wherein the composition comprises a targeting portion, the targeting portion selectively mediates receptor-mediated endocytosis to cells in a selected cell population without cell selection or purification. [Invention 1109] The method according to any one of the present invention 1100 to 1108, wherein the nanoparticles contain one or more cyclic RNA polynucleotides. [Invention 1110] The aforementioned conditions include acute lymphoblastic leukemia; acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B-cell malignancies; bladder cancer (e.g., bladder carcinoma); bone cancer; brain cancer (e.g., medulloblastoma); breast cancer; cancer of the anus, anal canal, or anorectum; eye cancer; intrahepatic bile duct cancer; joint cancer; neck cancer; gallbladder cancer; pleural cancer; cancer of the nose, nasal cavity, or middle ear; oral cancer; vulvar cancer; chronic lymphoblastic leukemia; chronic myeloid carcinoma; colon cancer; esophageal cancer; cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumors; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin's lymphoma; hypopharyngeal cancer; kidney cancer; laryngeal cancer; and leukemia. A method according to any one of the 1100 to 1109 of the present invention, having a cancer selected from the group consisting of: humoral tumors; liver cancer; lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma); lymphoma; mesothelioma; mast cell tumor; melanoma; multiple myeloma; nasopharyngeal cancer; non-Hodgkin lymphoma; chronic lymphocytic leukemia B; hairy cell leukemia; acute lymphoblastic leukemia (ALL); Burkitt lymphoma; ovarian cancer; pancreatic cancer; peritoneal cancer; omental cancer; mesenteric cancer; pharyngeal cancer; prostate cancer; rectal cancer; kidney cancer; skin cancer; small intestine cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; testicular cancer; thyroid cancer; and ureteral cancer. [Invention 1111] The method according to any one of the present invention 1100 to 1109, wherein the subject has an autoimmune disorder selected from systemic autoimmune diseases typically represented by scleroderma, Graves' disease, Crohn's disease, Sjögren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyglandular endocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and human lupus. [Invention 1112] A vector for producing circular RNA polynucleotides, comprising, in the following order: a 5' double helix formation region, a 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, a 5' group I intron fragment, and a 3' double helix formation region. [Invention 1113] A vector according to the present invention 1112, comprising a polynucleotide sequence encoding a cleavage site between the first expression sequence and the second expression sequence. [Invention 1114] The vector according to the present invention 1112 or 1113, wherein the cutting portion is a self-cutting spacer. [Invention 1115] A vector according to any of invention 1112 to 1114, wherein the self-cleaving spacer is a 2A self-cleaving peptide. [Invention 1116] A vector according to any one of the invention 1112 to 1115, comprising a first spacer between the 5' double-strand forming region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-strand forming region. [Invention 1117] A vector according to any of the inventions 1112 to 1116, wherein the first and second spacers each have a length of about 5 to about 60 nucleotides. [Invention 1118] A vector according to any one of the inventions 1112 to 1117, wherein the first and second spacers each include an unstructured region of at least 5 nucleotides in length. [Invention 1119] A vector according to any one of the inventions 1112 to 1118, wherein the first and second spacers each include a structuring region of at least 7 nucleotides in length. [Invention 1120] A vector according to any one of the invention 1112 to 1119, wherein the first and second double-strand forming regions each have a length of approximately 9 to 50 nucleotides. [Invention 1121] A codon-optimized vector according to any of the inventions 1112 to 1120. [Invention 1122] A vector according to any of the invention 1112 to 1121, lacking at least one microRNA binding site present in equivalent pre-optimization polynucleotides. [Invention 1123] A eukaryotic cell containing any of the circular RNA polynucleotides 1001 to 1092 of the present invention. [Invention 1124] A human cell, a eukaryotic cell according to the present invention 1123. [Invention 1125] An immune cell, a eukaryotic cell according to the present invention 1124. [Invention 1126] A T cell, which is a eukaryotic cell according to the present invention 1125. [Brief explanation of the drawing]

[0090] [Figure 1A] This shows the luminescence in the supernatant of HEK293 cells 24 hours after transfection with circular RNA containing a gaussial alciferase expression sequence and various IRES sequences. [Figure 1B] This shows luminescence in the supernatant of HepG2 cells 24 hours after transfection with circular RNA containing a Gaussial ciferase expression sequence and various IRES sequences. [Figure 1C] This study shows luminescence in the supernatant of 1C1C7 cells 24 hours after transfection using circular RNA containing a Gaussial ciferase expression sequence and various IRES sequences. [Figure 1D] This shows the luminescence in the supernatant of HEK293 cells 24 hours after transfection with circular RNA containing a gaussial alciferase expression sequence and various IRES sequences. [Figure 1E] This shows the luminescence in the supernatant of HEK293 cells 24 hours after transfection with circular RNA containing a gaussial alciferase expression sequence and various IRES sequences.

[0091] [Figure 2] The images show luminescence in the supernatant of HEK293(A), HepG2(B), or 1C1C7(C) cells 24 hours after transfection with circular RNA containing a Gaussial alciferase expression sequence and various IRES sequences of different lengths.

[0092] [Figure 3] This shows the stability of selected IRES constructs in HepG2(A) or 1C1C7(B) cells over 3 days, as measured by luminescence.

[0093] [Figure 4] A and B show protein expression from selected IRES constructs in Jurkat cells, as measured by luminescence from secreted Gaussial Alciferase in the cell supernatant.

[0094] [Figure 5] A and B show the stability of selected IRES constructs in Jurkat cells over 3 days, as measured by luminescence.

[0095] [Figure 6A] This shows a comparison of 24-hour luminescence of modified linear, unpurified circular, and purified circular RNA encoding Gaussial ciferase. [Figure 6B] This shows a comparison of relative luminescence over three days for modified linear, unpurified circular, and purified circular RNA encoding Gausial ciferase.

[0096] [Figure 7A] This shows the induction of IFNγ transcripts in Jurkat cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 7B] This shows the induction of IL-6 transcripts after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA. [Figure 7C] This shows the induction of IL-2 transcripts after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA. [Figure 7D] This shows the transcriptional induction of RIG-I after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA. [Figure 7E]This shows the induction of IFN-β1 transcripts in Jurkat cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 7F] This shows the induction of TNFα transcripts after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA.

[0097] [Figure 8A] This shows a comparison of the luminescence of circular RNA encoding Gaussial ciferase and modified linear RNA in human primary monocytes. [Figure 8B] This shows a comparison of the luminescence of circular RNA encoding Gaussial ciferase and modified linear RNA in macrophages. [Figure 8C] This shows a comparison of the luminescence of circular RNA encoding Gaussial ciferase and modified linear RNA in macrophages.

[0098] [Figure 9] The images show relative luminescence over 3 days (A) or 24 hours (B) in the supernatant of primary T cells transduced using circular RNA containing Gaussial ciferase expression sequences and various IRES sequences.

[0099] [Figure 10A] This shows 24-hour luminescence in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence. [Figure 10B] This shows relative luminescence over 3 days in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence. [Figure 10C] This shows 24-hour luminescence in PBMCs in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial ciferase expression sequence.

[0100] [Figure 11]The HPLC chromatograms (A) and cyclization efficiency (B) of RNA constructs with different permutation sites are shown.

[0101] [Figure 12] The HPLC chromatograms (A) and cyclization efficiency (B) of RNA constructs with different intron and / or permutation sites are shown.

[0102] [Figure 13A] The HPLC chromatograms of three RNA constructs, with and without homology arms, are shown. [Figure 13B] This shows the circularization efficiency of three RNA constructs, with and without homology arms.

[0103] [Figure 14] This shows the cyclization efficiency of three RNA constructs, either without homologous arms or with homologous arms of varying lengths and GC content.

[0104] [Figure 15] A and B show HPLC chromatograms illustrating the contribution of strong homologous arms to improved splicing efficiency, the relationship between cyclization efficiency and nicking in the selected constructs, and combinations of permutation substitution sites and homologous arms assumed to demonstrate improved cyclization efficiency.

[0105] [Figure 16] The images show fluorescence images of T cells co-cultured with Raji cells expressing GFP and firefly luciferase, either electroporated with mock electroporation (left) or electroporated with CAR-encoding circular RNA (right).

[0106] [Figure 17] Bright-field (left), fluorescence (center), and overlay (right) images of T cells co-cultured with Raji cells expressing GFP and firefly luciferase, either electroporated with mock electroporation (top) or electroporated with CAR-encoding circular RNA (bottom).

[0107] [Figure 18] This shows specific lysis of Raji target cells by T cells electroporated with mock electroporation or with circular RNA encoding a different CAR sequence.

[0108] [Figure 19] The images show luminescence in the supernatant of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction using linear or circular RNA containing Gaussial ciferase expression sequences and various IRES sequences (A), and relative luminescence over 3 days (B).

[0109] [Figure 20A] This shows the induction of IFN-β1 transcripts in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 20B] This shows the transcriptional induction of RIG-I in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 20C] This shows the induction of IL-2 transcripts in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 20D] This shows the induction of IL-6 transcripts in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 20E] This shows the induction of IFNγ transcripts in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA. [Figure 20F] This shows the induction of TNFα transcripts in human CD3+ T cells after electroporation using modified linear, unpurified circular, or purified circular RNA.

[0110] [Figure 21A]This shows the specific lysis of Raji target cells by human primary CD3+ T cells electroporated with CAR-encoding circRNA, as determined by detection of firefly bioluminescence. [Figure 21B] This shows the induction of IFNγ transcripts 24 hours after electroporation using different amounts of circular or linear RNA encoding CAR sequences.

[0111] [Figure 22] This shows the specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding CAR at different E:T ratios (A and B), as determined by detection of firefly bioluminescence.

[0112] [Figure 23] This shows the specific lysis of target cells by human CD3+ T cells electroporated with CAR-encoding RNA, 1, 3, 5, and 7 days after electroporation.

[0113] [Figure 24] This demonstrates the specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding CD19 or BCMA target CARs.

[0114] [Figure 25] Table 10b shows the total flux of organs taken from CD-1 mice administered with circular RNA encoding FLuc, formulated with 50% lipid 15, 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0115] [Figure 26] The images show enhanced luminescence in organs isolated from CD-1 mice administered with circular RNA encoding FLuc, formulated with 50% lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0116] [Figure 27A] Table 10a shows the molecular characteristics of lipids 26 and 27. The proton nuclear magnetic resonance (NMR) spectrum of lipid 26 is shown. [Figure 27B] Table 10a shows the molecular characteristics of lipids 26 and 27. The retention time of lipid 26, measured by liquid chromatography-mass spectrometry (LC-MS), is also shown. [Figure 27C] Table 10a shows the molecular characteristics of lipids 26 and 27. The mass spectrum of lipid 26 is shown. [Figure 27D] The molecular characteristics of lipids 26 and 27 from Table 10a are shown. The proton NMR spectrum of lipid 27 is shown. [Figure 27E] Table 10a shows the molecular characteristics of lipids 26 and 27. The retention time of lipid 27, measured by LC-MS, is also shown. [Figure 27F] The molecular characteristics of lipids 26 and 27 from Table 10a are shown. The mass spectrum of lipid 27 is shown.

[0117] [Figure 28A] This document describes the molecular characteristics of lipid 22-S14 and its synthetic intermediates. The NMR spectrum of 2-(tetradecylthio)ethane-1-ol is also shown. [Figure 28B] This document describes the molecular characteristics of lipid 22-S14 and its synthetic intermediates. The NMR spectrum of 2-(tetradecylthio)ethyl acrylate is also shown. [Figure 28C] This shows the molecular characteristics of lipid 22-S14 and its synthetic intermediate. The NMR spectrum of bis(2-(tetradecylthio)ethyl)3,3'-((3-(2-methyl-1H-imidazole-1-yl)propyl)azanegyl)dipropionate (lipid 22-S14) is shown.

[0118] [Figure 29] The NMR spectrum of bis(2-(tetradecylthio)ethyl)3,3'-((3-(1H-imidazole-1-yl)propyl)azanegyl)dipropionate (lipid 93-S14) is shown.

[0119] [Figure 30A] This shows the molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 54 from Table 10a). The proton NMR spectrum of lipid 54 is shown. [Figure 30B] This shows the molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 54 from Table 10a). The retention time of lipid 54 measured by LC-MS is shown. [Figure 30C] The molecular characteristics of heptadecan-9-yl8-((3-(2-methyl-1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 54 from Table 10a) are shown. The mass spectrum of lipid 54 is shown.

[0120] [Figure 31A] This shows the molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 53 from Table 10a). The proton NMR spectrum of lipid 53 is shown. [Figure 31B] This shows the molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 53 from Table 10a). The retention time of lipid 53 measured by LC-MS is shown. [Figure 31C] The molecular characteristics of heptadecan-9-yl8-((3-(1H-imidazole-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 53 from Table 10a) are shown. The mass spectrum of lipid 53 is shown.

[0121] [Figure 32]A shows the total flux from the spleen and liver of CD-1 mice administered with circular RNA encoding firefly luciferase (FLuc), formulated with the ionizable lipid of interest, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. B shows the mean brightness for the in vivo distribution of protein expression.

[0122] [Figure 33] Image A shows an image highlighting the luminescence of organs taken from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Image B shows a whole-body IVIS image of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0123] [Figure 34] Image A shows an image highlighting the luminescence of organs taken from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Image B shows a whole-body IVIS image of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0124] [Figure 35] Image A shows an image highlighting the luminescence of organs taken from CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Image B shows a whole-body IVIS image of CD-1 mice administered with circular RNA encoding FLuc, formulated with ionizable lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0125] [Figure 36A] This image shows an enhanced bioluminescence in organs isolated from c57BL / 6J mice administered with FLuc-encoding, circular RNA encapsulated in lipid nanoparticles formed from lipid 15 (Figure 36A) from Table 10b. [Figure 36B] This image shows the luminescence of organs isolated from c57BL / 6J mice that were administered FLuc-encoded and encapsulated in lipid nanoparticles formed from lipid 53 (Figure 36B) from Table 10a, with circular RNA being highlighted. [Figure 36C] This image shows the luminescence of organs isolated from c57BL / 6J mice that were administered FLuc-encoded and encapsulated in lipid nanoparticles formed from lipid 54 (Figure 36C) from Table 10a. [Figure 36D] PBS was used as a control (Figure 36D).

[0126] [Figure 37] This shows the relative luminescence in the lysate of human PBMCs after 24 hours of incubation with test lipid nanoparticles containing circular RNA encoding firefly luciferase.

[0127] [Figure 38]This shows the expression of GFP(A) and CD19 CAR(B) in human PBMCs after incubation with test lipid nanoparticles containing circular RNA encoding either GFP or CD19 CAR.

[0128] [Figure 39] This shows the expression of anti-mouse CD19 CAR in 1C1C7 cells lipotransfected with circular RNA containing anti-mouse CD19 CAR expression sequences and various IRES sequences.

[0129] [Figure 40] This demonstrates cytotoxicity of mouse T cells by anti-mouse CD19 CAR. The CD19 CAR is encoded and expressed in mouse T cells by electroporated circular RNA.

[0130] [Figure 41A] Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR. [Figure 41B] Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR. [Figure 41C] Figures 40A and 40B and 40B show the number of B cells in peripheral blood (Figure 40A and 40B) or spleen (Figure 40C) of C57BL / 6J mice injected every other day with test lipid nanoparticles containing circular RNA encoding anti-mouse CD19 CAR.

[0131] [Figure 42] The expression levels of anti-human CD19 CAR expressed from circular RNA are being compared with those expressed from linear mRNA.

[0132] [Figure 43]The cytotoxic effect of anti-human CD19 CAR expressed from circular RNA is being compared with that expressed from linear mRNA.

[0133] [Figure 44] This shows cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMA CAR) expressed from a single circular RNA in T cells.

[0134] [Figure 45A] Representative FACS plots are shown along with the frequency of tdTomato expression in various spleen immune cell subsets after treatment with LNPs formed from lipids 27 or 26 from Table 10a or lipid 15 from Table 10b. [Figure 45B] This shows the quantitative proportion of myeloid cells, B cells, and T cells expressing tdTomato, equivalent to the proportion of each cell population successfully transfected with Cre circular RNA (mean + standard deviation, n=3). [Figure 45C] This shows the percentage of additional spleen immune cell populations, including NK cells, classical monocytes, non-classical monocytes, neutrophils, and dendritic cells, expressing tdTomato after treatment with lipids 27 and 26 (mean + standard deviation, n=3).

[0135] [Figure 46A] This shows an exemplary RNA construct design with a built-in polyA sequence in the intron. [Figure 46B] This shows the chromatographic trace of unpurified circular RNA. [Figure 46C] This shows the chromatographic trace of affinity-purified circular RNA. [Figure 46D] This document describes the immunogenicity of circular RNA prepared under various IVT conditions and purification methods. (Commercial = Commercial IVT mix; Custom = Customized IVT mix; Aff = Affinity purification; Enz = Enzyme purification; GMP:GTP ratio = 8, 12.5, or 13.75).

[0136] [Figure 47]A shows an exemplary RNA construct design with a specific binding sequence as an alternative to poly(A) for hybridization purification. B shows a chromatographic trace of unpurified circular RNA. C shows a chromatographic trace of affinity-purified circular RNA.

[0137] [Figure 48] A shows the chromatographic trace of unpurified circular RNA encoding dystrophin. B shows the chromatographic trace of enzyme-purified circular RNA encoding dystrophin.

[0138] [Figure 49] The expression (Figure 49A) and stability (Figure 49B) of purified circRNAs with different 5' spacers between the 3' intron fragment / 5' inner double-strand region and IRES in Jurkat cells are compared. (AC = only A and C were used in the spacer sequence; UC = only U and C were used in the spacer sequence).

[0139] [Figure 50] This shows the level and stability of luminescence expression in primary T cells from circular RNAs containing the original or modified IRES elements.

[0140] [Figure 51] This shows the luminescence expression levels and expression stability in HepG2 cells from circular RNAs containing the original or modified IRES elements shown.

[0141] [Figure 52] This shows the luminescence expression levels and expression stability in 1C1C7 cells from circular RNAs containing the original or modified IRES elements shown.

[0142] [Figure 53]This shows the luminescence expression levels and expression stability in HepG2 cells from circular RNAs containing IRES elements with inserted untranslated regions (UTRs) or hybrid IRES elements. "Scr" refers to the scramble used as a control.

[0143] [Figure 54] This study demonstrates the luminescence expression levels and expression stability in 1C1C7 cells from circular RNA containing an IRES operably ligated to a sequence encoding Gaussial ciferase and a variable stop codon cassette.

[0144] [Figure 55] This shows the luminescence expression level and expression stability in 1C1C7 cells from circular RNA containing an IRES and a variable untranslated region (UTR) inserted before the start codon of the sequence encoding Gaussial ciferase.

[0145] [Figure 56] This shows the expression level of human erythropoietin (hEPO) in Huh7 cells from a circular RNA containing two miR-122 target sites downstream of the hEPO-coding sequence.

[0146] [Figure 57] This image shows the CAR expression levels in peripheral blood (A) and spleen (B) after treatment with LNPs containing circular RNA expressing anti-CD19 CARs. Anti-CD20 (aCD20) and circular RNA encoding luciferase (oLuc) were used for comparison.

[0147] [Figure 58]This shows the overall frequency of anti-CD19 CAR expression, the frequency of anti-CD19 CAR expression on the cell surface, and the impact of IRES-specific circular RNA encoding anti-CD19 CAR on T cells on the antitumor response. A shows the geometric mean fluorescence intensity of anti-CD19 CAR, B shows the percentage of anti-CD19 CAR expression, and C shows the percentage of target cell lysis performed by anti-CD19 CAR. (CK = goat's gall virus; AP = apodems spicorniavirus; CK* = codon-optimized goat's gall virus; PV = parabovirus; SV = Salivirus).

[0148] [Figure 59] This shows the CAR expression levels of A20 FLuc target cells after treatment with an IRES-specific circular RNA construct.

[0149] [Figure 60] This shows the levels of luminescence expression of cytoplasmic (A) and surface (B) proteins from circular RNA in primary human T cells.

[0150] [Figure 61A] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61B] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61C]This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61D] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61E] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61F] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells. [Figure 61G] This shows luminescence expression in human T cells treated with an IRES-specific circular construct. Expression in the circular RNA construct was compared with that of linear mRNA. Figures 61A, 61B, and 61G show Gaussian luciferase expression in multiple donor cells. Figures 61C, 61D, 61E, and 61F show firefly luciferase expression in multiple donor cells.

[0151] [Figure 62]This study demonstrates the expression of anti-CD19 CAR(A and B) and anti-BCMA CAR(B) in human T cells after treatment with lipid nanoparticles containing circular RNA encoding either anti-CD19 or anti-BCMA CAR in K562 cells expressing firefly luciferase.

[0152] [Figure 63] This shows the anti-CD19 CAR expression levels resulting from in vitro electroporation delivery of circular RNA encoding anti-CD19 CAR in a specific antigen-dependent manner. A shows Nalm6 cell lysis by anti-CD19 CAR. B shows K562 cell lysis by anti-CD19 CAR.

[0153] [Figure 64A] This shows transfection of LNPs expressing green fluorescent protein (GFP) via the use of ApoE3 in a solution containing LNPs and circular RNA. Figure 64A shows the viability results. Figures 64B, 61C, 61D, and 64E provide expression frequencies from multiple donors. [Figure 64B] This shows transfection of LNPs expressing green fluorescent protein (GFP) via the use of ApoE3 in a solution containing LNPs and circular RNA. Figure 64A shows the viability results. Figures 64B, 61C, 61D, and 64E provide expression frequencies from multiple donors. [Figure 64C] This shows transfection of LNPs expressing green fluorescent protein (GFP) via the use of ApoE3 in a solution containing LNPs and circular RNA. Figure 64A shows the viability results. Figures 64B, 61C, 61D, and 64E provide expression frequencies from multiple donors. [Figure 64D] This shows transfection of LNPs expressing green fluorescent protein (GFP) via the use of ApoE3 in a solution containing LNPs and circular RNA. Figure 64A shows the viability results. Figures 64B, 61C, 61D, and 64E provide expression frequencies from multiple donors. [Figure 64E]This shows transfection of LNPs expressing green fluorescent protein (GFP) via the use of ApoE3 in a solution containing LNPs and circular RNA. Figure 64A shows the viability results. Figures 64B, 61C, 61D, and 64E provide expression frequencies from multiple donors. [Modes for carrying out the invention]

[0154] Detailed explanation The present invention provides, in particular, methods and compositions for treating autoimmune disorders or cancer based on circular RNA therapy. Specifically, the present invention provides a method for treating autoimmune disorders or cancer by administering a composition comprising RNA encoding two therapeutic proteins to a subject in need of treatment in an effective dose and dosing interval such that the intensity, severity, or frequency of at least one symptom or feature of the autoimmune disorder or cancer is reduced or the onset is delayed.

[0155] In certain embodiments, provided herein is a vector for constructing circular RNA, the vector comprising an optional 5' double helix region, a 3' group I intron fragment, an optional first spacer, an intrasequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, an optional second spacer, a 5' group I intron fragment, and an optional 3' double helix region. In certain embodiments, provided herein is a vector for constructing circular RNA, the vector comprising an optional 5' double helix region, a 3' group I intron fragment, an optional first spacer, a first intrasequence ribosome entry site (IRES), a first expression sequence, a second IRES, a second expression sequence, an optional second spacer, a 5' group I intron fragment, and an optional 3' double helix region. In some embodiments, these elements are arranged in the vector in the order described above. In some embodiments, the polynucleotide comprises a 3' double helix region and a 5' double helix region. In some embodiments, the vector further includes an internal 5' double helix formation region between the 3' group I intron fragment and the IRES, and an internal 3' double helix formation region between the expression sequence and the 5' group I intron fragment. In some embodiments, the internal double helix formation regions can form a double helix between themselves but not with the external double helix formation region. In some embodiments, the internal double helix formation regions are part of first and second spacers. Additional embodiments include circular RNA polynucleotides comprising circular RNA polynucleotides prepared using the vectors provided herein, compositions comprising such circular RNA, cells comprising such circular RNA, and methods for using and preparing such vectors, circular RNA, compositions, and cells.

[0156] In some embodiments, methods are provided herein that include administering cyclic RNA polynucleotides provided herein to cells for therapeutic or useful protein production. In some embodiments, the method is advantageous in that it provides the production of a desired polypeptide in eukaryotic cells with a longer half-life than linear RNA because the cyclic RNA is resistant to ribonucleases.

[0157] Because circular RNA polynucleotides lack the free ends required for exonuclease-mediated degradation, they offer resistance to several mechanisms of RNA degradation and a longer half-life compared to equivalent linear RNA. Circularization can stabilize RNA polynucleotides, which generally have short half-lives, potentially improving the overall efficacy of exogenous mRNA in various applications. In one embodiment, the functional half-life of the circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells such as human cells), as evaluated by protein synthesis, is at least 20 hours (e.g., at least 80 hours).

[0158] 1.Definition As used herein, the terms "circRNA," "cyclic polyribonucleotide," or "cyclic RNA" are interchangeable and refer to polyribonucleotides that form a cyclic structure through covalent bonds.

[0159] As used herein, the term “3' group I intron fragment” refers to a sequence having 75% or greater similarity to the 3' proximal end of a natural group I intron, including a splice site dinucleotide and optionally a stretch of the natural exon sequence.

[0160] As used herein, the term “5' group I intron fragment” refers to a sequence having 75% or greater similarity to the 5' proximal end of a natural group I intron, including a splice site dinucleotide and optionally a stretch of the natural exon sequence.

[0161] As used herein, the term “permutation site” refers to a site within a group I intron where a cleavage occurs prior to the permutation of the intron. This cleavage produces 3' and 5' group I intron fragments, which are permuted onto either side of the stretch of the circularized precursor RNA.

[0162] As used herein, the term “splice site” refers to a dinucleotide that is partially or completely contained within a group I intron, between which a phosphodiester bond is cleaved during RNA cyclization.

[0163] Expression sequences in polynucleotide constructs can be separated by "cleavage site" sequences, which allow the polypeptide encoded by the expression sequence to be expressed separately by the cell once translated.

[0164] A "self-cleaving peptide" refers to a peptide that is translated without a peptide bond between two adjacent amino acids, or functions to be immediately cleaved or separated into distinct first and second polypeptides without any external cleavage activity when a polypeptide containing a protein and a self-cleaving peptide is produced.

[0165] As used herein, the term “therapeutic protein” means any protein that, when administered directly or indirectly to a subject in the form of translated nucleic acid, has therapeutic, diagnostic, and / or prophylactic effects, and / or elicits desirable biological and / or pharmacological effects.

[0166] The α and β chains of the αβ TCR are generally considered to each have two domains or regions, namely a variable domain and a constant domain / region. The variable domain consists of a linkage of a variable region and a binding region. Therefore, in this specification and the claims, the term "TCR alpha variable domain" refers to the linkage of the TRAV and TRAJ regions, and the term "TCR alpha constant domain" refers to the extracellular TRAC region or C-terminally cleaved TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the linkage of the TRBV and TRBD / TRBJ regions, and the term "TCR beta constant domain" refers to the extracellular TRBC region or C-terminally cleaved TRBC sequence.

[0167] As used herein, the term “immunogenic” refers to the potential to induce an immune response to a substance. An immune response, referring to the possibility of inducing an immune response, can be induced when an organism’s immune system or certain types of immune cells are exposed to an immunogenic substance. The term “non-immunogenic” refers to exceeding a detectable threshold for an immune response to a substance. An immune response, referring to the absence or non-existence of an immune response, is not detectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic substance. In some embodiments, non-immunogenic cyclic polyribonucleotides, such as those provided herein, do not induce an immune response exceeding a predetermined threshold when measured by an immunogenic assay. In some embodiments, an innate immune response is not detectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic cyclic polyribonucleotide, such as those provided herein. In some embodiments, an adaptive immune response is not detectable when an organism’s immune system or certain types of immune cells are exposed to a non-immunogenic cyclic polyribonucleotide, such as those provided herein.

[0168] As used herein, the term "cyclization efficiency" refers to a measured value of the resulting cyclic polyribonucleotide compared to its linear starting material.

[0169] As used herein, the term “translation efficiency” refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of transcript encoding a protein or peptide.

[0170] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include species including purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, and pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Nucleotide analogs include nucleotides having modifications to the chemical structure of a base, sugar, and / or phosphate, including 5'-pyrimidine modifications, 8'-purine modifications, cytosine extra-ring amine modifications, and 5-bromouracil substitutions; and 2'-sugar modifications, including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH group is substituted with a group such as H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN (where R is an alkyl moiety as defined herein). Nucleotide analogs are also intended to include nucleotides accompanied by bases, such as inosine, quasine, and xanthine; sugars, such as 2'-methylribose; and unnatural phosphodiester linkages, such as methylphosphonic acid, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseuduridine, and 6-methyladenosine.

[0171] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and describe polymers composed of nucleotides, such as deoxyribonucleotides or ribonucleotides, of any length, e.g., more than about 2 nucleotides, more than about 10 nucleotides, more than about 100 nucleotides, more than about 500 nucleotides, more than 1000 nucleotides, or up to about 10,000 nucleotides or more, which can be produced enzymatically or synthetically (as described, e.g., in U.S. Patent No. 5,948,902 and the references cited herein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids, e.g., participate in Watson-Crick base-pairing interactions. Naturally occurring nucleic acids are composed of nucleotides containing guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0172] As used herein, the terms “ribonucleic acid” and “RNA” mean polymers composed of ribonucleotides.

[0173] As used herein, the terms “deoxyribonucleic acid” and “DNA” mean polymers composed of deoxyribonucleotides.

[0174] "Isolated" or "purified" generally refers to the isolation of a substance (e.g., in some embodiments, a compound, polynucleotide, protein, polypeptide, polynucleotide composition, or polypeptide composition) such that it constitutes a significant percentage (e.g., more than 1%, more than 2%, more than 5%, more than 10%, more than 20%, more than 50%, or more, typically up to about 90%–100%) of the sample in which it is present. In certain embodiments, substantially purified components constitute at least 50%, 80%–85%, and 90%–95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-dependent precipitation. Generally, a substance is purified when it is present in the sample in amounts greater than naturally occurring compared to other components of the sample.

[0175] As used herein, the terms “double-stranded,” “double-stranded,” or “hybridized” refer to nucleic acids formed by the hybridization of two single-stranded nucleic acids containing complementary sequences. In most cases, genomic DNA is double-stranded. Sequences can be fully complementary or partially complementary.

[0176] As used herein, “unstructured” with respect to RNA refers to an RNA sequence that is not predicted by RNAFold software or similar predictive tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease-protected assays.

[0177] As used herein, “structured” with respect to RNA means an RNA sequence that is predicted by RNAFold software or similar predictive tools to form a structure (e.g., a hairpin loop) with itself or with other sequences within the same RNA molecule.

[0178] As used herein, two “double-chain forming regions,” “homologous arms,” or “homologous regions” are complementary or complementary to each other if the two regions share a sufficient level of sequence identity with respect to each other’s reverse complement in order to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences have “homology” if they are identical or share sequence identity with respect to their reverse complement or “complementary” sequence. The percentage of sequence identity between a double-chain forming region and the reverse complement of the corresponding double-chain forming region can be any percentage of sequence identity that allows hybridization to occur. In some embodiments, an internal double-chain forming region of a polynucleotide of the present invention may form a double chain with another internal double-chain forming region but not with an external double-chain forming region.

[0179] Linear nucleic acid molecules are said to have a "5' end" and a "3' end" because nucleic acid phosphodiester linkages occur at the 5' and 3' carbon atoms of the sugar portion of the substituted mononucleotide. The terminal nucleotide of a polynucleotide where the new linkage is at the 5' carbon is its 5' terminal nucleotide. The terminal nucleotide of a polynucleotide where the new linkage is at the 3' carbon is its 3' terminal nucleotide. As used herein, a terminal nucleotide is a nucleotide located at the 3' or 5' end.

[0180] "Transcription" means the formation or synthesis of RNA molecules by RNA polymerase using DNA molecules as templates. The present invention is not limited to the RNA polymerase used for transcription. For example, in some embodiments, T7 type RNA polymerase can be used.

[0181] "Translation" refers to the formation of polypeptide molecules by ribosomes based on an RNA template.

[0182] It should be understood that the terms used herein are intended solely to describe and not to limit a particular embodiment. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “cell” includes a combination of two or more cells, or an entire cell culture; a reference to “polynucleotide” practically includes many copies of that polynucleotide. Where used herein, the term “or” is understood to be inclusive unless specifically stated or evident from the context. Unless defined herein and in the remainder of this specification, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the invention pertains.

[0183] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerance range in the art, for example, within two standard deviations of the mean. “About” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “about.”

[0184] As used herein, the term “encode” broadly refers to any process that uses information about a polymer macromolecule to direct the creation of a second molecule distinct from a first molecule. The second molecule may have a chemical structure different from the chemical properties of the first molecule.

[0185] "Co-administration" means administering the therapeutic agent provided herein in combination with one or more additional therapeutic agents, at a time interval that is sufficiently close to the time that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa.

[0186] As used herein, the terms “treat” and “prevent,” and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, the degree to which a person skilled in the art would recognize a treatment or prevention as having potential benefit or therapeutic effect varies, and is provided in the manner disclosed herein. Treatment or prevention may include treatment or prevention of one or more conditions or symptoms of a disease. Also, for the purposes of this specification, “prevention” may include delaying the onset of a disease, or its symptoms or conditions.

[0187] As used herein, “autoimmunity” is defined as a persistent and progressive immune response to non-infectious autoantigens, distinct from infectious non-self antigens of bacterial, viral, fungal, or parasitic origin that invade and persist within mammals and humans. Autoimmune diseases include scleroderma, Graves' disease, Crohn's disease, Sjögren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyglandular endocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, and thyroiditis, as well as systemic autoimmune diseases typically represented by human lupus. As used herein, “autoantigen” or “self-antigen” refers to an antigen or epitope that is specific to a mammal and is immunogenic in that mammal.

[0188] As used herein, the term “expression sequence” may refer to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide, a regulatory nucleic acid, or a non-coding nucleic acid. An exemplary expression sequence encoding a peptide or polypeptide may contain multiple nucleotide triads, each of which may encode an amino acid and be referred to as a “codon.”

[0189] As used herein, “spacer” refers to a region of a polynucleotide sequence ranging from one nucleotide to hundreds or thousands of nucleotides that separates two other elements along the polynucleotide sequence. The sequence may be defined or random. Spacers are typically non-coding. In some embodiments, the spacer includes a double-strand forming region.

[0190] As used herein, “intrasequence ribosome entry site” or “IRES” refers to an RNA sequence or structural element in the size range of 10 nt to 1000 nt or more that can initiate polypeptide translation in the absence of a typical RNA cap structure. IRESs are typically about 500 nt to 700 nt in length.

[0191] As used herein, “miRNA site” refers to a stretch of nucleotides within a polynucleotide that can form a double helix with a native miRNA sequence of at least eight nucleotides.

[0192] As used herein, “endonuclease site” refers to a stretch of nucleotides within a polynucleotide that can be recognized and cleaved by an endonuclease protein.

[0193] As used herein, “bisistronic RNA” refers to a polynucleotide containing two expression sequences encoding two different proteins. These expression sequences are often separated by cleavable peptides such as 2A sites or IRES sequences. They can be separated by ribosome skipping elements or protease cleavage.

[0194] As used herein, the term “ribosome skipping element” refers to a nucleotide sequence encoding a short peptide sequence capable of causing the generation of two peptide chains from the translation of one RNA molecule. While we do not wish to be bound by theory, it is assumed that ribosome skipping elements function by (1) terminating the translation of the first peptide chain and restarting the translation of the second peptide chain, or (2) cleaving peptide bonds in the peptide sequence encoded by the ribosome skipping element, either by the intrinsic protease activity of the encoded peptide or by another protease in the environment (e.g., cytosol).

[0195] As used herein, the term “co-formulation” refers to a nanoparticle formulation comprising two or more nucleic acids or nucleic acids and other active drugs. Typically, the ratio is equimolar, or it is defined by the ratio measurement of two or more nucleic acids or nucleic acids and other active drugs.

[0196] As used herein, “transport vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients, etc., that are generally intended for use in connection with the administration of biologically active substances, including nucleic acids.

[0197] As used herein, the term "lipid nanoparticles" refers to a transport vehicle comprising one or more lipids (for example, in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0198] As used herein, the term "cationic lipid" refers to any of many lipid species that have a net positive charge at a selected pH, such as physiological pH.

[0199] As used herein, the term "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid.

[0200] As used herein, the term "anionic lipid" refers to any of the many lipid species that have a net negative charge at a selected pH, such as physiological pH.

[0201] As used herein, the term "ionizable lipid" refers to any of many lipid species that have a net positive charge at a selected pH, such as physiological pH 4, and a neutral charge at other pH levels, such as physiological pH 7.

[0202] In some embodiments, the lipids disclosed herein, e.g., ionizable lipids, include one or more cleavable groups. The terms “cleaved” and “cleavable,” as used herein, mean that one or more chemical bonds (e.g., one or more of covalent bonds, hydrogen bonds, van der Waals forces, and / or ionic interactions) within or between adjacent atoms of the functional group in question can be broken (e.g., hydrolyzed) or broken upon exposure to selected conditions (e.g., enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in certain embodiments, a disulfide group that can be cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional group that can be cleaved upon exposure to selected biological conditions. For example, a disulfide group can be cleaved enzymatically or by hydrolysis, oxidation, or reduction. Upon cleavage of such a disulfide functional group, one or more functional moieties or groups (e.g., one or more of the head group and / or tail group) attached to it may be released. Exemplary cleavable groups may include, but are not limited to, disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl and aryl esters). In certain embodiments, the cleavable group is not an ester or ether group. In some embodiments, the cleavable group is bonded to one or more functional moieties or groups (e.g., at least one head group and at least one tail group) (e.g., bonded by one or more of hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds). In certain embodiments, at least one of the functional moieties or groups is hydrophilic (e.g., a hydrophilic head group comprising one or more of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino, and pyridyl).

[0203] As used herein, the term "hydrophilic" is used to qualitatively indicate that a functional group prefers water and typically such a group is water-soluble. For example, compounds are disclosed herein that include a cleavable disulfide (S-S) functional group attached to one or more hydrophilic groups (e.g., hydrophilic head groups), and such hydrophilic groups include, or are selected from, the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl.

[0204] In certain embodiments, at least one of the functional groups of a moiety that includes a compound disclosed herein is essentially hydrophobic (e.g., a hydrophobic tail group that includes a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used to qualitatively indicate that a functional group dislikes water and typically such a group is not water-soluble. For example, compounds are disclosed herein that include a cleavable functional group (e.g., a disulfide (S-S) group) attached to one or more hydrophobic groups, and such hydrophobic groups include one or more naturally occurring lipids, such as cholesterol, and / or optionally substituted, variably saturated or unsaturated C6-C 20 alkyl and / or optionally substituted, variably saturated or unsaturated C6-C 20 acyl.

[0205] The compounds described herein may also include one or more isotope substitutions. For example, H may be in any isotopic form that includes 1 H, 2 H (D or deuterium), and 3 H (T or tritium); C may be in any isotopic form that includes 12 C, 13 C, and 14 C; O may be in any isotopic form that includes 16 O and 18 O; F may be in any isotopic form that includes 18 F and 19 F.

[0206] In describing the present invention, which may include compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms, where present, have the following meanings unless otherwise indicated. It should also be understood that, as used herein, any of the parts defined below may be substituted with various substituents, and that each definition is intended to include substituted parts within the ranges set forth below. Unless otherwise specified, the term “substituted” is defined as set forth below. It should also be understood that, as used herein, the terms “group” and “radical” may be considered interchangeable.

[0207] When a range of values ​​is enumerated, it is intended that each value and sub-range within that range be included. For example, "C 1-6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 It is intended to include alkyl groups.

[0208] In certain embodiments, the compounds disclosed herein include, for example, at least one hydrophilic head group and at least one hydrophobic tail group, each bonded to at least one cleavable group, thereby making such compounds amphiphilic. When used herein to describe a compound or composition, the term “amphiphilic” means the ability to dissolve in both polar (e.g., water) and nonpolar (e.g., lipid) environments. For example, in certain embodiments, the compounds disclosed herein include at least one lipophilic tail group (e.g., cholesterol or C6-C 20It comprises an alkyl group and at least one hydrophilic head group (e.g., imidazole), each bonded to a cleavable group (e.g., disulfide).

[0209] It should be noted that the terms “head group” and “tail group” used are used to describe the compounds of the present invention and, in particular, the functional groups comprising such compounds, and to facilitate reference in describing the orientation of one or more functional groups relative to other functional groups. For example, in certain embodiments, a hydrophilic head group (e.g., guanidinium) is bonded to a cleavable functional group (e.g., a disulfide group) (e.g., by one or more of the following: hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds), which then bonds to a hydrophilic tail group (e.g., cholesterol).

[0210] As used herein, the term "alkyl" refers to both linear and branched C1-C1 chains. 40 Hydrocarbons (e.g., C6-C) 20 This refers to hydrocarbons and includes both saturated and unsaturated hydrocarbons. In certain embodiments, alkyl may include one or more cyclic alkyls and / or one or more heteroatoms, e.g., oxygen, nitrogen, or sulfur, and may be optionally substituted with substituents (e.g., one or more of alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide). In certain embodiments, the alkyl intended includes (9Z,12Z)-octadeca-9,12-diene. For example, "C6-C 20 The use of names such as "alkyl" is intended to refer to alkyl groups having the described range of carbon atoms (e.g., linear or branched, including alkenes and alkyls). In some embodiments, alkyl groups have 1 to 10 carbon atoms ("C"). 1-10 (alkyl). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C"). 1-9 (alkyl). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C"). 1-8 (alkyl). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C"). 1-7("alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C"). 1-6 (alkyl). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C"). 1-5 (alkyl). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C"). 1-4 (alkyl). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C"). 1-3 Alkyl). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C"). 1-2 ("Alkyl"). In some embodiments, the alkyl group has one carbon atom ("C1 alkyl"). 1-6 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, and hexyl.

[0211] As used herein, “alkenyl” refers to a radical of a linear or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) ("C 2-20 ("Alkenyl"). In certain embodiments, the alkenyl does not contain any triple bond. In some embodiments, the alkenyl has 2 to 10 carbon atoms ("C"). 2-10 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C"). 2-9 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C"). 2-8 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C"). 2-7 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C"). 2-6 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C"). 2-5("Alkenyl"). In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C"). 2-4 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C"). 2-3 "Alkenyl"). In some embodiments, the alkenyl group has two carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). 2-4 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), and butadienyl (C4). 2-6 Examples of alkenyl groups include the aforementioned C 2-4 This includes alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Additional examples of alkenyl groups include heptenyl (C7), octenyl (C8), octatrienyl (C8), etc.

[0212] As used herein, “alkynyl” refers to a radical of a linear or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) ("C 2-20 ("Alkynyl"). In certain embodiments, the alkynyl does not contain any double bonds. In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("C"). 2-10 In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C"). 2-9 In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C"). 2-8 In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C"). 2-7 In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C"). 2-6In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C"). 2-5 In some embodiments, the alkynyl group has 2 to 4 carbon atoms ("C"). 2-4 In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C"). 2-3 "Alkynyl"). In some embodiments, the alkynyl group has two carbon atoms ("C2 alkynyl"). One or more carbon-carbon triple bonds may be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). 2-4 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), and 2-butynyl (C4). 2-6 Examples of alkenyl groups include the aforementioned C 2-4 This includes alkynyl groups as well as pentynyl (C5), hexynyl (C6), and others. Examples of additional alkynyl groups include heptynyl (C7) and octinyl (C8).

[0213] As used herein, “alkylene,” “alkenylene,” and “alkynylene” refer to the divalent radicals of alkyl, alkenyl, and alkynyl groups, respectively. Where a range or number of carbon atoms is provided for a particular “alkylene,” “alkenylene,” or “alkynylene” group, it is understood that the range or number refers to the range or number of carbon atoms in a linear divalent carbon chain. The “alkylene,” “alkenylene,” and “alkynylene” groups may or may not be substituted with one or more substituents described herein.

[0214] As used herein, the term “aryl” refers to an aromatic group (e.g., monocyclic, bicyclic, and tricyclic structures) containing 6 to 10 carbon atoms in the ring portion. The aryl group may optionally be substituted via available carbon atoms and, in certain embodiments, may contain one or more heteroatoms such as oxygen, nitrogen, or sulfur. In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl," e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C6 aryl"). 10 "Aryl" (e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl).

[0215] As used herein, “heteroaryl” refers to a radical of a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in a cyclic arrangement) having a ring carbon atom and 1 to 4 ring heteroatoms provided to the aromatic ring system, each heteroatom independently selected from nitrogen, oxygen, and sulfur (“5- to 10-membered heteroaryl”). In a heteroaryl group containing one or more nitrogen atoms, the bond site may be a carbon or nitrogen atom, as long as the valence allows. A heteroaryl bicyclic ring system may contain one or more heteroatoms in one or both rings. “Heteroaryl” includes a ring system, where the heteroaryl ring as defined above is condensed with one or more carbocyclyl or heterocyclyl groups, and the bond site is on the heteroaryl ring, in such examples the number of ring members continues to specify the number of ring members in the heteroaryl ring system. "Hyperaryl" also includes a ring system, where the heteroaryl ring defined above is fused with one or more aryl groups, and the bond site is on either the aryl or heteroaryl ring, in which case the number of ring members specifies the number of ring members in the fused (aryl / heteroaryl) ring system. In a bicyclic heteroaryl group where one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl), the bond site can be on either ring, i.e., the ring with a heteroatom (e.g., 2-indolyl) or the ring without a heteroatom (e.g., 5-indolyl).

[0216] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group with 3-12, 3-8, 4-8, or 4-6 carbon atoms, and in this specification, for example, "C" derived from cycloalkanes. 4-8 These are referred to as "cycloalkyl" compounds. Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane.

[0217] As used herein, “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 10-membered non-aromatic ring system having a ring carbon atom and 1- to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3- to 10-membered heterocyclyl”). In heterocyclyl groups containing one or more nitrogen atoms, the bond sites may be carbon or nitrogen atoms, as long as the valence allows. Heterocyclyl groups may be monocyclic (“monocyclic heterocyclyl”) or bicyclic (“bicyclic heterocyclyl”) systems, or condensed, bridging, or spirocyclic systems, and may be saturated or partially unsaturated. A heterocyclyl bicyclic ring system may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes a ring system, where the heterocyclyl ring defined above is fused with one or more carbocykyl groups, with the bond point located on either the carbocykyl or heterocyclyl ring or ring system, or where the heterocyclyl ring defined above is fused with one or more aryl or heteroaryl groups, with the bond point located on the heterocyclyl ring, in which case the number of ring members continues to specify the number of ring members in the heterocyclyl ring system. The terms "heterocyclyl," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic part," and "heterocyclic radical" may be used interchangeably.

[0218] As used herein, "cyano" refers to -CN.

[0219] As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iod, I). In certain embodiments, the halo group is either fluoro or chloro.

[0220] As used herein, the term "alkoxy" refers to an alkyl group bonded to another part via an oxygen atom (-O(alkyl)). Non-limiting examples include, for example, methoxy, ethoxy, propoxy, and butoxy.

[0221] As used herein, "oxo" refers to -C=O.

[0222] Generally, the term “substitution” means that at least one hydrogen atom present on a group (e.g., a carbon or nitrogen atom) is replaced by an acceptable substituent, such as a substituent that, upon substitution, produces a stable compound, such as one that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reactions. Unless otherwise indicated, a “substituted” group has substituents at one or more substituted positions on the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position.

[0223] As used herein, “pharmaceutically acceptable salts” refer to salts that, within reasonable medical judgment, are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and that balance with a reasonable benefit / risk ratio. Pharmacochemically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmacochemically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphor salt, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and rhynchophosphate. These include ctobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivaphosphates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, and valersates. Pharmaceutically acceptable salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium and N + (C 1-4Alkyl) tetra salts are included. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfons, and aryl sulfons.

[0224] In typical embodiments, the present invention is intended to encompass the compounds disclosed herein, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomers, polymorphs, and prodrugs of such compounds. In some embodiments, the present invention includes pharmaceutically acceptable addition salts, pharmaceutically acceptable esters, solvates (e.g., hydrates) of addition salts, tautomers, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (as pure or racemic or non-racemic mixtures) of the compounds described herein.

[0225] The compounds described herein may contain one or more chiral centers and thus may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of racemic mixtures and stereoisomer mixtures including one or more stereoisomer-rich mixtures. The isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or preferred isomers may be prepared by asymmetric synthesis. For example, see Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively as mixtures of various isomers.

[0226] In certain embodiments, compounds and transport vehicles (e.g., lipid nanoparticles) in which such compounds are components exhibit an improved (e.g., increased) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally include the step of contacting one or more target cells with the compounds and / or pharmaceutical compositions disclosed herein so that one or more target cells are transfected with circular RNA encapsulated therein. As used herein, the terms “transfect” or “transfection” refer to the intracellular introduction of one or more encapsulated substances (e.g., nucleic acids and / or polynucleotides) into cells, or preferably into target cells. The term “transfection efficiency” refers to the relative amount of such encapsulated substances (e.g., polynucleotides) taken up by the target cells being transfected, introduced into the target cells, and / or expressed by the target cells. In some embodiments, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cells after transfection. In some embodiments, transport vehicles have high transfection efficiency. In some embodiments, the transport vehicle has a transfection efficiency of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0227] As used herein, the term “liposome” generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayers. In certain embodiments, the liposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprising one or more of the ionizable lipid compounds disclosed herein). Such a liposome may be a monolayer or multilayer vesicle having a membrane formed from a lipophilic substance, as well as an aqueous interior containing encapsulated circRNA to be delivered to one or more target cells, tissues, and organs. In certain embodiments, the compositions described herein comprise one or more lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that may be used to form the intended liposomes and lipid nanoparticles include one or more of the compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and / or HGT4005). Such liposomes and lipid nanoparticles may also include additional ionizable lipids, such as C12-200, DLin-KC2-DMA, and / or HGT5001, helper lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, ClinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0228] As used herein, the terms “noncationic lipid,” “noncationic helper lipid,” and “helper lipid” are interchangeable and refer to any neutral, zwitterionic, or anionic lipid.

[0229] As used herein, the term "anionic lipid" refers to any of the many lipid species that have a net negative charge at a selected pH, such as physiological pH.

[0230] As used herein, the terms "biodegradable lipid" or "degradable lipid" refer to any of many lipid species that are broken down in the host environment within minutes, hours, or days, ideally becoming less toxic and less likely to accumulate in the host over time. Common modifications to lipids include ester bonds, and especially disulfide bonds, which enhance the biodegradability of lipids.

[0231] As used herein, the terms "biodegradable PEG lipid" or "degradable PEG lipid" refer to any of many lipid species in which the PEG molecule is cleaved from the lipid within minutes, hours, or days in the host environment, ideally resulting in reduced immunogenicity. Common modifications to PEG lipids include ester bonds, and especially disulfide bonds, which enhance the biodegradability of the lipid.

[0232] In certain embodiments of the present invention, a transport vehicle (e.g., lipid nanoparticles) is prepared to encapsulate one or more substances or therapeutic agents (e.g., circRNA). The process of incorporating a desired therapeutic agent (e.g., circRNA) into the transport vehicle is referred to herein as “loading” or “encapsulating” (Lasic, et al., FEBS Lett., 312:255-258, 1992). The substance (e.g., circRNA) loaded or encapsulated into the transport vehicle may be positioned entirely or partially within the internal space of the transport vehicle, within the bilayer membrane of the transport vehicle, or accompanying the external surface of the transport vehicle.

[0233] As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties.

[0234] As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols.

[0235] As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties.

[0236] As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer.

[0237] As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on its surface.

[0238] As used herein, "phospholipid" refers to a lipid comprising a phosphate group and one or more carbon chains, such as an unsaturated fatty acid chain.

[0239] All nucleotide sequences disclosed herein may represent RNA sequences or corresponding DNA sequences. It is understood that deoxythymidine (dT or T) in DNA is transcribed to uridine (U) in RNA. Thus, "T" and "U" are used interchangeably in nucleotide sequences herein.

[0240] As used herein, “sequence identity” or, for example, “sequences that are 50% identical to” refers to the degree to which sequences are identical nucleotide-wise or amino acid-wise across a comparison window. Thus, “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exist to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The specified materials include nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the reference sequences described herein, and typically the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0241] The term “antibody” (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain may comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three constant domains, CH1, CH2, and CH3. Each light chain may comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL may contain three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of Ab may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, manipulated antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as “antibody complexes”), heterocomplex antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain variable fragments (scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-bonded variable fragments (sdFv), anti-idiotype (anti-id) antibodies (e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), and any of the antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a polyclonal antibody population.

[0242] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term “antibody” includes, by example, both naturally occurring and non-naturally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-stranded Abs. Non-human Abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and indicated otherwise in the context, the term “antibody” also includes antigen-binding fragments or moieties of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or moieties, and single-stranded Abs.

[0243] The terms “antigen-binding molecule,” “antigen-binding portion,” or “antibody fragment” refer to any molecule containing the antigen-binding portion (e.g., CDR) of an antibody from which the molecule originates. An antigen-binding molecule may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to BCMA. In further embodiments, the antigen-binding molecule is an antibody fragment containing one or more of its complementarity-determining regions (CDRs) that specifically bind to an antigen. In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule includes or consists of an avimer.

[0244] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the 110–120 amino acids at the amino-terminus of the mature heavy chain, and approximately 90–115 amino acids in the mature light chain, which vary significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called framework regions (FRs). While we do not wish to be bound by any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily involved in antibody-antigen interaction and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region includes rodent or mouse CDR and primate (e.g., non-human primate) framework regions (FR).

[0245] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0246] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0247] Several definitions of CDR are commonly used in Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is an intermediate between the two, used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on the analysis of available composite crystal structures. The terms "Kabat numbering" and similar terms are recognized in the art and refer to a system of numbered amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding molecule. In certain embodiments, the CDR of an antibody may be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31–35, which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50–65 (CDR2), and amino acid positions 95–102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), amino acid positions 50–56 (CDR2), and amino acid positions 89–97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.In certain embodiments, the CDR of an antibody may be determined according to the Chothia numbering scheme, which points to the position of the immunoglobulin structural loop (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al, (1997), J Mol Biol 273:927-948; Chothia C et al., (1992), J Mol Biol 227:799-817; Tramontano A et al, (1990), J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using Kabat numbering rules, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34, the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56, the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102, while the Chothia CDR-L1 loop is located at light chain amino acids 24-34, the Chothia CDR-L2 loop is located at light chain amino acids 50-56, and the Chothia CDR-L3 loop is located at light chain amino acids 89-97. When numbered using the Kabat numbering rules, the termination of the Chothia CDR-HI loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if 35A and 35B are not present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; and if both 35A and 35B are present, the loop terminates at 34). In certain embodiments, the CDR of the antibody described herein is determined according to the Chothia numbering scheme.

[0248] As used herein, the terms “constant region” and “constant domain” are interchangeable and have the common meanings in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chain of the antibody moiety, for example, which is not directly involved in the binding of the antibody to an antigen but can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0249] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by its dissociation constant (KD or Kd). Affinity can be measured and / or expressed by many methods known to those skilled in the art, including but not limited to the equilibrium dissociation constant (KD) and equilibrium association constant (KA or Ka). KD is calculated from the quotient of koff / kon, and KA is calculated from the quotient of kon / koff. kon refers, for example, to the association rate constant of an antibody to an antigen, and koff refers, for example, to the dissociation of an antibody to an antigen. Kon and koff can be determined by techniques known to those skilled in the art, such as BIACORE® or KiNExA.

[0250] As used herein, “conservative amino acid substitution” refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, etc.), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within a CDR(or CDR) or within a framework region(or CDR) of an antibody or its antigen-binding molecule may be replaced with amino acid residues having similar side chains.

[0251] As used herein, the term “heterogeneous” means derived from any source other than naturally occurring sequences.

[0252] As used herein, “epitope” is a term used in the art and refers to a local region of an antigen to which an antibody can specifically bind. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be composed of, for example, two or more discontinuous regions of a polypeptide (conformity, nonlinear, discontinuous, or discontinuous epitope). In some embodiments, the epitope to which the antibody binds may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenic mapping (e.g., site-directed mutagenic mapping). With regard to X-ray crystallography, crystallization can be achieved using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350, McPherson A (1990) Eur J Biochem 189:1-23, Chayen NE (1997) Structure 5:1269-1274, McPherson A (1976) J Biol Chem 251:6300-6303).Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be purified using computer software such as X-PLOR (Yale University, 1992, popularized by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., U.S. Patent Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1):37-60, Bricogne G (1997) Meth Enzymol 276A:361-423, eds Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10):1316-1323).

[0253] When used herein, an antigen-binding molecule, antibody, or their antigen-binding molecule "cross-competes" with a reference antibody or their antigen-binding molecule if the interaction between the antigen and the first binding molecule, antibody, or their antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or their antigen-binding molecule to interact with the antigen. Cross-competition can be complete, for example, the binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, for example, the binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In some embodiments, the antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, the antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. To determine whether one antigen-binding molecule competes with another, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., 1983, Methods, Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J.Immunol.137:3614-3619), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA using 1-125 labeling (Morel et al., 1988, Molec.Immunol.25:7-15), solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology Numerous types of competitive binding assays can be used, including 176:546-552 and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0254] As used herein, the terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are analogous terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) in such a way that such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides at a lower affinity, as determined by, for example, an immunoassay, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen binds to the antigen at a KA of at least 2log, 2.5log, 3log, 4log or more than KA when the molecule binds to another antigen.

[0255] An "antigen" refers to any molecule that can trigger an immune response or be bound by an antibody or antigen-binding molecule. An immune response may involve antibody production, activation of specific immune-qualified cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. Antigens may be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens may be specific to certain tissues, such as cancer cells, or they may be broadly expressed. In addition, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0256] The term “self” refers to any substance derived from the same individual from which it is later reintroduced. For example, the manipulated autologous cell therapy (eACT®) method described herein involves the collection of lymphocytes from a patient, which are then manipulated to express, for example, a CAR construct, and then administered to the same patient.

[0257] The term "allogeneic" refers to any substance that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T cell transplantation.

[0258] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein may be used to treat, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethral cancer, penile cancer, chronic or It may be used to reduce the tumor size of tumors resulting from acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environment-induced cancers including those induced by asbestos, other B cell malignancies, and combinations of these cancers.In some embodiments, the methods disclosed herein apply to, for example, sarcomas and carcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteogenic sarcomas, Kaposi's sarcoma, soft tissue sarcomas, other sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancers, pancreatic cancers, breast cancers, ovarian cancers, prostate cancers, hepatocellular carcinomas, lung cancers, colorectal cancers, squamous cell carcinomas, basal cell carcinomas, adenocarcinomas (e.g., adenocarcinomas of the pancreas, colon, ovaries, lungs, breasts, stomachs, prostates, cervix, or esophagus), and sweat gland carcinomas. It may be used to reduce the tumor size of tumors originating from sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, renal pelvis carcinoma, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, etc.). Certain cancers may respond to chemotherapy or radiotherapy, or the cancer may be refractory. Refractory cancer refers to cancer that is not suitable for surgical intervention and which does not initially respond to chemotherapy or radiotherapy, or which becomes unresponsive over time.

[0259] As used herein, “antitumor effect” refers to a biological effect that may manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall survival or progression-free survival, an increase in lifespan, or an improvement in various physiological symptoms associated with tumor. The antitumor effect may also refer to the prevention of tumor development, such as through vaccination.

[0260] As used herein, “cytokines” refer to non-antibody proteins released by one cell in response to contact with a specific antigen, which then interact with a second cell to mediate a response in that second cell. As used herein, “cytokines” means proteins released by a population of cells that act on another cell as intercellular mediators. Cytokines can be endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-a, IL-lb, IL-6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), TGF-beta, IL-35, and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0261] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that is a major component of the innate immune system. NK cells reject tumor and virus-infected cells. They function through apoptosis, or programmed cell death. They are called “natural killers” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. Helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, also known as CD8+ T cells or killer T cells), memory T cells ((i) stem memory cells (TSCMs), like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, exhibiting many functional attributes specific to memory cells), (ii) stem There are many types of T cells, including (iii) effector memory cells (TEMs), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ or CD4+FoxP3+ regulatory T cells), natural killer T cells (NKTs), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies, function as antigen-presenting cells (APCs), and, after activation by antigen interaction, can transform into memory B cells and plasma cells, both short-lived and long-lived. In mammals, immature B cells are formed in the bone marrow.

[0262] The terms “genetic manipulation” or “manipulation” refer to methods of modifying a cell’s genome, including but not limited to deleting coding or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In some embodiments, the cells to be modified are lymphocytes, such as T cells, which can be obtained from either a patient or a donor. The cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which are incorporated into the cell’s genome.

[0263] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including alpha, cytokines, and complement) produced by these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of vertebrates of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0264] As used herein, “co-stimulatory signal” refers to, but is not limited to, a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in a T cell response such as the proliferation and / or upregulation or downregulation of key molecules.

[0265] As used herein, “costimulatory ligands” include molecules on antigen-presenting cells that specifically bind to homologous costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation. For example, costimulatory ligands induce signals in addition to the primary signals provided by stimulatory molecules, such as by binding of a T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule. Co-stimulatory ligands include, but are not limited to, Toll-like receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta receptors, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or 3 / TR6, 4-IBB ligands that bind to programmed death (PD)LI, agonists, or antibodies. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0266] "Co-stimulatory molecules" are congenital binding partners on T cells that specifically bind to costimulatory ligands, thereby mediating costimulatory responses by T cells, such as proliferation, but are not limited to these.Co-stimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD33, CD45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3(alpha CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD1-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD1 This includes, but is not limited to, la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules, SLAM (SLAMF1;CD150;IPO-3), SLAMF4 (CD244;2B4), SLAMF6 (NTB-A;Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or their fragments, truncations, or combinations.

[0267] As used herein, “sequence identity” or, for example, “sequences that are 50% identical to” refers to the degree to which sequences are identical nucleotide-wise or amino acid-wise across a comparison window. Thus, the “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, U) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exist to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The specified materials include nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to any of the reference sequences described herein, and typically, in the case of polypeptides, the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0268] As used herein, “vaccine” refers to a composition for generating immunity for the prevention and / or treatment of a disease. Therefore, a vaccine is a drug containing an antigen and intended for use in humans or animals to generate specific protective and defensive substances upon administration to humans or animals.

[0269] As used herein, “nascent antigen” refers to a class of tumor antigens arising from tumor-specific mutations in expressed proteins.

[0270] 2. Vectors, precursor RNA, and circular RNA Circular RNA, precursor RNA that can be circularized into circular RNA, and vectors (e.g., DNA vectors) that can be transcribed into precursor RNA or circular RNA are also provided herein.

[0271] In certain embodiments, circular RNA polynucleotides comprising a post-splicing 3' group I intron fragment, optionally a first spacer, an intra-sequence ribosome entry site (IRES), an expression sequence, optionally a second spacer, and a post-splicing 5' group I intron fragment are provided herein. In some embodiments, these regions are in this order. In some embodiments, the circular RNA is prepared by the method provided herein or from a vector provided herein.

[0272] In certain embodiments, transcription of a vector provided herein (e.g., comprising a 5' double-strand formation region, a 3' group I intron fragment, an optional first spacer, an intra-sequence ribosome entry site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, an optional second spacer, a 5' group I intron fragment, and a 3' double-strand formation region) results in the formation of a precursor linear RNA polynucleotide that can be cyclized. In some embodiments, this precursor linear RNA polynucleotide is cyclized when incubated in the presence of a guanosine nucleotide or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg2+).

[0273] In some embodiments, the vectors and precursor RNA polynucleotides provided herein include a first (5') double-stranding region and a second (3') double-stranding region. In certain embodiments, the first and second double-stranding regions may form a complete or incomplete double helix. Thus, in certain embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second double-stranding regions may base-pair with each other. In some embodiments, the double-stranding regions are expected to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base-pairing with unintended sequences in the RNA (e.g., non-double-stranding region sequences). In some embodiments, the double-stranding region is located at the end of the precursor RNA strand and is adjacent to or very close to a group I intron fragment, thereby bringing the group I intron fragments closer together and increasing splicing efficiency. In some embodiments, the double-stranding region is 3 to 100 nucleotides long (e.g., 3 to 75 nucleotides, 3 to 50 nucleotides, 20 to 50 nucleotides, 35 to 50 nucleotides, 5 to 25 nucleotides, 9 to 19 nucleotides). In some embodiments, the double-stranding region is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the double-stranding region has a length of about 9 to about 50 nucleotides. In some embodiments, the double-stranding region has a length of about 9 to about 19 nucleotides. In some embodiments, the double-stranding region has a length of about 20 to about 40 nucleotides. In certain embodiments, the double-stranding region has a length of about 30 nucleotides.

[0274] Two types of spacers were designed to improve precursor RNA cyclization and / or gene expression from circular RNA. The first type of spacer is an external spacer, i.e., present in the precursor RNA but removed upon cyclization. While not wishing to be constrained by theory, the external spacer is intended to improve ribozyme-mediated cyclization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements from interfering with its folding and function. The second type of spacer is an internal spacer, i.e., present in the precursor RNA and retained in the resulting circular RNA. While not wishing to be constrained by theory, the internal spacer is intended to improve ribozyme-mediated cyclization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements, particularly adjacent IRESs and coding regions, from interfering with their folding and function. The internal spacer is also intended to improve protein expression from IRESs by preventing adjacent sequence elements, particularly intron elements, from hybridizing with sequences within IRESs and inhibiting their ability to fold into their most preferred and active conformation.

[0275] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include a first (5') and / or a second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES can preserve secondary structure in those regions by preventing them from interacting and thus increasing splicing efficiency. In some embodiments, the first (between the 3' group I intron fragment and the IRES) and second (between the expression sequence and the 5' group I intron fragment) spacers include additional base-pairing regions that are not expected to base-pair with respect to the first and second double-strand formation regions. In some embodiments, such spacer base-pairing brings the group I intron fragments closer together, further increasing splicing efficiency. In addition, in some embodiments, the combination of base-pairing between the first and second double-strand formation regions, and separately, base-pairing between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragment adjacent to the base-pairing region. A typical spacer is a contiguous sequence containing one or more of the following qualities: 1) expected to avoid interference with proximal structures, e.g., IRES, expression sequences, or introns; 2) at least 7 nt in length and no more than 100 nt; 3) located after and adjacent to a 3' intron fragment and / or before and adjacent to a 5' intron fragment; and 4) below: a) an unstructured region of at least 5 nt in length; b) a base-pairing region of at least 5 nt in length in a distal sequence containing another spacer; and c) a structured region of at least 7 nt in length, limited to the sequence of the spacer. A spacer may have several regions, including an unstructured region, a base-pairing region, a hairpin / structured region, and combinations thereof. In some embodiments, the spacer has a structured region with a high GC content. In some embodiments, a region within a spacer base pairs with another region within the same spacer. In some embodiments, a region within a spacer base pairs with a region within another spacer.In some embodiments, the spacer comprises one or more hairpin structures. In some embodiments, the spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In some embodiments, an additional spacer is present between the 3' group I intron fragment and the IRES. In some embodiments, this additional spacer prevents, or reduces to the extent that, the structuring region of the IRES interferes with the folding of the 3' group I intron fragment. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides or less long. In some embodiments, the 5' spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and / or 25 and 35 nucleotides in length. In certain embodiments, the 5' spacer sequence is between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a poly-AC sequence. In one embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% poly-AC content. In another embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0276] In certain embodiments, the 3' group I intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 3' proximal fragment of a natural group I intron, which includes a 3' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. Typically, a 5' group I intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5' proximal fragment of a natural group I intron, which includes a 5' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. As described by Umekage et al. (2012), the outer portions of the 3' group I intron fragment and the 5' group I intron fragment are removed during circulation, so that the circular RNA provided herein contains only portions of the 3' group I intron fragment formed by any exon sequence of at least 1 nt in length and the 5' group I intron fragment formed by any exon sequence of at least 1 nt in length, provided that such sequences are present in the non-circularized precursor RNA. The portion of the 3' group I intron fragment retained by the circular RNA is referred herein to as the "post-splicing 3' group I intron fragment." The portion of the 5' group I intron fragment retained by the circular RNA is referred herein to as the "post-splicing 5' group I intron fragment."

[0277] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include intrasequence ribosome entry sites (IRESs). The inclusion of IRESs enables the translation of one or more open reading frames (e.g., open reading frames forming an expression sequence) from the circular RNA. The IRES elements attract the eukaryotic ribosome translation initiation complex, thereby promoting translation initiation. For example, see Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161).

[0278] A large number of IRES sequences are available, including sequences derived from a wide variety of viruses, such as leader sequences from picornaviruses like encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), polio leader sequences, hepatitis A virus leaders, hepatitis C virus IRESs, human rhinovirus type 2 IRESs (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), and giardiavirus IRESs (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397).

[0279] In some embodiments, IRES is Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated stink bug enterovirus, Casimir wasp virus, human rhinovirus 2, Homalodisca coagulatavirus-1, human immunodeficiency virus type 1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, white-spotted oak picorna-like virus, encephalomyocarditis virus, Sho Fruit fly C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antenapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast TFIID, budding yeast YAP1, tobacco etch virus, cabbage claw virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sarivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A1220, Pacivirus A 3. IRES sequences of aptamers for saperovirus, rosavirus B, bakunsavirus, tremovirus A, porcine pacivirus 1, PLV-CHN, pacivirus A, sisinivirus, hepacivirus K, hepacivirus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Hupey picorna-like virus, CRPV, sarivirus A BN5, sarivirus A BN2, sarivirus A 02394, sarivirus A GUT, sarivirus A CH, sarivirus A SZ1, sarivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0280] To induce protein expression, the circular RNA contains an IRES functionally ligated to a protein-coding sequence. Exemplary IRES sequences are provided in Table 17 below. In some embodiments, the circular RNA disclosed herein contains an IRES sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the IRES sequences in Table 17. In some embodiments, the circular RNA disclosed herein contains an IRES sequence in Table 17. Modification of the IRES and associated sequences is disclosed herein to increase or decrease IRES activity, for example, by cleaving the 5' and / or 3' ends of the IRES, adding a spacer to the 5' end of the IRES, modifying the 6 nucleotides at 5' of the translation initiation site (Kozak sequence), modifying alternative translation initiation sites, and generating chimeric / hybrid IRES sequences. In some embodiments, the IRES sequences in the circular RNA disclosed herein include one or more of these modifications to the native IRES (e.g., the native IRESs disclosed in Table 17).

[0281] A large number of IRES sequences are available, including sequences derived from a wide variety of viruses, such as leader sequences from picornaviruses like encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), polio leader sequences, hepatitis A virus leaders, hepatitis C virus IRESs, human rhinovirus type 2 IRESs (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), and giardiavirus IRESs (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397).

[0282] In some embodiments, IRES is Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant virus 1, wheat aphid virus, reticuloendotheliosis virus, human poliovirus 1, brown marmorated stink bug enterovirus, Casimir wasp virus, human rhinovirus 2, Homalodisca coagulatavirus-1, human immunodeficiency virus type 1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, white-spotted oak picorna-like virus, encephalomyocarditis virus, Sho Fruit fly C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antenapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast TFIID, budding yeast YAP1, tobacco etch virus, cabbage claw virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Sarivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A1220, Pacivirus A 3. The IRES sequence of an aptamer for saperovirus, rosavirus B, bakunsavirus, tremovirus A, porcine pacivirus 1, PLV-CHN, pacivirus A, sisinivirus, hepacivirus K, hepacivirus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Hupey picorna-like virus, CRPV, sarivirus A BN5, sarivirus A BN2, sarivirus A 02394, sarivirus A GUT, sarivirus A CH, sarivirus A SZ1, sarivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0283] In some embodiments, the polynucleotides herein comprise one or more expression sequences. In some embodiments, the circular RNA is a bicistronic RNA. Sequences encoding two or more polypeptides may be separated by nucleotide sequences encoding ribosome skipping elements or protease cleavage sites. In certain embodiments, the ribosome skipping elements encode Tosea signalavirus 2A peptide (T2A), porcine tesiovirus-1 2A peptide (P2A), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), cytoplasmic polyhedron disease virus 2A peptide (BmCPV 2A), or silkworm (B. mori) soft rot virus 2A peptide (BmIFV 2A).

[0284] In certain embodiments, the vectors provided herein include a 3'UTR. In some embodiments, the 3'UTR is derived from human betaglobin, human alphaglobin xenops betaglobin, xenops alphaglobin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFα, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorophyll ring-shaped virus, woodchuck hepatitis virus, hepatitis virus post-translational regulatory element, Sindbis virus, turnip crinkle virus, tobacco etching virus, or Venezuelan encephalitis virus.

[0285] In some embodiments, the vectors provided herein include a 5'UTR. In some embodiments, the 5'UTR is derived from human betaglobin, African clawed frog betaglobin, human alphaglobin, African clawed frog alphaglobin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etching virus, cabbage virus, or adenovirus ternary leader.

[0286] In some embodiments, the vectors provided herein include polyA regions outside the 3' and / or 5' group I intron fragments. In some embodiments, the polyA regions are at least 15, 30, or 60 nucleotides long. In some embodiments, one or both polyA regions are 15–50 nucleotides long. In some embodiments, one or both polyA regions are 20–25 nucleotides long. The polyA sequence is removed during cyclization. Therefore, oligonucleotides that hybridize with the polyA sequence, such as deoxythymine oligonucleotides (oligo(dT)) compounded onto a solid surface (e.g., resin), can be used to separate the circular RNA from its precursor RNA. Other sequences may also be positioned at 5' relative to the 3' group I intron fragment or at 3' relative to the 5' group I intron fragment, and complementary sequences can similarly be used for circular RNA purification.

[0287] In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are of lengths of 300-15000, 300-14000, 300-13000, 300-12000, 300-11000, 300-10000, 400-9000, 500-8000, 600-7000, 700-6000, 800-5000, 900-5000, 1000-5000, 1100-5000, 1200-5000, 1300-5000, 1400-5000, and / or 1500-5000 nucleotides. In some embodiments, the polynucleotide is at least 300nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, 1100nt, 1200nt, 1300nt, 1400nt, 1500nt, 2000nt, 2500nt, 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, 10000nt, 11000nt, 12000nt, 13000nt, 14000nt, or 15000nt in length. In some embodiments, the polynucleotides are 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, 10000nt, 11000nt, 12000nt, 13000nt, 14000nt, 15000nt, or 16000nt or less in length. In some embodiments, the lengths of the DNA, linear RNA, and / or circular RNA polynucleotides provided herein are approximately 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10000 nt, 11000 nt, 12000 nt, 13000 nt, 14000 nt, or 15000 nt.

[0288] In some embodiments, vectors are provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' double helix formation region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) an optional second spacer sequence, i) a 5' group I intron fragment, and g) a 3' double helix formation region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' double helix formation region.

[0289] In some embodiments, vectors are provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' double-strand forming region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) an optional second spacer sequence, i) a 5' group I intron fragment, and g) a 3' double-strand forming region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' double-strand forming region.

[0290] In some embodiments, precursor RNA is provided herein. In certain embodiments, the precursor RNA is linear RNA produced by in vitro transcription of the vector provided herein. In some embodiments, the precursor RNA comprises, in the following order: a) any 5' double helix formation region, b) a 3' group I intron fragment, c) any first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) any second spacer sequence, i) a 5' group I intron fragment, and j) any 3' double helix formation region. In some embodiments, the precursor RNA comprises, in the following order: a) a 5' double helix formation region, b) a 3' group I intron fragment, c) any first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) any second spacer sequence, i) a 5' group I intron fragment, and j) a 3' double helix formation region. Precursor RNA can be unmodified, partially modified, or completely modified.

[0291] In certain embodiments, circular RNA is provided herein. In certain embodiments, the circular RNA is circular RNA produced by the vector provided herein. In some embodiments, the circular RNA is circular RNA produced by the cyclization of the precursor RNA provided herein. In certain embodiments, transcription of the vector provided herein results in the formation of a precursor linear RNA that can be cyclized. In some embodiments, this precursor linear RNA polynucleotide is a guanosine nucleotide or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg 2+It becomes circular when incubated in the presence of ). In some embodiments, the circular RNA comprises the following sequences: a) a first spacer sequence, b) an IRES, c) a first expression sequence, d) a polynucleotide sequence encoding a cleavage site, e) a second expression sequence, and f) an optional second spacer sequence. In some embodiments, the circular RNA comprises the following sequences: a) a post-splicing 3' group I intron fragment, b) a first spacer sequence, c) an IRES, d) a first expression sequence, e) a polynucleotide sequence encoding a cleavage site, f) a second expression sequence, and g) a second spacer sequence, and h) a post-splicing 5' group I intron fragment. In some embodiments, the circular RNA comprises the following sequences: a) a first spacer sequence, b) a first IRES, c) a first expression sequence, d) a second IRES, e) a second expression sequence, and f) an optional second spacer sequence. In some embodiments, the circular RNA further comprises a portion of a 3' group I intron fragment that is the 3' of the 3' splice site. In some embodiments, the circular RNA further comprises a portion of a 5' group I intron fragment that is the 5' of the 5' splice site. In some embodiments, the circular RNA is of a size of at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 nucleotides. The circular RNA can be unmodified, partially modified, or fully modified.

[0292] In some embodiments, the vectors and precursor RNA polynucleotides provided herein include a first (5') double-stranding region and a second (3') double-stranding region. In certain embodiments, the first and second homologous regions may form a complete or incomplete double helix. Thus, in certain embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second double-stranding regions may base-pair with each other. In some embodiments, the double-stranding regions are expected to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base-pairing with unintended sequences in the RNA (e.g., non-double-stranding region sequences). In some embodiments, the double-stranding region is located at the end of the precursor RNA strand and is adjacent to or very close to a group I intron fragment, thereby bringing the group I intron fragments closer together and increasing splicing efficiency. In some embodiments, the double-stranding region is 3 to 100 nucleotides long (e.g., 3 to 75 nucleotides, 3 to 50 nucleotides, 20 to 50 nucleotides, 35 to 50 nucleotides, 5 to 25 nucleotides, 9 to 19 nucleotides). In some embodiments, the double-stranding region is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the double-stranding region has a length of about 9 to about 50 nucleotides. In some embodiments, the double-stranding region has a length of about 9 to about 19 nucleotides. In some embodiments, the double-stranding region has a length of about 20 to about 40 nucleotides. In certain embodiments, the double-stranding region has a length of about 30 nucleotides.

[0293] In some embodiments, the circular RNAs provided herein have higher functional stability than mRNAs containing the same expression sequence.

[0294] In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of 5–80, 10–70, 15–60, and / or 20–50 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a longer functional half-life (e.g., at least 1.5 times longer, at least 2 times longer) than equivalent linear RNA polynucleotides encoding the same protein. In some embodiments, the functional half-life can be evaluated through detection of functional protein synthesis.

[0295] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include a first (5') and / or second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES can preserve secondary structure in those regions by preventing them from interacting and thus increasing splicing efficiency. In some embodiments, the first (between the 3' group I intron fragment and the IRES) and second (between the expression sequence and the 5' group I intron fragment) spacers include additional base-pairing regions that are expected to base-pair each other, but not with respect to the first and second double-stranding regions. In other embodiments, the first (between the 3' group I intron fragment and the IRES) and second (between one of the expression sequences and the 5' group I intron fragment) spacers include additional base-pairing regions that are expected to base-pair each other, but not with respect to the first and second double-stranding regions. In some embodiments, such spacer base-pairing brings the group I intron fragments closer together, further increasing splicing efficiency. In addition, in some embodiments, the combination of base pairing between the first and second double-strand forming regions, and separately, base pairing between the first and second spacers, facilitates the formation of a splicing bubble containing a group I intron fragment adjacent to the base-pairing region. A typical spacer is a contiguous sequence comprising one or more of the following qualities: 1) expected to avoid interference with proximal structures, e.g., IRES, expression sequences, or introns; 2) at least 7 nt in length and no more than 100 nt; 3) located after and adjacent to the 3' intron fragment and / or before and adjacent to the 5' intron fragment; and 4) below: a) an unstructured region of at least 5 nt in length; b) a base-pairing region of at least 5 nt in length in a distal sequence containing another spacer; and c) a structured region of at least 7 nt in length, limited to the sequence of the spacer. A spacer may have several regions, including an unstructured region, a base-pairing region, a hairpin / structured region, and a combination thereof.In some embodiments, the spacer has a structuring region with a high GC content. In some embodiments, a region within the spacer bases pairs with another region within the same spacer. In some embodiments, a region within the spacer bases pairs with a region within another spacer. In some embodiments, the spacer includes one or more hairpin structures. In some embodiments, the spacer includes one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In some embodiments, an additional spacer is present between the 3' group I intron fragment and the IRES. In some embodiments, this additional spacer prevents or reduces the degree to which the structuring region of the IRES interferes with the folding of the 3' group I intron fragment. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides or less in length. In some embodiments, the 5' spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and / or 25 and 35 nucleotides in length. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a poly-A sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0296] In certain embodiments, the 3' group I intron fragment is a contiguous sequence that is at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the 3' proximal fragment of a natural group I intron, which includes a 3' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. Typically, a 5' group I intron fragment is a contiguous sequence that is at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the 5' proximal fragment of a native group I intron, which includes a 5' splice site dinucleotide and optionally at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt in length) and adjacent exon sequences up to the length of an exon. As described by Umekage et al. (2012), the outer portions of the 3' group I intron fragment and the 5' group I intron fragment are removed during circulation, so that the circular RNA provided herein contains only portions of the 3' group I intron fragment formed by any exon sequence of at least 1 nt in length and the 5' group I intron fragment formed by any exon sequence of at least 1 nt in length, provided that such sequences are present in the non-circularized precursor RNA. The portion of the 3' group I intron fragment retained by the circular RNA is referred herein to as the post-splicing 3' group I intron fragment. The portion of the 5' group I intron fragment retained by the circular RNA is referred herein to as the post-splicing 5' group I intron fragment.

[0297] In some embodiments, the circular RNAs provided herein may have a higher degree of expression than equivalent linear mRNAs, for example, a higher degree of expression 24 hours after RNA administration to cells. In some embodiments, the circular RNAs provided herein have a higher degree of expression than mRNAs containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail.

[0298] In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a functional half-life of 5–80, 10–70, 15–60, and / or 20–50 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a longer functional half-life (e.g., at least 1.5 times longer, at least 2 times longer) than equivalent linear RNA polynucleotides encoding the same protein. In some embodiments, the functional half-life can be evaluated through detection of functional protein synthesis.

[0299] In some embodiments, the cyclic RNA polynucleotides provided herein have a half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a half-life of 5–80, 10–70, 15–60, and / or 20–50 hours. In some embodiments, the cyclic RNA polynucleotides provided herein have a longer half-life (e.g., at least 1.5 times longer, at least 2 times longer) than equivalent linear RNA polynucleotides encoding the same protein. In some embodiments, the cyclic RNA polynucleotide, or its pharmaceutical composition thereof, has a functional half-life in human cells that is longer than or equivalent to that of a predetermined threshold. In some embodiments, the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, in which the activity of Gaussial luciferase (GLuc) is measured in the culture medium of human cells (e.g., HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay, in which the level of the protein encoded by the expression sequence of the circular RNA polynucleotide is measured every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days in patient serum or tissue samples. In some embodiments, the default threshold is the functional half-life of a reference linear RNA polynucleotide containing the same expression sequence as the circular RNA polynucleotide.

[0300] In some embodiments, the circular RNAs provided herein may have a higher degree of expression than equivalent linear mRNAs, for example, a higher degree of expression 24 hours after RNA administration to cells. In some embodiments, the circular RNAs provided herein have a higher degree of expression than mRNAs containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail.

[0301] In some embodiments, the circular RNAs provided herein may be less immunogenic than equivalent mRNAs when exposed to the immune system of an organism or a particular type of immune cell. In some embodiments, the circular RNAs provided herein are associated with the regulation of cytokine production when exposed to the immune system of an organism or a particular type of immune cell. For example, in some embodiments, the circular RNAs provided herein are associated with reduced production of TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferons, e.g., IFN-β1, when exposed to the immune system of an organism or a particular type of immune cell, compared to mRNAs containing the same expression sequence. In some embodiments, the circular RNAs provided herein are associated with less transcription induction of TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferons, e.g., IFN-β1, when exposed to the immune system of an organism or a particular type of immune cell, compared to mRNAs containing the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs containing the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail.

[0302] In some embodiments, the compositions and methods described herein provide RNA (e.g., circRNA) that has higher stability or functional stability than equivalent linear RNA without requiring nucleoside modification. In some embodiments, methods for producing nucleoside-deficient RNA produce a higher proportion of full-length transcripts than methods for producing RNA-containing nucleoside modifications resulting from a reduction in sterile transcription. In some embodiments, the compositions and methods described herein can produce large (e.g., 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, or 15kb) RNA constructs without adding sterile transcription associated with nucleoside-modified RNA.

[0303] In certain embodiments, the circular RNAs provided herein can be transfected directly into cells or transfected in the form of a DNA vector and transcribed intracellularly. Transcription of circular RNAs from the transfected DNA vector can occur via an additional polymerase or a polymerase encoded by a nucleic acid transfected into the cell, or preferably via an endogenous polymerase.

[0304] In certain embodiments, the circular RNA polynucleotides provided herein include modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleoside is m 5 C (5-methylcytidine). In another embodiment, the modified nucleoside is m 5 U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A (N 6 -methyladenosine). In another embodiment, the modified nucleoside is s 2 U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2′-O-methyluridine). In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2’-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio-N 6 -isopentenyladenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A (2-methylthio-N 6-(cis-Hydroxyisopentenyl)adenosine);g 6 A(N 6 -Glycylcarbamoyladenosine);t 6 A(N 6 -Threonylcarbamoyladenosine);ms 2 t 6 A(2-Methylthio-N 6 -Threonylcarbamoyladenosine);m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyladenosine);hn 6 A(N 6 -Hydroxynorvalylcarbamoyladenosine);ms 2 hn 6 A(2-Methylthio-N 6 -Hydroxynorvalylcarbamoyladenosine);Ar(p)(2’-O-Ribosyladenosine(phosphate));I(Inosine);m 1 I(1-Methylinosine);m 1 Im(1,2’-O-Dimethylinosine);m 3 C(3-Methylcytidine);Cm(2’-O-Methylcytidine);s 2 C(2-Thiocytidine);ac 4 C(N 4 -Acetylcytidine);f 5 C(5-Formylcytidine);m 5 Cm(5,2′-O-Dimethylcytidine);ac 4 Cm(N 4 -Acetyl-2’-O-Methylcytidine);k 2 C(Lysidine);m 1 G(1-Methylguanosine);m 2 G(N 2 -Methylguanosine);m 7 G(7-Methylguanosine);Gm(2′-O-Methylguanosine);m 2 2G(N 2 ,N 2 -Dimethylguanosine);m 2 Gm(N 2 ,2’-O-Dimethylguanosine);m 2 2Gm(N2 ,N 2 ,2'-O-trimethylguanosine);Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(unmodified hydroxywybutosine);imG(wyosine);mimG(methylwyosine);Q(keuosine);oQ(epoxykeuosine);galQ(galactosyl-keuosine);manQ(mannosyl-keuosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Alkaeosin); D (Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine); m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine); mo 5 U(5-methoxyuridine); cmo 5 U (uridine 5-oxyacetic acid); mcmo 5 U (Uridine 5-oxyacetate methyl ester); chm 5 U(5-(carboxyhydroxymethyl)uridine)); mchm 5 U(5-(carboxyhydroxymethyl)uridinemethyl ester); mcm 5 U(5-methoxycarbonylmethyluridine); mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine);nm 5 S 2 U(5-aminomethyl-2-thiouridine); mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2U(5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine); ncm 5 U(5-carbamoylmethyluridine); ncm 5 Um(5-carbamoylmethyl-2′-O-methyluridine); cmnm 5 U(5-carboxymethylaminomethyluridine); cmnm 5 Um(5-carboxymethylaminomethyl-2′-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m 6 2A(N 6 ,N 6 -dimethyladenosine);Im(2'-O-methylinosine);m 4 C(N 4 -methylcytidine); m 4 Cm(N 4 ,2'-O-dimethylcytidine); hm 5 C(5-hydroxymethylcytidine); m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine); m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine);m 2,7 G(N 2 ,7-dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine); f 5 Cm(5-formyl-2'-O-methylcytidine);m 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine); τm 5 U(5-taurinomethyluridine); τm5 s 2 U(5-taurinomethyl-2-thiouridine)); imG-14(4-demethylyosin); imG2(isoyosin); or ac 6 A(N 6 -Acetyladenosine)

[0305] In some embodiments, the modified nucleosides include pyridine-4-onribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, and 5-taurinomethyl-2-thiouridine. , 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseuduridine, 4-methoxy-2-thio- Pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine N, Zebralin, 5-Aza-Zebralin, 5-Methyl-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopurine, 7-Deaza-Adenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-Aminopurine, 7-Deaza-8-Aza-2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-Hydroxyisopentenyl)adenosine, 2-Methylthio-N6-(cis-Hydroxyisopentenyl)adenosine, N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-Adenine, 2-Methoxy-Adenine, Inosine, 1-Methyl-Inosine, Wyosin, Wyobutosin, 7-Deaza-Guano Compounds may be selected from the group consisting of cin, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In another embodiment, the modification is independently selected from the group consisting of 5-methylcytosine, pseudouridine, and 1-methylpseudridine.

[0306] In some embodiments, the modified ribonucleoside includes 5-methylcytidine, 5-methoxyuridine, 1-methylpseudridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified nucleosides provide further stability and resistance to immunoactivation.

[0307] In certain embodiments, polynucleotides can be codon-optimized. A codon-optimized sequence may be a sequence in which the codons of a polynucleotide encoding a polypeptide are substituted to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variations in the degree of codon bias within an organism, gene, or set of genes; (iii) phylogenetic variations of codons, including context; (iv) variations in codons according to their decoded tRNAs; (v) variations in codons according to GC% at either the whole of a triplet or at one position; (vi) variations in similarity to a reference sequence, such as a naturally occurring sequence; (vii) variations in codon frequency cutoffs; (viii) structural characteristics of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of a DNA sequence based on the design of a codon substitution set; and / or (x) phylogenetic variations of the codon set of each amino acid. In some embodiments, codon-optimized polynucleotides can minimize ribozyme collisions and / or limit structural interference between the expression sequence and IRES.

[0308] In certain embodiments, the circular RNA provided herein is produced inside a cell. In some embodiments, the precursor RNA is transcribed in the cytoplasm by bacteriophage RNA polymerase or in the nucleus by host RNA polymerase II using a DNA template (for example, using a vector provided herein in some embodiments) and then circularized.

[0309] In certain embodiments, the circular RNA provided herein is injected into an animal (e.g., a human) so that the polypeptide encoded by the circular RNA molecule is expressed inside the animal.

[0310] 3. Payload In some embodiments, the first expression sequence encodes a therapeutic protein. In some embodiments, the second expression sequence encodes a therapeutic protein. In some embodiments, one or both of the therapeutic proteins are selected from the proteins listed in the table below. TIFF0007851254000001.tif218166TIFF0007851254000002.tif240166TIFF000 7851254000003.tif245166TIFF0007851254000004.tif244166TIFF00078512540 00005.tif244166TIFF0007851254000006.tif244166TIFF0007851254000007.t if244166TIFF0007851254000008.tif244166TIFF0007851254000009.tif134166

[0311] In some embodiments, at least one of the expression sequences encodes a therapeutic protein. In some embodiments, the first or second expression sequence encodes a cytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN-β, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. In some embodiments, the first or second expression sequence encodes an immune checkpoint inhibitor. In some embodiments, the first or second expression sequence encodes an agonist (e.g., a TNFR family member, e.g., CD137L, OX40L, ICOSL, LIGHT, or CD70). In some embodiments, the first or second expression sequence encodes a chimeric antigen receptor. In some embodiments, the first or second expression sequence encodes an inhibitory receptor agonist (e.g., PDL1, PDL2, galectin-9, VISTA, B7H4, or MHCII) or an inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3, or TIM3). In some embodiments, the first or second expression sequence encodes an inhibitory receptor antagonist. In some embodiments, the first or second expression sequence encodes one or more TCR chains (alpha and beta chains or gamma and delta chains). In some embodiments, the first or second expression sequence encodes a secretory T cell or immune cell engager (e.g., a bispecific antibody such as BiTE targeting CD3, CD137, or CD28 and tumor expression proteins, e.g., CD19, CD20, or BCMA). In some embodiments, the first or second expression sequence encodes a transcription factor (e.g., FOXP3, HELIOS, TOX1, or TOX2). In some embodiments, the first or second expression sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39 / CD73). In some embodiments, the first or second expression sequence encodes GvHD (e.g., anti-HLA-A2 CAR-Treg).

[0312] In some embodiments, the first and second expression sequences encode the alpha and beta chains of the T cell receptor (TCR). In some embodiments, the first and second expression sequences encode the gamma and delta chains of the TCR. The present invention includes a method for treating a subject with cancer, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding the TCR alpha and TCR beta chain or the TCR gamma and TCR delta chain.

[0313] In some embodiments, the first and second expression sequences encode a chimeric antigen receptor (CAR) and a PD1 or PDL1 antagonist. In some embodiments, the first and second expression sequences encode a chimeric antigen receptor (CAR) and a cytokine. In some embodiments, the cytokine is IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN-β, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. The present invention includes a method for treating a subject with cancer, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding a CAR and a PD1 or PDL1 antagonist. The present invention includes a method for treating a subject with cancer, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding a CAR and a cytokine.

[0314] In some embodiments, the first and second expression sequences encode a transcription factor and a cytokine. In some embodiments, the transcription factor is FOXP3, STAT5B, or HELIOS, and the cytokine is IL10, IL12, or TGF beta. The present invention includes a method for treating a subject suffering from an autoimmune disorder, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding a transcription factor, e.g., FOXP3, and a cytokine.

[0315] In some embodiments, the first and second expression sequences encode a transcription factor and a CAR. In some embodiments, the transcription factor is FOXP3, STAT5B, or HELIOS. The present invention includes a method for treating a subject suffering from an autoimmune disorder, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding a transcription factor, for example, FOXP3 and a CAR.

[0316] In some embodiments, the first and second expression sequences encode a cytokine and an antigen. In some embodiments, the cytokine is IFNγ. In some embodiments, the antigen is a nascent antigen. The present invention includes a method for treating a subject with cancer, comprising administering a therapeutically effective amount of a composition comprising a cytokine, for example, IFNγ, and a cyclic RNA polynucleotide encoding a tumor antigen or a fragment thereof.

[0317] In some embodiments, a first expression sequence encodes a first chimeric antigen receptor (CAR), and a second expression sequence encodes a second CAR. In some embodiments, the first CAR is specific to a first antigen and includes a costimulatory domain and an intracellular signaling domain, and the second CAR is specific to a second antigen and includes a costimulatory domain and an intracellular signaling domain. In some embodiments, expressing CARs that target multiple tumor antigens provides a more effective treatment for tumors with heterologous antigen expression. The present invention includes a method for treating a subject with cancer, comprising administering a therapeutically effective amount of a composition comprising a cyclic RNA polynucleotide encoding a first CAR and a second CAR.

[0318] In some embodiments, the first expression sequence encodes a first cytokine, and the second expression sequence encodes a second cytokine. In some embodiments, the first and second cytokines belong to the group IL-10, TGFβ, and IL-35. In some embodiments, the first and second cytokines belong to the group IFNγ, IL-2, IL-7, IL-15, and IL-18.

[0319] In some embodiments, polynucleotides encode proteins composed of subunits encoded by one or more genes. For example, a protein may be a heterodimer in which each chain or subunit of the protein is encoded by distinct genes. It is possible that one or more circRNA molecules are delivered by transport vehicles, with each circRNA encoding a distinct subunit of the protein. Alternatively, a single circRNA may be manipulated to encode more than one subunit. In certain embodiments, distinct circRNA molecules encoding individual subunits may be administered by distinct transport vehicles.

[0320] 3.1 Cytokines Descriptions and / or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-27β, IFNγ, and / or TGFβ1 are provided herein and in the www.uniprot.org database under accession numbers: P60568(IL-2), P29459(IL-12A), P29460(IL-12B), P13232(IL-7), P22301(IL-10), P40933(IL-15), Q14116(IL-18), Q14213(IL-27β), P01579(IFNγ), and / or P01137(TGFβ1).

[0321] 3.2 PD-1 and PD-L1 Antagonists In some embodiments, the PD-1 inhibitor is pembrolizumab, pizilizumab, or nivolumab. In some embodiments, nivolumab is described in International Patent Publication WO2006 / 121168. In some embodiments, pembrolizumab is described in W02009 / 114335. In some embodiments, pizilizumab is described in International Patent Publication WO2009 / 101611. Additional anti-PD1 antibodies are described in U.S. Patent No. 8,609,089, U.S. Patent Publications US2010028330 and US20120114649, and International Patent Publications WO2010 / 027827 and WO2011 / 066342.

[0322] In some embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, BMS-936559, or CK-301.

[0323] Descriptions of the heavy and light chains and / or amino acid sequences of PD-1 and / or PD-L1 antibodies are provided herein and in the www.drugbank.ca database under accession numbers: DB09037 (pembrolizumab), DB09035 (nivolumab), DB15383 (pizilizumab), DB11595 (atezolizumab), DB11945 (avelumab), and DB11714 (durvalumab).

[0324] 3.3 Chimeric Antigen Receptors Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be inserted into and expressed by immune cells, including T cells, via circular RNA as described herein. Using CARs, a single receptor can be programmed so that both recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells possessing that antigen. If these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells. In some embodiments, a CAR encoded by a polynucleotide comprises (i) an antigen-binding molecule that specifically binds to the target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activation domain.

[0325] In some embodiments, the orientation of a CAR according to this disclosure includes an antigen-binding domain (such as scFv) in tandem with a co-stimulatory domain and an activating domain. The co-stimulatory domain may include one or more of the extracellular, transmembrane, and intracellular portions. In other embodiments, multiple co-stimulatory domains may be utilized in tandem.

[0326] antigen-binding domain CARs can be engineered to bind to antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. In some embodiments, the antigen-binding molecule is its antibody fragment, for example, one or more single-chain antibody fragments (scFv). An scFv is a single-chain antibody fragment having variable regions of the heavy and light chains of linked antibodies. See, for example, U.S. Patents 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. The scFv retains the ability of the parent antibody to specifically interact with the target antigen. Since scFvs can be engineered to be expressed as part of a single chain together with other CAR components, they are useful in chimeric antigen receptors. See also Id. Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It will be understood that the antigen-binding molecule is typically contained within the extracellular portion of the CAR so that it can recognize and bind to the antigen of interest. Bispecific and multispecific CARs are intended within the scope of the present invention and involve specificity to more than one target of interest.

[0327] In some embodiments, the antigen-binding molecule comprises a single chain, with a heavy chain variable region and a light chain variable region connected by a linker. In some embodiments, VH is located at the N-terminus of the linker and VL is located at the C-terminus of the linker. In other embodiments, VL is located at the N-terminus of the linker and VH is located at the C-terminus of the linker. In some embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids.

[0328] In some embodiments, the antigen-binding molecule comprises a nanobody. In some embodiments, the antigen-binding molecule comprises DARPin. In some embodiments, the antigen-binding molecule comprises antikalin or other synthetic protein capable of specifically binding to a target protein.

[0329] In some embodiments, CAR is a group of CD19, CD123, CD22, CD30, CD171, CS-1, type C lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesoserine, interleukin-13 Ixocyte 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, HER2, HER3, mucin 1, cell surface-related (MUC1), epidermal growth factor receptor (EGFR), neuronal adhesion molecule (NCAM), prostaglandins -ase, prostatic acid phosphatase (PAP), elongation factor 2 mutation (ELF2M), ephrin B2, fibroblast-activating protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonate anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Abelson mouse leukemia virus oncogene Oncogene fusion protein (bcr-abl) consisting of homolog 1 (Abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R),Claudin 6 (CLDN6), Thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), Chromosome X open reading frame 61 (CXORF61), CD97, CD179a, Anaplastic lymphoma kinase (ALK), Polysialic acid, Placenta-specific receptor 1 (PLAC1), Hexasaccharide moiety of globoH glycoceramide (GloboH), Mammary gland differentiation antigen (NY-BR-1), Uloplakin 2 (UPK2), Hepatitis A virus cell receptor 1 (HAVCR1), Adrenergic receptor beta 3 (ADRB3), Panexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, gene locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma surrogate leading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3 and MAGE-A4), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2) Melanoma carcinoma testicular antigen-1 (MAD-CT-1), Melanoma carcinoma testicular antigen-2 (MAD-CT-2), Fos-related antigen 1, Tumor protein p53 (p53), p53 variant, Prostein, Survivin, Telomerase, Prostate cancer tumor antigen-1, Melanoma antigen recognized by T cell 1, Rat sarcoma (Ras) variant, Human telomerase reverse transcriptase (hTERT), Sarcoma translocation breakpoint, Apoptosis-mediated melanoma inhibitor (ML-IAP), ER G (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired-box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytoplasmosis virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC binding factor (zinc finger protein)-like,Squamous cell carcinoma antigen recognized by T cell 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase-fixed protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous protein 1 (RU1), renal ubiquitous protein 2 (RU2), regmine, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutation (mut). hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, ανβθ integrin, αvβ6 integrin, alpha-fetoprotein (AFP), B7-H6, ca -125, CA9, CD44, CD44v7 / 8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate-binding protein (FBP), kinase insertion domain receptor (KDR), k-light chain, L1 cell adhesion molecule, MUC18, NKG2D, carcinoembryonic antigen (h5T4), tumor / testicular antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), hepatitis B surface antigen-binding protein (HBsAg), viral capsid antigen (VCA), initial antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen,It includes antigen-binding domains specific to antigens selected from large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, hemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, human metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lymphotrophic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, and Kaposi's sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and KSHV latent nuclear antigen. In some embodiments, the antigen-binding domain includes SEQ ID NOs: 321 and / or 322.

[0330] Hinge / Spacer Domain In some embodiments, the CAR of the Disclosure includes a hinge or spacer domain. In some embodiments, the hinge / spacer domain may include a truncated hinge / spacer domain (THD), where the THD domain is a truncated version of the full hinge / spacer domain ("CHD"). In some embodiments, the extracellular domain may include ErbB2, glycophorin A (GpA), CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T, CDl 1a (IT GAL), CDl 1b (IT GAM), CDl 1c (IT GAX), CDl 1d (ITGAD), CD18(ITGB2), CD19(B4), CD27(TNFRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48( SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d CD66e (CEACAM3), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2D) L1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(K IR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247 (CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG 2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRT AM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CDl1a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor, and their fragments or combinations (e.g., all of them or their fragments). Hinge or spacer domains may be derived from either natural or synthetic sources.

[0331] In some embodiments, the hinge or spacer domain is located between the antigen-binding molecule (e.g., scFv) and the transmembrane domain. In this orientation, the hinge / spacer domain provides distance between the antigen-binding molecule and the surface of the cell membrane on which the CAR is expressed. In some embodiments, the hinge or spacer domain is from or derived from immunoglobulin. In some embodiments, the hinge or spacer domain is selected from the hinge / spacer regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM or fragments thereof. In some embodiments, the hinge or spacer domain is from or derived from CD8 alpha, including its hinge / spacer region. In some embodiments, the hinge or spacer domain is from or derived from CD28, including its hinge / spacer region. In some embodiments, the hinge or spacer domain comprises a fragment of the CD8 alpha hinge / spacer region or a fragment of the CD28 hinge / spacer region, the fragment being any smaller than the entire hinge / spacer region. In some embodiments, the CD8 alpha hinge / spacer region fragment or the CD28 hinge / spacer region fragment comprises an amino acid sequence that excludes 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 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, or at least 20 amino acids at the N-terminus or C-terminus or both of the CD8 alpha hinge / spacer region or the CD28 hinge / spacer region.

[0332] transmembrane domain The CARs of this disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to fuse with the extracellular domain of the CAR. Similarly, it may fuse with the intracellular domain of the CAR. In some embodiments, a naturally associated transmembrane domain is used for one of the domains within the CAR. In some examples, the transmembrane domain may be selected or modified (e.g., by amino acid substitution) to avoid binding of such domain to transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex. The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-binding protein or transmembrane protein.

[0333] The transmembrane region contains receptor tyrosine kinases (e.g., ErbB2), glycophorin A (GpA), 4-1BB / CD137, activated NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 ld, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, ligands that specifically bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPL G(CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM(SLAMF1;CD150;IPO-3), SLAMF4(CD244;2B4), SLAMF6(NTB-A;Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or their fragments, truncations, or combinations may be derived from (i.e., include).

[0334] In some embodiments, preferred intracellular signaling domains include, but are not limited to, activated macrophage / myeloid cell receptors CSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4 / 80, CD91, C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, lactoferrin, or their fragments, truncations, or combinations.

[0335] In some embodiments, receptor tyrosine kinases include insulin receptor (InsR), insulin-like growth factor I receptor (IGF1R), insulin receptor-related receptor (IRR), platelet-derived growth factor receptor alpha (PDGFRa), platelet-derived growth factor receptor beta (PDGFRfi), KIT oncogene receptor tyrosine kinase (Kit), colony-stimulating factor 1 receptor (CSFR), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 1 (VEGFR-1), kinase insertion domain receptor (VEGFR-2), and fm s-related tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine kinase-like orphan receptor 2 ( ROR2), muscle-associated receptor tyrosine kinase (MuSK), MET oncogene, receptor tyrosine kinase (MET), macrophage-stimulated receptor 1 (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin-like and EGF-like domain 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphAl), EPH receptor A2 (EphA2), (EPH receptor A3)Eph A3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphAl0), EPH receptor B1 (EphBl), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret proto-oncogene (Ret), receptor-like tyrosine kinase (RYK), discoidin domain receptor tyrosine kinase 1 (DDR1),It may be derived from (for example, including) discoidin domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis-related tyrosine kinase (Lmrl), lemur tyrosine kinase 2 (Lmr2), lemur tyrosine kinase 3 (Lmr3), leukocyte receptor tyrosine kinase (LTK), ALK receptor tyrosine kinase (ALK), or serine / threonine / tyrosine kinase 1 (STYK1).

[0336] Co-stimulatory domain In certain embodiments, the CAR includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain includes 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In preferred embodiments, the co-stimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (ζ). 4-1BB, CD28, CD3 zeta, or any of these may be present in smaller quantities than 4-1BB, CD28, or CD3 zeta as a whole. Chimeric antigen receptors may incorporate co-stimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N.Engl. J. Med. 365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0337] In some embodiments, the co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 318 or 320.

[0338] Intracellular signal transduction domains The manipulated intracellular (signaling) domains of T cells disclosed herein may provide signaling to an activation domain, which then activates at least one of the normal effector functions of the immune cell. For example, the effector function of the T cell may be cytolytic activity or helper activity, such as cytokine secretion.

[0339] In some embodiments, preferred intracellular signaling domains include 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96(tactile), CD1 la, CD1 lb, CD1 lc, CD1 Id, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAL, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that specifically binds to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Lyl08), lymphocyte function-associated antigen-1 (LFA-1; CDl-la / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), This includes, but is not limited to, signaling lymphocyte-activated molecules (SLAM proteins), SLAM(SLAMF1;CD150;IPO-3), SLAMF4(CD244;2B4), SLAMF6(NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or their fragments, truncations, or combinations.

[0340] CD3 is an element of the T cell receptor on native T cells and has been shown to be an important intracellular activation element in CAR. In some embodiments, CD3 is CD3 zeta. In some embodiments, the activation domain includes an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide sequence of SEQ ID NO: 319.

[0341] 3.4 T cell receptor TCRs are represented using the International Immunogenetics (IMGT) TCR nomenclature and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimer TCRs have an alpha chain and a beta chain. Generally, each chain may contain variable, binding, and constant regions, and the beta chain also usually contains a short diversity region between the variable and binding regions, although this diversity region is often considered part of the binding region. Each variable region may contain three CDRs (complementarity-determining regions) embedded in the framework sequence, one of which is a hypervariable region named CDR3. There are several types of alpha-chain variable (Vα) regions and several types of beta-chain variable (Vβ) regions, distinguished by their framework, CDR1 and CDR2 sequences, and partially defined by the CDR3 sequence. Vα types are designated by their own TRAV number in IMGT nomenclature. Therefore, "TRAV21" defines a TCR Vα region having a unique framework and CDR1 and CDR2 sequences, as well as a CDR3 sequence that is partially defined by amino acid sequences conserved across TCRs but also includes amino acid sequences that vary across TCRs. Similarly, "TRBV5-1" defines a TCR Vβ region having a unique framework and CDR1 and CDR2 sequences, but with a partially defined CDR3 sequence.

[0342] The binding region of the TCR is similarly defined by its own IMGT TRAJ and TRBJ nomenclature, while the constant region is defined by the IMGT TRAC and TRBC nomenclature.

[0343] In IMGT nomenclature, the beta-chain diversity region is abbreviated as TRBD, and as mentioned, linked TRBD / TRBJ regions are often considered together as a binding region.

[0344] The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the field of TCR. For example, they can be found in the IMGT public database. "T cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but due to its publication date and the resulting time lag, the information contained therein must be verified by referencing the IMGT database.

[0345] Native TCRs exist in heterodimer αβ or γδ forms. However, recombinant TCRs consisting of αα or ββ homodimers have been previously shown to bind to peptide MHC molecules. Therefore, the TCRs of the present invention may be heterodimer αβ TCRs or αα or ββ homodimer TCRs.

[0346] For use in adoptive therapy, the αβ heterodimer TCR can be transfected, for example, as a full-length chain having both cytoplasmic and transmembrane domains. In certain embodiments, the TCR of the present invention may have disulfide bonds introduced between residues of each constant domain, as described, for example, in WO2006 / 000830.

[0347] The TCR of the present invention, particularly the alpha-beta heterodimer TCR, may comprise an alpha-chain TRAC constant domain sequence and / or a beta-chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta-chain constant domain sequences may be modified by cleavage or substitution to delete the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The alpha and / or beta-chain constant domain sequence(s) may also be modified by substitution at cysteine ​​residues at Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, where the cysteine ​​forms a disulfide bond between the alpha and beta-chain constant domains of the TCR.

[0348] Binding affinity (equilibrium constant K D The binding half-life (expressed as T1 / 2), which is inversely proportional to the TCR, can be determined by any suitable method. Doubling the affinity of the TCR gives K D It is understood that it becomes half. T1 / 2 is calculated as ln2 divided by the off-rate (koff). Therefore, doubling T1 / 2 halves koff. K for TCR D The koff value is typically measured for the soluble form of the TCR, i.e., the form in which cytoplasmic and transmembrane domain residues have been removed. Therefore, it should be understood that a given TCR has improved binding affinity and / or binding half-life compared to the parent TCR if its soluble form has the aforementioned characteristics. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g., three or more times, using the same assay protocol, and the average of the results is obtained.

[0349] Since the TCR of the present invention has practical applications in adoptive therapy, the present invention includes cells, particularly T cells, that do not exist naturally and / or are purified and / or manipulated, that exhibit the TCR of the present invention. Numerous suitable methods exist for transfecting T cells with nucleic acids (such as DNA, cDNA, or RNA) encoding the TCR of the present invention (see, for example, Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCR of the present invention are suitable for use in adoptive therapy-based treatment of cancer (such as pancreatic and hepatic cancer). As is known to those skilled in the art, numerous suitable methods exist in which adoptive therapy can be performed (see, for example, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0350] As is well known in the art, the TCR of the present invention may be subjected to post-translational modification when expressed by transfected cells. Glycosylation is one such modification, which may involve covalent bonding of oligosaccharide sites to defined amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are well known sites for oligosaccharide bonding. The glycosylation state of a particular protein depends on a number of factors, including the protein sequence, protein conformation, and availability of a given enzyme. Furthermore, the glycosylation state (i.e., oligosaccharide type, covalent bond, and total number of bonds) can affect protein function. Therefore, when producing recombinant proteins, it is often desirable to control glycosylation. Glycosylation of a transfected TCR can be controlled by mutations in the transfected gene (Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations are also included in the present invention.

[0351] TCR is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MA GE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX -4, SSX-5, SCP-1, CT-7, Alpha-Actinin-4, Bcr-Abl fusion protein, Casp-8, Beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, Myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triose phosphate isomeras, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, Lage-2, SP17, and TRP2-Int2, (MART-I), gp100(Pmel 17) TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO(LAGE), SCP-1, Hom / Mel-40, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, Beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, Telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA27, 29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1 It may be specific to antigens in the following groups: CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, and TPS.

[0352] 3.5 Transcription Factors Regulatory T cells (Tregs) are important for maintaining homeostasis, controlling the degree and duration of inflammatory responses, and preventing autoimmune and allergic reactions.

[0353] Tregs are generally thought to be primarily involved in suppressing immune responses and partially function as a "self-check" of the immune system to prevent excessive reactions. In particular, Tregs are involved in maintaining tolerance to harmless substances such as autoantigens, pollen, or food, and in preventing autoimmune diseases.

[0354] Treg cells are found throughout the body, including, but not limited to, the intestines, skin, lungs, and liver. Treg cells may also be found in certain compartments of the body not directly exposed to the external environment, such as the spleen, lymph nodes, and even adipose tissue. Each of these Treg cell populations is known or suspected to possess one or more unique features, and additional information can be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294):1-10, the disclosure of which is incorporated herein in its entirety.

[0355] Typically, Tregs are known to require TGF-β and IL-2 for proper activation and development. Tregs expressing abundant amounts of the IL-2 receptor (IL-2R) are dependent on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β (both potent immunosuppressive cytokines). Tregs are also known to inhibit the ability of antigen-presenting cells (APCs) to stimulate T cells. One proposed mechanism for APC inhibition is via CTLA-4 expressed by Foxp3+ Tregs. It is thought that CTLA-4 binds to B7 molecules on APCs, blocking or internalizing them to remove them, thereby reducing the availability of B7 and preventing it from providing adequate co-stimulation for the immune response. Further discussion on the origin, differentiation, and function of Treg cells can be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4(253):1-11.

[0356] Descriptions and / or amino acid sequences of FOXP3, STAT5B, and / or HELIOS are provided herein and in the www.uniprot.org database under accession numbers: Q9BZS1(FOXP3), P51692(STAT5b), and / or Q9UKS7(HELIOS).

[0357] Foxp3 In some embodiments, the transcription factor is the forkhead box P3 transcription factor (Foxp3). Foxp3 has been shown to be an important regulator in Treg differentiation and activity. In practice, loss-of-function mutations in the Foxp3 gene have been shown to result in a fatal IPEX syndrome (immunodysregulation, polyglandular endocrine disorder, intestinal disease, X-linked). Patients with IPEX suffer from severe autoimmune reactions, persistent eczema, and colitis. Treg cells expressing Foxp3 play a crucial role in limiting inflammatory responses in the intestine (Josefowicz, SZet al. Nature, 2012, 482, 395-U1510).

[0358] STAT The members of the transcription (STAT) protein family, including signaling and activating factors, are intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis, and differentiation. They are primarily activated by membrane receptor-associated Janus kinases (JAKs). Dysregulation of this pathway is frequently observed in primary tumors, resulting in increased angiogenesis, increased tumor survival, and immunosuppression. Gene knockout studies have provided evidence that STAT proteins are involved in the development and function of the immune system and play a role in maintaining immune tolerance and tumor surveillance.

[0359] There are seven identified mammalian STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B), and STATE.

[0360] Extracellular binding of cytokines or growth factors induces activation of receptor-associated Janus kinases, which phosphorylate specific tyrosine residues within STAT proteins that promote dimerization via their SH2 domains. The phosphorylated dimers are then actively transported to the nucleus via the importin α / β ternary complex. Originally, STAT proteins were described as potential cytoplasmic transcription factors because phosphorylation was thought to be required for nuclear retention. However, unphosphorylated STAT proteins also travel back and forth between the cytosol and the nucleus, playing a role in gene expression. Once STAT reaches the nucleus, it binds to a consensus DNA-recognition motif called gamma-activation (GAS) in the promoter region of cytokine-inducible genes, activating transcription. STAT proteins can be dephosphorylated by nuclear phosphatases, which results in inactivation of STAT and subsequent transport out of the nucleus by the exportin-RanGTP complex.

[0361] In some embodiments, the STAT proteins of this disclosure may be STAT proteins that include modifications that regulate their expression level or activity. In some embodiments, such modifications include, among other things, mutations that result in STAT dimerization, binding of the STAT protein to a signaling partner, STAT protein localization, or STAT protein degradation. In some embodiments, the STAT proteins of this disclosure are constitutively active. In some embodiments, the STAT proteins of this disclosure are constitutively active by constitutive dimerization. In some embodiments, the STAT proteins of this disclosure are constitutively active by constitutive phosphorylation, as described in Onishi, M. et al., Mol. Cell. Biol. July 1998 vol. 18 no. 7 3871-3879, the whole of which is incorporated herein by reference.

[0362] 3.6 Vaccines In some embodiments, one or more expression sequences encode an antigen, such as a tumor antigen, or a fragment thereof. In some embodiments, the expression of such sequences produces an immunogenic composition, such as a vaccine composition that can elicit a specific T-cell response. In some embodiments, the antigen is a nascent antigen.

[0363] 4. Cutting site In some embodiments, two or more expression sequences in a polynucleotide construct may be separated by one or more cleavage site sequences.

[0364] The cleavage site can be any sequence that allows two or more polypeptides to be separated. The cleavage site may be self-cleaving, such that once polypeptides are produced, they are immediately cleaved into individual polypeptides without requiring external cleavage activity.

[0365] The cleavage site may be a furin cleavage site.

[0366] Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process potential precursor proteins into biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta-1 precursor, proalbumin, probeta-secretase, membrane type 1 matrix metalloproteinase, pronervation growth factor beta subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of a basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg) and is enriched in the Golgi apparatus.

[0367] The cleavage site may encode a self-cleaving peptide.

[0368] The cleavage site may operate by ribosome skipping, such as skipping the glycylpropyl bond at the C-terminus of the 2A self-cleaving peptide. In some embodiments, steric hindrance causes ribosome skipping. In some embodiments, the 2A self-cleaving peptide comprises the sequence GDVEXNPGP (SEQ ID NO: 324), where X is E or S. In some embodiments, the protein encoded upstream of the 2A self-cleaving peptide is bound to the 2A self-cleaving peptide, except for post-translational proline at the C-terminus. In some embodiments, the protein encoded downstream of the 2A self-cleaving peptide is bound to proline at the post-translational N-terminus.

[0369] Self-cleaving peptides can be 2A self-cleaving peptides derived from aftviruses or cardioviruses. Major 2A / 2B cleavage in aftviruses and cardioviruses is mediated by 2A cleavage at their C-terminus. In aptoviruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short segment of about 18 amino acids that, together with the N-terminal residue (a conserved proline residue) of protein 2B, represents an autonomous element that can mediate cleavage at its C-terminus (Donelly et al. (2001)).

[0370] 2A-like sequences have been found in picornaviruses other than aptoviruses or cardioviruses, "picornavirus-like" insect viruses, rotavirus type C, and repeat sequences within Trypanosoma and bacterial sequences (Donnelly et al. (2001)). The cleavage site may include one of these 2A-like sequences, such as those listed in Table 8.

[0371] In some embodiments, the self-cleaving peptide is F2A. In some embodiments, the self-cleaving peptide is derived from foot-and-mouth disease virus. In some embodiments, the self-cleaving peptide is E2A. In some embodiments, the self-cleaving peptide is derived from equine rhinitis A virus. In some embodiments, the self-cleaving peptide is P2A. In some embodiments, the self-cleaving peptide is derived from porcine tescovirus-1. In some embodiments, the self-cleaving peptide is T2A. In some embodiments, the self-cleaving peptide is derived from Thosea asigna virus. In some embodiments, the self-cleaving peptide has the sequences listed in Table 8.

[0372] In one embodiment, the expression sequences encoding the therapeutic protein separated by the cleavage site have the same level of protein expression.

[0373] In some embodiments, self-cleaving peptides are described in Liu, Z., Chen, O., Wall, JBJ et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017).

[0374] 5. Polynucleotides containing the second IRES In some embodiments, the expression ratio of the therapeutic protein encoded by the first and second expression sequences may be controlled or influenced by the IRES used in the circRNA and whether the cleavage site or the second IRES separates the first and second expression sequences. If equivalent expression of the proteins encoded by the first and second expression sequences is desired, the circRNA may encode a cleavage site between the first and second expression sequences, e.g., a 2A self-cleaving peptide. If greater expression of the protein encoded by the first expression sequence is desired, the circRNA may encode a first IRES and a second IRES, with the first IRES associated with greater expression than the second IRES, or the second IRES is an intergenetic region (IGR) IRES. If greater expression of the protein encoded by the second expression sequence is desired, the circRNA may encode a first IRES and a second IRES, with the second IRES associated with greater expression than the first IRES.

[0375] In some embodiments, the RNA polynucleotide comprises the first IRES and the second IRES described herein. In some embodiments, the DNA vector encodes the first IRES and the second IRES described herein.

[0376] In some embodiments, the first IRES and the second IRES have the same sequence. In some embodiments, the first IRES and the second IRES have different sequences. In some embodiments, the first IRES is an IRES having the sequences listed in Table 1 (sequence numbers 1 to 72). In some embodiments, the first IRES is a Salivirus IRES. In some embodiments, the first IRES is a Salivirus SZ1 IRES. In some embodiments, the second IRES is an IRES having the sequences listed in Table 1 (sequence numbers 1 to 72). In some embodiments, the second IRES is a Salivirus IRES. In some embodiments, the first IRES is a Salivirus SZ1 IRES.

[0377] In some embodiments, the first IRES is associated with greater expression than the second IRES (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more expression when compared using a construct containing a single IRES). In some embodiments, the second IRES is associated with greater expression than the first IRES (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more expression when compared using a construct containing a single IRES). In some embodiments, the second IRES is an intergenetic region (IGR) IRES.

[0378] Expressing two proteins using a single circRNA polynucleotide offers advantages over expression using multiple polynucleotides. In some embodiments, the expression of two proteins from the circRNA polynucleotide of the present invention results in a more consistent expression ratio than expression from multiple polynucleotides. In some embodiments, the expression of two proteins from the circRNA polynucleotide of the present invention results in transient expression, which may be preferable to sustained expression of DNA.

[0379] 6. Production of polynucleotides The vectors provided herein can be prepared using standard molecular biology techniques. For example, various elements of the vectors provided herein can be obtained using recombinant methods, for example, by screening cDNA and genomic libraries from cells, or by inducing polynucleotides from vectors known to contain polynucleotides.

[0380] The various elements of the vectors provided herein can also be generated synthetically rather than by cloning, based on known sequences. The complete sequence can be assembled from duplicate oligonucleotides prepared by standard methods. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:631¹.

[0381] Therefore, specific nucleotide sequences can be obtained from vectors containing the desired sequence, or synthesized entirely or partially using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques, where appropriate. One method for obtaining a nucleotide sequence encoding a desired vector element is to anneal a complementary set of duplicated synthetic oligonucleotides produced by a conventional automated polynucleotide synthesizer, followed by ligation with a suitable DNA ligase, and then amplification of the ligated nucleotide sequence via PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. In addition, oligonucleotide-specific synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-specific mutagenesis of existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of gap-filled oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0382] The precursor RNA provided herein can be generated by incubating the vector provided herein under conditions that allow transcription of the precursor RNA encoded by the vector. For example, in some embodiments, the precursor RNA is synthesized by incubating the vector provided herein, which includes an RNA polymerase promoter upstream of its 5' double-strand formation region and / or expression sequence, with a compatible RNA polymerase enzyme under conditions that allow in vitro transcription. In some embodiments, the vector is incubated intracellularly by bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II.

[0383] In certain embodiments, a method for generating precursor RNA is provided herein by performing in vitro transcription using a vector provided herein as a template (for example, a vector provided herein having an RNA polymerase promoter located upstream of the 5' double-strand formation region).

[0384] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., circular RNA polynucleotides provided herein) by incubating it in the presence of magnesium ions and guanosine nucleotides or nucleosides at a temperature at which RNA cyclization occurs (e.g., 20°C to 60°C).

[0385] Therefore, in certain embodiments, a method for producing circular RNA is provided herein. In certain embodiments, the method comprises synthesizing a precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a post-splicing 3' group I intron fragment, an intra-sequence ribosome entry site (IRES), an expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, and a 5' group I intron fragment) as a template, and incubating the obtained precursor RNA in the presence of a divalent cation (e.g., a magnesium ion) and GTP to circulate it and form circular RNA. In some embodiments, the precursor RNA disclosed herein can be circulated in the absence of magnesium ions and GTP, and / or without the incubation step with magnesium ions and GTP. It has been found that circular RNA has reduced immunogenicity compared to the corresponding mRNA, at least in part, because the mRNA contains an immunogenic 5' cap. When a DNA vector is transcribed from a given promoter (e.g., a T7 promoter) to produce precursor RNA, it is understood that the 5' end of the precursor RNA is G. To reduce the immunogenicity of circular RNA compositions containing low levels of contaminating linear mRNA, excess GMP relative to GTP may be provided during transcription so that most transcripts contain 5'GMP that cannot be capped. Therefore, in some embodiments, transcription is carried out in the presence of excess GMP. In some embodiments, transcription is carried out with a GMP concentration to GTP concentration ratio in the range of about 3:1 to about 15:1, for example, about 3:1 to about 10:1, about 3:1 to about 5:1, about 3:1, about 4:1, or about 5:1.

[0386] In some embodiments, the composition containing circular RNA is purified. The circular RNA can be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in the order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse-phase HPLC. In some embodiments, the purified composition contains less double-stranded RNA, DNA sprints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes, and / or nicked RNA than the unpurified RNA. In some embodiments, the purified composition is less immunogenic than the unpurified composition. In some embodiments, immune cells exposed to the purified composition produce less TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferon, such as IFN-β1, than immune cells exposed to the unpurified composition.

[0387] 7. Nanoparticles In certain embodiments, what is provided herein is a pharmaceutical composition comprising a circular RNA provided herein. In certain embodiments, such a pharmaceutical composition is formulated with nanoparticles to facilitate delivery.

[0388] In certain embodiments, the circular RNA provided herein may be delivered to and / or targeted to cells in a transport vehicle, such as nanoparticles or a composition containing nanoparticles. In some embodiments, the circular RNA may also be delivered to a target in a transport vehicle or a composition containing a transport vehicle. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, solid lipid nanoparticles, polymer core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the transport vehicle contains or is coated with one or more cationic lipids, non-cationic lipids, ionic lipids, PEG-modified lipids, polyglutamic acid polymers, hyaluronic acid polymers, poly-β-aminoesters, poly-beta-aminopeptides, or positively charged peptides.

[0389] In one embodiment, a transport vehicle can be selected and / or prepared to optimize the delivery of circRNA to target cells. For example, if the target cells are hepatocytes, the properties of the transport vehicle (e.g., size, charge, and / or pH) can be optimized to effectively deliver such vehicle to the target cells, reduce immune clearance, and / or promote retention in the target cells.

[0390] The use of a transport vehicle to facilitate the delivery of nuc...

Claims

1. A circular RNA polynucleotide comprising, in the following order, a post-splicing 3' group I intron fragment, a first intra-sequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, and a post-splicing 5' group I intron fragment, wherein the first expression sequence encodes a CD19 or BCMA antigen-binding molecule and / or the second expression sequence encodes a CD19 or BCMA antigen-binding molecule.

2. The circular RNA polynucleotide according to claim 1, further comprising a cleavage site between the first expression sequence and the second expression sequence.

3. The cyclic RNA polynucleotide according to claim 2, wherein the cleavage site is a self-cleaving spacer or a 2A self-cleaving peptide.

4. The cyclic RNA polynucleotide according to claim 1, comprising a second IRES between the first expression sequence and the second expression sequence.

5. The cyclic RNA polynucleotide according to claim 4, wherein the first IRES and / or the second IRES consists of or includes any sequence of SEQ ID NOs: 1 to 72.

6. The cyclic RNA polynucleotide according to any one of claims 1 to 5, wherein the CD19 antigen-binding molecule and / or BCMA antigen-binding molecule is an antibody or an antigen-binding fragment thereof, or a chimeric antigen receptor (CAR).

7. The cyclic RNA polynucleotide according to claim 6, wherein the antibody or its antigen-binding fragment is selected from scFv, a single-domain antibody, and a camelized antibody.

8. (a) The first expression sequence encodes a CD19 antigen-binding molecule, and the second expression sequence encodes a therapeutic protein. (b) The first expression sequence encodes a therapeutic protein, and the second expression sequence encodes a CD19 antigen-binding molecule. (c) The first expression sequence encodes a BCMA antigen-binding molecule, and the second expression sequence encodes a therapeutic protein, or (d) The first expression sequence encodes a therapeutic protein, and the second expression sequence encodes a BCMA antigen-binding molecule. A cyclic RNA polynucleotide according to any one of claims 1 to 7.

9. The expression sequence encoding the aforementioned therapeutic protein is (a) T cell receptor (TCR), (b) Chimeric antigen receptor (CAR), (c) PD1 or PDL1 antagonist, (d) Cytokines, (e) Chemokines, (f) Tumor antigen, (g) immunosuppressive enzymes; (h) Transcription factor, (i) BCL-XL, (j) Chaperone protein, (k) signaling protein, or (l) An enzyme that can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), A cyclic RNA polynucleotide according to claim 8, which codes for the cyclic RNA polynucleotide.

10. (a) The tumor antigen is a newly generated antigen, (b) The cytokine is selected from IFNγ, IL-2, IL-7, IL-12, IL-15, IL-18, IL-10, IL-35, and TGFβ. (c) The chemokine is CC chemokine, CXC chemokine, C chemokine, CX3C chemokine, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, C From CL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1, (d) The transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and cd25. (e) The chaperone protein is selected from Skp, Spy, FkpA, SurA, Hsp60, Hsp70, GroEL, GroES, Hsp90, HtpG, Hsp100, ClpA, ClpX, ClpP, and Hsp104, and / or (f) The enzyme that can convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) is cytosine deaminase. The cyclic RNA polynucleotide according to claim 9.

11. A cyclic RNA polynucleotide according to any one of claims 1 to 10, comprising, in the following order: a 5' double helix formation region, a post-splicing 3' group I intron fragment, a first intrasequential ribosome entry site (IRES), a first expression sequence, a second expression sequence, a post-splicing 5' group I intron fragment, and a 3' double helix formation region.

12. The cyclic RNA polynucleotide according to claim 11, comprising a first spacer between the 5' double helix formation region and the post-splicing 3' group I intron fragment, and a second spacer between the post-splicing 5' group I intron fragment and the 3' double helix formation region.

13. The cyclic RNA polynucleotide according to claim 12, wherein the first and second spacers each have a length of 10 to 60 nucleotides or 9 to 19 nucleotides.

14. The cyclic RNA polynucleotide according to claim 12 or 13, wherein the 5' double-strand forming region and the 3' double-strand forming region each have a length of 30 nucleotides.

15. The aforementioned IRES includes Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliopathy virus, human poliovirus 1, brown marmorated bug (Platia stalli) enterovirus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, pygmy kite P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, and Ectropis obliqua) picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen bee brood virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n. myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, budding yeast (S. cerevisiae) TFIID, budding yeast YAP1, tobacco etch virus, cabbage crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCVQC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, human parechovirus 1, black hivirus B, Yc-3, rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, human parechovirus 5, Aichivirus, hepatitis A virus HA16, fopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, pegivirus A1220, Pacivirus A 3. Saperovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine pacivirus 1, PLV-CHN, Pacivirus A, Sisinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border disease virus, BVDV2, CSFV-PK15C, SF573 disicisthovirus, Houpei picorna-like virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A A circular RNA polynucleotide according to any one of claims 1 to 14, having an IRES sequence from an aptamer for SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

16. A cyclic RNA polynucleotide according to any one of claims 1 to 15, comprising natural nucleotides.

17. The circular RNA polynucleotide according to any one of claims 1 to 16, wherein the expression sequence is codon-optimized.

18. (a) At least one microRNA binding site present in the pre-optimized cyclic RNA polynucleotide, (b) At least one endonuclease-sensitive site present in the pre-optimized cyclic RNA polynucleotide, and / or (c) At least one RNA editing sensitive site present in the pre-optimized cyclic RNA polynucleotide A circular RNA polynucleotide according to any one of claims 1 to 17, which is optimized to lack a certain characteristic.

19. A circular RNA polynucleotide according to any one of claims 1 to 18, having a length of 100 nucleotides to 15 kilobases.

20. A cyclic RNA polynucleotide according to any one of claims 1 to 19, having a duration of therapeutic effect in vivo in humans of at least 20 hours, and / or a functional half-life of at least 20 hours.

21. (a) Having a duration of therapeutic effect in human cells that is longer than or equal to that of linear RNA polynucleotides containing the same expression sequence, (b) Having a functional half-life in human cells that is longer than or equal to that of a linear RNA polynucleotide containing the same expression sequence, (c) Having a longer duration of in vivo therapeutic effect in humans than that of linear RNA polynucleotides having the same expression sequence, and / or (d) Having a longer functional half-life in vivo in humans than that of linear RNA polynucleotides with the same expression sequence, A cyclic RNA polynucleotide according to any one of claims 1 to 20.

22. A pharmaceutical composition comprising one or more cyclic RNA polynucleotides and nanoparticles according to any one of claims 1 to 21, wherein the one or more cyclic RNA polynucleotides are encapsulated by the nanoparticles.

23. The pharmaceutical composition according to claim 22, wherein the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles.

24. The pharmaceutical composition according to claim 22 or 23, comprising a targeting portion, wherein the targeting portion mediates receptor-mediated endocytosis or direct fusion of the nanoparticles to selected cells in a selected cell population or tissue without cell isolation or purification, and the targeting portion is operably connected to the nanoparticles.

25. The pharmaceutical composition according to claim 24, wherein the targeting portion is an scFv, a nanobody, a peptide, a minibody, a polynucleotide aptamer, a heavy chain variable region, a light chain variable region, or a fragment thereof.

26. (a) Less than 1% by weight of the polynucleotide in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA, and / or (b) Less than 1% by weight of the polynucleotide in the pharmaceutical composition is a phosphatase protein, a protein ligase, and a capping enzyme. A pharmaceutical composition according to any one of claims 22 to 25.

27. (a) One or more cationic lipids, ionizable lipids, or polyβ-aminoesters, (b) One or more noncationic lipids, (c) One or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids, (d) Cholesterol, and / or (e) Arachidonic acid or oleic acid A pharmaceutical composition according to any one of claims 22 to 26, comprising [the specified element].

28. The pharmaceutical composition according to any one of claims 22 to 27, wherein the nanoparticles encapsulate one or more of the cyclic RNA polynucleotides.

29. A pharmaceutical composition for treating a target requiring treatment, comprising a therapeutically effective amount of a cyclic RNA polynucleotide according to any one of claims 1 to 21, or the pharmaceutical composition according to any one of claims 22 to 28.

30. The aforementioned subject is, (a) Acute lymphoblastic leukemia; acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B-cell malignancies; bladder cancer (e.g., bladder carcinoma); bone cancer; brain cancer (e.g., medulloblastoma); breast cancer; cancer of the anus, anal canal, or anorectum; eye cancer; intrahepatic bile duct cancer; joint cancer; neck cancer; gallbladder cancer; pleural cancer; cancer of the nose, nasal cavity, or middle ear; oral cancer; vulvar cancer; chronic lymphoblastic leukemia; chronic myeloid carcinoma; colon cancer; esophageal cancer; cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumors; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin's lymphoma; hypopharyngeal cancer; kidney cancer; laryngeal cancer; leukemia; humoral neoplasms; liver cancer; Lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma); lymphoma; mesothelioma; mast cell tumor; melanoma; Multiple myeloma; nasopharyngeal carcinoma; non-Hodgkin lymphoma; chronic lymphocytic leukemia B; hairy cell leukemia; acute lymphoblastic leukemia (ALL); Burkitt lymphoma; ovarian cancer; Pancreatic cancer; peritoneal cancer; Cancer of the omentum; mesenteric cancer; pharyngeal cancer; prostate cancer; rectal cancer; renal cancer; skin cancer; Having a cancer selected from the group consisting of small intestine cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; testicular cancer; thyroid cancer; and ureteral cancer, and / or (b) Having an autoimmune disorder selected from systemic autoimmune diseases typically represented by scleroderma, Graves' disease, Crohn's disease, Sjögren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyglandular endocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and human lupus, The pharmaceutical composition according to claim 29.

31. A vector for producing circular RNA polynucleotides, comprising, in the following order: a 5' double helix formation region, a 3' group I intron fragment, an intrasequence ribosome entry site (IRES), a first expression sequence, a second expression sequence, a 5' group I intron fragment, and a 3' double helix formation region. (a) A polynucleotide sequence encoding a cleavage site between the first expression sequence and the second expression sequence, and / or (b) A first spacer between the 5' double-chain forming region and the 3' group I intron fragment, and a second spacer between the 5' group I intron fragment and the 3' double-chain forming region. Includes, The first expression sequence encodes a CD19 or BCMA antigen-binding molecule, and / or the second expression sequence encodes a CD19 or BCMA antigen-binding molecule. The aforementioned vector.

32. A eukaryotic cell comprising a cyclic RNA polynucleotide as described in any one of claims 1 to 21, which is a human cell, an immune cell, and / or a T cell.

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