Circular RNA compositions and methods

Abstract

To provide pharmaceutical compositions capable of delivering a circular RNA polynucleotide to human immune cells.SOLUTION: The disclosed pharmaceutical composition comprises: a. a circular RNA polynucleotide comprising 3' group I intron fragment, an internal ribosome entering site (IRES), an expression sequence encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex proteins, and 5' group I intron fragment; and b. a transport vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG modified lipids, where the transport vehicle can transport the circular RNA polynucleotide to human immune cells present in a human subject so that the CAR is translated in the human immune cells and expressed on the surface of the human immune cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 851,548, filed May 22, 2019; U.S. Provisional Patent Application No. 62 / 857,121, filed June 4, 2019; International Patent Application No. PCT / US2019 / 035531, filed June 5, 2019; U.S. Provisional Patent Application No. 62 / 943,796, filed December 4, 2019; U.S. Provisional Patent Application No. 62 / 943,779, filed December 4, 2019; and U.S. Provisional Patent Application No. 62 / 972,194, filed February 10, 2020, the disclosures of which are incorporated herein by reference in their entireties for all purposes. [Background technology]

[0002] background Traditional gene therapy involves the use of DNA to insert desired genetic information into host cells. DNA introduced into cells typically integrates to some degree into the genome of one or more transfected cells, allowing for long-term function of the introduced genetic material within the host. While such long-lasting function can have substantial benefits, integration of exogenous DNA into the host genome can also have many adverse effects. For example, the introduced DNA may insert into an intact gene, resulting in a mutation that interferes with or even completely eliminates the function of the endogenous gene. Thus, DNA-based gene therapy can result in fatal impairment of gene function in the treated host, such as the elimination or harmful reduction of production of essential enzymes or the interference with genes critical for cell growth control, leading to uncontrolled or cancerous cell proliferation. In addition, traditional DNA-based gene therapy requires the inclusion of strong promoter sequences for effective expression of the desired gene product, which can also result in undesirable changes in the control of normal gene expression within the cell. DNA-based genetic material can also induce unwanted anti-DNA antibodies, which can trigger potentially fatal immune responses. Gene therapy approaches using viral vectors can also lead to adverse immune responses. In some circumstances, 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 introduced genetic 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 can be limited for these various reasons.

[0003] In contrast to DNA, the use of RNA as a gene therapy agent is substantially safer, since there is no risk of RNA being stably integrated into the genome of the transfected cell, thus eliminating the concern that the introduced genetic material will disrupt the normal function of essential genes or cause mutations that result in harmful or oncogenic effects; and exogenous promoter sequences are not required for the effective translation of the encoded protein, again avoiding possible 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 stable forms of RNA. Circularization of RNA molecules provides advantages for studying RNA structure and function, especially for molecules that tend to fold into inactive conformations (Wang and Ruffner, 1998). Circular RNA is also particularly interesting and may be useful for in vivo applications, especially in the research field of RNA-based control of gene expression and therapeutic agents, including protein replacement therapy and vaccination.

[0005] The use of T cells genetically modified to express chimeric antigen receptors (CARs) and recombinant T cell receptors (TCRs) that target antigens on cancer cells is an attractive therapeutic strategy for the treatment of cancer. However, current methods of modifying T cells to express CARs and TCRs, and the resulting therapies, are associated with toxicity in the form of cytokine release syndrome (CRS) and other complications. Safer methods of engineering cells to express CARs and recombinant TCRs remain needed.

[0006] Prior to the present invention, there were three major techniques for generating circularized RNA in vitro: splint-mediated, permuted intron-exon, and RNA ligase-mediated. However, existing methodologies are limited by the size of the RNA that can be circularized, thereby limiting their therapeutic applications. Summary of the Invention

[0007] overview

[0010] In one aspect, provided herein is a pharmaceutical composition 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 T cell receptor (TCR) complex protein, and a 5' group I intron fragment; and a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structured lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a human immune cell present in a human subject, such that the CAR is translated in the human immune cell and expressed on the surface of the human immune cell.

[0008] In some embodiments, the pharmaceutical composition is formulated for intravenous administration to a human subject in need thereof. 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 site of the intact intron. In certain embodiments, the 3' and 5' intron fragments are defined by the L8-2 permutation site of the intact intron.

[0010] In some embodiments, the IRES is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, 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, Little 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, 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, dog 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, HCV QC64, 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, pasivirus A, pasivirus 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, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus 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 an aptamer against eIF4G.

[0011] In some embodiments, the IRES comprises the 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 the sarivirus 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 protein is selected from the group: 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 mutation (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrant) 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 antibody comprises an antigen-binding domain specific for an antigen selected from 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.

[0013] In some embodiments, the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a costimulatory 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 comprises a CAR comprising a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

[0014] In some embodiments, the CAR or TCR complex protein comprises 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 comprising 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 group TCR alpha, TCR beta, TCR gamma, and TCR delta. In some embodiments, the delivery vehicle comprises a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymeric nanoparticle, or a biodegradable polymeric nanoparticle.

[0016] In some embodiments, the pharmaceutical composition further comprises a targeting moiety. In certain embodiments, the targeting moiety mediates receptor-mediated endocytosis or direct fusion to selected cells in a selected cell population or tissue without cell isolation or purification. In certain embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CD5, CD7, PD-1, 4-1BB, CD28, C1q, and CD2. In certain embodiments, the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen. In certain embodiments, the targeting moiety comprises an scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region, or fragments thereof.

[0017] In some embodiments, the pharmaceutical composition is administered in an amount effective to treat cancer in a human subject. In some embodiments, the pharmaceutical composition has an improved safety profile when compared to a pharmaceutical composition comprising a T cell or vector containing exogenous DNA encoding the same CAR.

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

[0019] In some embodiments, 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. In some embodiments, the delivery vehicle comprises more than one circular RNA polynucleotide.

[0020] In another aspect, the disclosure provides a circular RNA polynucleotide comprising, in the following order: a 3' Group I intron fragment, an internal ribosome entry site (IRES), an expressible 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 the intact intron. In certain embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 permutation site of the intact intron. In certain embodiments, the IRES comprises the 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 comprises the 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 comprises a first internal spacer between the 3' Group I intron fragment and the IRES, and a second internal spacer between the expressed sequence and the 5' Group I intron fragment.

[0023] In certain embodiments, 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 is composed of natural 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 protein is selected from the group: 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, iPSC, IL-16, IL-16-26, IL-16-28, IL-16-29 ... 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 mutation (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrant) 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 antibody comprises an antigen-binding domain specific for an antigen selected from 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.

[0026] In some embodiments, the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a costimulatory 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 comprises a CAR comprising a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

[0027] In some embodiments, the CAR or TCR complex protein comprises 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 comprising antigen-binding domains for at least two different antigens, hi some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the group TCR alpha, TCR beta, TCR gamma, and TCR delta.

[0029] In some embodiments, the circular RNA polynucleotide consists of naturally occurring 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 that is present in the equivalent pre-optimized polynucleotide.In some embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site that is present in the equivalent pre-optimized polynucleotide.In some embodiments, the circular RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site that is present in the 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 the functional half-life of an equivalent linear RNA polynucleotide comprising 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 equal to or longer than the duration of therapeutic effect of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the circular RNA polynucleotide has a functional half-life in human cells that is equal to or longer than the functional half-life of an equivalent linear RNA polynucleotide comprising the same expression sequence.

[0032] In another aspect, the disclosure provides a DNA vector comprising, in the following order: a 5' duplex forming region, an Anabaena 3' Group I intron fragment and 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 and a second permutation site, and a 3' duplex forming 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 an L9a-5 permutation site in the intact intron. In some embodiments, the 3' intron fragment and the 5' intron fragment are defined by an L8-2 permutation site in the intact intron. In some embodiments, the IRES comprises 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 the 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' duplex forming region, a 3' group I intron fragment, an internal ribosome entry site (IRES), an expressed sequence encoding a chimeric antigen receptor (CAR) or a TCR complex protein, a 5' group I intron fragment, and a 3' duplex forming region. In some embodiments, the 5' duplex forming region and the 3' duplex forming region each have about 70% GC nucleotides. In some embodiments, the 5' duplex forming region and the 3' duplex forming region each have a length of about 30 nucleotides.

[0035] In some embodiments, the DNA vector comprises a first external spacer between the 5' duplex-forming region and the 3' Group I intron fragment, and a second external spacer between the 5' Group I intron fragment and the 3' duplex-forming region. In some embodiments, the first and second external spacers each have a length of about 10 to about 60 nucleotides. In some embodiments, the 5' duplex-forming region is directly adjacent to the 3' Group I intron fragment, and the 5' Group I intron fragment is directly adjacent to the 3' duplex-forming region. In some embodiments, the DNA vector 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 some embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

[0036] In some embodiments, the CAR or TCR complex protein is selected from the group: 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, iPSC, IL-16, IL-16-26, IL-16-28, IL-16-29 ... 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 mutation (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrant) 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 antibody comprises an antigen-binding domain specific for an antigen selected from 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.

[0037] In some embodiments, the CAR or TCR complex protein comprises a CAR comprising an antigen-binding domain specific for CD19. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a costimulatory 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 comprises a CAR comprising a CD3 zeta signaling domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

[0038] In some embodiments, the CAR or TCR complex protein comprises 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 comprising antigen-binding domains for at least two different antigens, hi some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the group TCR alpha, TCR beta, TCR gamma, and TCR delta.

[0040] In another aspect, the disclosure provides a eukaryotic cell 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. In some embodiments, the eukaryotic cell comprises a human cell. In some embodiments, the eukaryotic cell comprises an immune cell. In some embodiments, the eukaryotic cell comprises a T cell.

[0041] In another aspect, the disclosure provides a population of eukaryotic cells 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 TCR complex protein, and a 5' Group I intron fragment, wherein the population of eukaryotic cells express the CAR or TCR complex protein encoded by the circular RNA polynucleotide on their cell surface.

[0042] In some embodiments, the population of cells comprises NK cells, NKT cells, macrophages, dendritic cells, alpha beta T cells, gamma delta T cells, or a combination thereof. In some embodiments, the population of cells comprises T cells. In some embodiments, the population comprises CD3+ T cells. In some embodiments, the population comprises CD4+ T cells. In some embodiments, the population comprises CD8+ T cells. In some embodiments, the population of eukaryotic cells is administered in an amount effective to treat cancer in a human subject in need thereof. In some embodiments, 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.

[0043] In another aspect, provided herein is a method of producing a population of eukaryotic cells, the method comprising contacting cells in the 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, wherein the transfer vehicle comprises: (i) an ionizable lipid, (ii) a structured lipid, and (iii) a PEG-modified lipid, wherein the transfer 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.

[0044] In another aspect, provided herein is a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising, in the following order: 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 a TCR complex protein, and a 5' group I intron fragment; and a transfer vehicle comprising (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer 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.

[0045] In some embodiments, 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, The patient has a cancer selected from: ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0046] In another aspect, provided herein is 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.

[0047] In some embodiments, the RNA polynucleotide comprises a 5' duplex-forming region, an Anabaena 3' Group I intron fragment and 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 and a second permutation site, and a 3' duplex-forming region. In some embodiments, the RNA polynucleotide comprises 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. In some embodiments, the self-circularization element is a Group I intron fragment. In some embodiments, the 3' Group I intron fragment and a 5' intron fragment. 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 the intact intron. In some embodiments, the 3' intron fragment and the 5' intron fragment are defined by the L8-2 permutation site of the intact intron. In some embodiments, the RNA polynucleotide is capable of circularization in the absence of an enzyme. In some embodiments, the RNA polynucleotide is composed of naturally occurring nucleotides.

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

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

[0050] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0051] [Figure 1A] Luminescence in the supernatant of HEK293 (A), HepG2 (B), or 1C1C7 (C) cells 24 hours after transfection with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences is shown. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] Luminescence in the supernatants of HEK293 (A), HepG2 (B), and 1C1C7 (C) cells 24 hours after transfection with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences of different lengths is shown. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3] 3A shows the stability of selected IRES constructs in HepG2 (3A) or 1C1C7 (3B) cells over a 3-day period, as measured by luminescence. [Figure 4A] A and B show protein expression from selected IRES constructs in Jurkat cells as measured by luminescence from secreted Gaussia luciferase in the cell supernatant. [Figure 4B] See legend to Figure 4A. [Figure 5] A and B show the stability of selected IRES constructs in Jurkat cells over a 3 day period as measured by luminescence. [Figure 6A]Comparison of 24-hour luminescence (A) or relative luminescence over 3 days (B) of modified linear, unpurified circular, or purified circular RNA encoding Gaussia luciferase. [Figure 6B] See legend to Figure 6A. [Figure 7A] Transcript induction of IFNγ (A), IL-6 (B), IL-2 (C), RIG-I (D), IFN-β1 (E), and TNFα (F) is shown after electroporation of Jurkat cells with modified linear, unpurified circular, or purified circular RNA. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8A] A comparison of the luminescence of circular and modified linear RNAs encoding Gaussia luciferase in human primary monocytes (A) and macrophages (B and C). [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 9A] Relative luminescence over 3 days (A) or 24-hour luminescence (B) in supernatants of primary T cells after transduction with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences. [Figure 9B] See legend to Figure 9A. [Figure 10A] Shown are 24-hour luminescence (A) or relative luminescence over 3 days (B) in supernatants of primary T cells after transduction with circular or modified linear RNA containing a Gaussia luciferase expression sequence, and 24-hour luminescence in PBMCs (C). [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A] HPLC chromatograms (A) and circularization efficiencies (B) of RNA constructs with different permutation sites are shown. [Figure 11B] See legend to Figure 11A. [Figure 12A] HPLC chromatograms (A) and circularization efficiencies (B) of RNA constructs with different introns and / or permutation sites are shown. [Figure 12B] See legend to Figure 12A. [Figure 13A] HPLC chromatograms (A) and circularization efficiencies (B) of three RNA constructs with and without homologous arms are shown. [Figure 13B] See legend to Figure 13A. [Figure 14] Circularization efficiency of three RNA constructs with no homologous arms or with homologous arms of various lengths and GC content is shown. [Figure 15] A and B show HPLC chromatograms illustrating the contribution of strong homology arms to improved splicing efficiency, the relationship between circularization efficiency and nicking in selected constructs, and combinations of permutation sites and homology arms hypothesized to show improved circularization efficiency. [Figure 16] Fluorescence images of T cells mock-electroporated (left) or electroporated with circular RNA encoding CAR (right) and co-cultured with Raji cells expressing GFP and firefly luciferase are shown. [Figure 17] Brightfield (left), fluorescent (center), and overlay (right) images of T cells mock-electroporated (top) or electroporated with circular RNA encoding CAR (bottom) and co-cultured with Raji cells expressing GFP and firefly luciferase are shown. [Figure 18] Specific lysis of Raji target cells by T cells mock-electroporated or electroporated with circular RNAs encoding different CAR sequences is shown. [Figure 19]A shows the luminescence in the supernatant of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction with linear or circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences. B shows the relative luminescence over a 3-day period. [Figure 20A] Transcript induction of IFN-β1 (A), RIG-I (B), IL-2 (C), IL-6 (D), IFNγ (E), and TNFα (F) is shown after electroporation of human CD3+ T cells with modified linear, unpurified circular, or purified circular RNA. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 20E] See legend to Figure 20A. [Figure 20F] See legend to Figure 20A. [Figure 21] A shows specific lysis of Raji target cells by human primary CD3+ T cells electroporated with CAR-encoding circRNA, as determined by detection of firefly luminescence. 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 specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding a CAR at different E:T ratios, as determined by detection of firefly luminescence. [Figure 23] Specific lysis of target cells by human CD3+ T cells electroporated with RNA encoding a CAR at 1, 3, 5, and 7 days after electroporation. [Figure 24] Specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding CD19- or BCMA-targeted CARs is shown. [Figure 25]RNAFold prediction of precursor RNA secondary structure for homology arm design. Base density indicates a higher probability of base pairing. Without homology arms, no base pairing is predicted to occur between the ends of the precursor molecule. [Figure 26] Agarose gel confirmation of precursor RNA circularization is shown. C: Precursor RNA (with strong homology arms) subjected to circularization conditions. C+R: Lane C, digested with RNase R. C+R+H: Lane C+R, digested with oligonucleotide-guided RNase H. U: Precursor RNA not subjected to circularization conditions. U+H: Lane U, digested with oligonucleotide-guided RNase H. [Figure 27] 26 shows the Sanger sequencing output of RT-PCR across the splice junction of the sample shown in lanes C+R from FIG. [Figure 28] RNAFold prediction of the secondary structure of the precursor RNA in the context of the designed spacer is shown. Secondary structures potentially important for ribozyme function are identified by black arrows. [Figure 29] Agarose gel demonstrating the effect of spacers on splicing is shown. (-): No spacer. D: Disruptive spacer. P1: Permissive spacer 1. P2: Permissive spacer 2. cRNAFold prediction of precursor RNA secondary structure for internal homology region design. Lack of significant internal homology (Anabaena 1.0) and introduced internal homology (Anabaena 2.0) are indicated by black arrows. Splicing bubble shown as the region between the homology arms and internal homology regions containing the splicing ribozyme. [Figure 30] FIG. 1 is a schematic diagram showing elements of the engineered self-splicing precursor RNA design. [Figure 31] Luminescence in the supernatants 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 is shown (n=4 HEK293, n=3 HeLa). [Figure 32]Luminescence in the supernatant of HEK293 cells starting 24 hours after transfection with CVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA and continuing for 6 days is shown (n=4). [Figure 33] An overview of precursor RNA design and self-splicing is shown. Shading indicates different regions of the RNA described herein. [Figure 34] Figure 1 shows cell viability, circRNA expression stability, and cytokine release from A549 cells transfected with different circRNA preparations (+RNase R, crude circRNA only digested with RNase R; +HPLC, crude circRNA purified by HPLC and then digested with RNase R; +Phos, crude 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 are presented as mean + SD; n = 3; *p < 0.05; ND, not detected). [Figure 35] A schematic diagram of the RNAs introduced and used for TLR experiments is shown. The linearized circRNA contains all the same sequence elements as the spliced ​​circRNA due to the deletion encompassing both the intron and the homologous arms. [Figure 36] Shows surface expression of anti-CD19 CAR 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] Shows the efficacy of mock-electroporated or electroporated T cells with circRNA or linear RNA encoding an anti-CD19 CAR in reducing the bioluminescence of luciferase-expressing CD19+ target cells and CD19- non-target cells. [Figure 39A]Efficacy of T cells electroporated with circRNA encoding an anti-CD19 CAR with different IRES and co-cultured with luciferase-expressing Raji or K562 cells 5 days (A) or 1 day (B) after electroporation. (Oro-B1 = circKymriah; CVB3 IRES, Oro-152 = circKymriah; Salivirus SZ1 IRES, L9a-5 permutation site) [Figure 39B] See legend to Figure 39A. [Figure 40A] Lysis of target and non-target cells by T cells electroporated with circRNA encoding an anti-CD19 CAR. (A) Lysis of CD19+ Raji cells and CD19- K562 cells by primary human T cells electroporated with circRNAs carrying an anti-CD19 CAR expression sequence and a CVB3 IRES or a salivirus SZ1 IRES. (B) Lysis of CD19+ Raji cells and CD19- K562 cells by primary human T cells electroporated with different ratios of circRNAs or linear mRNAs carrying an anti-CD19 CAR expression sequence. (C) Lysis of CD19+ Raji cells and CD19- K562 cells by different ratios of primary human T cells electroporated with circRNAs carrying an anti-CD19 CAR expression sequence. [Figure 40B] See legend to Figure 40A. [Figure 40C] See legend to Figure 40A. [Figure 41A]Figure 1 shows the efficacy of T cells electroporated with a circRNA encoding an anti-CD19 CAR in lysing CD19+ Raji cells compared to T cells transduced with a lentivirus encoding an anti-CD19 CAR. (A) Specific lysis of circRNA encoding an anti-CD19 CAR in lysing CD19+ Raji cells compared to T cells transduced with a lentivirus encoding an anti-CD19 CAR at a ratio of 10 Raji cells to 1 T cell. (B) Percentage of anti-CD19 CAR-expressing T cells mock-electroporated or electroporated with a lentivirus or circRNA encoding an anti-CD19 CAR. (C) Induction of interferon gamma mRNA in T cells electroporated with a circRNA encoding an anti-CD19 CAR co-cultured with or without CD19+ Raji cells. (Oro-B1 = circKymriah; CVB3 IRES, Oro-B6 = linKymriah) [Figure 41B] See legend to Figure 41A. [Figure 41C] See legend to Figure 41A. [Figure 42] Shows the stability of anti-CD19 CAR expression in primary human CD3+ T cells electroporated with circRNA or linear mRNA encoding the anti-CD19 CAR. [Figure 43] Shows the efficacy of THP-1 monocytes electroporated with CAR-encoding circRNA in lysing luciferase-expressing Raji cells in co-culture experiments. [Figure 44] Shows the efficacy of T cells electroporated with a circRNA encoding an anti-mouse CD19 CAR in lysing CD19+ A20 cells and CD19- K562 cells. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description Provided herein are pharmaceutical compositions and delivery vehicles, e.g., lipid nanoparticles, comprising circular RNA. The circular RNA provided herein can be delivered and / or targeted to cells in a delivery vehicle, e.g., nanoparticles, or a composition comprising the delivery vehicle. In some embodiments, the circular RNA can also be delivered to a subject in a delivery vehicle or a composition comprising the delivery vehicle. In some embodiments, the delivery vehicle is a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a non-lipid polymer core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the delivery vehicle comprises one or more ionizable lipids, PEG-modified lipids, helper lipids, and / or structural lipids.

[0053] In some embodiments, the transfer vehicle encapsulates the circular RNA and comprises an ionizable lipid, a structural lipid, and a PEG-modified lipid. In some embodiments, the transfer vehicle encapsulates the circular RNA and comprises an ionizable lipid, a structural lipid, a PEG-modified lipid, and a helper lipid.

[0054] Without wishing to be bound by theory, it is believed that the transport vehicles described herein protect the encapsulated circular RNA from degradation and provide effective delivery of the circular RNA to target cells in vivo and in vitro.

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

[0056] In one embodiment, the mole % of helper lipid can be about 1 mole % to about 50 mole %. In one embodiment, the mole % of helper lipid can be about 2 mole % to about 45 mole %. In one embodiment, the mole % of helper lipid can be about 3 mole % to about 40 mole %. In one embodiment, the mole % of helper lipid can be about 4 mole % to about 35 mole %. In one embodiment, the mole % of helper lipid can be about 5 mole % to about 30 mole %. In one embodiment, the mole % of helper lipid can be about 10 mole % to about 20 mole %. In some embodiments, the helper lipid mole % of a transport vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole %.

[0057] In one embodiment, the mole % of structured lipids can be from about 10 mole % to about 80 mole %. In one embodiment, the mole % of structured lipids can be from about 20 mole % to about 70 mole %. In one embodiment, the mole % of structured lipids can be from about 30 mole % to about 60 mole %. In one embodiment, the mole % of structured lipids can be from about 35 mole % to about 55 mole %. In one embodiment, the mole % of structured lipids can be from about 40 mole % to about 50 mole %. In some embodiments, the mole % of structured lipids in a transport vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole %.

[0058] In one embodiment, the mole % of PEG-modified lipid can be about 0.1 mole % to about 10 mole %. In one embodiment, the mole % of PEG-modified lipid can be about 0.2 mole % to about 5 mole %. In one embodiment, the mole % of PEG-modified lipid can be about 0.5 mole % to about 3 mole %. In one embodiment, the mole % of PEG-modified lipid can be about 1 mole % to about 2 mole %. In one embodiment, the mole % of PEG-modified lipid can be about 1.5 mole %. In some embodiments, the mole % of PEG-modified lipid in a delivery vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole %.

[0059] Also contemplated are pharmaceutical compositions, and in particular transport vehicles, that comprise one or more of the compounds disclosed herein.In certain embodiments, this transport vehicle comprises one or more of the PEG-modified lipids, ionizable lipids, helper lipids and / or structural lipids disclosed herein.Also contemplated are transport vehicles that comprise one or more of the compounds disclosed herein and further comprise one or more additional lipids.In certain embodiments, this transport vehicle is loaded with or otherwise encapsulates circular RNA.

[0060] The delivery vehicle of the present invention encapsulates a circular RNA. In certain embodiments, the polynucleotide encapsulated by the compound or pharmaceutical composition and liposome composition of the present invention includes an RNA encoding a protein or enzyme (e.g., a circRNA encoding phenylalanine hydroxylase (PAH)). The present invention contemplates the use of such polynucleotides as therapeutic agents that can be expressed by target cells for the production (and, in certain instances, 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 June 8, 2011 (the teachings of which are both incorporated herein by reference in their entireties). For example, in certain embodiments, upon expression of one or more polynucleotides by target cells, production of a functional enzyme or protein (e.g., a urea cycle enzyme or an enzyme associated with a lysosomal storage disorder) in which the subject is deficient can be observed. As another example, the circular RNA encapsulated by the delivery vehicle can encode a T cell receptor protein or a chimeric antigen receptor (CAR).

[0061] Also provided herein are methods for treating a disease in a subject by administering to the subject an effective amount of a composition comprising a circular RNA encoding a functional protein described herein and a delivery vehicle. In some embodiments, the circular RNA is encapsulated within the delivery vehicle. In certain embodiments, such methods may enhance (e.g., increase) expression of the polynucleotide and / or increase production and secretion of a functional polypeptide product in one or more target cells and tissues (e.g., hepatocytes). Generally, such methods include contacting target cells with one or more compounds and / or delivery vehicles that contain or otherwise encapsulate the circRNA.

[0062] In certain embodiments, delivery vehicles (e.g., lipid nanoparticles) are formulated based in part on their ability to facilitate transfection (e.g., of circular RNA) of target cells. In another embodiment, delivery vehicles (e.g., lipid nanoparticles) can be selected and / or prepared to optimize delivery of circular RNA to target cells, tissues, or organs. For example, if the target cells are hepatocytes, the properties (e.g., size, charge, and / or pH) of the pharmaceutical and / or liposomal compositions can be optimized to effectively deliver, reduce immune clearance, and / or facilitate retention in the target cells or organs of such compositions (e.g., lipid nanoparticles). Alternatively, if the target tissue is the central nervous system, the selection and preparation of the delivery vehicle must take into account the permeability of and retention within the blood-brain barrier, and / or the use of alternative means to directly deliver such compositions (e.g., lipid nanoparticles) to such target tissues (e.g., via intracerebrovascular administration). In certain embodiments, the transport vehicle may be combined with an agent that facilitates transport of the encapsulated substance across the blood-brain barrier (e.g., an agent that disrupts or improves the permeability of the blood-brain barrier, thereby improving the transport of circular RNA to target cells). While the transport vehicles (e.g., lipid nanoparticles) described herein 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 comprising the pharmaceutical composition can, in some cases, significantly improve the transfection efficiency of some types of transport vehicles by 2-28 fold 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). In some embodiments, the target cell is an immune cell. In some embodiments, the target cell is a T cell.

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

[0064] The transport vehicles described herein may allow the encapsulated polynucleotide to reach target cells, or may preferentially allow the encapsulated polynucleotide to differentially reach target cells or organs (e.g., the transport vehicle may concentrate in the liver or spleen of the subject to which such transport vehicle is administered). Alternatively, the transport vehicle may limit the delivery of the encapsulated polynucleotide to other non-target cells or organs where the presence of the encapsulated polynucleotide is undesirable or may be of limited utility.

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

[0066] In certain embodiments, provided herein are vectors for generating circular RNAs, the vectors comprising a 5' duplex-forming 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' duplex-forming region. In some embodiments, these elements are arranged in the above order within the vector. In some embodiments, the vector further comprises an internal 5' duplex-forming region between the 3' Group I intron fragment and the IRES, and an internal 3' duplex-forming region between the expression sequence and the 5' Group I intron fragment. In some embodiments, the internal duplex-forming regions can form duplexes with each other but not with external duplex-forming regions. In some embodiments, the internal duplex-forming regions are part of the first and second spacers. Additional embodiments include circular RNA polynucleotides, including circular RNA polynucleotides generated using the vectors provided herein, compositions comprising such circular RNAs, cells comprising such circular RNAs, and methods of using and making such vectors, circular RNAs, compositions, and cells.

[0067] In some embodiments, provided herein are methods that involve administering a circular RNA polynucleotide provided herein to a cell for therapy or for the production of a useful protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein. In some embodiments, the methods are advantageous in providing for the production of a desired polypeptide in a eukaryotic cell with a longer half-life than linear RNA due to the resistance of circular RNA to ribonucleases.

[0068] Circular RNA polynucleotides lack the free ends required for exonuclease-mediated degradation, and therefore, compared with comparable linear RNA, they are resistant to some mechanisms of RNA degradation and have a longer half-life.Circularization allows the stabilization of RNA polynucleotides, which generally have a short half-life, and improves the overall effectiveness of exogenous mRNA in various applications.In some embodiments, the half-life of the circular RNA polynucleotide 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" or "circular polyribonucleotide" or "circular RNA" are used interchangeably and refer to polyribonucleotides that form a circular 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 native Group I intron, including the splice site dinucleotide and optionally a stretch of native 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 native Group I intron, including the splice site dinucleotide and optionally a stretch of native exon sequence.

[0072] As used herein, the term "permutation site" refers to a site within a Group I intron where cleavage occurs prior to permutation of the intron, generating 3' and 5' Group I intron fragments that are permuted onto either side of the stretch of precursor RNA that is circularized.

[0073] As used herein, the term "splice site" refers to a dinucleotide that is partially or completely contained within a Group I intron and between which the phosphodiester bond is cleaved during RNA circularization.

[0074] As used herein, the term "therapeutic protein" refers to any protein that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered directly or indirectly in the form of a translated nucleic acid to a subject.

[0075] As used herein, the term "immunogenic" refers to the potential to induce an immune response against a substance. An immune response can be induced when an organism's immune system or a certain type of immune cell is exposed to an immunogenic substance. The term "non-immunogenic" refers to the lack or absence of an immune response above a detectable threshold against a substance. An immune response is not detected when an organism's immune system or a certain type of immune cell is exposed to a non-immunogenic substance. In some embodiments, non-immunogenic cyclic polyribonucleotides as provided herein do not induce an immune response above a predetermined threshold as measured by an immunogenicity assay. In some embodiments, an innate immune response is not detected when an organism's immune system or a certain type of immune cell is exposed to a non-immunogenic cyclic polyribonucleotide as provided herein. In some embodiments, an adaptive immune response is not detected when an organism's immune system or a certain type of immune cell is exposed to a non-immunogenic cyclic polyribonucleotide as provided herein.

[0076] As used herein, the term "circularization efficiency" refers to the measurement of the resulting circular 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 can be expressed as the amount of protein or peptide produced per given amount of transcript encoding the protein or peptide.

[0078] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include species containing purines, such as adenine, hypoxanthine, and guanine, and derivatives and analogs thereof, and pyrimidines, such as cytosine, uracil, and thymine, and derivatives and analogs thereof. Nucleotide analogs include nucleotides with modifications in the chemical structure of the base, sugar, and / or phosphate, including 5'-position pyrimidine modifications, 8'-position purine modifications, cytosine exocyclic amine modifications, and 5-bromo-uracil substitutions; and 2'-position sugar modifications, including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced with a group such as H, OR, R, halo, SH, SR, NH, NHR, NR, or CN (where R is an alkyl moiety as defined herein). Nucleotide analogs are also intended to include nucleotides with bases such as inosine, queosine, and xanthine; sugars such as 2'-methylribose; and non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0079] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or up to about 10,000 bases or more, which can be produced enzymatically or synthetically (e.g., as described in U.S. Pat. No. 5,948,902 and references cited therein), which can hybridize, e.g., participate in Watson-Crick base pairing interactions, with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids. Naturally occurring nucleic acids are composed of nucleotides including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0080] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0081] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer 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 the substance constitutes a significant percentage (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100%) of the sample in which it is present. In certain embodiments, a substantially purified component comprises at least 50%, 80%-85%, or even 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 sedimentation according to density. Generally, a substance is purified when it is present in a sample in an amount greater than that found in nature relative to other components of the sample.

[0083] As used herein, the terms "duplex," "double-stranded," or "hybridized" refer to a nucleic acid formed by hybridization of two single strands of nucleic acid containing complementary sequences. In most cases, genomic DNA is double-stranded. The 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 prediction tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease protection assays.

[0085] As used herein, "structured" with respect to RNA refers to an RNA sequence that is predicted by RNAFold software or similar prediction tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule.

[0086] As used herein, two "duplex-forming regions," "homologous arms," ​​or "homologous regions" complement or are complementary to each other 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" sequences. The percent sequence identity between a homologous region and the reverse complement of a corresponding homologous region can be any percent sequence identity that allows hybridization to occur. In some embodiments, an internal duplex-forming region of a polynucleotide of the invention can form a duplex with another internal duplex-forming region but not with an external duplex-forming region.

[0087] Linear nucleic acid molecules are said to have a "5' end" (5' end) and a "3' end" (3' end) because the nucleic acid phosphodiester linkages occur at the 5' and 3' carbons of the sugar moieties of the substituent mononucleotides. 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 the nucleotide at the end position of either the 3' or 5' end.

[0088] "Transcription" refers to the formation or synthesis of an RNA molecule by RNA polymerase using a DNA molecule as a template. The present invention is not limited by 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 a polypeptide molecule by ribosomes from an RNA template.

[0090] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a combination of two or more cells, or an entire culture of cells; a reference to a "polynucleotide" includes, as a practical matter, many copies of that polynucleotide. Unless specifically stated or clear from the context, the term "or" as used herein is understood to be inclusive. Unless defined herein and in the remainder of the specification below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] Unless specifically stated or clear from the context, the term "about" as used herein is understood to mean within a normal range of tolerance in the art, for example, within two standard deviations of the mean. "About" can 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 clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0092] As used herein, the term "encoding" refers broadly to any process that uses information in a polymeric macromolecule to direct the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.

[0093] By "co-administered" is meant administration of a therapeutic agent provided herein in conjunction with one or more additional therapeutic agents sufficiently close in time 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 mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of skill in the art would recognize as having potential benefit or therapeutic effect. The treatment or prevention provided by the methods disclosed herein can include treatment or prevention of one or more conditions or symptoms of a disease. Also, for purposes of this specification, "prevention" can encompass delaying the onset of a disease, or its symptoms or conditions.

[0095] As used herein, the term "expressed sequence" can refer to a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, a regulatory nucleic acid, or a non-coding nucleic acid. An exemplary expressed sequence that encodes a peptide or polypeptide can include multiple nucleotide triads, each of which can encode an amino acid, and are 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 can be defined or can be random. Spacers are typically non-coding. In some embodiments, a spacer comprises a duplex-forming region.

[0097] As used herein, "splice site" refers to one or more dinucleotides between which cleavage of a phosphodiester bond occurs in a splicing reaction. A "5' splice site" refers to the naturally occurring 5' dinucleotide of an intron, e.g., a Group I intron, while a "3' splice site" refers to the naturally occurring 3' dinucleotide of an intron.

[0098] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA sequence or structural element ranging in size from 10 nt to over 1000 nt that can initiate translation of a polypeptide in the absence of a typical RNA cap structure. IRESs are typically about 500 nt to about 700 nt in length.

[0099] As used herein, an "miRNA site" refers to a stretch of nucleotides within a polynucleotide that can form a duplex with a native miRNA sequence of at least 8 nucleotides.

[0100] As used herein, an "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, "bicistronic RNA" refers to a polynucleotide that contains two expressed sequences that encode two different proteins, often separated by a cleavable peptide, such as a 2A site or IRES sequence.

[0102] As used herein, the term "co-formulation" refers to a nanoparticle formulation containing two or more nucleic acids or a nucleic acid and another active agent. Typically, the ratio is defined as an equimolar or ratiometric amount of the two or more nucleic acids or a nucleic acid and another active agent.

[0103] As used herein, "delivery vehicle" includes any of the standard pharmaceutical carriers, diluents, excipients, etc. generally intended for use in connection with the administration of biologically active agents, including nucleic acids.

[0104] As used herein, the phrase "lipid nanoparticle" refers to a delivery vehicle comprising one or more lipids (e.g., in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0105] As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0106] As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid.

[0107] As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0108] As used herein, the phrase "ionizable lipid" refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH 4, and a neutral charge at other pHs, such as physiological pH 7.

[0109] In some embodiments, the lipids disclosed herein, e.g., ionizable lipids, comprise one or more cleavable groups. The terms "cleavage" and "cleavable," as used herein, mean that one or more chemical bonds (e.g., one or more of covalent bonds, hydrogen bonds, van der Waals forces, and / or ionic interactions) between atoms within or adjacent to the functional group of interest can be broken (e.g., hydrolyzed) or broken upon exposure to selected conditions (e.g., upon exposure to 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, the disulfide group can be cleaved enzymatically or by hydrolysis, oxidation, or reduction reactions. Upon cleavage of such a disulfide functional group, one or more functional moieties or groups (e.g., one or more of the head group and / or tail group) attached thereto can 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 attached (e.g., attached by one or more of hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds) to one or more functional moieties or groups (e.g., at least one head group and at least one tail group). In certain embodiments, at least one of the functional moieties or groups is hydrophilic (e.g., a hydrophilic head group comprising one or more of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino, and pyridyl).

[0110] As used herein, the term "hydrophilic" is used qualitatively to indicate that a functional group is water-loving, typically such that such group is water-soluble. For example, disclosed herein are compounds that include a cleavable disulfide (SS) functional group attached to one or more hydrophilic groups (e.g., hydrophilic head groups), where 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 the moiety comprising the compounds disclosed herein is hydrophobic in nature (e.g., a hydrophobic tail group comprising a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used qualitatively to indicate that the functional group dislikes water, and typically, such groups are not water-soluble. For example, compounds are disclosed herein that include a cleavable functional group (e.g., a disulfide (SS) group) attached to one or more hydrophobic groups, where such hydrophobic groups are derived from one or more naturally occurring lipids, e.g., cholesterol, and / or optionally substituted, variably saturated or unsaturated C6-C 20 Alkyl and / or optionally substituted, variably saturated or unsaturated C-C 20 Contains acyl.

[0112] In certain embodiments, compounds disclosed herein include, for example, at least one hydrophilic head group and at least one hydrophobic tail group, each attached to at least one cleavable group, thereby rendering such compounds amphiphilic. As used herein to describe a compound or composition, the term "amphiphilic" refers to the ability to dissolve in both polar (e.g., water) and non-polar (e.g., lipid) environments. For example, in certain embodiments, compounds disclosed herein include at least one lipophilic tail group (e.g., cholesterol or C6-C 20alkyl) and at least one hydrophilic head group (e.g., imidazole), each linked to a cleavable group (e.g., disulfide).

[0113] It should be noted that the terms "head group" and "tail group" as used are used for ease of reference to describe the compounds of the invention, and in particular the functional groups that comprise such compounds, and to describe the orientation of one or more functional groups relative to other functional groups. For example, in certain embodiments, a hydrophilic head group (e.g., guanidinium) is attached (e.g., by one or more of hydrogen bonding, van der Waals forces, ionic interactions, and covalent bonding) to a cleavable functional group (e.g., a disulfide group), which is in turn attached to a hydrophilic tail group (e.g., cholesterol).

[0114] As used herein, the term "alkyl" refers to straight and branched chain C-C alkyl groups. 40 Hydrocarbons (e.g., C6-C 20 "Alkyl" refers to both saturated and unsaturated hydrocarbons, including both saturated and unsaturated hydrocarbons. In certain embodiments, alkyl may contain 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, contemplated alkyls include (9Z,12Z)-octadeca-9,12-diene. For example, "C6-C 20 " is intended to refer to an alkyl (e.g., straight or branched chain, including alkenes and alkyls) having the specified range of carbon atoms.

[0115] As used herein, the term "aryl" refers to aromatic groups containing 6 to 10 carbons in the ring portion (e.g., monocyclic, bicyclic, and tricyclic structures). Aryl groups can be optionally substituted through available carbon atoms and, in certain embodiments, can contain one or more heteroatoms, such as oxygen, nitrogen, or sulfur.

[0116] In certain embodiments, compounds and delivery vehicles (e.g., lipid nanoparticles) of which such compounds are components exhibit improved (e.g., increased) ability to transfect one or more target cells. Accordingly, methods of transfecting one or more target cells are also provided herein. Such methods generally include contacting one or more target cells with a compound and / or pharmaceutical composition disclosed herein, such that the one or more target cells are transfected with the circular RNA encapsulated therein. As used herein, the term "transfect" or "transfection" refers to the intracellular introduction of one or more encapsulated substances (e.g., nucleic acids and / or polynucleotides) into a cell, or preferably into a target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated substances (e.g., polynucleotides) taken up by, introduced into, and / or expressed by a target cell subject to transfection. In some embodiments, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cell after transfection. In some embodiments, the delivery vehicle has high transfection efficiency. In some embodiments, the delivery 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., amphipathic 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 liposomes may be unilamellar or multilamellar vesicles with a membrane formed from a lipophilic substance and an aqueous interior containing encapsulated circRNAs 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 can be used to form contemplated 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 "non-cationic lipid," "non-cationic helper lipid," and "helper lipid" are used interchangeably and refer to any neutral, zwitterionic, or anionic lipid.

[0119] As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0120] As used herein, the phrase "biodegradable lipid" or "degradable lipid" refers to any of a number of lipid species that degrade in the host environment within minutes, hours, or even days, ideally making them less toxic and less likely to accumulate within the host over time. Common modifications to lipids include ester bonds and, particularly, disulfide bonds, which enhance the biodegradability of the lipid.

[0121] As used herein, the phrase "biodegradable PEG lipid" or "degradable PEG lipid" refers to any of a number of lipid species in which the PEG molecule is cleaved from the lipid within minutes, hours, or even days in the host environment, ideally resulting in low immunogenicity. Common modifications to PEG lipids include ester bonds and, among others, disulfide bonds, which enhance the biodegradability of the lipid.

[0122] In certain embodiments of the present invention, a transport vehicle (e.g., lipid nanoparticle) 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 a 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 in the transport vehicle can be located completely or partially in the interior space of the transport vehicle, within the bilayer membrane of the transport vehicle, or attached to the outer surface of the transport vehicle.

[0123] As used herein, the term "structured lipid" refers to a sterol and also to lipids that contain a sterol moiety.

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

[0125] As used herein, the term "structured lipid" refers to a sterol and also to lipids that contain a sterol moiety.

[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 the lipid.

[0128] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.

[0129] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. 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 can be further subdivided into regions of hypervariability, termed complementarity-specific regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin 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 can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0130] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of 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 can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain antibodies.

[0131] An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that comprises the antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may comprise an antigen-complementarity specific region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or 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, comprising one or more of its complementarity specific regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule comprises or consists of an avimer.

[0132] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 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 among antibodies and are used to determine the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine 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 comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

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

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

[0135] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two and is used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures. The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding molecule. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). When using the Kabat numbering system, the CDRs in an 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 (designated 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 in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.In certain embodiments, the CDRs of an antibody may be determined according to the Chothia numbering scheme, which refers to the position of the immunoglobulin structural loops (see, e.g., 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, using the Kabat numbering convention, 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 convention, 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 are 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 CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

[0136] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meanings in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0137] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD or Kd). Affinity can be measured and / or expressed in many ways 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 koff / koff, while Ka is calculated from the quotient koff / koff. koff refers to the association rate constant of, for example, an antibody to an antigen, and koff refers to the dissociation rate of, for example, an antibody to an antigen. koff and koff can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0138] As used herein, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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(s) or framework region(s) of an antibody or antigen-binding molecule thereof may be replaced with an amino acid residue having a similar side chain.

[0139] As used herein, the term "heterologous" means derived from any source other than the naturally occurring sequence.

[0140] As used herein, "epitope" is a term of art that refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be composed of, for example, two or more non-contiguous regions of one or more polypeptides (a conformational, non-linear, interrupted, or discontinuous epitope). In some embodiments, the epitope to which an antibody binds can be determined by, for example, 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 mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of 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 probed using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., 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 antigen-binding molecule thereof "cross-competes" with a reference antibody or its antigen-binding molecule when the interaction between the antigen and a first binding molecule, antibody, or antigen-binding molecule thereof 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, e.g., 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, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In some embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a 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 antigen-binding molecule, for example, solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods 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 label (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 "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex), as understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with a lower affinity, as determined by, for example, immunoassays, a BIACORE®, a 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 K A of at least 2 logs, 2.5 logs, 3 logs, 4 logs, or more than the K A for binding to another antigen.

[0143] "Antigen" refers to any molecule that can induce an immune response or be bound by an antibody or antigen-binding molecule. The immune response can include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or they can be widely expressed. In addition, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0144] The term "autologous" refers to any material derived from the same individual to which the material is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves collecting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered back to the same patient.

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

[0146] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade nearby tissues and may even metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can 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 white blood cell malignancies. In some embodiments, the methods disclosed herein can be used to treat or prevent the development of cancers including, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's 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), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, and the like. The compounds may reduce tumor size in 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), childhood solid tumors, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the foregoing cancers.In some embodiments, the methods disclosed herein can be used to treat or prevent the development of tumors, such as sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, sarcomas of soft tissue and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g., of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, breast cancer, and other cancers. The tumor size of tumors derived from cranial adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, renal pelvic carcinoma, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma) may be reduced. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer refers to cancer that is not suitable for surgical intervention, or the cancer does not respond to chemotherapy or radiation therapy initially or becomes unresponsive over time.

[0147] As used herein, "anti-tumor effect" refers to a biological effect that can 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 tumors.Anti-tumor effect can also refer to the prevention of tumor development, such as a vaccine.

[0148] As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen; the cytokine interacts with a second cell and mediates a response in the second cell. As used herein, "cytokine" is intended to refer to a protein released by one cell population that acts as an intercellular mediator on another cell. Cytokines can be endogenously expressed by cells or administered to a subject. 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 various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effector, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response. 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 (cytotoxic) lymphocyte and represent a major component of the innate immune system. NK cells reject tumors and cells infected with viruses. They function through the process of apoptosis, or programmed cell death. They are named "natural killer" 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 receptors (TCRs) distinguish between themselves and 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, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory T cells like naive cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they contain large amounts of CD95, IL-2R[T (ii) central memory T cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ or CD4+FoxP3+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. Meanwhile, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and, after activation by antigen interaction, develop into both short-lived and long-lived memory B cells and plasma cells. In mammals, immature B cells are formed in the bone marrow, hence their name.

[0150] The term "genetically engineered" or "engineered" refers to methods of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the cell to be modified is a lymphocyte, e.g., a T cell, which may be obtained from a patient or a donor. The cell may be modified to express an exogenous construct, such as, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is integrated into the genome of the cell.

[0151] "Immune response" refers to the actions 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, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0152] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, e.g., TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or upregulation or downregulation of key molecules.

[0153] As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The costimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule, for example, by binding of the T cell receptor (TCR) / CD3 complex with a peptide-loaded major histocompatibility complex (MHC) molecule. Costimulatory ligands may include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds 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. Costimulatory ligands include, but are not limited to, ligands that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, and 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 cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecules" are cognate binding partners on a T cell that specifically bind to a costimulatory ligand and thereby mediate a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory 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, and CD3 (alpha ; beta; delta; epsilon; gamma; zeta), 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, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, IT GAD, 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 1a / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, 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 fragments, truncations, or combinations thereof.

[0155] As used herein, "sequence identity" or a reference including, for example, "a sequence that is 50% identical to" refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotide and polypeptide variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, and typically the polypeptide variants retain at least one biological activity of the reference polypeptide.

[0156] 2. Vector, precursor RNA, and circular RNA In certain aspects, provided herein is 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. In some embodiments, these regions are in this order. In some embodiments, the circular RNA is produced by the methods provided herein or from the vectors provided herein.

[0157] In certain embodiments, transcription of a vector provided herein (e.g., comprising a 5' homology 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' homology region) results in the formation of a precursor linear RNA polynucleotide that can be circularized. In some embodiments, this precursor linear RNA polynucleotide circularizes 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 comprise a first (5') duplex-forming region and a second (3') duplex-forming region. In certain embodiments, the first and second homologous regions may form a perfect or imperfect duplex. 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 duplex-forming regions may be base-paired to each other. In some embodiments, the duplex-forming regions are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, or less than 25%) base-pairing with unintended sequences (e.g., non-duplex-forming region sequences) within the RNA. In some embodiments, such duplex-forming regions are included at the ends of the precursor RNA strands adjacent to or very close to the Group I intron fragments, bringing the Group I intron fragments into close proximity with each other and increasing splicing efficiency. In some embodiments, the duplex-forming regions are 3-100 nucleotides in length (e.g., 3-75 nucleotides in length, 3-50 nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides in length, 5-25 nucleotides in length, 9-19 nucleotides in length). In some embodiments, the duplex-forming region is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the duplex-forming region is about 9 to about 50 nucleotides in length. In some embodiments, the duplex-forming region is about 9 to about 19 nucleotides in length. In some embodiments, the duplex-forming region is about 20 to about 40 nucleotides in length. In certain embodiments, the duplex-forming region is about 30 nucleotides in length.

[0159] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein comprise a first (5') and / or second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES may preserve secondary structure in those regions by preventing them from interacting, 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 contain additional base-pairing regions that are predicted to base-pair with each other and not with the first and second duplex-forming regions. In some embodiments, such spacer base-pairing brings the group I intron fragments into close proximity with each other, further increasing splicing efficiency. Additionally, in some embodiments, the combination of base-pairing between the first and second duplex-forming regions, and separately, base-pairing between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragment bordered by adjacent regions of base-pairing (Figure 25). A typical spacer has the following qualities: 1) is predicted to avoid interference with proximal structures, such as an IRES, expressed sequence, or intron; 2) is at least 7 nt in length and no more than 100 nt; 3) is located after and adjacent to a 3' intron fragment and / or before and adjacent to a 5' intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt in length, b) a region of base-pairing to a distal sequence containing another spacer at least 5 nt in length, and c) a structured region at least 7 nt in length bounded by the sequence of the spacer. A spacer can have several regions, including unstructured regions, base-paired 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 4-12 nucleotides and a loop of 2-10 nucleotides. In some embodiments, an additional spacer is present between the 3' group I intron fragment and the IRES. In some embodiments, this additional spacer prevents or reduces the extent to which the structured region of the IRES interferes with folding of the 3' group I intron fragment. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is no longer than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and / or 25 and 35 nucleotides in length. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, the spacer comprises about 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 native Group I intron that includes the 3' splice site dinucleotide and optionally adjacent exon sequence 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 the 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 a 5' proximal fragment of a native Group I intron that includes the 5' splice site dinucleotide and optionally adjacent exon sequence 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 the exon. As described by Umekage et al. (2012) and shown in Figure 33, the outer portions of the 3' group I intron fragment and the 5' group I intron fragment are removed during circularization, such that the circular RNAs provided herein contain only the 3' group I intron fragment formed by optional exon sequences at least 1 nt in length and the portions of the 5' group I intron fragment formed by optional exon sequences at least 1 nt in length, if 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 to herein 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 to herein as the post-splicing 5' group I intron fragment.

[0161] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein contain an internal ribosome entry site (IRES). The inclusion of an IRES allows translation of one or more open reading frames (e.g., open reading frames that form an expression sequence) from the circular RNA. The IRES element attracts the eukaryotic ribosomal translation initiation complex and facilitates translation initiation. See, e.g., 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] Numerous IRES sequences are available, including those derived from a wide variety of viruses, including picornavirus leader sequences such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660), polio leader sequences, hepatitis A virus leader, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), an IRES element from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), and the Giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397).

[0163] In some embodiments, the IRES is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali enterovirus, Kashmir wasp virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, encephalomyocarditis virus, Drosophila C virus, human coxsackievirus B3, crucifer tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute honeybee paralysis virus, hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human mouse AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1alpha, 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 etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, 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, pasivirus A, pasivirus 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 The IRES sequences are those of K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsavirus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus 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 an aptamer against eIF4G.

[0164] In some embodiments, the polynucleotide herein comprises an expression sequence. In some embodiments, the expression sequence encodes a CAR. In some embodiments, the polynucleotide comprises more than one expression sequence, for example, 2, 3, 4, or 5 expression sequences. In some embodiments, one such expression sequence encodes a CAR and another encodes another therapeutic protein, for example, a checkpoint inhibitor, for example, an inhibitor of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the polynucleotide comprises a first expressed sequence encoding a CAR and a second expressed sequence encoding an inhibitor of 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 LAGS. In some embodiments, the inhibitor is nivolumab, pembrolizumab, ipilimumab, or atezolizumab. In some embodiments, the expressed sequence encodes a protein that is cleaved into two or more functional units, for example, a CAR and another therapeutic protein.

[0165] In certain embodiments, the polynucleotides provided herein comprise 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 an artificial TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, and / or CD8 variant. In some embodiments, the CAR or TCR complex protein encodes a naturally occurring TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, and / or CD8 variant. 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 comprise a 3'UTR. In some embodiments, the 3'UTR is derived from human beta globin, human alpha globin Xenopus beta globin, Xenopus alpha globin, human prolactin, human GAP-43, human eEF11, 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 ringspot virus, woodchuck hepatitis virus post-translational control element, Sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

[0167] In some embodiments, the vectors provided herein comprise a 5' UTR, which in some embodiments is derived from human beta globin, Xenopus beta globin, human alpha globin, Xenopus alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or adenovirus tripartite leader.

[0168] In some embodiments, the vectors provided herein comprise a polyA region. In some embodiments, the polyA region is at least 30 nucleotides in length or at least 60 nucleotides in length.

[0169] In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are 300-10,000, 400-9,000, 500-8,000, 600-7,000, 700-6,000, 800-5,000, 900-5,000, 1,000-5,000, 1,100-5,000, 1,200-5,000, 1,300-5,000, 1,400-5,000, and / or 1,500-5,000 nucleotides in length. In some embodiments, a 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, a polynucleotide is no longer than 3000nt, 3500nt, 4000nt, 4500nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, or 1000nt in length. In some embodiments, the length of the DNA, linear RNA, and / or circular RNA polynucleotides provided herein is about 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 generating circular RNA polynucleotides. Such polynucleotides include a CAR or TCR complex protein-encoding domain. Certain current CAR and recombinant TCR treatments engineer cells with DNA encoding a CAR or recombinant TCR, which causes greater toxicity and poses the risk of harmful mutagenesis compared to transient forms of CAR or recombinant TCR expression. An alternative method is linear RNA encoding a CAR or recombinant TCR complex protein. However, linear RNA has a short half-life in vivo, limiting treatment efficacy. In certain embodiments, the circular RNA polynucleotides provided herein encoding a CAR or recombinant TCR complex protein offer the toxicity advantages of transient expression while increasing the therapeutic efficacy of treatment compared to linear RNA. The methods of circularizing RNA described herein, including the addition of homologous regions flanking a group I intron fragment, enable high circularization efficiency and circularization of large RNA polynucleotides.

[0171] In some embodiments, a vector is provided herein. In certain embodiments, the vector comprises, in the following order: a) a 5' homology 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' homology region. In some embodiments, the vector comprises a transcription promoter upstream of the 5' homology region.

[0172] In some embodiments, precursor RNAs are provided herein. In certain embodiments, the precursor RNA is a 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' homology 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' homology region. The precursor RNA can be unmodified, partially modified, or fully modified.

[0173] In certain embodiments, circular RNAs are provided herein. In certain embodiments, the circular RNA is a circular RNA produced by a vector provided herein. In some embodiments, the circular RNA is a circular RNA produced by circularizing a precursor RNA provided herein. In some embodiments, the circular RNA comprises the following sequence: 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 the 3' group I intron fragment that is 3' of the 3' splice site. In some embodiments, the circular RNA further comprises a portion of the 5' group I intron fragment that is 5' of the 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. The circular RNA can be unmodified, partially modified, or fully modified.

[0174] In some embodiments, the circular RNAs provided herein have greater functional stability than mRNAs comprising the same expressed sequence, hi some embodiments, the circular RNAs provided herein have greater functional stability than mRNAs comprising the same expressed sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails.

[0175] In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 hours. In some embodiments, the circular 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 circular RNA polynucleotides provided herein have a longer (e.g., at least 1.5-fold longer, at least 2-fold longer) functional half-life than an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the functional half-life can be assessed through detection of functional protein synthesis.

[0176] In some embodiments, the circular RNA polynucleotides provided herein have a half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 hours. In some embodiments, the circular 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 circular RNA polynucleotides provided herein have a longer half-life (e.g., at least 1.5-fold longer, at least 2-fold longer) than an equivalent linear RNA polynucleotide encoding the same protein.

[0177] In some embodiments, the circular RNAs provided herein may have a higher degree of expression than comparable linear mRNAs, for example, a higher degree of expression 24 hours after administration of the RNA to cells. In some embodiments, the circular RNAs provided herein may have a higher degree of expression than mRNAs containing the same expression sequence, 5moU modification, optimized UTRs, caps, and / or polyA tails. In some embodiments, the circular RNAs provided herein may have a higher stability than comparable linear mRNAs. In some embodiments, this may be demonstrated by measuring the presence and density of receptors in vitro or in vivo after electroporation, measured at time points over a 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 an organism's immune system or a specific type of immune cell. In some embodiments, the circular RNAs provided herein are associated with modulating cytokine production when exposed to an organism's immune system or a specific 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 an organism's immune system or a specific type of immune cell, compared to mRNA containing the same expression sequence. In some embodiments, the circular RNAs provided herein are associated with less induction of IFN-β1, RIG-1, IL-2, IL-6, IFNγ, and / or TNFα transcripts when exposed to an organism's immune system or a specific type of immune cell, compared to mRNA containing the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNA containing the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs containing the same expression sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails. In certain embodiments, the circular RNAs provided herein may result in lower toxicity than DNA-based viral, e.g., lentiviral, engineering when expressing CAR or TCR complex proteins on immune cells, e.g., T cells, due to cytokine release syndrome (CRS). In some embodiments, this may be demonstrated by measuring in vitro cytokine, e.g., IL6, release post-infection / transfection, as assessed by ELISA and / or qPCR.

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

[0180] In some embodiments, the circular RNA provided herein can cause less toxicity than DNA-based viruses, such as lentivirus manipulation, when expressing CAR or TCR complex proteins on immune cells, such as T cells, due to the lack of insertion mutagenesis caused by circular RNA.In some embodiments, this can be demonstrated by demonstrating that circular RNA is not integrated into the genome, while the DNA delivered by lentivirus is integrated into the genome, by sequencing after administering circular RNA or DNA.In certain embodiments, the circular RNA provided herein can be directly transfected into cells, or can be transfected in the form of a DNA vector and transcribed in cells.The transcription of circular RNA from transfected DNA vector can be mediated by additional polymerase or the polymerase encoded by nucleic acid transfected into cells, or preferably by 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 nucleosides include 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 In another embodiment, the modified nucleoside is s2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine). In 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 -glycinylcarbamoyl adenosine);t 6 A(N 6 -threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -threonylcarbamoyl adenosine);m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyl adenosine);hn 6 A(N 6 -hydroxynorvalylcarbamoyl adenosine);ms 2 hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; 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(lycidin);m 1 G(1-methylguanosine);m 2 G(N 2 -methylguanosine);m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 ,N 2 -dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine(phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW * (unmodified hydroxywybutosine); imG (wybutosine); mimG (methylwybutosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0 (7-cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G + (Archaeosin); 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-hydroxyacetic 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-demethylwyosine); imG2(isowyosine); or ac 6 A(N 6 -acetyladenosine).

[0182] In some embodiments, the modified nucleoside is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -Taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-sh Douridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine , Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 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, wyosine, wyobutosine, 7-deaza-guanosine In another embodiment, the modifications may include compounds selected from the group consisting of 5-methylcytosine, 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 modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine, and 1-methylpseudouridine.

[0183] In some embodiments, modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and / or pseudouridine, hi some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.

[0184] In certain embodiments, polynucleotides may be codon-optimized. A codon-optimized sequence can be a sequence in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons containing context; (iv) variation of codons according to their decoding tRNAs; (v) variation of codons according to GC % either globally or at a single position in a triplet; (vi) variation in similarity to a reference sequence, such as a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence based on which the codon substitution set is designed; and / or (x) systematic variation of the codon set for each amino acid. In some embodiments, the codon-optimized polynucleotide may minimize ribozyme conflicts and / or limit structural interference between the expression sequence and the IRES.

[0185] In certain embodiments, the circular RNAs provided herein are produced inside cells. In some embodiments, precursor RNAs are transcribed using a DNA template (e.g., in some embodiments, using a vector provided herein) in the cytoplasm by a bacteriophage RNA polymerase or in the nucleus by host RNA polymerase II, and then circularized.

[0186] In certain embodiments, the circular RNAs provided herein are injected into an animal (e.g., a human) such that the polypeptide (e.g., a CAR or TCR complex protein) encoded by the circular RNA molecule is expressed inside the animal.

[0187] 3. Payload In some embodiments, the expressed 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 TIFF2025072522000001.tif201166TIFF2025072522000002.tif230166TIFF2025072522000003.tif240166TIFF2025072522000004.tif23516 6TIFF2025072522000005.tif239166TIFF2025072522000006.tif240166TIFF2025072522000007.tif240166TIFF2025072522000008.tif69166

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

[0190] 4. Ionizable lipids In certain embodiments, disclosed herein are lipids that can be used as components of transport vehicles to facilitate or enhance the delivery and release of circular RNA into one or more target cells (e.g., by penetrating or fusing with the lipid membranes of such target cells).

[0191] In some embodiments, the lipid or transport vehicle is selected from the group consisting of lipids and nucleotides described in International Patent Application No. PCT / US2010 / 061058, pages 32-55; U.S. Application Publication No. US2019 / 0314524, paragraphs 86-117; International Patent Application No. PCT / US2018 / 058555, pages 43-146; International Patent Application No. PCT / US2018 / 053569, pages 46-51; International Patent Application No. PCT / US2017 / 028981, paragraphs 195-217; U.S. Application Publication No. US2019 / 032148 9, paragraphs 82-95; U.S. Application Publication No. US2019 / 0314284, paragraphs 5-19 and / or 38-77; Tables 1-4 of International Patent Application No. PCT / US2019 / 025246; U.S. Application Publication No. 20190091164, paragraphs 92-107; and International Patent Application No. PCT / US2019 / 015913, pages 78-97, 109-164, and / or 190-217 (the contents of each 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), which, for example, allow the hydrophilic functional head group of the compound to dissociate 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 represented by Formula 1 or is 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 is 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 lipid is 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 of which are incorporated herein by reference in their entirety.

[0193] In some embodiments, the lipid or transport vehicle is selected from the group consisting of lipids and soluble lipids, and lipids described 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 S2019 / 016362, U.S. Application 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 entireties.

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

[0195] Table 2: Exemplary lipids TIFF2025072522000009.tif48170TIFF2025072522000010.tif238170TIFF2025072522000011.tif209170TIFF2025072522000012.tif199170TIFF2025072522000013.tif217170TIFF2025072522000014.tif237170TIFF2025072522000015.tif223170TIFF2025072522000016.tif207170TIFF2025072522000017.tif221170TIFF2025072522000018.tif223170TIFF2025072522000019.tif239170TIFF2025072522000020.tif216170TIFF2025072522000021.tif233170TIFF2025072522000022.tif141170TIFF2025072522000023.tif223170TIFF2025072522000024.tif189170TIFF2025072522000025.tif194170TIFF2025072522000026.tif64170

[0196] 5. PEG lipid The use and inclusion 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), in the liposomes and pharmaceutical compositions described herein, preferably in combination with one or more of the compounds and lipids disclosed herein, is contemplated. Contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6 to C20 length. In some embodiments, the PEG-modified lipid employed in the compositions and methods of the present invention is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (2000 MW PEG) "DMG-PEG2000." The addition of PEG-modified lipids to lipid delivery vehicles can prevent complex aggregation, prolong circulation life, and provide a means of increasing delivery of lipid-polynucleotide compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected to rapidly exchange from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C14 or C18). PEG-modified phospholipids and derivatized lipids of the present invention can comprise a molar ratio 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 liposomal lipid nanoparticles.

[0197] In some embodiments, the PEG-modified lipids are described in International Patent Application PCT / US2019 / 015913. In some embodiments, the delivery 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-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0199] In some embodiments, 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-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloctylpropyl-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 about C 14 ~About C 22 , for example, about C 14 ~About C 16In 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. Pat. No. 8,158,601 and International Publication No. WO / 2015 / 130584 A2, which are incorporated herein by reference in their entireties.

[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 may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids may 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 may be 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, which has the following structure: I have TIFF2025072522000027.tif19128.

[0207] In one embodiment, the PEG lipid useful in the present invention can be a PEGylated lipid described in International Publication No. WO2012099755 (the contents of which are incorporated herein by reference in their entirety).All of these exemplary PEG lipids described herein can be modified to include hydroxyl groups on the PEG chain.In certain embodiments, the PEG lipid is a PEG-OH lipid.In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes 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): TIFF2025072522000028.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 in R are independently selected from C 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4-10 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 by S(O)2O-; R N Each instance of is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0209] For example, R is a C17 alkyl. For example, a PEG lipid can be represented by the formula (P1-a): TIFF2025072522000029.tif12128, or a salt or isomer thereof, wherein r is an integer between 1 and 100.

[0210] For example, a PEG lipid may have the following formula: The compound is TIFF2025072522000030.tif12128.

[0211] 6. Helper lipids In some embodiments, the transport vehicles (e.g., LNPs) described herein comprise one or more non-cationic 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 can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

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

[0213] The fatty acid moiety may be selected from the 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 analog, oleic acid, an oleic acid analog, or a DSPC substitute.

[0217] In some embodiments, the helper lipid is described in PCT / US2018 / 053569. Helper lipids suitable for use in the lipid compositions of the present disclosure include, for example, various 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), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine ( The fatty acids may include, but are not limited to, 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-diecocenoyl-sn-glycero-3-phosphocholine (DEPC), paimitoioieoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the helper lipid can be distearoylphosphatidylcholine (DSPC) or dimyristoylphosphatidylethanolamine (DMPE).In another embodiment, the helper lipid can be distearoylphosphatidylcholine (DSPC). The helper lipid functions to stabilize and improve 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 an ionizable lipid, the helper lipid can comprise a molar ratio of 5% to about 90%, or about 10% to about 70%, of the total lipid present in the lipid nanoparticle.

[0218] 7. Structured lipids In some embodiments, the structured lipids are described in International Patent Application PCT / US2019 / 015913.

[0219] The transport vehicle described herein comprises one or more structured lipids. The incorporation of structured lipids into lipid nanoparticles can help reduce the aggregation of other lipids in the particles. The structured lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, basicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid comprises cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, hydrocortisone, etc.), or a combination thereof.

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

[0221] The transport vehicle described herein comprises one or more structured lipids. The incorporation of structured lipids into transport vehicles, such as lipid nanoparticles, can help reduce the aggregation of other lipids in the particles. In certain embodiments, the structured lipid comprises cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0222] In some embodiments, the structured lipid is a sterol. The structured lipid can include, but is not limited to, a sterol (e.g., a phytosterol or a zoosterol).

[0223] In certain embodiments, the structural lipid is a steroid. For example, the sterol can include, but is not limited to, cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, 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 and expressed by immune cells, including T cells, via circular RNA as described herein. CARs allow a single receptor to be programmed to both recognize a specific antigen and, upon binding to that antigen, activate the immune cell to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. In some embodiments, the CAR encoded by the polynucleotide comprises (i) an antigen-binding molecule that specifically binds to the target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activation domain.

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

[0226] CARs are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain. Characteristics of CARs include the ability to redirect T cell specificity and reactivity to at least one selected target (e.g., in a non-MHC-restricted manner) by utilizing the antigen-binding properties of monoclonal antibodies. The ability of CARs to recognize non-MHC-restricted antigens gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby circumventing a major mechanism of tumor escape. Bispecific CARs are specific for two different antigens. In certain embodiments, certain polynucleotides provided herein encode bispecific CARs.

[0227] In some embodiments, the CAR comprises a transmembrane domain. In certain embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the CAR comprises a CD8α (CD8 alpha) hinge and transmembrane domain. In preferred embodiments, the CD8 is human. The CAR may comprise less than the entire CD8 protein. (..) In certain embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD28 hinge and transmembrane domain. In preferred embodiments, the CD28 is human. The CAR may comprise less than the entire CD28 protein.

[0228] In some embodiments, a CAR orientation according to the present disclosure comprises an antigen-binding domain (such as an scFv) in tandem with a costimulatory domain and an activation domain. The costimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains may be utilized in tandem.

[0229] In some embodiments, the CAR comprises a CAR protein spacer. The CAR protein spacer can be between any of the aforementioned domains. In certain embodiments, the CAR comprises an IgG heavy chain constant domain (CH2CH3) spacer. In further embodiments, the CAR protein spacer can be between the scFv and transmembrane domains. 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 forms part of a 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 forms part of a 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 comprises an artificial binding domain and / or costimulatory domain.

[0231] In certain embodiments, the TCR complex protein comprises 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 sequence of a naturally occurring TCR Va, Vβ, Ca and / or Cβ. In some embodiments, each CDR or TCR complex protein comprises zero changes or at most one, two, or three changes from a TCR or fragment or derivative thereof 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 an antigen-binding molecule that interacts with the target antigen. In some embodiments, the antigen-binding molecule is an antibody fragment thereof, such as one or more single-chain antibody fragments (scFvs). An scFv is a single-chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked to each other. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. An 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 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 an 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 contemplated within the scope of the present invention, with specificity for more than one target of interest. In some embodiments, the antigen-binding domain is an aptamer or nanobody specific for the target antigen.

[0233] In some embodiments, the antigen-binding molecule comprises a single chain, and the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N-terminus of the linker, and the VL is located at the C-terminus of the linker. In other embodiments, the VL is located at the N-terminus of the linker, and the 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, the CAR or TCR is selected from the group CD19, CD123, CD22, CD30, CD171, CS-1, C-type 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, mesothelin, iPSC, IL-16, IL-16-2 ... 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 mutation (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrant) 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),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 differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), and pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading 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 cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis 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 cells 1, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (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 myelocytomatosis viral 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 cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchoring protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mutated) 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, ανβ6 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 insert domain receptor (KDR), k-light chain, L1 cell adhesion molecule, MUC18, NKG2D, carcinoembryonic antigen (h5T4), tumor / testis 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), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen,The antibody comprises an antigen-binding domain specific for an antigen selected from HHV-6B U94 latent antigen, HHV-6B p98 late 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 lymphotropic 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, a CAR of the present disclosure comprises a hinge or spacer domain. In some embodiments, the hinge / spacer domain may comprise a truncated hinge / spacer domain (THD), which is a shortened version of the complete hinge / spacer domain ("CHD"). In some embodiments, the extracellular domain comprises 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 (CEACAM3), CD66e (CEACAM5), 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, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof (e.g., including all or fragments thereof). Hinge or spacer domains can be derived from either natural or synthetic sources.

[0236] In some embodiments, a hinge or spacer domain is positioned 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 an immunoglobulin. In some embodiments, the hinge or spacer domain is selected from the hinge / spacer regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, or fragments thereof. In some embodiments, the hinge or spacer domain comprises, is from, or is derived from the hinge / spacer region of CD8 alpha. In some embodiments, the hinge or spacer domain comprises, is from, or is derived from the hinge / spacer region of CD28. In some embodiments, the hinge or spacer domain comprises a fragment of the hinge / spacer region of CD8alpha or a fragment of the hinge / spacer region of CD28, wherein the fragment is any fragment smaller than the entire hinge / spacer region. In some embodiments, the fragment of the CD8alpha hinge / spacer region or the fragment of the CD28 hinge / spacer region 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, C-terminus, or both, of the CD8alpha hinge / spacer region or CD28 hinge / spacer region.

[0237] Transmembrane domain The CAR of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. Similarly, it may be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain may be selected or modified (e.g., by amino acid substitution) to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0238] Transmembrane regions include receptor tyrosine kinases (e.g., ErbB2), glycophorin A (GpA), 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 c, 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. GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, ligand specifically binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPL G (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof.

[0239] In some embodiments, the receptor tyrosine kinase is selected from the group consisting of 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 proto-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 insert domain receptor (VEGFR-2), fms-related tyrosine kinase 2 (FLT3), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 1 (VEGFR-1), fms-related tyrosine kinase 2 (VEGFR-2), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 2 (VEGFR-2), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 1 (VEGFR-1), fms-related tyrosine kinase 2 (VEGFR-2), fms-related tyrosine kinase 3 (FLT3 ...3 (FLT3), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 3 (VEGFR-1), fms-related tyrosine kinase 2 (VEGFR-2), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 3 (FLT3), fms-related Vesicle tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), OR2), muscle-associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage-stimulating 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), EPH receptor A3 (EphA4), 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),The gene may be derived from (e.g., comprise) 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] Costimulatory domain In certain embodiments, the CAR comprises a costimulatory domain. In some embodiments, the costimulatory domain includes 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In a preferred embodiment, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (ζ). Less than the entire 4-1BB, CD28, CD3 zeta, or any of these may be included. Chimeric antigen receptors may incorporate costimulatory (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. (supra), 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 costimulatory domain comprises the amino acid sequence of SEQ ID NO: 318 or 320.

[0242] Intracellular signaling domains The intracellular (signaling) domain of the engineered T cells disclosed herein can provide signaling to the activation domain, which in turn activates at least one of the normal effector functions of an immune cell. For example, the effector function of a T cell can be cytolytic activity or a helper activity, such as the secretion of cytokines.

[0243] In some embodiments, suitable intracellular signaling domains include 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, 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 la, CD1 lb, CD1 lc, CD1 Id, CDS, CEACAM1, 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 specific binding ligand, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162) , signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof.

[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 CARs. In some embodiments, the CD3 is CD3 zeta. In some embodiments, the activation domain comprises 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. Polynucleotide Production The vectors provided herein can be produced using standard techniques of molecular biology. For example, various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving the polynucleotide from a vector known to contain the polynucleotide.

[0246] Various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on known sequences. Complete sequences can be assembled from overlapping oligonucleotides prepared by standard methods and then assembled into complete sequences. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:631 1.

[0247] Thus, a specific nucleotide sequence can be obtained from a vector harboring the desired sequence, or can be synthesized, in whole or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques, as appropriate. One method for obtaining a nucleotide sequence encoding a desired vector element is by annealing a complementary set of overlapping synthetic oligonucleotides produced in a conventional automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase, and amplifying the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-directed 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 gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0248] The precursor RNAs provided herein can be produced by incubating a vector provided herein under conditions that allow transcription of the precursor RNA encoded by the vector. For example, in some embodiments, precursor RNAs are synthesized by incubating a vector provided herein that includes an RNA polymerase promoter upstream of its 5' duplex-forming 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 inside a cell with a bacteriophage RNA polymerase or in the nucleus of a cell with host RNA polymerase II.

[0249] In certain embodiments, provided herein are methods for generating precursor RNA by in vitro transcription using a vector provided herein as a template (e.g., a vector provided herein having an RNA polymerase promoter positioned upstream of a 5' homologous region).

[0250] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., a circular RNA polynucleotide provided herein) by incubating it in the presence of magnesium ions and guanosine nucleotides or nucleosides at a temperature at which RNA circularization occurs (e.g., between 20°C and 60°C).

[0251] Thus, 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' homology 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' homology region) as a template, and incubating the resulting precursor RNA in the presence of divalent cations (e.g., magnesium ions) and GTP to circularize it to form a circular RNA. In some embodiments, the precursor RNA of the present invention can be circularized in the absence of magnesium ions and GTP and / or without the step of incubation with magnesium ions and GTP. In some embodiments, transcription is performed in the presence of excess GMP.

[0252] In some embodiments, the composition comprising circular RNA is purified. 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 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 splints, triphosphorylated RNA, phosphatase protein, protein ligase, capping enzyme, and / or nicked RNA than 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 aspects, provided herein are pharmaceutical compositions comprising the circular RNAs provided herein. In certain embodiments, such pharmaceutical compositions are formulated with nanoparticles to facilitate delivery.

[0254] In certain embodiments, the circular RNA provided herein can be delivered and / or targeted to cells in a delivery vehicle, such as a nanoparticle or a composition comprising a nanoparticle. In some embodiments, the circular RNA can also be delivered to a subject in a delivery vehicle or a composition comprising a delivery vehicle. In some embodiments, the delivery vehicle is a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a non-lipid polymer core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the delivery vehicle comprises one or more cationic lipids, non-cationic lipids, ionizable lipids, PEG-modified lipids, polyglutamic acid lipids, hyaluronic acid lipids, poly-β-amino esters, poly-β-amino peptides, or positively charged peptides.

[0255] In one embodiment, the transport vehicle can be selected and / or prepared to optimize the delivery of circRNA to target cells. For example, if the target cells are hepatocytes, the properties of the transport vehicle (e.g., size, charge, and / or pH) can be optimized to effectively deliver the transport vehicle to the target cells, reduce immune clearance, and / or promote retention in the target cells. Alternatively, if the target cells are the central nervous system (e.g., circRNAs 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 the permeability of the blood-brain barrier; retention within the blood-brain barrier; and / or the use of alternative means to directly deliver the transport vehicle to the target cells. In one embodiment, the compositions of the present invention can 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 present invention contemplates the use of transport vehicles to facilitate the delivery of nucleic acids to target cells. Liposomes (e.g., liposomal lipid nanoparticles) are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as transport vehicles for diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol. Rev., 51:691-743, 1999), and are typically characterized as microscopic vesicles with an internal aqueous space separated 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 the liposome can also be formed by an amphiphilic polymer and a surfactant (eg, polymerosome, niosome, etc.).

[0257] In the context of the present invention, a delivery vehicle typically serves to transport circRNAs to target cells. For the purposes of the present invention, the delivery vehicle is prepared to contain a desired nucleic acid. The process of incorporating a desired entity (e.g., a 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 a liposome may be located completely or partially in the interior space of the liposome, within the bilayer membrane of the liposome, or in association with the outer surface of the liposome membrane. The purpose of incorporating a circRNA into a delivery vehicle such as a liposome is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade the nucleic acid and / or from systems or receptors that cause rapid excretion of the nucleic acid. Therefore, in certain embodiments of the present invention, the selected delivery vehicle can improve the stability of the circRNA contained therein. Liposomes can allow the encapsulated circRNA to reach target cells and / or can allow the encapsulated circRNA to reach target cells, or alternatively, limit the delivery of such circRNA to other sites or cells where the presence of the 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, delivery vehicles are prepared to encapsulate one or more desired circRNAs so that the compositions exhibit 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 can facilitate the transfection of some types of cationic liposomes and, in some cases, can significantly improve the transfection efficiency of some types of cationic liposomes by 2-28 fold 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 an embodiment of the present invention, the delivery vehicle is formulated as a lipid nanoparticle. In some embodiments, the lipid nanoparticle is 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 delivery vehicles, alone or in combination with other delivery vehicles, is also contemplated. Suitable polymers may include, for example, polyacrylate, polyalkylcyanoacrylate, polylactide, polylactide-polyglycolide copolymer, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, dendrimer, and polyethyleneimine. In some embodiments, the delivery vehicle is formulated as a lipid, as described in U.S. Patent Application No. 16 / 065,067 (incorporated herein in its entirety). In one embodiment, a delivery vehicle is selected based on its ability to facilitate transfection of the circRNA into target cells.

[0260] The present invention contemplates the use of lipid nanoparticles containing cationic lipids as transport vehicles to encapsulate circRNA and / or enhance its delivery to target cells, which can serve as a depot for protein production. The contemplated lipid nanoparticles can be prepared by incorporating 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 invention include those described in International Patent Publication WO 2010 / 053572 and / or U.S. Patent Application No. 15 / 809,680, such as C12-200. In certain embodiments, the compositions and methods of the invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5000), The present invention employs lipid nanoparticles comprising ionizable cationic lipids described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (incorporated herein by reference), such as (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[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. Pat. No. 4,897,355). DOTMA, alone or in combination with the neutral lipid, dioleoylphosphatidyl-ethanolamine or "DOPE," or other cationic or non-cationic lipids, can be formulated into transport vehicles or lipid nanoparticles, and such liposomes can be used to enhance 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-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or "DOSPA" (Behr et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). Acad. Sci. 86, 6982 (1989); U.S. Patent Nos. 5,171,678; 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP," 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP." Contemplated cationic lipids 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-aminopropane, and 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane. Aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoyl)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoyl)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilin ... Also included are 2,2-dilinoleyl-4-dimethylaminopropane or "DLincarbDAP," 1,2-dilinoleoylcarbamyl-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-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (see WO 2010 / 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 present invention also contemplates the use of cholesterol-based cationic lipids.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-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)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 for improving transfection efficiency are commercially available, suitable examples of which include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, CA), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE 2000 (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, such as those described in US Pat. No. 10,413,618, are also contemplated.

[0266] In other embodiments, the compositions and methods described herein relate to lipid nanoparticles comprising one or more cleavable lipids, e.g., one or more cationic lipids or compounds comprising a cleavable disulfide (SS) functional group (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and HGT4005), as further described in U.S. Provisional Application No. 61 / 494,745, the entire teachings of which are incorporated herein by reference in their entirety.

[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), alone or in combination with other lipids comprising delivery vehicles (e.g., lipid nanoparticles), is also contemplated by the present invention. Contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids with alkyl chain(s) of C6 to C20 length. The addition of such components may prevent complex aggregation, prolong circulation life, and provide a means of increasing 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 exchanged from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C14 or C18). PEG-modified phospholipids and derivatized lipids of the present invention may comprise a molar ratio 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 lipid present in the delivery vehicle.

[0268] The present invention also contemplates the use of non-cationic lipids, including those described in U.S. Patent Application No. 15 / 809,680, including distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine (D ... Non-cationic lipids include, but are not limited to, diolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such non-cationic lipids can be used alone or in combination with other excipients, such as cationic lipids. When used in combination with cationic lipids, the non-cationic lipid can comprise a molar ratio of 5 to about 90%, or about 10% to about 70%, of the total lipid present in the transport vehicle.

[0269] Delivery vehicles (e.g., lipid nanoparticles) can be prepared by combining multiple lipid and / or polymer components. For example, delivery vehicles can be prepared using C12-200, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol, and DMG-PEG2K in a molar ratio of 18:56:20:6, 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 that make up the lipid nanoparticles, as well as the relative molar ratios of such lipids to each other, is based on the characteristics of the selected lipid(s), the nature of the intended target cells, and the characteristics of the circRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipid(s). Thus, the molar ratio can be adjusted accordingly. For example, in some embodiments, the percentage of cationic lipids in the lipid nanoparticles can 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 the lipid nanoparticles can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in the lipid nanoparticles can be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipids in the lipid nanoparticles can be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0270] The transport vehicle for use in the composition of the present invention can be prepared by various techniques currently known in the art.Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example, by depositing selected lipids on the inner wall of a suitable container or vessel, dissolving the lipids in a suitable solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying.Then, aqueous phase is added to the vessel using vortexing, thereby forming MLVs.Then, unilamellar vesicles (ULVs) can be formed by homogenizing, sonicating or extruding the multilamellar vesicles.In addition, ULVs can be formed by detergent removal technology.

[0271] In a specific embodiment of the present invention, the composition of the present invention comprises a transport vehicle, and circRNA is associated with both surfaces of the transport vehicle and is encapsulated in the same transport vehicle.For example, during the preparation of the composition of the present invention, cationic transport vehicles can be associated with circRNA through electrostatic interaction.

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

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

[0274] Various alternative methods known in the art are available for sizing the population of delivery vehicles. One such sizing method is described in U.S. Pat. No. 4,737,323 (incorporated herein by reference). Sonicating a liposome suspension, either by bath or probe sonication, results in a gradual size reduction to small ULVs with diameters less than about 0.05 microns. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller ones. In a typical homogenization process, MLVs are recirculated through a standard emulsion homogenizer until a selected liposome size, typically about 0.1 to 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 assessment to guide efficient liposome synthesis.

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

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

[0277] The present invention also contemplates 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 a transport vehicle in vivo, without relying on the use of additional excipients or means to enhance the recognition of the transport vehicle by target cells. For example, transport vehicles that are subject to phagocytosis by cells of the reticuloendothelial system are likely to accumulate in the liver or spleen, thus providing a means to passively direct the delivery of compositions to such target cells.

[0278] Alternatively, the present invention contemplates active targeting, which involves using a targeting moiety that can be bound (either covalently or non-covalently) to a transport vehicle to facilitate localization of such a transport vehicle at a specific target cell or tissue. For example, targeting can be mediated by including one or more endogenous targeting moieties in or on the transport vehicle to facilitate distribution to the target cell or tissue. Recognition of the targeting moiety by the target tissue actively facilitates tissue distribution and cellular uptake of the transport vehicle and / or its contents in the target cells and tissues (e.g., including an apolipoprotein E targeting ligand in or on the transport vehicle facilitates recognition and binding of the transport vehicle to the endogenous low-density lipoprotein receptor expressed by hepatocytes). As provided herein, the composition can include a moiety that can improve the affinity of the composition for target cells. The targeting moiety can be linked to the outer bilayer of the lipid particle during or after formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers, such as PEAA, hemagglutinin, other lipopeptides (see U.S. Patent Application Nos. 08 / 835,281 and 60 / 083,294, incorporated herein by reference), and other properties useful for in vivo and / or intracellular delivery. In some other embodiments, the compositions of the present invention demonstrate improved transfection efficiency and / or exhibit increased selectivity for the intended target cell or tissue. 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 content for the target cell or tissue are contemplated. Suitable moieties may optionally be bound or linked to the surface of the delivery vehicle. In some embodiments, the targeting moiety may extend over the surface of the delivery vehicle or be encapsulated within the delivery vehicle. Suitable moieties are selected based on their physical, chemical, or biological properties (e.g., selective affinity and / or recognition of target cell surface markers or features). Cell-specific targeting moieties and their corresponding targeting ligands can vary widely.Suitable targeting moieties are selected to take advantage of the unique characteristics of target cells, thereby enabling the composition to distinguish between target cells and non-target cells. For example, the compositions of the present invention may include a surface marker (e.g., apolipoprotein B or apolipoprotein E) that selectively enhances hepatocyte recognition or affinity (e.g., by receptor-mediated recognition of and binding to such surface markers). For example, the use of galactose as a targeting moiety can be expected to direct the compositions of the present invention to hepatocytes, or alternatively, the use of mannose containing sugar residues as a targeting ligand can be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that can preferentially bind to the asialoglycoprotein receptor present on hepatocytes). (See Hillery AM, et al. "Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists" (2002) Taylor & Francis, Inc.). The presentation of such targeting moieties conjugated to moieties present in the delivery vehicle (e.g., lipid nanoparticles) thus facilitates recognition and uptake of the compositions of the 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 comprises a targeting moiety. In some embodiments, the targeting moiety mediates receptor-mediated endocytosis selectively 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 macrocycle, 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 antibody is selected from the group consisting of an antibody, an anti-CD35 antibody, an anti-CD40 antibody, an anti-CD45RA antibody, an anti-CD45RO antibody, an anti-CD52 antibody, an anti-CD56 antibody, an anti-CD62L antibody, an anti-CD68 antibody, an anti-CD80 antibody, an anti-CD95 antibody, an anti-CD117 antibody, an anti-CD127 antibody, an anti-CD133 antibody, an anti-CD137 (4-1BB) antibody, an anti-CD163 antibody, an anti-C1q antibody, an anti-F4 / 80 antibody, an anti-IL-4Rα antibody, an anti-Sca-1 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-GARP antibody, an anti-LAP antibody, an anti-granzyme B antibody, an anti-LFA-1 antibody, an anti-transferrin receptor antibody, and fragments thereof.

[0280] In some embodiments, the circular RNA is formulated according to the process described in U.S. Patent Application Serial No. 15 / 809,680. In some embodiments, the present invention provides a process for encapsulating circular RNA in lipid nanoparticles, which involves 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 RNA formulations with higher potency (peptide or protein expression) and higher efficacy (improvements in biologically relevant endpoints) both in vitro and in vivo, potentially with better tolerability, compared to the same RNA formulations prepared without preforming lipid nanoparticles (e.g., by directly combining lipids with RNA). In some embodiments, the targeting moiety is a small molecule binding agent of an ectoenzyme on lymphocytes. Small molecule binding agents of ectoenzymes include A2A inhibitors, CD73 inhibitors, CD39, or adenosine receptors A2aR and A2bR. Potential small molecules include AB928.

[0281] In some embodiments, the delivery 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 delivery vehicle is comprised of three lipid types. In some embodiments, the delivery vehicle is comprised of four lipid types. In some embodiments, the delivery vehicle is comprised of five lipid types. In some embodiments, the delivery vehicle is comprised of six lipid types.

[0282] In the case of certain cationic lipid nanoparticle formulations of RNA, the RNA in a buffer (e.g., citrate buffer) must be heated to achieve high RNA encapsulation. In these processes or methods, heating after formulation (after nanoparticle formation) does not increase the efficiency of RNA encapsulation in lipid nanoparticles, so heating must be performed before the formulation process (i.e., separate components are heated). In contrast, in some embodiments of the process of the present invention, the order in which the RNA is heated does not appear to affect the RNA encapsulation percentage. In some embodiments, heating 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., they are maintained at ambient temperature).

[0283] RNA can be provided in a solution that can be mixed with lipid solution, so that RNA can be encapsulated in lipid nanoparticles.Suitable RNA solution can be any aqueous solution that contains the RNA to be encapsulated at various concentrations.For example, suitable RNA solution can contain RNA at a concentration of about 0.01mg / ml, 0.05mg / ml, 0.06mg / ml, 0.07mg / ml, 0.08mg / ml, 0.09mg / ml, 0.1mg / ml, 0.15mg / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml, 0.9mg / ml or more than 1.0mg / ml. In some embodiments, suitable RNA solutions comprise RNA at concentrations of about 0.01-1.0 mg / ml, 0.01-0.9 mg / ml, 0.01-0.8 mg / ml, 0.01-0.7 mg / ml, 0.01-0.6 mg / ml, 0.01-0.5 mg / ml, 0.01-0.4 mg / ml, 0.01-0.3 mg / ml, 0.01-0.2 mg / ml, 0.01-0.1 mg / ml, 0.05-1.0 mg / ml, 0.05-0.9 mg / ml , 0.05 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, a suitable concentration of buffer may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 mM to 12 mM.

[0285] Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, a suitable concentration of salt in an 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 suitable RNA solution may have a pH in the range of about 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 the RNA solution suitable for the present invention.In some embodiments, RNA can be directly dissolved in the buffer solution described herein.In some embodiments, RNA solution can be produced by mixing RNA stock solution with buffer solution before mixing with lipid solution for encapsulation.In some embodiments, RNA solution can be produced by mixing RNA stock solution with buffer solution just before mixing with lipid solution for encapsulation.

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

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

[0290] Any desired lipids can be mixed in any ratio suitable for encapsulating RNA.In some embodiments, 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, 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 a nucleic acid to hepatocytes, hepatocytes represent the target cells. In some embodiments, the compositions of the present invention differentially transfect target cells (i.e., do not transfect non-target cells). The compositions of the present invention can 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, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal cells, 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, cardiac myocytes, 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 the heart, lung, kidney, liver, and spleen. In some embodiments, the compositions of the present invention distribute to liver cells, facilitating delivery and subsequent expression of the circRNA contained in the composition by liver cells (e.g., hepatocytes). Targeted cells can function as biological "reservoirs" or "depots" 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 hepatocytes upon delivery. In certain embodiments, following transfection of the target hepatocytes, the circRNA loaded into the vehicle is translated, producing a functional protein product that is excreted and distributed throughout the body. In other embodiments, cells other than hepatocytes (e.g., cells of the lung, spleen, heart, eye, or central nervous system) can serve as depots for protein production.

[0293] In one embodiment, the compositions of the present invention facilitate the endogenous production of one or more functional proteins and / or enzymes in a subject. In certain embodiments of the present invention, the delivery vehicle comprises a circRNA encoding a missing protein or enzyme. Upon distribution of such a composition to a target tissue and subsequent transfection of such target cells, the exogenous circRNA loaded into the delivery 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., a protein or enzyme missing in the subject). Thus, the compositions of the present invention utilize the subject's ability to translate exogenously or recombinantly prepared circRNA to produce an endogenously translated protein or enzyme, thereby producing (and, if applicable, excreting) the functional protein or enzyme. The expressed or translated protein or enzyme may also be characterized in vivo by containing native post-translational modifications that may often not be present in recombinantly prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.

[0294] Administration of circRNAs encoding defective proteins or enzymes avoids the need to deliver nucleic acids to specific organelles within target cells. Rather, upon transfection of the target cell and delivery of the nucleic acid to the cytoplasm of the target cell, the circRNA content of the transport vehicle can be translated and a functional protein or enzyme can be expressed.

[0295] In some embodiments, the circular RNA comprises one or more miRNA binding sites. In some embodiments, the circular RNA comprises one or more miRNA binding sites 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 comprises one or more miRNA binding sites 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). It is believed that miRNAs function by pairing with complementary sequences within RNA molecules, causing gene silencing.

[0296] 12. Pharmaceutical Compositions In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutic agent provided herein. In some embodiments, the therapeutic agent is a circular 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 a circular RNA or vector provided herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the compositions provided herein comprise a therapeutic agent provided herein in combination with other pharmaceutically active agents or drugs, for example, an anti-inflammatory drug or antibody capable of targeting a B cell antigen, for example, an anti-CD20 antibody, for example, rituximab, a chemotherapeutic agent, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises a cell or population thereof provided herein.

[0297] For pharmaceutical compositions, pharmaceutically acceptable carriers can be any commonly used, limited only by chemical and physical considerations, such as solubility and lack of reactivity with the active agent(s), and the route of administration. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. Preferably, pharmaceutically acceptable carriers are chemically inert to the therapeutic agent(s) and have no adverse side effects or toxicity under the conditions of use.

[0298] The choice of carrier will be determined in part by the particular therapeutic agent, as well as the particular method used to administer the therapeutic agent. Accordingly, there are a variety of 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 optionally be used. 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 therapeutic agent in a pharmaceutical composition can vary, e.g., be less than about 1% by weight, or 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 primarily by fluid volume and viscosity according to the particular mode of administration selected.

[0302] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are exemplary only and are in no way limiting. More than one route can be used to administer the therapeutic agents provided herein, and in certain instances, a particular route can provide a more rapid and effective response than another route.

[0303] Formulations suitable for oral administration can comprise or consist of (a) a liquid solution, e.g., an effective amount of the therapeutic agent dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and pastilles, each containing a predetermined amount of the active ingredient as a solid or granules; (c) powders; (d) suspensions in a suitable liquid; and (e) suitable emulsions. Liquid formulations can contain diluents, e.g., water and alcohols, e.g., ethanol, benzyl alcohol, and polyethylene alcohol, with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be, for example, the ordinary hard- or soft-shelled gelatin type containing surfactants, lubricants, and inert fillers, e.g., lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can contain one or more of 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 other excipients, coloring agents, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can contain a therapeutic agent with a flavoring agent, usually sucrose, acacia, or tragacanth. Pastilles can contain a therapeutic agent with an inert base, 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 which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. In some embodiments, the therapeutic agents provided herein can be administered in a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids comprising water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants, such as soaps or detergents, suspending agents, such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants.

[0305] Oils can be used in some embodiments in parenteral formulations, including petroleum, animal, vegetable, 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] Suitable soaps for use in certain embodiments of the parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include: (a) cationic detergents, such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents, such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, such as 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 solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants, for example, having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations will typically range, for example, from 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 with propylene glycol. Parenteral formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid vehicle, such as water, immediately prior to use for injection. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind described above.

[0308] In certain embodiments, injectable formulations are provided herein.The requirements of effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott 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 suitable in certain embodiments provided herein for application to the skin.In some embodiments, the therapeutic agent, alone or in combination with other suitable components, can be made into an aerosol formulation that is administered via inhalation.These aerosol formulations can be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc.They can also be formulated as pharmaceuticals for non-pressurized preparations, such as nebulizers or atomizers.Such spray formulations can also be used to spray mucous membranes.

[0310] In certain embodiments, the therapeutic agents provided herein can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes can help target therapeutic agents to specific tissues. Liposomes can also be used to extend the half-life of therapeutic agents. Many methods are available for preparing liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0311] In some embodiments, the therapeutic agents provided herein are formulated in a time-release, delayed-release, or sustained-release delivery system so that delivery of the composition occurs sufficiently in advance of sensitization of the treatment site. Such a system can avoid repeated administration of the therapeutic agent, thereby increasing convenience for the subject and the physician, and may be particularly suitable for certain composition embodiments provided herein. In one embodiment, the compositions of the present invention are formulated to be suitable for sustained release of the circRNA contained therein. Such sustained-release compositions can be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice daily, daily, or every other day. In some embodiments, the compositions of the present invention are administered to a subject twice weekly, once weekly, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, once monthly, 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 for a sustained period of time. For example, the protein may be produced for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration. In some embodiments, the polypeptide is expressed at a peak level about 6 hours after administration. In some embodiments, the expression of the polypeptide is sustained at at least a therapeutic level. In some embodiments, the polypeptide is expressed at at least a therapeutic level for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in the patient's serum or tissues (e.g., liver or lung). In some embodiments, the detectable polypeptide level is due to continuous expression from the circRNA composition for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration.

[0313] In certain embodiments, the protein encoded by the polynucleotide of the present invention is produced at a level that exceeds normal physiological levels. The protein level can 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 with a deficiency in the relevant protein or polypeptide or a population of individuals with a deficiency in the relevant protein or polypeptide. In some embodiments, the control can be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the expression level of the polypeptide at one or more comparable time points during other therapeutic interventions, such as direct injection of the corresponding polypeptide.

[0314] In certain embodiments, levels of proteins encoded by polynucleotides of the invention are detectable 3, 4, 5 days, or 1 week or more after administration. Increased levels of secreted proteins can be observed in serum and / or tissues (e.g., liver or lung).

[0315] In some embodiments, this method provides the protein encoded by the polynucleotide of the present invention with a sustained circulating half-life.For example, protein can be detected for a longer period of time or days than the half-life observed through subcutaneous injection of protein or mRNA encoding protein.In some embodiments, the half-life of protein is 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week or more.

[0316] Many types of release delivery systems are available and known to those skilled in the art. These include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Drug-containing microcapsules of the aforementioned polymers are described, for example, in U.S. Patent No. 5,075,109. Delivery systems also include non-polymeric systems, including lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral lipids 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 implants; and the like. Specific examples include, but are not limited to, (a) erosion systems, in which the active composition is contained in a matrix form, such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) diffusion systems, in which the active ingredient permeates at a controlled rate from a polymer, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0317] In some embodiments, the therapeutic agent can be directly or indirectly conjugated to the targeting moiety through a linking moiety. Methods for conjugating a therapeutic agent to a targeting moiety 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 a depot form so that the manner in which the therapeutic agent is released into the body to which it is administered can be controlled with respect to time and location within the body (see, e.g., U.S. Pat. No. 4,450,150). A depot form of a therapeutic agent can be, for example, an implantable composition comprising a therapeutic agent and a porous or non-porous material, such as a polymer, where the therapeutic agent is encapsulated by or diffuses throughout the material and / or degrades the non-porous material. The depot is then implanted at a desired location within the body, and the therapeutic agent is released from the implant at a predetermined rate.

[0319] 13.Treatment method In certain aspects, provided herein are methods for treating and / or preventing a condition, e.g., cancer, comprising introducing a pharmaceutical composition provided herein into a subject in need thereof (e.g., a subject with cancer). In some embodiments, the pharmaceutical composition comprises a circular 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) comprising a polynucleotide provided herein (e.g., a circular RNA or vector provided herein).

[0320] Thus, in certain embodiments, provided herein are methods of treating and / or preventing disease in a subject (e.g., a mammalian subject, e.g., a human subject). Without being bound by a particular theory or mechanism, CAR and TCR complex proteins have a biological activity, e.g., the ability to recognize an antigen, e.g., CD19, such that, when expressed by a cell, the CAR or TCR can mediate an immune response against cells expressing the antigen for which the CAR or TCR is specific, e.g., CD19. In this regard, certain embodiments provided herein provide methods of treating or preventing cancer in a mammal, the methods comprising administering to the mammal a therapeutic agent thereof and / or a pharmaceutical composition provided herein in an amount effective to treat or prevent cancer in the mammal.

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

[0322] In certain embodiments, the therapeutic agent is a cell or a population of cells that contains the circular RNA or vector provided herein, and expresses the 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 comprises lymphodepleting the subject prior to administering the therapeutic agent. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0324] In some embodiments, the subject is a mammal. In some embodiments, the mammal referred to herein can be any mammal, including, but not limited to, rodent mammals, such as mice and hamsters, or logomorpha mammals, such as rabbits. The mammal can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). The mammal can be from the order Artiodactyla, including Bovidae (cattle) and Suidae (pigs), or Perissodactyla, including Equidae (horses). The mammal can be from the order Primates, Ceboids, or Simoides (monkeys), or Anthropoids (humans and apes). Preferably, the mammal is a human.

[0325] 14. Array (Table 3) Exemplary IRES sequences. TIFF2025072522000031.tif177160TIFF2025072522000032.tif241160TIFF2025072522000033.tif240160TIFF2025072522000034.t if240160TIFF2025072522000035.tif241160TIFF2025072522000036.tif240160TIFF2025072522000037.tif240160TIFF20250725220 00038.tif241160TIFF2025072522000039.tif241160TIFF2025072522000040.tif241160TIFF2025072522000041.tif241160TIFF202 5072522000042.tif240160TIFF2025072522000043.tif240160TIFF2025072522000044.tif240160TIFF2025072522000045.tif128160

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

[0327] (Table 4) Anabaena permutation site 5' intron fragment sequence. TIFF2025072522000046.tif69160TIFF2025072522000047.tif241160TIFF2025072522000048.tif242160TIFF2025072522000049.tif71160

[0328] In some embodiments, the 5' intron fragment is a fragment having a sequence listed in Table 4. Typically, a construct containing a 5' intron fragment listed in Table 4 will contain a corresponding 3' intron fragment as listed in Table 5 (e.g., both representing fragments with the L9a-8 permutation site).

[0329] (Table 5) Anabaena permutation site 3' intron fragment sequence. TIFF2025072522000050.tif124160TIFF2025072522000051.tif241160TIFF20250725220 00052.tif241160TIFF2025072522000053.tif241160TIFF2025072522000054.tif177160

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

[0331] (Table 6) Non-Anabaena permutation site 5' intron fragment sequences. TIFF2025072522000055.tif188160

[0332] In some embodiments, the 5' intron fragment is a fragment having a sequence listed in Table 6. A construct containing a 5' intron fragment listed in Table 6 will contain a corresponding 3' intron fragment as listed in Table 7 (e.g., both represent fragments with the Azop1 intron).

[0333] (Table 7) Non-Anabaena permutation site 3' intron fragment sequences. TIFF2025072522000056.tif26160TIFF2025072522000057.tif242160TIFF2025072522000058.tif30160

[0334] In some embodiments, the 3' intron fragment is a fragment having a sequence listed in Table 7. A construct containing a 3' intron fragment listed in Table 7 will contain a corresponding 5' intron fragment as listed in Table 6 (e.g., both represent fragments with the Azop1 intron).

[0335] (Table 8) Spacer and Anabear 5' intron fragment sequences. TIFF2025072522000059.tif168160TIFF2025072522000060.tif241160TIFF2025072522000061.tif241160 TIFF2025072522000062.tif249166TIFF2025072522000063.tif242160TIFF2025072522000064.tif114160

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

[0337] (Table 9) Spacer and Anabear 3' intron fragment sequences. TIFF2025072522000065.tif99160TIFF2025072522000066.tif241160TIFF2025 072522000067.tif241160TIFF2025072522000068.tif241160TIFF20250725220 00069.tif241160TIFF2025072522000070.tif241160TIFF2025072522000071.t if241160TIFF2025072522000072.tif241160TIFF2025072522000073.tif153160

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

[0339] (Table 10) CAR sequences. TIFF2025072522000074.tif162160TIFF2025072522000075.tif157160TIFF2025072522000076.t if205160TIFF2025072522000077.tif186160TIFF2025072522000078.tif205160TIFF2025072522 000079.tif205160TIFF2025072522000080.tif201160TIFF2025072522000081.tif239160TIFF20 25072522000082.tif64160TIFF2025072522000083.tif239160TIFF2025072522000084.tif74160

[0340] In some embodiments, the CAR has a sequence as listed in Table 10.

[0341] (Table 11) CAR domain sequences. TIFF2025072522000085.tif104160

[0342] In some embodiments, the CAR domain encoded by the polynucleotides of the 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 upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [Example]

[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 entireties.

[0345] The present invention is further described in detail by reference to the following examples, which are not intended to be limiting, but which include any and all variations of the examples intended to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention.

[0346] Example 1 Example 1A: External regions of homology allow circularization of long precursor RNAs using a permuted intron exon (PIE) circularization strategy. A 1.1-kb sequence containing the full-length encephalomyocarditis virus (EMCV) IRES, a Gaussia luciferase (GLuc) expression sequence, and two short exon fragments of a permuted intron-exon (PIE) construct were inserted between the 3' and 5' introns of the permuted group I catalytic intron of the thymidylate synthase (Td) gene of T4 phage. Precursor RNA was synthesized by runoff transcription. Circularization was attempted by heating the precursor RNA in the presence of magnesium ions and GTP, but no spliced ​​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. Addition of these homologous arms increased splicing efficiency, as assessed by the disappearance of the precursor RNA band, from 0 to 16% for the 9-nucleotide homologous region and to 48% for the 19-nucleotide homologous region.

[0348] The spliced ​​products were treated with RNase R. Sequencing across the putative splice junction of the RNase R-treated splicing reaction revealed the ligated exon, and digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band, in contrast to the two bands obtained by RNase H-digested linear precursors. This indicates that circular RNA is the major product of splicing reactions of precursor RNAs containing external homologous regions 9 or 19 nucleotides long.

[0349] Example 1B: Spacers that preserve the secondary structure of the IRES and PIE splice sites increase the efficiency of circularization. A series of spacers were designed and inserted between the 3' PIE splice site and the IRES. These spacers were designed to preserve or disrupt secondary structure within the IRES, 3' PIE splice site, and / or the intronic sequence of the 5' splice site. Addition of spacer sequences designed to preserve secondary structure resulted in 87% splicing efficiency, while addition of disruptive spacer sequences resulted in no detectable splicing.

[0350] Example 2 Example 2A: Internal homologous regions in addition to external homologous regions create splicing bubbles, allowing translation of some expressed sequences. The spacers were designed to be non-homologous to the unstructured, intronic, and IRES sequences and contain spacer-spacer homology regions. These were inserted between the 5' exon and IRES and between the 3' exon and expression sequences of constructs containing external homology regions, the EMCV IRES, and expression sequences for Gaussia luciferase (total length: 1289 nt), firefly luciferase (2384 nt), eGFP (1451 nt), human erythropoietin (1313 nt), and Cas9 endonuclease (4934 nt). Circularization of all five constructs was achieved. Circularization of constructs using the T4 phage and Anabaena introns was nearly equivalent. Circularization efficiency was higher with shorter sequences. Each construct was transfected into HEK293 cells to measure translation. Gaussia and firefly luciferase transfected cells showed strong responses as measured by luminescence, human erythropoietin was detectable in the medium of cells transfected with erythropoietin circRNA, and EGFP fluorescence was observed from cells transfected with EGFP circRNA. Co-transfection of Cas9 circRNA and sgRNA against GFP into cells constitutively expressing GFP abolished fluorescence in up to 97% of cells compared to sgRNA-only controls.

[0351] Example 2B: Use of the CVB3 IRES increases protein production. Constructs were made with different IRES constructs containing internal and external homology regions and either Gaussia 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: Use of polyA or polyAC spacers increases protein production. A 30-nucleotide poly(A) or poly(AC) spacer was added between the IRES and splice junction of each IRES-containing construct used to produce proteins in Example 2B. Gaussia luciferase 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 a spacer.

[0353] Example 3 HEK293 or HeLa cells transfected with circular RNA produce more protein than cells transfected with equivalent unmodified or modified linear RNA. We compared HPLC-purified Gaussia luciferase-encoding circRNA (CVB3-GLuc-pAC) with standard unmodified 5'-methylguanosine cap and 3'-poly(A) tail linear GLuc mRNA and commercially available nucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA (Trilink). Luminescence measurements 24 h after transfection revealed that the circRNA produced 811.2% more protein than unmodified linear mRNA and 54.5% more protein than modified mRNA in HEK293 cells. Similar results were obtained in HeLa cells and in comparison with an 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 with 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 with 44 hours for unmodified linear mRNA and 49 hours for modified linear mRNA. CircRNAs produced substantially more protein than both unmodified and modified linear mRNAs over their lifespan in both cell types.

[0355] Example 4 Example 4A: Purification of circRNA by RNase digestion, HPLC purification, and phosphatase treatment reduces immunogenicity. Fully purified circular RNA is significantly less immunogenic than unpurified or partially purified circular RNA. Protein expression stability and cell viability depend on the cell type and circular RNA purity. Human embryonic kidney 293 (HEK293) and human lung cancer A549 cells were a. Unpurified GLuc circular RNA splicing reaction product; b. Products of RNase R digestion of the splicing reaction; c. The product of RNase R digestion and HPLC purification of the splicing reaction, or d. Products of RNase digestion, HPLC purification, and phosphatase treatment of splicing reactions was transfected with

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

[0357] The addition of HPLC purification was also insufficient to prevent cytokine release, but it resulted in a significant decrease in interleukin-6 (IL-6) and a significant increase in interferon-α1 (IFN-α1) compared to the unpurified splicing reaction.

[0358] Addition of 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 undetectable or reduced to untransfected baseline levels.

[0359] There was no substantial cytokine release in HEK293 cells. A549 cells transfected with more highly purified circular RNA showed increased GLuc expression stability and cell viability. Fully purified circular RNA had a stability phenotype similar to that of transfected 293 cells.

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

[0361] A549 cells were A. Unpurified circular RNA, b. high molecular weight (linear and circularly linked) RNA; C. circular (nicked) RNA, d. The initial fraction of purified circular RNA (which has a high overlap with the nicked RNA peak), e. late fraction of purified circular RNA (less overlap with the nicked RNA peak), f. an intron excised during circularization, or g. Vehicle (i.e., non-transfected control) was transfected with

[0362] Precursor RNA was synthesized and purified separately in the form of a splice site deletion mutant (DS) due to the difficulty of obtaining suitably pure linear precursor RNA from the splicing reaction. Cytokine release and cell viability were measured in each case.

[0363] Strong IL-6, RANTES, and IP-10 release was observed in response to most species present in the splicing reaction, as well as precursor RNA. The early circRNA fraction induced cytokine responses comparable to those of other non-circRNA fractions, indicating that even relatively small amounts of linear RNA contaminants can induce substantial cellular immune responses in A549 cells. The late circRNA fraction did not induce cytokine responses greater than those from untransfected controls. 36 hours after transfection, A549 cell viability was significantly greater with the late circRNA fraction compared to all other fractions.

[0364] We analyzed the induction of RIG-I and IFN-β1 transcripts upon transfection of A549 cells with late circRNA HPLC fractions, precursor RNA, or unpurified splicing reactions. Induction of both RIG-I and IFN-β1 transcripts was weaker with the late circRNA fraction than with precursor RNA and unpurified splicing reactions. RNase R treatment of the splicing reactions alone was not sufficient to eliminate this effect. Addition of very small amounts of RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-I transcription. In HeLa cells, transfection of RNase R-digested splicing reactions induced RIG-I and IFN-β1, but not 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 reactions or fully purified circRNAs revealed no transient response to circRNAs.Purified circRNAs similarly failed to induce pro-inflammatory transcripts in RAW264.7 murine macrophages.

[0366] A549 cells were transfected with purified circRNA containing the EMCV IRES and EGFP expression sequence, which failed to produce substantial induction of pro-inflammatory transcripts. These data demonstrate that the non-circular components of the splicing reaction are responsible for the immunogenicity observed in previous studies and that circRNAs are not natural ligands of RIG-I.

[0367] Example 5 Circular RNA evades 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] Linearized RNAs were constructed by deleting the intron and homology arm sequences, and then treated with phosphatase (after capping in the case of capped RNA) and purified by HPLC.

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

[0370] In the second experiment, circRNAs were linearized using two methods: heat treatment of circRNAs in the presence of magnesium ions and DNA oligonucleotide-induced RNase H digestion. Both methods yielded mostly full-length linear RNAs and a small amount of intact circRNAs. 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 h posttransfection. 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 mock transfection. Despite activation of TLR3 by in vitro-transcribed linearized RNA, no activation was observed in TLR3 and TLR7 reporter cells under degraded or intact conditions.

[0371] Example 6 Unmodified circular RNA produces increased and sustained in vivo protein expression over linear RNA. Unmodified and m1ψ-modified human erythropoietin (hEpo) linear mRNA and circRNA were used to inject mice and transfect HEK293 cells. Equimolar transfection of m1ψ-mRNA and unmodified circRNA resulted in robust protein expression in HEK293 cells. hEpo linear mRNA and circRNA showed similar relative protein expression patterns and cell viability compared with GLuc linear mRNA and circRNA upon equal weight transfection of HEK293 and A549 cells.

[0372] In mice, hEpo was detected in serum after injection of hEpo circRNA or linear mRNA into the visceral fat layer. The 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 declined rapidly after 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 uniform multilamellar structures with average size, polydispersity index, and encapsulation efficiency similar to those of particles containing a commercially available control linear mRNA modified with 5 moU.

[0374] Purified hEpo circRNAs, when encapsulated in LNPs and added to HEK293 cells, showed higher expression than 5moU-mRNA. The 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 was able to 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 adipose injection of unmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, and the delay in serum expression decay was similar to that observed in vitro. However, 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 IRES-mediated expression and functional stability in HEK293, HepG2, and 1C1C7 cells. Constructs containing the Anabaena intron / exon region, Gaussia luciferase expression sequence, and various IRESs were circularized. 100 ng of each circularization reaction was transfected separately into 20,000 HEK293, HepG2, and 1C1C7 cells using Lipofectamine MessengerMax. Luminescence in each supernatant was assessed after 24 hours as a measure of protein expression. In HEK293 cells, constructs containing the Kurohivirus B, Sarivirus FHB, Aichivirus, Sarivirus HG-J1, and Enterovirus J IRESs produced the most luminescence at 24 hours (Figure 1A). In HepG2 cells, constructs containing the Aichivirus, Sarivirus FHB, EMCV-Cf, and CVA3 IRESs produced the most luminescence at 24 hours (Figure 1B). In 1C1C7 cells, constructs containing sarivirus FHB, Aichivirus, sarivirus NG-J1, and sarivirus A SZ-1 IRES produced high luminescence at 24 h (Fig. 1C ).

[0377] A trend was observed in which larger IRESs produced greater luminescence at 24 hours. Because shorter total sequence lengths tend to increase circularization efficiency, high expression and the selection of a relatively short IRES may result in improved constructs. In HEK293 cells, constructs using the Kurohivirus B IRES produced the highest luminescence, especially 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 Gaussia luciferase in the supernatant was measured every 24 hours after transfection of 20,000 cells with 100 ng of each circularization reaction, followed by a complete medium change. Sarivirus A GUT and Sarivirus FHB exhibited the highest functional stability in HepG2 cells, while Sarivirus N-J1 and Sarivirus FHB produced the most stable expression in 1C1C7 cells (Figures 3A and 3B).

[0379] Example 9 IRES-mediated expression and functional stability in Jurkat cells. Two sets of constructs containing the Anabaena intron / exon region, Gaussia luciferase expression sequences, and a subset of previously tested IRES constructs were circularized. 60,000 Jurkat cells were electroporated with 1 μg of each circularization reaction. 24 hours after electroporation, luminescence from secreted Gaussia luciferase in the supernatant was measured. For comparison between the sets and with the efficacy of previously defined IRES constructs, the CVB3 IRES construct was included in both sets. The CVB1 and Salivirus A SZ1 IRES constructs produced the most expression at 24 hours. Data can be seen in Figures 4A and 4B.

[0380] The functional stability of the IRES constructs in each round of electroporated Jurkat cells was measured over a 3-day period: luminescence from secreted Gaussia luciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 μg of each circularization reaction, followed by a complete medium change (Figures 5A and 5B).

[0381] The Sarivirus A SZ1 and Sarivirus A BN2 IRES constructs had high functional stability compared to the other constructs.

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

[0383] Luminescence from secreted Gaussia luciferase in the supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 μg of each RNA species, followed by a complete medium change. A comparison of the functional stability data of modified mRNAs and circRNAs in Jurkat cells over a 3-day period is shown in Figure 6B.

[0384] IFNγ (Fig. 7A), IL-6 (Fig. 7B), IL-2 (Fig. 7C), RIG-I (Fig. 7D), IFN-β1 (Fig. 7E), and TNFα (Fig. 7F) transcript induction 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, a known RIG-I agonist).

[0385] Example 11 Expression of circular and linear RNAs in monocytes and macrophages. Constructs containing the Anabaena intron / exon region, Gaussia luciferase expression sequence, and Salivirus FHB IRES were circularized. mRNA containing the Gaussia luciferase expression sequence and a 150-nt poly(A) tail, as well as mRNA modified to replace 100% of uridines with 5-methoxyuridine (5moU), were purchased from Trilink. Expression of circularized and modified mRNAs was measured in human primary monocytes (Figure 8A) and human primary macrophages (Figure 8B). Luminescence from secreted Gaussia luciferase in the supernatant was measured 24 h after electroporation of 60,000 cells with 1 μg of each RNA species. Luminescence was also measured 4 days after electroporation in human primary macrophages, with the medium replaced every 24 h (Figure 8C). Differences in luminescence were statistically significant in both cases (p<0.05).

[0386] Example 12 IRES-mediated expression and functional stability in primary T cells. Constructs containing the Anabaena intron / exon region, Gaussia luciferase expression sequence, and a subset of previously tested IRES constructs were circularized, 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 Gaussia luciferase in the supernatant was measured 24 hours after electroporation (Figure 9A). The Aichi virus and CVB3 IRES constructs were most highly expressed at 24 hours.

[0387] Luminescence was also measured every 24 hours for 3 days after electroporation to compare the functional stability of each construct (Fig. 9B). The construct with the 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. Constructs containing the Anabaena intron / exon region, Gaussia luciferase expression sequence, and the Sarivirus A SZ1 IRES or Sarivirus FHB IRES were circularized. mRNA containing the Gaussia luciferase expression sequence and a 150-nt poly(A) tail, as well as those modified to replace 100% of uridines with 5-methoxyuridine (5moU), were purchased from Trilink. Expression of Sarivirus A SZ1 IRES HPLC-purified circular and modified mRNA was measured in human primary CD3+ T cells. Expression of Sarivirus FHB HPLC-purified circular, unpurified circular, and modified mRNA was measured in human PBMCs. Luminescence from secreted Gaussia luciferase 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. The differences in expression between purified circular RNA and unpurified circular or linear RNA were significant in all cases (p<0.05).

[0389] Luminescence from secreted Gaussia luciferase in primary T cell supernatants was measured every 24 hours for 3 days after electroporation to compare the functional stability of the constructs. Data are shown in Figure 10B. The difference in relative luminescence from measurements on day 1 between purified circular and linear RNA was significant on both days 2 and 3 for primary T cells.

[0390] Example 14 Circularization efficiency by permutation site in the Anabaena intron. An RNA construct containing the CVB3 IRES, Gaussia luciferase expression sequence, Anabaena intron / exon region, spacer, internal homology region, and homology arms was generated. The circularization efficiency of constructs using the conventional Anabaena intron permutation site and five consecutive permutation sites in P9 was measured by HPLC. The HPLC chromatogram for the five consecutive permutation sites in P9 is shown in Figure 11A.

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

[0392] The circular RNAs in this example were circularized by in vitro transcription (IVT) and then purified via spin columns. Circularization efficiency was measured using Mg for all constructs. 2+ The inclusion of an additional step of incubation with ribonucleotides and guanosine nucleotides could potentially increase the cost; however, the removal of this step allowed for comparison between and optimization of circular RNA constructs. This level of optimization is particularly useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

[0393] Example 15 Circularization efficiency of alternative introns. Precursor RNAs containing permuted group 1 introns or permutation sites of various species origins and several constant elements, including the CVB3 IRES, Gaussia luciferase expression sequence, spacer, internal homology region, and homology arms, were generated. Circularization data can be found in Figure 12. Figure 12A shows chromatograms resolving the precursor, CircRNA, and intron. Figure 12B provides ranked quantification of circularization efficiency based on the chromatograms shown in Figure 12A as a function of intron construct.

[0394] In this example, circular RNAs were circularized by in vitro transcription (IVT) followed by spin column purification. Circularization efficiency was measured for all constructs using Mg 2+However, the removal of this step allows for comparison between and optimization of circular RNA constructs. This level of optimization is particularly useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

[0395] Example 16 Circularization efficiency depending on the presence or length of homologous arms. RNA constructs were generated containing the CVB3 IRES, Gaussia luciferase expression sequence, Anabaena intron / exon region, spacer, and internal homology region. Constructs representing the three Anabaena intron permutation sites were tested with 30 nt, 25% GC homology arms or without homology arms ("NA"). These constructs were used in the Mg 2+ Circularization was possible without an incubation step. Circularization efficiencies were measured and compared. The data can be seen in Figure 13. Circularization efficiency was higher for each construct lacking homologous arms. Figure 13A provides a ranked quantification of circularization efficiency; Figure 13B provides a chromatogram resolving the precursor, circRNA, and intron.

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

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

[0398] Figure 15A (right) shows HPLC chromatograms demonstrating increased splicing efficiency coupled with increased nicking, appearing as shoulders on the circRNA peak. Top right: 75% GC content, 10 nt homologous arm. Center right: 75% GC content, 20 nt homologous arm. Bottom right: 75% GC content, 30 nt homologous arm.

[0399] FIG. 15B (left) shows selected combinations of permutation sites and homology arms that were hypothesized to demonstrate improved circularization efficiency.

[0400] FIG. 15B (right) shows selected combinations of permutation sites and homologous arms hypothesized to demonstrate improved circularization efficiency upon treatment with E. coli polyA polymerase.

[0401] In this example, circular RNAs were circularized by in vitro transcription (IVT) followed by spin column purification. Circularization efficiency was evaluated for all constructs by adding guanosine nucleotides and additional Mg 2+ The inclusion of an incubation step could potentially be expensive; however, the removal of this step allowed for comparison between and optimization of circular RNA constructs. This level of optimization is particularly useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

[0402] Example 17 Circular RNA encoding CAR A construct containing the Anabaena intron / exon region, the Kymriah chimeric antigen receptor (CAR) expression sequence, and the CVB3 IRES was circularized. 100,000 human primary CD3+ T cells were electroporated with 500 ng of circRNA and co-cultured with Raji cells stably expressing GFP and firefly luciferase for 24 hours at an effector-to-target ratio (E:T ratio) of 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 mock-electroporated or electroporated with 1 μg of circRNA, then co-cultured with Raji cells stably expressing GFP and firefly luciferase for 48 hours at an E:T ratio of 10:1 (Figure 17).

[0404] Quantification of specific lysis of Raji target cells was determined by detecting firefly luminescence (Figure 18). 100,000 human primary CD3+ T cells, mock-electroporated or electroporated with circRNAs encoding different CAR sequences, were cocultured with Raji cells stably expressing GFP and firefly luciferase for 48 hours. The percentage of specific lysis was defined as 1 - [CAR-conditioned luminescence] / [mock-conditioned luminescence]. The E:T...

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 structured lipid; and (iii) a PEG-modified lipid. A pharmaceutical composition comprising: The pharmaceutical composition, wherein the delivery vehicle is capable of delivering the circular RNA polynucleotide to a human immune cell present in a human subject, such that the CAR is translated in the human immune cell and expressed on the surface of the human immune cell.

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

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the 3' Group I intron fragment and the 5' Group I intron fragment are Anabaena Group I intron fragments.

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

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

6. The IRES is capable of binding to any of the following viruses: Taura syndrome virus, triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, 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 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, Pasivirus A, Pasivirus 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, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Swine pasivirus 1, PLV-CHN, Pasivirus 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 an aptamer against eIF4G.

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 disposed 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. 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), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetran) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine 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),The pharmaceutical composition according to 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 that comprises 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 combinations 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 polymeric nanoparticle, or a biodegradable polymeric 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. The pharmaceutical composition of any one of claims 1 to 26, in an amount effective to treat cancer in the human subject.

28. 28. The pharmaceutical composition of any one of claims 1 to 27, having 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. 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 expressed 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 an L9a-5 permutation site within an intact intron.

35. 34. The circular RNA polynucleotide of claim 33, wherein the 3' and 5' intron fragments are defined by an L8-2 permutation site 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 mutant 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 mutant 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 disposed between the expression sequence and the 5' Group I intron fragment.

40. A circular RNA polynucleotide according to 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 naturally occurring nucleotides.

43. 43. The circular RNA polynucleotide of any one of claims 32 to 42, further comprising a second expressed 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), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetran) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine 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),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. 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 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 according to any one of claims 32 to 51, comprising a CAR comprising:

53. 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 expressed 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 so as to lack at least one endonuclease sensitive site present in a comparable pre-optimised polynucleotide.

59. 59. A circular RNA polynucleotide according to 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. A circular RNA polynucleotide according to any one of claims 32 to 59, having an in vivo functional half-life in humans that is greater 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 a comparable 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 a human cell that is equal to or greater than the functional half-life of a comparable 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 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.

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 an L9a-5 permutation site within an intact intron.

69. 68. The DNA vector of claim 67, wherein the 3' and 5' intron fragments are defined by an L8-2 permutation site 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 expressed 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' and 3' duplex forming regions 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 disposed 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 disposed 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), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetran) subunit, beta type, 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Averso Oncogene fusion protein (bcr-abl) consisting of murine 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),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 of CD28, 4-1BB, OX40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations 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. A eukaryotic cell according to 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 said CAR or TCR complex protein encoded by said circular RNA polynucleotide on their cell surface.

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 according to 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 structured 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 ureteral 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 an L9a-5 permutation site within an intact intron.

113. 112. The RNA polynucleotide of claim 111, wherein the 3' and 5' intron fragments are defined by an L8-2 permutation site 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. An RNA polynucleotide according to 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. 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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