Circular RNA composition

Abstract

Provided herein are circular RNA constructs comprising an IRES and at least one expressed sequence encoding a binding molecule, compositions thereof, and methods of treatment, including treatment for cancer and autoimmune diseases. In particular, circular RNAs comprising an IRES and a CD19-, HER2-, or BCMA-conjugate, optionally combined with a delivery vehicle, are provided. Also described herein are precursor polynucleotides comprising an IRES and at least one expressed sequence encoding a CAR construct.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 423,760, filed November 8, 2022, U.S. Provisional Application No. 63 / 501,820, filed May 12, 2023, and U.S. Provisional Application No. 63 / 509,361, filed June 21, 2023, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided in file entitled "01318-0002-00PCT_SL.xml," created on October 31, 2023, and is 213,220 bytes in size. The information in this Sequence Listing in electronic format is incorporated herein by reference in its entirety. [Background technology]

[0003] Circular RNA (circRNA or oRNA™) is a known stable form of RNA that offers structural and functional advantages over linear RNA, particularly in molecules that tend to fold into inactive higher-order structures (Wang and Ruffner, 1998). Circular RNA polynucleotides lack the free ends necessary for exonuclease-mediated degradation, making them resistant to several RNA degradation mechanisms and resulting in a longer half-life compared to corresponding linear RNAs. Circularization may stabilize RNA polynucleotides, which generally suffer from a short half-life, and may improve the overall efficacy of exogenous mRNA in various applications. Circular RNAs are also particularly interesting and useful for in vivo applications, particularly in research areas of RNA-based control of gene expression and therapeutic approaches, including protein replacement therapy and vaccination.

[0004] Adoptive T cell immunotherapy is a rapidly growing field, particularly in the treatment of cancer. Generally, when chimeric antigen receptor (CAR) T cells, or "CAR-T," engage cancer cells expressing CD19, the T cells become activated, proliferate, and secrete inflammatory cytokines and chemokines, resulting in tumor cell lysis. However, while CAR-T therapy has become an important tool in cancer treatment, it has toxic side effects and complex procedures. Treatment with CAR-T can result in the massive and rapid release of cytokines into the blood, which can cause cytokine release syndrome (CRS) or CAR-T cell-associated encephalopathy syndrome (CRES), also known as CAR-T-associated neurotoxicity. CRS is the most common and well-described toxicity associated with CAR-T therapy, occurring in over 90% of patients of all grades. It is characterized by hyperthermia, hypotension, hypoxia, and / or multi-organ toxicity and can lead to death. Neurotoxicity is characterized by damage to nervous tissue, which can cause tremors, encephalopathy, dizziness, or seizures. Additionally, patients typically undergo lymphocyte depletion therapy prior to infusion. Lymphocyte depletion therapy is known to enhance CAR-T cell proliferation and improve the efficacy of infused CAR-T cells, for example, by altering the tumor phenotype and microenvironment. However, lymphocyte depletion agents often cause side effects in patients. For example, lymphocyte depletion therapy can cause neutropenia, anemia, thrombocytopenia, and immunosuppression, and can increase the risk of infection, among other toxicities. In addition to the toxicity associated with targeted CAR-T therapy, generating modified lymphocytes requires procedures, specialized equipment, and costs. CAR-T therapy requires a combination of protocols to isolate, genetically modify, and selectively expand the reprogrammed cells, and then infuse them back into the patient.

[0005] In compassionate-use anti-CD19 CAR T-cell therapy for refractory systemic lupus erythematosus, autologous T cells from five SLE patients "were transduced with a lentiviral anti-CD19 CAR vector, expanded...and reinfused into the patients after lymphodepletion with fludarabine and cyclophosphamide. The in vivo expanded CAR T cells resulted in intense B-cell depletion, improvement in clinical symptoms, and normalization of laboratory parameters, including seroconversion to anti-double-stranded DNA antibodies. All five patients achieved remission of SLE according to the DORIS criteria after 3 months, and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2)." See Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nature Medicine (2022), and also Nunez et al., Cytokine and reactivity profiles in SLE patients following anti-CD19 CART therapy, Molecular Therapy (2023). Because circRNAs are more stable and can be expressed in a tissue-specific manner, and because their use can avoid the lymphocyte depletion step of conventional therapies, circRNAs offer an attractive alternative to traditional CAR therapy and other therapies. Accordingly, provided herein are circular RNA constructs comprising an internal ribosome entry site (IRES) and at least one expression sequence encoding a binding molecule. In certain embodiments, the binding molecule encodes a CAR targeting a cancer antigen for use in cancer treatment. To facilitate and / or enhance the delivery and release of circRNAs into one or more target cells, circular RNAs can be formulated with a transport vehicle. Thus, lipid nanoparticles (LNPs) or other transport vehicles containing ionized lipids may be used to deliver the circular RNAs described herein, for example, to patients in need of treatment. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mackensen et al.,Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus,Nature Medicine(2022) [Non-patent document 2] Nunez et al.,Cytokine and reactivity profiles in SLE patients following anti-CD19 CART therapy,Molecular Therapy(2023) Summary of the Invention

[0007] The present disclosure provides a circular RNA encoding a cancer-binding polypeptide paired with a lipid transport vehicle for use in treating cancer. In particular, the present disclosure provides a circular RNA comprising an IRES and a nucleic acid encoding a binding molecule, where the IRES and the nucleic acid encoding the binding molecule are paired to optimally express the polypeptide-binding molecule. Surprisingly, it has been found that certain IRES and nucleic acid combinations work better than others for optimal expression. It has also been discovered that certain IRES / binding molecule combinations work better with certain transport vehicles. Thus, particularly preferred combinations of IRES / nucleic acid encoding a binding molecule / transport vehicle are provided herein. In some embodiments, the circular RNAs provided herein can be used to treat or prevent cancer. In some embodiments, the circular RNAs provided herein can be used to treat or prevent autoimmune diseases, such as B-cell-mediated autoimmune diseases (e.g., lupus).

[0008] In some embodiments, an engineered chimeric antigen receptor (CAR) is encoded by a circular RNA and delivered via a lipid transfer vehicle. The engineered circular RNA (circRNA or oRNA) can then be inserted into and expressed by immune cells, including T cells, NK cells, macrophages, and the like. In some embodiments, the CAR recognizes a specific antigen (e.g., CD19, HER2, or BCMA), and upon binding to the antigen, can activate the immune cell to attack and destroy the cell. Thus, the circular RNAs, compositions, and methods herein are useful for mitigating known side effects associated with CAR-T therapy by programming circulating immune cells (e.g., T cells) with tumor-recognition capabilities and by using lipid transfer vehicles (e.g., LNPs) to deliver circular RNA constructs that can efficiently introduce CAR genes into immune cells. Also provided are methods for producing such circularized RNA constructs, as well as methods for treating subjects in need of treatment using circular RNA. A linear precursor RNA polynucleotide is provided to generate a circular RNA construct containing core functional elements, including a translation initiation element (TIE). The TIE may comprise an untranslated region (UTR), an aptamer complex, or a combination thereof. The UTR may be derived entirely or in part from a viral or eukaryotic mRNA. The UTR may comprise a viral or eukaryotic internal ribosome entry site (IRES). Regarding the linear precursor and circular RNA constructs, pharmaceutical compositions are also provided, including an IRES, an expression sequence, and, optionally, a delivery vehicle. In certain embodiments, the circular RNA construct comprises an expression sequence encoding a CAR construct targeting a cancer antigen. The pharmaceutical compositions of the present disclosure are particularly suitable for efficient protein expression in immune cells in vivo. The delivery vehicle may include, for example, an ionizable lipid, a PEG-modified lipid, a helper lipid, and / or a structural lipid capable of encapsulating the circular RNA.

[0009] Thus, the following embodiments are provided: Embodiment 1. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a binding molecule; A circular RNA construct comprising: Embodiment 2. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expression sequence encoding a chimeric antigen receptor (CAR) that targets a cancer antigen; A circular RNA construct comprising: Embodiment 3. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; A circular RNA construct comprising: Embodiment 4. The circular RNA construct of embodiment 3, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 5. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A circular RNA construct comprising: Embodiment 6. The circular RNA construct of embodiment 5, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 7. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a binding molecule; A circular RNA construct comprising: Embodiment 8. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expression sequence encoding a chimeric antigen receptor (CAR) that targets a cancer antigen; A circular RNA construct comprising: Embodiment 9. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; A circular RNA construct comprising: Embodiment 10. The circular RNA construct of embodiment 9, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 11. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A circular RNA construct comprising: Embodiment 12. The circular RNA construct of embodiment 11, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 13. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a binding molecule; and (B) transportation medium; 10. A pharmaceutical composition comprising: Embodiment 14. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) transportation medium; 10. A pharmaceutical composition comprising: Embodiment 15. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressible sequence encoding a binding molecule; and (B) transportation medium; A pharmaceutical composition comprising: Embodiment 16. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) transportation medium; A pharmaceutical composition comprising: Embodiment 17. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a binding molecule; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 18. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 19. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 20. The pharmaceutical composition of embodiment 19, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 21. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 22. The pharmaceutical composition of embodiment 21, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 23. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressible sequence encoding a binding molecule; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 24. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 25. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 26. The pharmaceutical composition of embodiment 25, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 27. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) a transport vehicle containing ionized lipids; A pharmaceutical composition comprising: Embodiment 28. The pharmaceutical composition of embodiment 27, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 29. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a binding molecule; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 30. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 31. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 32. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 33. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 34. The following: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-BCMA conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 35. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressible sequence encoding a binding molecule; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 36. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expression sequence encoding a CAR construct that targets a cancer antigen; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 37. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 38. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-CD19 conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl;30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 39. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 40. The following: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-BCMA conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115; and (B) A transport medium, including: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] A pharmaceutical composition comprising: Embodiment 41. The pharmaceutical composition of any one of embodiments 1-4, 7-10, 13-20, 23-26, 29-32, and 35-38, wherein the CAR construct comprises a CD19 conjugate and the circular RNA comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61. Embodiment 42. The pharmaceutical composition of any one of embodiments 1-4, 7-10, 13-20, 23-26, 29-32, and 35-38, wherein the CAR construct comprises a CD19 conjugate and the circular RNA comprises a sequence selected from any one of SEQ ID NOs: 50-61. Embodiment 43. The pharmaceutical composition of embodiment 42, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59. Embodiment 44. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and wherein the circular RNA comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61. Embodiment 45. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expression sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50-61. Embodiment 46. The pharmaceutical composition of embodiment 45, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59. Embodiment 47. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, wherein the circular RNA comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61, and wherein the delivery vehicle comprises one of the following: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] The pharmaceutical composition comprising: Embodiment 48. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expression sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and the circular RNA comprises a sequence selected from any one of SEQ ID NOs: 50-61, and the delivery vehicle comprises one of the following: (i) an ionizable lipid of formula (I) [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] The pharmaceutical composition comprising: Embodiment 49. The pharmaceutical composition of embodiment 48, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59. Embodiment 50. The circular RNA construct or pharmaceutical composition of any one of embodiments 1-49, wherein said circular RNA construct comprises SEQ ID NO: 50. Embodiment 51. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 51. Embodiment 52. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 52. Embodiment 53. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 54. Embodiment 54. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 55. Embodiment 55. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 56. Embodiment 56. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 58. Embodiment 57. The circular RNA construct or pharmaceutical composition of any one of embodiments 1 to 49, wherein the circular RNA construct comprises SEQ ID NO: 59. Embodiment 58. The pharmaceutical composition of any one of embodiments 5-6, 21-22, 27, 28, 33-34, or 39-40, wherein the IRES comprises the sequence of SEQ ID NO: 8, and the CAR construct comprises a BCMA conjugate, and the BCMA conjugate comprises a sequence selected from any one of SEQ ID NOs: 104-115. Embodiment 59. The pharmaceutical composition of any one of embodiments 13-58, wherein the delivery vehicle comprises an ionizable lipid of formula (I). Embodiment 60. The pharmaceutical composition of embodiment 59, wherein the delivery vehicle comprises a helper lipid, a structural lipid, and a PEG lipid. Embodiment 61. The pharmaceutical composition of any one of embodiments 59-60, wherein the delivery vehicle is formulated with a lipid molar ratio as set forth in Table 4b. Embodiment 62. The pharmaceutical composition of any one of embodiments 13-58, wherein the delivery vehicle comprises an ionizable lipid of formula (II). Embodiment 63. The ionizable lipid is selected from the group consisting of: [ka] 63. The pharmaceutical composition of embodiment 62, wherein the ionizable lipid is selected from the group consisting of: Embodiment 64. The ionizable lipid is selected from the group consisting of: [ka] 64. The pharmaceutical composition of embodiment 63, wherein Embodiment 65. The pharmaceutical composition of any one of embodiments 13-64, wherein the delivery vehicle further comprises at least one lipid selected from a helper lipid, a structural lipid, and a PEG-modified lipid. Embodiment 66. The pharmaceutical composition of embodiment 65, wherein the delivery vehicle comprises PEG-DSPC. Embodiment 67. The pharmaceutical composition of any one of embodiments 13-66, wherein the delivery vehicle is a lipid nanoparticle. Embodiment 68. The pharmaceutical composition of any one of embodiments 13-67, wherein the delivery vehicle further comprises a targeting moiety. Embodiment 69. The pharmaceutical composition of embodiment 68, wherein the targeting moiety is a small molecule, scFv, nanobody, peptide, cyclic peptide, di- or tricyclic peptide, minibody, polynucleotide aptamer, engineered scaffold protein, heavy chain variable region, light chain variable region, or fragment thereof. Embodiment 70. The pharmaceutical composition of any one of embodiments 13-69, further comprising a pharmaceutical salt, a buffer, a diluent, or a combination thereof. Embodiment 71. The circular RNA construct or pharmaceutical composition of any one of the preceding embodiments, wherein said circular RNA further comprises a polyA tract. Embodiment 72. The circular RNA construct or pharmaceutical composition of any one of the preceding embodiments, wherein said circular RNA further comprises at least one miRNA binding site. Embodiment 73. The circular RNA construct or pharmaceutical composition of embodiment 72, wherein said circular RNA comprises at least one miR-122 binding site. Embodiment 74. The circular RNA construct or pharmaceutical composition of any one of the preceding embodiments, wherein at least one expressed sequence encoding the CAR is codon-optimized. Embodiment 75. The circular RNA construct or pharmaceutical composition of any one of the preceding embodiments, wherein said RNA construct further comprises a 5'-enhanced intron element, a 5'-enhanced exon element, a 3'-enhanced exon element, and a 3'-enhanced intron fragment. Embodiment 76. A method for preparing a circular RNA construct or a pharmaceutical composition according to any one of the preceding embodiments. Embodiment 77. A method for treating cancer in a subject by administering an effective amount of a composition comprising the circular RNA construct or the pharmaceutical composition of any one of embodiments 1-75, thereby treating the cancer, and a method for treating an autoimmune disease in a subject by administering an effective amount of a composition comprising the circular RNA construct or the pharmaceutical composition of any one of embodiments 1-75, thereby treating the autoimmune disease. Embodiment 78. Use of a composition comprising the circular RNA construct or the pharmaceutical composition of any one of embodiments 1-75 for the treatment of cancer, and use of a composition comprising the circular RNA construct or the pharmaceutical composition of any one of embodiments 1-75 for the treatment of autoimmune diseases. Embodiment 79. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a binding molecule; A linear precursor RNA polynucleotide comprising: Embodiment 80. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expression sequence encoding a CAR construct that targets a cancer antigen; A linear precursor RNA polynucleotide comprising: Embodiment 81. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; A linear precursor RNA polynucleotide comprising: Embodiment 82. The linear precursor RNA polynucleotide of embodiment 81, wherein said expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 83. The following: (A) an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A linear precursor RNA polynucleotide comprising: Embodiment 84. The linear precursor RNA polynucleotide of embodiment 83, wherein said expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 85. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a binding molecule; A linear precursor RNA polynucleotide comprising: Embodiment 86. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expression sequence encoding a CAR construct that targets a cancer antigen; A linear precursor RNA polynucleotide comprising: Embodiment 87. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; A linear precursor RNA polynucleotide comprising: Embodiment 88. The linear precursor RNA polynucleotide of embodiment 87, wherein said expressed sequence comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34. Embodiment 89. The following: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A linear precursor RNA polynucleotide comprising: Embodiment 90. The linear precursor RNA polynucleotide of embodiment 89, wherein said expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115. Embodiment 91. The linear precursor RNA polynucleotide of any one of embodiments 79 to 90, wherein the expressed sequence is codon-optimized. Embodiment 92. The linear precursor RNA polynucleotide of any one of embodiments 79 to 91, further comprising a 5'-enhanced intronic element, a 5'-enhanced exon element, a 3'-enhanced exon element, and a 3'-enhanced intron fragment. Embodiment 93. In the following order: (A) the 5' enhanced intron element; (B) the 5'-enhanced exon element; (C) a core functional element comprising the IRES, at least one expression sequence encoding a CAR construct targeting a cancer antigen, and optionally a stop codon or stop cassette; (D) the 3'-enhanced exon element, and (E) the 3' enhanced intron element; 93. The linear precursor RNA polynucleotide of embodiment 92, comprising: Embodiment 94. The linear precursor RNA polynucleotide of any one of embodiments 79 to 93, further comprising at least one miRNA binding site. Embodiment 95. The linear precursor RNA polynucleotide of embodiment 94, wherein said precursor RNA comprises at least one miR-122 binding site. Embodiment 96. A DNA vector encoding an RNA polynucleotide according to any one of embodiments 79-95. Embodiment 97. A method for preparing a circular RNA construct, comprising incubating a linear RNA polynucleotide according to any one of embodiments 79-95 under conditions suitable for circularization. Embodiment 98. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 1, 2, 4, and 8, and (ii) at least one expression sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate comprising a sequence selected from any one of SEQ ID NOs: 19 and 20, and the delivery vehicle is a lipid nanoparticle. Embodiment 99. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 8, 16, 17, and 18, and (ii) at least one expression sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate comprising SEQ ID NO: 115, and the delivery vehicle is a lipid nanoparticle. Embodiment 100. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 8, 16, 17, and 18, and (ii) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a HER2 conjugate comprising a nucleotide sequence selected from any one of SEQ ID NOs: 132 or 133, and the delivery vehicle is a lipid nanoparticle. Embodiment 101. The lipid nanoparticles comprise: (i) an ionized lipid of formula (I): [ka] In the formula, n is an integer between 1 and 4, R a is hydrogen or hydroxyl, R1 and R2 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C6-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 is heteroalkyl, or (ii) an ionizable lipid of formula (II) [ka] wherein each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3; R1 and R3 are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight or branched C9-C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 20 Alkyl or C9-C 20 is alkenyl, R2 is selected from the group consisting of: [ka] 101. The pharmaceutical composition according to any one of embodiments 98 to 100, comprising: Embodiment 102. The lipid nanoparticle delivery vehicle comprises an ionizable lipid, wherein the ionizable lipid is [ka] 102. The pharmaceutical composition of embodiment 101, wherein Embodiment 103. The pharmaceutical composition according to any one of embodiments 98-102, wherein the lipid nanoparticle delivery vehicle further comprises at least one lipid selected from a helper lipid, a structural lipid, and a PEG-modified lipid. Embodiment 104. A method of treating cancer, comprising administering to a human subject in need thereof a pharmaceutical composition of any one of embodiments 98-103. Embodiment 105. A method of treating an autoimmune disease, comprising administering to a human subject in need thereof a pharmaceutical composition of any one of embodiments 98-103. Embodiment 106. Use of a composition comprising the circular RNA construct for the treatment of cancer, comprising administering to a human subject in need thereof the pharmaceutical composition of any one of embodiments 98-103. Embodiment 107. Use of a composition comprising the circular RNA construct for the treatment of an autoimmune disease, comprising administering to a human subject in need thereof the pharmaceutical composition of any one of embodiments 98-103. Embodiment 108. The method of embodiment 77, or embodiment 104 or 105, or the use of embodiment 106 or 107, wherein the administration is performed daily, every other day, twice weekly, weekly, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, monthly, every 6 weeks, every 8 weeks, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or yearly. [Brief explanation of the drawings]

[0010] [Figure 1A] Schematic diagram of the sequence insertion site of an exemplary IRES / codon plasmid. The IRES and codon (expression sequence) were synthesized together and inserted into a circular RNA comprising a plasmid "backbone" containing bacterial sequences and 5' and 3' complex accessory elements. Accessory elements may include, but are not limited to, promoters, introns, exons, internal and external spacers, internal duplex regions, and polyA stretches. [Figure 1B] The general sequence construct of a linear precursor RNA polynucleotide (10) is shown. The sequence shown is exemplified, in 5' to 3' order, by 5'-enhanced intronic elements (20), 5'-enhanced exonic elements (30), core functional elements (40), 3'-enhanced exonic elements (50), and 3'-enhanced intronic elements (60). [Figure 1C] An exemplary linear precursor RNA polynucleotide (10) is shown, comprising, in 5' to 3' order: leading untranslated sequence (21), 5' affinity tag (22), 5' external spacer (26), 3' intron fragment (28), 3' exon fragment (32), 5' internal duplex region (34), 5' internal spacer (36), TIE (42), coding element (46), termination region (48), 3' internal spacer (52), 3' internal duplex region (54), 5' exon fragment (56), 5' intron fragment (62), 3' external spacer (64), 3' affinity tag (68), and terminal untranslated sequence (69). [Figure 1D] Exemplary locations of accessory elements (70) (e.g., miRNA binding sites) contained in a linear RNA polynucleotide located within a core functional element (40) are illustrated, e.g., 42 is a TIE (translation initiation element), 46 is a coding region, 47 is a non-coding region, and 48 is a termination region (stop codon or termination cassette).

[0011] [Figure 2] A and B represent a schematic representation of the preliminary process of selecting IRES and codon combinations for circRNA constructs.

[0012] [Figure 3A]The effect of three different codon optimization algorithms is shown. CD19 CAR+ expression (gMFI) was assessed by flow cytometry for each construct in donor 4003, and all sequences are plotted in rank order and sorted by codon optimization algorithm. The open bar on the right indicates the expression of the non-codon-optimized CD19 CAR sequence (positive control). [Figure 3B] The effect of three different codon optimization algorithms is shown. CD19 CAR+ expression (gMFI) was assessed by flow cytometry for each construct in donor 609C, and all sequences are plotted in rank order and sorted by codon optimization algorithm. The open bar on the right indicates the expression of the non-codon-optimized CD19 CAR sequence (positive control).

[0013] [Figure 4A] The effect of three different codon optimization algorithms is shown. MFI (total T cells) was assessed over time after electroporation using the three algorithms compared to positive and mock-negative controls. [Figure 4B] The effect of three different codon optimization algorithms is shown. The percentage of CD3+ cells (CAR-T cell frequency) was assessed over time after electroporation using the three algorithms compared to positive and mock-negative controls. [Figure 4C] The effect of three different codon optimization algorithms is shown. Total cell number (number of CAR-T cells) was assessed over time after electroporation using the three algorithms, compared to a positive control and a mock-negative control.

[0014] [Figure 5A]For circular RNA constructs containing a combination of an IRES and an expression sequence, the T cell MFI (expression) over time (day 1) by IRES is shown for donor 4003. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5B] For circular RNA constructs containing a combination of IRES and expression sequences, the time course (day 2) of T cell MFI (expression) by IRES is shown for donor 4003. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5C] For circular RNA constructs containing a combination of IRES and expression sequences, the IRES-driven T cell MFI (expression) over time (day 3) is shown for donor 4003. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5D] For circular RNA constructs containing a combination of IRES and expression sequences, the time course (day 4) of T cell MFI (expression) by IRES is shown for donor 4003. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5E] For circular RNA constructs containing a combination of IRES and expression sequences, the T cell MFI (expression) over time (day 5) by IRES is shown for donor 4003. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5F] Figure 1 shows T cell MFI (expression) over time (day 2) for IRES-driven circular RNA constructs containing combinations of IRES and expression sequences in donor 609C. Each point on the x-axis represents an IRES from Table 1A, and each point represents a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5G]Circular RNA constructs containing IRES-expression sequences are shown for T cell MFI (expression) over time (day 1) in donor 609C. Each point on the x-axis represents an IRES from Table 1A, and each point represents a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5H] Figure 1 shows T cell MFI (expression) over time (day 3) for IRES-driven circular RNA constructs containing combinations of IRES and expression sequences in donor 609C. Each point on the x-axis represents an IRES from Table 1A, and each point represents a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5I] Circular RNA constructs containing IRES-expression sequences are shown in donor 609C (day 4) for T cell MFI (expression) over time. Each point on the x-axis represents an IRES from Table 1A, and each point represents a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 5J] Circular RNA constructs containing IRES-expression sequences are shown for T cell MFI (expression) over time (day 5) in donor 609C. Each point on the x-axis represents an IRES from Table 1A, and each point represents a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A.

[0015] [Figure 6A] Figure 1 shows the % CAR positive cells (i.e., percent of expressing cells or average signal of such cells over time (day 1)) by IRES over time in donor 4003 for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6B]Figure 1 shows the % CAR positive cells (i.e., percent of expressing cells or average signal of such cells over time (day 2)) by IRES over time in donor 4003 for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6C] Figure 1 shows the % CAR positive cells (i.e., percent of expressing cells or average signal of such cells over time (day 3)) by IRES over time in donor 4003 for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6D] Figure 1 shows the % CAR positive cells (i.e., percent of expressing cells or average signal of such cells over time (day 4)) by IRES over time in donor 4003 for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6E] Figure 1 shows the % CAR positive cells (i.e., percent of expressing cells or average signal of such cells over time (day 5)) by IRES over time in donor 4003 for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6F] Figure 1 shows the % CAR-positive cells (i.e., percent of expressing cells or average signal of those cells over time (day 1)) by IRES in donor 609C for circular RNA constructs containing a combination of IRES and expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6G]Figure 1 shows the % CAR-positive cells (i.e., the percentage of expressing cells or the average signal of those cells over time (day 2)) by IRES in donor 609C for circular RNA constructs containing a combination of an IRES and an expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6H] Figure 1 shows the % CAR-positive cells (i.e., the percentage of expressing cells or the average signal of those cells over time (day 3)) by IRES in donor 609C for circular RNA constructs containing a combination of an IRES and an expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6I] Figure 1 shows the % CAR-positive cells (i.e., the percentage of expressing cells or the average signal of those cells over time (day 4)) by IRES in donor 609C for circular RNA constructs containing a combination of an IRES and an expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A. [Figure 6J] Figure 1 shows the % CAR-positive cells (i.e., the percentage of expressing cells or the average signal of those cells over time (day 5)) by IRES in donor 609C for circular RNA constructs containing a combination of an IRES and an expression sequence. Each point on the x-axis is an IRES from Table 1A, and each point is a different expression sequence (codon-optimized, anti-CD19 28-ζ) from Table 2A.

[0016] [Figure 7A]The effect of IRES modification on Nalm6 % lysis data is shown at 24 and 48 hours. Various constructs containing the base CD19 codon (3276) were created in combination with various IRESs, including IRES numbers 1-1, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, and 1-15 (including SEQ ID NOs: 1, 4-15) from Table 1A, and compared to a mock negative control, the base CD19 CAR control IRES, and Nalm6 alone in donor 4003. [Figure 7B] The effect of IRES modification on Nalm6 % lysis data at 24 and 48 hours is shown. Various constructs containing the base CD19 codon (3276) were created in combination with various IRESs, including IRES numbers 1-1, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, and 1-15 (including SEQ ID NOs: 1, 4-15) from Table 1A, and compared to a mock negative control, the base CD19 CAR control IRES, and Nalm6 alone in donor 609C.

[0017] [Figure 8A] Cytotoxicity data at 24 and 48 hours in donor 609C for 69 CD19 CAR oRNA constructs, identified by IRES / CO construct number in Table 5, are shown and ranked compared to mock negative control, base CD19 CAR control, and Nalm6 alone. [Figure 8B] Cytotoxicity data at 24 and 48 hours in donor 4003 for 69 CD19 CAR oRNA constructs, identified by IRES / CO construct number in Table 5, are shown and ranked compared to mock negative control, base CD19 CAR control, and Nalm6 alone.

[0018] [Figure 9A]Similar to Figure 8A, this figure reflects the Nalm6 killing cytotoxicity % data for CD19 CAR oRNA constructs for day 1 for donor 609C, but presented in a different visual format. Each point on the x-axis represents an IRES containing the sequence of SEQ ID NO: 1-15 (listed in Table 1A, IRES numbers 1-1 to 1-15). Each point represents a different codon containing the sequence of SEQ ID NO: 19-23 (listed in Table 2A, codon numbers 2A-19 to 2A-23, codon-optimized, anti-CD19 28-ζ). The control is the IRES of the base CD19 CAR control (3276). [Figure 9B] Similar to Figure 8A, this figure reflects the Nalm6 killing cytotoxicity % data for CD19 CAR oRNA constructs for day 2 for donor 609C, but presented in a different visual format. Each point on the x-axis represents an IRES containing the sequence of SEQ ID NO: 1-15 (listed in Table 1A, IRES numbers 1-1 to 1-15). Each point represents a different codon containing the sequence of SEQ ID NO: 19-23 (listed in Table 2A, codon numbers 2A-19 to 2A-23, codon-optimized, anti-CD19 28-ζ). The control is the IRES of the base CD19 CAR control (3276). [Figure 9C] Similar to Figure 8B, this figure reflects the % Nalm6 killing cytotoxicity data for CD19 CAR oRNA constructs for day 1 for donor 4003, but presented in a different visual format. Each point on the x-axis represents an IRES containing the sequence of SEQ ID NO: 1-15 (listed in Table 1A, IRES numbers 1-1 to 1-15). Each point represents a different codon containing the sequence of SEQ ID NO: 19-23 (listed in Table 2A, codon numbers 2A-19 to 2A-23, codon-optimized, anti-CD19 28-ζ). The control is the IRES of the base CD19 CAR control (3276). [Figure 9D]Similar to Figure 8B, this figure reflects the Nalm6 killing cytotoxicity % data for CD19 CAR oRNA constructs for day 2 for donor 4003, but presented in a different visual format. Each point on the x-axis represents an IRES containing the sequence of SEQ ID NO: 1-15 (listed in Table 1A, IRES numbers 1-1 to 1-15). Each point represents a different codon containing the sequence of SEQ ID NO: 19-23 (listed in Table 2A, codon numbers 2A-19 to 2A-23, codon-optimized, anti-CD19 28-ζ). The control is the IRES of the base CD19 CAR control (3276).

[0019] [Figure 10A] Reflects IFNγ expression 24 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 609C. [Figure 10B] Reflects IFNγ expression 48 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 609C. [Figure 10C] Reflects IFNγ expression 24 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 4003. [Figure 10D] Reflects IFNγ expression 48 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 4003. [Figure 10E] Reflects IL-2 expression 24 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 609C. [Figure 10F] Reflects IL-2 expression 48 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 609C. [Figure 10G]Reflects IL-2 expression 24 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 4003. [Figure 10H] Reflects IL-2 expression 48 hours after electroporation for 69 CD19 CAR circular RNA constructs in donor 4003.

[0020] [Figure 11] AnnexinV+Nalm6 (% of Nalm6) for the CD19 CAR construct of IRES / CO clone #37 (SEQ ID NO: 52) is shown compared to the base CD19 CAR construct (3276) containing the non-optimized CAR sequence, the HER2 circular RNA construct (including HER2_9), a mock negative control, and Nalm6 alone.

[0021] [Figure 12A] Shown are the % T cells over 96 hours for the 12 CD19 CAR constructs (identified by IRES / CO clone number) in Table 6 for donor 9003. [Figure 12B] CAR+ MFI over 48 hours post-electroporation for 12 constructs for donor 9003 is shown. [Figure 12C] AnnexinV+Nalm6 over 72 hours is shown compared to a basal CD19 CAR control (3276), a mock negative control, Nalm6 alone, and HER2.

[0022] [Figure 13] A and B show IRES expression by luminescence for 12 different IRES in 293 and Jurkat cell lines.

[0023] [Figure 14]Figure 1 shows the in vivo antitumor efficacy of CD19 oCAR constructs containing IRES / CO clone numbers 7, 37, and 87 (SEQ ID NOs: 50, 52, and 55, respectively) at doses of 1.0 mg / kg, 0.3 mg / kg, and 0.1 mg / kg compared to the base CD19 CAR control (3276), PBS, and HER2 control. Multiple Mann-Whitney test with Holm-Sidak correction, *p≦0.05, **p≦0.01, ***p≦0.001.

[0024] [Figure 15] Figure 1 shows the in vivo antitumor efficacy of CD19 oCAR constructs containing IRES / CO clone numbers 97, 17, 164, and 87 (SEQ ID NOs: 56, 51, 58, and 55, respectively) at doses of 1.0 mg / kg, 0.3 mg / kg, and 0.1 mg / kg compared to the base CD19 CAR control (3276), PBS, and HER2 control.

[0025] [Figure 16] The total flux (photons / second) after four doses of LNP / oCAR is shown for various lipid compositions containing HER2 and CD19 circular RNA constructs compared to a control. The HER2 and CD19 lipid compositions contain ionized lipids 126, 128, 16, 45, and 86 from Table 3. Ionized lipids 126 and 128 from Table 3 are lipids of Formula II, while ionized lipids 16, 45, and 86 from Table 3 are lipids of Formula I. The designation " / 3" indicates that the lipid composition contains a PEG-modified lipid. Lipids (3-128) / 3, (3-16) / 3, (3-45) / 3, and (3-86) / 3 contain a PEG-modified lipid. Lipid (3-128) / 3L contains ionized lipid 128 from Table 3 and a PEG-modified lipid.

[0026] [Figure 17]% cytotoxicity (IncuCyte cytotoxicity assay) over time for circular RNA constructs containing HER2_9 and HER2_10 compared to the base CD19 CAR control (3276) and mock negative control.

[0027] [Figure 18A] Shown is the % target lysis for the HER2.BBζ oCAR construct and the HER2 28ζ oCAR construct compared to the CD19 oCAR construct, the base CD19 CAR control, and the mock-negative control, assessed using a FACS-based cytotoxicity assay after 24 hours of co-culture with the engineered HER2 / K562 cell line. [Figure 18B] Shown is the % target lysis for the HER2.BBζ oCAR construct and the HER2 28ζ oCAR construct compared to the CD19 oCAR construct, the base CD19 CAR control, and the mock-negative control, assessed using a FACS-based cytotoxicity assay after 24 hours of co-culture with engineered CD19 / K562 cells. [Figure 18C] Shown is the % target lysis for the HER2.BBζ oCAR construct and the HER2 28ζ oCAR construct compared to the CD19 oCAR construct, the base CD19 CAR control, and the mock-negative control, assessed using a FACS-based cytotoxicity assay after 24 hours of co-culture with engineered Nalm6 (CD19+ / HER2-) cells.

[0028] [Figure 19A] Target-specific cytotoxicity for oCAR constructs containing the BCMA_16 sequence is shown compared to the base CD19 CAR (3276) and a mock-negative control in MM.1S cells. [Figure 19B]Target-specific cytotoxicity for oCAR constructs containing the BCMA_16 sequence is shown compared to the base CD19 CAR (3276) and a mock-negative control in U266B1 cells. [Figure 19C] Target-specific cytotoxicity for oCAR constructs containing the BCMA_16 sequence is shown compared to the base CD19 CAR (3276) in Nalm6 cells.

[0029] [Figure 20] Following Nalm6 implantation, tumor control measured by total flux (photons / sec) is shown following weekly dosing at 0.1 mg / kg (mpk) and 0.3 mg / kg (mpk).

[0030] [Figure 21A] Tumor control measured by total flux (photons / second) after Nalm6 implantation was demonstrated following administration of 0.1 mg / kg and 0.3 mg / kg biweekly (every other week, or q2w). These oRNA CAR constructs used lipid 86 ("3-86") from Table 3. [Figure 21B] Tumor control measured by total flux (photons / second) after Nalm6 implantation was demonstrated when administered biweekly (every other week, or q2w) at 0.3 mg / kg. These oRNA CAR constructs used lipid 86 ("3-86") from Table 3. [Figure 21C] Tumor control measured by total flux (photons / second) after Nalm6 implantation was demonstrated when administered biweekly (every other week, or q2w) at 0.1 mg / kg. These oRNA CAR constructs used lipid 86 ("3-86") from Table 3.

[0031] [Figure 22] Figure 1 shows in vivo tumor control when administered at 0.1 mg / kg and 0.3 mg / kg every other week.

[0032] [Figure 23A]Quantitative tumor measurements are shown over time following Nalm6 implantation, with grey circles representing control responses and black squares representing responses following treatment with the CD19 oRNA CAR construct described herein. [Figure 23B] Quantitative tumor measurements are shown over time after Nalm6 implantation. Whole-body images are shown over time for untreated and treated mice.

[0033] [Figure 24A-1] Figure 1 shows the expression of BCMA CAR detected in soluble BCMA.PE after electroporation of an exemplary circular RNA (circRNA) encoding the BCMA-41BBζ CAR at doses of 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, compared to "mock" control T cells not electroporated with any circRNA. Expression was analyzed in T cells 24, 48, and 72 hours after circular RNA introduction. "A," "B," and "C" correspond to "template DNA A," "template DNA B," and "template DNA C," respectively, in Table α1 (i.e., circular RNA construct "A" contains the IRES sequence of template DNA A and the BCMA sequence of template DNA A; circular RNA construct "B" contains the IRES sequence of template DNA B and the BCMA sequence of template DNA B; circular RNA construct "C" contains the IRES sequence of template DNA C and the BCMA sequence of template DNA C, etc.). [Figure 24A-2]Figure 1 shows the expression of BCMA CAR detected in soluble BCMA.PE after electroporation of an exemplary circular RNA (circRNA) encoding the BCMA-41BBζ CAR at doses of 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, compared to "mock" control T cells not electroporated with any circRNA. Expression was analyzed in T cells 24, 48, and 72 hours after circular RNA introduction. "A," "B," and "C" correspond to "template DNA A," "template DNA B," and "template DNA C," respectively, in Table α1 (i.e., circular RNA construct "A" contains the IRES sequence of template DNA A and the BCMA sequence of template DNA A; circular RNA construct "B" contains the IRES sequence of template DNA B and the BCMA sequence of template DNA B; circular RNA construct "C" contains the IRES sequence of template DNA C and the BCMA sequence of template DNA C, etc.). [Figure 24B] Geometric mean fluorescent intensity (gMFI) activity is used to quantify BCMA CAR expression over 24 hours following transfection of circular RNA encoding BCMA-41BBζ CAR at doses of 10 ng, 30 ng, or 100 ng per 0.1x10 T cells. "A," "B," and "C" correspond to "template DNA A," "template DNA B," and "template DNA C," respectively, in Table 1 (i.e., circular RNA construct "A" contains the IRES sequence from template DNA A and the BCMA sequence from template DNA A; circular RNA construct "B" contains the IRES sequence from template DNA B and the BCMA sequence from template DNA B; circular RNA construct "C" contains the IRES sequence from template DNA C and the BCMA sequence from template DNA C, etc.).

[0034] [Figure 25A-1]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," and "C" correspond to "Template DNA A," "Template DNA B," and "Template DNA C," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, and template DNA C were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the percent CAR expression detected by a soluble BCMA PE detection reagent over a 24-72 hour period after electroporation is shown. [Figure 25A-2] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," and "C" correspond to "Template DNA A," "Template DNA B," and "Template DNA C," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, and template DNA C were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the percent CAR expression detected by a soluble BCMA PE detection reagent over a 24-72 hour period after electroporation is shown. [Figure 25B-1]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," and "C" correspond to "Template DNA A," "Template DNA B," and "Template DNA C," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, and template DNA C were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells. Figure 1 shows the geometric mean fluorescence intensity (gMFI) of T cells detected with a soluble BCMA PE detection reagent over a 24-72 hour period after electroporation. [Figure 25B-2] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," and "C" correspond to "Template DNA A," "Template DNA B," and "Template DNA C," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, and template DNA C were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells. Figure 1 shows the geometric mean fluorescence intensity (gMFI) of T cells detected with a soluble BCMA PE detection reagent over a 24-72 hour period after electroporation. [Figure 25C]Figure 25 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," and "C" correspond to "Template DNA A," "Template DNA B," and "Template DNA C," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. Figure 25 shows fluorescence-activated cell sorting (FACS) imaging 24 hours after transfection of T cells with a 30 ng dose of the circular RNA depicted in Figures 25A and 25B. [Figure 25D-1] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the figures represent the percent CAR expression detected by a soluble BCMA PE detection reagent over a 24-96 hour period after electroporation. [Figure 25D-2]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the figures represent the percent CAR expression detected by a soluble BCMA PE detection reagent over a 24-96 hour period after electroporation. [Figure 25E-1] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the figures represent the percent CAR expression detected by anti-Whitlow PE detection reagent over a 24-96 hour period after electroporation. [Figure 25E-2]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the figures represent the percent CAR expression detected by anti-Whitlow PE detection reagent over a 24-96 hour period after electroporation. [Figure 25F-1] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the percent CAR expression detected by anti-G4S detection reagent over a 24-96 hour period after electroporation is shown. [Figure 25F-2]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at either 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the percent CAR expression detected by anti-G4S detection reagent over a 24-96 hour period after electroporation is shown. [Figure 25G-1] Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the average MFI (%) of T cells detected with a soluble BCMA PE detection reagent was measured over a 24-96 hour period after electroporation. [Figure 25G-2]Figure 1 shows anti-BCMA chimeric antigen receptor (CAR) expression for an exemplary circular RNA encoding the BCMA-41BBζ CAR after electroporation into T cells. "A," "B," "C," "D," and "E" correspond to "Template DNA A," "Template DNA B," "Template DNA C," "Template DNA D," and "Template DNA E," respectively, in Table 1. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. CircRNAs formed from template DNA A, template DNA B, template DNA C, template DNA D, and template DNA E were administered into T cells at 10 ng, 30 ng, or 100 ng per 0.1×10 T cells, and the average MFI (%) of T cells detected with a soluble BCMA PE detection reagent was measured over a 24-96 hour period after electroporation.

[0035] [Figure 26] Figure 1 depicts an exemplary gating method used to analyze flow cytometry results for T cells electroporated with circular RNA encoding BCMA CAR at a dose of 10 ng x 10. BCMA CAR expression was detected with either soluble BCMA PE, anti-Whitlow PE, or anti-G4S linker.

[0036] [Figure 27] Target protein expression in multiple myeloma positive cells (eg, MM1S, NCI-H929, and RPMI-8226) and negative target cell lines (eg, Nalm6 target cell line) is shown.

[0037] [Figure 28]The percentage of viable T cells collected 24 hours after electroporation with circular RNA containing BCMA-41BBζ CAR or CD19-CD28ζ CAR is shown compared to a "mock" solution containing only electroporation buffer without circular RNA. "F," "C," "G," "H," "A," "I," and "J" correspond to "template DNA F," "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to form the circular RNA.

[0038] [Figure 29A] The gMFI collected from various circular RNA constructs encoding BCMA-41BBζ or BCMA-CD28ζ CARs or CD19-CD28ζ CARs electroporated into T cells at a dose of 50 ng per 0.1×10 T cells is presented compared to "mock" control T cells (containing only electroporation buffer) lacking any circular RNA. Each circular RNA solution was subjected to either soluble BCMA (sBCMA-PE), anti-Whitlow-PE, or anti-G4S linker PE (G4S-AF647) detection reagents. Histograms of gMFI collected from cells are shown. "F," "C," "G," "H," "A," "I," and "J" correspond to "template DNA F," "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to generate the circular RNAs. [Figure 29B]The gMFI collected from various circular RNA constructs encoding BCMA-41BBζ or BCMA-CD28ζ CARs or CD19-CD28ζ CARs electroporated into T cells at a dose of 50 ng per 0.1×10 T cells is presented compared to "mock" control T cells (containing only electroporation buffer) that lacked any circular RNA. Each circular RNA solution was subjected to soluble BCMA (sBCMA-PE) detection reagent. The gMFI collected for each cell using the sBCMA-PE detection reagent is presented. "F," "C," "G," "H," "A," "I," and "J" correspond to "template DNA F," "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 29C] The gMFI collected from various circular RNA constructs encoding BCMA-41BBζ or BCMA-CD28ζ CAR or CD19-CD28ζ CAR electroporated into T cells at a dose of 50 ng per 0.1×10 T cells is presented compared to "mock" control T cells (containing only electroporation buffer) that lacked any circular RNA. Each circular RNA solution was subjected to anti-Whitlow-PE detection reagent. The gMFI collected for each cell using anti-Whitlow-PE detection reagent is presented. "C," "G," and "H" correspond to "template DNA C," "template DNA G," and "template DNA H" used to generate the circular RNA. [Figure 29D]The gMFI collected from various circular RNA constructs encoding BCMA-41BBζ or BCMA-CD28ζ CAR or CD19-CD28ζ CAR electroporated into T cells at a dose of 50 ng per 0.1×10 T cells is presented compared to "mock" control T cells (containing only electroporation buffer) that lacked any circular RNA. Each circular RNA solution was subjected to anti-G4S linker PE (G4S-AF647) detection reagent. The gMFI collected for each cell using the G4S-AF647 detection reagent is presented. "F," "A," "I," and "J" correspond to "template DNA F," "template DNA A," "template DNA I," and "template DNA J" used to generate the circular RNA.

[0039] [Figure 30] Figure 1 depicts an exemplary gating process for oCAR-T cells 24 hours after electroporation. The top boxes (left to right) present FACS imaging of lymphocytes, CD3-negative cells, live T cells, and BCMA-positive cells. The bottom two boxes are histograms of soluble BCMA or BCMA CAR detected by either anti-Whitlow detection reagent (bottom left) or anti-G4S-PE fluorescence (bottom right).

[0040] [Figure 31A]Figures represent the percent expression of the detection reagent used (i.e., soluble BCMA PE (indicated as "sBCMA"). Percent expression was calculated from the presence of the relevant detection reagent 24 hours after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or HER2 CAR, gating on live T cells. "F," "C," "G," "H," "A," "I," and "J" correspond to "template DNA F," "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to form the circular RNA. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. [Figure 31B] The figures represent the percent expression of the detection reagent used (i.e., anti-Whitlow-PE (indicated by "Whitlow"). Percent expression was calculated from the presence of the relevant detection reagent 24 hours after gating on live T cells and electroporation with circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or HER2 CAR. "C," "G," and "H" correspond to "template DNA C," "template DNA G," and "template DNA H" used to form the circular RNA. "Mock" in the figure represents data for control T cells not electroporated with circular RNA. [Figure 31C] Figures represent the percent expression of the detection reagent used (i.e., anti-G4S linker PE (indicated by "G4S"). Percent expression was calculated from the presence of the relevant detection reagent 24 hours after gating on live T cells and electroporation with circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or HER2 CAR. "F," "A," "I," and "J" correspond to "template DNA F," "template DNA A," "template DNA I," and "template DNA J" used to form the circular RNA. "Mock" in the figure represents data for control T cells not electroporated with circular RNA.

[0041] [Figure 32A] BCMA expression by gMFI after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD-CD28ζ, or CD19-CD28ζ is shown, gated on CD3+ cells. "Mock" indicates T cell solutions not electroporated with circular RNA constructs. Histograms are presented for detection of soluble BCMA-PE or anti-Whitlow.PE for circular RNA constructs collected at 24 and 48 hours. [Figure 32B] BCMA expression by gMFI after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD-CD28ζ, or CD19-CD28ζ is shown, gated on CD3+ cells. "Mock" indicates a T cell solution without electroporation of the circular RNA construct. gMFI for each construct is shown over 24-72 hours after co-culture of CD3+ cells containing circular RNA with multiple myeloma (MM1S) cells. [Figure 32C] The percent soluble BCMA PE detection (indicated as "% sBCMA-PE") after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD-CD28ζ, or CD19-CD28ζ is shown, gated on CD3+ cells. "Mock" indicates T cell solution without electroporation of circular RNA constructs. The % sBCMA-PE expression over 24-72 hours for each construct is presented after co-culture of CD3+ cells containing circular RNA with multiple myeloma (MM1S) cells. [Figure 32D]BCMA expression by gMFI after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD-CD28ζ, or CD19-CD28ζ is shown, gated on CD3+ cells. "Mock" indicates a T cell solution without electroporation of the circular RNA construct. The gMFI for each construct is shown 72 hours after electroporation after coculture of CD3+ cells containing circular RNA with multiple myeloma (MM1S) cells, NCI-H929 (indicated as "H929" in the figure), Nalm6, or K562.CD19 cells. "C," "G," "H," "A," "I," and "J" correspond to "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to generate the circular RNA. "Mock" in the figure represents data for control T cells without electroporation of circular RNA. [Figure 32E] The percent soluble BCMA PE detection (indicated as "%sBCMA-PE") after electroporation of circular RNA encoding BCMA-41BBζ, BCMA-CD-CD28ζ, or CD19-CD28ζ is shown, gated on CD3+ cells. "Mock" indicates a T cell solution without electroporation of the circular RNA construct. The % sBCMA-PE expression is shown for each construct 72 hours after electroporation after coculture of CD3+ cells containing circular RNA with multiple myeloma (MM1S), NCI-H929 (indicated as "H929" in the figure), Nalm6, or K562.CD19 cells. "C," "G," "H," "A," "I," and "J" correspond to "template DNA C," "template DNA G," "template DNA H," "template DNA A," "template DNA I," and "template DNA J" used to generate the circular RNA. "Mock" in the figure represents data for control T cells that were not electroporated with circular RNA.

[0042] [Figure 33A] Figure 1 shows the cytotoxicity of circular RNA constructs encoding the BCMA-41BBζ chimeric antigen receptor (CAR) to MM1S cells over a 0-72 hour period. The circular RNAs contain a BCMA sequence and an IRES sequence derived from either the α1, β, or γ CD19-CD28ζ CAR or the HER2-CD28ζ CAR. Cytotoxicity of each circular RNA encoding a CAR construct to MM1S cells at a dose of either 10 or 30 ng per 0.1 x 10 T cells is shown. Mock T cells (i.e., T cells not electroporated with circular RNA, designated "Mock" in the figure) and MM1S cells not cocultured with T cells, designated "MM1S," were used as controls. Percent cytotoxicity was calculated by (green area + red area / green area) obtained by live cell analysis portfolio system imaging. "A," "B," "C," "F," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," "template DNA F," and "template DNA K" used to form the circular RNA. [Figure 33B]Figure 1 shows the cytotoxicity of circular RNA constructs encoding the BCMA-41BBζ chimeric antigen receptor (CAR) to Nalm6 cells over a 0-72 hour period after co-culture. The circular RNAs contain the BCMA sequence and an IRES sequence derived from the α1, β, or γ CD19-CD28ζ CAR or HER2-CD28ζ CAR. Cytotoxicity of each circular RNA encoding a CAR construct to Nalm6 cells at a dose of either 10 or 30 ng per 0.1 x 10 T cells is shown. Mock T cells (i.e., T cells not electroporated with circular RNA, designated "Mock" in the figure) and Nalm6 cells not co-cultured with T cells, designated "Nalm6," were used as controls. Percent cytotoxicity was calculated by (green area + red area / green area) obtained by live cell analysis portfolio system imaging. "A," "B," "C," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," and "template DNA K" used to form the circular RNA. [Figure 33C] Figure 1 shows the cytotoxicity of circular RNA constructs encoding the BCMA-41BBζ chimeric antigen receptor (CAR) against CD19 T stable cell lines over a 0-96 hour period. The circular RNAs contain a BCMA sequence and an IRES sequence derived from either the α1, β, or γ CD19-CD28ζ CAR or the HER2-CD28ζ CAR. Cytotoxicity of each circular RNA encoding a CAR construct against CD19 T stable cell lines is shown at a dose of 20 ng per 0.1 x 10 T cells. Mock T cells (i.e., tumor T cells not electroporated with circular RNA, designated "Mock" in the figure) and CD19 T stable cells not cocultured with T cells, designated "Tumor," were used as controls. Percent cytotoxicity was calculated by (green area + red area / green area) obtained by live cell analysis portfolio system imaging. "A," "B," "C," and "F" correspond to "template DNA A," "template DNA B," "template DNA C," and "template DNA F" used to form the circular RNA.

[0043] [Figure 34A] Figure 1 shows the cytotoxicity analysis of various engineered circular RNAs across multiple cell types. FACS imaging of cells (e.g., lymphocytes, CD3-negative cells, viable cells, and BCMA-positive cells) 24 hours after co-culture with oCAR-T cells formed by transfection of a circular RNA containing the 3' Anabaena exon, a caprine kobuvirus internal ribosome entry site (IRES), a BCMA-41BBζ CAR, and a 5' Anabaena exon is shown. [Figure 34B] Figure 1 shows the cytotoxicity analysis of various engineered circular RNAs against MM1S. Percent cytotoxicity against MM1S obtained from circular RNAs encoding BCMA-41BBζ, CD19-CD28ζ, or HER2-CD28ζ CARs is shown. "MM1S+Mock" and "MM1S" refer to MM1S cells cocultured with T cells not transfected with circular RNA. "A," "B," "C," "F," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," "template DNA F," and "template DNA K" used to generate the circular RNAs. [Figure 34C] Figure 1 shows the cytotoxicity analysis of various engineered circular RNAs against Nalm6. Percent cytotoxicity against Nalm6 obtained from circular RNAs encoding BCMA-41BBζ, CD19-CD28ζ, or HER2-CD28ζ CARs is shown. "Nalm6+Mock" and "Nalm6" refer to Nalm6 cells cocultured with T cells not transfected with circular RNA. "A," "B," "C," "F," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," "template DNA F," and "template DNA K" used to generate the circular RNAs.

[0044] [Figure 35A]Figure 1 shows FACS imaging of "Mock+MM1S" (i.e., MM1S tumor cells co-cultured with T cells not electroporated with circular RNA), "Mock+Nalm6" (i.e., Nalm6 tumor cells co-cultured with T cells not electroporated with circular RNA), "Mock+H929" (i.e., NCI-H929 tumor cells co-cultured with T cells not electroporated with circular RNA), and "Mock+K562.CD19" (i.e., K562.CD19 tumor cells co-cultured with T cells not electroporated with circular RNA). [Figure 35B] 1 shows FACS imaging for CD19+CD3+ cells.

[0045] [Figure 36A-1] Table 1 shows the percentage of target cell viability (top row) and target cell death (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (MM1S). "Mock + MM1S" (i.e., MM1S tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36A-2]Table 1 shows the percentage of target cell viability (top row) and target cell death (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (MM1S). "Mock + MM1S" (i.e., MM1S tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36B-1] Table 1 shows the % target cell viability (top row) and % target cell killing (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (NCI-H929 (represented as "H929" in the figure)). Also shown are "Mock+H929" (i.e., NCI-H929 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36B-2]Table 1 shows the % target cell viability (top row) and % target cell killing (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (NCI-H929 (represented as "H929" in the figure)). Also shown are "Mock+H929" (i.e., NCI-H929 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36C-1] Table 1 shows the percentage of target cell viability (top row) and percentage of target cell death (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (Nalm6). "Mock + Nalm6" (i.e., Nalm6 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36C-2]Table 1 shows the percentage of target cell viability (top row) and percentage of target cell death (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (Nalm6). "Mock + Nalm6" (i.e., Nalm6 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36D-1] Table 1 shows the percentage of target cell viability (top row) and target cell killing (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (K562.CD19). "Mock + K562.CD19" (i.e., K562.CD19 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 36D-2]Table 1 shows the percentage of target cell viability (top row) and target cell killing (bottom row) after 24 hours (left) or 48 hours (right) of coculture for T cells electroporated with circular RNA derived from α1, β, and / or γ1 template DNA and then cocultured with target cells (K562.CD19). "Mock + K562.CD19" (i.e., K562.CD19 tumor cells cocultured with T cells not electroporated with circular RNA). "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA.

[0046] [Figure 37A] Figure 1 shows the INγ cytokine secretion in MM1S cells resulting from circular RNA encoding BCMA-41BBζ, CD19-CD28ζ, and HER2-CD28ζ CARs after co-culture of T cells containing circular RNA at doses of 10, 30, or 100 ng per 0.1×10 T cells with MM1S. Cytotoxicity levels were calculated using a cytokine and chemokine kit (e.g., MSD). "MM1S+Mock" refers to MM1S tumor cells co-cultured with T cells that were not electroporated with circular RNA. "MM1S" refers to tumor cells not co-cultured with T cells. "A," "B," "C," "F," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," "template DNA F," and "template DNA K" used to generate the circular RNA. [Figure 37B]Figure 1 shows the INγ cytokine secretion in Nalm6 cells induced by circular RNA encoding BCMA-41BBζ, CD19-CD28ζ, and HER2-CD28ζ CARs after co-culture of T cells containing circular RNA at doses of 10, 30, or 100 ng per 0.1×10 T cells with Nalm6. Cytotoxicity levels were calculated using a cytokine and chemokine kit (e.g., MSD). "Nalm6+Mock" refers to Nalm6 tumor cells co-cultured with T cells that were not electroporated with circular RNA. "Nalm6" refers to tumor cells not co-cultured with T cells. "A," "B," "C," "F," and "K" correspond to "template DNA A," "template DNA B," "template DNA C," "template DNA F," and "template DNA K" used to generate the circular RNA.

[0047] [Figure 38A] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include MM1S. The figure displays INFγ cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38B]Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include NCI-H929 (designated as "H929"). The figure displays INFγ cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38C] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells contain Nalm6. The figure displays INFγ cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38D] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include K562.CD19. The figure displays INFγ cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38E]Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include MM1S. The figure displays TNFα cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38F] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include NCI-H929 (designated as "H929"). The figure displays TNFα cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38G] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells contain Nalm6. The figure displays TNFα cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38H]Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include K562.CD19. The figure displays TNFα cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38I] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include MM1S. The figure displays IL-2 cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38J] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include NCI-H929 (designated as "H929"). The figure displays IL-2 cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38K]Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells contain Nalm6. The figure displays IL-2 cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38L] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include K562.CD19. The figure displays IL-2 cytokine levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38M] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include MM1S. The figure shows GM-CSF levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38N]Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include NCI-H929 (designated as "H929"). The figure shows GM-CSF levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38O] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells contain Nalm6. The figure shows GM-CSF levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA. [Figure 38P] Cytokine levels (pg / mL) are shown at 24 and 48 hours (left and right of the figure, respectively) in T cells containing circular RNA encoding BCMA-41BBζ, BCMA-CD28ζ, or CD19-CD28ζ CARs cocultured with target cells. Target cells include K562.CD19. The figure shows GM-CSF levels. "A," "G," "C," "F," "H," "I," and "J" correspond to "template DNA A," "template DNA G," "template DNA C," "template DNA F," "template DNA H," "template DNA I," and "template DNA J" used to generate the circular RNA.

[0048] [Figure 39]Figure 1 shows the percent apoptosis (e.g., % apoptotic target cells = (green area + red area) / green area) of target cells (e.g., Nalm6) collected from a live cell analysis portfolio system (e.g., IncuCyte) over 72 hours after introduction of circular RNA encoding a HER2 CAR. The green area indicates the target cells. The red area indicates Annexin V reagent present in the apoptotic cells. Nalm6 without circular RNA was used as a control.

[0049] [Figure 40A] Figure 1 shows Annexin V / phase % after circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CAR was introduced into activated PBMC T cells and co-cultured with BT474 HER2-positive cells. For comparison, activated PBMC T cells without any circular RNA were used (indicated as "mock"). The "Annexin V / phase %" referred to in the figure relates to the percentage of apoptotic cells per phase. "K," "L," and "M" correspond to "template DNA K," "template DNA L," and "template DNA M" used to form the circular RNA. [Figure 40B] Figure 1 shows Annexin V / phase % after circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CAR was introduced into activated PBMC T cells and co-cultured with SKBR3 HER2-positive cells. For comparison, activated PBMC T cells without any circular RNA were used (indicated as "mock"). The "Annexin V / phase %" referred to in the figure relates to the percentage of apoptotic cells per phase. "K," "L," and "M" correspond to "template DNA K," "template DNA L," and "template DNA M" used to form the circular RNA. [Figure 40C]Figure 1 shows Annexin V / phase % after circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CAR was introduced into activated PBMC T cells and co-cultured with JIMT1 HER2-positive cells. For comparison, activated PBMC T cells without any circular RNA were used (indicated as "mock"). The "Annexin V / phase %" referred to in the figure relates to the percentage of apoptotic cells per phase. "K," "L," and "M" correspond to "template DNA K," "template DNA L," and "template DNA M" used to form the circular RNA.

[0050] [Figure 41A] Figure 1 shows CAR expression of three different circular RNA constructs encoding HER2 CAR delivered by frozen and fresh LNP. "Mock" cells were T cells given empty LNP (no circular RNA). "K" and "L" correspond to "template DNA K" and "template DNA L" used to form the circular RNA. "Fresh" indicates that the LNP was not previously frozen. "Frozen" indicates that the LNP was previously frozen. [Figure 41B] T cells containing circular RNA constructs encoding HER2-28ζ, HER2-BBζ, or CD19-28ζ CARs were co-cultured with BT-474 target cells at a 1:1 E:T ratio and analyzed using a live cell analysis portfolio system (e.g., IncuCyte) to present the % cytotoxicity collected, where the circular RNA was delivered either fresh or frozen LNPs. Fresh and frozen LNPs were composed of ionizable lipids from Table 3. "K," "L," and "F" correspond to "template DNA K," "template DNA L," and "template DNA F" used to form the circular RNAs. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 41C]Cytokine release (top graph: INFγ, bottom graph: TNFα) produced by T cells co-cultured with BT-474 is shown for each circular RNA construct. "K," "L," and "F" correspond to "template DNA K," "template DNA L," and "template DNA F" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen.

[0051] [Figure 42A] Figure 1 shows anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. "L" corresponds to the "template DNA L" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42B] Figure 1 shows anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. "L" corresponds to the "template DNA L" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42C] Figures 42A-B show anti-HER2 expression of circular RNAs encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. Spider plots of collected data are shown. "L" corresponds to the "template DNA L" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42D]Figures 42A-B show anti-HER2 expression of circular RNAs encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. Spider plots of collected data are shown. "K" corresponds to "template DNA K" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42E] Figures 42A-B show anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. Spider plots of collected data are shown. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42F] Figures 42A-B show anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in the JIMT-1 mouse model. Spider plots of collected data are shown. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42G] Figure 1 shows anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. "L" and "F" correspond to the "template DNA L" and "template DNA F" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42H]Figure 1 shows anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. "K" and "F" correspond to "template DNA K" and "template DNA F" used to form the circular RNAs. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42I] Figures 42G and 42H show anti-HER2 expression of circular RNAs encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. Spider plots of data collected in Figures 42G and 42H. "L" corresponds to the "template DNA L" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42J] Figures 42G and 42H show anti-HER2 expression of circular RNAs encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. Spider plots of data collected in Figures 42G and 42H. "K" corresponds to the "template DNA K" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42K] Figures 42G and 42H show anti-HER2 expression of circular RNAs encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. Spider plots of data collected in Figures 42G and 42H. "F" corresponds to the "template DNA F" used to form the circular RNA. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen. [Figure 42L]Figures 42G and 42H show anti-HER2 expression of circular RNA encoding HER2.28ζ, HER2.BBζ, or CD19.28ζ CARs delivered intravenously using lipid nanoparticles in a BT-474 mouse model. Spider plots of data collected in Figures 42G and 42H. "Fresh" indicates that the LNPs were not previously frozen. "Frozen" indicates that the LNPs were previously frozen.

[0052] [Figure 43A] CAR expression after electroporation with circular RNA constructs (HER2_9, HER2_1, HER2_3, HER2_4 as described herein in Table 9) containing an IRES sequence, anti-HER2 CAR, and a 28z domain is shown. [Figure 43B] CAR expression after electroporation with circular RNA constructs (HER2_9, HER2_1, HER2_3, HER2_4 as described herein in Table 9) containing an IRES sequence, anti-HER2 CAR, and a 28z domain is shown. [Figure 43C] CAR expression after electroporation with circular RNA constructs (HER2_9, HER2_1, HER2_3, HER2_4 as described herein in Table 9) containing an IRES sequence, anti-HER2 CAR, and a 28z domain is shown. [Figure 43D] CAR expression after electroporation with circular RNA constructs (HER2_9, HER2_1, HER2_3, HER2_4 as described herein in Table 9) containing an IRES sequence, anti-HER2 CAR, and a 28z domain is shown.

[0053] [Figure 44A] CAR expression after electroporation with circular RNA constructs containing an IRES sequence, anti-HER2 CAR, and a BBz domain (HER2_10, HER2_5, HER2_7, HER2_8 as described herein in Table 9) is shown. [Figure 44B]CAR expression after electroporation with circular RNA constructs containing an IRES sequence, anti-HER2 CAR, and a BBz domain (HER2_10, HER2_5, HER2_7, HER2_8 as described herein in Table 9) is shown. [Figure 44C] CAR expression after electroporation with circular RNA constructs containing an IRES sequence, anti-HER2 CAR, and a BBz domain (HER2_10, HER2_5, HER2_7, HER2_8 as described herein in Table 9) is shown. [Figure 44D] CAR expression after electroporation with circular RNA constructs containing an IRES sequence, anti-HER2 CAR, and a BBz domain (HER2_10, HER2_5, HER2_7, HER2_8 as described herein in Table 9) is shown.

[0054] [Figure 45A] The performance of circular RNA constructs containing an IRES sequence, anti-HER2 CAR, and a 28z domain in BT474 target cells is shown compared to controls (3273 (base) and mock). [Figure 45B] The performance of circular RNA constructs (HER2_10, HER2_5, HER2_7, HER2_8) containing an IRES sequence, an anti-HER2 CAR, and a BBz domain in BT474 target cells is shown compared to controls (3273 (base) and mock).

[0055] [Figure 46] An exemplary method is shown for assessing the ability of circular RNA containing an anti-CD19 CAR (in situ CAR or isCAR™) to deplete human B cells in a CD34+ engrafted humanized mouse model.

[0056] [Figure 47] 1 shows an exemplary flow cytometry panel used in autoimmune studies.

[0057] [Figure 48A-1] 1 shows B cell depletion mediated by circular RNA containing anti-CD19 CAR. [Figure 48A-2] 1 shows B cell depletion mediated by circular RNA containing anti-CD19 CAR. [Figure 48B] 1 shows B cell depletion mediated by circular RNA containing anti-CD19 CAR. [Figure 48C] 1 shows B cell depletion mediated by circular RNA containing anti-CD19 CAR.

[0058] [Figure 49] AC shows that splenic B cells were depleted in mice treated with circular RNA encoding a reporter (mWasabi) encapsulated in lipid nanoparticles as described herein.

[0059] [Figure 50] 1 shows an exemplary method for assessing RAJI regulation of NK cells using circular RNA containing an anti-CD19 CAR in NOG-IL15 mice.

[0060] [Figure 51] The data show that: On day 0, the CD19+ Raji-luc cell line was transplanted into NOG-IL15 mice. On day 3, primary human NK cells were purified from peripheral blood and transplanted into recipient animals. On day 8, mice were left untreated or treated iv with vehicle, LNP-1 mg / kg mOX40L CAR, or 1 mg / kg LNP-CD19 CAR. Mice were treated every two days for 10 treatments. Tumor burden was imaged using IVIS imaging. The data show that mice treated with LNP-CD19 CAR exhibited tumor control up to day 24, the study endpoint.

[0061] [Figure 52]1 shows an exemplary method for assessing circular RNA in macrophages.

[0062] [Figure 53] An exemplary FACS gating strategy for establishing circular RNA delivery to monocytes, as applied elsewhere herein, is shown.

[0063] [Figure 54A] 1 shows the expression of mOX40L in myeloid cells in bone marrow. [Figure 54B] 1 shows the expression of mOX40L in myeloid cells in bone marrow. [Figure 54C] 1 shows the expression of mOX40L in myeloid cells in bone marrow. [Figure 54D] 1 shows the expression of mOX40L in myeloid cells in bone marrow.

[0064] [Figure 55A] 1 shows the expression of mOX40L in CD33+CD14+ and CD14- cells in bone marrow. [Figure 55B] 1 shows the expression of mOX40L in CD14+ cells in bone marrow. [Figure 55C] 1 shows the expression of mOX40L in CD14 − cells in bone marrow. [Figure 55D] 1 shows the expression of mOX40L in CD14+ cells in bone marrow. [Figure 55E] 1 shows the expression of mOX40L in CD14 − cells in bone marrow. [Figure 55F] 1 shows the expression of mOX40L in CD14+ cells in bone marrow. [Figure 55G] 1 shows the expression of mOX40L in CD14 − cells in bone marrow.

[0065] [Figure 56A] 1 shows the expression of mOX40L in CD33+CD64+ and CD64- cells in bone marrow. [Figure 56B] 1 shows the expression of mOX40L in CD64+ cells in bone marrow. [Figure 56C] 1 shows the expression of mOX40L in CD64 − cells in bone marrow. [Figure 56D] 1 shows the expression of mOX40L in CD64+ cells in bone marrow. [Figure 56E] 1 shows the expression of mOX40L in CD64 − cells in bone marrow. [Figure 56F] 1 shows the expression of mOX40L in CD64+ cells in bone marrow. [Figure 56G] 1 shows the expression of mOX40L in CD64 − cells in bone marrow.

[0066] [Figure 57A] 1 shows the expression of mOX40L in myeloid cells in the spleen. [Figure 57B] 1 shows the expression of mOX40L in myeloid cells in the spleen. [Figure 57C] 1 shows the expression of mOX40L in myeloid cells in the spleen. [Figure 57D] 1 shows the expression of mOX40L in myeloid cells in the spleen.

[0067] [Figure 58A] 1 shows the expression of mOX40L in CD33+CD14+ and CD14- cells in the spleen. [Figure 58B] 1 shows the expression of mOX40L in CD14+ cells in the spleen. [Figure 58C] 1 shows the expression of mOX40L in CD14 − cells in the spleen. [Figure 58D] 1 shows the expression of mOX40L in CD14+ cells in the spleen. [Figure 58E] 1 shows the expression of mOX40L in CD14 − cells in the spleen. [Figure 58F] 1 shows the expression of mOX40L in CD14+ cells in the spleen. [Figure 58G]1 shows the expression of mOX40L in CD14 − cells in the spleen.

[0068] [Figure 59A] 1 shows the expression of mOX40L in CD33+CD64+ and CD64- cells in the spleen. [Figure 59B] 1 shows the expression of mOX40L in CD64+ cells in the spleen. [Figure 59C] 1 shows the expression of mOX40L in CD64 − cells in the spleen. [Figure 59D] 1 shows the expression of mOX40L in CD64+ cells in the spleen. [Figure 59E] 1 shows the expression of mOX40L in CD64 − cells in the spleen. [Figure 59F] 1 shows the expression of mOX40L in CD64+ cells in the spleen. [Figure 59G] 1 shows the expression of mOX40L in CD64 − cells in the spleen.

[0069] [Figure 60A] Figure 1 shows in vivo tumor control by circular RNA encoding BCMA CARs (BCMA oCAR, BCMA_7 and BCMA_3) compared to HER2 and untreated controls when administered EOD to donor 1. [Figure 60B] Figure 1 shows in vivo tumor control by circular RNA encoding BCMA CARs (BCMA oCAR, BCMA_7 and BCMA_3) compared to HER2 and untreated controls when administered EOD to donor 2.

[0070] [Figure 61A] Figure 1 shows in vivo tumor control by circular RNA encoding BCMA CAR (BCMA oCAR, BCMA_7) when administered once weekly (QW) to donor 1 compared to HER2 and untreated controls. [Figure 61B]Figure 1 shows in vivo tumor control by circular RNA encoding BCMA CAR (BCMA oCAR, BCMA_7) when administered once weekly (QW) to donor 2 compared to HER2 and untreated controls.

[0071] [Figure 62] Exemplary IVIS images show in vivo tumor control by circular RNA encoding BCMA CARs (BCMA oCAR, BCMA_7 and BCMA_3) when administered every other day (EOD) and once a week (QW) compared to HER2 and untreated controls. DETAILED DESCRIPTION OF THE INVENTION

[0072] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the appended claims and the included embodiments.

[0073] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific configurations or process steps, as such may vary. It should be noted that as used in this specification and 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 guide" includes plural guides, a reference to "a cell" includes plural cells, and so forth.

[0074] Numerical ranges are inclusive of the numbers defining the range. Measurements and measurable values ​​are understood to be approximations taking into account significant digits and error associated with measurement. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general and detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0075] Unless otherwise indicated herein, embodiments herein reciting "comprising" various components can also be construed as "consisting of" or "consisting essentially of" the recited components, and embodiments herein reciting "consisting of" various components can also be construed as "comprising" or "consisting essentially of," and embodiments herein reciting "consisting essentially of" various components can also be construed as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in its inclusive sense (i.e., equivalent to "and / or") unless the context clearly dictates otherwise.

[0076] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined herein or any other explicit content related to this specification, the present specification shall control. While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. To the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0077] I. Definition Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0078] As used herein, the terms "circRNA," "circular polyribonucleotide," "circular RNA," "circularized RNA," or "oRNA" are used interchangeably and refer to a single-stranded RNA polynucleotide in which the 3' and 5' ends, as typically present in a linear RNA polynucleotide, are joined to each other.

[0079] As used herein, the term "template DNA" refers to a DNA sequence from which a linear RNA polynucleotide can be transcribed. For example, but not limited to, template DNA can include a DNA vector, a PCR product, or a plasmid.

[0080] 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 naturally occurring Group I intron, including the splice site dinucleotide and, optionally, a stretch of naturally occurring exon sequence. In some embodiments, the circular RNA comprises a spliced ​​3' Group I intron fragment. In some embodiments, the spliced ​​3' Group I intron fragment within the circular RNA is a stretch of spliced ​​exon sequence. In some embodiments, the circular RNA further comprises a desired expression sequence, wherein the stretch of spliced ​​exon sequence is part of, adjacent to, and / or in-frame with (e.g., designed to be) the desired expression sequence.

[0081] 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 naturally occurring Group I intron, including the splice site dinucleotide and optionally a stretch of naturally occurring exon sequence. In some embodiments, the circular RNA comprises a spliced ​​5' Group I intron fragment. In some embodiments, the spliced ​​5' Group I intron fragment within the circular RNA is a stretch of spliced ​​exon sequence. In some embodiments, the circular RNA further comprises a desired expression sequence, wherein the stretch of spliced ​​exon sequence is part of, adjacent to, and / or in-frame with (e.g., designed to be) the desired expression sequence.

[0082] As used herein, the term "permutation site" refers to a site within a Group I intron that is cut prior to intron permutation. This cut generates 3' and 5' Group I intron fragments that become permuted on either side of the precursor RNA stretch that is circularized.

[0083] As used herein, the term "splice site" refers to a dinucleotide contained partially or completely within a Group I intron, where the phosphodiester bond between the dinucleotides is cleaved during RNA circularization. (As used herein, "splice site" refers to the dinucleotide(s) where the phosphodiester bond between the dinucleotides is cleaved during the splicing reaction. A "5' splice site" refers to the 5' dinucleotide of a naturally occurring intron (e.g., a Group I intron), and a "3' splice site" refers to the 3' dinucleotide of a naturally occurring intron.)

[0084] As used herein, the term "expressed sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide, a regulatory nucleic acid, or a non-coding nucleic acid. An exemplary expressed sequence that encodes a peptide or polypeptide may contain multiple nucleotide triplets, each of which can encode an amino acid, and are referred to as a "codon."

[0085] As used herein, a "coding element" or "coding region" is a region located within an expressed sequence that encodes one or more proteins or polypeptides (e.g., therapeutic proteins).

[0086] As used herein, a "non-coding element," "non-coding region," or "non-coding nucleic acid" is a region located within an expressed sequence that does not itself encode a protein or polypeptide, but may have other regulatory functions, including, but not limited to, allowing the entire polynucleotide to act as a biomarker or adjuvant for specific cells.

[0087] As used herein, the term "therapeutic protein" refers to any protein that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered directly or indirectly to a subject in the form of a translated nucleic acid.

[0088] As used herein, the term "immunogenic" refers to the potential to induce an immune response against an object. When an organism's immune system or a certain type of immune cell is exposed to an immunogenic object, an immune response can be induced. The term "non-immunogenic" refers to the lack or absence of an immune response above a detectable threshold against an object. When an organism's immune system or a certain type of immune cell is exposed to a non-immunogenic object, no immune response is detected. In some embodiments, the non-immunogenic cyclic polyribonucleotides provided herein do not induce an immune response above a predetermined threshold as measured by an immunogenicity assay. In some embodiments, when an organism's immune system or a certain type of immune cell is exposed to the non-immunogenic cyclic polyribonucleotides provided herein, no innate immune response is detected. In some embodiments, when an organism's immune system or a certain type of immune cell is exposed to the non-immunogenic cyclic polyribonucleotides provided herein, no adaptive immune response is detected.

[0089] 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.

[0090] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include species containing purines (e.g., adenine, hypoxanthine, guanine, and derivatives and analogs thereof) and pyrimidines (e.g., cytosine, uracil, thymine, and derivatives and analogs thereof). Nucleotide analogs include nucleotides with modifications within the chemical structure of the base, sugar, and / or phosphate (including, but not limited to, 5'-position pyrimidine modifications, 8'-position purine modifications, modifications at the exocyclic amine of cytosine, and substitution of 5-bromo-uracil), and nucleotides with 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, queusine, xanthine, sugars such as 2'-methylribose, and unnatural phosphodiester linkages such as methylphosphonates, phosphorothioates, and peptide bonds. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0091] "Polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along the backbone, including polymers of traditional RNA, DNA, mixed RNA-DNA, and analogs thereof. The terms can be used to describe polymers 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 greater than about 10,000 bases) composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), which can be produced enzymatically or synthetically (e.g., as described in U.S. Pat. No. 5,948,902 and references cited therein), and which can hybridize (e.g., engage 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. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA, PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds that are substituted (e.g., 2' methoxy or 2' halide substituted).The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others), a derivative of inosine, purine, or pyrimidine (e.g., N4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), purine bases having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O4-alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT Publication No. WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992. Nucleic acids may contain one or more "abasic" residues, where the backbone does not contain a nitrogenous base at one or more polymer positions (U.S. Pat. No. 5,585,481). Nucleic acids may be composed exclusively of conventional RNA or DNA sugars, bases, and linkages, or may contain both conventional building blocks and substituents (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). All nucleotide sequences disclosed herein may represent RNA sequences or the corresponding DNA sequences. It is understood that deoxythymidine (dT or T) in DNA is transcribed to uridine (U) in RNA. Therefore, in nucleotide sequences, "T" and "U" are used interchangeably herein.

[0092] An "oligonucleotide" is a polynucleotide containing fewer than 1000 nucleotides, for example, fewer than 500 nucleotides or fewer than 100 nucleotides.

[0093] As used herein, the terms "monotron," "monotron sequence," or "monotron element" are used interchangeably to refer to a segment of a precursor RNA polynucleotide located at either the 5' or 3' end of the polynucleotide (i.e., either 5' or 3' from the intervening region). A monotron element refers to a sequence having 70% or greater similarity to a naturally occurring Group I or Group II intron containing a splice site dinucleotide. In some embodiments, a monotron can contribute to ribozyme activity, allowing for enzymatic self-cleavage. In some embodiments, a monotron can form a phosphodiester bond with a termination sequence (i.e., a sequence containing a splice site dinucleotide and optionally a naturally occurring exon sequence or a fragment thereof). In some embodiments, in a linear precursor, the termination sequence is upstream of the monotron. In some embodiments, in a linear precursor, the monotron sequence is upstream of the termination sequence. When a termination sequence is upstream of the monotron in a linear precursor, the monotron can sequentially perform two transesterification reactions, e.g., self-cleavage and phosphodiester bond formation with the termination sequence. In embodiments in which a termination sequence is upstream of the monotron in a linear precursor, (a) the monotron can interact with a nucleophile capable of cleaving at the splice site dinucleotide at or near the 5' end of the monotron, and (b) the cleavage product of (a) (i.e., e.g., the 5' splice site nucleotide bearing a 3' hydroxyl group) can engage in a transesterification reaction (cleavage) at the splice site nucleotide of the termination sequence, resulting in a circular RNA or oRNA. In such embodiments, the monotron interacts with the nucleophile (e.g., guanosine, e.g., a free guanosine introduced into the precursor) by forming a binding pocket with the nucleophile, and the linear precursor can adopt a conformation that allows the nucleophile to approach and cleave the splice site dinucleotide at or near the 5' end of the monotron. When the monotron is upstream of the termination sequence in a linear precursor, the monotron can also perform two transesterification reactions.In embodiments in which the monotron is upstream of the termination sequence in a linear precursor, (a) the monotron can interact with a nucleophile that can cleave at the splice site nucleotide of the termination element, and (b) the cleavage product of (a) (i.e., e.g., the 5' splice site nucleotide with a 3' hydroxyl group) can engage in transesterification (cleavage) at the splice site dinucleotide at or near the 3' end of the monotron, resulting in a circular RNA or oRNA. In such embodiments, the monotron interacts with the nucleophile (e.g., a guanosine, e.g., a free guanosine introduced into the precursor) by forming a binding pocket with the nucleophile, and the linear precursor can adopt a conformation that allows the nucleophile to approach and cleave the splice site nucleotide of the termination element.

[0094] In some embodiments, the monotron comprises the 5'-proximal end of a naturally occurring Group I or Group II intron, including the splice site dinucleotide and optionally the naturally occurring exon sequence or a fragment thereof. In some embodiments, the 5' end of the monotron refers to nucleotides within the 5' half of the monotron. In some embodiments, the 3' end of the monotron refers to nucleotides within the 3' half of the monotron. In some embodiments, at or near the 5' end of the monotron refers to nucleotides within the 5' half of the monotron. In some embodiments, at or near the 5' end of the monotron refers to within the first 10 5' positions of the monotron. In some embodiments, at the 5' end of the monotron refers to the first 5' position(s) of the monotron. In some embodiments, at or near the 3' end of the monotron refers to within the 3' half of the monotron. In some embodiments, at or near the 3' end of the monotron refers to within the last 10 3' positions of the monotron. In some embodiments, at the 3' end of the monotron refers to the last 3' position(s) of the monotron.

[0095] As used herein, the terms "termination sequence" or "termination element" are used interchangeably to refer to an RNA sequence capable of complexing with a monotron sequence or a monotron element. A termination sequence comprises a splice site nucleotide derived from a naturally occurring Group I or Group II intron present in a monotron. In some embodiments, a termination sequence further comprises a naturally occurring exon or fragment thereof and / or a synthetic sequence.

[0096] The term "nucleophile" refers to a nucleophilic nucleotide or nucleoside capable of nucleophilic attack at a splice site and / or initiating a transesterification reaction (cleavage) at a splice site.

[0097] As used herein, "poly A" refers to a polynucleotide or a portion of a polynucleotide consisting of nucleotides containing adenine. As used herein, "poly T" refers to a polynucleotide or a portion of a polynucleotide consisting of nucleotides containing thymine. As used herein, "poly AC" refers to a polynucleotide or a portion of a polynucleotide consisting of nucleotides containing adenine or cytosine.

[0098] "Isolated" or "purified" generally refers to the isolation of an entity (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., more than 1%, more than 2%, more than 5%, more than 10%, more than 20%, more than 50%, or more, typically up to about 90%-100%) of the sample in which it is present. In certain embodiments, a substantially purified component constitutes at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of the sample. In further embodiments, a substantially purified component constitutes about 80%-85%, or 90%-95%, 95-99%, 96-99%, 97-99%, or 95-100% 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 by density. Generally, an entity 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.

[0099] As used herein, "unstructured" with respect to RNA refers to an RNA sequence that is not predicted by RNA structure prediction means to form a structure (e.g., a hairpin loop) by itself or with other sequences within the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease protection assays.

[0100] As used herein, "structured" with respect to RNA refers to an RNA sequence that is predicted by RNAFold software or similar predictive means to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule.

[0101] As used herein, two "duplex sequences," "duplex region(s)," "homology arms," ​​or "homology regions" can be any two regions that are thermodynamically favorable for cross-pairing in a sequence-specific interaction. In some embodiments, two duplex sequences, duplex regions, homology arms, or homology regions share a sufficient level of sequence identity to each other's reverse complementary sequences to act as substrates for a hybridization reaction. As used herein, a polynucleotide sequence has "homology" if it is identical to or shares sequence identity with its reverse complementary or "complementary" sequence. The percent sequence identity between a homologous region and the reverse complementary sequence of the corresponding homologous region can be any percent sequence identity that allows hybridization to occur. In some embodiments, an internal duplex region of a polynucleotide of the invention can form a duplex with another internal duplex region but not with an external duplex region.

[0102] As used herein, an "affinity sequence" or "affinity tag" is a region of a polynucleotide sequence, ranging from one nucleotide to hundreds or thousands of nucleotides, that contains a repeating set of nucleotides intended to aid in the purification of the polynucleotide sequence. For example, affinity sequences can include, but are not limited to, polyA or polyAC sequences. In some embodiments, affinity tags are used in purification methods referred to herein as "affinity purification," in which a binder selectively binds to molecules containing the affinity tag, facilitating their separation from molecules that do not contain the affinity tag. In some embodiments, the affinity purification method is a "negative selection" purification method, in which undesired species, such as linear RNA, are selectively bound and removed, and desired species, such as circular RNA, are eluted and separated from the undesired species.

[0103] 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 random. Spacers are typically non-coding. In some embodiments, a spacer comprises a double-stranded region.

[0104] Linear nucleic acid molecules are said to have a "5'-terminus" (5' end) and a "3'-terminus" (3' end) because the nucleic acid phosphodiester bonds occur at the 5' and 3' carbons of the sugar moiety of the substituent mononucleotide. The terminal nucleotide of a polynucleotide at which a new bond would occur at the 5' carbon is the 5'-terminal nucleotide of that polynucleotide. The terminal nucleotide of a polynucleotide at which a new bond would occur at the 3' carbon is the 3'-terminal nucleotide of that polynucleotide. As used herein, a terminal nucleotide is the nucleotide at the end position of the 3'-terminus or 5'-terminus.

[0105] As used herein, a "lead untranslated sequence" is a region of a polynucleotide sequence located at the 5'-most end of the polynucleotide sequence, ranging from one nucleotide to several hundred nucleotides. The sequence can be defined or random. The lead untranslated sequence is non-coding.

[0106] As used herein, a "terminal untranslated sequence" is a region of a polynucleotide sequence located at the very 3' end of the polynucleotide sequence, ranging from one nucleotide to several hundred nucleotides. The sequence can be defined or random. Terminal untranslated sequences are non-coding.

[0107] "Transcription" refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The present disclosure is not limited with respect to the RNA polymerase used for transcription. For example, in some embodiments, a T7-type RNA polymerase may be used.

[0108] "Translation" refers to the formation of a polypeptide molecule by ribosomes from an RNA template.

[0109] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA sequence or structural element, ranging in size from 10 nt to 1000 nt or more, that can initiate translation of a polypeptide in the absence of a typical RNA cap structure. Exemplary IRESs can be about 500 nt to about 700 nt in length.

[0110] As used herein, the term "about" or "approximately" refers to the acceptable error of a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined. Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance range in the art (e.g., within 2 standard deviations of the mean). Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0111] As used herein, the term "encoding" broadly refers to any process that uses information within a polymeric macromolecule to lead to 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.

[0112] As used herein, "aptamer" generally refers to either a single oligonucleotide of defined sequence or a mixture of such nucleotides that retains the property of specifically binding to a target molecule (e.g., eukaryotic initiation factors, 40S ribosomes, poly C-binding protein, poly A-binding protein, polypyrimidine tract-binding protein, Argonaute protein family, heterogeneous nuclear ribonucleoproteins K and La, and related RNA-binding proteins). Thus, as used herein, "aptamer" refers to both a single nucleotide sequence and multiple nucleotide sequences, as defined above. The term "aptamer" refers to a single- or double-stranded nucleic acid capable of binding to a protein or other molecule. Generally, aptamers preferably contain about 10 to about 100 nucleotides, preferably about 15 to about 40 nucleotides, and more preferably about 20 to about 40 nucleotides. In this regard, oligonucleotides within these ranges in length are readily prepared by conventional techniques. Optionally, an aptamer can further comprise the minimum of approximately 6 nucleotides, preferably 10, more preferably 14 or 15 nucleotides necessary to achieve specific binding.

[0113] As used herein, "miRNA site" or "miRNA binding site" refers to a stretch of nucleotides within a polynucleotide that can form a duplex with at least 8 nucleotides of a naturally occurring miRNA sequence.

[0114] As used herein, "bicistronic RNA" refers to a polynucleotide containing two expressed sequences encoding two different proteins. These expressed sequences may be separated by a nucleotide sequence encoding a cleavable peptide, such as a protease cleavage site. They may also be separated by a ribosomal skipping element.

[0115] As used herein, the term "ribosomal skipping element" refers to a nucleotide sequence that encodes a short peptide sequence that can generate two peptide chains from the translation of one RNA molecule. Without wishing to be bound by theory, it is hypothesized that the ribosomal skipping element functions by (1) terminating the translation of the first peptide chain and restarting the translation of the second peptide chain, or (2) cleaving the peptide bond in the peptide sequence encoded by the ribosomal skipping element by the endogenous protease activity of the encoded peptide or by another protease in the environment (e.g., cytosol).

[0116] 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 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 undergoing transfection. In some embodiments, transfection efficiency may be estimated by the amount of reporter polynucleotide product produced by a target cell after transfection. In some embodiments, the transfection efficiency of a delivery vehicle is high. In some embodiments, the transfection efficiency of a delivery vehicle is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0117] As used herein, "delivery vehicles" generally include any of the standard pharmaceutical carriers, diluents, excipients, and the like intended for use in connection with the administration of biologically active agents, including nucleic acids.

[0118] As used herein, the phrase "nanoparticle" refers to a delivery or transport vehicle, e.g., less than about 1000 nm in diameter. The nanoparticles may be "lipid nanoparticles," and in certain instances herein, the terms are used interchangeably herein.

[0119] As used herein, the phrase "LNP" or "lipid nanoparticle" refers to a delivery or transport vehicle comprising one or more cationic or ionizable lipids, stabilizing lipids, structural lipids, and helper lipids.

[0120] As used herein, the phrase "cationic lipid" or "ionizable lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH (such as physiological pH 4) and a neutral charge at other pHs (e.g., physiological pH 7).

[0121] In some embodiments, the lipids (e.g., ionizable lipids) disclosed herein comprise one or more cleavable groups. The terms "cleave" and "cleavable" are used herein to 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., 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 may be cleaved enzymatically or by hydrolysis, oxidation, or reduction. Upon cleavage of such a disulfide functional group, one or more functional moieties or groups (e.g., one or more of the head group and / or tail group) attached thereto may be liberated. Exemplary cleavable groups include, but are not limited to, disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl esters and aryl esters). In certain embodiments, the cleavable group is not an ester group or an ether group. In some embodiments, the cleavable group is attached (e.g., by one or more of hydrogen bonding, 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).

[0122] As used herein, the term "liposome" generally refers to a vesicle composed of lipids (e.g., amphipathic lipids) arranged in one or more spherical bilayer(s). Such liposomes may be unilamellar or multilamellar vesicles with a membrane formed from a lipophilic substance and an aqueous interior containing encapsulated circRNA to be delivered to one or more target cells, tissues, and organs. In certain embodiments, the compositions described herein comprise one or more lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that 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.

[0123] As used herein, the phrase "biodegradable lipid" or "degradable lipid" refers to any of a number of lipid species that decompose in the host environment in a matter of minutes, hours, or days, ideally resulting in low toxicity and low likelihood of accumulation within the host over time. Common modifications to lipids include ester bonds, particularly disulfide bonds, to enhance the biodegradability of the lipid.

[0124] As used herein, the term "structured lipid" refers to a sterol and also to a lipid that contains a sterol moiety.

[0125] As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols.

[0126] As used herein, the term "PEG" refers to any polyethylene glycol or other polyalkylene ether polymer. 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. 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 in the host environment within a matter of minutes, hours, or days, ideally resulting in reduced immunogenicity. Common modifications to PEG lipids include ester bonds, particularly disulfide bonds, to enhance the biodegradability of the lipid.

[0127] As used herein, the term "hydrophilic" is used qualitatively to indicate that a functional group is water-loving, typically such a group is water-soluble. For example, disclosed herein are compounds comprising 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.

[0128] In certain embodiments, at least one of the functional groups of the moieties constituting 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 repels water and is typically 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, wherein the hydrophobic group comprises one or more naturally occurring lipids such as cholesterol, and / or one or more optionally substituted, variably saturated or unsaturated C6-C20 alkyls and / or one or more optionally substituted, variably saturated or unsaturated C6-C20 acyls.

[0129] The compounds described herein may also contain one or more isotopic substitutions. For example, H can be any isotopic form including 1H, 2H (D or deuterium), and 3H (T or tritium), C can be any isotopic form including 12C, 13C, and 14C, O can be any isotopic form including 16O and 18O, F can be any isotopic form including 18F and 19F, and so forth.

[0130] As used and appearing herein, the following terms have the following meanings unless otherwise indicated. It is also to be understood that, as described herein, any of the moieties defined below may be substituted with various substituents, and that each definition is intended to include such substituted moieties within the scope as set forth below. Unless otherwise stated, the term "substituted" will be defined as set forth below. It is further to be understood that, as used herein, the terms "group" and "radical" can be considered interchangeable.

[0131] When a range of values ​​is listed, it is intended that each value and subrange be encompassed within that range. For example, "Ci_6 alkyl" is intended to include C, C2, C3, C4, C5, C6, Ci_6, C1_5, C1_4, C1_3, C1_2, C2_6, C2_5, C2_4, C2_3, C3_6, C3_5, C3_4, C4_6, C4_5, and C5_6 alkyl.

[0132] As used herein, the term "alkyl" refers to both straight-chain and branched-chain C1-C40 hydrocarbons (e.g., C6-C20 hydrocarbons), including both saturated and unsaturated hydrocarbons. In certain embodiments, alkyl can include one or more cyclic alkyls and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur), and can be optionally 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, the use of a designation such as "C6-C20" is intended to refer to an alkyl (e.g., straight-chain or branched, including alkenes and alkyls) having the specified range of carbon atoms. In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-10 alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C1-9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("C alkyl"). Examples of C alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[0133] As used herein, "alkenyl" refers to the radical of a straight-chain or branched-chain hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) ("C2-20 alkenyl"). In certain embodiments, the alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-10 alkenyl"). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C2-9 alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-8 alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C2-7 alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as 2-butenyl) or terminal (such as 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include, in addition to the aforementioned C2-4 alkenyl groups, pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.

[0134] As used herein, the term "aryl" refers to aromatic groups (e.g., monocyclic, bicyclic, and tricyclic structures) containing 6 to 10 carbons in the ring portion. Aryl groups may be optionally substituted through available carbon atoms and, in certain embodiments, may contain one or more heteroatoms such as oxygen, nitrogen, or sulfur. In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl," e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl).

[0135] As used herein, "heteroaryl" refers to the radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in the cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms present in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom, valence permitting. Heteroaryl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring is fused to one or more carbocyclyl or heterocyclyl groups, as defined above, and the point of attachment is on the heteroaryl ring; in such instances, the number of ring members is designated as that of the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring is fused to one or more aryl groups, as defined above, and the point of attachment is on either the aryl or heteroaryl ring; in such cases, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, and the like), the point of attachment can be on either ring (i.e., the ring with the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

[0136] As used herein, "heterocyclyl" or "heterocycle" refers to the radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom, where valence permits. Heterocyclyl groups can be either monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, such as bicyclic systems ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring is fused to one or more carbocyclyl groups, as defined above, and the point of attachment is on either the carbocyclyl ring or the heterocyclyl ring, or in which a heterocyclyl ring is fused to one or more aryl or heteroaryl groups, as defined above, and the point of attachment is on the heterocyclyl ring; in such instances, the number of ring members designates the number of ring members in the heterocyclyl ring system. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" may be used interchangeably.

[0137] As used herein, "cyano" refers to --CN.

[0138] As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iodo, I). In certain embodiments, a halo group is either fluoro or chloro.

[0139] As used herein, the term "alkoxy" refers to an alkyl group that is attached to another moiety through an oxygen atom (-O(alkyl)). Non-limiting examples include, for example, methoxy, ethoxy, propoxy, and butoxy.

[0140] As used herein, "oxo" refers to -C=O.

[0141] In general, the term "substituted," whether preceded by the term "optionally" or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with an acceptable substituent, e.g., a substituent that upon substitution gives rise to a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, other reaction, etc.). Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position is substituted in any given structure, the substituents are either the same or different at each position.

[0142] As used herein, "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals, without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactate, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N(C1-4 alkyl) salts.Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include those formed, where appropriate, with non-toxic ammonium, quaternary ammonium, and amine cations with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0143] The term "composition" or "formulation" refers to a preparation that is in a form that effectively potentiates the biological activity of the active ingredients contained therein and that does not contain any additional components that are unacceptably toxic to the subject to which the composition will be administered.

[0144] As used herein, "antigen" refers to any molecule that can elicit an immune response or be bound by an antibody or antigen-binding molecule. The immune response may involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will readily understand that any macromolecule, including virtually any protein or peptide, can serve 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 broadly expressed. In addition, fragments of larger molecules can behave as antigens. In some embodiments, the antigen is a tumor antigen.

[0145] As used herein, "treatment" (and variations thereof, such as "treat" or "treating") refers to any administration or application of a therapeutic agent for a disease or disorder in a subject, and includes inhibiting the disease or onset of the disease (which may occur before or after the disease is formally diagnosed, e.g., when a subject has a genotype that is likely to result in the development of the disease or is likely to result in the development of the disease), preventing the onset of the disease, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease. As used herein, "treatment" can include administering a therapy or therapeutic regimen, including optional adjuvant therapy or pretreatment regimens, to achieve a therapeutic or prophylactic benefit. As used herein, "treatment" also encompasses "ameliorating," which refers to any beneficial effect on a phenotype or symptom, such as reducing its severity, delaying or delaying its onset, preventing its onset, or partially or completely reversing or eliminating it.

[0146] As used herein, "cancer" refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors, which can invade adjacent tissues and metastasize to distant parts of the body via 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 are used to treat cancers of the following cancers: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin'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), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer. , 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, bladder cancer, kidney or ureter cancer, central nervous system (CNS) tumors, 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 asbestos-induced cancer, other B-cell malignancies, and tumors resulting from combinations of the above cancers.In some embodiments, the methods disclosed herein are directed to the treatment of tumors, e.g., sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, Kaposi's sarcoma, soft tissue sarcomas, 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., adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), and other tumors. ), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, renal pelvis cancer, CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. A refractory cancer refers to a cancer that is not amenable to surgical intervention, either because the cancer does not respond to chemotherapy or radiation therapy initially, or because the cancer becomes refractory over time.

[0147] As used herein, "autoimmune disease" refers to a disease or disorder directed against and / or arising from a subject's own tissues and / or organs. Clinical and laboratory markers of autoimmune disease are known in the art. Exemplary markers include, but are not limited to, high levels of autoantibodies, deposition of antigen-antibody complexes (e.g., in a subject's tissue(s)), lymphoid cell aggregates in affected tissues, and hypergammaglobulinemia. Exemplary autoimmune diseases include, but are not limited to, lupus, e.g., systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis (LN), antisynthetase syndrome, multifocal motor neuropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, and systemic sclerosis. In some embodiments, the autoimmune disease is B cell-mediated. Autoimmunity can be associated with autoantibody production, immune complex formation, dendritic cell activation, T cell activation, cytokine synthesis, and / or chemokine release. For example, SLE is a life-threatening autoimmune disease characterized by adaptive immune system activation, double-stranded DNA autoantibody formation, and organ inflammation. Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nature Medicine (2022). SLE can be assessed using the Systemic Lupus Erythematosus Disease Activity Index and / or the DORIS criteria (ibid.).

[0148] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors.Anti-tumor effect can also refer to the prevention of tumor development (e.g., vaccines).

[0149] As used herein, the term "administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of agents disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, agents disclosed herein can be administered by a non-parenteral route (e.g., orally). Other non-parenteral routes include topical, epithelial, or mucosal administration routes (e.g., intranasal, vaginal, rectal, sublingual, or topical). The phrase "systemic injection" as used herein non-exclusively relates to intravenous, intraperitoneal, subcutaneous, via nasal submucosa, lingual, via a bronchoscope, intravenous, intraarterial, intramuscular, intraocular, intrastriatal, subcutaneous, intradermal, via a transdermal patch, via a skin patch, via a patch, into the cerebrospinal fluid, into the portal vein, into the brain, into the lymphatic system, intrapleural, retroorbital, intradermal, intrasplenic, intralymphatic, among others.

[0150] The terms "genetically engineered" or "engineered" refer 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 cells to be modified are lymphocytes (e.g., T cells), which can be obtained from either the patient or a donor. The cells can be modified to express an exogenous construct, such as, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the genome of the cell.

[0151] 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" refers to a protein released by one cell population that acts on another cell as an intercellular mediator. Cytokines can be expressed endogenously by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effector proteins, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote immune cell survival and proliferation, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell 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).

[0152] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that represents a major component of the innate immune system. NK cells eliminate tumors and cells infected with viruses. They act through the process of apoptosis, or programmed cell death. NK cells are called "natural killers" because they do not need to be activated to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). T cell receptors (TCRs) distinguish T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. There are many types of T cells, including 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), and memory T cells (i.e., stem cell memory (TSCM) T cells, which, like naive T cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and have numerous other features characteristic of memory cells). (ii) central memory cells (TCMs) express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory cells (TCMs) 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 (NKTs), and gamma delta T cells. On the other hand, B cells play a major role in humoral immunity (antibody-mediated immunity). After activation by interaction with antigen, B cells can produce antibodies, act as antigen-presenting cells (APCs), and differentiate into both short-lived and long-lived memory B cells and plasma cells. In mammals, immature B cells are formed in the bone marrow.

[0153] "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 either by these cells or the liver, which result in the selective targeting, binding, damaging, destroying, and / or eliminating invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues in a vertebrate body.

[0154] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to a T cell response, such as, but not limited to, proliferation and / or upregulation or downregulation of key molecules.

[0155] 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 a costimulatory ligand provides signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, and the like. Costimulatory ligands induce signals that add to the primary signal provided by stimulatory molecules, e.g., by binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Costimulatory ligands can include, but are not limited to, 3 / TR6, 4-IBB ligand, an agonist or antibody that binds to a Toll-like receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I-related chain A protein (MICA), MHC class I-related chain B protein (MICB), OX40 ligand, PD-L2, or programmed death (PD)LI. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells (such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT)).

[0156] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, and which binding mediates a costimulatory response by the T cell, such as, but not limited to, proliferation.Examples of "costimulatory molecules" include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (A CD4, CD5, CD6, CD8, CD9, CD96 (Tactile), CD1-la, CD1-lb, CD1-lc, CD1-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA- 1 (CD11a / 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.

[0157] As used herein, a "subject" can be a mammal, such as a primate, an ungulate (e.g., cow, pig, horse), cat, dog, household pet, or domesticated mammal. In some cases, the mammal can be a rabbit, pig, horse, sheep, cow, cat, or dog, or a human. In some embodiments, the subject is a human. In some embodiments, the subject is an adult human. In some embodiments, the subject is a juvenile human.

[0158] II. Circular RNA and Compositions Thereof Provided herein are related pharmaceutical compositions comprising circular RNA constructs and delivery vehicles, which can be delivered in vivo to immune cells for protein therapy or protein production. According to the present disclosure, the circular RNAs provided herein can be injected into an animal (e.g., a human) such that a polypeptide encoded by the circular RNA molecule is expressed in the animal by, for example, immune cells and T cells.

[0159] In certain embodiments, the circular RNA construct comprises an IRES. In certain embodiments, the circular RNA construct comprises at least one expressed sequence encoding a binding molecule, wherein the binding molecule binds to or associates with a tumor cell antigen. In certain embodiments, the circular RNA construct comprises an IRES and at least one expressed sequence encoding a binding molecule.

[0160] In some embodiments, provided herein is a circular RNA polynucleotide comprising a spliced ​​3' Group I intron fragment (e.g., a stretch of exon sequence), optionally a first spacer, an internal ribosome entry site (IRES), an expression sequence, optionally a second spacer, and a spliced ​​5' Group I intron fragment (e.g., a stretch of exon sequence), in some embodiments, these regions in that order.

[0161] In certain embodiments, the circular RNA construct is incorporated into a pharmaceutical composition.In certain embodiments, the pharmaceutical composition comprises a transport vehicle.In certain embodiments, the circular RNA construct comprising an IRES and at least one expression sequence encoding a binding molecule is incorporated into a pharmaceutical composition comprising a transport vehicle.

[0162] In certain embodiments, pharmaceutical compositions are disclosed that include a circular RNA construct comprising an IRES and at least one expression sequence encoding a binding molecule, and a delivery vehicle, which in certain embodiments facilitates and / or enhances delivery and release of the circular RNA into one or more target cells.

[0163] In certain embodiments, the circular RNA constructs and related pharmaceutical compositions comprise an IRES and at least one expression sequence encoding a therapeutic protein, wherein the IRES is capable of promoting expression of the protein when delivered in vivo.

[0164] In certain embodiments, the circular RNA construct comprises an IRES and at least one expression sequence encoding a cytokine, an immune checkpoint inhibitor, an agonist, a chimeric antigen receptor (CAR), an inhibitory receptor agonist, one or more T cell receptors, and / or a B cell receptor.

[0165] In some embodiments, the polynucleotide encodes a protein composed of subunits encoded by two or more genes. For example, the protein may be a heterodimer, with each chain or subunit of the protein encoded by a separate gene. Two or more circular RNA molecules may be delivered in a delivery vehicle, with each circular RNA encoding a separate subunit of the protein. Alternatively, a single circular RNA may be engineered to encode two or more subunits. In certain embodiments, separate circular RNA molecules encoding individual subunits may be administered in separate delivery vehicles.

[0166] In certain embodiments, the circular RNA comprises an IRES and at least one expression sequence encoding a CAR construct. In some embodiments, the CAR targets a cancer antigen. In some embodiments, the CAR may be programmed to both recognize a specific antigen and, upon binding to the antigen, activate immune cells to attack and destroy the cell. In certain embodiments, the payload encoded by the circular RNA polynucleotide may be optimized through the use of a specific internal ribosome entry site (IRES) within a translation initiation element (TIE). The TIE may comprise an untranslated region (UTR), an aptamer complex, or a combination thereof. The UTR may be derived in whole or in part from a viral or eukaryotic mRNA. In some embodiments, the IRES specificity within the circular RNA may significantly enhance expression of a specific protein encoded within the coding element.

[0167] Circular RNA is generated by transcription of a template DNA, resulting in the formation of a linear precursor RNA polynucleotide that can be circularized. The linear precursor RNA polynucleotide is provided for generating circular RNA constructs and related pharmaceutical compositions. The template DNA shares the same sequence as the linear precursor RNA polynucleotide before splicing. The template DNA shares the same sequence (e.g., 3'-enhanced intron elements, 3'-enhanced exon elements, core functional elements, and 5'-enhanced exon elements, 5'-enhanced intron elements) as the linear precursor RNA polynucleotide before splicing. In some embodiments, the linear precursor RNA polynucleotide is spliced ​​during the circularization process, removing the 3'-enhanced intron elements and 5'-enhanced intron elements. In some embodiments, the resulting circular RNA polynucleotide lacks the 3'-enhanced intron fragment and the 5'-enhanced intron fragment, but maintains the 3'-enhanced exon fragment, core functional elements, and 5'-enhanced exon elements. Circularization strategies are known in the art and are described elsewhere herein. In certain embodiments, the resulting circular RNA may contain a PIE (permuted intron-exon) region, a translated region (IRES and coding / non-coding elements), and a PIE region. The resulting permuted intron-exon (PIE) region covalently links the 5' and 3' ends of the RNA, allowing the formation of a circular RNA.

[0168] In some embodiments, the precursor RNA polynucleotide comprises, in the following order: (a) a terminal element, (b) an intervening region, and (c) a monotron element. In some embodiments, the terminal sequence is upstream of the monotron sequence within the precursor RNA polynucleotide. In such embodiments, (i) the terminal element comprises a splice site nucleotide, (ii) the monotron element comprises a splice site dinucleotide at or near the 5' end of the monotron, (iii) the monotron element is capable of interacting with a nucleophile capable of cleaving at the splice site dinucleotide at or near the 5' end of the monotron, and the cleavage product of (iii) comprises a 5' splice site nucleotide capable of cleaving at the splice site nucleotide of the terminal element. In some embodiments, the nucleophile is a free nucleophile that is introduced into the precursor RNA polynucleotide (e.g., not in cis and / or covalently linked to the precursor RNA polynucleotide). In some embodiments, the nucleophile is a guanosine that is capable of cleaving at the splice site dinucleotide at or near the 5' end of the monotron. In some embodiments, the guanosine is a free guanosine that is introduced into the precursor RNA polynucleotide (e.g., not in cis and / or covalently linked to the precursor RNA polynucleotide). In some embodiments, the cleavage product of (iii) comprises a 5' splice site nucleotide having a 3' hydroxyl group that can be cleaved at the splice site nucleotide of the terminal element.

[0169] In some embodiments, the precursor RNA polynucleotide comprises, in the following order: (a) a monotron element, (b) an intervening region, and (c) a terminal element. In some embodiments, the monotron sequence is upstream of the terminal sequence within the precursor RNA polynucleotide. In such embodiments, (i) the monotron element comprises a splice site dinucleotide at or near the 3' end of the monotron, (ii) the terminal element comprises a splice site nucleotide, (iii) the monotron element is capable of interacting with a nucleophile capable of cleaving at the splice site nucleotide of the terminal element, and the cleavage product of (iii) comprises a 5' splice site nucleotide capable of cleaving at the splice site dinucleotide at or near the 3' end of the monotron. In some embodiments, the nucleophile is a free nucleophile introduced into the precursor RNA polynucleotide (e.g., not in cis and / or covalently linked to the precursor RNA polynucleotide). In some embodiments, the nucleophile is a guanosine capable of cleaving at the splice site nucleotide of the terminal element. In some embodiments, the guanosine is a free guanosine that is introduced into the precursor RNA polynucleotide (e.g., not in cis and / or covalently linked to the precursor RNA polynucleotide). In some embodiments, the cleavage product of (iii) comprises a 5' splice site nucleotide having a 3' hydroxyl group that can be cleaved at the splice site nucleotide of the terminal element.

[0170] In some embodiments, the linear precursor RNA polynucleotide comprises one or more guanosine nucleotides or nucleosides (e.g., GTP) and divalent cations (e.g., Mg 2+ In some embodiments, the 3'-enhanced exonic elements, 5'-enhanced exonic elements, and / or core functional elements promote, in whole or in part, circularization of a linear precursor RNA polynucleotide to form a circular RNA polynucleotide provided herein.

[0171] In certain embodiments, the circular RNAs provided herein are generated within cells. In some embodiments, precursor RNAs are transcribed by bacteriophage RNA polymerase in the cytoplasm or by host RNA polymerase II in the nucleus using template DNA (e.g., in some embodiments, using vectors provided herein), and then circularized.

[0172] In certain embodiments, the circular RNAs provided herein are injected into an animal (e.g., a human) such that the polypeptide encoded by the circular RNA molecule is expressed in the animal.

[0173] In some embodiments, the length of the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein is 300 to 10,000, 400 to 9,000, 500 to 8,000, 600 to 7,000, 700 to 6,000, 800 to 5,000, 900 to 5,000, 1,000 to 5,000, 1,100 to 5,000, 1,200 to 5,000, 1,300 to 5,000, 1,400 to 5,000, and / or 1,500 to 5,000 nucleotides. In some embodiments, the length of 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 some embodiments, the length of a polynucleotide is 3000nt or less, 3500nt or less, 4000nt or less, 4500nt or less, 5000nt or less, 6000nt or less, 7000nt or less, 8000nt or less, 9000nt or less, or 10000nt or less. 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.

[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, modified nucleotides (e.g., 5moU modifications), optimized UTRs, caps, and / or polyA tails.

[0175] In some embodiments, the functional half-life of the circular RNA polynucleotides provided herein is at least 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 hours. In some embodiments, the functional half-life of the circular RNA polynucleotides provided herein is 5-80, 10-70, 15-60, and / or 20-50 hours. In some embodiments, the functional half-life of the circular RNA polynucleotides provided herein is longer (e.g., at least 1.5-fold longer, at least 2-fold longer) than the functional half-life of 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 half-life of the circular RNA polynucleotides provided herein is at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the half-life of the circular RNA polynucleotides provided herein is 5-80 hours, 10-70 hours, 15-60 hours, and / or 20-50 hours. In some embodiments, the half-life of the circular RNA polynucleotides provided herein is longer (e.g., at least 1.5-fold longer, at least 2-fold longer) than the half-life of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the functional half-life of the circular RNA polynucleotide, or pharmaceutical composition thereof, in human cells is equal to or exceeds a predetermined threshold value. In some embodiments, the functional half-life is determined by a functional protein assay. In exemplary embodiments, the functional half-life is determined by an in vitro luciferase assay in which Gaussia luciferase (GLuc) activity is measured in the culture medium of human cells (e.g., HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours for 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay in which the level of the protein encoded by the expressed sequence of the circular RNA polynucleotide is measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours for 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the predetermined threshold is the functional half-life of a reference linear RNA polynucleotide comprising the same expressed sequence as the circular RNA polynucleotide.

[0177] In some embodiments, the circular RNAs provided herein are expressed at higher levels than comparable linear mRNAs, e.g., may be expressed at higher levels 24 hours after administration of the RNA to cells. In some embodiments, the circular RNAs provided herein are expressed at higher levels than mRNAs that contain the same expression sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails.

[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 particular 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 particular type of immune cell. For example, in some embodiments, the circular RNAs provided herein are associated with reduced production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to an organism's immune system or a particular type of immune cell, compared to mRNA containing the same expression sequence. In some embodiments, the circular RNAs provided herein are associated with reduced transcriptional induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to an organism's immune system or a particular 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, modified nucleotides (e.g., 5moU modifications), optimized UTRs, caps, and / or polyA tails.

[0179] In some embodiments, the circular RNA provided herein can be encapsulated in a transport vehicle (e.g., LNP) capable of delivering the circular RNA construct. By encapsulating the circular RNA in a transport vehicle, for example, a CAR gene can be efficiently introduced into T cells. The transport vehicle can include, for example, an ionizable lipid, a PEG-modified lipid, a helper lipid, and / or a structural lipid capable of encapsulating the circular RNA. Regarding the circular RNA construct, a pharmaceutical composition is provided that includes an IRES, an expression sequence, and a transport vehicle.

[0180] In certain embodiments, the circular RNA constructs provided herein can be directly transfected into cells, or can be transfected in the form of a DNA vector and transcribed within the cell. The transcription of the circular RNA from the transfected DNA vector can be via an added polymerase or a polymerase encoded by a nucleic acid transfected into the cell, or preferably via an endogenous polymerase. Thus, the present invention also provides eukaryotic cells comprising the circular RNA polynucleotides provided herein. In some embodiments, the eukaryotic cells are human cells. In some embodiments, the eukaryotic cells are immune cells. In some embodiments, the eukaryotic cells are T cells, dendritic cells, macrophages, B cells, neutrophils, or basophils. Also provided herein are prokaryotic cells comprising the circular RNA polynucleotides provided herein.

[0181] In some embodiments, provided herein are T cells (e.g., human T cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are helper T cells (e.g., human helper T cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are cytotoxic T cells (e.g., human cytotoxic T cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are NK cells (e.g., human NK cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are macrophages (e.g., human macrophages) comprising a circular RNA construct provided herein. In some embodiments, provided herein are monocytes (e.g., human monocytes) comprising a circular RNA construct provided herein. In some embodiments, provided herein are bone marrow cells (human monocytes) comprising a circular RNA construct provided herein. In some embodiments, the cells are present in the bone marrow. In some embodiments, the cells are present in the spleen. In some embodiments, the cells are present in the blood (e.g., peripheral blood).

[0182] In some embodiments, provided herein are CD3+ cells (e.g., human CD3+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD4+ cells (e.g., human CD4+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD8+ cells (e.g., human CD8+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD14+ cells (e.g., human CD14+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD16+ cells (e.g., human CD16+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD56+ cells (e.g., human CD56+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD11B+ cells (e.g., human CD11B+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD33+ cells (e.g., human CD33+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD33+CD14+ cells (e.g., human CD33+CD14+ cells) comprising a circular RNA construct provided herein. In some embodiments, provided herein are CD33+CD14+ cells (e.g., human CD33+CD64+ cells) comprising a circular RNA construct provided herein. In some embodiments, the cells are present in the bone marrow. In some embodiments, the cells are present in the spleen. In some embodiments, the cells are present in the blood (e.g., peripheral blood).

[0183] The circular RNA may be unmodified, partially modified, or fully modified. In one embodiment, the circular RNA contains at least one nucleoside modification. In one embodiment, up to 100% of the nucleosides of the circular RNA are modified. In one embodiment, the at least one nucleoside modification is a uridine modification or an adenosine modification. In one embodiment, the at least one nucleoside modification is selected from N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methoxyuridine (5moU). In one embodiment, the precursor RNA is modified with methylpseudouridine (m1ψ).

[0184] In certain embodiments, a provided polynucleotide (e.g., a template DNA, a precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises modified nucleotides and / or modified nucleosides. In some embodiments, the modified nucleosides are 5 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 s 2 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 -isopentenyl adenosine), ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine), io 6 A(N6 -(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 3 C(3-methylcytidine), Cm(2'-O-methylcytidine), s 2 C(2-thiocytidine), ac 4 C(N 4 -acetylcytidine), f 5 C(5-formylcytidine), m 5 Cm (5,2'-O-dimethylcytidine), ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine), k 2 C (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 ,N2 -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), ho 5 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 2U (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 3 U (3-methyluridine), cm 5 U (5-carboxymethyluridine), m 6 Am(N 6 ,2'-O-dimethyladenosine), m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine), m 2,7 G(N 2 ,7-dimethylguanosine), m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine), m 3 Um (3,2'-O-dimethyluridine), m 5 D (5-methyldihydrouridine), f 5 Cm (5-formyl-2'-O-methylcytidine), m 1Gm (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).

[0185] In some embodiments, modified nucleosides include those from the following group: pyridin-4-one ribonucleosides, 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-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2- Thio-pseudouridine, 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 Cytidine, 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, wybutosine, 7-deaza-guanosine , 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.

[0186] In some embodiments, modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and / or pseudouridine, which in some embodiments provide additional stability and resistance to immune activation.

[0187] Various circular RNAs, circular RNA constructs, compositions comprising circular RNAs, precursor RNAs, and related methods are described, for example, in WO2019236673, WO2020237227, WO2021113777, WO2021226597, WO2021189059, WO2021236855, WO2022261490, WO2023056033, and WO2023081526 (each of which is incorporated by reference in its entirety).

[0188] A. Enhanced intronic and exonic elements The circular RNAs provided herein may contain enhanced intron elements or fragments and enhanced exon elements or fragments. In certain embodiments, the enhanced intron elements and enhanced exon elements provided herein may include spacers, duplex regions, affinity sequences, intron fragments, exon fragments, and various untranslated elements. These sequences within the enhanced intron elements or enhanced exon elements are positioned to optimize circularization or protein expression.

[0189] In certain embodiments, the template DNA, linear precursor RNA polynucleotides, and circular RNAs provided herein comprise a first (5') spacer and / or a second (3') spacer. In some embodiments, the template DNA or linear precursor RNA polynucleotide comprises one or more spacers within an enhanced intron element. In some embodiments, the template DNA or linear precursor RNA polynucleotide comprises one or more spacers within an enhanced exon element. In certain embodiments, the template DNA or linear RNA polynucleotide comprises a spacer within a 3'-enhanced intron fragment and a spacer within a 5'-enhanced intron fragment. In certain embodiments, the template DNA, linear precursor RNA polynucleotide, or circular RNA comprises a spacer within a 3'-enhanced exon fragment and another spacer within a 5'-enhanced exon fragment to aid in circularization or protein expression due to the symmetry created in the overall sequence.

[0190] In some embodiments, including a spacer between the 3' Group I intron fragment and the core functional element may preserve the secondary structure of these regions by preventing their interaction, thus potentially improving splicing efficiency. In some embodiments, the first spacer (between the 3' Group I intron fragment and the core functional element) and the second spacer (between the two expression sequences and the core functional element) contain additional base-pairing regions that are predicted to base-pair to each other, rather than to the first and second duplex regions. In other embodiments, the first spacer (between the 3' Group I intron fragment and the core functional element) and the second spacer (between one of the core functional elements and the 5' Group I intron fragment) contain additional base-pairing regions that are predicted to base-pair to each other, rather than to the first and second duplex regions. In some embodiments, such spacer base-pairing brings the Group I intron fragments into close proximity with each other, further improving splicing efficiency. Additionally, in some embodiments, the combination of base pairing between the first and second duplex regions and the separate base pairing between the first and second spacers promotes the formation of a splicing bubble containing a group I intron fragment flanked by flanking base-paired regions. A typical spacer is a contiguous sequence with one or more of the following characteristics: 1) predicted to avoid interference with proximal structures (e.g., IRES, expression sequences, aptamers, or introns); 2) at least 7 nt and no more than 100 nt in length; 3) located adjacent to the 3' intron fragment and / or adjacent to the 5' intron fragment; and 4) containing one or more of the following: a) an unstructured region at least 5 nt in length, b) a base-paired region at least 5 nt in length to a distal sequence comprising another spacer, and c) a structured region at least 7 nt in length confined within the spacer sequence. The spacer can have several regions, including unstructured regions, base-paired regions, hairpin / structured regions, and combinations thereof.In some embodiments, the spacer has a structured region with 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 having a stem of 4-12 nucleotides and a loop of 2-10 nucleotides. In some embodiments, there is an additional spacer between the 3' group I intron fragment and the core functional element. In some embodiments, this additional spacer prevents or reduces the extent to which the structured region of the IRES or aptamer of the TIE interferes with folding of the 3' group I intron fragment. In some embodiments, the length of 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 some embodiments, the 5' spacer sequence is 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides or less in length. In some embodiments, the 5' spacer sequence is 5 to 50, 10 to 50, 20 to 50, 20 to 40, and / or 25 to 35 nucleotides in length. In certain embodiments, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In one embodiment, the 5' spacer sequence is a poly(A) sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer 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.

[0191] In some embodiments, the template DNA and linear precursor RNA polynucleotides and circular RNA polynucleotides provided herein comprise a first (5') duplex region and a second (3') duplex region. In certain embodiments, the template DNA and linear precursor RNA polynucleotides comprise a 5' external duplex region located within the 3'-enhanced intron fragment and a 3' external duplex region located within the 5'-enhanced intron fragment. In some embodiments, the template DNA, linear precursor RNA polynucleotides and circular RNA polynucleotides comprise a 5' internal duplex region located within the 3'-enhanced exon fragment and a 3' internal duplex region located within the 5'-enhanced exon fragment. In some embodiments, the DNA polynucleotides and linear precursor RNA polynucleotides comprise a 5' external duplex region, a 5' internal duplex region, a 3' internal duplex region, and a 3' external duplex region.

[0192] In certain embodiments, the first and second duplex 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 regions may be base-paired to each other. In some embodiments, the duplex regions are predicted to base-pair less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, or less than 25%) with unintended sequences (e.g., non-duplex region sequences) within the RNA. In some embodiments, including such duplex regions at the ends of the precursor RNA strands, adjacent to or very close to the group I intron fragments, brings the group I intron fragments into close proximity with each other, improving splicing efficiency. In some embodiments, the length of the duplex region is between 3 and 100 nucleotides (e.g., 3-75 nucleotides long, 3-50 nucleotides long, 20-50 nucleotides long, 35-50 nucleotides long, 5-25 nucleotides long, 9-19 nucleotides long). In some embodiments, the length of the duplex 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 some embodiments, the length of the duplex region is about 9 to about 50 nucleotides. In one embodiment, the length of the duplex region is about 9 to about 19 nucleotides. In some embodiments, the length of the duplex region is about 20 to about 40 nucleotides. In certain embodiments, the length of the duplex region is about 30 nucleotides.

[0193] In other embodiments, the template DNA, linear precursor RNA polynucleotide, or circular RNA polynucleotide does not contain any double-stranded region to optimize translation or circularization.

[0194] In certain embodiments, as provided herein, a template DNA or linear precursor RNA polynucleotide may comprise an affinity tag. In some embodiments, the affinity tag is located within the 3'-enhanced intron element. In some embodiments, the affinity tag is located within the 5'-enhanced intron element. In some embodiments, both (3' and 5') enhanced intron elements each comprise an affinity tag. In one embodiment, the affinity tag of the 3'-enhanced intron element is of similar length to the affinity tag in the 5'-enhanced intron element. In some embodiments, the affinity tag of the 3'-enhanced intron element is of the same sequence as the affinity tag in the 5'-enhanced intron element. In some embodiments, the affinity sequence is positioned to optimize oligo dT purification.

[0195] In some embodiments, one or more affinity tags present in the linear precursor RNA polynucleotide are removed upon circularization. In some embodiments, after RNA circularization, affinity tags are added to the remaining linear RNA. In some such embodiments, affinity tags are enzymatically added to the linear RNA. The presence of one or more affinity tags on the linear RNA and their absence on the circular RNA can facilitate the purification of the circular RNA. In some embodiments, such purification is performed using negative selection or affinity purification methods. In some embodiments, such purification is performed using a binding agent that preferentially or specifically binds to the affinity tag.

[0196] In some embodiments, the affinity tag comprises a polyA region. In some embodiments, the length of the polyA region is at least 15, 30, or 60 nucleotides. In some embodiments, the affinity tag comprising a polyA region is present at two locations within the linear precursor RNA. In some embodiments, the length of one or both polyA regions is 15-50 nucleotides. In some embodiments, the length of one or both polyA regions is 20-25 nucleotides. The polyA sequence(s) are removed upon circularization. Thus, the circular RNA can be separated from its precursor RNA using oligonucleotides that hybridize to polyA sequences, such as deoxythymidine oligonucleotides (oligo(dT)) conjugated to a solid surface (e.g., a resin).

[0197] In some embodiments, the affinity tag comprises a sequence that is not present in the circular RNA product. In some such embodiments, the sequence that is not present in the circular RNA product is a dedicated binding site (DBS). In some embodiments, the DBS is an unstructured sequence (i.e., a sequence that does not form a defined structural element such as a hairpin loop, a continuous dsRNA region, or a triple helix). In some embodiments, the DBS sequence forms a random coil. In some embodiments, the DBS comprises at least 25% GC content, at least 50% GC content, at least 75% GC content, or at least 100% GC content. In some embodiments, the DBS comprises at least 25% AC content, at least 50% AC content, at least 75% AC content, or 100% AC content. In some embodiments, the DBS is at least 15, 30, or 60 nucleotides in length. In some embodiments, the affinity tag comprising a DBS is present at two locations within the linear precursor RNA. In some embodiments, the DBS sequences are each independently 15-50 nucleotides in length. In some embodiments, the DBS sequences are each independently 20-25 nucleotides in length.

[0198] In some embodiments, the DBS sequence(s) are removed upon circularization.Therefore, a binder comprising an oligonucleotide comprising a sequence complementary to DBS can be used to facilitate the purification of circular RNA.For example, the binder can comprise an oligonucleotide complementary to DBS conjugated to a solid surface (e.g., a resin).

[0199] In some embodiments, an affinity sequence or other type of affinity handle (such as biotin) is added to the linear RNA by ligation. In some embodiments, an oligonucleotide comprising an affinity sequence is ligated to the linear RNA. In some embodiments, an oligonucleotide conjugated to an affinity handle is ligated to the linear RNA. In some embodiments, a solution comprising a linear RNA ligated to an affinity sequence or handle and a circular RNA without an affinity sequence or handle is contacted with a binder comprising an oligonucleotide complementary to the affinity sequence or a solid support conjugated to a binding partner of the affinity handle, whereby the linear RNA binds to the binder and the circular RNA is eluted or separated from the solid support.

[0200] Any of the methods for purifying circular RNA described herein may include one or more buffer exchange steps. In some embodiments, buffer exchange is performed after in vitro transcription (IVT) and before additional purification steps. In some such embodiments, the IVT reaction solution is buffer exchanged into a buffer containing Tris. In some embodiments, the IVT reaction solution is buffer exchanged into a buffer containing greater than 1 mM or greater than 10 mM of one or more monovalent salts (such as NaCl or KCl), and optionally EDTA. In some embodiments, buffer exchange is performed after circular RNA purification is complete. In some embodiments, buffer exchange is performed after IVT and after circular RNA purification. In some embodiments, buffer exchange performed after circular RNA purification comprises exchanging the circular RNA buffer for water or a storage buffer. In some embodiments, the storage buffer comprises 1 mM sodium citrate (pH 6.5).

[0201] In certain embodiments, the 3'-enhanced intron element comprises a leader untranslated sequence. In some embodiments, the leader untranslated sequence is at the 5' end of the 3'-enhanced intron fragment. In some embodiments, the leader untranslated sequence comprises the last nucleotide of a transcription start site (TSS). In some embodiments, the TSS is selected from viral, bacterial, or eukaryotic template DNA. In one embodiment, the leader untranslated sequence comprises the last nucleotide of the TSS and 0 to 100 additional nucleotides. In some embodiments, the TSS is a terminal spacer. In one embodiment, the leader untranslated sequence contains a guanosine at the 5' end upon translation by RNA T7 polymerase.

[0202] In certain embodiments, the 5'-enhanced intron element comprises a trailing untranslated sequence. In some embodiments, the 5'-trailing untranslated sequence is located at the 3' end of the 5'-enhanced intron element. In some embodiments, the trailing untranslated sequence is a partial restriction digest sequence. In one embodiment, the trailing untranslated sequence is, in whole or in part, a restriction digest site used to linearize the template DNA. In some embodiments, the restriction digest site is, in whole or in part, derived from a naturally occurring viral, bacterial, or eukaryotic template DNA. In some embodiments, the trailing untranslated sequence is a terminal restriction site fragment.

[0203] 1. Enriched Intron Fragments In certain embodiments, as provided herein, the 3'-enhanced intron element and the 5'-enhanced intron element each comprise an intron fragment. In certain embodiments, the 3'-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 naturally occurring Group I intron that includes the 3'-splice site dinucleotide. Typically, the 5'-intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5'-proximal fragment of a naturally occurring Group I intron that includes the 5'-splice site dinucleotide. In some embodiments, the 3' intron fragment comprises the first nucleotide of a 3' Group I splice site dinucleotide. In some embodiments, the 5' intron fragment comprises the first nucleotide of a 5' Group I splice site dinucleotide. In other embodiments, the 3' intron fragment comprises the first and second nucleotides of a 3' Group I intron fragment splice site dinucleotide, and the 5' intron fragment comprises the first and second nucleotides of a 3' Group I intron fragment dinucleotide.

[0204] 2. Enriched Exon Fragments In certain embodiments, as provided herein, the template DNA, linear precursor RNA polynucleotide, and circular RNA polynucleotide each comprise an enhanced exon fragment. In some embodiments, the 3'-enhanced exon element is located upstream of the core functional element in 5'-3' order. In some embodiments, the 5'-enhanced intron element is located downstream of the core functional element in 5'-3' order.

[0205] According to the present disclosure, the 3'-enhanced exonic element and the 5'-enhanced exonic element each comprise an exonic fragment. In some embodiments, the 3'-enhanced exonic element comprises a 3'-exonic fragment. In some embodiments, the 5'-enhanced exonic element comprises a 5'-exonic fragment. In certain embodiments, as provided herein, the 3'-exonic fragment and the 5'-exonic fragment comprise 1-100 nucleotides of a Group I intron fragment and exonic sequence, respectively. In certain embodiments, the 3'-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 naturally occurring Group I intron that includes the 3'-splice site dinucleotide. Typically, the 5' Group I intron fragment is a contiguous sequence that is at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous) to the 5'-proximal fragment of a naturally occurring Group I intron that includes the 5' splice site dinucleotide. In some embodiments, the 3' exon fragment includes the second nucleotide of the 3' Group I intron splice site dinucleotide and 1-100 nucleotides of exon sequence. In some embodiments, the 5' exon fragment includes the first nucleotide of the 5' Group I intron splice site dinucleotide and 1-100 nucleotides of exon sequence. In some embodiments, the exon sequence includes some or all of the naturally occurring exon sequence derived from a viral, bacterial, or eukaryotic DNA vector. In other embodiments, the exon sequences further comprise synthetic exon sequences, genetically altered exon sequences (eg, containing modified nucleotides), or other engineered exon sequences.

[0206] In one embodiment, when the 3' intron fragment contains both nucleotides of the 3' Group I splice site dinucleotide and the 5' intron fragment contains both nucleotides of the 5' Group I splice site dinucleotide, the exon fragment located within the 5' enhanced exon element and the 3' enhanced exon element does not contain the Group I splice site dinucleotide.

[0207] 3. Exemplary reordering of enriched intronic elements & enriched exonic elements By way of example and not limitation, in some embodiments, a 3'-enhanced intron element comprises, in 5' to 3' order: a leading untranslated sequence, a 5' affinity tag, an optional 5' external duplex region, a 5' external spacer, and a 3' intron fragment. In the same embodiment, a 3'-enhanced exon element comprises, in 5' to 3' order: a 3' exon fragment, an optional 5' internal duplex region, an optional 5' internal duplex region, and a 5' internal spacer. In the same embodiment, a 5'-enhanced exon element comprises, in 5' to 3' order: a 3' internal spacer, an optional 3' internal duplex region, and a 5' exon fragment. In further embodiments, a 3'-enhanced intron element comprises, in 5' to 3' order: a 5' intron fragment, a 3' external spacer, an optional 3' external duplex region, a 3' affinity tag, and a trailing untranslated sequence.

[0208] B. Core Functional Element - IRES In some embodiments, the template DNA, linear precursor RNA polynucleotide, and circular RNA polynucleotide comprise core functional elements. In some embodiments, the core functional elements comprise coding and / or non-coding elements. In some embodiments, the core functional elements further comprise a translation initiation element (TIE) and / or a termination element upstream of the coding or non-coding element.

[0209] In some embodiments, the core functional element comprises a termination element. In some embodiments, the termination sequence comprises a stop codon. In one embodiment, the termination sequence comprises a stop cassette. In some embodiments, the termination cassette comprises at least two stop codons. In some embodiments, the termination cassette comprises at least two stop codon frames. In some embodiments, the stop codon frames within the termination cassette each comprise one, two, or more stop codons. In some embodiments, the termination cassette comprises a LoxP or RoxStopRox, or frt-flanked termination cassette. In some embodiments, the termination cassette comprises a lox-stop-lox termination cassette.

[0210] In some embodiments, the polynucleotides herein comprise coding or non-coding elements, or a combination of both. In some embodiments, the coding elements comprise expression sequences. In some embodiments, the coding elements encode at least one therapeutic protein. In some embodiments, the circular RNA encodes two or more polypeptides.

[0211] In some embodiments, the core functional element comprises at least one translation initiation element (TIE). The TIE is designed to increase the translation efficiency of the encoded protein. In some embodiments, a core functional element comprising one or more coding elements will further comprise one or more TIEs. In some embodiments, the translation initiation element (TIE) comprises a synthetic TIE. In some embodiments, the synthetic TIE comprises an aptamer complex, a synthetic IRES, or other engineered TIES capable of initiating translation of a linear or circular RNA polynucleotide.

[0212] In some embodiments, TIE comprises untranslated region (UTR) or its fragment, aptamer complex or its fragment, or a combination thereof.In certain embodiments, TIE contains modified nucleotide.In certain embodiments, TIE provided herein comprises internal ribosome entry site (IRES).In certain embodiments, IRES comprises one or more modified nucleotides compared with wild-type virus IRES or eukaryotic IRES.For example, see WO2022 / 261490 (its entirety is incorporated herein by reference).

[0213] Since the discovery of viral IRESs, their differences have made their classification difficult. It is recognized that there is no common mechanism for the function of all IRESs. In addition, specific structural elements shared by all IRESs have not been found, and their sequences lack significant homology. See Nikonov, Biochemistry (Moscow), 2017, Vol. 82, No. 13, pp. 1615-1631. According to one author, four IRES classes have been defined. Type I and II IRESs are found in picornaviruses and can be around 400-500 nt in length. Type III IRESs are associated with Flaviviridae (including HCV) and HCV-like picornaviruses and are characterized by the presence of a pseudoknot upstream of the AUG codon and the requirement of the first 30 nt of the coding sequence. Type IV IRES are intergenic region (IGR) IRES, first identified in cricket paralysis virus (CrPV), which can function in the absence of any start codon and in which translation begins with a GCU triplet. See Godet, Int. J. Mol. Sci. 2019, 20, 924; doi:10.3390 / ijms20040924.

[0214] The inclusion of an IRES allows for translation of one or more open reading frames (e.g., open reading frames forming an expression sequence) from the circular RNA. The IRES element attracts the eukaryotic ribosomal translation initiation complex and promotes 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. In some embodiments, the IRES can promote expression of a protein encoded by a precursor RNA in a cell. In some embodiments, the IRES can promote protein expression such that the level of protein expression is similar to or higher than when a control IRES is used.

[0215] Numerous IRES sequences are available, including those derived from a wide variety of viruses, such as picornavirus leader sequences such as the encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63:1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, the human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), the foot-and-mouth disease virus IRES element (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), the giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. The ability of various IRES sequences to drive protein expression varies, and the ability of any particular identified or predicted IRES sequence to express a protein from a linear mRNA or circular RNA construct is unknown and unpredictable. In certain embodiments, potential IRES sequences can be identified bioinformatically based on their sequence location within the viral sequence. However, the activity of such sequences has not previously been characterized. As demonstrated herein, such IRES sequences may have different protein expression capabilities depending on the cell type (e.g., in T cells, hepatocytes, or muscle cells). In some embodiments, the novel IRES sequences described herein may increase expression in a particular cell type by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100-fold compared to previously described EMCV IRES sequences.

[0216] In some embodiments, the IRES is selected from the group consisting of Aarivirus, Ailurivirus, Ampivirus, Anachivirus, Aphthovirus, Akuamavirus, Abihepatovirus, Abyssivirus, Bucepivirus, Bopivirus, Cursilivirus, Cardiovirus, Cosavirus, Krahelivirus, Kurohivirus, Danipivirus, Dicipivirus, Diresapivirus, Enterovirus, Erbovirus, Felipivirus, Fipivirus, Gallivirus, Gruhelivirus, Grusopivirus, Harkavirus, Hemipivirus, Hepatovirus, Hunnivirus, Kobuvirus, Kunsaguivirus, Limnipivirus, Ribpivirus, Ludopivirus, Malagavirus, and others. Civirus, Marspivirus, Meghrivirus, Missivirus, Mosavirus, Mupivirus, Miropivirus, Orivirus, Osthivirus, Parabovirus, Parechovirus, Pasivirus, Passerivirus, Pemapivirus, Poesivirus, Potamipivirus, Paigosepivirus, Rabovirus, Rafivirus, Lajidapivirus, Rohelivirus, Rosavirus, Sakobuvirus, Sarivirus, Sapelovirus, Senecavirus, Shambavirus, Citinivirus, Simapivirus, Teschovirus, Torchvirus, Tottorivirus, Tremovirus, Tropivirus, Hepacivirus, Pegivirus, Pestivirus, Flavivirus IRES. In some embodiments herein, the IRES is selected from enterovirus, kobuvirus, parechovirus, Hunnivirus, Passerivirus, Missivirus, and cardiovirus.

[0217] In some embodiments, the IRES is selected from the group consisting of Taura syndrome virus, Triatomine bug 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 bee 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 brood virus, aphid fatal paralysis virus, chicken 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-IAPl, 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, phopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pacivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pacivirus 1, PLV-CHN, Pacivirus A, Citinivirus, 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 The IRES sequence of an aptamer for SZ1, sarivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0218] In some embodiments, the IRES comprises, in whole or in part, a eukaryotic or cellular IRES. In certain embodiments, the IRES is derived from a human gene, such as ABCF1, ABCG1, ACAD10, ACOT7, ACSS3, ACTG2, ADCYAP1, ADK, AGTR1, AHCYL2, AHI1, AKAP8L, AKR1A1, ALDH3A1, ALDOA, ALG13, AMMECR1L, ANGPTL4, ANK3, AOC3, AP4B1, AP4E1, APAF1, APBB1, APC, APH1A, APOBEC3D, APOM, APP, AQP4, ARHGAP36, ARL13B, A RMC8, ARMCX6, ARPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURK A, B3GALNT1, B3GNTL1, B4GALT3, BAAT, BAG1, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, B TN3A2, C12orf43, C14orf93, C17orf62, C1orf226, C21orf62, C2orf15, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP 2, CAV1, CBX5, CCDC120, CCDC17, CCDC186, CCDC51, CCN1, CCND1, CCNT1, CD2BP2, CD9, CDC25C, CDC42, CDC7, CDCA7L, CDIP1, CDK1, CDK11A, CDKN1B , CEACAM7, CEP295NL, CFLAR, CHCHD7, CHIA, CHIC1, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, COG4, C OMMD1, COMMD5, CPEB1, CPS1, CRACR2B, CRBN, CREM, CRYBG1, CSDE1, CSF2RA, CSNK2A1, CSTF3, CTCFL, CTH, CTNNA3, CTNNB1, CTNNB1, CTNND1, CTSL,CUTA、CXCR5、CYB5R3、CYP24A1、CYP3A5、DAG1、DAP3、DAP5、DAXX、DCAF4、DCAF7、DCLRE1A、DCP1A、 DCTN1、DCTN2、DDX19B、DDX46、DEFB123、DGKA、DGKD、DHRS4、DHX15、DIO3、DLG1、DLL4、DMDUTR、DMD ex5、DMKN、DNAH6、DNAL4、DUSP13、DUSP19、DYNC1I2、DYNLRB2、DYRK1A、ECI2、ECT2、EIF1AD、EIF2B4、EIF4G1、EIF4G2、EIF4G3、EL ANE、ELOVL6、ELP5、EMCN、ENO1、EPB41、ERMN、ERVV-1、ESRRG、ET FB、ETFBKMT、ETV1、ETV4、EXD1、EXT1、EZH2、FAM111B、FAM157A、F AM213A、FBXO25、FBXO9、FBXW7、FCMR、FGF1、FGF1、FGF1A、FGF2、 FGF2、FGF-9、FHL5、FMR1、FN1、FOXP1、FTH1、FUBP1、G3BP1、GABBR 1、GALC、GART、GAS7、ガストリン、GATA1、GATA4、GFM2、GHR、GJB2、GLI 1、GLRA2、GMNN、GPAT3、GPATCH3、GPR137、GPR34、GPR55、GPR89A、 GPRASP1、GRAP2" B4、HMBS、HMGA1、HNRNPC、HOPX、HOXA2、HOXA3、HPCAL1、HR、HSP9 0AB1、HSPA1A、HSPA4L、HSPA5、HYPK、IFFO1、IFT74、IFT81、IGF1、 IGF1R, IGF1R, IGF2, IL11, IL17RE, IL1RL1, IL1RN, IL32, IL6, ILF2, ILVBL, INSR, INTS13, IP6K1, ITGA4, ITGAE, KCNE4, KERA, KIAA0355, KIAA0895L, KIAA1324, KIAA1522, KIAA1683, KIF2C, KIZ, KLHL31, KLK7, KRR1, KRT14, KRT17, KRT33A, KRT6A, KRTAP10-2KRTAP13-3、KRTAP13-4、KRTAP5-11、KRTCAP2、LACRT、LAMB1、LAMB3、LANCL1 、LBX2、LCAT、LDHA、LDHAL6A、LEF1、LINC-PINT、LMO3、LRRC4C、LRRC7、LRTOM T、LSM5、LTB4R、LYRM1、LYRM2、MAGEA11、MAGEA8、MAGEB1、MAGEB16、MAGEB3、 MAPT、MARS、MC1R、MCCC1、METTL12、METTL7A、MGC16025、MGC16025、MIA2、MIA 2、MITF、MKLN1、MNT、MORF4L2、MPD6、MRFAP1、MRPL21、MRPS12、MSI2、MSLN、M SN、MT2A、MTFR1L、MTMR2、MTRR、MTUS1、MYB、MYC、MYCL、MYCN、MYL10、MYL3、M YLK、MYO1A、MYT2、MZB1、NAP1L1、NAV1、NBAS、NCF2、NDRG1、NDST2、NDUFA7、N DUFB11、NDUFC1、NDUFS1、NEDD4L、NFAT5、NFE2L2、NFE2L2、NFIA、NHEJ1、NHP2 、NIT1、NKRF、NME1-NME2、NPAT、NR3C1、NRBF2、NRF1、NTRK2、NUDCD1、NXF2、N XT2、ODC1、ODF2、OPTN、OR10R2、OR11L1、OR2M2、OR2M3、OR2M5、OR2T10、OR4C 15、OR4F17、OR4F5、OR5H1、OR5K1、OR6C3、OR6C75、OR6N1、OR7G2、p53、P2RY4 、PAN2、PAQR6、PARP4、PARP9、PC、PCBP4、PCDHGC3、PCLAF、PDGFB、PDZRN4、PEL O、PEMT、PEMT、PFKM、PGBD4、PGLYRP3、PHLDA2、PHTF1、PI4KB、PIGC、PIM1、PK D2L1、PKM、PLCB4、PLD3、PLAY1、PLEKHB1、PLS3、PML、PNMA5、PNN、POC1A、PO C1B、POLD2、POLD4、POU5F1、PPIG、PQBP1、PRAME、PRPF4、PRR11、PRRT1、PRSS 8、PSMA2、PSMA3、PSMA4、PSMD11、PSMD4、PSMD6、PSME3、PSMG3、PTBP3、PTCH1、PTHLH, PTPRD, PUS7L, PVRIG, QPRT, RAB27A, RAB7B, RABGGTB, RAET1E, RALGDS, RALYL, RARB, RCVRN, REG3G, RFC5, RGL4, RGS19, RGS3, RHD, RINL, RIPOR2, RITA1, RMDN2, RNASE1, RNASE4, RNF4, RPA2, RPL17, RPL21, RPL26L1, RPL28, RPL29, RPL41, RPL9, RPS11, RPS13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RUNX1T 1, RUNX1T1, RUNX2, RUSC1, RXRG, S100A13, S100A4, SAT1, SCHIP1, SCMH1, SEC14L1, SEMA4A, SERPINA1, SERPINB4, SERTAD3, SFTPD, SH3D19, SHC1, SHMT1, SHPRH, SIM1, SIRT5, SLC11A2, SLC12A4, SLC16A1, SLC25A3, SLC26A9, SLC5A11, SLC6A12, SLC6A19, SLC7A1, SLFN11, SLIRP, SMAD5, SMARCAD1, SMN1, SNCA , SNRNP200, SNRPB2, SNX12, SOD1, SOX13, SOX5, SP8, SPARCL1, SPATA12, SPATA31C2, SPN, SPOP, SQSTM1, SRBD1, SRC, SREBF1, SRPK2, SSB, SSB, SSBP1, ST3GAL6, STAB1, STAMBP, STAU1, STAU1, STAU1, STAU1, STK16, STK24, STK38, STMN1, STX7, SULT2B1, SYK, SYNPR, TAF1C, TAGLN, TANK, TAS2R40, TBC1D 15、TBXAS1、TCF4、TDGF1、TDP2、TDRD3、TDRD5、TESK2、THAP6、THBD、THTPA、TIAM2、TKFC、TKTL1、TLR10、TM9SF2、TMC6、TMCO2、TMED10、TMEM116、TMEM126A 、TMEM159、TMEM208、TMEM230、TMEM67、TMPRSS13、TMUB2、TNFSF4、TNIP3、TP53、TP53、TP73、TRAF1、TRAK1、TRIM31、TRIM6、TRMT1、TRMT2B、TRPM7、TRPM8、TSPEAR, TTC39B, TTLL11, TUBB6, TXLNB, TXNIP, TXNL1, TXNRD1, TYROBP, U2AF1, UBA1, UBE2D3, UBE2I, UBE2L3, UB E2V1, UBE2V2, UMPS, UNG, UPP2, USMG5, USP18, UTP14A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS29, V SIG10L, WDHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY1AP1, ZBTB32, ZNF146, ZNF250, ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF70, or ZNRD1.

[0219] In some embodiments, the cell is a myotube cell. In some embodiments, the IRES is derived from a bopivirus, an ostivirus, a hunnivirus, a passerivirus, a missivirus, a kobuvirus, an enterovirus, a cardiovirus, a sarivirus, a rabovirus, a parechovirus, a gallivirus, or a citiniivirus. In some embodiments, the IRES is derived from a hunnivirus, a passerivirus, a kobuvirus, a bopivirus, or an enterovirus. In some embodiments, the IRES is derived from an enterovirus I, an enterovirus F, an enterovirus E, an enterovirus J, an enterovirus C, an enterovirus A, an enterovirus B, an aichivirus B, a parechovirus A, a cardiovirus F, a cardiovirus B, or a cardiovirus E.

[0220] In some embodiments, the cell is a hepatocyte. In some embodiments, the IRES is derived from an enterovirus, bopivirus, missivirus, gallivirus, ostivirus, cardiovirus, kobuvirus, rabovirus, sarivirus, parechovirus, hunnivirus, tottorivirus, passerivirus, cosavirus, or citiniivirus. In some embodiments, the IRES is derived from an enterovirus, missivirus, kobuvirus, bopivirus, or gallivirus. In some embodiments, the IRES is derived from enterovirus B, enterovirus A, enterovirus D, enterovirus J, enterovirus C, rhinovirus B, enterovirus H, enterovirus I, enterovirus E, enterovirus F, aichivirus B, aichivirus A, parechovirus A, cardiovirus F, cardiovirus E, or cardiovirus B.

[0221] In some embodiments, the cell is a T cell. In some embodiments, the IRES is derived from a Passerivirus, a Bopivirus, a Hunnivirus, a Missivirus, an Enterovirus, a Kobuvirus, a Rabovirus, a Tottorivirus, a Salivirus, a Cardiovirus, a Parechovirus, a Meghrivirus, an Alexivirus, an Ostichivirus, or a Shambavirus. In some embodiments, the IRES is derived from a Passerivirus, a Hunnivirus, a Missivirus, an Enterovirus, or a Kobuvirus. In some embodiments, the IRES is derived from an Enterovirus I, an Enterovirus D, an Enterovirus C, an Enterovirus A, an Enterovirus J, an Enterovirus H, an Aichivirus B, a Parechovirus A, or a Cardiovirus B.

[0222] To drive protein expression, the circular RNA comprises an IRES operably linked to the protein-coding sequence. Exemplary IRES sequences are shown in Table 1A. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the IRES sequence in Table 1A or any of the constructs of SEQ ID NOs: 50-61 or construct AP in Table 1B. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence in Table 1A or any of the constructs of SEQ ID NOs: 50-61 or construct AP in Table 1B. Disclosed herein are modifications of IRES and accessory sequences to increase or decrease IRES activity, for example, by truncating the 5' and / or 3' ends of the IRES, adding a spacer 5' to the IRES, modifying the 6 nucleotides 5' to the translation start site (Kozak sequence), modifying alternative translation start sites, and creating chimeric / hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNA constructs and related pharmaceutical compositions disclosed herein comprises one or more of these modifications compared to the native IRES.

[0223] In certain embodiments, the circular RNA constructs disclosed herein comprise an IRES and at least one expressed sequence encoding a binding molecule. In certain embodiments, the IRES sequence is an exemplary IRES sequence shown in Table 1A below, or an IRES from any of the constructs of SEQ ID NOs: 50-61 or construct AP in Table 1B. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 1A, or an IRES from any of the constructs of SEQ ID NOs: 50-61 or construct AP in Table 1B. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence in Table 1A, or an IRES from any of the constructs of SEQ ID NOs: 50-61 or construct AP in Table 1B, and at least one expressed sequence encoding a binding molecule. [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4] [Table 1A-5] [Table 1A-6] [Table 1A-7] [Table 1A-8] [Table 1A-9] [Table 1A-10] [Table 1A-11] [Table 1A-12] [Table 1A-13]

[0224] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Table 1A, the constructs of SEQ ID NOs: 50-61, or any of the constructs AP in Table 1B shown below, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of any of the constructs AP in Table 1B shown below. In some embodiments, the circular RNA further comprises a CD28z or 4-1BB costimulatory domain described herein.

[0225] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct A, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct A. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Construct A, and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of Construct A. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0226] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct B, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct B. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct B, and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct B. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0227] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct C, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct C. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct C, and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct C. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0228] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct D, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct D. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct D and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct D. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0229] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Construct E, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of Construct E. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Construct E and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of Construct E. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0230] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct F, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct F. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct F and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct F. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0231] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct G, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct G. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct G and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct G. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to an appropriate control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to an appropriate control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to an appropriate control having an alternative costimulatory domain.

[0232] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct H, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct H. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct H and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct H. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0233] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Construct I, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of Construct I. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of Construct I, and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of Construct I. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further comprises a CD28z costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain. In some embodiments, the circular RNA further comprises a 4-1BB costimulatory domain described herein, and optionally exhibits increased activity compared to a suitable control having an alternative costimulatory domain.

[0234] In some embodiments, the circular RNA comprises an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct J, and a CAR sequence encoding a polypeptide having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct J. In some embodiments, the circular RNA comprises an IRES sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the IRES sequence of construct J and a CAR sequence encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CAR of construct J. In some embodiments, the circular RNA exhibits increased expression and / or activity compared to a suitable control having an alternative IRES. In some embodiments, the circular RNA further compr...

Claims

1. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a binding molecule; A circular RNA construct comprising:

2. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a chimeric antigen receptor (CAR) that targets a cancer antigen; A circular RNA construct comprising:

3. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; A circular RNA construct comprising:

4. 4. The circular RNA construct of claim 3, wherein the expression sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

5. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A circular RNA construct comprising:

6. 6. The circular RNA construct of claim 5, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

7. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a binding molecule; A circular RNA construct comprising:

8. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a chimeric antigen receptor (CAR) that targets a cancer antigen; A circular RNA construct comprising:

9. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; A circular RNA construct comprising:

10. 10. The circular RNA construct of claim 9, wherein the expression sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

11. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A circular RNA construct comprising:

12. 12. The circular RNA construct of claim 11, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

13. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a binding molecule, and (B) a transportation medium; A pharmaceutical composition comprising:

14. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transportation medium; A pharmaceutical composition comprising:

15. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a binding molecule, and (B) a transportation medium; A pharmaceutical composition comprising:

16. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transportation medium; A pharmaceutical composition comprising:

17. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a binding molecule, and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

18. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

19. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

20. 20. The pharmaceutical composition of claim 19, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

21. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

22. 22. The pharmaceutical composition of claim 21, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

23. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a binding molecule, and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

24. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

25. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

26. 26. The pharmaceutical composition of claim 25, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

27. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) a transport vehicle comprising an ionizable lipid; A pharmaceutical composition comprising:

28. 28. The pharmaceutical composition of claim 27, wherein the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

29. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a binding molecule, and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical Engineering 201】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical Engineering 202】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical 203】 A pharmaceutical composition comprising:

30. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressible sequence encoding a CAR that targets a cancer antigen; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 204】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 205】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical 206】 A pharmaceutical composition comprising:

31. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 207】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 208】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical Engineering 209】 A pharmaceutical composition comprising:

32. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 210】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 211】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical Engineering 212】 A pharmaceutical composition comprising:

33. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemistry 213】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 214】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical 215】 A pharmaceutical composition comprising:

34. below: (A) A circular RNA construct comprising: i. an IRES comprising a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-BCMA conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 216】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 217】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical 218】 A pharmaceutical composition comprising:

35. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a binding molecule, and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 219】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 220】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 221】 A pharmaceutical composition comprising:

36. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressible sequence encoding a CAR construct that targets a cancer antigen; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemistry 222】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 223】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 224】 A pharmaceutical composition comprising:

37. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemical 225】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 226】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 227】 A pharmaceutical composition comprising:

38. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-CD19 conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemistry 228】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 229】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 230】 A pharmaceutical composition comprising:

39. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemistry 231】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 232】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemical 233】 A pharmaceutical composition comprising:

40. below: (A) A circular RNA construct comprising: i. an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus, and ii. at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises an anti-BCMA conjugate, and the expressed sequence comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115; and (B) A transport medium comprising: (i) an ionizable lipid of formula (I) 【Chemistry 234】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 235】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 236】 A pharmaceutical composition comprising:

41. 39. The pharmaceutical composition of any one of claims 1-4, 7-10, 13-20, 23-26, 29-32, and 35-38, wherein the CAR construct comprises a CD19 conjugate and the circular RNA comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61.

42. 39. The pharmaceutical composition of any one of claims 1-4, 7-10, 13-20, 23-26, 29-32, and 35-38, wherein the CAR construct comprises a CD19 conjugate and the circular RNA comprises a sequence selected from any one of SEQ ID NOs: 50-61.

43. 43. The pharmaceutical composition of claim 42, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59.

44. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expressed sequence encoding a CAR construct that targets a cancer antigen, the CAR construct comprising a CD19 binder, and the circular RNA comprises a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61.

45. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expression sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder, and the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50-61.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59.

47. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expressed sequence encoding a CAR construct that targets a cancer antigen, the CAR construct comprising a CD19 binder, the circular RNA comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 50-61, and the delivery vehicle comprises one of the following: (i) an ionizable lipid of formula (I) 【Chemistry 237】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemical 238】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 239】 The pharmaceutical composition comprising:

48. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises an IRES and at least one expression sequence encoding a CAR construct that targets a cancer antigen, the CAR construct comprising a CD19 binder, the circular RNA comprising a sequence selected from any one of SEQ ID NOs: 50-61, and the delivery vehicle comprising: (i) an ionizable lipid of formula (I) 【Chemistry 240】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 241】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 242】 The pharmaceutical composition comprising:

49. 49. The pharmaceutical composition of claim 48, wherein the circular RNA construct comprises a sequence selected from any one of SEQ ID NOs: 50, 51, 52, 54, 55, 56, 58, and 59.

50. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1-49, wherein the circular RNA construct comprises SEQ ID NO:

50.

51. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

51.

52. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

52.

53. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

54.

54. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

55.

55. 50. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

56.

56. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

58.

57. The circular RNA construct or pharmaceutical composition of any one of claims 1 to 49, wherein the circular RNA construct comprises SEQ ID NO:

59.

58. 41. The pharmaceutical composition of any one of claims 5-6, 21-22, 27, 28, 33-34, or 39-40, wherein the IRES comprises the sequence of SEQ ID NO: 8, the CAR construct comprises a BCMA conjugate, and the BCMA conjugate comprises a sequence selected from any one of SEQ ID NOs: 104-115.

59. 59. The pharmaceutical composition of any one of claims 13-58, wherein the delivery vehicle comprises an ionizable lipid of formula (I).

60. 60. The pharmaceutical composition of claim 59, wherein the delivery vehicle comprises a helper lipid, a structural lipid, and a PEG lipid.

61. 61. The pharmaceutical composition of any one of claims 59-60, wherein the delivery vehicle is formulated with a lipid molar ratio as set forth in Table 4b.

62. 59. The pharmaceutical composition of any one of claims 13-58, wherein the delivery vehicle comprises an ionizable lipid of formula (II).

63. The ionizable lipid may be: 【Chemistry 243】 63. The pharmaceutical composition of claim 62, wherein the ionizable lipid is selected from the group consisting of:

64. The ionizable lipid may be: 【Chemistry 244】 64. The pharmaceutical composition of claim 63, wherein:

65. 65. The pharmaceutical composition of any one of claims 13-64, wherein the delivery vehicle further comprises at least one lipid selected from a helper lipid, a structural lipid, and a PEG-modified lipid.

66. 66. The pharmaceutical composition of claim 65, wherein the delivery vehicle comprises PEG-DSPC.

67. 67. The pharmaceutical composition of any one of claims 13-66, wherein the delivery vehicle is a lipid nanoparticle.

68. 68. The pharmaceutical composition of any one of claims 13-67, wherein the delivery vehicle further comprises a targeting moiety.

69. 69. The pharmaceutical composition of claim 68, wherein the targeting moiety is a small molecule, scFv, nanobody, peptide, cyclic peptide, di- or tricyclic peptide, minibody, polynucleotide aptamer, engineered scaffold protein, heavy chain variable region, light chain variable region, or fragment thereof.

70. 70. The pharmaceutical composition of any one of claims 13-69, further comprising a pharmaceutical salt, a buffer, a diluent, or a combination thereof.

71. Circular RNA construct or pharmaceutical composition according to any one of the preceding claims, wherein said circular RNA further comprises a polyA region.

72. 10. The circular RNA construct or pharmaceutical composition of any one of the preceding claims, wherein the circular RNA further comprises at least one miRNA binding site.

73. 73. The circular RNA construct or pharmaceutical composition of claim 72, wherein the circular RNA comprises at least one miR-122 binding site.

74. 10. The circular RNA construct or pharmaceutical composition of any one of the preceding claims, wherein at least one expressed sequence encoding said CAR is codon-optimized.

75. 10. The circular RNA construct or pharmaceutical composition of any one of the preceding claims, wherein the RNA construct further comprises a 5'-enhanced intron element, a 5'-enhanced exon element, a 3'-enhanced exon element, and a 3'-enhanced intron fragment.

76. A method for preparing a circular RNA construct or a pharmaceutical composition according to any one of the preceding claims.

77. 76. A method of treating cancer or an autoimmune disorder in a subject by administering an effective amount of a composition comprising said circular RNA construct or the pharmaceutical composition of any one of claims 1-75, thereby treating said cancer or autoimmune disorder.

78. Use of a composition comprising said circular RNA construct or a pharmaceutical composition according to any one of claims 1-75 for the treatment of said cancer or autoimmune disorder.

79. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a binding molecule; A linear precursor RNA polynucleotide comprising:

80. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressible sequence encoding a CAR construct that targets a cancer antigen; A linear precursor RNA polynucleotide comprising:

81. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; A linear precursor RNA polynucleotide comprising:

82. 82. The linear precursor RNA polynucleotide of claim 81, wherein the expressed sequence comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

83. below: (A) an IRES comprising a sequence that is at least 80% identical to a sequence selected from any one of SEQ ID NOs: 1-18; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A linear precursor RNA polynucleotide comprising:

84. 84. The linear precursor RNA polynucleotide of claim 83, wherein the expressed sequence comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

85. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a binding molecule; A linear precursor RNA polynucleotide comprising:

86. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressible sequence encoding a CAR construct that targets a cancer antigen; A linear precursor RNA polynucleotide comprising:

87. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 binder; A linear precursor RNA polynucleotide comprising:

88. 88. The linear precursor RNA polynucleotide of claim 87, wherein the expressed sequence comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 19-34.

89. below: (A) an IRES selected from enterovirus, kobuvirus, parechovirus, hunnivirus, passerivirus, missivirus, and cardiovirus; and (B) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate; A linear precursor RNA polynucleotide comprising:

90. 90. The linear precursor RNA polynucleotide of claim 89, wherein the expressed sequence comprises a sequence at least 80% identical to a sequence selected from any one of SEQ ID NOs: 103-115.

91. 91. The linear precursor RNA polynucleotide of any one of claims 79-90, wherein the expression sequence is codon-optimized.

92. 92. The linear precursor RNA polynucleotide of any one of claims 79-91, further comprising a 5' enhanced intron element, a 5' enhanced exon element, a 3' enhanced exon element, and a 3' enhanced intron fragment.

93. In the following order: (A) the 5' enhanced intron element; (B) the 5'-enhanced exon element; (C) a core functional element comprising the IRES, at least one expression sequence encoding a CAR construct targeting a cancer antigen, and optionally a stop codon or stop cassette; (D) the 3'-enhanced exon element; and (E) the 3' enhanced intron element; 93. The linear precursor RNA polynucleotide of claim 92, comprising:

94. 94. The linear precursor RNA polynucleotide of any one of claims 79 to 93, further comprising at least one miRNA binding site.

95. 95. The linear precursor RNA polynucleotide of claim 94, wherein the precursor RNA comprises at least one miR-122 binding site.

96. A DNA vector encoding the RNA polynucleotide of any one of claims 79-95.

97. 96. A method for preparing a circular RNA construct, comprising incubating a linear RNA polynucleotide according to any one of claims 79-95 under conditions suitable for circularization.

98. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 1, 2, 4, and 8, and (ii) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a CD19 conjugate comprising a sequence selected from any one of SEQ ID NOs: 19 and 20, and the delivery vehicle is a lipid nanoparticle.

99. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 8, 16, 17, and 18, and (ii) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a BCMA conjugate comprising SEQ ID NO: 115, and the delivery vehicle is a lipid nanoparticle.

100. 1. A pharmaceutical composition comprising a circular RNA construct and a delivery vehicle, wherein the circular RNA construct comprises (i) an IRES comprising a sequence selected from any one of SEQ ID NOs: 8, 16, 17, and 18, and (ii) at least one expressed sequence encoding a CAR construct that targets a cancer antigen, wherein the CAR construct comprises a HER2 conjugate comprising a nucleotide sequence selected from any one of SEQ ID NOs: 132 or 133, and the delivery vehicle is a lipid nanoparticle.

101. The lipid nanoparticles comprise: (i) an ionizable lipid of formula (I): 【Chemistry 245】 wherein n is an integer between 1 and 4; R ａ is hydrogen or hydroxyl, R １ and R ２ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ６ -C ３０ Alkyl, C ６ -C ３０ Alkenyl, or C ６ -C ３０ is heteroalkyl, or (ii) an ionizable lipid of formula (II) 【Chemistry 246】 wherein each n is independently an integer from 2 to 15; L １ and L ３ are each independently -OC(O)-* or -C(O)O-*, where * represents R １ or R ３ indicates the attachment point to R １ and R ３ are each independently optionally selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl a straight-chain or branched C alkyl group substituted by one or more substituents selected from the group consisting of alkyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; ９ -C ２０ Alkyl or C ９ -C ２０ is alkenyl, R ２ is selected from the group consisting of: 【Chemistry 247】 The pharmaceutical composition according to any one of claims 98 to 100, comprising:

102. The lipid nanoparticle delivery vehicle comprises an ionizable lipid, the ionizable lipid comprising: 【Chemistry 248】 102. The pharmaceutical composition of claim 101, wherein

103. The pharmaceutical composition of any one of claims 98-102, wherein the lipid nanoparticle delivery vehicle further comprises at least one lipid selected from a helper lipid, a structural lipid, and a PEG-modified lipid.

104. 104. A method of treating cancer, comprising administering to a human subject in need thereof the pharmaceutical composition of any one of claims 98-103.

105. 104. A method for treating an autoimmune disease, comprising administering to a human subject in need thereof a pharmaceutical composition according to any one of claims 98-103.

106. 104. Use of a composition comprising said circular RNA construct for the treatment of said cancer, said use comprising administering to a human subject in need thereof the pharmaceutical composition of any one of claims 98-103.

107. 104. Use of a composition comprising said circular RNA construct for the treatment of said autoimmune disease, said use comprising administering to a human subject in need thereof the pharmaceutical composition of any one of claims 98-103.

108. 108. The method of claim 77, or claim 104 or 105, or the use of claim 106 or 107, wherein the administration is daily, every other day, twice a week, weekly, every 10 days, every 2 weeks, every 3 weeks, every 4 weeks, monthly, every 6 weeks, every 8 weeks, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or yearly.

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