Circular RNA Compositions and Methods

Abstract

Circular RNAs and delivery vehicles are described herein, along with related compositions and methods. In some embodiments, the circular RNAs of the present invention comprise a group I intron fragment, a spacer, an IRES, a duplex-forming region, and an expression sequence. In some embodiments, the expression sequence encodes a chimeric antigen receptor (CAR). 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 benefit of and priority over U.S. Provisional Patent Application No. 62 / 851,548 filed on 22 May 2019; U.S. Provisional Patent Application No. 62 / 857,121 filed on 4 June 2019; International Patent Application No. PCT / US2019 / 035531 filed on 5 June 2019; U.S. Provisional Patent Application No. 62 / 943,796 filed on 4 December 2019; U.S. Provisional Patent Application No. 62 / 943,779 filed on 4 December 2019; and U.S. Provisional Patent Application No. 62 / 972,194 filed on 10 February 2020 (the disclosures thereof are incorporated herein by reference in their entirety 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 detrimental 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 detrimental 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; US2004 / 0110709), these approaches may 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] The use of genetically modified T cells expressing chimeric antigen receptors (CARs) and recombinant T cell receptors (TCRs) that target antigens on cancer cells is an attractive therapeutic strategy for treating cancer. However, current methods for modifying T cells to express CARs and TCRs, and the resulting therapies, are toxic in the form of cytokine release syndrome (CRS) and other complications. Safer methods for manipulating cells to express CARs and recombinant TCRs are still needed.

[0006] 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 Initiative]

[0007] overview In one embodiment, the following pharmaceutical composition is provided herein, comprising a cyclic RNA polynucleotide containing a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein, and a 5' group I intron fragment, in the order described below, and a transport vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, the transport vehicle capable of delivering the cyclic RNA polynucleotide to human immune cells present in a human subject, thereby translating the CAR in the human immune cells and expressing it on the surface of the human immune cells.

[0008] In some embodiments, the pharmaceutical composition is formulated for intravenous administration to human subjects requiring it. In some embodiments, the 3' group I intron fragment and the 5' group I intron fragment are anabaena group I intron fragments.

[0009] In certain embodiments, the 3' and 5' intron fragments are defined by the L9a-5 permutation substitutive sites of the intact intron. In certain embodiments, the 3' and 5' intron fragments are defined by the L8-2 permutation substitutive sites of the intact intron.

[0010] In some embodiments, IRES is 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 cell 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-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, S.Cerevisiae YAP1, Tobacco HTC virus, Kabukurinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirnavirus, HCV QC64, Hitocosavirus E / D, Hitocosavirus F, Hitocosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A virus HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C Derived from aptamers for K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Saperovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Cissinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Disicisthovirus, Hubei 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.

[0011] In some embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof, or the IRES comprises a sequence according to SEQ ID NO: 65. In some embodiments, the IRES comprises a Salivirus SZ1 IRES or a fragment or variant thereof. In certain embodiments, the IRES comprises a sequence according to SEQ ID NO: 63. In some embodiments, the pharmaceutical composition comprises a first internal spacer between the 3' group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5' group I intron fragment. In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

[0012] In some embodiments, the CAR or TCR complex proteins include: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family members, B cell maturation antigen (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, and Interleukin-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), nerve cell adhesion molecule (N CAM), prostase, 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 Averso Oncogene fusion protein (bcr-abl) consisting of mouse leukemia virus oncogene 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),It contains antigen-binding domains specific to antigens selected from tumor endothelial marker 7 (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

[0013] In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD19-specific antigen-binding domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing a co-stimulatory domain selected from the group CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing CH2CH3, CD28, and / or CD8 spacer domains. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD28 or CD8 transmembrane domain.

[0014] In some embodiments, the CAR or TCR complex protein includes a CAR comprising an antigen-binding domain, a spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular T cell signaling domain.

[0015] In some embodiments, the CAR or TCR complex protein comprises a multispecific CAR having antigen-binding domains for at least two different antigens. In some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the groups TCR alpha, TCR beta, TCR gamma, and TCR delta. In some embodiments, the transport vehicle comprises lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles.

[0016] In some embodiments, the pharmaceutical composition further comprises a targeted moiety. In certain embodiments, the targeted moiety mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue in the absence of cell isolation or purification. In certain embodiments, the targeted moiety can bind to proteins selected from the group CD3, CD4, CD8, CD5, CD7, PD-1, 4-1BB, CD28, C1q, and CD2. In certain embodiments, the targeted moiety comprises antibodies specific to macrophages, dendritic cells, NK cells, NKT, or T cell antigens. In certain embodiments, the targeted moiety comprises scFv, nanobodies, peptides, minibodies, polynucleotide aptamers, heavy chain variable regions, light chain variable regions, or fragments thereof.

[0017] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat cancer in human subjects. In some embodiments, the pharmaceutical composition has an improved safety profile compared to pharmaceutical compositions containing T cells or vectors containing exogenous DNA encoding the same CAR.

[0018] In some embodiments, less than 1% by weight of the polynucleotides in the composition is double-stranded RNA, DNA sprint, or triphosphorylated RNA.

[0019] In some embodiments, less than 1% by weight of polynucleotides and proteins in the pharmaceutical composition are double-stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes. In some embodiments, the transport vehicle contains more than one cyclic RNA polynucleotide.

[0020] In another embodiment, the present disclosure provides a circular RNA polynucleotide comprising, in the following order: a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, and a 5' group I intron fragment.

[0021] In some embodiments, the 3' group I intron fragment and the 5' group I intron fragment are anabaena group I intron fragments. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L9a-5 permutation site of an intact intron. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 permutation site of an intact intron. In certain embodiments, the IRES includes a CVB3 IRES or a fragment or variant thereof. In certain embodiments, the IRES has a sequence according to SEQ ID NO: 65. In some embodiments, the IRES includes a Salivirus SZ1 IRES or a fragment or variant thereof. In certain embodiments, the IRES has a sequence according to SEQ ID NO: 63.

[0022] In some embodiments, the circular RNA polynucleotide includes a first internal spacer between the 3' group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5' group I intron fragment.

[0023] In a particular embodiment, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

[0024] In some embodiments, the circular RNA polynucleotide consists of native nucleotides. In some embodiments, the circular RNA polynucleotide further comprises a second expression sequence encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a checkpoint inhibitor. In certain embodiments, the therapeutic protein comprises a cytokine.

[0025] In some embodiments, the CAR or TCR complex proteins include: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disiaroganglioside GD2, disiaroganglioside GD3, TNF receptor family members, B-cell maturation antigen (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, and Interleukin-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), nerve cell adhesion molecule (N CAM), prostase, 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 Averso Oncogene fusion protein (bcr-abl) consisting of mouse leukemia virus oncogene 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),It contains antigen-binding domains specific to antigens selected from tumor endothelial marker 7 (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

[0026] In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD19-specific antigen-binding domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing a co-stimulatory domain selected from the group CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing CH2CH3, CD28, and / or CD8 spacer domains. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD28 or CD8 transmembrane domain.

[0027] In some embodiments, the CAR or TCR complex protein includes a CAR comprising an antigen-binding domain, a spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular T cell signaling domain.

[0028] In some embodiments, the CAR or TCR complex protein comprises a multispecific CAR having antigen-binding domains for at least two different antigens. In some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the groups TCR alpha, TCR beta, TCR gamma, and TCR delta.

[0029] In some embodiments, the cyclic RNA polynucleotide consists of native nucleotides.

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

[0031] In some embodiments, the circular RNA polynucleotide has an in vivo functional half-life in humans that is longer than that of a comparable linear RNA polynucleotide having the same expression sequence. In some embodiments, the circular RNA polynucleotide has a length of about 100 nucleotides to about 10 kilobases. In some embodiments, the circular RNA polynucleotide has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in human cells of at least about 20 hours. In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in human cells that is longer than or equal to that of a comparable linear RNA polynucleotide containing the same expression sequence. In some embodiments, the circular RNA polynucleotide has a functional half-life in human cells that is longer than or equal to that of a comparable linear RNA polynucleotide containing the same expression sequence.

[0032] In another embodiment, the present disclosure provides a DNA vector comprising, in the following order: a 5' double-strand formation region, a first permutation substitution site for an anabaena 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide, a second permutation substitution site for an anabaena 5' group I intron fragment, and a 3' double-strand formation region.

[0033] In some embodiments, the 3' group I intron fragment and the 5' group I intron fragment are anabaena group I intron fragments. In some embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L9a-5 permutation site of an intact intron. In some embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 permutation site of an intact intron. In some embodiments, the IRES includes a CVB3 IRES or a fragment or variant thereof.

[0034] In some embodiments, the IRES encodes a sequence according to SEQ ID NO: 65. In some embodiments, the IRES comprises a Salivirus SZ1 IRES or a fragment or variant thereof. In some embodiments, the IRES encodes a sequence according to SEQ ID NO: 63. In some embodiments, the circular RNA polynucleotide comprises, in the following order: a 5' double-stranding region, a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, a 5' group I intron fragment, and a 3' double-stranding region. In some embodiments, the 5' double-stranding region and the 3' double-stranding region each have about 70% GC nucleotides. In some embodiments, the 5' double-stranding region and the 3' double-stranding region each have about 30 nucleotides in length.

[0035] In some embodiments, the DNA vector includes a first external spacer between the 5' double-strand formation region and the 3' group I intron fragment, and a second external spacer between the 5' group I intron fragment and the 3' double-strand formation region. In some embodiments, the first and second external spacers are each about 10 to about 60 nucleotides long. In some embodiments, the 5' double-strand formation region is directly adjacent to the 3' group I intron fragment, and the 5' group I intron fragment is directly adjacent to the 3' double-strand formation region. In some embodiments, the DNA vector includes a first internal spacer between the 3' group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5' group I intron fragment. In some embodiments, the first and second internal spacers are each about 10 to about 60 nucleotides long.

[0036] In some embodiments, the CAR or TCR complex proteins include: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disiaroganglioside GD2, disiaroganglioside GD3, TNF receptor family members, B-cell maturation antigen (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, and Interleukin-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), nerve cell adhesion molecule (N CAM), prostase, 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 Averso Oncogene fusion protein (bcr-abl) consisting of mouse leukemia virus oncogene 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),It contains antigen-binding domains specific to antigens selected from tumor endothelial marker 7 (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

[0037] In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD19-specific antigen-binding domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing a co-stimulatory domain selected from the group CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein includes a CAR containing CH2CH3, CD28, and / or CD8 spacer domains. In some embodiments, the CAR or TCR complex protein includes a CAR containing a CD28 or CD8 transmembrane domain.

[0038] In some embodiments, the CAR or TCR complex protein includes a CAR comprising an antigen-binding domain, a spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular T cell signaling domain.

[0039] In some embodiments, the CAR or TCR complex protein comprises a multispecific CAR having antigen-binding domains for at least two different antigens. In some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the groups TCR alpha, TCR beta, TCR gamma, and TCR delta.

[0040] In another embodiment, the disclosure provides a eukaryotic cell comprising, in the following order: a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, and a circular RNA polynucleotide comprising a 5' group I intron fragment. In some embodiments, the eukaryotic cell includes human cells. In some embodiments, the eukaryotic cell includes immune cells. In some embodiments, the eukaryotic cell includes T cells.

[0041] In another aspect, the disclosure provides a population of eukaryotic cells comprising, in the following order, a cyclic RNA polynucleotide comprising a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, and a 5' group I intron fragment, the population of eukaryotic cells expressing the CAR or TCR complex protein encoded by the cyclic RNA polynucleotide on its cell surface.

[0042] In some embodiments, the cell population includes NK cells, NKT cells, macrophages, dendritic cells, alpha-beta T cells, gamma-delta T cells, or a combination thereof. In some embodiments, the cell population includes T cells. In some embodiments, the population includes CD3+ T cells. In some embodiments, the population includes CD4+ T cells. In some embodiments, the population includes CD8+ T cells. In some embodiments, the eukaryotic cell population is administered in an effective amount to treat cancer in human subjects requiring it. In some embodiments, the cell population kills tumor cells more effectively or for longer than an equivalent population of eukaryotic cells containing the same CAR-encoding linear RNA.

[0043] In another embodiment, a method for producing a population of eukaryotic cells is provided herein, comprising contacting the cells in the population with a transport vehicle comprising a circular RNA polynucleotide comprising a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, and a 5' group I intron fragment, in the following order, wherein the transport vehicle comprises (i) ionizable lipids, (ii) structural lipids, and (iii) PEG-modified lipids, and the transport vehicle can deliver the circular RNA polynucleotide to human immune cells, thereby translating the CAR in the human immune cells and expressing it on the surface of the human immune cells.

[0044] In another embodiment, the Specified Method provides a method for treating a subject requiring treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising, in the following order, a cyclic RNA polynucleotide comprising a 3' group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or TCR complex protein, and a 5' group I intron fragment, and a transport vehicle comprising (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, the transport vehicle being able to deliver the cyclic RNA polynucleotide to human immune cells, thereby translating the CAR in the human immune cells and expressing it on the surface of the human immune cells.

[0045] In some embodiments, the subjects include: group cancer, acute lymphoblastic carcinoma, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), 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, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic lymphocytic 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 The patient has cancers selected from: tumors, 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 cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL), and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and 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.

[0046] In another embodiment, the Specified Information provides an RNA polynucleotide comprising an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide, and at least one auto-circular element.

[0047] In some embodiments, the RNA polynucleotide comprises a 5' double-stranding region, an anabaena 3' group I intron fragment and a first permutation substitution site, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide, an anabaena 5' group I intron fragment and a second permutation substitution site, and a 3' double-stranding region. In some embodiments, the RNA polynucleotide comprises a 5' double-stranding region, a first permutation substitution site, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide, a second permutation substitution site, and a 3' double-stranding region. In some embodiments, the self-cyclic element is a group I intron fragment. In some embodiments, the 3' group I intron fragment and the 5' intron fragment are anabaena group I intron fragments. In some embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L9a-5 permutation substitution site of an intact intron. In some embodiments, the 3' and 5' intron fragments are defined by the L8-2 permutation substitution site of the intact intron. In some embodiments, the RNA polynucleotide can be cyclized in the absence of an enzyme. In some embodiments, the RNA polynucleotide consists of native nucleotides.

[0048] In another embodiment, the present disclosure provides a DNA vector suitable for the synthesis of one of the above embodiments of RNA polynucleotides.

[0049] In some embodiments, the cyclic RNA polynucleotides of this disclosure are delivered to target cells in non-lipid polymer core-shell nanoparticles.

[0050] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is explicitly and individually incorporated herein by reference. [Brief explanation of the drawing]

[0051] [Figure 1] The images show luminescence in the supernatant of HEK293(A), HepG2(B), or 1C1C7(C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences. [Figure 2] The images show luminescence in the supernatant of HEK293 (A), HepG2 (B), and 1C1C7 (C) cells 24 hours after transfection with circular RNA containing Gaussial alciferase expression sequences and various IRES sequences of different lengths. [Figure 3] This shows the stability of selected IRES constructs in HepG2(3A) or 1C1C7(3B) cells over 3 days, as measured by luminescence. [Figure 4] A and B show protein expression from selected IRES constructs in Jurkat cells, as measured by luminescence from secreted Gaussial Luciferase in the cell supernatant. [Figure 5] A and B show the stability of selected IRES constructs in Jurkat cells over 3 days, as measured by luminescence. [Figure 6] This shows a comparison of 24-hour luminescence (A) or relative luminescence over 3 days (B) of modified linear, unpurified circular, or purified circular RNA encoding Gausial ciferase. [Figure 7] This shows the transcriptional induction of IFNγ (A), IL-6 (B), IL-2 (C), RIG-I (D), IFN-β1 (E), and TNFα (F) after electroporation of Jurkat cells using modified linear, unpurified circular, or purified circular RNA. [Figure 8]This shows a comparison of the luminescence of circular RNA and modified linear RNA encoding Gaussial luciferase in human primary monocytes (A) and macrophages (B and C). [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. [Figure 10] The images show 24-hour luminescence (A) or relative luminescence over 3 days (B) in the supernatant of primary T cells transduced using circular RNA or modified linear RNA containing a Gaussial luciferase expression sequence, and 24-hour luminescence in PBMCs (C). [Figure 11] The HPLC chromatograms (A) and cyclization efficiency (B) of RNA constructs with different permutation substitution sites are shown. [Figure 12] The HPLC chromatograms (A) and cyclization efficiencies (B) of RNA constructs with different intron and / or permutation sites are shown. [Figure 13] The HPLC chromatograms (A) and cyclization efficiencies (B) of three RNA constructs with and without homologous arms are shown. [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. [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 selected constructs, and combinations of permutation substitution sites and homologous arms assumed to demonstrate improved cyclization efficiency. [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). [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). [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. [Figure 19] A shows 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 alciferase expression sequences and various IRES sequences. B shows relative luminescence over 3 days. [Figure 20] This shows the transcriptional induction of IFN-β1 (A), RIG-I (B), IL-2 (C), IL-6 (D), IFNγ (E), and TNFα (F) after electroporation of human CD3+ T cells using modified linear, unpurified circular, or purified circular RNA. [Figure 21] A 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. B shows IFNγ transcript induction 24 hours after electroporation with different amounts of circular or linear RNA encoding the CAR sequence. [Figure 22] A and B show 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, as determined by detection of firefly bioluminescence. [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. [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. [Figure 25] This shows the RNAFold prediction of the precursor RNA secondary structure for homologous arm design. A higher concentration of bases indicates a higher probability of base pairing. In the absence of homologous arms, base pairing is predicted not to occur between the ends of the precursor molecule. [Figure 26] This shows agarose gel confirmation of precursor RNA cyclization. C: Precursor RNA subjected to cyclization conditions (having strong homologous arms). C+R: Lane C, digested with RNase R. C+R+H: Lane C+R, digested with oligonucleotide-induced RNase H. U: Precursor RNA not subjected to cyclization conditions. U+H: Lane U, digested with oligonucleotide-induced RNase H. [Figure 27] The Sanger sequencing output of RT-PCR across the splice junction of the sample shown in lanes C+R from Figure 26 is shown. [Figure 28] This shows the RNAFold prediction of the secondary structure of precursor RNA in relation to the designed spacer. Secondary structures potentially important for ribozyme function are identified by black arrows. [Figure 29] Agarose gels demonstrating the effect of spacers on splicing are shown. (-): No spacer. D: Destructive spacer. P1: Tolerant spacer 1. P2: Tolerant spacer 2. cRNAFold prediction of precursor RNA secondary structure for internal homology region design. Significant absence of internal homology (anabaena 1.0) and introduced internal homology (anabaena 2.0) are indicated by black arrows. Splicing bubbles are shown as regions between homologous arms containing splicing ribozymes and internal homology regions. [Figure 30] This is a schematic diagram showing the elements of the engineered self-splicing precursor RNA design. [Figure 31]The image shows luminescence in the supernatant of HEK293 (left, black outline) and HeLa (right, gray outline) cells 24 hours after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA (n=4 HEK293, n=3 HeLa). [Figure 32] The supernatant of HEK293 cells showed luminescence starting 24 hours after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA and continuing for 6 days (n=4). [Figure 33] This section outlines precursor RNA design and self-splicing. Shaded areas indicate different regions of the RNA described herein. [Figure 34] This study shows cell viability, circRNA expression stability, and cytokine release from A549 cells transfected with different circRNA preparations (+RNase R, unpurified circRNA digested only with RNase R; +HPLC, unpurified circRNA purified by HPLC and then digested with RNase R; +Phos, unpurified circRNA purified by HPLC, treated with phosphatase, and then digested with RNase R). Cell viability was assessed 3 days after transfection. Cytokine release was assessed 24 hours after transfection (data expressed as mean + SD; n=3; *p<0.05; ND, not detected). [Figure 35] A schematic diagram of the RNA introduced and used for the TLR experiment is shown. The linearized circRNA contains all the same sequence elements as the spliced ​​circRNA due to deletions that include both introns and homologous arms. [Figure 36] Surface expression of anti-CD19 CARs was shown on primary human T cells isolated from four donors and electroporated with circRNA. [Figure 37] A shows the percentage of CAR+ live T cells electroporated with circRNA. B shows the percentage of CD4 and CD8-positive T cells in the four human donors in Figure 36. [Figure 38]This study demonstrates the effectiveness of mock-electroporated or T cells electroporated with circRNA or linear RNA encoding anti-CD19 CARs in reducing bioluminescence in CD19+ target cells and CD19-non-target cells expressing luciferase. [Figure 39] The efficacy of T cells co-cultured with luciferase-expressing Raji or K562 cells 5 days (A) or 1 day (B) after electroporation with circRNA encoding anti-CD19 CARs having different IRESs is demonstrated. (Oro-B1 = circ Kymriah; CVB3 IRES, Oro-152 = circ Kymriah; Salivirus SZ1 IRES, L9a-5 permutation site) [Figure 40] This shows the lysis of target and non-target cells by T cells electroporated with circRNA encoding anti-CD19 CAR. A shows the lysis of CD19+ Raji cells and CD19-K562 cells by primary human T cells electroporated with circRNA containing an anti-CD19 CAR expression sequence and CVB3 IRES or Salivirus SZ1 IRES. B shows the lysis of CD19+ Raji cells and CD19-K562 cells by primary human T cells electroporated with different ratios of circRNA or linear mRNA containing an anti-CD19 CAR expression sequence. C shows the lysis of CD19+ Raji cells and CD19-K562 cells by different ratios of primary human T cells electroporated with circRNA containing an anti-CD19 CAR expression sequence. [Figure 41]This shows the efficacy of T cells electroporated with anti-CD19 CAR-encoding circRNA in the lysis of CD19+ Raji cells compared to T cells transduced with a lentivirus encoding anti-CD19 CAR. A shows the specific lysis of anti-CD19 CAR-encoding circRNA in the lysis of CD19+ Raji cells compared to T cells transduced with a lentivirus encoding anti-CD19 CAR, at a ratio of 10 Raji cells to 1 T cell. B shows the percentage (%) of anti-CD19 CAR-expressing T cells electroporated with mock electroporation or with an anti-CD19 CAR-encoding lentivirus or circRNA. C shows the induction of interferon-gamma mRNA in T cells electroporated with anti-CD19 CAR-encoding circRNA in co-culture with or without CD19+ Raji cells. (Oro-B1=circ Kymriah; CVB3 IRES, Oro-B6=lin Kymriah) [Figure 42] This demonstrates the stability of anti-CD19 CAR expression in primary human CD3+ T cells electroporated with circRNA or linear mRNA encoding anti-CD19 CARs. [Figure 43] This study demonstrates the effectiveness of electroporating THP-1 monocytes with CAR-encoding circRNA in the lysis of luciferase-expressing Raji cells in co-culture experiments. [Figure 44] This study demonstrates the efficacy of electroporated T cells with circRNA encoding anti-mouse CD19 CAR in the lysis of CD19+A20 cells and CD19-K562 cells. [Modes for carrying out the invention]

[0052] Detailed explanation Pharmaceutical compositions and transport vehicles, such as lipid nanoparticles, comprising circular RNA are provided herein. The circular RNA provided herein may be delivered and / or targeted to cells by transport vehicles, such as nanoparticles, or compositions comprising transport vehicles. In some embodiments, the circular RNA may also be delivered to a target by transport vehicles or compositions comprising transport vehicles. In some embodiments, the transport vehicle is nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, non-lipid polymer core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the transport vehicle comprises one or more ionizable lipids, PEG-modified lipids, helper lipids, and / or structural lipids.

[0053] In some embodiments, the transport vehicle encapsulates circular RNA and includes ionizable lipids, structural lipids, and PEG-modified lipids.

[0054] While we do not wish to be bound by theory, the transport vehicles described herein are thought to protect encapsulated circular RNA from degradation and provide effective delivery of circular RNA to target cells in vivo and in vitro.

[0055] Embodiments of this disclosure provide lipid compositions described according to the respective molar ratios of component lipids in the formulation. In one embodiment, the molar percentage of ionizable lipids may be about 10 mol% to about 80 mol%. In one embodiment, the molar percentage of ionizable lipids may be about 20 mol% to about 70 mol%. In one embodiment, the molar percentage of ionizable lipids may be about 30 mol% to about 60 mol%. In one embodiment, the molar percentage of ionizable lipids may be about 35 mol% to about 55 mol%. In one embodiment, the molar percentage of ionizable lipids may be about 40 mol% to about 50 mol%. In some embodiments, the molar percentage of ionizable lipids in a transport vehicle batch may be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage. In certain embodiments, the lot-to-lot variation of the transport vehicle may be less than 15%, less than 10%, or less than 5%.

[0056] In one embodiment, the molar percentage of helper lipids may be about 1 mol% to about 50 mol%. In one embodiment, the molar percentage of helper lipids may be about 2 mol% to about 45 mol%. In one embodiment, the molar percentage of helper lipids may be about 3 mol% to about 40 mol%. In one embodiment, the molar percentage of helper lipids may be about 4 mol% to about 35 mol%. In one embodiment, the molar percentage of helper lipids may be about 5 mol% to about 30 mol%. In one embodiment, the molar percentage of helper lipids may be about 10 mol% to about 20 mol%. In some embodiments, the molar percentage of helper lipids in the transport vehicle batch may be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0057] In one embodiment, the molar percentage of structural lipids may be about 10 mol% to about 80 mol%. In one embodiment, the molar percentage of structural lipids may be about 20 mol% to about 70 mol%. In one embodiment, the molar percentage of structural lipids may be about 30 mol% to about 60 mol%. In one embodiment, the molar percentage of structural lipids may be about 35 mol% to about 55 mol%. In one embodiment, the molar percentage of structural lipids may be about 40 mol% to about 50 mol%. In some embodiments, the molar percentage of structural lipids in the transport vehicle batch may be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0058] In one embodiment, the molar percentage of PEG-modified lipids may be about 0.1 mol% to about 10 mol%. In one embodiment, the molar percentage of PEG-modified lipids may be about 0.2 mol% to about 5 mol%. In one embodiment, the molar percentage of PEG-modified lipids may be about 0.5 mol% to about 3 mol%. In one embodiment, the molar percentage of PEG-modified lipids may be about 1 mol% to about 2 mol%. In one embodiment, the molar percentage of PEG-modified lipids may be about 1.5 mol%. In some embodiments, the molar percentage of PEG-modified lipids in the transport vehicle batch may be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage.

[0059] Pharmaceutical compositions comprising one or more of the compounds disclosed herein, and in particular transport vehicles, are also intended. In certain embodiments, such transport vehicles comprise one or more of the PEG-modified lipids, ionizable lipids, helper lipids, and / or structural lipids disclosed herein. Transport vehicles comprising one or more of the compounds disclosed herein, and further comprising one or more additional lipids, are also intended. In certain embodiments, such transport vehicles load or otherwise encapsulate circular RNA.

[0060] The transport vehicle of the present invention encapsulates circular RNA. In certain embodiments, the polynucleotides encapsulated by the compounds or pharmaceutical compositions and liposome compositions of the present invention include RNA encoding a protein or enzyme (for example, circRNA encoding phenylalanine hydroxylase (PAH)). The present invention envisions the use of such polynucleotides as therapeutic agents that can be expressed by target cells for the production (and, in certain examples, excretion) of functional enzymes or proteins, as disclosed, for example, in International Application No. PCT / US2010 / 058457 and U.S. Provisional Patent Application No. 61 / 494,881 filed on June 8, 2011 (both teachings are incorporated herein by reference in their entirety). For example, in certain embodiments, upon expression of one or more polynucleotides by target cells, the production of a functional enzyme or protein deficient in the subject (e.g., urea cycle enzymes or enzymes associated with lysosomal storage disorders) may be observed. As another example, the circular RNA encapsulated by the transport vehicle may encode a T cell receptor protein or a chimeric antigen receptor (CAR).

[0061] Methods for treating a target disease are also provided herein by administering an effective amount of a composition comprising a circular RNA encoding a functional protein described herein and a transport vehicle to the target. In some embodiments, the circular RNA is encapsulated within the transport vehicle. In certain embodiments, such methods may enhance (e.g., increase) the expression of polynucleotides and / or increase the production and secretion of functional polypeptide products in one or more target cells and tissues (e.g., hepatocytes). Generally, such methods involve contacting target cells with one or more compounds and / or a transport vehicle containing or otherwise encapsulating the circRNA.

[0062] In certain embodiments, the transport vehicle (e.g., lipid nanoparticles) is formulated partly based on its ability to facilitate the transfection (e.g., of circular RNA) of target cells. In other embodiments, the transport vehicle (e.g., lipid nanoparticles) may be selected and / or prepared to optimize the delivery of circular RNA to target cells, tissues, or organs. For example, if the target cells are hepatocytes, the properties of the pharmaceutical composition and / or liposome composition (e.g., size, charge, and / or pH) may be optimized to facilitate the effective delivery of such composition (e.g., lipid nanoparticles) to target cells or organs, reduce immune clearance, and / or retention in the target cells or organs. Alternatively, if the target tissue is the central nervous system, the selection and preparation of the transport vehicle must consider blood-brain barrier permeability and retention within the blood-brain barrier, and / or the use of alternative means to directly deliver such composition (e.g., lipid nanoparticles) to such target tissue (e.g., via intravascular administration to the brain). In certain embodiments, the transport vehicle may be combined with an active agent that facilitates the transport of encapsulated material across the blood-brain barrier (e.g., an active agent that disrupts or improves the permeability of the blood-brain barrier, thereby enhancing the transport of circular RNA to target cells). While the transport vehicles described herein (e.g., lipid nanoparticles) can facilitate the introduction of circRNA into target cells, the addition of polycations (e.g., poly-L-lysine and protamine) as copolymers to one or more of the lipid nanoparticles constituting the pharmaceutical composition can, in some cases, significantly improve the transfection efficiency of several types of transport vehicles by 2 to 28 times in many cell lines, both in vitro and in vivo (see NJ Caplen, et al., Gene Ther. 1995; 2:603; S. Li, et al., Gene Ther. 1997; 4, 891). In some embodiments, the target cells are immune cells. In some embodiments, the target cells are T cells.

[0063] In certain embodiments, the transport vehicles described herein (e.g., lipid nanoparticles) are prepared by combining several lipid components (e.g., one or more of the compounds disclosed herein) with one or more polymer components. For example, lipid nanoparticles may be prepared using HGT4003, DOPE, cholesterol, and DMG-PEG2000. Lipid nanoparticles may consist of combinations of additional lipids in various ratios, including, for example, HGT4001, DOPE, and DMG-PEG2000. The selection of ionizable lipids, helper lipids, structural lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, as well as the relative molar ratios of such lipids, are based on the characteristics of the selected lipids, the properties of the intended target cells or tissues, and the characteristics of the substance or polynucleotide to be delivered by the lipid nanoparticles. Additional considerations include, for example, alkyl chain saturation, as well as the size, charge, pH, pKa, fusionability, and toxicity of the selected lipids.

[0064] The transport vehicles described herein may enable encapsulated polynucleotides to reach target cells, or may preferentially enable encapsulated polynucleotides to reach target cells or organs selectively (for example, the transport vehicle may concentrate on the liver or spleen of the target to which such transport vehicle is administered). Alternatively, the transport vehicle may restrict the delivery of encapsulated polynucleotides to other non-target cells or organs where the presence of the encapsulated polynucleotides is undesirable or may have limited utility.

[0065] Loading or encapsulating polynucleotides, such as circRNAs, into a transport vehicle can help protect them from environments (e.g., serum) that may contain enzymes or chemicals that degrade such polynucleotides, and / or from systems or receptors that cause rapid excretion of such polynucleotides. Therefore, in some embodiments, the compositions described herein can improve the stability of the encapsulated polynucleotide(s), particularly with respect to the environment to which such polynucleotides may be exposed.

[0066] In certain embodiments, a vector for producing a circular RNA is provided herein, the vector comprising a 5' double-stranding region, a 3' group I intron fragment, optionally a first spacer, an internal ribosome entry site (IRES), an expression sequence, optionally a second spacer, a 5' group I intron fragment, and a 3' double-stranding region. In some embodiments, these elements are arranged in the vector in the order described above. In some embodiments, the vector further comprises an internal 5' double-stranding region between the 3' group I intron fragment and the IRES, and an internal 3' double-stranding region between the expression sequence and the 5' group I intron fragment. In some embodiments, the internal double-stranding regions can form a double helix between themselves but not with an external double-stranding region. In some embodiments, the internal double-stranding regions are part of the first and second spacers. Additional embodiments include circular RNA polynucleotides comprising a circular RNA polynucleotide produced using the vector provided herein, compositions comprising such circular RNA, cells comprising such circular RNA, and methods for using and producing such vectors, circular RNA, compositions, and cells.

[0067] In some embodiments, methods are provided herein that involve administering cyclic RNA polynucleotides provided herein to cells for therapeutic purposes or for the production of useful proteins such as chimeric antigen receptor (CAR) or T cell receptor (TCR) complex proteins. In some embodiments, the methods are advantageous in that they provide the production of desired polypeptides in eukaryotic cells with a longer half-life than linear RNA because cyclic RNA is resistant to ribonucleases.

[0068] Because circular RNA polynucleotides lack the free ends required for exonuclease-mediated degradation, they exhibit resistance to several mechanisms of RNA degradation and have a longer half-life compared to equivalent linear RNA. Circularization allows for the stabilization of RNA polynucleotides, which generally have short half-lives, improving the overall efficacy of exogenous mRNA in various applications. In one embodiment, the half-life of the circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells such as human cells) is at least 20 hours (e.g., at least 80 hours).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] As used herein, the term “splice site” refers to a dinucleotide that is partially or completely contained within a group I intron and in which a phosphodiester bond is cleaved during RNA cyclization.

[0074] 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.

[0075] As used herein, the term “immunogenic” refers to the potential to induce an immune response to a substance. An immune response may be induced when an organism’s immune system or certain types of immune cells are exposed to an immunogenic substance. The term “non-immunogenic” refers to the absence or non-existence of an immune response to a substance that exceeds a detectable threshold. 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.

[0076] As used herein, the term "cyclization efficiency" refers to a measured value of the resulting cyclic polyribonucleotide compared to its linear starting material.

[0077] 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 may be expressed as the amount of protein or peptide produced per given amount of transcript encoding a protein or peptide.

[0078] 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.

[0079] 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).

[0080] As used herein, the terms “ribonucleic acid” and “RNA” mean polymers composed of ribonucleotides.

[0081] As used herein, the terms “deoxyribonucleic acid” and “DNA” mean polymers composed of deoxyribonucleotides.

[0082] "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.

[0083] 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.

[0084] 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 other sequences within the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease-protected assays.

[0085] 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.

[0086] As used herein, two “double-chain forming regions,” “homologous arms,” or “homologous regions” are complementary or complementary if the two regions share a sufficient level of sequence identity with each other’s reverse complement to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences have “homology” if they are identical to or share sequence identity with their reverse complement or “complementary” sequence. The percentage of sequence identity between homologous regions and the reverse complement of the corresponding homologous regions 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.

[0087] 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.

[0088] "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.

[0089] "Translation" refers to the formation of polypeptide molecules by ribosomes based on an RNA template.

[0090] It should be understood that the terms used herein are intended solely to describe and not to limit specific embodiments. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “cell” includes a combination of two or more cells, or an entire cell culture; a reference to “polynucleotide” practically includes many copies of that polynucleotide. Where used herein, the term “or” is understood to be inclusive unless specifically stated or evident from the context. Unless defined herein and in the remainder of this specification, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the invention pertains.

[0091] 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.”

[0092] 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.

[0093] "Co-administration" means administering the therapeutic agent provided herein together with one or more additional therapeutic agents at sufficiently close intervals such that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa.

[0094] 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 treatment or prevention is perceived by a person skilled in the art as having potential benefits or therapeutic effects varies. Treatments or preventions provided by the methods disclosed herein may include treatment or prevention of one or more conditions or symptoms of a disease. Furthermore, for the purposes of this specification, “prevention” may include delaying the onset of a disease, or its symptoms or conditions.

[0095] 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.”

[0096] 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, spacers include a double-strand forming region.

[0097] As used herein, “splice site” refers to one or more dinucleotides in a splicing reaction product in which a phosphodiester bond is cleaved between them. “5' splice site” refers to the natural 5' dinucleotide of an intron, for example, a group I intron, while “3' splice site” refers to the natural 3' dinucleotide of an intron.

[0098] As used herein, “internal 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.

[0099] 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.

[0100] As used herein, “endonuclease site” refers to a stretch of nucleotides within a polynucleotide that can be recognized and cleaved by an endonuclease protein.

[0101] As used herein, “bisistronic RNA” refers to a polynucleotide containing two expression sequences that encode two different proteins. These expression sequences are often separated by a cleavable peptide, such as a 2A site or an IRES sequence.

[0102] 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.

[0103] 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.

[0104] 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).

[0105] 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.

[0106] As used herein, the term "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid.

[0107] 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.

[0108] 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.

[0109] 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., 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 head groups and / or tail groups) 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 including one or more of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino, and pyridyl).

[0110] As used herein, the term "hydrophilic" is used qualitatively to indicate that a functional group prefers water and is typically water-soluble. For example, compounds comprising a cleavable disulfide (SS) functional group bonded to one or more hydrophilic groups (e.g., hydrophilic head groups) are disclosed herein, 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.

[0111] In certain embodiments, at least one of the functional groups of a portion of a compound disclosed herein is inherently hydrophobic (e.g., a hydrophobic tail group including a naturally occurring lipid such as cholesterol). As used herein, the term “hydrophobic” is used qualitatively to indicate that a functional group is averse to water and is typically not water-soluble. For example, compounds disclosed herein include a cleavable functional group (e.g., a disulfide (SS) group) bonded to one or more hydrophobic groups, where such hydrophobic groups are one or more naturally occurring lipids, such as cholesterol, and / or optionally substituted with variable saturated or unsaturated C6-C6 groups. 20 Variable saturated or unsaturated C6-C, which are alkyl and / or optionally substituted. 20 Contains acyl

[0112] 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).

[0113] 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 a particular embodiment, 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).

[0114] As used herein, the term "alkyl" refers to linear and branched C1-C 40 Hydrocarbons (e.g., C6-C) 20 This refers to both hydrocarbons and includes both saturated and unsaturated hydrocarbons. In certain embodiments, the alkyl may consist of one or more cyclic alkyls and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and may optionally be 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 may consist of (9Z,12Z)-octadeca-9,12-diene. For example, "C6-C 20 The use of designations such as "[...]" is intended to refer to alkyl groups (e.g., linear or branched, including alkenes and alkyls) having carbon atoms within the specified range.

[0115] 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 be optionally substituted through the available carbon atoms and, in certain embodiments, may contain one or more heteroatoms, such as oxygen, nitrogen, or sulfur.

[0116] 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 may 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%.

[0117] 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.

[0118] As used herein, the terms “noncationic lipid,” “noncationic helper lipid,” and “helper lipid” are interchangeable and refer to any neutral, zwitterionic, or anionic lipid.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties.

[0124] As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols.

[0125] As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties.

[0126] As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer.

[0127] 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.

[0128] 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.

[0129] 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 comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions may be further subdivided into more conserved regions called framework regions (FR) and interspersed, highly variable regions called complementary specific regions (CDR). Each VH and VL contains 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 the antigen. The constant region of the antibody can 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 (which may be referred to herein as "antibody conjugates"), heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-id) antibodies (for example, including anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (which may be referred to herein as "antibody mimetic"), and any of the antigen-binding fragments described above. In some embodiments, the antibodies described herein refer to a polyclonal antibody population.

[0130] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG subclass is also well known to those skilled in the art and includes, but is not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an antibody 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 antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated otherwise and the context otherwise specifies, the term "antibody" also includes the antigen-binding fragment or antigen-binding moiety of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or moieties, as well as single-chain antibodies.

[0131] 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-complementary specific region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and 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 that specifically binds to an antigen, containing one or more of its complementary specific regions (CDRs). In further embodiments, the antigen-binding molecule is a single-stranded variable fragment (scFv). In some embodiments, the antigen-binding molecule includes or consists of an avimer.

[0132] As used herein, the terms “variable region” or “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 amino-terminal approximately 110–120 amino acids of the mature heavy chain and approximately 90–115 amino acids of 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 a region called the complementarity-specific region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to be constrained 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).

[0133] The terms "VL" and "VL domain" are used interchangeably and refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0134] The terms "VH" and "VH domain" are used interchangeably and refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0135] Many definitions of CDR are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two, used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures. The terms "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy chain 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). When using the Kabat numbering system, the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31–35 (CDR1), which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme), amino acid positions 50–65 (CDR2), and amino acid positions 95–102 (CDR3). When using the Kabat numbering system, the CDRs within the 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 refers 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, and 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 end of the Chothia CDR-H1 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 neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). In certain embodiments, the CDR of the antibody described herein is determined according to the Chothia numbering scheme.

[0136] As used herein, the terms “constant region” and “constant domain” are interchangeable and have the general meanings in the art. The constant region is the antibody portion, e.g., the carboxyl-terminal portion of the light and / or heavy chain, which does not directly participate in the binding of the antibody to the antigen but may 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.

[0137] "Binding affinity" generally refers to the strength of the combined 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 specified, 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 for 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 in the art, including but not limited to the equilibrium dissociation constant (KD) and the equilibrium association constant (KA or Ka). KD is calculated from the quotient of koff / kon, while 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.

[0138] As used herein, “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having 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), 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 the CDR(y) or framework region(y) of an antibody or its antigen-binding molecule may be replaced by amino acid residues having a similar side chain.

[0139] As used herein, the term “heterogeneous” means derived from any source other than naturally occurring sequences.

[0140] As used herein, “epitope” is a term of the art and refers to a localized 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 one or more polypeptides (conformative, nonlinear, discontinuous, or discontinuous epitopes). In some embodiments, the epitope to which the antibody binds may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange and mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenic mapping (e.g., site-directed mutagenic mapping). In X-ray crystallography, crystallization can be achieved using any of the methods known 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 examined using computer software such as X-PLOR (distributed by Yale University, 1992, Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; US2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323).

[0141] As used herein, an antigen-binding molecule, antibody, or its antigen-binding molecule "cross-competes" with a reference antibody or its antigen-binding molecule if the interaction between the antigen and the first binding molecule, antibody, or its antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or its 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 the reference antigen-binding molecule binds to the same or overlapping epitopes as the reference antigen-binding molecule. In other embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. Numerous types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another, e.g., solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J.Immunol. 137:3614-3619); solid-phase direct label assay, solid-phase direct label sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using 1-125 labels (Morel et al., 1988, Molec.Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al. al., 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0142] As used herein, the terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are synonymous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex), such binding being understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with generally lower affinity, as determined by immunoassays, 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 with a KA of at least 2, 2.5, 3, 4, or greater than the KA of the molecule when it binds to another antigen.

[0143] 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 include antibody production, activation of specific immunologically competent 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 can 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.

[0144] The term “self” refers to any substance originating from the same individual in which any substance is subsequently reintroduced. For example, the method of engineered autologous cell therapy (eACT®) described herein includes collecting lymphocytes from a patient, then manipulating them to express, for example, a CAR construct, and then administering and returning them to the same patient.

[0145] The term "allogeneic" refers to any substance that originates from one individual and is then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0146] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells within the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade neighboring tissues and may further metastasize to distant parts of the body through 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 are 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. It may reduce the tumor size of tumors resulting from: cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), pediatric solid tumors, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial 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 the aforementioned cancers.In some embodiments, the methods disclosed herein are used to, for example, sarcomas and carcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteogenic sarcomas, Kaposi's sarcoma, soft tissue sarcomas, and 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), sweat gland carcinomas, sebaceous gland carcinomas, papillary carcinomas, and breast cancers. It may reduce the tumor size of tumors originating from cephalic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choroid carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, carcinoma of the Fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the renal pelvis, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). 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 the cancer does not respond to chemotherapy or radiotherapy from the beginning, or becomes unresponsive over time.

[0147] As used herein, “antitumor effect” refers to a biological effect that may be presented as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physical symptoms associated with the tumor. Antitumor effect may also refer to the prevention of tumor development, such as a vaccine.

[0148] As used herein, “cytokines” refers to non-antibody proteins released by a cell in response to contact with a specific antigen, which interact with a second cell to mediate a response in that second cell. As used herein, “cytokines” is intended to refer to proteins released by a population of cells that act as intercellular mediators on another cell. Cytokines can be endogenously expressed by cells or administered to a target. Cytokines can be released by immune cells, including macrophages, dendritic cells, B cells, T cells, 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, can promote immune cell survival and proliferation, 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-1a, IL-1b, 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).

[0149] 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 represent the major components of the innate immune system. NK cells reject virus-infected tumors and cells. They function through a process of apoptosis or programmed cell death. They are named “natural killers” because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). Their T cell receptor (TCR) distinguishes them from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells, so-called: 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), and memory T cells ((i) stem memory T cells like naive cells, such as CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but they also have large amounts of CD95, IL-2R[T] CXCR3 and LFA-1 are also expressed, and they exhibit numerous functional attributes specific to memory cells; (ii) Central memory TCM cells express L-selectin and CCR7, and they secrete IL-2 but not IFNy or IL-4, and (iii) Effector memory TEM cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ or CD4+FoxP3+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and after being activated by antigen interaction, become both short-lived and long-lived memory B cells and plasma cells. In mammals, immature B cells are formed in the bone marrow, which is the origin of the name.

[0150] The terms “genetically engineered” or “engineered” refer to methods of modifying a cell’s genome, including but not limited to deleting coding regions or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In some embodiments, the cells being modified are lymphocytes, such as T cells, which may be obtained from 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.

[0151] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of a vertebrate of invading pathogens, pathogen-infected cells or tissues, cancerous cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0152] As used herein, “co-stimulatory signal” refers to a signal combined with a primary signal, such as TCR / CD3 ligation, which results in a T cell response, for example, the proliferation and / or upregulation or downregulation of key molecules.

[0153] As used herein, “costimulatory ligand” includes molecules on antigen-presenting cells that specifically bind to a congeneral costimulatory molecule on a T cell. The binding of a costimulatory ligand provides a signal that mediates a T cell response, including but not limited to proliferation, activation, and differentiation. The costimulatory ligand induces a signal in addition to the primary signal provided by the stimulating molecule, for example, by the binding of the T cell receptor (TCR) / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule. Co-stimulatory ligands may include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, 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 receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed cell death (PD)L1. Co-stimulatory ligands include, but are not limited to, ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, and CD83, as well as antibodies that specifically bind to costimulatory molecules present on T cells, such as 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).

[0154] A "costimulatory molecule" is a congenital binding partner on a T cell that mediates a costimulatory response by the T cell, such as proliferation, by specifically binding to a costimulatory ligand. A "costimulatory molecule" is a congenital binding partner on a T cell that mediates a costimulatory response by the T cell, such as proliferation, by specifically binding to a costimulatory ligand.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), CD1-1a, CD1-1b, CD1-1c, CD1-1d, CDDS, 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, 1a / 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;Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or their fragments, truncations, or combinations.

[0155] 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, 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 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 variants maintain at least one biological activity of the reference polypeptide.

[0156] 2. Vectors, precursor RNA, and circular RNA In certain embodiments, a circular RNA polynucleotide comprising a 3' post-splicing group I intron fragment, optionally a first spacer, an internal ribosome entry site (IRES), an expression sequence, optionally a second spacer, and a 5' post-splicing group I intron fragment is provided herein. In some embodiments, these regions are in this order. In some embodiments, the circular RNA is prepared by the method provided herein or from a vector provided herein.

[0157] In certain embodiments, transcription of the vector provided herein (e.g., comprising a 5' homologous region, a 3' group I intron fragment, optionally a first spacer, an internal ribosome entry site (IRES), an expression sequence, optionally a second spacer, a 5' group I intron fragment, and a 3' homologous 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+).

[0158] 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 to each other 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 a particular embodiment, the double-stranding region has a length of about 30 nucleotides.

[0159] 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 (Figure 25). 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 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 unstructured regions, base-pairing regions, hairpin / structured regions, and combinations thereof. In some embodiments, a 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.

[0160] 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 native group I intron, which includes a 5' splice site dinucleotide and, optionally, an adjacent exon sequence of 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 up to the length of an exon. As described by Umekage et al. (2012) and as shown in Figure 33, 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 and the 5' group I intron fragment formed by an optional 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.

[0161] In certain embodiments, the vector, precursor RNA, and circular RNA provided herein include an internal ribosome entry site (IRES). The inclusion of the IRES 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 element attracts the eukaryotic ribosome translation initiation complex, thereby facilitating translation initiation. For example, see Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161).

[0162] 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).

[0163] In some embodiments, IRES is Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliopathy virus, human poliovirus 1, Plautia stali enteric virus, Casimir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, Ectropis Obliqua picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, Brassicaceae tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ring spot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, Hi AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, 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, dog Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, S.Cerevisiae YAP1, Tobacco HTC virus, Kabukurinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picovirnavirus, HCV QC64, Hitocosavirus E / D, Hitocosavirus F, Hitocosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shambavirus A, Pacivirus A, Pacivirus A2, Echovirus E14, Human Parechovirus 5, Aichivirus, Hepatitis A virus HA16, Fopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C These are IRES sequences for aptamers of K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Saperovirus, Rosavirus B, Vakunsavirus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Sissinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Disicisthovirus, Hubei 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.

[0164] In some embodiments, the polynucleotides herein include expression sequences. In some embodiments, the expression sequences encode CARs. In some embodiments, the polynucleotides include more than one expression sequence, for example, two, three, four, or five expression sequences. In some embodiments, some such expression sequences encode a CAR, while others encode another therapeutic protein, for example, a checkpoint inhibitor, such as a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the polynucleotide includes a first expression sequence encoding CAR and a second expression sequence encoding programmed cell death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), TIM-3, CEACAM (e.g., CEACAM-1, -3 and / or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GALS, adenosine, TGFR (e.g., TGFR beta), B7-H1, B7-H4 (VTCN1), OX-40, CD137, CD40, or an inhibitor of LAGS. In some embodiments, the inhibitor is nivolumab, pembrolizumab, ipilimumab, or atezolizumab. In some embodiments, the expression sequence encodes two or more functional units, e.g., a CAR and a protein that is cleaved into another therapeutic protein.

[0165] In certain embodiments, the polynucleotides provided herein include a CAR or TCR complex protein-coding region. The CAR or TCR complex protein-coding region is a sequence encoding a chimeric antigen receptor (CAR) or any T cell receptor (TCR) complex protein. In some embodiments, the CAR or TCR complex protein encodes a CAR. In some embodiments, the CAR or TCR complex protein-coding region encodes two CARs in a bicistronic construct. In some embodiments, the CAR or TCR complex protein encodes TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, and / or CD8. In some embodiments, the CAR or TCR complex protein encodes artificial TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, and / or CD8 variants. In some embodiments, the CAR or TCR complex protein encodes native TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, and / or CD8 variants. In some embodiments, the CAR or TCR complex coding region ends with a stop codon. In some embodiments, the CAR or TCR complex coding region ends with a stop cassette.

[0166] 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 chlorotic rinsgspot virus, woodchuck hepatitis virus posttranslational regulatory element, Sindbis virus, turnip crinkle virus, tobacco etching virus, or Venezuelan encephalitis virus.

[0167] 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.

[0168] In some embodiments, the vectors provided herein include a poly-A region. In some embodiments, the poly-A region is at least 30 nucleotides long or at least 60 nucleotides long.

[0169] In some embodiments, the DNA (e.g., vectors), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are 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 length. 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, or 5000nt in length. In some embodiments, the polynucleotide is 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, or 1000nt 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, or 10000 nt.

[0170] In certain embodiments, the polynucleotides provided herein are circular RNA polynucleotides, or are useful for constructing circular RNA polynucleotides. Such polynucleotides contain a CAR or TCR complex protein-coding domain. Certain current CAR and recombinant TCR treatments manipulate cells with DNA encoding the CAR or recombinant TCR, causing greater toxicity and posing a risk of harmful mutagenesis compared to transient forms of CAR or recombinant TCR expression. An alternative method is linear RNA encoding the CAR or recombinant TCR complex protein. However, linear RNA has a short half-life in vivo, limiting the efficacy of the treatment. In certain embodiments, the circular RNA polynucleotides provided herein encoding the CAR or recombinant TCR complex protein offer the toxic advantages of transient expression while increasing the therapeutic efficacy of the treatment compared to linear RNA. The RNA cyclization method described herein, including the addition of homologous regions adjacent to group I intron fragments, enables high cyclization efficiency and cyclization of large RNA polynucleotides.

[0171] In some embodiments, vectors are provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' homologous region, b) a 3' group I intron fragment, c) optionally a first spacer sequence, d) an IRES, e) an expression sequence (e.g., a CAR or TCR complex protein coding region), f) optionally a second spacer sequence, g) a 5' group I intron fragment, and h) a 3' homologous region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' homologous region.

[0172] 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) a 5' homologous region, b) a 3' group I intron fragment, c) optionally a first spacer sequence, d) an IRES, e) an expression sequence (e.g., a CAR or TCR complex protein-coding region), f) optionally a second spacer sequence, g) a 5' group I intron fragment, and h) a 3' homologous region. The precursor RNA may be unmodified, partially modified, or fully modified.

[0173] In certain embodiments, circular RNA is provided herein. In certain embodiments, the circular RNA is circular RNA produced by a vector provided herein. In some embodiments, the circular RNA is circular RNA produced by cyclization of a precursor RNA provided herein. In some embodiments, the circular RNA comprises the following sequences: a) a first spacer sequence, b) an IRES, c) an expression sequence (e.g., a CAR or TCR complex protein-coding region), and d) a 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 a 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 a 5' splice site. In some embodiments, the circular RNA is at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides in size. Circular RNA can be unmodified, partially modified, or fully modified.

[0174] In some embodiments, the circular RNA provided herein has higher functional stability than mRNA containing the same expression sequence.

[0175] 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.

[0176] 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 to 80, 10 to 70, 15 to 60, and / or 20 to 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.

[0177] In some embodiments, the circular RNA provided herein may have a higher degree of expression than equivalent linear mRNA, for example, a higher degree of expression 24 hours after RNA administration to cells. In some embodiments, the circular RNA provided herein may have a higher degree of expression than mRNA containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail. In some embodiments, the circular RNA provided herein may have higher stability than equivalent linear mRNA. In some embodiments, this may be demonstrated by measuring the presence and density of the receptor in vitro or in vivo after electroporation, measured over a period of one week. In some embodiments, this may be demonstrated by measuring the presence of RNA via qPCR or ISH.

[0178] 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 IFN-β1, RIG-1, IL-2, IL-6, IFNγ, and / or TNFα 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 IFN-β1, RIG-1, IL-2, IL-6, IFNγ, and / or TNFα 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. In certain embodiments, the circular RNAs provided herein may result in lower toxicity for cytokine release syndrome (CRS) when expressing CAR or TCR complex proteins on immune cells, e.g., T cells, than viruses using DNA, e.g., lentiviral manipulation. In some embodiments, this may be demonstrated by measuring cytokines in vitro, e.g., post-IL-6 release infection / transfection, by evaluation by ELISA and / or qPCR.

[0179] In certain embodiments, the circular RNAs provided herein can result in lower toxicity than viral, e.g., lentiviral engineering using DNA when expressing CAR or TCR complex proteins on immune cells, e.g., T cells, due to cytokine release syndrome (CRS). In some embodiments, this can be shown by measuring post-infection / transfection cytokine, e.g., IL6 release in vitro, evaluated by ELISA and / or qPCR.

[0180] In some embodiments, the circular RNAs provided herein can result in lower toxicity than viral, e.g., lentiviral engineering using DNA when expressing CAR or TCR complex proteins on immune cells, e.g., T cells, due to the lack of insertional mutagenesis by the circular RNAs. In some embodiments, this can be shown by demonstrating that circular RNAs are not integrated into the genome while DNA delivered by lentiviruses is integrated into the genome by sequencing after administration of circular RNA or DNA. 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 transfected DNA vectors can occur via additional polymerase or polymerase encoded by nucleic acids transfected into the cells, or preferably via endogenous polymerase.

[0181] In certain embodiments, the circular RNA polynucleotides provided herein comprise 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 s2 In another embodiment, the modified nucleoside is U(2-thiouridine). In yet another embodiment, the modified nucleoside is Ψ(pseudolidine). In yet another embodiment, the modified nucleoside is Um(2'-O-methyluridine). In yet another embodiment, 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 -Glycinylcarbamoyladenosine); t 6 A(N 6 -Threonylcarbamoyladenosine); ms 2 t 6 A(2-methylthio-N) 6 -Threonylcarbamoyladenosine);m 6 t 6 A(N 6 -methyl-N 6 -Threonylcarbamoyladenosine);hn 6 A(N 6 -Hydroxynorvalylcarbamoyladenosine); ms 2 hn 6 A(2-methylthio-N) 6 -Hydroxynorvalylcarbamoyladenosine);Ar(p)(2'-O-ribosyladenosine (phosphate));I(inosine);m 1 I(1-methylinosine); m 1 Im(1,2'-O-dimethylinosine);m 3C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine); ac 4 C(N 4 -acetylcytidine);f 5 C(5-formylcytidine); m 5 Cm(5,2'-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lysidine); m 1 G(1-methylguanosine); m 2 G(N 2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine); m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine);Gr(p)(2'-O-ribosylguanosine (phosphate));yW(weibutosine);o2yW(peroxyweibutosine);OHyW(hydroxyweibutosine);OHyW * (Unmodified hydroxywybutosin); imG (wyosin); mimG (methylwyosin); Q (queosin); oQ (epoxyqueosin); galQ (galactosylqueosin); manQ (mannosylqueosin); 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);ho5 U (5-Hydroxyuridine); mo 5 U (5-Methoxyuridine); cmo 5 U (Uridine 5-Oxyacetic acid); mcmo 5 U (Uridine 5-Oxyacetic acid Methyl Ester); chm 5 U (5-(Carboxyhydroxymethyl)uridine)); mchm 5 U (5-(Carboxyhydroxymethyl)uridine Methyl Ester); mcm 5 U (5-Methoxycarbonylmethyluridine); mcm 5 Um (5-Methoxycarbonylmethyl-2’-O-methyluridine); mcm 5 s 2 U (5-Methoxycarbonylmethyl-2-thiouridine); nm 5 S 2 U (5-Aminomethyl-2-thiouridine); mnm 5 U (5-Methylaminomethyluridine); mnm 5 s 2 U (5-Methylaminomethyl-2-thiouridine); mnm 5 se 2 U (5-Methylaminomethyl-2-selenouridine); ncm 5 U (5-Carbamoylmethyluridine); ncm 5 Um (5-Carbamoylmethyl-2’-O-methyluridine); cmnm 5 U (5-Carboxymethylaminomethyluridine); cmnm 5 Um (5-Carboxymethylaminomethyl-2’-O-methyluridine); cmnm 5 s 2 U (5-Carboxymethylaminomethyl-2-thiouridine); m 6 2A (N 6 ,N 6 -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 3U(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); τm 5 s 2 U(5-taurinomethyl-2-thiouridine)); imG-14(4-demethylyosin); imG2(isoyosin); or ac 6 A(N 6 -Acetyladenosine)

[0182] In some embodiments, the modified nucleoside is 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, 5-taurinomethyl-2-thiouridine, 1 -Taurinomethyl-4-thiouridine, 5-methyluridine, 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-thiouridine, 4-methoxy-pseuduridine, 4-methoxy-2-thio-sulfuridine Douridine, 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolocytidine, 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 , 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 The compounds may include those 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.

[0183] 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.

[0184] 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 cutoff; (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 in 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.

[0185] 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.

[0186] 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 (e.g., a CAR or TCR complex protein) is expressed inside the animal.

[0187] 3. Payload In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the proteins listed in Table 1.

[0188] (Table 1) Protein expression sequences and delivery formulations TIFF2022533796000001.tif201166TIFF2022533796000002.tif230166TIFF2022533796000003.tif240166TIFF2022533796000004.tif23516 6TIFF2022533796000005.tif239166TIFF2022533796000006.tif240166TIFF2022533796000007.tif240166TIFF2022533796000008.tif69166

[0189] In some embodiments, polynucleotides encode proteins composed of subunits encoded by more than one gene. For example, a protein may be a heterodimer in which each chain or subunit of the protein is encoded by a distinct gene. More than one circRNA molecule can be delivered by transport vehicle, with each circRNA encoding a distinct subunit of the protein. Alternatively, a single circRNA can be manipulated to encode more than one subunit. In certain embodiments, distinct circRNA molecules encoding individual subunits may be administered by separate transport vehicles.

[0190] 4. Ionizable lipids In certain embodiments, lipids that can be used as components of a transport vehicle to facilitate or improve the delivery and release of circular RNA to one or more target cells (for example, by penetrating or fusing with the lipid membrane of such target cells) are disclosed herein.

[0191] In some embodiments, the lipid or transport vehicle is described in pages 32–55 of International Patent Application No. PCT / US2010 / 061058, paragraphs 86–117 of U.S. Patent Publication No. US2019 / 0314524, pages 43–146 of International Patent Application No. PCT / US2018 / 058555, pages 46–51 of International Patent Application No. PCT / US2018 / 053569, paragraphs 195–217 of International Patent Application No. PCT / US2017 / 028981, and U.S. Patent Publication No. US2019 / 032148. Lipids such as those described in paragraphs 82–95 of 9, paragraphs 5–19 and / or 38–77 of U.S. Patent Publication No. US2019 / 0314284, Tables 1–4 of International Patent Application No. PCT / US2019 / 025246, paragraphs 92–107 of U.S. Patent Publication No. 20190091164, and pages 78–97, 109–164, and / or pages 190–217 of International Patent Application No. PCT / US2019 / 015913 (the contents of which are incorporated herein by reference in their entirety).

[0192] In some embodiments, the lipid or transport vehicle is an ionizable lipid. In certain embodiments, the ionizable lipid comprises one or more cleavable functional groups (e.g., disulfides) that enable, for example, the dissociation of the hydrophilic functional head group of the compound from the lipophilic functional tail group (e.g., upon exposure to oxidative, reductive, or acidic conditions), thereby facilitating a phase transition in the lipid bilayer of one or more target cells. In some embodiments, the ionizable lipid is a lipid represented by formula 1 or listed in Table 1 or 2 of U.S. Patent No. 9,708,628 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the ionizable lipid is described on pages 7-13 of U.S. Patent No. 9,765,022 or represented by formula 1 of U.S. Patent No. 9,765,022 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the ionizable lipids are described on pages 12–24 of International Patent Application No. PCT / US2019 / 016362, or represented by Formula 1 of International Patent Application No. PCT / US2019 / 016362 (the contents thereof being incorporated herein by reference in their entirety).

[0193] In some embodiments, the lipid or transport vehicle is specified in International Patent Application Nos. PCT / US2010 / 061058, PCT / US2018 / 058555, PCT / US2018 / 053569, PCT / US2017 / 028981, PCT / US2019 / 025246, PCT / US2019 / 015913, PCT / US2019 / 016362, PCT / U Lipids such as those described in S2019 / 016362, U.S. Patent Publication Nos. US2019 / 0314524, US2019 / 0321489, US2019 / 0314284, and US2019 / 0091164, and U.S. Patent Nos. 9,708,628 and 9,765,022 (the contents of which are incorporated herein by reference in their entirety).

[0194] In some embodiments, the lipids that can be used as components of a transport vehicle to facilitate or improve the delivery and release of circular RNA to one or more target cells may be one or more lipids listed in Table 2.

[0195] (Table 2) Exemplary lipids TIFF2022533796000009.tif48170TIFF2022533796000010.tif238170TIFF2022533796000011.tif209170TIFF2022533796000012.tif199170TIFF2022533796000013.tif217170TIFF2022533796000014.tif237170TIFF2022533796000015.tif223170TIFF2022533796000016.tif207170TIFF2022533796000017.tif221170TIFF2022533796000018.tif223170TIFF2022533796000019.tif239170TIFF2022533796000020.tif216170TIFF2022533796000021.tif233170TIFF2022533796000022.tif141170TIFF2022533796000023.tif223170TIFF2022533796000024.tif189170TIFF2022533796000025.tif194170TIFF2022533796000026.tif64170

[0196] 5. PEG lipid The use and inclusion of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramide (PEG-CER), such as N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide), in liposomes and pharmaceutical compositions described herein, preferably in combination with one or more of the compounds and lipids disclosed herein, is intended. The intended PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids accompanied by C6-C20 length alkyl chains(s). In some embodiments, the PEG-modified lipid employed in the compositions and methods of the present invention is 1,2-dimiristoyl-sn-glycerol, methoxypolyethylene glycol (2000MW PEG) "DMG-PEG2000". The addition of PEG-modified lipids to lipid delivery vehicles can prevent complex aggregation, extend circulating life, and provide a means to increase the delivery of lipid-polynucleotide compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected to be rapidly replaced from the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful replaceable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention may constitute molar ratios of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in liposomal lipid nanoparticles.

[0197] In one embodiment, the PEG-modified lipid is described in international patent application PCT / US2019 / 015913. In another embodiment, the transport vehicle comprises one or more PEG-modified lipids.

[0198] Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also called PEGylated lipids. For example, PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0199] In some embodiments, the PEG lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxulpropyl-3-amine (PEG-c-DMA).

[0200] In one embodiment, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0201] In some embodiments, the lipid portion of the PEG lipid is approximately C 14 ~About C 22 For example, about C 14 ~About C 16Those having a length are included. In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG lipid is PEG2k-DMG.

[0202] In one embodiment, the lipid nanoparticles described herein can include a PEG lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[0203] PEG lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publication No. WO / 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[0204] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein can be synthesized as described in International Patent Application No. PCT / US2016 / 000129, which is incorporated herein by reference in its entirety.

[0205] The lipid component of the lipid nanoparticle composition can include one or more molecules including polyethylene glycol such as PEG or a PEG-modified lipid. Such species can alternatively be referred to as pegylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0206] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. PEG-DMG has the following structure: It has TIFF2022533796000027.tif19128.

[0207] In one embodiment, the PEG lipids useful in the present invention can be the PEGylated lipids described in International Publication No. WO2012099755, the content of which is incorporated herein by reference in its entirety. Any of these exemplary PEG lipids described herein can be modified to contain a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments, the PEG-OH lipid contains one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid contains an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0208] In some embodiments, the PEG lipid has the formula (PI): It is a compound of TIFF2022533796000028.tif10128 or a salt or isomer thereof, wherein: r is an integer between 1 and 100; R is C 10-40 alkyl, C 10-40 alkenyl, or C 10-40 alkynyl; optionally one or more methylene groups of R are independently C 3-10 carbocyclylene, 4- to 10-membered heterocyclylene, C 6-10 arylene, 4- to 10-membered heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NRN )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-,-N(R N )S(O)N(R N )-,-OS(O)N(R N )-,-N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-,-N(R N )S(O)2N(R N )-,-OS(O)2N(R N )-, or -N(R N Replaced with S(O)2O-; R N Each example independently involves hydrogen, C 1-6 It is an alkyl group or a nitrogen protecting group.

[0209] For example, R is a C17 alkyl group. For example, PEG lipids have the formula (P1-a): The compound TIFF2022533796000029.tif12128, or a salt or isomer thereof, where r is an integer between 1 and 100.

[0210] For example, PEG lipids are given by the following formula: This is the compound TIFF2022533796000030.tif12128.

[0211] 6. Helper lipids In some embodiments, the transport vehicle described herein (e.g., LNP) comprises one or more noncationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or replacement. In some embodiments, the phospholipid or phospholipid substitute may be, for example, one or more saturated or (poly)unsaturated phospholipids, or a phospholipid substitute, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0212] The phospholipid portion can be selected from a non-limited group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0213] The fatty acid portion can be selected from a non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0214] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids such as sphingomyelin.

[0215] In some embodiments, the helper lipid is a 1,2-distearoyl-177-glycero-3-phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[0216] In some embodiments, the helper lipid is a non-phosphatidylcholine (PC) zwitterionic lipid, a DSPC analogue, oleic acid, an oleic acid analogue, or a DSPC substitute.

[0217] In some embodiments, helper lipids are described in PCT / US2018 / 053569. Suitable helper lipids for use in the lipid compositions of this disclosure include, for example, a variety of neutral, uncharged, or zwitterionic lipids. Such helper lipids are preferably used in combination with one or more of the compounds and lipids disclosed herein. Examples of helper lipids include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauriroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-pymitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine ( Examples include, but are not limited to, PSPC, 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-diecocenoyl-sn-glycero-3-phosphocholine (DEPC), paimitoioylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the helper lipid may be distearoyl phosphatidylcholine (DSPC) or dimyristoyl phosphatidylethanolamine (DMPE).In another embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC). The helper lipid functions to stabilize and improve the processing of the transport vehicle. Such helper lipids are preferably used in combination with other excipients, such as one or more of the ionizable lipids disclosed herein. In some embodiments, when used in combination with ionizable lipids, the helper lipids may constitute a molar ratio of 5% to about 90% or about 10% to about 70% of the total lipids present in the lipid nanoparticles.

[0218] 7. Structured lipids In one embodiment, the structural lipid is described in international patent application PCT / US2019 / 015913.

[0219] The transport vehicles described herein include one or more structural lipids. Incorporation of structural lipids into lipid nanoparticles may help reduce the aggregation of other lipids within the particles. Structural lipids may include, but are not limited to, cholesterol, fecosterol, ergosterol, basicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0220] In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is a cholesterol analog. In certain embodiments, the structural lipid is alpha-tocopherol.

[0221] The transport vehicle described in this specification contains one or more structural lipids. Incorporation of structural lipids into transport vehicles, such as lipid nanoparticles, can help reduce the aggregation of other lipids in the particles. In certain embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone, etc.), or combinations thereof.

[0222] In some embodiments, the structural lipid is a sterol. The structural lipid can include, but is not limited to, sterols (such as phytosterol or zymosterol).

[0223] In certain embodiments, the structural lipid is a steroid. For example, sterols can include, but are not limited to, cholesterol, β-sitosterol, fucosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatin, ursolic acid, or alpha-tocopherol.

[0224] 8. Chimeric Antigen Receptor Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be inserted into immune cells, including T cells, via circular RNAs as described herein and can be expressed by the immune cells. Using CARs, a single receptor can be programmed to activate immune cells to attack and destroy cells bearing a particular antigen when both recognize and bind to that antigen. When 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 includes (i) an antigen-binding molecule that specifically binds to a target antigen, (ii) a hinge domain, transmembrane domain, and intracellular domain, and (iii) an activation domain.

[0225] In certain embodiments, vectors, precursor RNAs, and cyclic RNA polynucleotides comprising protein-coding regions encoding chimeric antigen receptor (CAR) or T cell receptor (TCR) complex proteins are provided herein.

[0226] A CAR is an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain (e.g., a single-strand variable fragment (scFv)) linked to a T cell signaling domain. Features of CARs include the ability to leverage the antigen-binding properties of a monoclonal antibody to redirect T cell specificity and reactivity to at least one selected target (e.g., in a non-MHC restriction manner). The ability of a CAR to recognize a non-MHC restriction antigen allows T cells expressing the CAR to acquire the ability to recognize the antigen independently of antigen processing, thus circumventing a major mechanism of tumor escape. A bispecific CAR is specific to two different antigens. In some embodiments, certain polynucleotides provided herein encode a bispecific CAR.

[0227] In some embodiments, the CAR includes a transmembrane domain. In some embodiments, the transmembrane domain includes the CD8 transmembrane domain. In some embodiments, the CAR includes the CD8α (CD8 alpha) hinge and the transmembrane domain. In preferred embodiments, CD8 is human. The CAR may be present in amounts less than the total CD8 protein. (...) In some embodiments, the transmembrane domain includes the CD28 transmembrane domain. In some embodiments, the CAR includes the CD28 hinge and the transmembrane domain. In preferred embodiments, CD28 is human. The CAR may be present in amounts less than the total CD28 protein.

[0228] 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.

[0229] In some embodiments, the CAR includes a CAR protein spacer. The CAR protein spacer may be between any of the aforementioned domains. In some embodiments, the CAR includes an IgG heavy chain constant domain (CH2CH3) spacer. In further embodiments, the CAR protein spacer may be between the scFv and the transmembrane domain. In preferred embodiments, the sequence of the spacer, e.g., CH2CH3, is human.

[0230] In some embodiments, the CAR or TCR complex protein is a TCR complex protein (i.e., a protein that constitutes part of the TCR complex). In some embodiments, the TCR complex protein is a recombinant, naturally occurring protein. In some embodiments, the TCR complex protein is an artificial version of a protein that constitutes part of the TCR complex. In some embodiments, the TCR complex protein is TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 epsilon, CD3 gamma, CD delta, CD3 zeta, CD4, or CD8. In some embodiments, the TCR complex protein includes an artificial binding domain and / or a co-stimulatory domain.

[0231] In certain embodiments, the TCR complex protein contains a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequences of the native TCR Vα, Vβ, Cα, and / or Cβ. In some embodiments, each CDR or TCR complex protein contains zero changes or at most one, two, or three changes from the TCR or its fragment or derivative that specifically binds to the target of interest.

[0232] 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 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. Because 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. In some embodiments, the antigen-binding domain is an aptamer or nanobody specific to the target antigen.

[0233] 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.

[0234] In some embodiments, CAR or TCR is group 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, i Interleukin-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), nerve cell adhesion molecule (N CAM), prostase, 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 Averso Oncogene fusion protein (bcr-abl) consisting of mouse leukemia virus oncogene 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), claudin 18.2 (CLDN18.2), 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 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 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 mutant, Prostein, Survivin, Telomerase, Prostate cancer tumor antigen-1, Melanoma antigen 1 recognized by T cell 1, Rat sarcoma (Ras) mutant, Human telomerase reverse transcriptase (hTERT), Sarcoma translocation breakpoint, Apoptosis melanoma inhibitor (ML -IAP), ERG (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 myelocytoma 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), enteric 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 initial antigen,It contains antigen-binding domains specific to antigens selected from HHV-6B U94 latent antigen, HHV-6B p98 late-stage antigen, cytomegalovirus (CMV) antigen, 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.

[0235] 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, D4, CD7, CD8a, CD8[T, CD1 1a (IT GAL), CD1 1b (IT GAM), CD1 1c (IT GAX), CD1 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 (CD11a / 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.

[0236] 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 their fragments. 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.

[0237] 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.

[0238] The transmembrane region contains receptor tyrosine kinases (e.g., ErbB2), glycophorin A (GpA), 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, 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 1a, CD1 1b, CD1 1c, CD1 1d, CDS, CEACAM1, 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; CD1-1a / 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;Ly108), 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).

[0239] 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 fms-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 (R OR2), muscle-related 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)EphA 3. EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphA10), EPH receptor B1 (EphB1), 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).

[0240] 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 the total 4-1BB, CD28, or CD3 zeta. 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. (see 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).

[0241] In some embodiments, the co-stimulatory domain includes the amino acid sequence of SEQ ID NO: 318 or 320.

[0242] 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.

[0243] 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, CD8 alpha, CD8 beta, CD96(Tactile), CD1 1a, CD1 1b, CD1 1c, 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, ligands that specifically bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162) This includes, but is not limited to, signaling lymphocyte-activating 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.

[0244] 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.

[0245] 9. 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.

[0246] 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.

[0247] 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 amplifying 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.

[0248] 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.

[0249] 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' homologous region).

[0250] 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., between 20°C and 60°C).

[0251] Therefore, in certain embodiments, a method for producing circular RNA is provided herein. In certain embodiments, the method includes synthesizing a precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a vector comprising, in the following order, a 5' homologous region, a 3' group I intron fragment, a first spacer, an internal ribosome entry site (IRES), an expression sequence, a second spacer, a 5' group I intron fragment, and a 3' homologous region) as a template, and cyclizing the resulting precursor RNA in the presence of a divalent cation (e.g., magnesium ion) and GTP to form circular RNA. In some embodiments, the precursor RNA of the present invention can be cyclized in the absence of magnesium ions and GTP, and / or without the incubation step with magnesium ions and GTP. In some embodiments, transcription is carried out in the presence of excess GMP.

[0252] 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 includes one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification includes the following steps in the order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification includes 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 IFN-β1, RIG-1, IL-2, IL-6, IFNγ, and / or TNFα than immune cells exposed to the unpurified composition.

[0253] 10. Nanoparticles In certain embodiments, pharmaceutical compositions comprising the cyclic RNA provided herein are provided herein. In certain embodiments, such pharmaceutical compositions are formulated with nanoparticles to facilitate delivery.

[0254] In certain embodiments, the circular RNA provided herein may be delivered to and / or targeted to cells by a transport vehicle, such as nanoparticles, or a composition containing nanoparticles. In some embodiments, the circular RNA may also be delivered to a target by a transport vehicle or a composition containing a transport vehicle. In some embodiments, the transport vehicle is nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, non-lipid polymer core-shell nanoparticles, or biodegradable nanoparticles. In some embodiments, the transport vehicle includes one or more cationic lipids, non-cationic lipids, ionizable lipids, PEG-modified lipids, polyglutamic acid lipids, hyaluronic acid lipids, poly-β-aminoesters, poly-beta-aminopeptides, or positively charged peptides.

[0255] In one embodiment, the transport vehicle may 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) may be optimized to effectively deliver such transport vehicle to the target cells, reduce immune clearance, and / or promote retention in those target cells. Alternatively, if the target cells are the central nervous system (e.g., circRNA administered for the treatment of neurodegenerative diseases may specifically target brain or spinal cord tissue), the selection and preparation of the transport vehicle must take into account blood-brain barrier permeability; retention within the blood-brain barrier and / or the use of alternative means to directly deliver such transport vehicle to such target cells. In one embodiment, the compositions of the present invention may be combined with agents that facilitate the transport of exogenous circRNA (e.g., agents that disrupt or improve the permeability of the blood-brain barrier, thereby improving the transport of exogenous circRNA to target cells).

[0256] The use of a transport vehicle to facilitate the delivery of nucleic acids to target cells is intended by the present invention. Liposomes (e.g., liposomal lipid nanoparticles) are generally useful for a variety of applications in research, industry, and medicine, particularly for their use as transport vehicles for in vivo diagnostic or therapeutic compounds (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol. Rev., 51:691-743, 1999), and are typically characterized as microscopic vesicles having an internal aqueous space isolated from the external medium by one or more bilayer membranes. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0257] In the context of the present invention, a transport vehicle typically serves to transport circRNA to target cells. For the purposes of the present invention, the transport vehicle is prepared to contain a desired nucleic acid. The process of incorporating a desired entity (e.g., nucleic acid) into a liposome is often referred to as loading (Lasic, et al., FEBS Lett., 312:255-258, 1992). The nucleic acid incorporated into the liposome may be located entirely or partially within the internal space of the liposome, within the bilayer membrane of the liposome, or relative to the outer surface of the liposome membrane. The purpose of incorporating circRNA into a transport vehicle such as a liposome is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade the nucleic acid and / or systems or receptors that cause rapid excretion of the nucleic acid. Therefore, in certain embodiments of the present invention, a selected transport vehicle can improve the stability of the circRNA contained therein. Liposomes can enable encapsulated circRNA to reach target cells and / or may enable encapsulated circRNA to reach target cells, or restrict the delivery of such circRNA to other sites or cells where the presence of administered circRNA may be unhelpful or undesirable. Furthermore, incorporating circRNA into a transport vehicle, such as such cationic liposomes, also facilitates the delivery of such circRNA to target cells.

[0258] Ideally, the transport vehicle is prepared to encapsulate one or more desired circRNAs so that the composition exhibits high transfection efficiency and improved stability. While liposomes can facilitate the introduction of nucleic acids into target cells, the addition of polycations (e.g., poly-L-lysine and protamine) as copolymers facilitates the transfection of several types of cationic liposomes, and in some cases, the transfection efficiency of several types of cationic liposomes can be significantly improved by 2 to 28 times in many cell lines, both in vitro and in vivo. (See N J. Caplen, et al., Gene Ther. 1995; 2:603; S. Li, et al., Gene Ther. 1997; 4, 891.)

[0259] In embodiments of the present invention, the transport vehicle is formulated as lipid nanoparticles. In some embodiments, the lipid nanoparticles are formulated to deliver one or more circRNAs to one or more target cells. Examples of suitable lipids include phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). The use of polymers as transport vehicles, either alone or in combination with other transport vehicles, is also intended. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, dendrimers, and polyethyleneimines. In some embodiments, the transport vehicle is formulated as a lipid as described in U.S. Patent Application No. 16 / 065,067 (which is incorporated herein by reference in its entirety). In one embodiment, the transport vehicle is selected based on its ability to facilitate the transfection of circRNA into target cells.

[0260] The present invention envisions the use of lipid nanoparticles containing cationic lipids as transport vehicles to improve the delivery of circRNA to target cells that can encapsulate circRNA and / or function as depots for protein production. The envisioned lipid nanoparticles may be prepared by comprising a multi-component lipid mixture in various ratios employing one or more cationic lipids, non-cationic lipids, and PEG-modified lipids. Several cationic lipids have been described in the literature, and many of them are commercially available.

[0261] Cationic lipids suitable for use in the compositions and methods of the present invention include those described in International Patent Publication WO2010 / 053572 and / or U.S. Patent Application No. 15 / 809,680, for example, C12-200. In certain embodiments of the present invention, the compositions and methods of the present invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000) and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (H Lipid nanoparticles containing ionizable cationic lipids, such as GT5001 and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), as described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (incorporated herein by reference).

[0262] In some embodiments, the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used (Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355). DOTMA can be incorporated alone or in combination with neutral lipids, dioleoylphosphatidylethanolamine or "DOPE" or other cationic or non-cationic lipids into transport vehicles or lipid nanoparticles, such liposomes can be used to improve the delivery of nucleic acids to target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycine dioctadecylamide or "DOGS", 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propanaminonium or "DOSPA" (Behr et al. Proc. Nat'l Acad.Sci.86,6982 (1989); U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP", 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP". Cationic lipids intended include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3- Minopropane or "DLinDMA", 1,2-Dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-Dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-Distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-Dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-Dimethylamino-2-(Cholesta-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-Octadecadieneoxy)propane or "CLinDMA", 2-[5'-(Cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-Octadecadieneoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamyl-3-dimeth This also includes diaminopropane or "DLincarbDAP", 1,2-dilinoleylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethaneamine (DLin-KC2-DMA)) (see WO2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107:276-287(2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8):1003-1007(2005); PCT Publication WO2005 / 121348A1). ,

[0263] The use of cholesterol-based cationic lipids is also intended by the present invention. Such cholesterol-based cationic lipids can be used alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, GL67, DC-Chol (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179,280 (1991); Wolf et al. BioTechniques 23,139 (1997); U.S. Patent No. 5,744,335), or ICE.

[0264] In addition, several reagents are commercially available to improve transfection efficiency. Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, CA), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000 (Invitrogen), FUGENE (Promega, Madison, WI), TRANSFECTAM (DOGS) (Promega), and EFFECTENE (Qiagen, Valencia, CA).

[0265] Cationic lipids, such as dialkylamino-based, imidazole-based, and guanidinium-based lipids, as described in U.S. Patent No. 10,413,618, are also intended.

[0266] In other embodiments, the compositions and methods described herein relate to lipid nanoparticles comprising one or more cleavable lipids, for example, one or more cationic lipids or compounds containing cleavable disulfide (SS) functional groups (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and HGT4005), as further described in U.S. Provisional Application No. 61 / 494,745 (the entire teaching thereof is incorporated herein by reference).

[0267] The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide), either alone or in combination with other lipids constituting a transport vehicle (e.g., lipid nanoparticles), is also envisioned by the present invention. The envisioned PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids accompanied by C6-C20 alkyl chains(s). The addition of such components may prevent complex aggregation, extend circulating life, and provide means to increase the delivery of lipid-nucleic acid compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly replaced from the formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful replaceable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention may constitute molar ratios of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in the transport vehicle.

[0268] The present invention also intends to utilize noncationic lipids, including those described in U.S. Patent Application No. 15 / 809,680. Noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine. These include, but are not limited to, 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such noncationic lipids may be used alone or in combination with other excipients, such as cationic lipids. When used in combination with cationic lipids, the noncationic lipids may constitute a molar ratio of 5 to about 90%, or about 10% to about 70%, of the total lipids present in the transport vehicle.

[0269] Transport vehicles (e.g., lipid nanoparticles) can be prepared by combining multiple lipid and / or polymer components. For example, a transport vehicle may be prepared using C12-200, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 18:56:20:6, or HGT5000, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:20:35:5, or HGT5001, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:20:35:5. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, as well as the relative molar ratios of such lipids, are based on the characteristics of the selected lipids, the properties of the target cells of interest, and the characteristics of the circRNA to be delivered. Additional considerations include, for example, alkyl chain saturation, as well as the size, charge, pH, pKa, fusionability, and toxicity of the selected lipid(s). Therefore, the molar ratio can be adjusted accordingly. For example, in some embodiments, the percentage of cationic lipids in lipid nanoparticles may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The percentage of non-cationic lipids in lipid nanoparticles may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in lipid nanoparticles may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipids in lipid nanoparticles may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0270] Transport vehicles for use in the compositions of the present invention can be prepared by various techniques currently known in the art. Multilamellar vesicles (MLVs) can be prepared using conventional techniques, for example, by depositing selected lipids onto the inner wall of a suitable container or vessel, dissolving the lipids in a suitable solvent, then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. Next, an aqueous phase is added to the vessel using a vortex motion, thereby forming the MLV. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, ULVs can be formed by detergent removal techniques.

[0271] In certain embodiments of the present invention, the composition comprises a transport vehicle, in which circRNA is associated on both surfaces of the transport vehicle and encapsulated within the same transport vehicle. For example, during the preparation of the composition of the present invention, a cationic transport vehicle may associate with circRNA via electrostatic interactions.

[0272] In certain embodiments, the compositions of the present invention may be loaded with diagnostic radionuclides, fluorescent substances, or other substances detectable in both in vitro and in vivo applications. For example, suitable diagnostic substances for use in the present invention may include rhodamine-dioleoylphosphatidylethanolamine (Rh-PE), green fluorescent protein circRNA (GFP circRNA), sea urchin luciferase circRNA, and firefly luciferase circRNA.

[0273] In some embodiments, the selection of an appropriate size for the transport vehicle takes into account the site of the target cell or tissue, and to some extent, the intended use for which the liposome is constructed. In some embodiments, it may be desirable to restrict the transfection of circRNA to certain cells or tissues. For example, to target hepatocytes, the transport vehicle may be sized such that its dimensions are smaller than the openings in the endothelial layer lining the hepatic sinusoids of the liver. Thus, an appropriately sized transport vehicle can easily penetrate such endothelial openings and reach the target hepatocytes. Alternatively, the transport vehicle may be sized such that the diameter of the liposome is sufficient to restrict or explicitly avoid distribution to certain cells or tissues. For example, the transport vehicle may be sized such that its dimensions are larger than the openings in the endothelial layer lining the hepatic sinusoids, thereby restricting the distribution of the transport vehicle to hepatocytes. Generally, the size of the transport vehicle is in the range of approximately 25–250 nm. In some embodiments, the size of the transport vehicle is approximately 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or less than 10 nm.

[0274] Various alternative methods known in the art are available for sizing a population of transport vehicles. One such sizing method is described in U.S. Patent No. 4,737,323 (incorporated herein by reference). Sonication of liposome suspensions by either bath-type or probe-type sonication results in a gradual size reduction to small ULVs with a diameter of less than approximately 0.05 microns. Homogenization is another method that relies on shear energy to fragment larger liposomes into smaller liposomes. In a typical homogenization process, MLVs are recirculated through a standard emulsion homogenizer until a selected liposome size, typically about 0.1–0.5 microns, is observed. The size of liposome vesicles can be determined by quasi-elastic light scattering (QELS), as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421–450 (1981) (incorporated herein by reference). The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles may be alternated with QELS evaluation to facilitate efficient liposome synthesis.

[0275] In addition, in certain embodiments, the circular RNA provided herein may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the circular RNA may be formulated with lipid nanoparticles, such as those described in International Publication No. WO2012170930 (which is incorporated herein in its entirety by reference). In one embodiment, the lipid may be a cleavable lipid, such as those described in International Publication No. WO2012170889 (which is incorporated herein in its entirety by reference). In one embodiment, the pharmaceutical composition of the circular RNA may comprise at least one of the PEGylated lipids described in International Publication No. 2012099755 (which is incorporated herein in its entirety by reference). In one embodiment, the lipid nanoparticle formulation may be formulated by the method described in International Publication No. WO2011127255 or WO2008103276 (which are incorporated herein in their entirety by reference, respectively). Lipid nanoparticles may be coated with or associated with copolymers, such as, for example, block copolymers, including, but not limited to, branched polyether-polyamide block copolymers described in International Publication No. WO2013012476 (which is incorporated herein by reference in its entirety). Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficiency of protein production induced by circular RNA. This is because these formulations may be able to increase cellular transfection with circular RNA, increase the in vivo or in vitro half-life of circular RNA, and / or enable controlled release.

[0276] In some embodiments, a polynucleotide encodes a protein composed of subunits encoded by more than one gene. For example, a protein may be a heterodimer in which each chain or subunit of the protein is encoded by a distinct gene. More than one circRNA molecule can be delivered by transport vehicle, with each circRNA encoding a distinct subunit of the protein. Alternatively, a single circRNA can be manipulated to encode more than one subunit (for example, in the case of a single-stranded Fv antibody). In certain embodiments, distinct circRNA molecules encoding individual subunits may be administered by separate transport vehicles.

[0277] The present invention also aims at the differential targeting of target cells and tissues by both passive and active targeting means. The phenomenon of passive targeting utilizes the natural distribution pattern of transport vehicles in vivo without relying on the use of additional excipients or means to improve the recognition of transport vehicles by target cells. For example, transport vehicles subjected to phagocytosis by cells of the reticuloendothelial system are likely to accumulate in the liver or spleen, and therefore, means can be provided to passively guide the delivery of compositions to such target cells.

[0278] Alternatively, the present invention aims at active targeting, which involves using a targeting moiety that can bind (either covalently or noncovalently) to a transport vehicle to facilitate the localization of such transport vehicle to a particular target cell or target tissue. For example, targeting may be mediated by including one or more endogenous targeting moieties in or on a transport vehicle to facilitate distribution to target cells or tissues. Recognition of the targeting moiety by the target tissue actively facilitates the tissue distribution and cellular uptake of the transport vehicle and / or its contents in the target cells and tissues (for example, including an apolipoprotein-E targeting ligand in or on a transport vehicle facilitates the recognition and binding of the transport vehicle to an endogenous low-density lipoprotein receptor expressed by hepatocytes). As provided herein, compositions may include moieties that can improve the affinity of the composition to target cells. Targeting moieties may be ligated to the outer bilayer of lipid particles during or after formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusion polymers, such as PEAA, hemagglutinins, other lipopeptides (see U.S. Patent Applications No. 08 / 835,281 and No. 60 / 083,294 (incorporated herein by reference)), and other properties useful for in vivo and / or intracellular delivery. In other embodiments, the compositions of the present invention demonstrate improved transfection efficiency and / or enhanced selectivity for target cells or tissues of interest. Thus, compositions comprising one or more moieties (e.g., peptides, aptamers, oligonucleotides, vitamins, or other molecules) that can improve the affinity of the composition and its nucleic acid contents to target cells or tissues are contemplated. Preferred moieties may optionally be bound or ligated to the surface of a transport vehicle. In some embodiments, the targeting moieties may spread across the surface of the transport vehicle or be encapsulated within the transport vehicle. Preferred moieties (and are) are selected based on their physical, chemical, or biological properties (e.g., selective affinity and / or recognition of target cell surface markers or features). Cell-specific target sites and their corresponding targeting ligands can vary considerably.A suitable targeting moiety is selected to take advantage of the unique characteristics of the target cell, thus enabling the composition to differentiate between target and non-target cells. For example, the composition of the present invention may include a surface marker (e.g., apolipoprotein B or apolipoprotein E) that selectively enhances the recognition of hepatocytes or affinity to hepatocytes (e.g., by receptor-mediated recognition of such surface markers and binding to such surface markers). For example, the use of galactose as the targeting moiety may be expected to direct the composition of the present invention to hepatocytes, or the use of mannose containing a sugar residue as a targeting ligand may be expected to direct the composition of the present invention to hepatic endothelial cells (e.g., mannose containing a sugar residue that can preferentially bind to asialoglycoprotein receptors present on hepatocytes). (See Hillery AM, et al. “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002), Taylor & Francis, Inc.). The presentation of such a targeting moiety, conjugated to a portion present on a transport vehicle (e.g., lipid nanoparticles), thus facilitates the recognition and uptake of the composition of the present invention in target cells and tissues. Examples of suitable targeting moieties include one or more peptides, proteins, aptamers, vitamins, and oligonucleotides.

[0279] In certain embodiments, the transport vehicle includes a targeting moiety. In some embodiments, the targeting moiety selectively mediates receptor-mediated endocytosis to a specific cell population. In some embodiments, the targeting moiety can bind to a T cell antigen. In some embodiments, the targeting moiety can bind to an NK, NKT, dendritic cell, or macrophage antigen. In some embodiments, the targeting moiety can bind to a protein selected from the group CD3, CD4, CD8, PD-1, 4-1BB, CD5, CD7, C1q, and CD2. In some embodiments, the targeting moiety is a single-chain Fv(scFv) fragment, a nanobody, a peptide, a peptide-based macrocyclic compound, a minibody, a heavy chain variable region, a light chain variable region, or a fragment thereof. In some embodiments, the targeting moiety is an anti-T cell receptor motif antibody, an anti-T cell alpha chain antibody, an anti-T cell beta chain antibody, an anti-T cell gamma chain antibody, an anti-T cell delta chain antibody, an anti-CCR7 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody. , anti-CD7 antibody, anti-CD8 antibody, anti-CD11b antibody, anti-CD11c antibody, anti-CD16 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD28 antibody, anti-CD34 antibody The antibodies are selected from the following: anti-CD35 antibody, anti-CD40 antibody, anti-CD45RA antibody, anti-CD45RO antibody, anti-CD52 antibody, anti-CD56 antibody, anti-CD62L antibody, anti-CD68 antibody, anti-CD80 antibody, anti-CD95 antibody, anti-CD117 antibody, anti-CD127 antibody, anti-CD133 antibody, anti-CD137(4-1BB) antibody, anti-CD163 antibody, anti-C1q antibody, anti-F4 / 80 antibody, anti-IL-4Rα antibody, anti-Sca-1 antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-GARP antibody, anti-LAP antibody, anti-granzyme B antibody, anti-LFA-1 antibody, anti-transferrin receptor antibody, and their fragments.

[0280] In some embodiments, the circular RNA is formulated according to the process described in U.S. Patent Application 15 / 809,680. In some embodiments, the present invention provides a process for encapsulating circular RNA in lipid nanoparticles, the process comprising the steps of forming lipids into preformed lipid nanoparticles (i.e., in the absence of RNA), and then combining the preformed lipid nanoparticles with RNA. In some embodiments, the novel formulation process results in an RNA formulation that has higher potency (peptide or protein expression) and higher efficacy (improvement of biologically relevant endpoints) both in vitro and in vivo, and potentially better tolerability, compared to the same RNA formulation prepared without the step of preforming lipid nanoparticles (e.g., by directly combining lipids with RNA). In some embodiments, the targeted moiety is a small molecule binding factor for ectoenzymes on lymphocytes. Small molecule binding factors for ectoenzymes include A2A inhibitors, CD73 inhibitors, CD39, or adesin receptors A2aR and A2bR. Potential small molecules include AB928.

[0281] In some embodiments, the transport vehicle is formulated and / or targeted as described in Shobaki N, Sato Y, Harashima H. ​​Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting. Int J Nanomedicine. 2018;13:8395-8410. Published December 10, 2018. In some embodiments, the transport vehicle consists of three lipid types. In some embodiments, the transport vehicle consists of four lipid types. In some embodiments, the transport vehicle consists of five lipid types. In some embodiments, the transport vehicle consists of six lipid types.

[0282] For certain cationic lipid nanoparticle formulations of RNA, heating of the RNA in a buffer (e.g., citrate buffer) is required to achieve high RNA encapsulation. In these processes or methods, heating after formulation (after nanoparticle formation) does not increase the efficiency of RNA encapsulation into lipid nanoparticles; therefore, heating must be performed before the formulation process (i.e., heating a separate component). In contrast, in some embodiments of the process of the present invention, the order of RNA heating does not appear to affect the RNA encapsulation percentage. In some embodiments, heating of one or more of the solution containing preformed lipid nanoparticles, the solution containing RNA, and the mixed solution containing RNA encapsulated in lipid nanoparticles does not need to occur before or after the formulation process (i.e., is maintained at ambient temperature).

[0283] RNA may be provided in a solution mixed with a lipid solution so that the RNA can be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain RNA at concentrations of approximately 0.01 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or greater than 1.0 mg / ml. In some embodiments, a suitable RNA solution contains RNA in concentrations of approximately 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, and 0.05-0.9 mg / ml. It may be contained in concentrations ranging from 0.05 to 0.8 mg / ml, 0.05 to 0.7 mg / ml, 0.05 to 0.6 mg / ml, 0.05 to 0.5 mg / ml, 0.05 to 0.4 mg / ml, 0.05 to 0.3 mg / ml, 0.05 to 0.2 mg / ml, 0.05 to 0.1 mg / ml, 0.1 to 1.0 mg / ml, 0.2 to 0.9 mg / ml, 0.3 to 0.8 mg / ml, 0.4 to 0.7 mg / ml, or 0.5 to 0.6 mg / ml.

[0284] Typically, a suitable RNA solution may also contain a buffer and / or salt. Generally, buffers may include HEPES, ammonium sulfate, Tris, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, or sodium phosphate. In some embodiments, suitable concentrations of buffers may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM.

[0285] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, preferred concentrations of the salt in the RNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.

[0286] In some embodiments, a preferred RNA solution may have a pH in the range of approximately 3.5–6.5, 3.5–6.0, 3.5–5.5, 3.5–5.0, 3.5–4.5, 4.0–5.5, 4.0–5.0, 4.0–4.9, 4.0–4.8, 4.0–4.7, 4.0–4.6, or 4.0–4.5.

[0287] Various methods can be used to prepare RNA solutions suitable for the present invention. In some embodiments, RNA may be dissolved directly in the buffers described herein. In some embodiments, the RNA solution may be prepared by mixing the RNA stock solution with the buffer before mixing it with the lipid solution for mounting. In some embodiments, the RNA solution may be prepared by mixing the RNA stock solution with the buffer immediately before mixing it with the lipid solution for mounting.

[0288] In accordance with the present invention, the lipid solution contains a mixture of lipids suitable for forming a transport vehicle for encapsulating RNA. In some embodiments, the preferred lipid solution is ethanol-based. For example, the preferred lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, the preferred lipid solution is isopropyl alcohol-based. In yet another embodiment, the preferred lipid solution is dimethyl sulfoxide-based. In yet another embodiment, the preferred lipid solution is a mixture of preferred solvents including, but not limited to, ethanol, isopropyl alcohol, and dimethyl sulfoxide.

[0289] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain the mixture of desired lipids at total concentrations ranging from approximately 0.1 to 100 mg / ml, 0.5 to 90 mg / ml, 1.0 to 80 mg / ml, 1.0 to 70 mg / ml, 1.0 to 60 mg / ml, 1.0 to 50 mg / ml, 1.0 to 40 mg / ml, 1.0 to 30 mg / ml, 1.0 to 20 mg / ml, 1.0 to 15 mg / ml, 1.0 to 10 mg / ml, 1.0 to 9 mg / ml, 1.0 to 8 mg / ml, 1.0 to 7 mg / ml, 1.0 to 6 mg / ml, or 1.0 to 5 mg / ml.

[0290] Any desired lipids can be mixed in any ratio suitable for RNA encapsulation. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and / or PEGylated lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol lipids), and one or more PEGylated lipids.

[0291] 11.Target cells In some embodiments, the target cells are deficient in the protein or enzyme of interest. For example, if it is desired to deliver nucleic acids to hepatocytes, then hepatocytes represent the target cells. In some embodiments, the compositions of the present invention selectively transfect target cells (i.e., do not transfect non-target cells). The compositions of the present invention may also be prepared to preferentially target a variety of target cells, including, but not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells (e.g., meninges, astrocytes, motor neurons, dorsal root ganglion cells and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial-lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, dendritic cells, macrophages, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0292] The compositions of the present invention can be prepared to preferentially distribute to target cells such as those in the heart, lungs, kidneys, liver, and spleen. In some embodiments, the compositions of the present invention are distributed to liver cells to facilitate the delivery and subsequent expression of circRNAs contained in the composition by liver cells (e.g., hepatocytes). Targeted cells can function as biological “storage” or “depot” that can produce and systemically excrete functional proteins or enzymes. Thus, in one embodiment of the present invention, the transport vehicle can target hepatocytes and / or preferentially distribute to liver cells upon delivery. In one embodiment, following transfection of target hepatocytes, the circRNA loaded into the vehicle is translated, producing functional protein products that are excreted and distributed systemically. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, eye, or central nervous system cells) can function as depot sites for protein production.

[0293] In one embodiment, the composition of the present invention facilitates the endogenous production of one or more functional proteins and / or enzymes of a target. In one embodiment of the present invention, the transport vehicle comprises circRNA encoding a deficiency protein or enzyme. During the distribution of such composition to a target tissue and subsequent transfection of such target cells, the exogenous circRNA loaded onto the transport vehicle (e.g., lipid nanoparticles) can be translated in vivo to produce a functional protein or enzyme encoded by the exogenously administered circRNA (e.g., the protein or enzyme that the target is deficient in). Thus, the composition of the present invention utilizes the ability of a target to translate exogenously or recombinantly prepared circRNA to produce an endogenously translated protein or enzyme, thereby producing (and, if applicable, excreting) a functional protein or enzyme. The expressed or translated protein or enzyme may also be characterized by containing innate post-translational modifications in vivo that are often not present in recombinantly prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.

[0294] Administration of circRNA encoding a deficient protein or enzyme avoids the need to deliver nucleic acids to specific organelles within target cells. Rather, during transfection of target cells and delivery of nucleic acids to the cytoplasm of target cells, the contents of the transport vehicle's circRNA can be translated, leading to the expression of functional proteins or enzymes.

[0295] In some embodiments, the circular RNA includes one or more miRNA binding sites. In some embodiments, the circular RNA includes one or more miRNA binding sites that are recognized by miRNAs present in one or more non-target cells or non-target cell types (e.g., Kupffer cells) and not present in one or more target cells or target cell types (e.g., hepatocytes). In some embodiments, the circular RNA includes one or more miRNA binding sites that are recognized by miRNAs present at increased concentrations in one or more non-target cells or non-target cell types (e.g., Kupffer cells) compared to one or more target cells or target cell types (e.g., hepatocytes). miRNAs are thought to function by pairing with complementary sequences within the RNA molecule, thereby causing gene silencing.

[0296] 12. Pharmaceutical Compositions In certain embodiments, compositions (e.g., pharmaceutical compositions) comprising a therapeutic agent provided herein are provided herein. In some embodiments, the therapeutic agent is a cyclic RNA polynucleotide provided herein. In some embodiments, the therapeutic agent is a vector provided herein. In some embodiments, the therapeutic agent is a cell (e.g., a human cell such as a human T cell) comprising the cyclic RNA or vector provided herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition provided herein comprises the therapeutic agent provided herein in combination with other pharmaceutically active agents or drugs, such as anti-inflammatory drugs or antibodies that can target B cell antigens, such as anti-CD20 antibodies, such as rituximab; chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In preferred embodiments, the pharmaceutical composition comprises cells or populations thereof provided herein.

[0297] With respect to pharmaceutical compositions, pharmaceutically acceptable carriers can be any of those conventionally used and are limited only by chemophysical considerations, such as solubility and lack of reactivity with the activator(s), and route of administration. The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are generally readily available. It is preferable that the pharmaceutically acceptable carrier(s) are chemically inert to the therapeutic agent(s) and do not have harmful side effects or toxicity under the conditions of use.

[0298] The choice of carrier will be determined in part by the specific therapeutic agent, as well as the specific method used to administer the therapeutic agent. Therefore, there are various suitable formulations of the pharmaceutical compositions provided herein.

[0299] In certain embodiments, the pharmaceutical composition includes a preservative. In certain embodiments, suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0300] In some embodiments, the pharmaceutical composition includes a buffer. In some embodiments, suitable buffers may include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffers may be used at the option of choice. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0301] In some embodiments, the concentration of the therapeutic agent in the pharmaceutical composition can vary, for example, to less than about 1% by weight, or to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% by weight or more, and can be selected mainly by fluid volume and viscosity according to a specific dosage mode selected.

[0302] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intra-arterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are merely illustrative and not limiting. More than one route may be used to administer the therapeutic agents provided herein, and in certain cases, a particular route may provide a faster and more effective response than another.

[0303] Formulations suitable for oral administration may include, or be derived from, (a) an effective amount of the therapeutic agent dissolved in a liquid solution, such as water, saline, or orange juice; (b) capsules, sachets, tablets, licks, and lozenges, each containing a predetermined amount of the active ingredient as a solid or granule; (c) powder; (d) suspension in a suitable liquid; and (e) a suitable emulsion. Liquid formulations may contain, or may not contain, a diluent, such as water, and an alcohol, such as ethanol, benzyl alcohol, and polyethylene alcohol, with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms may be of the usual hard-shell or soft-shell gelatin type, for example, containing a surfactant, a lubricant, and an inert filler, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms may contain lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and one or more of other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavorings, and other pharmacologically suitable excipients. Lozenge forms may contain therapeutic agents with flavorings, usually sucrose, acacia, or tragacanth. Lozenge may contain therapeutic agents having inert bases, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, etc., in addition to excipients known in the art.

[0304] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, antibacterial agents, and solutes to make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In some embodiments, the therapeutic agents provided herein may be administered with or without the addition of pharmaceutically acceptable surfactants, such as soaps or detergents, suspending agents, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants, in a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids containing water, physiological saline, dextrose aqueous solution and related sugar solution, alcohol, such as ethanol or hexadecyl alcohol, glycol, such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketal, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ether, poly(ethylene glycol) 400, oil, fatty acid, fatty acid ester or glyceride, or acetylated fatty acid glyceride.

[0305] Oils can be used in parenteral formulations in some embodiments and include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Fatty acids suitable for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0306] Soaps suitable for use in certain embodiments of parenteral formulations include fatty alkali metals, ammonium, and triethanolamine salts; suitable detergents include (a) cationic detergents, e.g., dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents, e.g., alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, e.g., fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, e.g., alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0307] In some embodiments, parenteral formulations will contain, for example, about 0.5% to about 25% by weight of the therapeutic agent in a solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain, for example, one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations will typically be in the range of, for example, about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol, sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide and propylene glycol. Parenteral formulations can be provided in sealed containers of unit or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid excipient, such as water, immediately before use for injection. Immediate injection solutions and suspensions can be prepared from the sterile powders, granules, and tablets of the types described above.

[0308] In certain embodiments, injectable formulations are provided herein. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed, pages 622-630 (1986)).

[0309] In some embodiments, topical formulations are provided herein. Topical formulations, including those useful for transdermal drug release, are preferred in the circumstances of certain embodiments provided herein for application to the skin. In some embodiments, the therapeutic agent can be made into an aerosol formulation administered by inhalation, either alone or in combination with other suitable ingredients. These aerosol formulations can be encapsulated in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, or nitrogen. They can also be formulated as pharmaceuticals for non-pressurized preparations such as nebulizers or atomizers. Such spray formulations can also be used for spraying mucous membranes.

[0310] In certain embodiments, the therapeutic agents provided herein may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes. Liposomes may help the therapeutic agent target specific tissues. Liposomes may also be used to extend the half-life of the therapeutic agent. Many methods are available for preparing liposomes, for example, as described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9,467 (1980) and U.S. Patents No. 4,235,871, No. 4,501,728, No. 4,837,028, and No. 5,019,369.

[0311] In some embodiments, the therapeutic agents provided herein are formulated using a time-release, delayed-release, or sustained-release delivery system such that delivery of the composition occurs with sufficient time to cause sensitization of the treated site before it occurs. Such systems can avoid repeated administration of the therapeutic agent, thereby increasing convenience for the subject and the physician, and may be particularly suitable for certain embodiments of the compositions provided herein. In one embodiment, the composition of the present invention is formulated to be suitable for the sustained release of the circRNA contained herein. Such a sustained-release composition can be conveniently administered to the subject with extended dosing intervals. For example, in one embodiment, the composition of the present invention is administered to the subject twice daily, daily, or every other day. In some embodiments, the composition of the present invention is administered to the subject twice weekly, once weekly, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, once a month, every 6 weeks, every 8 weeks, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or annually.

[0312] In some embodiments, the protein encoded by the polynucleotide of the present invention is produced by target cells over a sustained period of time. For example, the protein may be produced for longer than 1 hour, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after administration. In some embodiments, the polypeptide is expressed at a peak level about 6 hours after administration. In some embodiments, polypeptide expression is sustained at least at a therapeutic level. In some embodiments, the polypeptide is expressed at least at a therapeutic level for longer than 1 hour, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in the patient's serum or tissue (e.g., liver or lung). In some embodiments, the detectable level of polypeptide is from the sustained expression from the circRNA composition over a period of longer than 1 hour, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after administration.

[0313] In certain embodiments, the protein encoded by the polynucleotide of the present invention is produced at levels exceeding normal physiological levels. The protein level may be increased compared to a control. In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual or a population of individuals lacking the relevant protein or polypeptide. In some embodiments, the control may be the normal level of the relevant protein or polypeptide in an individual to which the composition is administered. In other embodiments, the control is the polypeptide expression level at one or more comparable time points in time, during other therapeutic interventions, for example, at direct injection of the corresponding polypeptide.

[0314] In certain embodiments, the level of the protein encoded by the polynucleotide of the present invention is detectable 3, 4, 5 days, or more than a week after administration. Increased levels of secreted proteins may be observed in serum and / or tissues (e.g., liver or lungs).

[0315] In some embodiments, the method results in a sustained cyclic half-life of the protein encoded by the polynucleotide of the present invention. For example, the protein may be detected over a longer period of time or days than the half-life observed via subcutaneous injection of the protein or the mRNA encoding the protein. In some embodiments, the half-life of the protein is 1 day, 2 days, 3 days, 4 days, 5 days, or more than 1 week.

[0316] Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems, such as poly(lactide-glycolides), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acids, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems, such as lipids including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono-di- and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused grafts; and the like. Specific examples, though not limited to these, include (a) erosion systems, where the active composition is contained in a matrix form such as those described in U.S. Patents No. 4,452,775, No. 4,667,014, No. 4,748,034, and No. 5,239,660, and (b) diffusion systems, where the active ingredient penetrates at a controlled rate from a polymer such as those described in U.S. Patents No. 3,832,253 and No. 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are suitable for implantation.

[0317] In some embodiments, the therapeutic agent can be directly or indirectly conjugated to the targeting portion through a conjugate portion. Methods for conjugating the therapeutic agent to the targeting portion are known in the art. See, for example, Wadwa et al., J, Drug Targeting 3:111 (1995) and U.S. Patent No. 5,087,616.

[0318] In some embodiments, the therapeutic agents provided herein are formulated into depot forms such that the manner in which the therapeutic agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The depot form of the therapeutic agent may be, for example, an implantable composition comprising the therapeutic agent and a porous or non-porous material such as a polymer, where the therapeutic agent is encapsulated by the material, diffused throughout the material, and / or decomposed by the non-porous material. The depot is then implanted at a desired location in the body, and the therapeutic agent is released from the implant at a predetermined rate.

[0319] 13.Treatment method In certain embodiments, methods for treating and / or preventing a condition, such as cancer, are provided herein, the methods comprising introducing a pharmaceutical composition provided herein into a subject in need thereof (e.g., a subject having cancer). In some embodiments, the pharmaceutical composition comprises a cyclic RNA polynucleotide provided herein. In some embodiments, the pharmaceutical composition comprises a vector provided herein. In some embodiments, the pharmaceutical composition comprises a cell (e.g., a human cell such as a human T cell) containing a polynucleotide (e.g., a cyclic RNA or vector provided herein).

[0320] Therefore, in certain embodiments, methods for treating and / or preventing diseases in subjects (e.g., mammalian subjects, e.g., human subjects) are provided herein. Not bound by any particular theory or mechanism, CAR and TCR complex proteins have biological activity, e.g., the ability to recognize antigens, e.g., CD19, and as a result, when expressed by cells, CAR or TCR can mediate an immune response against cells expressing a specific antigen, e.g., CD19. In this regard, certain embodiments provided herein provide a method for treating or preventing cancer in mammals, the method comprising administering to a mammal the therapeutic agent, and / or a pharmaceutical composition provided herein, in an amount effective for treating or preventing cancer in mammals.

[0321] In certain embodiments, the therapeutic agent provided herein is co-administered with one or more additional therapeutic agents (for example, in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the therapeutic agent provided herein may be administered first, followed by one or more additional therapeutic agents, or vice versa. Alternatively, the therapeutic agent provided herein and one or more additional therapeutic agents may be administered simultaneously. In some embodiments, additional therapeutic agents that can be co-administered with the therapeutic agent provided herein are T-cell activating cytokines, such as IL-2, IL-7, IL-15, and / or IL-21.

[0322] In certain embodiments, the therapeutic agent is a group of cells or cells containing the circular RNA or vector provided herein, expressing a CAR or TCR complex protein encoded by the circular RNA or vector. In some embodiments, the administered cells are allogeneic to the subject being treated. In some embodiments, the administered cells are autologous to the subject being treated.

[0323] In certain embodiments, the method further includes lymphocyte depletion of the subject before administration of the therapeutic agent. Examples of lymphocyte depletion include, but are not limited to, non-myeloablative lymphocyte depletion chemotherapy, myeloablative lymphocyte depletion chemotherapy, and total body irradiation.

[0324] In some embodiments, the subject is a mammal. In some embodiments, the mammals referred to herein may be any mammal, including but not limited to rodents such as mice and hamsters, or mammals of the order Logomorpha such as rabbits. The mammal may be from the order Carnivora, which includes the families Felidae (cats) and Canidae (dogs). The mammal may be from the order Artiodactyla, which includes the families Bovidae (cats) and Suidae (pigs), or the order Perissodactyla, which includes the family Equidae (horse). The mammal may be from the order Primates, Ceboidi, or Simoida (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0325] 14. Array (Table 3) Exemplary IRES sequences. TIFF2022533796000031.tif177160TIFF2022533796000032.tif241160TIFF2022533796000033.tif240160TIFF2022533796000034.t if240160TIFF2022533796000035.tif241160TIFF2022533796000036.tif240160TIFF2022533796000037.tif240160TIFF20225337960 00038.tif241160TIFF2022533796000039.tif241160TIFF2022533796000040.tif241160TIFF2022533796000041.tif241160TIFF202 2533796000042.tif240160TIFF2022533796000043.tif240160TIFF2022533796000044.tif240160TIFF2022533796000045.tif128160

[0326] In some embodiments, the IRES of the present invention is an IRES having the sequences listed in Table 3 (SEQ ID NOs: 1 to 72). In some embodiments, the IRES is a Salivirus IRES. In some embodiments, the IRES is a Salivirus SZ1 IRES.

[0327] (Table 4) Anabaena permutation site 5' intron fragment sequence. TIFF2022533796000046.tif69160TIFF2022533796000047.tif241160TIFF2022533796000048.tif242160TIFF2022533796000049.tif71160

[0328] In some embodiments, the 5' intron fragment is a fragment having the sequences listed in Table 4. Typically, a construct containing the 5' intron fragments listed in Table 4 would contain the corresponding 3' intron fragments listed in Table 5 (for example, both representing fragments with L9a-8 permutation sites).

[0329] (Table 5) Anabaena permutation site 3' intron fragment sequence. TIFF2022533796000050.tif124160TIFF2022533796000051.tif241160TIFF20225337960 00052.tif241160TIFF2022533796000053.tif241160TIFF2022533796000054.tif177160

[0330] In some embodiments, the 3' intron fragment is a fragment having the sequences listed in Table 5. In some embodiments, a construct containing the 3' intron fragments listed in Table 5 will contain the corresponding 5' intron fragments as listed in Table 4 (for example, both representing fragments with L9a-8 permutation sites).

[0331] (Table 6) Sequence of 5' intron fragments at non-anabaena permutation sites. TIFF2022533796000055.tif188160

[0332] In some embodiments, the 5' intron fragment is a fragment having the sequence listed in Table 6. A construct containing the 5' intron fragments listed in Table 6 would contain the corresponding 3' intron fragment as listed in Table 7 (for example, both representing fragments with an Azop1 intron).

[0333] (Table 7) Sequence of 3' intron fragments at non-anabaena permutation sites. TIFF2022533796000056.tif26160TIFF2022533796000057.tif242160TIFF2022533796000058.tif30160

[0334] In some embodiments, the 3' intron fragment is a fragment having the sequence listed in Table 7. A construct containing the 3' intron fragments listed in Table 7 would contain the corresponding 5' intron fragment as listed in Table 6 (for example, both representing fragments with an Azop1 intron).

[0335] (Table 8) Spacer and Anabear 5' intron fragment sequences. TIFF2022533796000059.tif168160TIFF2022533796000060.tif241160TIFF2022533796000061.tif241160 TIFF2022533796000062.tif249166TIFF2022533796000063.tif242160TIFF2022533796000064.tif114160

[0336] In some embodiments, the spacers and 5' intron fragments are spacers and fragments having the arrangements listed in Table 8.

[0337] (Table 9) Spacer and Anabear 3' intron fragment sequences. TIFF2022533796000065.tif99160TIFF2022533796000066.tif241160TIFF2022 533796000067.tif241160TIFF2022533796000068.tif241160TIFF20225337960 00069.tif241160TIFF2022533796000070.tif241160TIFF2022533796000071.t if241160TIFF2022533796000072.tif241160TIFF2022533796000073.tif153160

[0338] In some embodiments, the spacer and 3' intron fragment are spacer and intron fragment having the arrangements listed in Table 9.

[0339] (Table 10) CAR sequence. TIFF2022533796000074.tif162160TIFF2022533796000075.tif157160TIFF2022533796000076.t if205160TIFF2022533796000077.tif186160TIFF2022533796000078.tif205160TIFF2022533796 000079.tif205160TIFF2022533796000080.tif201160TIFF2022533796000081.tif239160TIFF20 22533796000082.tif64160TIFF2022533796000083.tif239160TIFF2022533796000084.tif74160

[0340] In some embodiments, the CARs have an arrangement as listed in Table 10.

[0341] (Table 11) CAR domain sequences. TIFF2022533796000085.tif104160

[0342] In some embodiments, the CAR domain encoded by the polynucleotide of the present invention has a sequence as listed in Table 11.

[0343] Preferred embodiments are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors anticipate that such variations will be appropriately used by those skilled in the art, and they intend that the invention will be practiced in a manner different from that specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the invention unless otherwise shown herein or otherwise clearly contradicted by context. [Examples]

[0344] Wesselhoeft et al. (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo. Molecular Cell. 74(3), 508-520 and Wesselhoeft et al. (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629 are incorporated herein by reference in their entirety.

[0345] The present invention will be described in more detail by reference to the following embodiments, but is not intended to be limited to these embodiments. These embodiments include any and all exemplary variations for the purpose of providing a complete disclosure and explanation to those skilled in the art of how to make and use the subject invention, and are not intended to limit the scope of what is considered the present invention.

[0346] Example 1 Example 1A: The external homologous region allows for the cyclization of long precursor RNA using a permutation substitution intron-exon (PIE) cyclization strategy. A 1.1 kb sequence containing the full-length encephalomyocarditis virus (EMCV) IRES, a Gaussial luciferase (GLuc) expression sequence, and two short exon fragments of a permutation-substitution intron-exon (PIE) construct were inserted between the 3' and 5' introns of the permutation-substitution group I catalytic intron of the T4 phage thymidylate synthase (Td) gene. Precursor RNA was synthesized by run-off transcription. Cyclization was attempted by heating the precursor RNA in the presence of magnesium ions and GTP, but no splicing product was obtained.

[0347] We designed perfectly complementary homologous regions of 9 and 19 nucleotides in length and added them to the 5' and 3' ends of the precursor RNA. The addition of these homologous arms increased splicing efficiency from 0 to 16% for the 9-nucleotide homologous region and to 48% for the 19-nucleotide homologous region, as assessed by the disappearance of the precursor RNA band.

[0348] The splicing product was treated with RNase R. Sequencing across the putative splice junction of the RNase R-treated splicing reaction revealed ligated exons. Digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band, in contrast to the two bands obtained from the linear precursor digested with RNase H. This indicates that circular RNA is the primary product of the splicing reaction of precursor RNA containing an external homologous region of 9 or 19 nucleotides in length.

[0349] Example 1B: Spacers that preserve the secondary structure of the IRES and PIE splice sites increase the efficiency of annularization. A series of spacers were designed and inserted between the 3'PIE splice site and the IRES. These spacers were designed to either preserve or disrupt secondary structures within the intron sequences of the IRES, 3'PIE splice site, and / or 5-splice site. Adding spacer sequences designed to preserve secondary structures resulted in an 87% splicing efficiency, while adding disruptive spacer sequences did not result in detectable splicing.

[0350] Example 2 Example 2A: In addition to the external homologous region, the internal homologous region creates a splicing bubble, enabling translation of several expression sequences. The spacers were designed to be non-homologous to unstructured, intronic, and IRES sequences, and to contain spacer-spacer homologous regions. These were inserted between the 5' exon and IRES, and between the 3' exon and expression sequences, into constructs containing an external homologous region, an EMCV IRES, and expression sequences of Gaussian luciferase (full length: 1289 nt), firefly luciferase (2384 nt), eGFP (1451 nt), human erythropoietin (1313 nt), and Cas9 endonuclease (4934 nt). Circularization was achieved for all five constructs. Circularization of constructs using T4 phage and anabena introns was nearly equivalent. Circularization efficiency was higher with shorter sequences. To measure translation, each construct was transfected into HEK293 cells. Cells transfected with Gaussia and firefly luciferase showed a strong response, as measured by luminescence; human erythropoietin was detectable in the culture medium of cells transfected with erythropoietin circRNA; and EGFP fluorescence was observed from cells transfected with EGFP circRNA. Co-transfection of cells constitutively expressing GFP with Cas9 circRNA and sgRNA for GFP resulted in the abolition of fluorescence in up to 97% of cells compared to a control with sgRNA alone.

[0351] Example 2B: Use of CVB3 IRES increases protein production. Constructs were created with different IRESs containing internal and external homologous regions, as well as either Gaussian luciferase or firefly luciferase expression sequences. Protein production was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. The coxsackievirus B3 (CVB3) IRES construct produced the most protein in both cases.

[0352] Example 2C: The use of poly-A or poly-AC spacers increases protein production. A 30-nucleotide poly-A or poly-AC spacer was added between the IRES and splice junction in each IRES-containing construct that produced protein in Example 2B. Gaussian alciferase activity was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. Both spacers improved expression in all constructs compared to the control construct without spacers.

[0353] Example 3 HEK293 or HeLa cells transfected with circular RNA produce more protein than cells transfected with equivalent unmodified or modified linear RNA. CircRNA encoding gausial ciferase (CVB3-GLuc-pAC), purified by HPLC, was compared with standard unmodified 5'-methylguanosine cap and 3'-poly A tail linear GLuc mRNA, as well as commercially available nucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA (Trilink). Luminescence was measured 24 hours after transfection, revealing that the circRNA produced 811.2% more protein than the unmodified linear mRNA and 54.5% more protein than the modified mRNA in HEK293 cells. Similar results were obtained in HeLa cells and in comparisons between optimized circRNA encoding human erythropoietin and linear mRNA modified with 5-methoxyuridine.

[0354] Luminescence data were collected over a 6-day period. In HEK293 cells, circRNA transfection resulted in a protein production half-life of 80 hours, compared to 43 hours for unmodified linear mRNA and 45 hours for modified linear mRNA. In HeLa cells, circRNA transfection resulted in a protein production half-life of 116 hours, compared to 44 hours for unmodified linear mRNA and 49 hours for modified linear mRNA. CircRNA produced substantially more protein over its lifetime than both unmodified and modified linear mRNA in both cell types.

[0355] Example 4 Example 4A: Purification of circRNA by RNase digestion, HPLC purification, and phosphatase treatment reduces immunogenicity. Completely purified circular RNA is significantly less immunogenic than unpurified or partially purified circular RNA. Protein expression stability and cell viability depend on cell type and circular RNA purity. Human fetal kidney 293 (HEK293) and human lung cancer A549 cells were used to extract human fetal kidney 293 (HEK293) and human lung cancer A549 cells. a. Products of unpurified GLuc circular RNA splicing reaction, b. Products of RNase R digestion of splice reactants, c. Products obtained by RNase R digestion and HPLC purification of the splice reaction, or d. Products obtained from RNase digestion, HPLC purification, and phosphatase treatment of the splicing reaction product. It was transfected.

[0356] RNase R digestion of the splicing reaction was insufficient to prevent cytokine release in A549 cells compared to untransfected controls.

[0357] Although additional HPLC purification was insufficient to prevent cytokine release, interleukin-6 (IL-6) levels were significantly reduced and interferon-α1 (IFN-α1) levels were significantly increased compared to the unpurified splicing reaction.

[0358] Adding phosphatase treatment after HPLC purification and before RNase R digestion dramatically reduced the expression of all upregulated cytokines evaluated in A549 cells. Secreted monocyte chemoattractant protein 1 (MCP1), IL-6, IFN-α1, tumor necrosis factor α (TNFα), and IFNγ-inducible protein-10 (IP-10) were reduced to undetectable or untransfected baseline levels.

[0359] There was virtually no cytokine release in HEK293 cells. Transfecting A549 cells with higher purity circular RNA increased GLuc expression stability and cell viability. Fully purified circular RNA exhibited a stable phenotype similar to that of transfected 293 cells.

[0360] Example 4B: The circular RNA did not cause significant immunogenicity and is not a RIG-I ligand. A549 cells were transfected with the product of the splicing reaction.

[0361] A549 cells, a. Unpurified circular RNA, b. High molecular weight (linear and cyclic linked) RNA, c. Circular (nicked) RNA, d. Initial fraction of purified circular RNA (often overlapping with nicked RNA peaks), e. Late fraction of purified circular RNA (with less overlap with nicked RNA peaks), f. Introns excised during circulation, or g. Vehicle (i.e., untransfected control) It was transfected.

[0362] Because it is difficult to obtain suitably pure linear precursor RNA from the splicing reaction product, the precursor RNA was separately synthesized and purified in the form of a splice site deletion mutant (DS). Cytokine release and cell viability were measured in each case.

[0363] Potent release of IL-6, RANTES, and IP-10 was observed in response to most species present in the splicing reaction product, as well as to the precursor RNA. The early circRNA fraction induced a cytokine response comparable to that of the other non-circRNA fractions. This indicates that even relatively small amounts of linear RNA contaminants can induce a substantial cellular immune response in A549 cells. The late circRNA fraction did not induced a cytokine response exceeding that of the untransfected control. A549 cell viability 36 hours after transfection was significantly higher in the late circRNA fraction compared to all other fractions.

[0364] The induction of RIG-I and IFN-β1 transcripts upon transfection of A549 cells using late circRNA HPLC fraction, precursor RNA, or unpurified splicing reaction product was analyzed. Induction of both RIG-I and IFN-β1 transcripts was weaker with the late circRNA fraction than with precursor RNA or unpurified splicing reaction product. RNase R treatment of the splicing reaction product alone was insufficient to eliminate this effect. Adding a very small amount of RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-I transcription. In HeLa cells, transfection with RNase R-digested splicing reaction product induced RIG-I and IFN-β1, but not with purified circRNA. Overall, HeLa cells were less sensitive to contaminating RNA species than A549 cells.

[0365] Time-course experiments monitoring RIG-I, IFN-β1, IL-6, and RANTES transcript induction within the first 8 hours after transfection of A549 cells with splicing reaction products or fully purified circRNA did not reveal a transient response to circRNA. Similarly, purified circRNA failed to induce pro-inflammatory transcripts in RAW264.7 mouse macrophages.

[0366] A549 cells were transfected with purified circRNA containing EMCV IRES and EGFP expression sequences. This failed to produce substantial induction of pro-inflammatory transcripts. These data demonstrate that the acyclic component of the splicing reaction is responsible for the immunogenicity observed in previous studies, and that circRNA is not a native ligand for RIG-I.

[0367] Example 5 Circular RNAs evade detection by TLRs. TLR3, 7, and 8 reporter cell lines were transfected with multiple linear or circular RNA constructs, and secreted embryonic alkaline phosphatase (SEAP) was measured.

[0368] Linear RNA was constructed by deleting intron and homologous arm sequences. The linear RNA constructs were then treated with phosphatase (or capped if capped RNA was present) and purified by HPLC.

[0369] None of the transfections attempted produced a response in TLR7 reporter cells. TLR3 and TLR8 reporter cells were activated by capped linear RNA, polyadenylated linear RNA, nicked circRNA HPLC fraction, and early circRNA fraction. Late circRNA fraction and m1ψ-mRNA did not induce a TLR-mediated response in any cell line.

[0370] In the second experiment, circRNA was linearized using two methods: treatment of circRNA with heat in the presence of magnesium ions, and DNA oligonucleotide-induced RNase H digestion. Both methods yielded mostly full-length linear RNA and a small amount of intact circRNA. TLR3, 7, and 8 reporter cells were transfected with circular RNA, heat-degraded circular RNA, or RNase H-degraded circular RNA, and SEAP secretion was measured 36 hours after transfection. TLR8 reporter cells secreted SEAP in response to both forms of degraded circular RNA, but did not produce a greater response to circular RNA transfection than to mock transfection. Despite activation of TLR3 by linearized RNA transcribed in vitro, no activation was observed in TLR3 and TLR7 reporter cells under degradation or intact conditions.

[0371] Example 6 Unmodified circular RNA produces sustained in vivo protein expression that is increased compared to linear RNA. Unmodified and m1ψ-modified human erythropoietin (hEpo) linear mRNA and circRNA were injected into mice, and HEK293 cells were transfected. Equimolar transfection with m1ψ-mRNA and unmodified circRNA resulted in potent protein expression in HEK293 cells. hEpo linear mRNA and circRNA showed similar relative protein expression patterns and cell viability compared to GLuc linear mRNA and circRNA after isoweight transfection of HEK293 and A549 cells.

[0372] In mice, hEpo was detected in serum after injection of hEpo circRNA or linear mRNA into visceral fat. hEpo detected after injection of unmodified circRNA decayed more slowly than that from unmodified or m1ψ-mRNA, and was still present 42 hours after injection. Serum hEpo decreased rapidly upon injection of unpurified circRNA splicing reaction or unmodified linear mRNA. Injection of unpurified splicing reaction produced a detectable cytokine response in serum, which was not observed with other RNAs, including purified circRNA.

[0373] Example 7 Circular RNA can be effectively delivered in vivo or in vitro via lipid nanoparticles. Purified circular RNA was formulated into lipid nanoparticles (LNPs) using the ionizable lipidoid cKK-E12 (Dong et al., 2014; Kauffman et al., 2015). The particles formed a uniform multilamellar structure with an average size, polydispersity index, and encapsulation efficiency similar to particles containing commercially available control linear mRNA modified with 5 moU.

[0374] Purified hEpo circRNA, when encapsulated in LNPs and added to HEK293 cells, showed higher expression than 5moU-mRNA. Expression stability from LNP-RNA in HEK293 cells was similar to that of RNA delivered by transfection reagents, except for a slight delay in decay for both 5moU-mRNA and circRNA. Neither unmodified circRNA nor 5moU-mRNA could activate RIG-I / IFN-β1 in vitro.

[0375] In mice, LNP-RNA was delivered by local injection into visceral adipose tissue or intravenous delivery to the liver. In both cases, 6 hours after delivery, serum hEpo expression from circRNA was low but comparable to that from 5moU-mRNA. Serum hEpo detected after fat injection of unmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, and the delayed decay of expression present in serum was similar to that observed in vitro, while serum hEpo after intravenous injection of LNP-circRNA or LNP-5moU-mRNA decayed at approximately the same rate. In neither of these cases was there an increase in serum cytokines or local RIG-I, TNFα, or IL-6 transcript induction.

[0376] Example 8 Expression and functional stability of HEK293, HepG2, and 1C1C7 cells mediated by IRES. Constructs containing anabenaintron / exon regions, Gaussial ciferase expression sequences, and various IRESs were cyclized. 100 ng of each cyclized reaction product was separately transfected into 20,000 HEK293, HepG2, and 1C1C7 cells using Lipofectamine MessengerMax. Luminescence in each supernatant was evaluated after 24 hours as a measure of protein expression. In HEK293 cells, constructs containing black hivirus B, Sarivirus FHB, Aichi virus, Sarivirus HG-J1, and Enterovirus J IRESs produced the most luminescence after 24 hours (Figure 1A). In HepG2 cells, constructs containing Aichi virus, Sarivirus FHB, EMCV-Cf, and CVA3 IRESs produced high luminescence after 24 hours (Figure 1B). In 1C1C7 cells, constructs containing Salivirus FHB, Aichivirus, Salivirus NG-J1, and Salivirus A SZ-1 IRES produced high luminescence within 24 hours (Figure 1C).

[0377] Larger IRESs tended to produce greater luminescence over 24 hours. Since shorter total sequence lengths tended to increase cyclization efficiency, high expression and selection of relatively short IRESs may lead to improved constructs. In HEK293 cells, constructs using blackhivirus B IRESs produced the highest luminescence, particularly compared to other IRESs of similar length (Figure 2A). Expression from IRES constructs in HepG2 and 1C1C7 cells, plotted against IRES size, is shown in Figures 2B and 2C.

[0378] The functional stability of selected IRES constructs in HepG2 and 1C1C7 cells was measured over a 3-day period. Luminescence from secreted Gaussian alciferase in the supernatant was measured every 24 hours after transfecting 20,000 cells with 100 ng of each cyclization reaction, followed by complete medium replacement. Salivirus A GUT and Salivirus FHB showed the highest functional stability in HepG2 cells, while Salivirus N-J1 and Salivirus FHB produced the most stable expression in 1C1C7 cells (Figures 3A and 3B).

[0379] Example 9 IRES-mediated expression and functional stability in Jurkat cells. Two sets of constructs containing anabenaintron / exon regions, Gaussian alciferase expression sequences, and subsets of previously tested IRESs were cyclized. 60,000 Jurkat cells were electroporated with 1 μg of each cyclized reaction. 24 hours after electroporation, luminescence from secreted Gaussian alciferase in the supernatant was measured. CVB3 IRES constructs were included in both sets for comparison between sets and with previously defined IRES efficacy. CVB1 and Salivirus A SZ1 IRES constructs produced the highest expression at 24 hours. Data can be found in Figures 4A and 4B.

[0380] The functional stability of the IRES construct in electroporated Jurkat cells was measured over a 3-day period for each round. Luminescence from secreted Gaussian alciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 μg of each cyclization reaction, followed by complete replacement of the culture medium (Figures 5A and 5B).

[0381] The Salivirus A SZ1 and Salivirus A BN2 IRES constructs exhibited higher functional stability compared to other constructs.

[0382] Example 10 Expression, functional stability, and cytokine release of circular and linear RNAs in Jurkat cells. The construct containing the anabena intron / exon region, the Gaussian alciferase expression sequence, and the Salivirus FHB IRES was cyclized. mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified versions with 100% uridine replaced by 5-methoxyuridine (5 moU), were commercially available and purchased from Trilink. 5 moU nucleotide modification has been shown to improve mRNA stability and expression (Bioconjug Chem. 2016 Mar 16;27(3):849-53). Expression of modified mRNA in Jurkat cells, the cyclization reaction product (unpurified), and circRNA (purified) purified by size exclusion HPLC were measured and compared (Figure 6A). Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 60,000 cells with 1 μg of each RNA species.

[0383] Luminescence from secreted Gaussial ciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 ug of each RNA species, followed by complete replacement of the culture medium. A comparison of functional stability data of modified mRNA and circRNA in Jurkat cells over 3 days is shown in Figure 6B.

[0384] Transcript induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) was measured 18 hours after electroporation of 60,000 Jurkat cells with 1 μg of each of the above RNA species and 3p-hpRNA (5' triphosphate hairpin RNA known as a RIG-I agonist).

[0385] Example 11 Expression of circular and linear RNAs in monocytes and macrophages. The construct containing the anabenaintron / exon region, the Gaussian alciferase expression sequence, and the Salivirus FHB IRES was circularized. mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified mRNA with 100% uridine replaced by 5-methoxyuridine (5 moU), were purchased from Trilink. Expression of the circular and modified mRNAs was measured in human primary monocytes (Figure 8A) and human primary macrophages (Figure 8B). Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 60,000 cells with 1 μg of each RNA species. Luminescence was also measured 4 days after electroporation of human primary macrophages, with the medium changed every 24 hours (Figure 8C). Differences in luminescence were statistically significant in all cases (p<0.05).

[0386] Example 12 IRES-mediated expression and functional stability in primary T cells. Constructs containing anabenaintron / exon regions, Gaussial alciferase expression sequences, and subsets of previously tested IRESs were cyclized, and the reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 1 μg of each circRNA. Luminescence from secreted Gaussial alciferase in the supernatant was measured 24 hours after electroporation (Figure 9A). Aichi virus and CVB3 IRES constructs were most highly expressed at 24 hours.

[0387] Luminescence was also measured every 24 hours after electroporation for 3 days to compare the functional stability of each construct (Figure 9B). The construct with Salivirus A SZ1 IRES was the most stable.

[0388] Example 13 Expression and functional stability of circular and linear RNAs in primary T cells and PBMCs. The construct containing the anabena intron / exon region, the Gaussian alciferase expression sequence, and either the Salivirus A SZ1 IRES or the Salivirus FHB IRES was circularized. The mRNA containing the Gaussian alciferase expression sequence and approximately 150 nt poly(A) tail, as well as modified versions with 100% uridine replaced by 5-methoxyuridine (5 moU), were purchased from Trilink. Expression of HPLC-purified circular and modified mRNA of the Salivirus A SZ1 IRES was measured in human primary CD3+ T cells. Expression of HPLC-purified circular, unpurified circular, and modified mRNA of the Salivirus FHB was measured in human PBMCs. Luminescence from secreted Gaussian alciferase in the supernatant was measured 24 hours after electroporation of 150,000 cells with 1 μg of each RNA species. Data for primary human T cells are shown in Figures 10A and 10B, and data for PBMCs are shown in Figure 10C. Differences in expression between purified circular RNA and unpurified circular RNA or linear RNA were significant in all cases (p<0.05).

[0389] To compare the functional stability of the constructs, luminescence from secreted Gaussial ciferase in primary T cell supernatant was measured every 24 hours after electroporation for 3 days. The data are shown in Figure 10B. The difference in relative luminescence between purified circular RNA and linear RNA from the measurement on day 1 was significant for primary T cells on both day 2 and day 3.

[0390] Example 14 Cyclization efficiency by permutation sites in anabenaintrons. RNA constructs containing CVB3 IRES, Gaussial ciferase expression sequences, anabenaintron / exon regions, spacers, internal homologous regions, and homologous arms were produced. The cyclization efficiency of constructs using conventional anabenaintron permutation sites and five consecutive permutation sites at P9 was measured by HPLC. The HPLC chromatograms for the five consecutive permutation sites at P9 are shown in Figure 11A.

[0391] Circulation efficiency was measured at various permutation sites. Circulation efficiency is defined as the area under the HPLC chromatogram curve for each circRNA / (circRNA + precursor RNA). Ranked quantifications of circulation efficiency at each permutation site are shown in Figure 11B. Three permutation sites (shown in Figure 11B) were selected for further investigation.

[0392] In this example, the circular RNA was cyclized by in vitro transcription (IVT) and then purified via a spin column. The cyclization efficiency was determined for all constructs by Mg 2+ The efficiency could potentially be higher if an additional incubation step with guanosine nucleotides were included; however, the elimination of this step allowed for comparison between circular RNA constructs and optimization of the circular RNA constructs. This level of optimization is particularly useful for maintaining high cyclization efficiency in large RNA constructs, such as those encoding chimeric antigen receptors.

[0393] Example 15 Circularization efficiency of alternative introns. Precursor RNAs were constructed containing permutation group 1 introns or permutation sites of various species origins, as well as several constant elements including CVB3 IRES, Gaussial ciferase expression sequences, spacers, internal homologous regions, and homologous arms. Circulation data can be found in Figure 12. Figure 12A shows chromatograms of the precursor, CircRNA, and intron degradation. Figure 12B provides a ranked quantification of circulation efficiency based on the chromatograms shown in Figure 12A, as a function of the intron construct.

[0394] In this example, the circular RNA was cyclized by in vitro transcription (IVT) and then purified by spin column. The cyclization efficiency was determined for all constructs by Mg 2+The efficiency may be higher if an additional incubation step with guanosine nucleotides is included; however, the removal of this step allows for comparison between circular RNA constructs and optimization of circular RNA constructs. This level of optimization is particularly useful for maintaining high cyclization efficiency in large RNA constructs, such as those encoding chimeric antigen receptors.

[0395] Example 16 Annularization efficiency due to the presence or length of homologous arms. RNA constructs containing CVB3 IRES, Gaussial ciferase expression sequences, anabenaintron / exon regions, spacers, and internal homologous regions were produced. Constructs representing three anabenaintron permutation sites were tested with 30 nt, 25% GC homologous arms, or without homologous arms ("NA"). These constructs were Mg 2+ Circulation was possible without an incubation step. Circulation efficiency was measured and compared. The data can be found in Figure 13. Circulation efficiency was higher in each construct lacking homologous arms. Figure 13A provides a ranked quantification of circulation efficiency; Figure 13B provides chromatograms of precursor, circRNA, and intron degradation.

[0396] For each of the three permutation sites, constructs were prepared with arm lengths of 10 nt, 20 nt, and 30 nt, and GC content of 25%, 50%, and 75%. The splicing efficiency of these constructs was measured and compared with constructs without homologous arms (Figure 14). Splicing efficiency was defined as the ratio of free introns to total RNA in the splicing reaction product.

[0397] Figure 15A (left) shows an HPLC chromatogram illustrating the contribution of strong homologous arms to improved splicing efficiency. Top left: 75% GC content, 10nt homologous arms. Center left: 75% GC content, 20nt homologous arms. Bottom left: 75% GC content, 30nt homologous arms.

[0398] Figure 15A (right) shows an HPLC chromatogram illustrating increased splicing efficiency paired with increased nicking, appearing as a shoulder of the circRNA peak. Top right: 75% GC content, 10nt homologous arm. Center right: 75% GC content, 20nt homologous arm. Bottom right: 75% GC content, 30nt homologous arm.

[0399] Figure 15B (left) shows the selected combinations of permutational substitution sites and homologous arms that are assumed to demonstrate improved cyclicization efficiency.

[0400] Figure 15B (right) shows selected combinations of permutation substitution sites and homologous arms that are assumed to demonstrate improved cyclization efficiency when treated with E. coli polyA polymerase.

[0401] In this example, the circular RNA was cyclized by in vitro transcription (IVT) and then purified by spin column. The cyclization efficiency was determined for all constructs by the addition of Mg with guanosine nucleotides. 2+ Including the incubation step could potentially increase the cost; however, removing this step allowed for comparison between circular RNA constructs and optimization of the circular RNA constructs. This level of optimization is particularly useful for maintaining high cyclization efficiency in large RNA constructs, such as those encoding chimeric antigen receptors.

[0402] Example 17 Circular RNA encoding CAR The construct, containing the anabena intron / exon region, Kymriah chimeric antigen receptor (CAR) expression sequence, and CVB3 IRES, was circularized. 100,000 human primary CD3+ T cells were electroporated with 500 ng of circRNA and co-cultured for 24 hours with Raji cells stably expressing GFP and firefly luciferase. The effector-to-target ratio (E:T ratio) was 0.75:1. 100,000 human primary CD3+ T cells were mock-electroporated and co-cultured as a control (Figure 16).

[0403] A set of 100,000 human primary CD3+ T cells was electroporated using mock electroporation or electroporation with 1 μg of circRNA, and then co-cultured for 48 hours with Raji cells that stably express GFP and firefly luciferase. The E:T ratio was 10:1 (Figure 17).

[0404] Quantitative lysis of Raji target cells was determined by detection of firefly bioluminescence (Figure 18). 100,000 human primary CD3+ T cells, either mock-electroporated or electroporated with circRNA encoding different CAR sequences, were co-cultured for 48 hours with Raji cells stably expressing GFP and firefly luciferase. Specific lysis rate (%) was defined as 1-[CAR-conditioned bioluminescence] / [mock-conditioned bioluminescence]. E:T ratio 10:1.

[0405] Example 18 Expression and functional stability of circular and linear RNAs in Jurkat cells and resting human T cells. Constructs containing anabenaintron / exon regions, gaussial ciferase expression sequences, and a subset of previously tested IRESs were cyclized, and the reaction products were purified by size exclusion HPLC. 150,000 Jurkat cells were electroporated with 1 μg of circular RNA or 5 moU-mRNA. Luminescence from secreted gaussial ciferase in the supernatant was measured 24 hours after electroporation (Figure 19A left). 150,000 resting primary human CD3+ T cells (10 days post-stimulation) were electroporated with 1 μg of circular RNA or 5 moU-mRNA. Luminescence from secreted gaussial ciferase in the supernatant was measured 24 hours after electroporation (Figure 19A right).

[0406] The luminescence from secreted Gaussial ciferase in the supernatant was measured every 24 hours after electroporation, after which the culture medium was completely replaced. Functional stability data are shown in Figure 19B. In all cases, circular RNA showed higher functional stability than linear RNA, with a more pronounced difference observed in Jurkat cells.

[0407] Example 19 Induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and TNFα transcripts in cells electroporated with linear RNA or various circular RNA constructs. Constructs containing anabena intron / exon regions, Gaussial ciferase expression sequences, and a subset of previously tested...

Claims

1. a. a circular RNA polynucleotide comprising, in the following order: a 3' Group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR) complex protein, and a 5' Group I intron fragment; and b. A delivery vehicle comprising at least one of: (i) an ionizable lipid; (ii) a structural lipid; and (iii) a PEG-modified lipid.

1. A pharmaceutical composition comprising: The pharmaceutical composition, wherein the transport vehicle is capable of delivering the circular RNA polynucleotide to human immune cells present in a human subject, such that the CAR is translated in the human immune cells and expressed on the surface of the human immune cells.

2. 10. The pharmaceutical composition of claim 1, formulated for intravenous administration to said human subject in need thereof.

3. 3. The pharmaceutical composition of claim 1, wherein the 3' Group I intron fragment and the 5' Group I intron fragment are Anabaena Group I intron fragments.

4. 4. The pharmaceutical composition of claim 3, wherein the 3' intron fragment and the 5' intron fragment are defined by an L9a-5 permutation site within an intact intron.

5. 4. The pharmaceutical composition of claim 3, wherein the 3' intron fragment and the 5' intron fragment are defined by an L8-2 permutation site within an intact intron.

6. The IRES is capable of binding to Taura syndrome virus, triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, fire ant (Solenopsis invicta) virus 1, wheat aphid (Rhopalosiphum padi) virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stali enteric virus, Kashmir wasp virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, Ectropis spp. obliqua) picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, crucifer tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ringspot 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-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n. myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCVQC64, human cosavirus E / D, human cosavirus F, human cosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, sarivirus A SH1, sarivirus FHB, sarivirus NG-J1, human parechovirus 1, Kurohivirus B, Yc-3, rosavirus M-7, shambavirus A, pacivirus A, pacivirus A 2, echovirus E14, human parechovirus 5, Aichi virus, 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 6. The pharmaceutical composition of claim 1, wherein the aptamer is derived from an aptamer against 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Swine Pathogenic Virus 1, PLV-CHN, Pacivirus A, Sisinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei 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.

7. 7. The pharmaceutical composition of claim 6, wherein the IRES comprises a CVB3 IRES or a fragment or variant thereof, or wherein the IRES comprises a sequence according to SEQ ID NO:

65.

8. The pharmaceutical composition of claim 6 , wherein the IRES comprises the salivirus SZ1 IRES or a fragment or mutant thereof.

9. The pharmaceutical composition of claim 8 , wherein the IRES comprises a sequence according to SEQ ID NO:

63.

10. a first internal spacer between the 3' Group I intron fragment and the IRES; and A second internal spacer is inserted between the expression sequence and the 5' Group I intron fragment. The pharmaceutical composition according to any one of claims 1 to 9, comprising

11. 11. The pharmaceutical composition of claim 10, wherein the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

12. The CAR or TCR complex protein is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disialoganglioside GD3, TNF receptor family members, B-cell maturation antigen (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, mesothelin, Interleukin-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 associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (N CAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrate) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine leukemia viral oncogene 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),The pharmaceutical composition of any one of claims 1 to 11, comprising an antigen-binding domain specific for an antigen selected from the group consisting of tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

13. The pharmaceutical composition of claim 12, wherein the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the CAR or TCR complex protein comprises a CAR comprising a costimulatory domain selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, MYD88, CD2, SLAM, and a combination thereof.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the CAR or TCR complex protein comprises a CAR comprising a CD3 zeta signaling domain.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

18. the CAR or TCR complex protein a. an antigen-binding domain; b. a spacer domain; c. a transmembrane domain; d. a costimulatory domain, and e. Intracellular T cell signaling domain The pharmaceutical composition according to any one of claims 1 to 17, comprising a CAR comprising:

19. The pharmaceutical composition of any one of claims 1 to 18, wherein the CAR or TCR complex protein comprises a multispecific CAR comprising antigen-binding domains for at least two different antigens.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein the CAR or TCR complex protein comprises a TCR complex protein selected from the group of TCR alpha, TCR beta, TCR gamma, and TCR delta.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the delivery vehicle comprises a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, or a biodegradable polymer nanoparticle.

22. The pharmaceutical composition of any one of claims 1 to 21, further comprising a targeting moiety.

23. 23. The pharmaceutical composition of claim 22, wherein the targeting moiety mediates receptor-mediated endocytosis or direct fusion into selected cells of a selected cell population or tissue in the absence of cell isolation or purification.

24. 23. The pharmaceutical composition of claim 22, wherein the targeting moiety is capable of binding to a protein selected from the group of CD3, CD4, CD8, CD5, CD7, PD-1, 4-1BB, CD28, C1q, and CD2.

25. 23. The pharmaceutical composition of claim 22, wherein the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen.

26. 26. The pharmaceutical composition of any one of claims 22-25, wherein the targeting moiety comprises 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.

27. 27. The pharmaceutical composition of any one of claims 1 to 26, in an amount effective to treat cancer in said human subject.

28. The pharmaceutical composition according to any one of claims 1 to 27, which has an improved safety profile when compared to a pharmaceutical composition comprising T cells or a vector comprising exogenous DNA encoding the same CAR.

29. 29. The pharmaceutical composition of any one of claims 1 to 28, wherein less than 1% by weight of the polynucleotides in the composition are double-stranded RNA, DNA splints, or triphosphorylated RNA.

30. 30. The pharmaceutical composition of any of claims 1 to 29, wherein less than 1% by weight of the polynucleotides and proteins in the pharmaceutical composition are double-stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes.

31. 31. The pharmaceutical composition of any one of claims 1 to 30, wherein the delivery vehicle comprises more than one circular RNA polynucleotide.

32. 1. A circular RNA polynucleotide comprising, in the following order: a 3′ Group I intron fragment; an internal ribosome entry site (IRES); an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein; and a 5′ Group I intron fragment.

33. 33. The circular RNA polynucleotide of claim 32, wherein the 3' Group I intron fragment and the 5' Group I intron fragment are Anabaena Group I intron fragments.

34. 34. The circular RNA polynucleotide of claim 33, wherein the 3' and 5' intron fragments are defined by L9a-5 permutation sites within an intact intron.

35. 34. The circular RNA polynucleotide of claim 33, wherein the 3' and 5' intron fragments are defined by L8-2 permutation sites within an intact intron.

36. 36. The circular RNA polynucleotide of any one of claims 32 to 35, wherein the IRES comprises a CVB3 IRES or a fragment or variant thereof.

37. 37. The circular RNA polynucleotide of claim 36, wherein the IRES has a sequence according to SEQ ID NO:

65.

38. 33. The circular RNA polynucleotide of claim 32, wherein the IRES comprises the salivirus SZ1 IRES or a fragment or variant thereof.

39. 39. The circular RNA polynucleotide of claim 38, wherein the IRES has a sequence according to SEQ ID NO:

63.

40. a first internal spacer between the 3' Group I intron fragment and the IRES; and A second internal spacer is inserted between the expression sequence and the 5' Group I intron fragment.

40. The circular RNA polynucleotide of any one of claims 32 to 39, comprising:

41. 41. The circular RNA polynucleotide of claim 40, wherein the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

42. 42. A circular RNA polynucleotide according to any one of claims 32 to 41, which consists of natural nucleotides.

43. 43. The circular RNA polynucleotide of any one of claims 32 to 42, further comprising a second expression sequence encoding a therapeutic protein.

44. 44. The circular RNA polynucleotide of claim 43, wherein the therapeutic protein comprises a checkpoint inhibitor.

45. 44. The circular RNA polynucleotide of claim 43, wherein the therapeutic protein comprises a cytokine.

46. The CAR or TCR complex protein is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family members, B-cell maturation antigen (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, mesothelin, Interleukin-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 associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (N CAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrate) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine leukemia viral oncogene 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),46. ​​The circular RNA polynucleotide of any one of claims 32 to 45, comprising an antigen-binding domain specific for an antigen selected from the group consisting of tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

47. The circular RNA polynucleotide of any one of claims 32 to 46, wherein the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19.

48. 48. The circular RNA polynucleotide of any one of claims 32 to 47, wherein the CAR or TCR complex protein comprises a CAR comprising a costimulatory domain selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof.

49. 49. The circular RNA polynucleotide of any one of claims 32 to 48, wherein the CAR or TCR complex protein comprises a CAR comprising a CD3 zeta signaling domain.

50. 50. The circular RNA polynucleotide of any one of claims 32 to 49, wherein the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain.

51. 51. The circular RNA polynucleotide of any one of claims 32 to 50, wherein the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

52. the CAR or TCR complex protein a. an antigen-binding domain; b. a spacer domain; c. a transmembrane domain; d. a costimulatory domain, and e. Intracellular T cell signaling domain The circular RNA polynucleotide of any one of claims 32 to 51, comprising a CAR comprising:

53. The circular RNA polynucleotide of any one of claims 32 to 52, wherein the CAR or TCR complex protein comprises a multispecific CAR comprising antigen-binding domains for at least two different antigens.

54. 54. The circular RNA polynucleotide of any one of claims 32 to 53, wherein the CAR or TCR complex protein comprises a TCR complex protein selected from the group of TCR alpha, TCR beta, TCR gamma, and TCR delta.

55. A circular RNA polynucleotide according to any one of claims 32 to 54, which consists of naturally occurring nucleotides.

56. 33. The circular RNA polynucleotide of claim 32, wherein the expression sequence is codon-optimized.

57. 57. A circular RNA polynucleotide according to any one of claims 32 to 56, which is optimized to lack at least one microRNA binding site present in a comparable pre-optimized polynucleotide.

58. 58. A circular RNA polynucleotide according to any one of claims 32 to 57, which is optimised to lack at least one endonuclease-sensitive site present in an equivalent pre-optimised polynucleotide.

59. 59. The circular RNA polynucleotide of any one of claims 32 to 58, which is optimized to lack at least one RNA editing sensitive site present in a comparable pre-optimized polynucleotide.

60. 60. The circular RNA polynucleotide of any one of claims 32 to 59, which has an in vivo functional half-life in humans that is longer than the functional half-life of a comparable linear RNA polynucleotide having the same expression sequence.

61. 61. A circular RNA polynucleotide according to any one of claims 32 to 60, having a length of from about 100 nucleotides to about 10 kilobases.

62. 62. A circular RNA polynucleotide according to any one of claims 32 to 61, having a functional half-life of at least about 20 hours.

63. 63. The circular RNA polynucleotide of any one of claims 32 to 62, having a duration of therapeutic effect in human cells of at least about 20 hours.

64. 64. A circular RNA polynucleotide according to any one of claims 32 to 63, having a duration of therapeutic effect in human cells that is equal to or greater than the duration of therapeutic effect of an equivalent linear RNA polynucleotide comprising the same expression sequence.

65. 65. A circular RNA polynucleotide according to any one of claims 32 to 64, having a functional half-life in human cells that is equal to or greater than the functional half-life of an equivalent linear RNA polynucleotide containing the same expression sequence.

66. 1. A DNA vector comprising, in the following order: a 5′ duplex-forming region; an Anabaena 3′ group I intron fragment having a first permutation site; an internal ribosome entry site (IRES); an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide; an Anabaena 5′ group I intron fragment having a second permutation site; and a 3′ duplex-forming region.

67. 67. The DNA vector of claim 66, wherein the 3' Group I intron fragment and the 5' Group I intron fragment are Anabaena Group I intron fragments.

68. 68. The DNA vector of claim 67, wherein the 3' and 5' intron fragments are defined by L9a-5 permutation sites within an intact intron.

69. 68. The DNA vector of claim 67, wherein the 3' and 5' intron fragments are defined by L8-2 permutation sites within an intact intron.

70. 70. The DNA vector of any one of claims 66 to 69, wherein the IRES comprises a CVB3 IRES or a fragment or mutant thereof.

71. 71. The DNA vector of claim 70, wherein the IRES encodes a sequence according to SEQ ID NO:

65.

72. 70. The DNA vector of any one of claims 66 to 69, wherein the IRES comprises the salivirus SZ1 IRES or a fragment or mutant thereof.

73. 73. The DNA vector of claim 72, wherein the IRES encodes a sequence according to SEQ ID NO:

63.

74. 74. The DNA vector of any one of claims 66 to 73, wherein the circular RNA polynucleotide comprises, in the following order: a 5' duplex-forming region, a 3' Group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein, a 5' Group I intron fragment, and a 3' duplex-forming region.

75. 75. The DNA vector of claim 74, wherein the 5' duplex-forming region and the 3' duplex-forming region each have about 70% GC nucleotides.

76. 76. The DNA vector of claim 74 or claim 75, wherein the 5' duplex-forming region and the 3' duplex-forming region each have a length of about 30 nucleotides.

77. a first external spacer between the 5' duplex forming region and the 3' Group I intron fragment; and A second external spacer is inserted between the 5' Group I intron fragment and the 3' duplex forming region. The DNA vector according to any one of claims 74 to 76, comprising

78. 78. The DNA vector of claim 77, wherein the first and second external spacers each have a length of about 10 to about 60 nucleotides.

79. 79. The DNA vector of any one of claims 74 to 78, wherein the 5' duplex-forming region is immediately adjacent to a 3' Group I intron fragment, and the 5' Group I intron fragment is immediately adjacent to a 3' duplex-forming region.

80. a first internal spacer between the 3' Group I intron fragment and the IRES; and A second internal spacer is inserted between the expression sequence and the 5' Group I intron fragment. The DNA vector according to any one of claims 66 to 79, comprising

81. 81. The DNA vector of claim 80, wherein the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

82. The CAR or TCR complex protein is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family members, B-cell maturation antigen (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, mesothelin, Interleukin-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 associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (N CAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrate) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine leukemia viral oncogene 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),The DNA vector according to any one of claims 66 to 81, comprising an antigen-binding domain specific for an antigen selected from the group consisting of tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

83. The DNA vector of any one of claims 66 to 82, wherein the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19.

84. The DNA vector of any one of claims 66 to 83, wherein the CAR or TCR complex protein comprises a CAR comprising a costimulatory domain selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and a combination thereof.

85. The DNA vector of any one of claims 66 to 84, wherein the CAR or TCR complex protein comprises a CAR comprising a CD3 zeta signaling domain.

86. The DNA vector of any one of claims 66 to 85, wherein the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain.

87. The DNA vector of any one of claims 66 to 86, wherein the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

88. the CAR or TCR complex protein a. an antigen-binding domain; b. a spacer domain; c. a transmembrane domain; d. a costimulatory domain, and e. Intracellular T cell signaling domain The DNA vector according to any one of claims 66 to 87, comprising a CAR comprising:

89. The DNA vector of any one of claims 66 to 88, wherein the CAR or TCR complex protein comprises a multispecific CAR comprising antigen-binding domains for at least two different antigens.

90. 90. The DNA vector of any one of claims 66 to 89, wherein the CAR or TCR complex protein comprises a TCR complex protein selected from the group of TCR alpha, TCR beta, TCR gamma, and TCR delta.

91. 1. A circular RNA polynucleotide comprising, in the following order: a 3′ Group I intron fragment; an internal ribosome entry site (IRES); an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein; and a 5′ Group I intron fragment. Eukaryotic cells, including:

92. 92. The eukaryotic cell of claim 91, comprising a human cell.

93. 93. A eukaryotic cell according to claim 91 or 92, comprising an immune cell.

94. 94. The eukaryotic cell of any one of claims 91 to 93, comprising a T cell.

95. 1. A circular RNA polynucleotide comprising, in the following order: a 3′ Group I intron fragment; an internal ribosome entry site (IRES); an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein; and a 5′ Group I intron fragment. A population of eukaryotic cells comprising: A population of said eukaryotic cells which express on their cell surface said CAR or TCR complex protein encoded by said circular RNA polynucleotide.

96. 96. The population of eukaryotic cells of claim 95, wherein the population of cells comprises NK cells, NKT cells, macrophages, dendritic cells, alpha beta T cells, gamma delta T cells, or a combination thereof.

97. 97. The population of eukaryotic cells of claim 95 or 96, wherein the population of cells comprises T cells.

98. 98. The population of eukaryotic cells of claim 97, wherein the population comprises CD3+ T cells.

99. 98. The population of eukaryotic cells of claim 97, wherein the population comprises CD4+ T cells.

100. 98. The population of eukaryotic cells of claim 97, wherein the population comprises CD8+ T cells.

101. 101. The population of eukaryotic cells of any one of claims 95 to 100, in an amount effective to treat cancer in a human subject in need thereof.

102. 102. The population of eukaryotic cells of any one of claims 95-101, wherein the population of cells kills tumor cells more effectively or for longer than a comparable population of eukaryotic cells comprising a linear RNA encoding the same CAR.

103. 1. A method for producing a population of eukaryotic cells, comprising: contacting cells in said population with a transfer vehicle comprising a circular RNA polynucleotide comprising, in the following order: a 3′ Group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein, and a 5′ Group I intron fragment; the transport vehicle comprises (i) an ionizable lipid, (ii) a structured lipid, and (iii) a PEG-modified lipid; the transport vehicle is capable of delivering the circular RNA polynucleotide to a human immune cell, such that the CAR is translated in the human immune cell and expressed on the surface of the human immune cell. The method.

104. 1. A method of treating a subject in need thereof, comprising: a. a circular RNA polynucleotide comprising, in the following order: a 3′ Group I intron fragment, an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein, and a 5′ Group I intron fragment; and b. A delivery vehicle comprising (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid. administering a therapeutically effective amount of a pharmaceutical composition comprising the transport vehicle is capable of delivering the circular RNA polynucleotide to a human immune cell, such that the CAR is translated in the human immune cell and expressed on the surface of the human immune cell. The method.

105. The subject is diagnosed with acute lymphocytic cancer; acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; 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; cancer of the intrahepatic bile duct; cancer of the joints; cancer of the neck, gallbladder, or pleura; cancer of the nose, nasal cavity, or middle ear; cancer of the oral cavity; cancer of the vulva; chronic lymphocytic leukemia; chronic myeloid carcinoma; colon cancer; esophageal cancer; cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumor; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin's lymphoma; hypopharyngeal cancer; kidney cancer; laryngeal cancer; leukemia; liquid tumors; 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's lymphoma; B-chronic lymphocytic leukemia; hairy cell leukemia; acute lymphocytic leukemia (ALL); and Burkitt's lymphoma; ovarian cancer; Pancreatic cancer; peritoneal, omental, and mesenteric cancer; pharyngeal cancer; prostate cancer; rectal cancer; kidney cancer; skin cancer; 105. The method of claim 104, wherein the patient has a cancer selected from the group consisting of small intestine cancer; soft tissue cancer; solid tumor; synovial sarcoma; gastric cancer; testicular cancer; thyroid cancer; and urinary tract cancer.

106. 1. An RNA polynucleotide comprising an internal ribosome entry site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) polypeptide, and at least one self-circularization element.

107. 107. The RNA polynucleotide of claim 106, comprising a 5' duplex forming region, an Anabaena 3' Group I intron fragment having a first permutation site, an internal ribosome entry site (IRES), an expressed sequence encoding a chimeric antigen receptor (CAR) polypeptide, an Anabaena 5' Group I intron fragment having a second permutation site, and a 3' duplex forming region.

108. 108. The RNA polynucleotide of Claim 106 or 107, comprising a 5' duplex forming region, a first permutation site, an internal ribosome entry site (IRES), an expressed sequence encoding a chimeric antigen receptor (CAR) polypeptide, a second permutation site, and a 3' duplex forming region.

109. 109. The RNA polynucleotide of any one of claims 106 to 108, wherein the self-circularization element is a group I intron fragment.

110. 110. The RNA polynucleotide of claim 108 or 109, comprising a 3' Group I intron fragment and a 5' intron fragment.

111. 111. The RNA polynucleotide of claim 110, wherein said 3' Group I intron fragment and said 5' Group I intron fragment are Anabaena Group I intron fragments.

112. 112. The RNA polynucleotide of claim 111, wherein the 3' and 5' intron fragments are defined by L9a-5 permutation sites within an intact intron.

113. 112. The RNA polynucleotide of claim 111, wherein the 3' and 5' intron fragments are defined by L8-2 permutation sites within an intact intron.

114. 114. The RNA polynucleotide of any one of claims 110 to 113, which is capable of circularization in the absence of an enzyme.

115. 115. The RNA polynucleotide of any one of claims 106 to 114, which consists of naturally occurring nucleotides.

116. A DNA vector suitable for synthesizing an RNA polynucleotide according to any one of claims 106 to 115.

117. 66. The circular RNA polynucleotide of any one of claims 32 to 65, wherein the circular RNA polynucleotide is delivered to a target cell in a non-lipid polymer core-shell nanoparticle.

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