Lipid nanoparticle compositions for delivery of circular polynucleotides

Abstract

Disclosed herein are novel lipids that can be used in combination with other lipid components, such as helper lipids, structural lipids, and cholesterol, to form lipid nanoparticles for delivery of therapeutic agents, such as nucleic acids (e.g., circular polynucleotides), both in vitro and in vivo.

Description

[Technical Field]

[0001] The present invention generally relates to novel lipids that can be used in combination with other lipid components, such as helper lipids, structural lipids, and cholesterol, to form lipid nanoparticles for the delivery of therapeutic agents, such as nucleic acids (e.g., circular polynucleotides), both in vitro and in vivo.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 277,055, filed November 8, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0003] Over the past few decades, nucleic acid therapeutics have rapidly expanded and become the basis for treating a wide variety of diseases. Available nucleic acid therapies include, but are not limited to, the use of DNA or viral vectors to insert desired genetic information into host cells and / or RNA engineered to encode therapeutic proteins. DNA and viral vector delivery have their own drawbacks and challenges that make them less desirable for RNA therapy. For example, introduced DNA can sometimes unintentionally insert into intact genes, resulting in mutations that disrupt or completely eliminate the function of endogenous genes, resulting in the elimination or harmful reduction of production of essential enzymes, or the disruption of genes important for regulating cell growth. Viral vector-based therapies can result in adverse immune responses. Compared to DNA or viral vectors, RNA is a substantially safer and more effective gene therapy agent due to its ability to encode proteins outside the nucleus and perform their functions. This eliminates the risk of RNA stably integrating into the genome of transfected cells.

[0004] RNA therapeutics have traditionally consisted of manipulating linear messenger RNA (mRNA). While linear mRNA is more effective than DNA or viral vectors, it presents its own set of challenges regarding stability, immunogenicity, translation efficiency, and delivery. Some of these challenges may result in size limitations and / or disruption of linear mRNA due to challenges present in the cap of linear mRNA. To overcome these limitations, circular polynucleotides, or circular RNA, may be used. Being a covalently closed continuous loop, circular RNA is useful for designing and producing stable forms of RNA. Circularization of RNA molecules offers advantages for studying RNA structure and function, especially for molecules that tend to fold into inactive conformations (Wang and Ruffner, 1998). Circular RNA may also be particularly interesting and useful for in vivo applications, particularly in the field of RNA-based control of gene expression and therapy, including protein replacement therapy and vaccination.

[0005] Additionally, nanoparticle delivery systems can be used to facilitate effective delivery of RNA polynucleotides. The inventions disclosed herein provide robust therapeutics using engineered polynucleotide and lipid nanoparticle compositions containing novel lipids. Summary of the Invention

[0006] The present application provides ionizable lipids and related transfer vehicles, compositions, and methods. The transfer vehicles can include ionizable lipids (e.g., ionizable lipids disclosed herein), PEG-modified lipids, and / or structured lipids, thereby forming lipid nanoparticles that encapsulate therapeutic agents (e.g., RNA polynucleotides, such as circular RNA). Pharmaceutical compositions containing such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. The present application also provides precursor RNAs and materials useful for producing precursor or circular RNAs with improved circularization efficiency and / or compatible with effective circular RNA purification methods.

[0007] In one embodiment, an ionizable lipid represented by formula (13*): [ka] or a pharmaceutically acceptable salt thereof, wherein: n * is an integer from 1 to 7; R a is hydrogen or hydroxyl; R b is hydrogen or C1-C6 alkyl; R1 and R2 are each independently 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, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl linear or branched C-C alkyl groups optionally substituted with one or more substituents selected from alkyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C2-C 30Alkenyl, or C1-C 30 is heteroalkyl; However, ionizable lipids are [ka] isn't it.

[0008] In some embodiments, R b is C1-C6 alkyl.

[0009] In some embodiments, R b is H, and the ionizable lipid is represented by formula (13): [ka] In the formula, n is an integer of 1 to 7.

[0010] In some embodiments, n is 1, 2, 3, or 4.

[0011] In some embodiments, R a is hydrogen. In some embodiments, the ionizable lipid is substituted by formula (13a-1), (13a-2), or (13a-3): [ka]

[0012] In some embodiments, R a is hydroxyl. In some embodiments, the ionizable lipid is substituted by formula (13b-1), formula (13b-2), or formula (13b-3): [ka]

[0013] In some embodiments, the ionizable lipid is substituted by the following formula (13b-4), (13b-5), (13b-6), (13b-7), (13b-8), or (13b-9): [ka]

[0014] In some embodiments, R and R are independently C-C 20 Alkoxy, C1-C 20 Alkyloxycarbonyl, C1-C 20 Alkylcarbonyloxy, C1-C 20 Alkyl carbonates, C2-C 20 Alkenyloxycarbonyl, C2-C 20 Alkenylcarbonyloxy, C2-C 20 Alkenyl carbonates, C2-C 20 Alkynyloxycarbonyl, C2-C 20 Alkynylcarbonyloxy and C2-C 20 linear or branched C-C alkyl groups optionally substituted with one or more substituents selected from alkynyl carbonates, 20 Alkyl, C2-C 20 Alkenyl, or C1-C 20 It is heteroalkyl.

[0015] In some embodiments, at least one of R and R is an unsubstituted straight or branched C-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 In some embodiments, at least one of R and R is -OC(O)R 6 , -C(O)OR 6 , or -OC(O)OR 6 Straight chain C1-C substituted with 12 alkyl, and each R 6 are independently linear or branched C1-C 20 Alkyl or C2-C 20In some embodiments, R and R are each independently -OC(O)R 6 , -C(O)OR 6 , or -OC(O)OR 6 Straight chain C1-C substituted with 12 alkyl, and each R 6 are independently linear or branched C1-C 20 Alkyl or C2-C 20 It is alkenyl.

[0016] In some embodiments, at least one of R1 and R2 is selected from the following: -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), in the formula, q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0017] In some embodiments, R1 and R2 are each independently selected from: -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9), and -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0018] In some embodiments, R is an unsubstituted straight or branched C-C 30 In some embodiments, R is -(CH) q C(O)O(CH2) r CH(R 8 )(R 9 In some embodiments, R1 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 In some embodiments, R1 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 In some embodiments, R2 is an unsubstituted straight or branched C6-C 30 In some embodiments, R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 In some embodiments, R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 In some embodiments, R2 is -(CH2) q OC(O)O(CH2) r CH(R8 )(R 9 )

[0019] In some embodiments, q is an integer from 1 to 6. In some embodiments, q is 3, 4, 5, or 6. In some embodiments, r is 0. In some embodiments, r is an integer from 1 to 6. In some embodiments, r is 1. In some embodiments, r is 2.

[0020] In some embodiments, R 8 is H. In some embodiments, R 8 is R 10 is.

[0021] In some embodiments, R 9 and R 10 are each independently an unsubstituted straight chain C1-C 12 In some embodiments, R 9 and R 10 are each independently an unsubstituted straight chain C-C alkyl. In some embodiments, R 9 and R 10 are each independently unsubstituted straight chain C6-C8 alkyl.

[0022] In some embodiments, R1 and R2 are each -(CH2) m -L-R', wherein m is an integer from 0 to 10; L is absent, -C(H)(R L )-*, -OC(O)-*, or -C(O)O-*, where "-*" indicates the point of attachment to R'; R' is C1-C 30 Alkyl, C2-C 30 Alkenyl, C1-C 30 alkoxy, 2- to 30-membered heteroalkylene, and 3- to 12-membered heterocyclyl, wherein the 2- to 30-membered heteroalkylene is optionally substituted with one or more R″, and the 3- to 12-membered heterocyclyl is selected from the group consisting of one or more C-C 30optionally substituted with alkyl; R L is C1-C 30 Alkyl, C2-C 30 Alkenyl, C1-C 30 alkoxy, 2- to 30-membered heteroalkylene, wherein the 3- to 12-membered heteroalkylene is optionally substituted with one or more R″; R" is independently oxo, C1-C 30 Alkoxy, -C(O)-C1-C 30 Alkyl, -C(O)-C1-C 30 Alkoxy, and -C(O)-C-C 30 Alkylene-C(O)-C1-C 30 alkoxy is selected from the group consisting of:

[0023] In some embodiments, R1 and R2 are each independently selected from the group consisting of: [ka] is selected from.

[0024] In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.

[0025] In some embodiments, the ionizable lipid is [ka] is selected from the group consisting of:

[0026] In some embodiments, the ionizable lipid is [ka] is selected from the group consisting of:

[0027] In some embodiments, the ionizable lipid is selected from Table 10e.

[0028] In another aspect, the present disclosure provides a pharmaceutical composition comprising a transfer vehicle, wherein the transfer vehicle comprises an ionizable lipid as described above.

[0029] In some embodiments, the pharmaceutical composition further comprises an RNA polynucleotide. In some embodiments, the RNA polynucleotide is a linear or circular RNA polynucleotide. In some embodiments, the RNA polynucleotide is a circular RNA polynucleotide.

[0030] In another aspect, the present disclosure provides a pharmaceutical composition comprising: a. an RNA polynucleotide that is a circular RNA polynucleotide, and b. A transfer vehicle comprising an ionizable lipid selected from the following: [ka] .

[0031] In some embodiments, the transfer vehicle comprises a nanoparticle, such as a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymeric nanoparticle, or a biodegradable polymeric nanoparticle.

[0032] In some embodiments, the RNA polynucleotide is encapsulated in a transfer vehicle. In some embodiments, the RNA polynucleotide is encapsulated in a transfer vehicle with an encapsulation efficiency of at least 80%.

[0033] In some embodiments, the circular RNA polynucleotide comprises a first expressible sequence. In some embodiments, the first expressible sequence encodes a therapeutic protein. In some embodiments, the first expressible sequence encodes a cytokine or a functional fragment thereof. In other embodiments, the first expressible sequence encodes a transcription factor. In other embodiments, the first expressible sequence encodes an immune checkpoint inhibitor. In other embodiments, the first expressible sequence encodes a chimeric antigen receptor (CAR).

[0034] In some embodiments, the circular RNA polynucleotide comprises, in the following order: (a) a 5'-enhancing exonic element, (b) a core functional element, and (c) a 3'-enhancing exonic element. In some embodiments, the core functional element comprises a translation initiation element (TIE). In some embodiments, the TIE comprises an untranslated region (UTR) or a fragment thereof. In some embodiments, the UTR or a fragment thereof comprises an IRES or a eukaryotic IRES. In some embodiments, the TIE comprises an aptamer complex, and optionally, the aptamer complex comprises at least two aptamers.

[0035] In some embodiments, the core functional elements comprise a coding region. In some embodiments, the coding region encodes a therapeutic protein. In some embodiments, the therapeutic protein is a chimeric antigen receptor (CAR).

[0036] In some embodiments, the core functional elements comprise non-coding regions.

[0037] In some embodiments, the RNA polynucleotide contained in the pharmaceutical compositions disclosed herein is from about 100 nt to about 10,000 nt in length, hi some embodiments, the RNA polynucleotide is from about 100 nt to about 15,000 nt in length.

[0038] In some embodiments, the transfer vehicle in the pharmaceutical compositions disclosed herein further comprises a structured lipid and a PEG-modified lipid.

[0039] In some embodiments, the structured lipid binds to C1q and / or enhances binding of the transfer vehicle comprising the lipid to C1q compared to a control transfer vehicle lacking the structured lipid, and / or increases uptake of the C1q-bound transfer vehicle into immune cells compared to a control transfer vehicle lacking the structured lipid. In some embodiments, the immune cell is a T cell, NK cell, NKT cell, macrophage, or neutrophil. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is beta-sitosterol. In some embodiments, the structured lipid is not beta-sitosterol.

[0040] In some embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. In some embodiments, the PEG-modified lipid is DSPE-PEG(2000).

[0041] In some embodiments, the transfer vehicle further comprises a helper lipid, hi some embodiments, the helper lipid is DSPC or DOPE.

[0042] In some embodiments, the transfer vehicle included in the pharmaceutical compositions disclosed herein comprises DSPC, cholesterol, and DMG-PEG(2000).

[0043] In some embodiments, the transfer vehicle comprises about 0.5% to about 4% PEG-modified lipid by molar ratio, hi some embodiments, the transfer vehicle comprises about 1% to about 2% PEG-modified lipid by molar ratio.

[0044] In some embodiments, the transfer vehicle comprises: a. An ionizable lipid selected from the following: [ka] or a mixture thereof, b. a helper lipid selected from DOPE or DSPC; Cholesterol, and d. A PEG-lipid selected from DSPE-PEG(2000) or DMG-PEG(2000).

[0045] In some embodiments, the transfer vehicle comprises an ionizable lipid, a helper lipid, cholesterol, and a PEG-lipid, wherein the molar ratio of ionizable lipid:helper lipid:cholesterol:PEG-lipid is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. In some embodiments, the molar ratios of each of the ionizable lipid, helper lipid, cholesterol, and PEG-lipid are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the recited values.

[0046] In some embodiments, the transfer vehicle comprises a helper lipid of DOPE and a PEG-lipid of DMG-PEG(2000), wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. In some embodiments, the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is about 62:4:33:1. In some embodiments, the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is about 53:5:41:1.

[0047] In some embodiments, the transfer vehicle comprises a helper lipid of DSPC and a PEG-lipid of DMG-PEG(2000), wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. In some embodiments, the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 50:10:38.5:1.5. In some embodiments, the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 41:12:45:2. In some embodiments, the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 45:9:44:2.

[0048] In some embodiments, the transfer vehicle comprises a helper lipid of DSPC and a PEG-lipid of DSPE-PEG(2000), and the molar ratio of ionizable lipid:DSPC:cholesterol:DSPE-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1.

[0049] In some embodiments, the transfer vehicle comprises a helper lipid of DOPE, the PEG-lipid is C14-PEG(2000), and the molar ratio of ionizable lipid:DOPE:cholesterol:C14-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1.

[0050] In some embodiments, the transfer vehicle comprises a helper lipid of DOPE and a PEG-lipid of DMG-PEG(2000), and the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1.

[0051] In some embodiments, the pharmaceutical compositions of the present disclosure have a lipid to phosphate (IL:P) molar ratio of about 3 to about 9, e.g., about 3, about 4, about 4.5, about 5, about 5.5, about 5.7, about 6, about 6.2, about 6.5, or about 7.

[0052] In some embodiments, the transfer vehicle is formulated for endosomal release of the RNA polynucleotide. In some embodiments, the transfer vehicle is capable of binding to apolipoprotein E (APOE) or is substantially free of APOE binding sites. In some embodiments, the transfer vehicle is capable of low-density lipoprotein receptor (LDLR)-dependent or LDLR-independent uptake into cells.

[0053] In some embodiments, the transfer vehicle has a diameter of less than about 120 nm and / or does not form aggregates having a diameter of more than 300 nm.

[0054] In some embodiments, the pharmaceutical compositions of the present disclosure are substantially free of linear RNA.

[0055] In some embodiments, the transfer vehicle further comprises a targeting moiety operably connected to the transfer vehicle. In some embodiments, the targeting moiety specifically or indirectly binds to an immune cell antigen, wherein the immune cell antigen is a T cell antigen selected from the group consisting of CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, and C1qR.

[0056] In some embodiments, the targeting moiety is a small molecule. In some embodiments, the small molecule is mannose, lectin, acivicin, biotin, or digoxigenin. In some embodiments, the small molecule binds to an exoenzyme on an immune cell, and the exoenzyme is selected from the group consisting of CD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. In some embodiments, the targeting moiety is a single-chain Fv (scFv) fragment, a nanobody, a peptide, a peptide-based macrocycle, a minibody, a small molecule ligand such as folate, arginylglycylaspartic acid (RGD), or phenol-soluble modulin alpha 1 peptide (PSMA1), a heavy chain variable region, a light chain variable region, or a fragment thereof.

[0057] In some embodiments, the pharmaceutical compositions of the present disclosure have less than 1% (by weight) of the polynucleotides in the composition that are double-stranded RNA, DNA splints, or triphosphorylated RNA. In some embodiments, the pharmaceutical composition has less than 1% (by weight) of the polynucleotides, and the protein in the pharmaceutical composition is double-stranded RNA, DNA splints, triphosphorylated RNA, a phosphatase protein, a protein ligase, or a capping enzyme.

[0058] In another aspect, provided herein is a method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition as described above and herein.

[0059] In another aspect, provided herein is a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition as described above and herein. [Brief explanation of the drawings]

[0060] [Figure 1] 1A-1E show luminescence in the supernatant of HEK293 cells, HepG2 cells (FIG. 1B), or 1C1C7 cells (FIG. 1C) 24 hours after transfection with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences.

[0061] [Figure 2] Figures 2A-2C show luminescence in the supernatants of HEK293 cells (Figure 2A), HepG2 cells (Figure 2B), or 1C1C7 cells (Figure 2C) 24 hours after transfection with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences of different lengths.

[0062] [Figure 3] Figures 3A and 3B show the stability of selected IRES constructs in HepG2 cells (Figure 3A) or 1C1C7 cells (Figure 3B) over a 3 day period as measured by luminescence.

[0063] [Figure 4] Figures 4A and 4B show protein expression from selected IRES constructs in Jurkat cells as measured by luminescence from secreted Gaussia luciferase in the cell supernatant.

[0064] [Figure 5] Figures 5A and 5B show the stability of selected IRES constructs in Jurkat cells over a 3 day period as measured by luminescence.

[0065] [Figure 6] Figures 6A and 6B show a comparison of the relative luminescence over 24 hours (Figure 6A) or over 3 days (Figure 6B) of modified linear RNA, unpurified circular RNA, or purified circular RNA encoding Gaussia luciferase.

[0066] [Figure 7] Figures 7A-7F show the transcriptional induction of IFNγ (Figure 7A), IL-6 (Figure 7B), IL-2 (Figure 7C), RIG-I (Figure 7D), IFN-β1 (Figure 7E), and TNFα (Figure 7F) after electroporation of Jurkat cells with modified linear RNA, unpurified circular RNA, or purified circular RNA.

[0067] [Figure 8] 8A-8C show a comparison of the luminescence of circular and modified linear RNAs encoding Gaussia luciferase in human primary monocytes (FIG. 8A) and macrophages (FIGS. 8B and 8C).

[0068] [Figure 9] Figures 9A and 9B show the relative luminescence over 3 days (Figure 9A) or 24 hours (Figure 9B) in the supernatants of primary T cells after transduction with circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences.

[0069] [Figure 10] Figures 10A-10C show the 24-hour luminescence in the supernatant of primary T cells (Figure 10A) after transduction with circular or modified linear RNA containing a Gaussia luciferase expression sequence, or the relative luminescence over 3 days (Figure 10B), and the 24-hour luminescence in PBMCs (Figure 10C).

[0070] [Figure 11] 11A and 11B show the HPLC chromatogram (FIG. 11A) and circularization efficiency (FIG. 11B) of RNA constructs with different permutation sites.

[0071] [Figure 12] 12A and 12B show the HPLC chromatograms (FIG. 12A) and circularization efficiencies (FIG. 12B) of RNA constructs with different introns and / or permutation sites.

[0072] [Figure 13] Figures 13A and 13B show the HPLC chromatograms (Figure 13A) and circularization efficiencies (Figure 13B) of three RNA constructs with and without homologous arms.

[0073] [Figure 14] FIG. 1 shows the circularization efficiency of three RNA constructs with no homologous arms or with homologous arms of various lengths and GC content.

[0074] [Figure 15] Figures 15A and 15B show HPLC chromatograms illustrating the contribution of strong homology arms to improved splicing efficiency, the relationship between circularization efficiency and nicking in selected constructs, and combinations of permutation sites and homology arms hypothesized to demonstrate improved circularization efficiency.

[0075] [Figure 16] Fluorescence images of T cells mock-electroporated (left) or electroporated (right) with circular RNA encoding a CAR and co-cultured with Raji cells expressing GFP and firefly luciferase are shown.

[0076] [Figure 17] Brightfield (left), fluorescent (center), and overlay (right) images of T cells mock-electroporated (top) or electroporated (bottom) with circular RNA encoding a CAR and co-cultured with Raji cells expressing GFP and firefly luciferase are shown.

[0077] [Figure 18] Specific lysis of Raji target cells by T cells mock-electroporated or electroporated with circular RNAs encoding different CAR sequences is shown.

[0078] [Figure 19] Figures 19A and 19B show the luminescence in the supernatants of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction with linear or circular RNAs containing a Gaussia luciferase expression sequence and various IRES sequences (Figure 19A), and the relative luminescence over a 3-day period (Figure 19B).

[0079] [Figure 20]Figures 20A-20F show the transcriptional induction of IFN-β1 (Figure 20A), RIG-I (Figure 20B), IL-2 (Figure 20C), IL-6 (Figure 20D), IFNγ (Figure 20E), and TNFα (Figure 20F) after electroporation of human CD3+ T cells with modified linear RNA, unpurified circular RNA, or purified circular RNA.

[0080] [Figure 21] Figures 21A and 21B show specific lysis of Raji target cells by human primary CD3+ T cells electroporated with CAR-encoding circRNA as determined by detection of firefly luminescence (Figure 21A), and induction of IFNγ transcription 24 hours after electroporation with different amounts of circular or linear RNA encoding the CAR sequence (Figure 21B).

[0081] [Figure 22] Figure 22A and Figure 22B show specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding a CAR at different E:T ratios as determined by detection of firefly luminescence.

[0082] [Figure 23] Specific lysis of target cells by human CD3+ T cells electroporated with RNA encoding a CAR at 1, 3, 5, and 7 days after electroporation.

[0083] [Figure 24] Specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding CD19- or BCMA-targeted CARs.

[0084] [Figure 25] Shown are the expression of GFP (Figure 25A) and CD19 CAR (Figure 25B) in human PBMCs after incubation with test lipid nanoparticles containing circular RNA encoding either GFP or CD19 CAR.

[0085] [Figure 26] 1 shows the expression of anti-mouse CD19 CAR in 1C1C7 cells lipotransfected with circular RNAs containing the anti-mouse CD19 CAR expression sequence and various IRES sequences.

[0086] [Figure 27]

[0033] Figure 1 shows the cytotoxicity of anti-mouse CD19 CAR against mouse T cells. The CD19 CAR is encoded by and expressed from circular RNA electroporated into mouse T cells.

[0087] [Figure 28] Figures 28A and 28B compare the expression levels of anti-human CD19 CAR expressed from circular RNA with that expressed from linear mRNA.

[0088] [Figure 29] Figures 29A and 29B compare the cytotoxic effect of anti-human CD19 CAR expressed from circular RNA to that expressed from linear mRNA.

[0089] [Figure 30] Figure 1 shows the cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMA CAR) expressed from a single circular RNA in T cells.

[0090] [Figure 31A] 1 shows an exemplary RNA construct design with a polyA sequence incorporated into an intron. [Figure 31B] Chromatography trace of crude circular RNA is shown. [Figure 31C] Chromatography trace of affinity purified circular RNA is shown. [Figure 46D]Immunogenicity of circular RNA prepared under various IVT conditions and purification methods is shown (Commercial = commercial IVT mix; Custom = customized IVT mix; Aff = affinity purified; Enz = enzyme purified; GMP:GTP ratio = 8, 12.5, or 13.75).

[0091] [Figure 32A] 1 shows an exemplary RNA construct design with a dedicated binding sequence as an alternative to polyA for hybridization purification. [Figure 32B] Chromatography trace of crude circular RNA is shown. [Figure 32C] Chromatography trace of affinity purified circular RNA is shown.

[0092] [Figure 33A] Chromatography trace of crude circular RNA encoding dystrophin is shown. [Figure 33B] Chromatography trace of enzyme-purified circular RNA encoding dystrophin is shown.

[0093] [Figure 34] Figure 34A and Figure 34B compare the expression (Figure 34A) and stability (Figure 34B) of purified circRNAs with different 5' spacers between the 3' intron fragment / 5' internal duplex region and the IRES in Jurkat cells. (AC = only A and C were used in the spacer sequence; UC = only U and C were used in the spacer sequence.)

[0094] [Figure 35] Figure 1 shows the luminescence expression levels and stability of expression in primary T cells from circular RNAs containing the original or modified IRES elements as indicated.

[0095] [Figure 36] 1 shows the luminescence expression levels and stability of expression in HepG2 cells from circular RNAs containing the original or modified IRES elements as indicated.

[0096] [Figure 37] 1 shows the luminescence expression levels and stability of expression in 1C1C7 cells from circular RNAs containing the original or modified IRES elements as indicated.

[0097] [Figure 38] Figure 1 shows the luminescence expression level and stability in HepG2 cells from circular RNAs containing an IRES element or a hybrid IRES element with an untranslated region (UTR) inserted. "Scr" stands for scrambled and was used as a control.

[0098] [Figure 39] 1 shows the luminescence expression level and stability of expression in 1C1C7 cells from a circular RNA containing an IRES and a variable stop codon cassette operably linked to a Gaussian luciferase coding sequence.

[0099] [Figure 40] 1 shows the luminescence expression level and stability of expression in 1C1C7 cells from a circular RNA containing an IRES and a variable untranslated region (UTR) inserted before the start codon of the Gaussian luciferase coding sequence.

[0100] [Figure 41] 1 shows the expression levels of human erythropoietin (hEPO) in Huh7 cells from circular RNA containing two miR-122 target sites downstream of the hEPO coding sequence.

[0101] [Figure 42] Figures 42A and 42B show CAR expression levels in peripheral blood (Figure 42A) and spleen (Figure 42B) upon treatment with LNPs encapsulating circular RNA expressing anti-CD19 CAR. Anti-CD20 (aCD20) and luciferase-encoding circular RNA (oLuc) were used for comparison.

[0102] [Figure 43] Figures 43A-43C show the overall frequency of anti-CD19 CAR expression, the frequency of anti-CD19 CAR expression on the cell surface, and the effect of IRES-specific circular RNA encoding anti-CD19 CAR on T cells on the anti-tumor response. Figure 43A shows the geometric mean fluorescence intensity of the anti-CD19 CAR, Figure 43B shows the percentage of anti-CD19 CAR expression, and Figure 43C shows the percentage of target cell lysis achieved by the anti-CD19 CAR. (CK = Caprine Kobuvirus; AP = Apodemus Picornavirus; CK* = codon-optimized Caprine Kobuvirus; PV = Parabovirus; SV = Salivirus.)

[0103] [Figure 44] CAR expression levels in A20 FLuc target cells upon treatment with IRES-specific circular RNA constructs are shown.

[0104] [Figure 45] Figures 45A and 45B show luminescence expression levels for cytosolic proteins (Figure 45A) and surface proteins (Figure 45B) from circular RNA in primary human T cells.

[0105] [Figure 46] Figures 46A-46F show luminescence expression in human T cells when treated with IRES-specific circular constructs. Expression in circular RNA constructs was compared to linear mRNA. Figures 46A, 46B, and 46G provide Gaussia luciferase expression in multiple donor cells. Figures 46C, 46D, 46E, and 46F provide Firefly luciferase expression in multiple donor cells.

[0106] [Figure 47]Figures 47A and 47B show the expression of anti-CD19 CAR and anti-BCMA CAR (Figure 47B) in human T cells after treatment of firefly luciferase-expressing K562 cells with lipid nanoparticles encapsulating circular RNA encoding either anti-CD19 CAR or anti-BCMA CAR.

[0107] [Figure 48] Figures 48A and 48B show anti-CD19 CAR expression levels resulting from in vitro electroporation delivery of circular RNA encoding the anti-CD19 CAR in a specific antigen-dependent manner. Figure 48A shows Nalm6 cell lysis by the anti-CD19 CAR. Figure 48B shows K562 cell lysis by the anti-CD19 CAR.

[0108] [Figure 49] Figures 49A-49E show transfection of LNP mediated by the use of ApoE3 in a solution containing LNP and circular RNA expressing green fluorescent protein (GFP). Figure 49A shows the survival and death results. Figures 49B, 49C, 49D, and 49E provide expression frequencies for multiple donors.

[0109] [Figure 50]Figures 50A-50C show the circularization efficiency of RNA molecules encoding the stabilized (double proline mutation) SARS-CoV2 spike protein. Figure 50A shows the in vitro transcription product of a circRNA encoding the approximately 4.5 kb SARS-CoV2 spike. Figure 50B shows a histogram of spike protein surface expression by flow cytometry after transfection of 293 cells with spike-encoding circRNA. Transfected 293 cells were stained with CR3022 primary antibody and APC-labeled secondary antibody 24 hours after transfection. Figure 50C shows a flow cytometry plot of spike protein surface expression in 293 cells after transfection with spike-encoding circRNA. Transfected 293 cells were stained with CR3022 primary antibody and APC-labeled secondary antibody 24 hours after transfection.

[0110] [Figure 51] Figures 51A and 51B provide multiple controlled adjuvant strategies. The circRNAs shown in the figures involve an in vitro unpurified sense circular RNA splicing reaction using GTP as an indicator molecule. 3p-circRNAs involve purified sense circular RNAs and mixed purified antisense circular RNAs containing triphosphorylated 5' ends. Figure 51A shows in vitro IFN-β induction in wild-type and MAVS knockout A549 cells, and Figure 51B shows the in vivo cytokine response to formulated circRNAs generated using the strategies shown.

[0111] [Figure 52] Figures 52A-52C illustrate intramuscular delivery of LNPs containing circular RNA constructs. Figure 52A provides the in vivo systemic flux after 6 hours, and Figure 52B provides the systemic IVIS after 6 hours of a 1 μg dose of LNP circular RNA construct. Figure 52C provides the ex vivo expression profile over 24 hours.

[0112] [Figure 53]Figures 53A and 53B show the expression of multiple circular RNAs from a single lipid formulation. Figure 53A provides the hEPO titers from single and mixed sets of LNPs containing circular RNA constructs, and Figure 53B provides the total flux of bioluminescence expression from single or mixed sets of LNPs containing circular RNA constructs.

[0113] [Figure 54] Figures 54A-54C show SARS-CoV2 spike protein expression of circular RNAs encoding spike SARS-CoV2 proteins. Figure 54A shows the frequency of spike CoV2 expression. Figure 54B shows the geometric mean fluorescence intensity (gMFI) of spike CoV2 expression. Figure 54C compares the gMFI expression of the constructs with the frequency of expression.

[0114] [Figure 55] The general sequence structure of a linear RNA polynucleotide precursor (10) is shown. The sequence provided is in 5' to 3' order: 5'-enhanced intronic element (20), 5'-enhanced exon (30), core functional element (40), 3'-enhanced exon element (50), and 3'-enhanced intron element (60).

[0115] [Figure 56] Various exemplary repeats of the 5'-enhancing exon element (20) are shown. As shown, one repeat of the 5'-enhancing exon element (20) includes, from 5' to 3', the following in this order: leading untranslated sequence (21), 5' affinity tag (22), 5' external duplex region (24), 5' external spacer (26), and 3' intron fragment (28).

[0116] [Figure 57] Various exemplary repeats of the 5'-enhancing exon element (30) are shown. As illustrated, one repeat of the 5'-enhancing exon element (30) includes, in 5' to 3' order: a 3' exon fragment (32), a 5' internal duplex region (34), and a 5' internal spacer (36).

[0117] [Figure 58] Various exemplary repeats of a core functional element (40) are shown. As shown, one repeat of the core functional element (40) includes a TIE (42), a coding region (46), and a termination region (e.g., a stop codon or stop cassette) (48). Another repeat is shown to illustrate a core functional element (47) that includes a non-coding region (47).

[0118] [Figure 59] Various exemplary repeats of the 3'-enhancing exon element (50) are shown. As shown, one repeat of the 3'-enhancing exon element (50) includes, in 5' to 3' order: a 3' internal spacer (52), a 3' internal duplex region (54), and a 5' exon fragment (56).

[0119] [Figure 60] Various exemplary repeats of a 3'-enhanced intron element (60) are shown. As shown, one repeat of a 3'-enhanced intron element (60) includes, in the following order: a 5' intron fragment (62), a 3' external spacer (64), a 3' external duplex region (66), a 3' affinity tag (68), and terminal untranslated sequence (69).

[0120] [Figure 61] Various exemplary repeats of the translation initiation element (TIE) (42) are shown. In one repeat, the exemplified TIE (42) sequence is an IRES (43) only. In another repeat, the TIE (42) is an aptamer (44). In two different repeats, the TIE (42) is a combination of an aptamer (44) and an IRES (43). In another repeat, the TIE (42) is an aptamer complex (45).

[0121] [Figure 62]An exemplary linear RNA polynucleotide precursor (10) is shown, including, in 5' to 3' order: leading untranslated sequence (21), 5' affinity tag (22), 5' external duplex region (24), 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' external duplex region (66), 3' affinity tag (68), and terminal untranslated sequence (69).

[0122] [Figure 63] An exemplary linear RNA polynucleotide precursor (10) is shown, including, in 5' to 3' order: leading untranslated sequence (21), 5' affinity tag (22), 5' external duplex region (24), 5' external spacer (26), 3' intron fragment (28), 3' exon fragment (32), 5' internal duplex region (34), 5' internal spacer (36), coding element (46), termination region (48), TIE (42), 3' internal spacer (52), 3' internal duplex region (54), 5' exon fragment (56), 5' intron fragment (62), 3' external spacer (64), 3' external duplex region (66), 3' affinity tag (68), and terminal untranslated sequence (69).

[0123] [Figure 64] An exemplary linear RNA polynucleotide precursor (10) is shown, including, in 5' to 3' order: leading untranslated sequence (21), 5' affinity tag (22), 5' external duplex region (24), 5' external spacer (26), 3' intron fragment (28), 3' exon fragment (32), 5' internal duplex region (34), 5' internal spacer (36), non-coding element (47), 3' internal spacer (52), 3' internal duplex region (54), 5' exon fragment (56), 5' intron fragment (62), 3' external spacer (64), 3' external duplex region (66), 3' affinity tag (68), and terminal untranslated sequence (69).

[0124] [Figure 65] The general structure of the circular RNA (8) formed after splicing is shown, which includes 5' exon elements (30), core functional elements (40), and 3' exon elements (50).

[0125] [Figure 66] Figures 66A-66E illustrate various ways in which accessory elements (70) (e.g., miRNA binding sites) can be included in linear RNA polynucleotides. Figure 66A illustrates a linear RNA polynucleotide including accessory elements (70) in the spacer region. Figure 66B illustrates a linear RNA polynucleotide including accessory elements (70) located between each external duplex region and an exon fragment. Figure 66C illustrates accessory elements (70) within the spacer. Figure 66D illustrates various repeats of accessory elements (70) located within a core functional element. Figure 66E illustrates accessory elements (70) located within an internal ribosome entry site (IRES).

[0126] [Figure 67] Figures 67A-67C show the screening of LNPs formulated with circular RNA encoding firefly luciferase and bearing a TIE in primary human (Figure 67A), mouse (Figure 67B), and cynomolgus monkey (Figure 67C) hepatocytes at various doses in vitro.

[0127] [Figure 68] Figures 68A-68C show the screening of LNPs formulated with circular RNA encoding firefly luciferase and bearing a TIE in primary human hepatocytes from three different donors at various doses in vitro.

[0128] [Figure 69] 1 shows the in vitro expression of LNPs formulated with circular RNA encoding GFP and bearing a TIE in HeLa, HEK293, and HUH7 human cell models.

[0129] [Figure 70] 1 shows the in vitro expression of LNPs formulated with circular RNA encoding the GFO protein and bearing a TIE in primary human hepatocytes.

[0130] [Figure 71] Figures 71A and 71B show the in vitro expression of a circular RNA encoding firefly luciferase and carrying a TIE in mouse myoblast (Figure 71A) and primary human myoblast (Figure 71B) cells.

[0131] [Figure 72] Figures 72A and 72B show in vitro expression of a circular RNA encoding firefly luciferase and carrying a TIE in myoblasts and differentiated primary human skeletal myotubes. Figure 72A provides data for cells received from human donor 1. Figure 72B provides data for cells received from human donor 2.

[0132] [Figure 73] Figures 73A and 73B show cell-free in vitro translation of circular RNAs of varying sizes. In Figure 73A, circular RNAs encoding firefly luciferase and linear mRNAs encoding firefly luciferase were tested for expression. In Figure 73B, human and mouse cells were fed circular RNAs encoding the ATP7B protein. Some of the circular RNAs tested were codon-optimized. Circular RNAs expressing firefly luciferase were used for comparison.

[0133] [Figure 74] Shows mOX40L expression in mouse spleens from LNPs containing circular RNA encoding mOX40L at a lipid to phosphate ratio (IL:P) of 5.7 (5.7A parameter formulation), either lipid 1 from Table 10e (10e-1) or lipid 15 from Table 10f (10f-15).

[0134] [Figure 75] Figure 1 shows circular RNA expression of firefly luciferase delivered using LNPs formulated with ionizable lipids containing various numbers of β-hydroxyl groups or a negative control (PBS). The ionizable lipids used, from left to right, include: Table 10e, lipid 85 (10e-85); Table 10e, lipid 89 (10e-89); Table 10f, lipid 22 (10f-22); Table 10e, lipid 86 (10e-86); and Table 10e, lipid 90 (10e-90).

[0135] [Figure 76] Figure 1 shows firefly luciferase oRNA expression in the spleen delivered using LNPs formulated with ionizable lipids from Table 10e (from left to right: lipids 1, 85, 38, 34, 45, 86, 88, 89, 90) after intravenous administration. Total luciferase flux was measured in the spleen.

[0136] [Figure 77] Shown is splenic T cell expression after intravenous administration of circular RNA encoding mOX40L delivered using LNPs containing ionizable lipids from Table 10e (from left to right: lipid 1, lipid 85, lipid 38, lipid 34, lipid 45, lipid 86, lipid 88, lipid 89, lipid 90).

[0137] [Figure 78] Figure 78 shows B cell depletion in mice treated with circular RNA encoding encapsulated CD-19 chimeric antigen receptor (CAR) protein. Circular RNA was delivered via LNPs containing ionizable lipids (1, 16, 85, 45, 86, or 90) from Table 10e. In Figure 78, B cell hypoplasia was observed in blood cells. The dotted line in the figure indicates Wasabi control B cell hypoplasia. % B cells were normalized to the Wasabi control.

[0138] [Figure 79] Table 10e shows tumor growth kinetics in the Nalm6 model following administration of LNP-oRNA constructs (lipid 16, 45, or 86). Total flux was measured in the tested mice. DETAILED DESCRIPTION OF THE INVENTION

[0139] The present invention provides, inter alia, ionizable lipids and related transfer vehicles, compositions, and methods. In some embodiments, the transfer vehicle comprises an ionizable lipid (e.g., an ionizable lipid disclosed herein), a PEG-modified lipid, and / or a structured lipid, thereby forming a lipid nanoparticle suitable for delivering a nucleic acid. In certain embodiments, the nucleic acid can be RNA, such as siRNA, mRNA, or circular RNA. The nucleic acid can encode a therapeutic agent. In some embodiments, the nucleic acid is encapsulated in the transfer vehicle.

[0140] Also disclosed herein are RNA therapeutics, along with related compositions and methods. In some embodiments, RNA therapeutics allow for, among other things, RNA stability, increased expression, and extended half-life.

[0141] Also disclosed herein are DNA templates (e.g., vectors) for generating circular RNA. In some embodiments, the DNA template comprises a 3'-enhanced intron fragment, a 3'-enhanced exon fragment, a core functional element, a 5'-enhanced exon fragment, and a 5'-enhanced intron fragment. In some embodiments, these elements are arranged in the DNA template in the above order.

[0142] Further embodiments include circular RNA polynucleotides comprising circular RNA polynucleotides generated using the DNA templates provided herein (e.g., circular RNAs comprising 3'-enhanced exonic elements, core functional elements, and 5'-enhanced exonic elements), compositions comprising such circular RNAs, cells comprising such circular RNAs, and methods of using and generating such DNA templates, circular RNAs, compositions, and cells.

[0143] In some embodiments, provided herein are methods comprising administering a circular RNA polynucleotide provided herein to a cell for therapeutic or useful protein production. In some embodiments, the methods are advantageous in providing for the production of a desired polypeptide in a eukaryotic cell that has a longer half-life than linear RNA due to the resistance of circular RNA to ribonucleases.

[0144] Circular RNA polynucleotides lack the free ends required for exonuclease-mediated degradation, making them resistant to some mechanisms of RNA degradation and extending their half-life compared to comparable linear RNA. Circularization can stabilize RNA polynucleotides, which generally have a short half-life, and can improve the overall effectiveness of exogenous mRNA in various applications. In one embodiment, the functional half-life of the circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells such as human cells), as assessed by protein synthesis, is at least 20 hours (e.g., at least 80 hours).

[0145] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0146] 1.Definition As used herein, the terms "circRNA" or "circular polyribonucleotide" or "circular RNA" or "oRNA" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.

[0147] As used herein, the term "DNA template" refers to a DNA sequence from which a linear RNA polynucleotide can be transcribed. For example, but not by way of limitation, a DNA template can include a DNA vector, a PCR product, or a plasmid.

[0148] 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.

[0149] 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.

[0150] As used herein, the term "permutation site" refers to the site in a Group I intron where cleavage occurs prior to intron permutation, generating permuted 3' and 5' Group I intron fragments that flank the stretch of precursor RNA to be circularized.

[0151] As used herein, the term "splice site" refers to a dinucleotide that is partially or completely contained in a Group I intron and between which the phosphodiester bond is cleaved during RNA circularization. (As used herein, "splice site" refers to one or more dinucleotides between which phosphodiester bond cleavage occurs during the splicing reaction. A "5' splice site" refers to the naturally occurring 5' dinucleotide of an intron, e.g., a Group I intron, and a "3' splice site" refers to the naturally occurring 3' dinucleotide of an intron.)

[0152] 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 can include multiple nucleotide 3 structures, each of which can encode an amino acid, referred to as a "codon."

[0153] 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).

[0154] As used herein, a "non-coding element" 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 overall polynucleotide to act as a biomarker or adjuvant for a particular cell.

[0155] 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.

[0156] As used herein, the term "immunogenic" refers to the potential to induce an immune response against a substance. An immune response can be induced when the immune system of an organism or a type of immune cell is exposed to an immunogenic substance. The term "non-immunogenic" refers to the lack or absence of an immune response above a detectable threshold against a substance. When the immune system of an organism or a type of immune cell is exposed to a non-immunogenic substance, 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 the immune system of an organism or a type of immune cell is exposed to the non-immunogenic cyclic polyribonucleotides provided herein, no innate immune response is detected. In some embodiments, when the immune system of an organism or a type of immune cell is exposed to the non-immunogenic cyclic polyribonucleotides provided herein, no adaptive immune response is detected.

[0157] As used herein, the term "circularization efficiency" refers to the measurement of the resulting circular polyribonucleotide compared to its linear starting material.

[0158] 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.

[0159] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include species containing purines, such as adenine, hypoxanthine, and guanine, and derivatives and analogs thereof, and pyrimidines, such as cytosine, uracil, and thymine, and derivatives and analogs thereof. Nucleotide analogs include nucleotides with modifications in the chemical structure of the base, sugar, and / or phosphate, including, but not limited to, 5'-position pyrimidine modifications, 8'-position purine modifications, modifications with the exocyclic amine of cytosine, and 5-bromo-uracil substitution; and 2'-position sugar modifications (including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced with a group such as H, OR, R, halo, SH, SR, NH, NHR, NR, or CN (where R is an alkyl moiety as defined herein)). Nucleotide analogs are also intended to include nucleotides having bases such as inosine, cucumber, and xanthine; sugars such as 2'-methylribose; and non-natural phosphodiester linkages, such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0160] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer 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 about 1000 bases, or up to about 10,000 bases or more, composed of nucleotides such as 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, e.g., hybridize with a naturally occurring nucleic acid in a sequence-specific manner similar to two naturally occurring nucleic acids, e.g., participate in Watson-Crick base pairing interactions. Naturally occurring nucleic acids are composed of nucleotides including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0161] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0162] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0163] "Isolated" or "purified" generally refers to the isolation of a substance (e.g., in some embodiments, a compound, polynucleotide, protein, polypeptide, polynucleotide composition, or polypeptide composition) such that the substance comprises a significant percentage (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, typically up to about 90%-100%) of the sample in which it is present. In particular embodiments, a substantially purified component comprises at least 50%, 80%-85%, or 90%-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation by density. Generally, a substance is purified when it is present in a sample in an amount greater than that found in nature relative to other components of the sample.

[0164] As used herein, the terms "duplex," "double stranded," or "hybridized" refer to a nucleic acid formed by the hybridization of two single strands of nucleic acid containing complementary sequences. In most cases, genomic DNA is double stranded. The sequences can be fully complementary or partially complementary.

[0165] As used herein, "unstructured" with respect to RNA refers to an RNA sequence that is not predicted by RNAFold software or similar prediction tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule. In some embodiments, nuclease protection assays can be used to functionally characterize unstructured RNA.

[0166] As used herein, "structured" with respect to RNA refers to an RNA sequence that is predicted by RNAFold software or similar prediction tools to form structures (e.g., hairpin loops) with itself or with other sequences within the same RNA molecule.

[0167] As used herein, two "duplex sequences," "duplex regions," "duplex regions," "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 with each other's reverse complements to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences have "homology" if they are identical or share sequence identity with their reverse complements or "complementary" sequences. The percent sequence identity between a homologous region and the reverse complement of the corresponding homologous region can be any percentage of 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.

[0168] 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 for the purpose of aiding in the purification of the polynucleotide sequence. For example, an affinity sequence can include, but is not limited to, a polyA or polyAC sequence.

[0169] 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.

[0170] Linear nucleic acid molecules are said to have a "5' end" (5' terminus) and a "3' end" (3' terminus) 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 where the new bond is to the 5' carbon is its 5' terminal nucleotide. The terminal nucleotide of a polynucleotide where the new bond is to the 3' carbon is its 3' terminal nucleotide. As used herein, a terminal nucleotide is the nucleotide at the end position of either the 3' or 5' terminus.

[0171] As used herein, a "first untranslated sequence" is a region of a polynucleotide sequence located at the top 5' end of the polynucleotide sequence, ranging from one nucleotide to several hundred nucleotides. The sequence can be defined or random. First untranslated sequences are non-coding.

[0172] As used herein, a "first untranslated sequence" is a region of a polynucleotide sequence located at the lowest 3' end of the polynucleotide sequence, ranging from one nucleotide to several hundred nucleotides. The sequence can be defined or random. First untranslated sequences are non-coding.

[0173] "Transcription" refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The present invention is not limited with respect to the RNA polymerase used for transcription. For example, in some embodiments, a T7-type RNA polymerase can be used.

[0174] "Translation" refers to the formation of a polypeptide molecule by ribosomes from an RNA template.

[0175] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a combination of two or more cells or a whole culture of cells; a reference to a "polynucleotide" includes, as a practical matter, many copies of that polynucleotide. As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive. Unless defined otherwise herein and in the remainder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0176] As used herein, unless otherwise specified or clear from the context, the term "about" is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0177] As used herein, the term "encoding" broadly refers to any process in which information in a polymer macromolecule is used to direct the production of a second molecule that is different from the first molecule. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.

[0178] By "co-administered" is meant that a therapeutic agent provided herein is administered in conjunction with one or more additional therapeutic agents sufficiently close in time that the therapeutic agent provided herein can potentiate the effect of the one or more additional therapeutic agents, or vice versa.

[0179] As used herein, the terms "treat" and "prevent," and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those of skill in the art recognize as having potential benefit or therapeutic effect. The treatment or prevention provided by the methods disclosed herein can include treatment or prevention of one or more symptoms or symptoms of a disease. For purposes of this specification, "prevention" can also encompass delaying the onset of a disease, or a symptom or symptom thereof.

[0180] 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. IRESs are typically about 500 nt to about 700 nt in length.

[0181] As used herein, "aptamer" generally refers to either a single oligonucleotide of a defined sequence or a mixture of such nucleotides, which mixture retains the property of specifically binding to a target molecule (e.g., eukaryotic initiation factor, 40S ribosome, 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). Therefore, as used herein, "aptamer" refers to both a single or multiple sequences of nucleotides as defined herein 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, and oligonucleotides within these lengths are readily prepared by conventional techniques. Optionally, aptamers can further contain a minimum of approximately 6 nucleotides, preferably 10 nucleotides, and more preferably 14 or 15 nucleotides, necessary to achieve specific binding.

[0182] "Eukaryotic initiation factor" or "eIF" refers to a protein or protein complex used to assemble the initiator tRNA, 40S and 60S ribosomal subunits required to initiate eukaryotic translation.

[0183] 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. IRESs are typically about 500 nt to about 700 nt in length.

[0184] As used herein, an "miRNA 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.

[0185] As used herein, an "endonuclease site" refers to a stretch of nucleotides within a polynucleotide that can be recognized and cleaved by an endonuclease protein.

[0186] 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 skip element.

[0187] As used herein, the term "ribosomal skipping element" refers to a nucleotide sequence encoding a short peptide sequence that can trigger the production of two peptide chains from the translation of one RNA molecule. Without wishing to be bound by theory, it is hypothesized that ribosomal skipping elements function by (1) terminating the translation of the first peptide chain and restarting the translation of the second peptide chain; or (2) cleaving the peptide bond in the peptide sequence encoded by the ribosomal skipping element by the inherent protease activity of the encoded peptide or by another protease in the environment (e.g., cytosol).

[0188] As used herein, the term "co-formulated" refers to a nanoparticle formulation comprising two or more nucleic acids or a nucleic acid and another active drug substance. Typically, the ratio is defined as an equimolar or ratiometric amount of the two or more nucleic acids or a nucleic acid and another active drug substance.

[0189] As used herein, "transfer vehicle" includes any of the standard pharmaceutical carriers, diluents, excipients, etc. generally intended for use in connection with the administration of biologically active agents, including nucleic acids.

[0190] As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle comprising one or more lipids (e.g., in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0191] As used herein, the phrase "ionizable lipid" refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH 4, and a neutral charge at other pHs, such as physiological pH 7.

[0192] In some embodiments, the lipids disclosed herein, e.g., ionizable lipids, comprise one or more cleavable groups. The terms "cleavage" 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 subject functional group are broken (e.g., hydrolyzed) or can be cleaved upon exposure to selected conditions (e.g., enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in specific embodiments, a disulfide group that can be cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional group that can be cleaved upon exposure to selected biological conditions. For example, the disulfide group can be cleaved enzymatically or by hydrolysis, oxidation, or reduction. When such a disulfide functional group is cleaved, one or more functional moieties or groups (e.g., one or more head groups and / or tail groups) attached thereto can be liberated. Exemplary cleavable groups can 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., attached 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).

[0193] As used herein, the term "hydrophilic" is used qualitatively to indicate that a functional group is water-loving, and typically, such groups are water-soluble. For example, disclosed herein are compounds that include a cleavable disulfide (SS) functional group attached to one or more hydrophilic groups (e.g., hydrophilic head groups), where such hydrophilic groups include or are selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino, e.g., dimethylamino), and pyridyl.

[0194] In certain embodiments, at least one of the functional groups of the moiety comprising the compounds disclosed herein is hydrophobic in nature (e.g., a hydrophobic tail group comprising a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used qualitatively to indicate that the functional group avoids water; typically, such groups are not water-soluble. For example, compounds are disclosed herein that include a cleavable functional group (e.g., a disulfide (SS) group) attached to one or more hydrophobic groups, where such hydrophobic groups include one or more naturally occurring lipids, such as cholesterol, and / or optionally substituted, variably saturated or unsaturated C6-C20 alkyl and / or optionally substituted, variably saturated or unsaturated C6-C20 acyl.

[0195] The compounds described herein may also contain one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; F may be in any isotopic form, including 18F and 19F, etc.

[0196] When describing the present invention, which may include compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms, when present, have the following meanings unless otherwise indicated. It should also 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 their scope as described below. Unless otherwise indicated, the term "substituted" should be defined as described below. It should further be understood that the terms "group" and "radical" can be considered interchangeable as used herein.

[0197] When a range of values ​​is listed, it is intended to encompass each value and subrange 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.

[0198] In certain embodiments, compounds disclosed herein include, for example, at least one hydrophilic head group and at least one hydrophobic tail group, each attached to at least one cleavable group, thereby rendering such compounds amphiphilic. As used herein to describe a compound or composition, the term "amphiphilic" refers to the ability to dissolve in both polar (e.g., aqueous) and non-polar (e.g., lipid) environments. For example, in certain embodiments, compounds disclosed herein include at least one lipophilic tail group (e.g., cholesterol or C6-C20 alkyl) and at least one hydrophilic head group (e.g., imidazole), each attached to a cleavable group (e.g., disulfide).

[0199] It should be noted that the terms "head group" and "tail group" as used are used to describe the compounds of the invention, particularly the functional groups that comprise such compounds, and are used for ease of reference to describe the orientation of one or more functional groups relative to other functional groups. For example, in certain embodiments, a hydrophilic head group (e.g., guanidinium) is attached (e.g., by one or more of hydrogen bonding, van der Waals forces, ionic interactions, and covalent bonding) to a cleavable functional group (e.g., a disulfide group), which is then attached to a hydrophobic tail group (e.g., cholesterol).

[0200] 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, alkyls can contain one or more cyclic alkyls and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can optionally be substituted with substituents (e.g., one or more of alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide). In certain embodiments, contemplated alkyls include (9Z,12Z)-octadeca-9,12-diene. For example, 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 recited 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 ("C1-8 alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-7 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-6 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1-5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). Examples of C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[0201] As used herein, "alkenyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) ("C2-20 alkenyl"). In certain embodiments, an 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 (e.g., 2-butenyl) or terminal (e.g., 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 the C2-4 alkenyl groups mentioned above as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), etc.

[0202] As used herein, "alkynyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) ("C2-20 alkynyl"). In certain embodiments, an alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-10 alkynyl"). In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C2-8 alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C2-7 alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2-6 alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4 alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C2-3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (e.g., 2-butynyl) or terminal (e.g., 1-butynyl). Examples of C2-4 alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the C2-4 alkynyl groups mentioned above as well as pentynyl (C5), hexynyl (C6), etc. Further examples of alkynyl include heptynyl (C7), octynyl (C8), etc.

[0203] As used herein, "alkylene," "alkenylene," and "alkynylene" refer to divalent radicals of alkyl, alkenyl, and alkynyl groups, respectively. When a range or number of carbons is provided for a particular "alkylene," "alkenylene," or "alkynylene" group, it is understood that the range or number refers to the range or number of carbons in a linear, divalent carbon chain. "Alkylene," "alkenylene," and "alkynylene" groups can be substituted or unsubstituted with one or more substituents described herein.

[0204] As used herein, the term "aryl" refers to aromatic groups containing 6 to 10 carbons in the ring portion (e.g., monocyclic, bicyclic, and tricyclic structures). Aryl groups can be optionally substituted through available carbon atoms and, in certain embodiments, can contain one or more heteroatoms such as oxygen, nitrogen, or sulfur. 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).

[0205] The term "heteroalkyl" refers to acyclic, stable, straight or branched chains, or combinations thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Exemplary heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)2, -S(O)-CH3, -S(O)2-CH2, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. A "heteroalkyl" may be listed, followed by a specific heteroalkyl group, e.g., -CHO, -NR B R C When the heteroalkyl and -CH2O or -NR2 are listed, B R C It will be understood that the terms "heteroalkyl" and "heteroalkyl-" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" includes -CH2O, -NR2O, -NR3O, -NR4O, -NR5O, -NR6O, -NR7O, -NR8O, -NR9O, -NR10O, -NR11O, -NR12O, -NR13O, -NR14O, -NR15O, -NR16O, -NR17O, -NR18O, -NR19O, -NR20O, -NR21O, -NR22O, -NR23O, -NR24O, -NR25O, -NR26O, -NR27O, -NR28O, -NR29O, -NR30O, -NR31O, -NR32O, -NR33O, -NR40O, -NR41O B R C Nothing herein should be construed as excluding specific heteroalkyl groups such as:

[0206] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, exemplified but not limited to, -CHO- and -CHCHO-. A heteroalkylene group may be described, for example, as a 2- to 7-membered heteroalkylene, where the term "member" refers to a non-hydrogen atom within the moiety. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- can represent both -C(O)R'- and -R'C(O)-.

[0207] As used herein, "heteroaryl" refers to a radical of a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in the cyclic array) having ring carbon atoms and 1 to 4 ring heteroatoms provided to the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 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, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring; in such cases, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, and the point of attachment is on either the aryl ring or the heteroaryl ring; in such cases, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be in either ring, i.e., the ring containing a heteroatom (e.g., 2-indolyl) or the ring without a heteroatom (e.g., 5-indolyl).

[0208] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon radical of 3 to 12, 3 to 8, 4 to 8, or 4 to 6 carbons, referred to herein as "C cycloalkyl," e.g., derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane.

[0209] As used herein, "heterocyclyl" or "heterocyclic" 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 (a "3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, valence permitting. Heterocyclyl groups may be either monocyclic (a "monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, e.g., bicyclic systems (a "bicyclic heterocyclyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more carbocyclyl groups, and the point of attachment is on either the carbocyclyl ring or the heterocyclyl ring, or in which a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, and the point of attachment is on the heterocyclyl ring, in which case the number of ring members continues to designate the number of ring members of the heterocyclyl ring system. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" may be used interchangeably.

[0210] As used herein, "cyano" refers to --CN.

[0211] The terms "halo" and "halogen," as used herein, 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.

[0212] The term "alkoxy," as used herein, 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.

[0213] As used herein, "oxo" refers to -C=O.

[0214] In general, the term "substituted," whether preceded by the term "optionally" or not, means that at least one hydrogen present on the group (e.g., a hydrogen bonded to a carbon or nitrogen atom of the group) is replaced with an acceptable substituent, e.g., a substituent that, upon substitution, results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position.

[0215] 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 reaction, etc., and that is 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 of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed 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 by using 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, and 2-hydroxy-ethanesulfonate. , lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, picrate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0216] In typical embodiments, the invention is intended to encompass the compounds disclosed herein, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomers, polymorphs, and prodrugs of such compounds. In some embodiments, the invention includes pharmaceutically acceptable addition salts, pharmaceutically acceptable esters, solvates (e.g., hydrates) of addition salts, tautomers, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (pure or as racemic or non-racemic mixtures) of the compounds described herein.

[0217] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., University of Notre Dame Press, Notre Dame, IN 1972). The present invention further encompasses the compounds described herein as individual isomers substantially free of other isomers or as mixtures of various isomers.

[0218] In certain embodiments, compounds and transfer vehicles (e.g., lipid nanoparticles) of which such compounds are components exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, methods of transfecting one or more target cells are also provided herein. Such methods generally involve contacting one or more target cells with the compounds and / or pharmaceutical compositions disclosed herein, such that the one or more target cells are transfected with the encapsulated circular RNA. As used herein, the term "transfect" or "transfection" refers to the intracellular introduction of one or more encapsulated materials (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 materials (e.g., polynucleotides) taken up, introduced, and / or expressed by a target cell undergoing transfection. In some embodiments, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cell after transfection. In some embodiments, the transfer vehicle has high transfection efficiency. In some embodiments, the transfer vehicle has a transfection efficiency of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0219] As used herein, the term "liposome" generally refers to a vesicle composed of lipids (e.g., amphipathic lipids) arranged in one or more spherical bilayers. In certain embodiments, the liposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprising one or more of the ionizable lipid compounds disclosed herein). Such liposomes may be unilamellar or multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior containing encapsulated circRNAs to be delivered to one or more target cells, tissues, and organs. In certain embodiments, the compositions described herein comprise one or more lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that can be used to form contemplated liposomes and lipid nanoparticles include one or more of the compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and / or HGT4005). Such liposomes and lipid nanoparticles may also include additional ionizable lipids such as C12-200, DLin-KC2-DMA, and / or HGT5001, helper lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0220] As used herein, the terms "non-cationic lipid," "non-cationic helper lipid," and "helper lipid" are used interchangeably and refer to any neutral, zwitterionic, or anionic lipid.

[0221] As used herein, the phrase "anionic lipid" refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH.

[0222] As used herein, the phrase "biodegradable lipid" or "degradable lipid" refers to any of several lipid species that are degraded in the host environment within minutes, hours, or days, ideally making them less toxic and less likely to accumulate in the host over time. Common modifications to lipids include ester and disulfide bonds, among others, to enhance the biodegradability of lipids.

[0223] As used herein, the phrase "biodegradable PEG lipid" or "degradable PEG lipid" refers to any of several lipid species in which the PEG molecule is cleaved from the lipid in a host environment within minutes, hours, or days, ideally reducing their immunogenicity. Common modifications to PEG lipids include, among others, ester and disulfide bonds to enhance the biodegradability of the lipid.

[0224] In certain embodiments of the present invention, transfer vehicles (e.g., lipid nanoparticles) are prepared to encapsulate one or more materials or therapeutic agents (e.g., circRNA). The process of incorporating a desired therapeutic agent (e.g., circRNA) into a transfer vehicle is referred to herein as "loading" or "encapsulation" (Lasic, et al., FEBS Lett., 312:255-258, 1992). The material (e.g., circRNA) loaded or encapsulated in the transfer vehicle may be located completely or partially within the interior space of the transfer vehicle, within the bilayer membrane of the transfer vehicle, or associated with the outer surface of the transfer vehicle.

[0225] As used herein, the term "structured lipids" also refers to sterols and lipids that contain sterol moieties.

[0226] "Sterols," as defined herein, are a subgroup of steroids consisting of steroid alcohols.

[0227] As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer.

[0228] 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.

[0229] As used herein, a "phospholipid" is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.

[0230] All nucleotide sequences disclosed herein can 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, "T" and "U" are used interchangeably in nucleotide sequences herein.

[0231] As used herein, "sequence identity" or a list including, for example, "a sequence 50% identical to" refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid over a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, where typically the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0232] Expression sequences in a polynucleotide construct may be separated by a "cleavage site" sequence that allows the polypeptides encoded by the expression sequences to be separately expressed by the cell upon translation.

[0233] A "self-cleaving peptide" refers to a peptide that functions such that when translated without a peptide bond between two adjacent amino acids or when a protein and a polypeptide comprising the self-cleaving peptide are produced, they are immediately cleaved or separated into different and distinct first and second polypeptides without the need for any external cleavage activity.

[0234] The α and β chains of the αβ TCR are generally considered to have two domains or regions each: a variable domain / region and a constant domain / region. The variable domain consists of the connection of the variable region and the joining region. Thus, in this specification and claims, the term "TCR alpha variable domain" refers to the connection of the TRAV region and the TRAJ region, and the term TCR alpha constant domain refers to the extracellular TRAC region or a C-terminal truncated TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the connection of the TRBV region and the TRBD / TRBJ region, and the term TCR beta constant domain refers to the extracellular TRBC region or a C-terminal truncated TRBC sequence.

[0235] As used herein, the terms "duplex," "double stranded," or "hybridized" refer to a nucleic acid formed by the hybridization of two single strands of nucleic acid containing complementary sequences. In most cases, genomic DNA is double stranded. The sequences can be fully complementary or partially complementary.

[0236] As used herein, "autoimmunity" is defined as a persistent and progressive immune response to non-infectious self-antigens, distinct from infectious non-self-antigens derived from bacteria, viruses, fungi, or parasites that invade and persist in mammals and humans. Autoimmune conditions include systemic autoimmune diseases such as scleroderma, Graves' disease, Crohn's disease, Sjogren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrine deficiency syndrome, type 1 diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveitis, polymyositis, colitis, and thyroiditis, as well as human lupus. As used herein, "autoantigen" or "self-antigen" refers to an antigen or epitope that is native to a mammal and immunogenic in that mammal.

[0237] As used herein, the phrase "cationic lipid" refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH.

[0238] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain may comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three constant domains, CH1, CH2, and CH3. Each light chain may comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. The VH and VL regions may be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL may contain three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain variable fragments (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked variable fragments (sdFv), anti-idiotypic (anti-id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0239] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain Abs.

[0240] An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that comprises an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may comprise an antigen-complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to BCMA. In further embodiments, the antigen-binding molecule is an antibody fragment comprising one or more of its complementarity-determining regions (CDRs) that specifically bind to an antigen. In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule comprises or consists of an avimer.

[0241] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-120 amino acids of the mature heavy chain and approximately 90-115 amino acids of the mature light chain, which vary significantly in sequence among antibodies and are responsible for the binding and specificity of a particular antibody to its particular antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with its antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

[0242] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding molecule thereof.

[0243] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding molecule thereof.

[0244] Several definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures. The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding molecule. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35, which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.In certain embodiments, the CDRs of an antibody may be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al, (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al, (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, using Kabat numbering, the Chothia CDR-H1 loop is located at amino acids 26-32, 33, or 34 in the heavy chain, the Chothia CDR-H2 loop is located at amino acids 52-56 in the heavy chain, and the Chothia CDR-H3 loop is located at amino acids 95-102 in the heavy chain, while the Chothia CDR-L1 loop is located at amino acids 24-34 in the light chain, the Chothia CDR-L2 loop is located at amino acids 50-56 in the light chain, and the Chothia CDR-L3 loop is located at amino acids 89-97 in the light chain. The end of the Chothia CDR-HI loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B. If neither 35A nor 35B are present, the loop ends at 32. If only 35A is present, the loop ends at 33. If both 35A and 35B are present, the loop ends at 34). In certain embodiments, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

[0245] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0246] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD or Kd). Affinity can be measured and / or expressed in many ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium association constant (KA or Ka). KD is calculated from the quotient koff / koff, and KA is calculated from the quotient koff / koff. koff refers to the association rate constant of, for example, an antibody to an antigen, and koff refers to the dissociation rate of, for example, an antibody to an antigen. koff and koff can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0247] As used herein, a "conservative amino acid substitution" refers to an amino acid residue being replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within the CDRs or framework regions of an antibody or antigen-binding molecule thereof may be replaced with an amino acid residue having a similar side chain.

[0248] As used herein, the term "heterologous" means from any source other than the naturally occurring sequence.

[0249] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or consecutive epitope), or an epitope can be, for example, composed of two or more non-contiguous regions of one or more polypeptides (a conformational epitope, a non-linear epitope, a discontinuous epitope, or a discontinuous epitope). In some embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303).Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114&115, eds. Wyckoff HW et al.; U.S. Patent Publication No. 2004 / 0014194) and BUSTER (see Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323).

[0250] As used herein, an antigen-binding molecule, antibody, or antigen-binding molecule thereof "cross-competes" with a reference antibody or its antigen-binding molecule if the interaction between the antigen and the first binding molecule, antibody, or its antigen-binding molecule prevents, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or its antigen-binding molecule to interact with the antigen. Cross-competition can be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In some embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same epitope as or an overlapping epitope with the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. To determine whether one antigen-binding molecule competes with another antigen-binding molecule, numerous types of competitive binding assays are available, including solid-phase direct or indirect radioimmunoassays (RIA); solid-phase direct or indirect enzyme immunoassays (EIA); sandwich competition assays (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assays, solid-phase direct label sandwich assays (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using 1 to 125 labels (Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) can be used.

[0251] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are analogous terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex), as such binding would be understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind with lower affinity to other peptides or polypeptides, as determined by, for example, immunoassays, a BIACORE®, a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen binds to the antigen with a K A that is at least 2 logs, 2.5 logs, 3 logs, 4 logs, or more greater than the K A for binding to another antigen.

[0252] "Antigen" refers to any molecule that can elicit an immune response or be bound by an antibody or antigen-binding molecule. The immune response can include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will readily understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or can be widely expressed. Additionally, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0253] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves collection of lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0254] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0255] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors that 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. Certain cancers can be responsive to chemotherapy or radiation therapy, or the cancer can be refractory. Refractory cancer refers to cancers that are not amenable to surgical intervention and that do not initially respond to chemotherapy or radiation therapy or that become unresponsive over time.

[0256] As used herein, "anti-tumor effect" refers to a biological effect that can be expressed as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall 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, for example, a vaccine.

[0257] As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where 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 endogenously expressed by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effector and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, can promote immune cell survival and proliferation, while pro-inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), TGF-beta, IL-35, and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0258] 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 reject tumor- and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). T cell receptors (TCRs) differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. Helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory cells (TSCM) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, similar to naive cells, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1). There are many types of T cells, including (i) central memory cells (TCMs), which express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4, and (ii) effector memory cells (TCMs), which 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 (involving antibodies). B cells can produce antibodies and act as antigen-presenting cells (APCs), and after activation by antigen interaction, they can become both short-lived and long-lived memory B cells and plasma cells. In mammals, immature B cells are formed in the bone marrow.

[0259] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes, e.g., T cells, which may be obtained from a patient or donor. The cells may be modified to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the genome of the cell.

[0260] An "immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced either by these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0261] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or upregulation or downregulation of key molecules.

[0262] As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The costimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule, for example, by binding of a peptide-loaded major histocompatibility complex (MHC) molecule to the T cell receptor (TCR) / CD3 complex. Costimulatory ligands may include, but are not limited to, 3 / TR6, 4-IBB ligand, agonists or antibodies that bind to Toll-like receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed 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).

[0263] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.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), CD 18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CD1-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, IT GAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1(CD1 la / 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; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0264] As used herein, "vaccine" refers to a composition for generating immunity for the prevention and / or treatment of disease. A vaccine is thus a pharmaceutical product containing an antigen, intended for use in humans or animals to generate specific defense and protection upon administration to humans or animals.

[0265] As used herein, "neoantigen" refers to a class of tumor antigens that arise from tumor-specific mutations of expressed proteins.

[0266] As used herein, a "fusion protein" is a protein having at least two domains encoded by separate genes that are linked to transcribe a single peptide.

[0267] 2. DNA template, precursor plexor RNA, and circular RNA According to the present invention, transcription of a DNA template provided herein (e.g., comprising a 3'-enhanced intron element, a 3'-enhanced exon element, a core functional element, a 5'-enhanced exon element, and a 5'-enhanced intron element) results in the formation of a precursor linear RNA polynucleotide that can be circularized. In some embodiments, the DNA template comprises a vector, a PCR product, a plasmid, a minicircle DNA, a cosmid, an artificial chromosome, a complementary DNA (cDNA), an extrachromosomal DNA (ecDNA), or a fragment thereof. In certain embodiments, the minicircle DNA may be linearized or non-linearized. In certain embodiments, the plasmid may be linearized or non-linearized. In some embodiments, the DNA template may be single-stranded. In other embodiments, the DNA template may be double-stranded. In some embodiments, the DNA template is composed, in whole or in part, of a viral, bacterial, or eukaryotic vector.

[0268] The present invention, as provided herein, includes DNA templates that share the same sequence as a precursor linear RNA polynucleotide prior to splicing of the precursor linear RNA polynucleotide (e.g., 3'-enhanced intronic elements, 3'-enhanced exonic elements, core functional elements and 5'-enhanced exonic elements, 5'-enhanced intronic elements). In some embodiments, the linear precursor RNA polynucleotide is spliced, and the 3'-enhanced intronic elements and 5'-enhanced intronic elements are removed during the circularization process. In some embodiments, the resulting circular RNA polynucleotide lacks the 3'-enhanced intronic and 5'-enhanced intronic fragments, but maintains the 3'-enhanced exonic fragments, core functional elements and 5'-enhanced exonic elements.

[0269] In some embodiments, the precursor linear 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'-enhancing exon elements, 5'-enhancing exon elements, and / or core functional elements, in whole or in part, promote circularization of a precursor linear RNA polynucleotide to form a circular RNA polynucleotide provided herein.

[0270] In certain embodiments, the circular RNAs provided herein are produced intracellularly. In some embodiments, precursor RNAs are transcribed and then circularized using a DNA template (e.g., in some embodiments, using a vector provided herein) in the cytoplasm by a bacteriophage RNA polymerase or in the nucleus by host RNA polymerase II.

[0271] 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 inside the animal.

[0272] In some embodiments, the DNA (e.g., vectors), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are 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 length. In some embodiments, a polynucleotide is at least 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, or 5000 nt in length, hi some embodiments, a polynucleotide is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10,000 nt in length. In some embodiments, the length of the DNA, linear RNA, and / or circular RNA polynucleotides provided herein is about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt.

[0273] In some embodiments, the circular RNAs provided herein have greater functional stability than mRNAs containing the same expressed sequence, hi some embodiments, the circular RNAs provided herein have greater functional stability than mRNAs containing the same expressed sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails.

[0274] In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 hours. In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of 5 to 80, 10 to 70, 15 to 60, and / or 20 to 50 hours. In some embodiments, the circular RNA polynucleotides provided herein have a half-life that 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 by detecting functional protein synthesis.

[0275] In some embodiments, the circular RNA polynucleotides provided herein have a half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some embodiments, the circular RNA polynucleotides provided herein have a half-life of 5 to 80 hours, 10 to 70 hours, 15 to 60 hours, and / or 20 to 50 hours. In some embodiments, the circular RNA polynucleotides provided herein have a half-life that is longer (e.g., at least 1.5 times longer, at least 2 times longer) than the half-life of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the circular RNA polynucleotide or pharmaceutical composition thereof has a functional half-life in human cells that is equal to or greater than a predetermined threshold. In some embodiments, the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, where the activity of Gaussia luciferase (GLuc) is measured every 1, 2, 6, 12, or 24 hours for 1, 2, 3, 4, 5, 6, 7, or 14 days in the culture medium of human cells (e.g., HepG2) expressing the circular RNA polynucleotide. In other embodiments, the functional half-life is determined by an in vivo assay, where the level of the protein encoded by the expressed sequence of the circular RNA polynucleotide is measured every 1, 2, 6, 12, or 24 hours in patient serum or tissue samples 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.

[0276] In some embodiments, the circular RNAs provided herein can have a higher magnitude of expression than a comparable linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of the RNA to a cell. In some embodiments, the circular RNAs provided herein have a higher magnitude of expression than an mRNA that includes the same expression sequence, 5moU modification, optimized UTR, cap, and / or polyA tail.

[0277] In some embodiments, the circular RNAs provided herein may be less immunogenic than equivalent mRNAs when exposed to the immune system of an organism or a type of immune cell. In some embodiments, the circular RNAs provided herein are associated with modulating cytokine production when exposed to the immune system of an organism or a 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 the immune system of an organism or a type of immune cell, compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNAs provided herein are associated with less transcriptional induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to the immune system of an organism or a type of immune cell, compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNA comprising the same expression sequence. In some embodiments, the circular RNAs provided herein are less immunogenic than mRNAs comprising the same expression sequence, 5moU modifications, optimized UTRs, caps, and / or polyA tails.

[0278] In certain embodiments, the circular RNAs provided herein can be transfected directly into cells or in the form of DNA vectors and transcribed within the cells. Transcription of the circular RNA from the transfected DNA vector can be carried out via an additional polymerase or polymerase encoded by a nucleic acid transfected into the cell, or preferably via an endogenous polymerase.

[0279] A. Enhanced Intronic and Exonic Elements As presented in the present invention, enhanced intronic and enhanced exonic elements can include spacers, duplex regions, affinity sequences, intron fragments, exon fragments, and various untranslated elements, which sequences within the enhanced intronic or enhanced exonic elements are positioned to optimize circularization or protein expression.

[0280] In certain embodiments, the DNA templates, precursor linear RNA polynucleotides, and circular RNAs provided herein comprise a first (5') and / or second (3') spacer. In some embodiments, the DNA template or precursor linear RNA polynucleotide comprises one or more spacers within an enhanced intron element. In some embodiments, the DNA template or precursor linear RNA polynucleotide comprises one or more spacers within an enhanced exon element. In certain embodiments, the DNA template or linear RNA polynucleotide comprises a spacer within the 3'-enhanced intron fragment and a spacer within the 5'-enhanced intron fragment. In certain embodiments, the DNA template, precursor linear RNA polynucleotide, or circular RNA comprises a spacer within the 3'-enhanced exon fragment and another spacer within the 5'-enhanced exon fragment to aid in circularization or protein expression due to the symmetry created across the sequence.

[0281] In some embodiments, including a spacer between the 3' Group I intron fragment and the core functional element can preserve the secondary structure of these regions by preventing them from interacting, thus increasing 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-paired regions that are predicted to base-pair with each other rather than with 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-paired regions that are predicted to base-pair with each other rather than with 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 increasing splicing efficiency. Furthermore, in some embodiments, the combination of base pairing between the first and second duplex regions and, separately, base pairing between the first and second spacers promotes the formation of a splicing bubble containing a group I intron fragment flanked by flanking regions of base pairing. A typical spacer is a contiguous sequence with one or more of the following properties: 1) predicted to avoid interference with proximal structures, such as an IRES, expression sequence, aptamer, or intron; 2) at least 7 nt in length and no more than 100 nt; 3) located adjacently after the 3' intron fragment and / or adjacently before the 5' intron fragment; and 4) comprising one or more of the following: a) an unstructured region at least 5 nt in length, b) a base-pairing 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 that is confined to the sequence of the spacer. A spacer can have several regions, including unstructured regions, base-paired regions, hairpin / structured regions, and combinations thereof. In one embodiment, a spacer has a structured region with a high GC content. In one embodiment, a region within a spacer base-pairs with another region within the same spacer.In one embodiment, a region within a spacer base pairs with a region within another spacer. In one embodiment, the spacer comprises one or more hairpin structures. In one embodiment, the spacer comprises one or more hairpin structures having a stem of 4-12 nucleotides and a loop of 2-10 nucleotides. In one embodiment, there is an additional spacer between the 3' Group I intron fragment and the core functional element. In one embodiment, this additional spacer prevents the structured region of the IRES or TIE aptamer from interfering with folding of the 3' Group I intron fragment, or reduces the extent to which this occurs. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is no longer than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the 5' spacer sequence is 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 polyA sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer comprises a poly AC content of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In one embodiment, the spacer comprises a poly pyrimidine (C / T or C / U) content of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0282] In some embodiments, the DNA templates and precursor linear RNA polynucleotides and circular RNA polynucleotides provided herein comprise a first (5') duplex region and a second (3') duplex region. In certain embodiments, the DNA templates and precursor linear 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 DNA templates, precursor linear 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 precursor linear RNA polynucleotides comprise a 5' external duplex region, a 5' internal duplex region, a 3' internal duplex region, and a 3' external duplex region.

[0283] 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 region is predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, or less than 25%) base-pairing with unintended sequences in the RNA (e.g., non-duplex region sequences). In some embodiments, such duplex regions are included at the ends of the precursor RNA strands, adjacent to or very close to the group I intron fragments, bringing the group I intron fragments into close proximity with each other and enhancing splicing efficiency. In some embodiments, the duplex region is 3 to 100 nucleotides in length (e.g., 3 to 75 nucleotides, 3 to 50 nucleotides, 20 to 50 nucleotides, 35 to 50 nucleotides, 5 to 25 nucleotides, 9 to 19 nucleotides in length). In some embodiments, 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 length. In some embodiments, the duplex region is about 9 to about 50 nucleotides in length. In one embodiment, the duplex region has a length of about 9 to about 19 nucleotides. In some embodiments, the duplex region has a length of about 20 to about 40 nucleotides. In certain embodiments, the duplex region has a length of about 30 nucleotides.

[0284] In other embodiments, the DNA template, precursor linear RNA polynucleotide, or circular RNA polynucleotide does not contain any double-stranded regions to optimize translation or circularization.

[0285] As provided herein, a DNA template or precursor linear RNA polynucleotide may comprise an affinity tag. In some embodiments, the affinity tag is located at the 3'-enhanced intron element. In some embodiments, the affinity tag is located at 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 the same length as the affinity tag of the 5'-enhanced intron element. In some embodiments, the affinity tag of the 3'-enhanced intron element is the same sequence as the affinity tag of the 5'-enhanced intron element. In some embodiments, the affinity sequence is positioned to optimize oligo-dT purification.

[0286] In some embodiments, the affinity tag comprises a polyA region. In some embodiments, the polyA region is at least 15, 30, or 60 nucleotides in length. In some embodiments, one or both polyA regions are 15-50 nucleotides in length. In some embodiments, one or both polyA regions are 20-25 nucleotides in length. The polyA sequence is removed during circularization. Thus, the circular RNA can be separated from its precursor RNA using oligonucleotides that hybridize to the polyA sequence, such as deoxythymine oligonucleotides (oligo(dT)) conjugated to a solid surface (e.g., a resin).

[0287] In certain embodiments, the 3'-enhanced intron element comprises a leading untranslated sequence. In some embodiments, the leading untranslated sequence is at the 5' end of the 3'-enhanced intron fragment. In some embodiments, the leading untranslated sequence comprises the last nucleotide of a transcription start site (TSS). In some embodiments, the TSS is selected from a viral, bacterial, or eukaryotic DNA template. In one embodiment, the leading 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 leading untranslated sequence comprises a guanosine at the 5' end upon translation by RNA T7 polymerase.

[0288] In certain embodiments, the 5'-enhanced intron element comprises a trailing untranslated sequence. In some embodiments, the 5'-enhanced intron element 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 a DNA template. In some embodiments, the restriction digest site is, in whole or in part, derived from a naturally occurring viral, bacterial, or eukaryotic DNA template. In some embodiments, the trailing untranslated sequence is a terminal restriction site fragment.

[0289] a. Enhanced intron fragment According to the present invention, 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.

[0290] b. Enhanced exon fragment In certain embodiments, the DNA templates, linear precursor RNA polynucleotides, and circular RNA polynucleotides provided herein each comprise an enhanced exon fragment. In some embodiments, the 3'-enhanced exon element is located upstream of the core functional element in 5' to 3' order. In some embodiments, the 5'-enhanced intron element is located downstream of the core functional element in 5' to 3' order.

[0291] According to the present invention, the 3'-enhanced exonic element and the 5'-enhanced exonic element each comprise an exon fragment. In some embodiments, the 3'-enhanced exonic element comprises a 3'-exon fragment. In some embodiments, the 5'-enhanced exonic element comprises a 5'-exon fragment. In certain embodiments, as provided herein, the 3'-exon fragment and the 5'-exon fragment comprise 1-100 nucleotides of a Group I intron fragment and exon sequence, respectively. In certain embodiments, the 3'-intron fragment is a contiguous sequence 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, including 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 is composed partially or entirely of naturally occurring exon sequence from a viral, bacterial, or eukaryotic DNA vector. In other embodiments, the exon sequences further comprise synthetic, genetically modified (eg, containing modified nucleotides), or other engineered exon sequences.

[0292] 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.

[0293] c. Exemplary permutations of enhanced intronic and exonic elements By way of example, and not intended to be limiting, in some embodiments, a 3'-enhanced intron element comprises, in 5' to 3' order: 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 yet the same embodiment, 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.

[0294] B. Core Functional Elements In some embodiments, the DNA template, the linear precursor RNA polynucleotide, and the circular RNA polynucleotide comprise a core functional element. In some embodiments, the core functional element comprises a coding element or a non-coding element. In certain embodiments, the core functional element can comprise both a coding element and a non-coding element. In some embodiments, the core functional element further comprises a translation initiation element (TIE) upstream of the coding element or the non-coding element. In some embodiments, the core functional element comprises a termination element. In some embodiments, the termination element is located downstream of the TIE and the coding element. In some embodiments, the termination element is located downstream of the coding element but upstream of the TIE. In certain embodiments, the coding element comprises a non-coding region, the core functional element lacks a TIE and / or a termination element.

[0295] a. Code or non-code element In some embodiments, the polynucleotides herein comprise coding elements 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.

[0296] In some embodiments, the circular RNA encodes two or more polypeptides. In some embodiments, the circular RNA is bicistronic. The sequences encoding the two or more polypeptides can be separated by a nucleotide sequence encoding a ribosome skipping element or a protease cleavage site. In certain embodiments, the ribosome skipping element encodes thosea asigna virus 2A peptide (T2A), porcine teschovirus-1 2A peptide (P2A), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2A peptide (BmCPV2A), or B. mori flacherie vims 2A peptide (BmIFV 2A).

[0297] b. Translation initiation element (TIE) As provided herein, in some embodiments, a core functional element comprises at least one translation initiation element (TIE). TIEs are designed to enable translational efficiency of the encoded protein. Thus, an optimal core functional element comprising only non-coding elements lacks any TIE. In some embodiments, a core functional element comprising one or more coding elements further comprises one or more TIEs.

[0298] In some embodiments, the TIE comprises an untranslated region (UTR). In certain embodiments, the TIE provided herein comprises an internal ribosome entry site (IRES). The inclusion of an IRES allows translation of one or more open reading frames (e.g., open reading frames that form an expression sequence) from the circular RNA. The IRES element attracts the eukaryotic ribosomal translation initiation complex and facilitates translation initiation. See, for example, 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.

[0299] i. Natural TIEs: viral and eukaryotic / cellular internal ribosome entry sites (IRES) Numerous IRES sequences are available, including 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 IRES element from the Foot and Mouth Disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), the Giardia virus IRES (Garlapati et al., J. Virol. (1996) 24:2697-2700), and the IRES from the Virus IRES (Garlapati et al., J. Virol. (1996) 24:2697-2700). These include sequences derived from a wide variety of viruses, such as those from the genus virulence filament virus (VVV) and genotypes (Gibber et al., J. Biol. Chem. (2004) 279(5):3389-3397).

[0300] To drive protein expression, the circular RNA comprises an IRES operably linked to a protein-coding sequence. Modifications of the IRES and accessory sequences are disclosed herein to increase or decrease IRES activity, for example, by truncating the 5' and / or 3' end of the IRES, adding a spacer 5' to the IRES, modifying the 6 nucleotides 5' to a translation start site (Kozak sequence), modifying an alternative translation start site, and creating a chimeric / hybrid IRES sequence. In some embodiments, the IRES sequence in the circular RNAs disclosed herein comprises one or more of these modifications compared to a naturally occurring IRES.

[0301] In some embodiments, the IRES is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, 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 1virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia 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, Canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus 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, Salivirus ASH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A2, 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 A1220, Pasivirus A3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus AThe IRES sequence of an aptamer against SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0302] In some embodiments, the IRES is composed, in whole or in part, of a eukaryotic or cellular IRES. In particular 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, ARMC 8, ARMCX6, ARPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURKA, B3GALNT1, B3GNTL1, B4GALT3, BAAT, BAG1, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, BTN 3A2, C12orf43, C14orf93, C17orf62, C1orf226, C21orf62, C2orf15, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP2 , 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、DCP 1A、DCTN1、DCTN2、DDX19B、DDX46、DEFB123、DGKA、DGKD、DHRS4、DHX15、DIO3、DLG1、DLL4、DMD UTR、DMD ex5、DMKN、DNAH6、DNAL4、DUSP13、DUSP19、DYNC1I2、DYNLRB2、DYRK1A、ECI2、ECT2、EIF1AD、EIF2B4、EIF4G1、EIF4G2、EIF4G3、E LANE、ELOVL6、ELP5、EMCN、ENO1、EPB41、ERMN、ERVV-1、ESRRG、ETFB、ETFBKMT、ETV1、ETV4、EXD1、EXT1、EZH2、FAM111B、FAM157A 、FAM213A、FBXO25、FBXO9、FBXW7、FCMR、FGF1、FGF1、FGF1A、FGF 2、FGF2、FGF-9、FHL5、FMR1、FN1、FOXP1、FTH1、FUBP1、G3BP1、GA BBR1、GALC、GART、GAS7、gastrin、GATA1、GATA4、GFM2、GHR、GJB2、GLI1、GLRA2、GMNN、GPAT3、GPATCH3、GPR137、GPR34、GPR55、G PR89A、GPRASP1、GRAP2、GSDMB、GSTO2、GTF2B、GTF2H4、GUCY1B2、HAX1、HCST、HIGD1A、HIGD1B、HIPK1、HIST1H1C、HIST1H3H、HK1 、HLA-DRB4、HMBS、HMGA1、HNRNPC、HOPX、HOXA2、HOXA3、HPCAL1、HR、HSP90AB1、HSPA1A、HSPA4L、HSPA5、HYPK、IFFO1、IFT74、IFT 81、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、KRTAP10-2、KRTAP13-3、KRTAP13-4、KRTAP5-11、KRTCAP2、LACRT、LAMB1、LA MB3、LANCL1、LBX2、LCAT、LDHA、LDHAL6A、LEF1、LINC-PINT、LMO3、LRRC4C、LR RC7、LRTOMT、LSM5、LTB4R、LYRM1、LYRM2、MAGEA11、MAGEA8、MAGEB1、MAGEB1 6、MAGEB3、MAPT、MARS、MC1R、MCCC1、METTL12、METTL7A、MGC16025、MGC16025 、MIA2、MIA2、MITF、MKLN1、MNT、MORF4L2、MPD6、MRFAP1、MRPL21、MRPS12、MS I2、MSLN、MSN、MT2A、MTFR1L、MTMR2、MTRR、MTUS1、MYB、MYC、MYCL、MYCN、MYL1 0、MYL3, MYLK, MYO1A, MYT2, MZB1, NAP1L1, NAV1, NBAS, NCF2, NDRG1, NDST2 NDUFA7、NDUFB11、NDUFC1、NDUFS1、NEDD4L、NFAT5、NFE2L2、NFE2L2、NFIA、NH EJ1、NHP2、NIT1、NKRF、NME1-NME2、NPAT、NR3C1、NRBF2、NRF1、NTRK2、NUDCD 1、NXF2、NXT2、ODC1、ODF2、OPTN、OR10R2、OR11L1、OR2M2、OR2M3、OR2M5、OR2T 10、OR4C15、OR4F17、OR4F5、OR5H1、OR5K1、OR6C3、OR6C75、OR6N1、OR7G2、p5 3、P2RY4、PAN2、PAQR6、PARP4、PARP9、PC、PCBP4、PCDHGC3、PCLAF、PDGFB、PDZ RN4、HAIR、PEMT、PEX2、PFKM、PGBD4、PGLYRP3、PHLDA2、PHTF1、PI4KB、PIGC、 PIM1、PKD2L1、PKM、PLCB4、PLD3、PLAKHA1、PLKHB1、PLS3、PML、PNMA5、PNN、P OC1A、POC1B、POLD2、POLD4、POU5F1、PPIG、PQBP1、PRAME、PRPF4、PRR11、PRRT 1、PRSS8、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, R IPOR2, RITA1, RMDN2, RNASE1, RNASE4, RNF4, RPA2, RPL17, RPL21, RPL26L1, RPL28, RPL29, RPL41, RPL9, RPS11, RPS13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RUNX1T1、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, SMN 1、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、STAU1、STK16、STK24、STK38、STMN1、STX7、SULT2B1、SYK、SYNPR、TAF1C、TAGLN、TANK、TAS2R40 、TBC1D15、TBXAS1、TCF4、TDGF1、TDP2、TDRD3、TDRD5、TESK2、THAP6、THBD、THTPA、TIAM2、TKFC、TKTL1、TLR10、TM9SF2、TMC6、TMCO2、TMED10、TMEM116、TM EM126A, 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, UBE2 L3, UBE2V1, UBE2V2, UMPS, UNG, UPP2, USMG5, USP18, UTP14A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS2 9, VSIG10L, WDHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY1AP1, ZBTB32, ZNF146, ZNF250, ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF707, or ZNRD1.

[0303] ii. Synthetic TIEs: Aptamer conjugates, modified nucleotides, IRES variants and other engineered TIEs As contemplated herein, in certain embodiments, a translation initiation element (TIE) comprises a synthetic TIE, hi 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.

[0304] In some embodiments, one or more aptamer sequences can bind to components of eukaryotic initiation factors to enhance or initiate translation. In some embodiments, aptamers can be used to enhance translation in vivo and in vitro by promoting specific eukaryotic initiation factors (eIFs) (e.g., the aptamers of WO2019081383 can bind to eukaryotic initiation factor 4F (eIF4F)). In some embodiments, an aptamer or aptamer complex can bind to EIF4G, EIF4E, EIF4A, EIF4B, EIF3, EIF2, EIF5, EIF1, EIF1A, 40S ribosome, PCBP1 (poly C binding protein), PCBP2, PCBP3, PCBP4, PABP1 (poly A binding protein), PTB, Argonaute protein family, HNRNPK (heterogeneous nuclear ribonucleoprotein K), or La protein.

[0305] c.Terminal sequence In certain embodiments, the core functional element comprises a termination sequence. In some embodiments, the termination sequence comprises a stop codon. In one embodiment, the termination sequence comprises a stop cassette. In some embodiments, the stop cassette comprises at least two stop codons. In some embodiments, the stop cassette comprises at least two stop codons in frame. In some embodiments, the frames of the stop codons in the stop cassette each comprise one, two, or more stop codons. In some embodiments, the stop cassette comprises a stop cassette flanked by LoxP or RoxStopRox, or frt. In some embodiments, the stop cassette comprises a lox-stop-lox stop cassette.

[0306] C. Variant In certain embodiments, the circular RNA polynucleotides provided herein comprise modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleosides are 5 C(5-methylcytidine). In another embodiment, the modified nucleoside is m 5U (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 -Isopentenyladenosine);ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine);io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -glycinylcarbamoyl adenosine);t 6 A(N 6 -threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -threonylcarbamoyl adenosine);m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyladenosine);hn 6 A(N 6 -hydroxynorvalylcarbamoylcarboxyadenosine);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 ,N 2 -dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(native hydroxywybutosine);imG(wybutosine);mimG(methylwybutosine);Q(cuosine);oQ(epoxycuosine);galQ(galactosyl-cuosine);manQ(mannosyl-cuosine);preQ 0( 7-cyano-7-deazaguanosine);preQ 1( 7-aminomethyl-7-deazaguanosine);G + (Archaeosin); D(Dihydrouridine); m 5 Um(5,2'-O-dimethyluridine);s 4U(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 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5 U(5-carbamoylmethyluridine);ncm 5 Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm 5 U(5-carboxymethylaminomethyluridine);cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m 6 2A(N 6 ,N 6-dimethyladenosine; Im (2'-O-methylinosine); m 4 C(N 4 -methylcytidine);m 4 Cm(N 4 ,2'-O-dimethylcytidine);hm 5 C(5-hydroxymethylcytidine);m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine);m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine);m 2,7 G(N 2 ,7-dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine);f 5 Cm(5-formyl-2'-O-methylcytidine);m 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine);τm 5 U(5-taurinomethyluridine);τm 5 s 2 U(5-taurinomethyl-2-thiouridine)); imG-14(4-demethylwyosine); imG2(isowyosine); or ac 6 A(N 6 -acetyladenosine).

[0307] In some embodiments, the modified nucleoside is pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-Taurinomethyl-4-thio-uridine, 5-Methyl-uridine, 1-Methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-Thio-1-methyl-pseudouridine, 1-Methyl-1-deaza-pseudouridine, 2-Thio-1-methyl-1-deaza-pseudouridine, Dihydrouridine, Dihydropseudouridine, 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-Methoxyuridine, 2-Methoxy-4-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 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 In another embodiment, the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine, and 1-methylpseudouridine.

[0308] In some embodiments, modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and / or pseudouridine, hi some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.

[0309] In specific embodiments, polynucleotides can be codon-optimized. A codon-optimized sequence can be one in which codons in a polynucleotide encoding a polypeptide are substituted to enhance the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of: (i) variation in codon bias between two or more organisms or genes or a synthetically constructed bias table; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons with context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC% across triplets or at single positions; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence underlying the design of the codon substitution set; and / or (x) systematic variation of the codon set for each amino acid. In some embodiments, codon-optimized polynucleotides may minimize ribozyme clashes and / or limit structural interference between the expression sequence and core functional elements.

[0310] 3. Payload In some embodiments, the expressed sequence encodes a therapeutic protein, hi some embodiments, the therapeutic protein is selected from the proteins listed in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

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

[0312] 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 in which each chain or subunit of the protein is encoded by a separate gene. Two or more circRNA molecules may be delivered in a transfer vehicle, with each circRNA encoding a separate subunit of the protein. Alternatively, a single circRNA may be engineered to encode two or more subunits. In certain embodiments, separate circRNA molecules encoding individual subunits may be administered in separate transfer vehicles.

[0313] A. Antigen-recognition receptor a. Chimeric antigen receptor (CARS) Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be inserted into immune cells, including T cells, via circular RNA as described herein and expressed by the immune cells. In a CAR, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate the immune cell to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. In some embodiments, the CAR encoded by the polynucleotide comprises (i) an antigen-binding molecule that specifically binds to the target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activation domain.

[0314] In some embodiments, a CAR orientation according to the present disclosure comprises an antigen binding domain (such as an scFv) in tandem with a costimulatory domain and an activation domain. The costimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains may be utilized in tandem.

[0315] i. antigen-binding domain CARs can be engineered to bind to antigens (such as cell surface antigens) by incorporating an antigen-binding molecule that interacts with the target antigen. In some embodiments, the antigen-binding molecule is an antibody fragment thereof, such as one or more single-chain antibody fragments (scFvs). An scFv is a single-chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. An scFv retains the ability of the parent antibody to specifically interact with the target antigen. Because scFvs can be engineered to be expressed as part of a single chain together with other CAR components, they are useful in chimeric antigen receptors. See also Id. Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It will be understood that an antigen-binding molecule is typically contained within the extracellular portion of the CAR so that it can recognize and bind to an antigen of interest. Bispecific and multispecific CARs having specificity for two or more desired targets are contemplated within the scope of the present invention.

[0316] In some embodiments, the antigen-binding molecule comprises a single chain, and the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N-terminus of the linker, and the VL is located at the C-terminus of the linker. In other embodiments, the VL is located at the N-terminus of the linker, and the VH is located at the C-terminus of the linker. In some embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids.

[0317] In some embodiments, the antigen binding molecule comprises a nanobody. In some embodiments, the antigen binding molecule comprises a DARPin. In some embodiments, the antigen binding molecule comprises an anticalin or other synthetic protein capable of specifically binding to a target protein.

[0318] In some embodiments, the CAR is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B-cell maturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin-13 receptor subunit alpha-2 ... Leukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, HER2, HER3, mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), pro prostatectomy enzymes, prostatic acid phosphatase (PAP), elongation factor 2 mutation (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (macropain) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), breakpoint cluster region (BCR), and Abelson matrix metalloproteinase (AMM) Oncogene fusion protein consisting of murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248),Tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor Glycoprotein receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3, and MAGE-A4), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen 1 (MAD-CT-1), melanoma cancer testis antigen 2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survival, telomerase, prostate cancer tumor antigen 1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (Transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1),CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein kinase (LCK), kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mutated) hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, ανβθ integrin, ανβ6 integrin, alpha-fetoprotein (AFP), B7-H6, ca-1 25, CA9, CD44, CD44v7 / 8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate-binding protein (FBP), kinase insert domain receptor (KDR), kappa light chain, L1 cell adhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4), tumor / testis antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), hepatitis B surface antigen-binding protein (HBsAg), viral capsid antigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen,The antigen-binding domain comprises an antigen specific for an antigen selected from the group consisting of a cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, hemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, human metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lymphotropic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, Kaposi's sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen, and KSHV latent nuclear antigen. In some embodiments, the antigen-binding domain comprises SEQ ID NO: 321 and / or 322.

[0319] ii. Hinge / spacer domain In some embodiments, a CAR of the present disclosure comprises a hinge domain or spacer domain. In some embodiments, the hinge / spacer domain may comprise a truncated hinge / spacer domain (THD), which is a truncated version of an intact hinge / spacer domain ("CHD"). In some embodiments, the extracellular domain is selected from ErbB2, glycophorin A (GpA), CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T CDl la (IT GAL), CDl lb (IT GAM), CDl lc (IT GAX), CDl ld (ITGAD), CD18(ITGB2), CD19(B4), CD27(TNFRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48( SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL 1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KI R2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247( CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG 2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRT AM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CDlla / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof. Hinge or spacer domains can be derived from either natural or synthetic sources.

[0320] In some embodiments, the hinge or spacer domain is located between the antigen binding molecule (e.g., scFv) and the transmembrane domain. In this orientation, the hinge / spacer domain provides distance between the antigen binding molecule and the surface of the cell membrane on which the CAR is expressed. In some embodiments, the hinge or spacer domain is from or derived from an immunoglobulin. In some embodiments, the hinge or spacer domain is selected from the hinge / spacer region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof. In some embodiments, the hinge or spacer domain comprises, is derived from, or is the hinge / spacer region of CD8 alpha. In some embodiments, the hinge or spacer domain comprises, is derived from, or is the hinge / spacer region of CD28. In some embodiments, the hinge or spacer domain comprises a fragment of the hinge / spacer region of CD8alpha or a fragment of the hinge / spacer region of CD28, wherein the fragment is less than the entire hinge / spacer region. In some embodiments, the fragment of the CD8alpha hinge / spacer region or the fragment of the CD28 hinge / spacer region comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids from the N-terminus or C-terminus, or both, of the CD8alpha hinge / spacer region or CD28 hinge / spacer region.

[0321] iii. Transmembrane domain The CAR of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. This may in turn be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some instances, the transmembrane domain may be selected or modified (e.g., by amino acid substitution) to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0322] Transmembrane regions include receptor tyrosine kinases (e.g., ErbB2), glycophorin A (GpA), 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 Id, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, ligand specifically binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-la / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, It may be derived from (i.e., comprise) SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or a fragment, truncated form or combination thereof.

[0323] In some embodiments, suitable intracellular signaling domains include, but are not limited to, activated macrophage / myeloid cell receptor CSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4 / 80, CD91, C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, lactoferrin, or fragments, truncations, or combinations thereof.

[0324] In some embodiments, the receptor tyrosine kinase may be derived from (e.g., include) insulin receptor (InsR), insulin-like growth factor I receptor (IGF1R), insulin receptor-related receptor (IRR), platelet-derived growth factor receptor alpha (PDGFRa), platelet-derived growth factor receptor beta (PDGFRfi), KIT proto-oncogene receptor tyrosine kinase (Kit), colony-stimulating factor 1 receptor (CSFR), fms-related tyrosine kinase 3 (FLT3), fms-related tyrosine kinase 1 (VEGFR-1), kinase insert domain receptor (VEGFR-2), fms-related tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase-like orphan receptor 1 (ROR1), tyrosine kinase-like orphan receptor 2 (ROR2), muscle-associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage-stimulated receptor 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene tyrosine kinase (Mer), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), EPH receptor A3 (EphA3), EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), hA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphAlO), EPH receptor B1 (EphBl), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret oncogene (Ret), receptor-like tyrosine kinase (RYK), discoidin domain receptor tyrosine kinase 1 (DDR1),Discoidin domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis-related tyrosine kinase (Lmrl), Lemur tyrosine kinase 2 (Lmr2), Lemur tyrosine kinase 3 (Lmr3), leukocyte receptor tyrosine kinase (LTK), ALK receptor tyrosine kinase (ALK), or serine / threonine / tyrosine kinase 1 (STYK1).

[0325] iv. Costimulatory domain In certain embodiments, the CAR comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In preferred embodiments, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (ζ). 4-1BB, CD28, and CD3 zeta may comprise less than the entire 4-1BB, CD28, or CD3 zeta, respectively. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to enhance their efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0326] In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 318 or 320.

[0327] v. Intracellular signaling domain The intracellular (signaling) domain of the engineered T cells disclosed herein can provide signaling to the activation domain, which then activates at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0328] In some embodiments, suitable intracellular signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 ld, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly08, lymphocyte function-associated antigen-1 (LFA-1; CD1-la / CD18), Comprises (e.g., consists of) an MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or a fragment, truncated form or combination thereof.

[0329] CD3 is a component of the T cell receptor on natural T cells and has been shown to be a key intracellular activation component in CARs. In some embodiments, the CD3 is CD3 zeta. In some embodiments, the activation domain comprises an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide sequence of SEQ ID NO:319.

[0330] bT cell receptor (TCR) TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, with links to the IMGT public database of TCR sequences. Naturally occurring alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Generally, each chain may contain a variable region, a joining region, and a constant region; beta chains usually also contain a short diversity region between the variable and joining regions, although this diversity region is often considered part of the joining region. Each variable region may contain three CDRs (complementarity-determining regions) embedded in framework sequences, one of which is a hypervariable region called CDR3. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Vα types are referenced by unique TRAV numbers in the IMGT nomenclature. Thus, "TRAV21" defines a TCR Vα region with unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by an amino acid sequence that is conserved from TCR to TCR, but also includes amino acid sequences that vary from TCR to TCR. Similarly, "TRBV5-1" defines a TCR Vβ region with unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence.

[0331] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature.

[0332] The beta chain diversity region is referred to by the abbreviation TRBD in the IMGT nomenclature, and as mentioned above, the linked TRBD / TRBJ region is often considered together as the linking region.

[0333] The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. "T cell Receptor Factsbook" (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but due to its publication date and the resulting time lag, it may be necessary to confirm the information therein with the IMGT database.

[0334] Naturally occurring TCRs exist in heterodimeric αβ or γδ forms. However, recombinant TCRs composed of αα or ββ homodimers have previously been shown to bind peptide-MHC molecules. Thus, the TCRs of the present invention may be heterodimeric αβ TCRs or αα or β homodimeric TCRs.

[0335] For use in adoptive therapy, αβ heterodimeric TCRs can be transfected, for example, as full-length chains having both cytoplasmic and transmembrane domains. In certain embodiments, the TCRs of the invention can have disulfide bonds introduced between residues of their respective constant domains, for example, as described in WO 2006 / 000830.

[0336] The TCRs of the present invention, particularly alpha-beta dimeric TCRs, may comprise an alpha chain TRAC constant domain sequence and / or a beta chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chain constant domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and / or beta chain constant domain sequences may also be modified by substitution of cysteine ​​residues Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, which form a disulfide bond between the alpha and beta constant domains of the TCR.

[0337] Binding affinity (equilibrium constant K D The binding half-life (expressed as T1 / 2, which is inversely proportional to K) and binding half-life (expressed as T1 / 2) can be determined by any suitable method. Doubling the affinity of the TCR results in a K D It will be understood that T1 / 2 is calculated as ln 2 divided by the off rate (koff). Therefore, doubling T1 / 2 halves koff. D and koff values ​​are typically measured for soluble forms of the TCR, i.e., forms truncated to remove cytoplasmic and transmembrane domain residues. It will therefore be understood that a given TCR will have improved binding affinity and / or binding half-life relative to the parent TCR if the soluble form of that TCR possesses such characteristics. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, for example, three or more times, using the same assay protocol and the results are averaged.

[0338] Because the TCRs of the present invention have utility in adoptive therapy, the present invention includes non-natural and / or purified and / or engineered cells, particularly T cells, that present the TCRs of the present invention. There are several suitable methods for transfecting T cells with nucleic acids (such as DNA, cDNA, or RNA) encoding the TCRs of the present invention (see, e.g., Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCRs of the present invention are suitable for use in adoptive therapy-based treatment of cancers, such as pancreatic and liver cancer. As known to those skilled in the art, there are several suitable methods by which adoptive therapy can be carried out (see, e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0339] As is well known in the art, the TCRs of the present invention may undergo post-translational modifications when expressed by transfected cells. Glycosylation is one such modification and can involve the covalent attachment of oligosaccharide moieties to selected amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are well-known positions for oligosaccharide attachment. The glycosylation status of a particular protein depends on many factors, including protein sequence, protein conformation, and the availability of specific enzymes. Furthermore, the glycosylation status (i.e., oligosaccharide type, covalent bonds, and total number of attachments) can affect protein function. Therefore, controlling glycosylation is often desirable when producing recombinant proteins. Glycosylation of transfected TCRs can be controlled by mutation of the transfected gene (Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations are also encompassed by the present invention.

[0340] TCR is MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE- 8, BAGE-1, RAGE-1, LB33 / MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL- 40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, Lage-1, Mag-C2, NA-88, Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA, NY-ESO (LAGE), SCP-1, Hom / Mel-40, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, and CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA It may be specific for antigens in the following group: 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

[0341] cB cell receptor (BCR) The B cell receptor (BCR), or B cell antigen receptor, is an immunoglobulin molecule that forms a type I transmembrane protein on the surface of B cells. The BCR can transmit activation signals to B cells after recognition of a specific antigen. Before a B cell binds to an antigen, the BCR remains in an unstimulated or "resting" stage. Binding of an antigen to the BCR results in a signal transduction that initiates a humoral immune response.

[0342] BCRs are expressed by mature B cells. These B cells cooperate with immunoglobulins (Ig) in the recognition and tagging of pathogens. A typical BCR contains membrane-bound immunoglobulins (e.g., mIgA, mIgD, mIgE, mIgG, and mIgM) along with the associated Igα / Igβ (CD79a / CD79b) heterodimer (α / β). These membrane-bound immunoglobulins are tetramers consisting of two identical heavy chains and two light chains. Within the BCR, the membrane-bound immunoglobulins respond to antigen binding by signaling across the plasma membrane, resulting in B cell activation, which can lead to clonal expansion and specific antibody production (Friess M et al. (2018), Front. Immunol. 2947(9)). The Igα / Igβ heterodimer is responsible for transmitting signals to the cell interior.

[0343] Igα / Igβ heterodimer signaling depends on the presence of immunoreceptor tyrosine-based activation motifs (ITAMs) located in each of the cytosolic tails of the heterodimer. ITAMs contain two tyrosine residues separated by 9–12 amino acids (e.g., tyrosine, leucine, and / or valine). Upon antigen binding, the tyrosines of the BCR ITAMs become phosphorylated by the Src family tyrosine kinases Blk, Fyn, or Lyn (Janeway C et al., Immunobiology: The Immune System in Health and Disease (Garland Science, 5th ed. 2001)).

[0344] d. Other chimeric proteins In addition to the chimeric proteins described above, the circular RNA polynucleotide can encode a variety of other chimeric proteins available in the art. Chimeric proteins can include recombinant fusion proteins, chimeric mutant proteins, or other fusion proteins.

[0345] B. Immunomodulatory Ligands In some embodiments, the circular RNA polynucleotide encodes an immunomodulatory ligand. In certain embodiments, the immunomodulatory ligand may be immunostimulatory, while in other embodiments, the immunomodulatory ligand may be immunosuppressive.

[0346] 1. Cytokines: interferons, chemokines, interleukins, growth factors, etc. In some embodiments, the circular RNA polynucleotide encodes a cytokine. In some embodiments, the cytokine includes chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Chemokines are chemotactic cytokines produced by various cell types in acute and chronic inflammation that recruit and activate leukocytes. Interferons comprise a family of secreted alpha-helical cytokines that are induced in response to specific extracellular molecules via stimulation of TLRs (Borden, Molecular Basis of Cancer (Fourth Edition) 2015). Interleukins are cytokines expressed by leukocytes.

[0347] Descriptions and / or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-27β, IFNγ and / or TGFβ1 are provided herein and in the database at www.uniprot.org under accession numbers: P60568 (IL-2), P29459 (IL-12A), P29460 (IL-12B), P13232 (IL-7), P22301 (IL-10), P40933 (IL-15), Q14116 (IL-18), Q14213 (IL-27β), P01579 (IFNγ), and / or P01137 (TGFβ1).

[0348] C. Transcription factors Regulatory T cells (Tregs) are important in maintaining homeostasis, controlling the magnitude and duration of inflammatory responses, and preventing autoimmune and allergic responses.

[0349] It is generally believed that Tregs are primarily involved in suppressing immune responses, functioning in part as a "self-check" to prevent the immune system from overreacting. In particular, Tregs are involved in maintaining tolerance to harmless agents such as self-antigens, pollen, or food, and in preventing autoimmune diseases.

[0350] Tregs are found throughout the body, including but not limited to the intestine, skin, lungs, and liver. Additionally, Treg cells can also be found in specific compartments of the body that are not directly exposed to the external environment, such as the spleen, lymph nodes, and even adipose tissue. Each of these Treg cell populations is known or suspected to have one or more unique characteristics; further information can be found in Lehtimaki and Lahesmaa, "Regulatory T cells control immune responses through their non-redundant tissue-specific features," 2013, FRONTIERS IN IMMUNOL., 4(294):1-10, the disclosure of which is incorporated herein in its entirety.

[0351] Typically, Tregs are known to require TGF-β and IL-2 for proper activation and development. Tregs, which express large amounts of IL-2 receptor (IL-2R), are dependent on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β, both of which are potent immunosuppressive cytokines. Furthermore, Tregs are known to inhibit the ability of antigen-presenting cells (APCs) to stimulate T cells. One proposed mechanism for APC inhibition is via CTLA-4, which is expressed by Foxp3+ Tregs. It is thought that CTLA-4 binds to B7 molecules on APCs and blocks or removes them by causing their internalization, reducing the availability of B7 and preventing them from providing appropriate costimulation for immune responses. Further discussion of the origin, differentiation, and function of Tregs can be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4(253):1-11, the disclosure of which is incorporated herein in its entirety.

[0352] D. Checkpoint Inhibitors and Agonists As provided herein, in certain embodiments, the coding element of the circular RNA encodes one or more checkpoint inhibitors or agonists.

[0353] In some embodiments, the immune checkpoint inhibitor is an inhibitor of programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T-cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1, CTLA4, PD-1, LAG3, PD-L1, TIM3, or a combination thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1.

[0354] As described herein, in at least one embodiment, the invention encompasses the use of immune checkpoint antagonists. Such immune checkpoint antagonists include antagonists of immune checkpoint molecules such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PDL-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain 3 (TIM-3). Antagonists of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 interfere with the function of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3, respectively. Such antagonists of CTLA-4, PD-1, PDL-1, LAG-3 and TIM-3 may include antibodies that specifically bind to CTLA-4, PD-1, PDL-1, LAG-3 and TIM-3, respectively, and inhibit and / or block the biological activity and function.

[0355] E. Other In some embodiments, the payload encoded within the one or more coding elements is a hormone, an FC fusion protein, an anticoagulant, a blood clotting factor, a protein associated with deficiency and genetic disease, a chaperone protein, an antimicrobial protein, an enzyme (e.g., a metabolic enzyme), a structural protein (e.g., a channel or nuclear pore protein), a protein variant, a small molecule, an antibody, a nanobody, an engineered non-body antibody, or a combination thereof.

[0356] 4. Additional accessory elements (array elements) As described herein, circular RNA polynucleotides, linear RNA polynucleotides, and / or DNA templates may further comprise accessory elements. In certain embodiments, these accessory elements may be included within the sequence of the circular RNA, linear RNA polynucleotide, and / or DNA template to enhance circularization, translation, or both. In certain embodiments, accessory elements are sequences that are specifically located between or within enhanced intronic elements, enhanced exonic elements, or core functional elements of the respective polynucleotides. By way of example, and not intended to be limiting, accessory elements include IRES transactivator regions, miRNA binding sites, restriction sites, RNA editing regions, structural or sequence elements, granule sites, zip code elements, RNA transport elements, or other specialized sequences found in the art that enhance and facilitate circularization and / or translation of proteins encoded within the circular RNA polynucleotide.

[0357] A. IRES trans-acting factors In certain embodiments, the accessory element comprises an IRES transacting factor (ITAF) region. In some embodiments, the IRES transacting factor region regulates translation initiation via binding to PCBP1-PCBP4 (polyC binding proteins), PABP1 (polyA binding protein), PTB (polypyrimidine tract binding), the Argonaute protein family, HNRNPK (heterogeneous nuclear ribonucleoprotein K protein), or La protein. In some embodiments, the IRES transacting factor region comprises a polyA, polyC, polyAC, or polypyrimidine tract.

[0358] In some embodiments, the ITAF region is located within a core functional element, hi some embodiments, the ITAF region is located within a TIE.

[0359] B. miRNA binding site In certain embodiments, the accessory element comprises an miRNA binding site, hi some embodiments, the miRNA binding site is located within a 5'-enhanced intron element, a 5'-enhanced exon element, a core functional element, a 3'-enhanced exon element, and / or a 3'-enhanced intron element.

[0360] In some embodiments, the miRNA binding site is located within a spacer within an enhanced intronic or exonic element, hi certain embodiments, the miRNA binding site comprises the entire spacer region.

[0361] In some embodiments, the 5'-enhanced intron element and the 3'-enhanced intron element each comprise the same miRNA binding site. In another embodiment, the miRNA binding site of the 5'-enhanced intron element comprises a miRNA binding site of a different length or nucleotides than the 3'-enhanced intron element. In one embodiment, the 5'-enhanced exon element and the 3'-enhanced exon element comprise the same miRNA binding site. In other embodiments, the 5'-enhanced exon element and the 3'-enhanced exon element comprise miRNA binding sites of different lengths or nucleotides.

[0362] In some embodiments, the miRNA binding sites are located adjacent to each other within the circular RNA polynucleotide, the linear RNA polynucleotide precursor, and / or the DNA template. In certain embodiments, the first nucleotide of one of the miRNA binding sites follows the first or last nucleotide of the second miRNA binding site.

[0363] In some embodiments, the miRNA binding site is located within the translation initiation element (TIE) of the core functional element. In one embodiment, the miRNA binding site is located before, after, or within the internal ribosome entry site (IRES). In another embodiment, the miRNA binding site is located before, after, or within the aptamer complex.

[0364] The unique sequences defined by the miRNA nomenclature are widely known and accessible to those working in the microRNA field, e.g., they can be found in the miRDB public database.

[0365] 5. Polynucleotide Production The DNA templates provided herein can be produced using standard techniques of molecular biology. For example, various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving polynucleotides from DNA templates known to contain them.

[0366] Various elements of the DNA template provided herein can also be synthetically produced based on known sequences, rather than by cloning.Complete sequences can be assembled from overlapping oligonucleotides prepared by standard methods, and assembled into complete sequences.See, for example, Edge, Nature (1981) 292: 756; Nambair et al., Science (1984) 223: 1299; and Jay et al., J.Biol.Chem. (1984) 259: 631 1.

[0367] Thus, specific nucleotide sequences can be obtained from a DNA template having the desired sequence, or can be synthesized in whole or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques, as needed. One method for obtaining a nucleotide sequence encoding a desired DNA template element is by annealing a complementary set of overlapping synthetic oligonucleotides produced in a conventional automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase, and amplifying the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-directed mutagenesis of existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0368] The precursor RNAs provided herein can be produced by incubating a DNA template provided herein under conditions that allow transcription of the precursor RNA encoded by the DNA template. For example, in some embodiments, precursor RNAs are synthesized by incubating a DNA template provided herein that includes an RNA polymerase promoter upstream of its 5' duplex sequence and / or expression sequence with a compatible RNA polymerase enzyme under conditions that allow in vitro transcription. In some embodiments, the DNA template is incubated inside a cell with a bacteriophage RNA polymerase or in the nucleus of a cell with host RNA polymerase II.

[0369] In certain embodiments, provided herein are methods for producing precursor RNA by in vitro transcription using a DNA template provided herein as a template (e.g., a vector provided herein having an RNA polymerase promoter positioned upstream of the 5' duplex region).

[0370] In certain embodiments, the resulting precursor RNA can be used to produce circular RNA (e.g., a circular RNA polynucleotide provided herein) by incubation in the presence of magnesium ions and guanosine nucleotides or nucleosides at a temperature at which RNA circularization occurs (e.g., 20°C to 60°C).

[0371] Thus, in certain embodiments, provided herein are methods for producing circular RNA. In certain embodiments, the method includes synthesizing a precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a 5'-enhanced intron element, a 5'-enhanced exon element, a core functional element, a 3'-enhanced exon element, and a 3'-enhanced intron element) as a template, and incubating the resulting precursor RNA in the presence of a divalent cation (e.g., magnesium ion) and GTP to circularize it to form a circular RNA. In some embodiments, the precursor RNA disclosed herein can be circularized in the absence of magnesium ion and GTP and / or without the incubation step with magnesium ion and GTP. It has been discovered that circular RNAs have reduced immunogenicity compared to the corresponding mRNA, at least in part because the mRNA contains an immunogenic 5' cap. When precursor RNAs are produced by transcribing a DNA vector from a specific promoter (e.g., a T7 promoter), it is understood that the 5' end of the precursor RNA is G. To reduce the immunogenicity of circular RNA compositions containing low levels of contaminating linear mRNA, excess GMP relative to GTP can be provided during transcription so that the majority of transcripts contain 5' GMP, which cannot be capped. Thus, in some embodiments, transcription is performed in the presence of excess GMP. In some embodiments, transcription is performed when the ratio of GMP concentration to GTP concentration is within the range of about 3:1 to about 15:1, e.g., about 3:1 to about 10:1, about 3:1 to about 5:1, about 3:1, about 4:1, or about 5:1.

[0372] In some embodiments, the composition comprising circular RNA is purified. Circular RNA can be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps, in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse-phase HPLC. In some embodiments, the purified composition contains less double-stranded RNA, DNA splints, triphosphorylated RNA, phosphatase protein, protein ligase, capping enzyme, and / or nicked RNA than unpurified RNA. In some embodiments, the purified composition is less immunogenic than the unpurified composition. In some embodiments, immune cells exposed to the purified composition produce less TNFα, RIG-I, IL-2, IL-6, IFNγ, and / or type 1 interferon, such as IFN-β1, than immune cells exposed to the unpurified composition.

[0373] 6. Overview of Transfer Vehicles and Other Delivery Mechanisms A. Ionizable lipids In certain embodiments, disclosed herein are ionizable lipids that can be used as components of transfer vehicles to facilitate or enhance the delivery and release of circular RNA into one or more target cells (e.g., by penetrating or fusing with the lipid membranes of such target cells). In certain embodiments, the ionizable lipids comprise one or more cleavable functional groups (e.g., disulfides) that allow, for example, a hydrophilic functional head group to dissociate from a lipophilic functional tail group of the compound (e.g., upon exposure to oxidizing, reducing, or acidic conditions), thereby facilitating a phase transition in the lipid bilayer of one or more target cells.

[0374] In various embodiments, the ionizable lipid of the present disclosure is a compound of formula (13*): [ka] wherein: n* is an integer from 1 to 7, R a is hydrogen or hydroxyl, R b is hydrogen or C1-C6 alkyl, R1 and R2 are each independently 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, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl linear or branched C-C alkyl groups optionally substituted with one or more substituents selected from alkyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C2-C 30 Alkenyl, or C1-C 30 It is heteroalkyl.

[0375] In some embodiments of formula (13*), R b is C1-C6 alkyl. In some embodiments of formula (13*), R b is methyl. In some embodiments of formula (13*), R b is ethyl.

[0376] In some embodiments of formula (13*), R b is H and the ionizable lipid is of formula (13): [ka] wherein n is an integer from 1 to 7. In some embodiments of formula (13), n is an integer from 1 to 4.

[0377] In some embodiments of Formula (13*) and Formula (13), R1 and R2 are the same. In some embodiments of Formula (13*) and Formula (13), R1 and R2 are different.

[0378] In some embodiments of Formula (13*) and Formula (13), R1 and R2 are each independently an optionally substituted linear or branched alkyl, alkenyl, or heteroalkyl, and the total number of carbon atoms in the optionally substituted linear or branched group is 30 or less, e.g., 6 to 30 carbon atoms, or 6 to 20 carbon atoms.

[0379] In some embodiments of Formula (13*) and Formula (13), at least one of R and R is an unsubstituted linear or branched C-C 30 Alkyl, C6-C 30 Alkenyl, or C6-C 30 It is heteroalkyl.

[0380] In some embodiments of Formula (13*) and Formula (13), R and R are each independently a linear or branched C-C alkyl group optionally substituted (e.g., as described above) with one or more substituents. 30 Alkyl, C6-C 30Alkenyl, or C9-C 20 In some embodiments of formula (13*) and formula (13), R and R are straight or branched C-C alkyl groups substituted with alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, or alkenylcarbonate. 30 Alkyl, C6-C 30 Alkenyl or C9-C 20 heteroalkyl.

[0381] In some embodiments of Formula (13*) and Formula (13), R and R are each independently a linear or branched C-C 20 Alkoxy, straight or branched C1-C 20 Alkyloxycarbonyl, linear or branched C1-C 20 Alkylcarbonyloxy, linear or branched C1-C 20 Alkyl carbonates, linear or branched C2-C 20 Alkenyloxycarbonyl, linear or branched C2-C 20 Alkenylcarbonyloxy, linear or branched C2-C 20 Alkenyl carbonates, linear or branched C2-C 20 Alkynyloxycarbonyl, linear or branched C2-C 20 Alkynylcarbonyloxy and straight or branched C2-C 20 linear or branched C-C alkyl groups optionally substituted with one or more substituents each independently selected from alkynyl carbonates, 20 Alkyl, C2-C 20 Alkenyl, or C1-C 20 It is heteroalkyl.

[0382] In some embodiments of Formula (13*) and Formula (13), at least one of R and R is —O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 Straight chain C1-C substituted with 12 alkyl, and each R 6are independently linear or branched C1-C 20 Alkyl or C2-C 20 In some embodiments of Formula (13*) and Formula (13), R and R are each independently -O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 Straight chain C1-C substituted with 12 alkyl, and each R 6 are independently linear or branched C1-C 20 Alkyl or C2-C 20 It is alkenyl.

[0383] In some embodiments, at least one of R and R is substituted with alkyloxycarbonyl. In some embodiments, alkyloxycarbonyl is -C(O)OR 6’ wherein R 6’ is an unsubstituted C6-C 30 Alkyl or C6-C 30 It is alkenyl.

[0384] In some embodiments, at least one of R and R is substituted with alkylcarbonyloxy. In some embodiments, alkylcarbonyloxy has the formula -OC(O)R 6’ wherein R 6’ is unsubstituted C6-C 30 Alkyl or C6-C 30 Alkenyl

[0385] In some embodiments, at least one of R and R is substituted with an alkyl carbonate. In some embodiments, the alkyl carbonate has the formula -O(CO)OR 6’ wherein R 6’ is unsubstituted C6-C 30 Alkyl or C6-C 30 Alkenyl

[0386] In some embodiments, R and R are each independently —O(CO)R 6’, -C(O)OR 6’ , or -O(CO)OR 6’ C1-C substituted with 12 alkyl, where R 6’ is unsubstituted C6-C 30 Alkyl or C6-C 30 In some embodiments, R and R are each -O(CO)R 6’ C1-C substituted with 12 In some embodiments, R and R are each —C(O)OR 6’ C1-C substituted with 12 In some embodiments, R and R are each —O(CO)OR 6’ C1-C substituted with 12 In some embodiments, R is —C(O)OR 6’ or -O(CO)R 6’ and R2 is -O(CO)OR 6’ In some embodiments, R is —O(CO)OR 6 and R2 is -C(O)OR 6 or -O(CO)R 6 is.

[0387] In some embodiments, at least one of R1 and R2 is selected from the following formulas: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is an integer from 0 to 12, r is an integer from 0 to 6, R8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0388] In some embodiments, each of R1 and R2 is independently selected from one of the following formulas: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0389] In some embodiments of any one of Formulas (i)-(iii), q is an integer from 1 to 6. In some embodiments of any one of Formulas (i)-(iii), q is 0. In some embodiments of any one of Formulas (i)-(iii), q is 1. In some embodiments of any one of Formulas (i)-(iii), q is 2. In some embodiments of any one of Formulas (i)-(iii), q is an integer from 3 to 12. In some embodiments of any one of Formulas (i)-(iii), q is an integer from 3 to 6.

[0390] In some embodiments of any one of Formulas (i)-(iii), r is 0. In some embodiments of any one of Formulas (i)-(iii), r is an integer from 1 to 6. In some embodiments of any one of Formulas (i)-(iii), r is 1. In some embodiments of any one of Formulas (i)-(iii), r is 2.

[0391] In some embodiments of formulas (i)-(iii), R 8 is H. In some embodiments of formulas (i)-(iii), R 8 is R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 is the same.

[0392] In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9is an unsubstituted straight chain C-C alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C5-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C6-C8 alkyl.

[0393] In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight-chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C2-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently unsubstituted straight chain C6-C8 alkyl.

[0394] In some embodiments, at least one of R and R is —(CH) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, R 1 and R 2At least one of the is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R and R is —(CH) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0395] In certain embodiments, at least one of R and R is —(CH) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In other embodiments, at least one of R and R is —(CH) q OC(O)(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R and R is —(CH) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R8 and R 9 is as defined above.

[0396] In certain embodiments, R1 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R is -(CH) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R is -(CH) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0397] In certain embodiments, R 1 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R8 and R 9 is as defined above. In certain embodiments, R is -(CH) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R2 is --(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R is -(CH) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0398] In certain embodiments, R1 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R is -(CH) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9is as defined above. In certain embodiments, R is -(CH) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R2 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0399] In some embodiments, R1 and R2 are each independently selected from the group consisting of: [ka] [ka]

[0400] In some embodiments, the ionizable lipid of formula (13) is substituted by formula (13a-1), formula (13a-2), or formula (13a-3): [ka]

[0401] In some embodiments, the ionizable lipid is substituted by formula (13b-1), formula (13b-2), or formula (13b-3): [ka]

[0402] In some embodiments, the ionizable lipid is substituted by the following formula (13b-4), (13b-5), (13b-6), (13b-7), (13b-8), or (13b-9): [ka]

[0403] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 are independently -O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 C1-C optionally substituted with 12 alkyl, and R 6 is an unsubstituted straight-chain or branched C1-C 20 Alkyl or C2-C 20 In some embodiments, R is alkenyl. 6 is an unsubstituted straight chain C1-C 20 In some embodiments, R 6 is an unsubstituted branched C6-C 20 In some embodiments, R 6 is an unsubstituted straight chain C6-C 20 In some embodiments, R 6 is an unsubstituted branched C6-C 20 It is alkyl.

[0404] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 is a straight or branched C6-C 30 Alkyl, straight or branched C6-C 30 Alkenyl, straight or branched C6-C 30 Heteroalkyl, -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) independently selected from: wherein q is 0 to 12, r is 0 to 6, and R 8is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 20 Alkyl or unsubstituted straight chain C2-C 20 In some embodiments, q is 1 to 8, for example, 1 to 6, or 2 to 6. In some embodiments, r is 0. In some embodiments, r is 1 to 6, for example, 1 to 3. In some embodiments, r is 1. In some embodiments, r is 2.

[0405] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 are different groups. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 are the same. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 One of the groups is a straight chain group, and R 1 and R 2 The other contains a branched group.

[0406] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of is selected from the following formulas: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0407] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 are each independently selected from one of the following formulas: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0408] In some embodiments of any one of Formulas (i)-(iii), q is an integer from 1 to 6. In some embodiments of any one of Formulas (i)-(iii), q is 1. In some embodiments of any one of Formulas (i)-(iii), q is 2. In some embodiments of any one of Formulas (i)-(iii), q is an integer from 3 to 12. In some embodiments of any one of Formulas (i)-(iii), q is an integer from 3 to 6.

[0409] In some embodiments of any one of Formulas (i)-(iii), r is 0. In some embodiments of any one of Formulas (i)-(iii), r is an integer from 1 to 6. In some embodiments of any one of Formulas (i)-(iii), r is 1. In some embodiments of any one of Formulas (i)-(iii), r is 2.

[0410] In some embodiments of formulas (i)-(iii), R 8 is H. In some embodiments of formulas (i)-(iii), R 8 is R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 is the same.

[0411] In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted linear C2-C8 alkyl. In some embodiments of formulas (i)-(iii), R8 is H and R 9 is an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C6-C8 alkyl.

[0412] In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight-chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C2-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently unsubstituted straight chain C6-C8 alkyl.

[0413] In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q OC(O)(CH2) r CH(R 8 )(R9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0414] In certain embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0415] In other embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments of formulas (13a-1) to (13b-9), R 1 and R 2 At least one of the is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0416] In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0417] In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)(CH2) r CH(R 8)(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0418] In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments of formulas (13a-1) to (13b-9), R 1 Ha-(CH2) q OC(O)O(CH2) rCH(R 8 )(R 9 ) and R 2 Ha-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0419] In some embodiments of Formulas (13a-1) through (13b-9), n is 1. In some embodiments of Formulas (13a-1) through (13b-9), n is an integer from 2 to 7. In some embodiments of Formulas (13a-1) through (13b-9), n is 2. In some embodiments of Formulas (13a-1) through (13b-9), n is an integer from 3 to 7. In some embodiments of Formulas (13a-1) through (13b-9), n is 3. In some embodiments of Formulas (13a-1) through (13b-9), n is an integer from 4 to 7. In some embodiments of Formulas (13a-1) through (13b-9), n is 4. In some embodiments of Formulas (13a-1) through (13b-9), n is 5. In some embodiments of Formulas (13a-1) through (13b-9), n is 6. In some embodiments of formulas (13a-1) through (13b-9), n is 7.

[0420] In some embodiments of Formula (13*), the ionizable lipid is of Formula (13c-1) or (13c-2): [ka] During the ceremony, n* and n are each an integer of 1 to 7; R a is hydrogen or hydroxyl, R b is hydrogen or C1-C6 alkyl, L A and L B are each independently a straight chain C1-C 12 is alkyl; ZA and Z B are each independently selected from absent or —C(O)O—, —OC(O)—, and —OC(O)O—; and R A and R B are independently linear or branched C1-C 20 Alkyl or C2-C 20 It is alkenyl.

[0421] In some embodiments of formulas (13c-1) and (13c-2), Z A is selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z B In some embodiments of formulas (13c-1) and (13c-2), Z B is selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z A does not exist.

[0422] In some embodiments of formula (13c-2), the ionizable lipid is of formula (13d-2): [ka] During the ceremony, q and q' are each independently an integer of 1 to 12, r and r' are each independently an integer of 0 to 6, R 8A is H or R 10A and R 8B is H or R 10B and R 9A , R 9B , R 10A , and R 10A are each independently an unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 It is alkenyl.

[0423] In some embodiments of formula (13d-2), R 9A , R9B , R 10A , and R 10A are each independently an unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 It is alkenyl.

[0424] In some embodiments of Formula (13d-2), q is an integer from 1 to 6. In some embodiments of Formula (13d-2), q is 0. In some embodiments of Formula (13d-2), q is 1. In some embodiments of Formula (13d-2), q is 2. In some embodiments of Formula (13d-2), q is 3 to 12. In some embodiments of Formula (13d-2), q is 3 to 6.

[0425] In some embodiments of Formula (13d-2), r is 0. In some embodiments of Formula (13d-2), r is an integer from 1 to 6. In some embodiments of Formula (13d-2), r is 1. In some embodiments of Formula (13d-2), r is 2.

[0426] In some embodiments of formula (13d-2), R a is hydrogen, and Z A is selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z B is absent. In some embodiments of formula (13d-2), R a is hydrogen, and Z B is a linking group selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z A does not exist.

[0427] In some embodiments of formula (13d-2), R a is hydroxyl and Z A is selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z B is absent. In some embodiments of formula (13d-2), R a is hydroxyl and Z Bis selected from —C(O)O—, —OC(O)—, and —OC(O)O—; Z A does not exist.

[0428] In some embodiments, the ionizable lipid of the present disclosure is of formula (13d-2), set forth in the compounds of Table 1 below, where any undefined variables are as described above. [Table 2-1] [Table 2-2]

[0429] In some embodiments, the ionizable lipids of the present disclosure are selected from the group consisting of: [ka] .

[0430] In some embodiments, the ionizable lipid is [ka] is selected from the group consisting of:

[0431] In some embodiments, the ionizable lipid is [ka] isn't it.

[0432] In some embodiments of formula (13c-1) and / or (13c-2), each R b is hydrogen.

[0433] In some embodiments of formula (13c-1) and / or (13c-2), only one R b is C1-C6 alkyl, and other R bIn some embodiments of formula (13c-1) and / or (13c-2), only one R group is present. b is methyl or ethyl. In some embodiments, only one R is C-C alkyl. b is attached to the carbon atom adjacent to the nitrogen atom of the ionizable lipid. In some embodiments of formula (13c-1) and / or (13c-2), n is 2 to 7, and only one R b is C1-C6 alkyl, and other R b The group, when present, is hydrogen.

[0434] In some embodiments, the ionizable lipid of the present disclosure is a lipid selected from Tables 10e-10h.

[0435] In some embodiments, the ionizable lipids of the present disclosure have a beta-hydroxylamine head group. In some embodiments, the ionizable lipids have a gamma-hydroxylamine head group.

[0436] In one embodiment, the ionizable lipids are described in U.S. Patent Publication No. US20170210697A1. In one embodiment, the ionizable lipids are described in U.S. Patent Publication No. US20170119904A1. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 4-1

Table 4-2

Table 4-3

Table 4-4

[0437] In some embodiments, the ionizable lipid has one of the structures set forth in Table 10 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12]

[0438] In some embodiments, the ionizable lipid is as described in International Patent Application PCT / US2020 / 038678.

[0001] In some embodiments, the ionizable lipid is represented by formula (14*): [ka] or a pharmaceutically acceptable salt thereof, wherein: L 1 is C2-C 11 Alkylene, C4-C 10 -alkenylene, or C4-C 10 -alkynylene; X 1 is OR 1 , S.R. 1 , or N(R 1 )2 and R 1 is independently H or unsubstituted C1-C6 alkyl; and R2 and R3 are each independently 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, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl linear or branched C-C alkyl groups optionally substituted with one or more substituents selected from alkyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; 30 Alkyl, C2-C 30 Alkenyl, or C1-C 30 Heteroalkyl.

[0002] In some embodiments, the ionizable lipid is represented by formula (14): [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 is C2-C 11 Alkylene, C4-C 10-alkenylene, or C4-C 10 -alkynylene; X 1 is OR 1 , S.R. 1 , or N(R 1 )2 and R 1 is independently H or unsubstituted C1-C6 alkyl; and R 2 and R 3 are independently C6-C 30 -Alkyl, C6-C 30 -alkenyl or C-C 30 -alkynyl.

[0003] In some embodiments, X 1 is OR 1 In some embodiments, X 1 is OH. In some embodiments, X 1 is SR 1 In some embodiments, X 1 is SH. In some embodiments, X 1 is N(R 1 )2. In some embodiments, X 1 is NH2.

[0004] In some embodiments, L 1 is C2-C 10 In some embodiments, L is alkylene. 1 is an unsubstituted C2-C 10 In some embodiments, L is alkylene. 1 is C4-C 10 In some embodiments, L is alkenylene. 1 is unsubstituted C4-C 10 In some embodiments, L is alkenylene. 1 is C4-C 10 In some embodiments, L is alkynylene. 1 is unsubstituted C4-C 10 It is alkynylene.

[0005] In some embodiments of formula (14), the lipid has a structure according to formula (14-2): [ka] or a pharmaceutically acceptable salt thereof (wherein n is an integer of 2 to 10).

[0006] In some embodiments, n is 2, 3, 4, or 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0007] In some embodiments of formula (14*) or formula (14-2), R2 and R3 are independently a linear or branched C1-C 20 Alkoxy, straight or branched C1-C 20 Alkyloxycarbonyl, linear or branched C1-C 20 Alkylcarbonyloxy, linear or branched C1-C 20 Alkyl carbonates, linear or branched C2-C 20 Alkenyloxycarbonyl, linear or branched C2-C 20 Alkenylcarbonyloxy, linear or branched C2-C 20 Alkenyl carbonates, linear or branched, C2-C 20 Alkynyloxycarbonyl, linear or branched C2-C 20 Alkynylcarbonyloxy and straight or branched C2-C 20 linear or branched C-C alkyl groups optionally substituted with one or more substituents each independently selected from alkynyl carbonates, 20 Alkyl, C2-C 20 Alkenyl, or C1-C 20 It is heteroalkyl.

[0008] In certain embodiments of formula (14*) or formula (14-2), R 2 and R 3 One or each of 30 -Alkyl, unsubstituted C-C 30 -alkenyl or unsubstituted C-C 30 -alkynyl. In certain embodiments, R 2 and R 3 Each of 30 -alkyl. In certain embodiments, R 2 and R 3 Each of 30 -alkenyl. In certain embodiments, R 2 and R 3 Each of 30 -alkynyl.

[0009] In some embodiments of formula (14*), the alkyloxycarbonyl substituent is of the formula —C(O)OR 6 wherein R 6 is unsubstituted C6-C 30 Alkyl or C6-C 30 In some embodiments of Formula (14*) or Formula (14-2), at least one of R2 and R3 is substituted with alkylcarbonyloxy. In some embodiments, alkylcarbonyloxy is of the formula -OC(O)R 6 wherein R 6 is unsubstituted C6-C 30 Alkyl or C6-C 30 In some embodiments that are alkenyl, at least one of R and R is substituted with an alkyl carbonate. In some embodiments, the alkyl carbonate has the formula -O(CO)OR 6 wherein R 6 is unsubstituted C6-C 30 Alkyl or C6-C 30 In some embodiments that are alkenyl, R and R are independently —O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 C1-C substituted with12 alkyl, where R 6 is unsubstituted C6-C 30 Alkyl or C6-C 30 In some embodiments, R and R are each -O(CO)R 6 C1-C substituted with 12 In some embodiments, R and R are each —C(O)OR 6 C1-C substituted with 12 In some embodiments, R and R are each —O(CO)OR 6 C1-C substituted with 12 In some embodiments, R2 is -C(O)OR 6 or -O(CO)R 6 and R3 is -O(CO)OR 6 In some embodiments, R2 is -O(CO)OR 6 and R3 is -C(O)OR 6 or -O(CO)R 6 is.

[0010] In some embodiments of formula (14*) or formula (14-2), at least one of R2 and R3 is selected from the following formulae: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is 0 to 12, r is 0 to 6; R 8 is H or R 10 and R9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0011] In some embodiments of Formula (14*) or Formula (14-2), each of R2 and R3 is independently selected from one of the following formulas: (i)-(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), (ii)-(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ), and (iii)-(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ), During the ceremony, q is 0 to 12, r is 0 to 6; R 8 is H or R 10 and R 9 and R 10 are independently unsubstituted straight-chain C1-C 12 Alkyl or unsubstituted straight chain C2-C 12 -alkenyl.

[0012] In some embodiments of any one of Formulas (i)-(iii), q is 1 to 6. In some embodiments of any one of Formulas (i)-(iii), q is 0. In some embodiments of any one of Formulas (i)-(iii), q is 1. In some embodiments of any one of Formulas (i)-(iii), q is 2. In some embodiments of any one of Formulas (i)-(iii), q is 3 to 12. In some embodiments of any one of Formulas (i)-(iii), q is 3 to 6.

[0013] In some embodiments of any one of Formulas (i)-(iii), r is 0. In some embodiments of any one of Formulas (i)-(iii), r is 1-6. In some embodiments of any one of Formulas (i)-(iii), r is 1. In some embodiments of any one of Formulas (i)-(iii), r is 2. In some embodiments of any one of Formulas (i)-(iii), r is 3. In some embodiments of any one of Formulas (i)-(iii), r is 4.

[0014] In some embodiments of formulas (i)-(iii), R 8 is H. In some embodiments of formulas (i)-(iii), R 8 is R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 In some embodiments of formulas (i)-(iii), R 9 and R 10 is the same.

[0015] In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is unsubstituted straight chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C-C alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9 is an unsubstituted straight chain C5-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 is H and R 9is an unsubstituted straight chain C6-C8 alkyl.

[0016] In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C1-C 12 Alkyl or unsubstituted straight chain C1-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight-chain C2-C 12 In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C2-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently an unsubstituted straight chain C4-C8 alkyl. In some embodiments of formulas (i)-(iii), R 8 and R 9 are each independently unsubstituted straight chain C6-C8 alkyl.

[0017] In some embodiments of formula (14*) or formula (14-2), at least one of R2 and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R2 and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R2 and R3 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R8 and R 9 is as defined above. In some embodiments of formula (14*) or formula (14-2), at least one of R2 and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ), wherein q is 3 to 12 (e.g., 6 to 12), r is 1 to 6 (e.g., 1, 2, or 3), and R 8 and R 9 are each independently unsubstituted straight-chain C4-C8 alkyl.

[0018] In certain embodiments of formula (14*) or formula (14-2), at least one of R2 and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In other embodiments, at least one of R2 and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R2 and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R9 is as defined above.

[0019] In other embodiments of formula (14*) or formula (14-2), at least one of R2 and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In some embodiments, at least one of R2 and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) or -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0020] In certain embodiments of formula (14*) or formula (14-2), R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R2 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R is -(CH) qC(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0021] In certain embodiments of formula (14*) or formula (14-2), R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R2 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0022] In certain embodiments of formula (14*) or formula (14-2), R2 is -(CH2)q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q C(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R2 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above. In certain embodiments, R2 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) and R3 is -(CH2) q OC(O)O(CH2) r CH(R 8 )(R 9 ) where q, r, R 8 and R 9 is as defined above.

[0023] In certain embodiments of formula (14*) or formula (14-2), one or each of R2 and R3 is an unsubstituted C6-C 22 -alkyl or R 2 and R 3 One or each of 22 -alkenyl. In certain embodiments, R 2 and R 3 Each of 22 In certain embodiments, each of R and R is an unsubstituted C-C 22 -alkenyl.

[0024] In certain embodiments, one or each of R2 and R3 is -CH 13 , -CH 15 , -CH 17 , -CH 19 , -C 10 H 21 , -C 11 H 23 , -C 12 H 25 , -C l3 H 27 , -C l4 H 29 , -C l5 H 3l , -C l6 H 33 , -C l7 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 21 H 43 , -C 22 H 45 , -C 23 H 47 , -C 24 H 49 , -C 25 H 51 is.

[0025] In certain embodiments, one or each of R2 and R3 is -(CH2)4CH=CH2, -(CH2)5CH=CH2, -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8SCH=CH2, -(CH2)9CH=CH2, -(CH2) 10 CH=CH2, -(CH2) 11 CH=CH2, -(CH2) 12 CH=CH2, -(CH2) 13 CH=CH2, -(CH2) 14 CH=CH2, -(CH2) 15 CH=CH2, -(CH2) 16 CH=CH2, -(CH2) 17 CH=CH2, -(CH2) 18CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)IICH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3.

[0026] In certain embodiments, one or each of R2 and R3 is -O(CO)R 6 or -C(O)OR 6 C6-C substituted with 12 alkyl, where R 6 is unsubstituted C6-C 14 In certain embodiments, R 6 is an unsubstituted straight chain C6-C 14 In certain embodiments, R 6 is an unsubstituted branched C6-C 14 It is alkyl.

[0027] In certain embodiments, one or each of R and R is (CH)C(O)O(CH)CH(CH 11 )2 or (CH2)8C(O)O(CH2)2CH(CH 11 )2. In certain embodiments, R 2 and R 3 One or each of [ka] is.

[0028] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] In certain embodiments, R 2 and R 3 One or each of [ka] is.

[0439] In some embodiments, the ionizable lipid is described in Table 10h. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0440] 5.1 Other ionizable lipids In some embodiments, one or more (e.g., two or more, or three or more) ionizable lipids are utilized in the transfer vehicles of the present disclosure. In some embodiments, the transfer vehicle comprises a first ionizable lipid (e.g., a lipid of formula (13*) or (14*), as described herein) and one or more additional ionizable lipids.

[0441] Lipids of interest, including ionizable lipids that can be incorporated into transfer vehicles of the present disclosure and used in combination with the first ionizable lipid described herein, include, but are not limited to, those described in International Application PCT / US2018 / 058555, International Application PCT / US2020 / 038678, U.S. Publication US2019 / 0314524, WO2019 / 152848, International Application PCT / US2010 / 061058, International Application PCT / US2017 / 028981, WO2015 / 095340, WO20 14 / 136086, US2019 / 0321489, WO2010 / 053572, U.S. Provisional Patent Application 61 / 617,468, International Patent Application PCT / US2019 / 025246, U.S. Patent Publications 2017 / 0190661 and 2017 / 0114010, U.S. Publication 20190314284, WO2015 / 095340, WO2019 / 152557, WO2019 / 152848, International Application PCT / US2019 / 015913, U.S. Patent No. 9,708,628, U.S. Patent No. 9,765,022; Wang et al., ACS Synthetic Biology, 1,403-07 (2012); WO 2008 / 042973, U.S. Patent No. 8,071,082, the disclosures of which are incorporated herein by reference in their entireties.

[0442] In some embodiments, the tail groups used in the lipids may be as described in WO2015 / 095340, WO2019 / 152557, and WO2019 / 152848, the disclosures of which are incorporated herein by reference in their entireties.

[0443] Lipid-like compounds can be prepared by methods well known in the art. See:

[0444] In some embodiments, the ionizable lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used (Felgner et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); US Pat. No. 4,897,355). DOTMA can be formulated with ionizable lipids (e.g., as described herein) and / or combined with the neutral lipid, dioleoylphosphatidylethanolamine or "DOPE," or other cationic or non-cationic lipids into lipid nanoparticles.

[0445] Other suitable lipids include, for example, ionizable cationic lipids, such as (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N, N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), C12-200 (described in WO 2010 / 053572), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanolamine (DLinKC2-DMA)) (WO 2010 / 042877; Semple et al., Nature Biotech.28:172-176 (2010)), 2-(2,2-di((9Z,2Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanolamine (DLin-KC2-DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5 001), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), 5-carboxyspermylglycine-dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al. Proc. Nat. Acad. Sci.86,6982 (1989); U.S. Patent Nos. 5,171,678; 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane or (DOTAP). Contemplated ionizable lipids include 1,2-distacyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DO DAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy) (cis)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl Also included are 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), or GL67, or mixtures thereof (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication WO 2005 / 121348 A1). The use of cholesterol-based ionizable lipids to formulate transfer vehicles (e.g., lipid nanoparticles) is also contemplated by the present invention. Such cholesterol-based ionizable lipids can be used alone or in combination with other lipids. Suitable cholesterol-based ionizable lipids include, for example, DC-cholesterol (N,N-dimethyl-N-ethylcarboxamidocholesterol) and 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al., Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335).

[0446] Also contemplated is cationic lipid, such as dialkylamino, imidazole and guanidinium lipid.For example, also contemplated is the use of ionizable lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), as disclosed in International Application No. PCT / US2010 / 058457, which is incorporated herein by reference.

[0447] Ionizable lipids are also contemplated, such as dialkylamino-based, imidazole-based and guanidinium-based lipids.For example, certain embodiments relate to compositions comprising one or more imidazole-based ionizable lipids, for example, imidazole cholesterol ester or "ICE" lipid, (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate.

[0448] Without wishing to be bound by any particular theory, the fusogenicity of imidazole-based cationic lipids (ICEs) is thought to be related to endosomal disruption, which is facilitated by the imidazole group, which has a lower pKa than conventional ionizable lipids. Endosome disruption then promotes osmotic swelling and rupture of the liposome membrane, subsequently facilitating transfection or intracellular release of the loaded nucleic acid(s) contents into target cells.

[0449] Imidazole-based ionizable lipids are also characterized by low toxicity compared to other ionizable lipids.

[0450] In certain embodiments, the transfer vehicle composition for delivering circular RNA comprises amine lipid.In certain embodiments, the ionizable lipid is amine lipid.In some embodiments, the amine lipid is described in International Patent Application PCT / US2018 / 053569.

[0451] In some embodiments, the amine lipid is lipid E, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate.

[0452] In certain embodiments, the amine lipid is an analog of lipid E. In certain embodiments, the lipid E ​​analog is an acetal analog of lipid E. In certain transfer vehicle compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In further embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

[0453] Amine lipids and other biodegradable lipids suitable for use in the transfer vehicle described herein, for example, lipid nanoparticles, are biodegradable in vivo. The amine lipids described herein have low toxicity (e.g., tolerated in animal models without adverse effects at doses of 10 mg / kg or more). In certain embodiments, transfer vehicles comprising amine lipids include those in which at least 75% of the amine lipid is removed from plasma within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days.

[0454] Biodegradable lipids include, for example, those of WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086.

[0455] Lipid clearance can be measured by methods known to those skilled in the art. See, for example, Maier, MA, et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78.

[0456] A transfer vehicle composition comprising an amine lipid can result in an increased clearance rate. In some embodiments, the clearance rate is the lipid clearance rate, e.g., the rate at which lipids are cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the RNA clearance rate, e.g., the rate at which circRNAs are cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which the transfer vehicle is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which the transfer vehicle is cleared from tissues such as liver tissue or spleen tissue. In certain embodiments, a high clearance rate results in a safety profile without substantial adverse effects. Amine lipids and biodegradable lipids can reduce transfer vehicle accumulation in the circulation and tissues. In some embodiments, reduced transfer vehicle accumulation in the circulation and tissues results in a safety profile without substantial adverse effects.

[0457] Lipids can be ionized depending on the pH of the medium they are in.For example, in a weakly acidic medium, lipids such as amine lipids can be protonated and therefore have positive charge.On the other hand, in a slightly basic medium such as blood, whose pH is approximately 7.35, lipids such as amine lipids can not be protonated and therefore have no charge.

[0458] The ability of a lipid to carry a charge is related to its inherent pKa. In some embodiments, the amine lipids of the present disclosure may each independently have a pKa ranging from about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each independently have a pKa ranging from about 5.1 to about 7.4. For example, the amine lipids of the present disclosure may each independently have a pKa ranging from about 5.8 to about 6.5. Lipids with a pKa ranging from about 5.1 to about 7.4 are effective for in vivo cargo delivery, for example, to the liver. Furthermore, lipids with a pKa ranging from about 5.3 to about 6.4 have been found to be effective for in vivo delivery, for example, to tumors. See, e.g., WO 2014 / 136086.

[0459] The lipids of the present disclosure may have -SS- (disulfide) bonds.

[0460] The lipid-like compounds of the present disclosure can be prepared using appropriate starting materials via synthetic routes known in the art. The methods may include additional steps of adding or removing appropriate protecting groups to ultimately allow synthesis of the lipid-like compounds. Furthermore, various synthetic steps can be performed in an alternative order or sequence to obtain the desired materials. Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful for synthesizing applicable lipid-like compounds are known in the art, including, for example, R. Larock, Comprehensive Organic Transformations (2nd Ed., VCH Publishers 1999); P.G.M. Buts and T.W. Greene, Greene's Protective Groups in Organic Synthesis (4th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd Ed., John Wiley and Sons 2009) and subsequent editions. Certain lipid-like compounds may contain non-aromatic double bonds and one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans-isomers, and all such isomeric forms are contemplated.

[0461] Methods for preparing the above compounds and compositions are described herein below and / or are known in the art.

[0462] Those skilled in the art will appreciate that in the processes described herein, it may be necessary to protect the functional groups of intermediate compounds with suitable protecting groups. Such functional groups include, for example, hydroxyl, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include, for example, trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include, for example, t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include, for example, -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include, for example, alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail, for example, in Green, T W and P G M Hutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one skilled in the art will appreciate, the protecting group may be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0463] It will also be understood by those skilled in the art that such protected derivatives of the compounds of the present invention may not have such pharmacological activity, but may be administered to a mammal and then metabolized in the body to form the pharmacologically active compounds of the present invention. Thus, such derivatives may be described as prodrugs. All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0464] Additionally, all compounds of the present invention that exist in a free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds of the present invention can also be converted to their free base or acid form by standard techniques.

[0465] It is understood that those skilled in the art may be able to prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art can prepare other compounds of formula (1) not specifically shown herein by using appropriate starting materials and modifying the synthetic parameters. In general, the starting materials can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as described in the present invention.

[0466] As mentioned above, these lipid-like compounds are useful for the delivery of pharmaceuticals. They can be preliminarily screened for their effectiveness in the delivery of pharmaceuticals by in vitro assays, and then confirmed by animal experiments and clinical trials. Other methods will be apparent to those skilled in the art.

[0467] Without being bound by any theory, the lipid-like compounds of the present disclosure can facilitate the delivery of pharmaceuticals by forming complexes, such as nanocomplexes and microparticles. The hydrophilic head of such lipid-like compounds is positively or negatively charged and binds to the oppositely charged portion of the pharmaceutical, while its hydrophobic portion binds to the hydrophobic portion of the pharmaceutical. Either bond can be covalent or non-covalent.

[0468] The above complexes can be prepared using procedures described in publications such as Wang et al., ACS Synthetic Biology, 1, 403-07 (2012). Typically, they are obtained by incubating a lipid-like compound and a pharmaceutical agent in a buffer solution such as sodium acetate buffer or phosphate-buffered saline ("PBS").

[0469] 5.2 Hydrophilic groups In certain embodiments, selected hydrophilic functional groups or moieties may alter or otherwise impart properties to compounds or transfer vehicles of which such compounds are components (e.g., by improving the transfection efficiency of lipid nanoparticles of which the compounds are components). For example, incorporation of guanidinium as a hydrophilic head group in the compounds disclosed herein may promote the fusogenicity of such compounds (or transfer vehicles of which such compounds are components) with the cell membrane of one or more target cells, thereby, for example, increasing the transfection efficiency of such compounds. The nitrogen from the hydrophilic guanidinium moiety is hypothesized to form a six-membered ring transition state that confers stability to the interaction, thus enabling cellular uptake of the encapsulated material (Wender, et al., Adv. Drug Del. Rev. (2008) 60:452-472). Similarly, incorporation of one or more amino groups or moieties (e.g., as a head group) into the disclosed compounds may further promote disruption of the endosomal / lysosomal membrane of target cells by exploiting the fusogenicity of such amino groups. This is based not only on the pKa of the amino group of the composition, but also on the ability of the amino group to undergo a hexagonal phase transition and fuse with the target cell surface, i.e., the vesicle membrane (Koltover, et al. Science (1998) 281:78-81). The result is thought to facilitate rupture of the vesicle membrane and release of the lipid nanoparticle contents into the target cell.

[0470] Similarly, in certain embodiments, for example, the incorporation of imidazole as a hydrophilic head group in the compounds disclosed herein can serve to facilitate endosomal or lysosomal release of contents encapsulated in the transfer vehicles (e.g., lipid nanoparticles) of the present invention. Such enhanced release can be achieved by one or both of a proton-sponge-mediated disruption mechanism and / or a fusogenicity enhancement mechanism. The proton-sponge mechanism is based on the ability of a compound, particularly a functional moiety or group of a compound, to buffer endosomal acidification. This can be manipulated or otherwise controlled by the pKa of the compound or one or more functional groups (e.g., imidazole) comprising such a compound. Thus, in certain embodiments, for example, the fusogenicity of imidazole-based compounds disclosed herein (e.g., HGT4001 and HGT4004) is related to their endosomal disruption properties, which are facilitated by the imidazole group's low pKa compared to other conventional ionizable lipids. Such endosome-disrupting properties then promote osmotic swelling and rupture of the liposome membrane, subsequently facilitating transfection or intracellular release of the loaded or encapsulated polynucleotide material within target cells. This phenomenon may be applicable to a variety of compounds that have desirable pKa profiles in addition to the imidazole moiety. Such embodiments also include polynitrogen-based functional groups such as polyamines, polypeptides (histidine), and nitrogen-based dendritic structures.

[0471] Exemplary ionizable lipids and / or cationic lipids are described in International PCT Patent Publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004 143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO20 12 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 086373, WO2011 / 071860, WO2009 / 1321 31, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO20 10 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 0001 07, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO20 05 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346 and WO2 013 / 086354, and U.S. Patent Publications US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926,US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 0128760, US2010 / 0324120 , US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 0338210, US2012 / 0101148, US2012 / 0027796, US2012 / 0058144, US2013 / 0323269, US2011 / 0117125, US2011 / 025617 5, US2012 / 0202871, US2011 / 0076335, US2006 / 0083780, US2013 / 0123338, US2015 / 0064242, US200 6 / 0051405, US2013 / 0065939, US2006 / 0008910, US2003 / 0022649, US2010 / 0130588, US2013 / 01163 Nos. 07, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the entire contents of which are incorporated herein by reference in their entirety. International Patent Application WO2019 / 131770 is also incorporated herein by reference in its entirety.

[0472] B.PEG lipid The use and inclusion of polyethylene glycol (PEG)-modified phospholipids, including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000], and derivatized lipids, such as derivatized ceramide (PEG-CER) (C8 PEG-2000 ceramide), in the liposome and pharmaceutical compositions described herein is contemplated, preferably in combination with one or more compounds and lipids disclosed herein. Contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids having C6-C20 alkyl chains. In some embodiments, the PEG-modified lipid used in the compositions and methods of the present invention is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (2000 MW PEG) "DMG-PEG2000." The addition of PEG-modified lipids to lipid delivery vehicles can provide a means to prevent complex aggregation, thereby extending circulatory lifetime and increasing delivery of lipid-polynucleotide compositions to target tissues (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected to rapidly exchange from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C14 or C18). PEG-modified phospholipids and derivatized lipids of the present invention can comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal lipid nanoparticles.

[0473] In one embodiment, the PEG-modified lipids are described in International Patent Application Nos. PCT / US2019 / 015913 or PCT / US2020 / 046407, which are incorporated herein by reference in their entireties. In one embodiment, the transfer vehicle comprises one or more PEG-modified lipids.

[0474] Non-limiting examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. In some further embodiments, the PEG-modified lipid can be e, g, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE.

[0475] In some still further embodiments, PEG-modified lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-l,2-dimyristylpropyl-3-amine (PEG-c-DMA).

[0476] In some still further embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. In some embodiments, the PEG-modified lipid is DSPE-PEG(2000).

[0477] In some embodiments, the PEG-modified lipid comprises a PEG moiety comprising 10-70 (e.g., 30-60) oxyethylene (-O-CH2-CH2-) units or portions thereof. In some embodiments, the PEG-modified lipid comprises (OCH2CH2) v -OR wwherein v is an integer of 0 to 70 (inclusive) (eg, an integer of 30 to 60), and w is hydrogen or alkyl.

[0478] In various embodiments, the PEG-modified lipid may also be referred to as a "PEGylated lipid" or a "PEG-lipid."

[0479] In one embodiment, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0480] In some embodiments, the lipid portion of the PEG-lipid is about C 14 ~About C 22 , for example, about C 14 ~About C 16 In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, about 2000, about 5000, about 10,000, about 15,000, or about 20,000 daltons. In one embodiment, the PEG-lipid is PEG2k-DMG.

[0481] In one embodiment, the lipid nanoparticles described herein can comprise lipids modified with non-diffusible PEGs, non-limiting examples of which include PEG-DSG and PEG-DSPE.

[0482] PEG-lipids are known in the art, for example, U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584A2, which are incorporated herein by reference in their entireties.

[0483] In various embodiments, the lipids described herein (e.g., PEG-lipids) are synthesized as described in International Patent Publication No. PCT / US2016 / 000129, which is incorporated by reference in its entirety.

[0484] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipids may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0485] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG, which has the following structure: [ka]

[0486] In some embodiments, the PEG-modified lipid is a modified form of PEG-C18 or PEG-1, which has the following structure: [ka]

[0487] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety. Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0488] In some embodiments, the PEG lipid is a compound of formula (P1): [ka] or a salt or isomer thereof, wherein: r is an integer from 1 to 100; R is C 10-40 Alkyl, C 10-40 Alkenyl, or C 10-40 alkynyl; and optionally, one or more methylene groups in R are independently selected from C 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6-10 Arylene, 4-10 membered heteroarylene, -N(R N )-, -O-, -S-, -C(O)-,-C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N)S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )- or -N(R N )S(O)2O-; and R N Each instance of 1-6 It is an alkyl or nitrogen protecting group.

[0489] For example, R is a C17 alkyl. For example, a PEG lipid can be prepared by the compound of formula (P1-a): [ka] or a salt or isomer thereof, wherein r is an integer of 1 to 100.

[0490] For example, a PEG lipid is a compound of the formula: [ka] .

[0491] C. Helper lipids In some embodiments, a transfer vehicle (e.g., LNP) described herein comprises one or more non-cationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a substitute or replacement for a phospholipid. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0492] The phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0493] The fatty acid moiety can be selected from the non-limiti...

Claims

1. An ionizable lipid represented by formula (13*): 【Chemistry 1】 During the ceremony: n ＊ is an integer from 1 to 7; R ａ is hydrogen; R ｂ is hydrogen or C １ -C ６ is alkyl; R １ and R ２ are each independently -(CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ), -(CH2)qOC(O)(CH2)rCH(R8)(R9), and -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), is selected from: q is an integer from 0 to 12; r is an integer from 0 to 6; R 8 is H or R 10 , where R 8 is R 10 for R 1 ; R ９ and R １０ are independently unsubstituted linear C １ -C １２ Alkyl or unsubstituted linear C ２ -C １２ -alkenyl, and optionally R 9 and R 10 are each independently unsubstituted straight chain C 4 -C 8 alkyl, preferably R 9 and R 10 are each independently unsubstituted straight chain C 6 -C 8 alkyl, or a pharmaceutically acceptable salt thereof.

2. R ｂ is C １ -C ６ alkyl, or R b is H and the ionizable lipid is represented by formula (13): 【Chemistry 2】 wherein n is an integer from 1 to 7, optionally n is 1, 2, 3, or 4, and preferably the ionizable lipid is represented by formula (13a-1), formula (13a-2), or formula (13a-3). The ionizable lipid of claim 1. 【Transformation 3】

3. 3. The ionizable lipid of claim 1, wherein q is an integer from 1 to 6, preferably q is 3, 4, 5, or 6, and / or r is 0 or an integer from 1 to 6, preferably r is 1 or 2.

4. R １ and R ２ are each independently selected from the group consisting of: 【Chemistry 4】

5. 2. The ionizable lipid of claim 1, wherein the ionizable lipid is selected from the group consisting of: 【Chemistry 5-1】 【Chemistry 5-2】

6. A pharmaceutical composition comprising a transfer vehicle, said transfer vehicle comprising an ionizable lipid according to any one of claims 1 to 5.

7. and / or 7. The pharmaceutical composition of claim 6, wherein the transfer vehicle comprises nanoparticles, such as lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymeric nanoparticles, or biodegradable polymeric nanoparticles.

8. 8. The pharmaceutical composition of claim 6 or 7, wherein the transfer vehicle further comprises a structured lipid and a PEG-modified lipid, and optionally the structured lipid binds to C1q and / or promotes binding of the transfer vehicle comprising the lipid to C1q compared to a control transfer vehicle lacking the structured lipid and / or increases uptake of the C1q-bound transfer vehicle into immune cells compared to a control transfer vehicle lacking the structured lipid, and further wherein the immune cells are preferably T cells, NK cells, NKT cells, macrophages or neutrophils.

9. 9. The pharmaceutical composition of claim 8, wherein the structured lipid is cholesterol, beta-sitosterol, or the structured lipid is not beta-sitosterol.

10. The pharmaceutical composition according to claim 8 or 9, wherein the PEG-modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1, and preferably the PEG-modified lipid is DSPE-PEG(2000).

11. 11. The pharmaceutical composition according to any one of claims 6 to 10, wherein the transfer vehicle further comprises a helper lipid, optionally wherein the helper lipid is DSPC or DOPE, preferably wherein the transfer vehicle comprises DSPC, cholesterol, and DMG-PEG(2000).

12. The transfer vehicle, a helper lipid which is DOPE or DSPC, Cholesterol, and PEG-lipids that are DSPE-PEG(2000) or DMG-PEG(2000) The pharmaceutical composition according to any one of claims 6 to 11, comprising:

13. 13. The pharmaceutical composition of claim 12, wherein the molar ratio of ionizable lipid:helper lipid:cholesterol:PEG-lipid is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1; and / or the transfer vehicle comprises the helper lipid DOPE and the PEG-lipid DMG-PEG(2000), and the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1, and optionally the PEG-lipid is C14-PEG(2000). (a) the transfer vehicle comprises the helper lipid DSPC and the PEG-lipid DMG-PEG(2000), and the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1; (b) the transfer vehicle comprises the helper lipid DSPC and the PEG-lipid DSPE-PEG(2000), wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DSPE-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1; or (c) the transfer vehicle comprises the helper lipid DOPE and the PEG-lipid DMG-PEG(2000), and the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1; The pharmaceutical composition according to any one of claims 6 to 13.

15. A pharmaceutical composition according to any one of claims 6 to 14 for use in a method for treating or preventing a disease, disorder, or symptom.

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