Circular RNA Compositions and Methods

Abstract

The present disclosure relates to circular RNAs and delivery vehicles, as well as related compositions and therapeutic methods. The circular RNAs may comprise Group I intron segments, spacer regions, IRESs, duplex-forming regions, and / or expression sequences, and thus have characteristics such as improved expression, functional stability, low immunogenicity, ease of manufacture, and / or extended half-life compared to linear RNAs. Pharmaceutical compositions comprising such circular RNAs and delivery vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful for generating the precursor or circular RNAs, which have improved cyclization efficiency and / or are compatible with an efficient circular RNA purification method.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 943,779, filed on December 4, 2019; U.S. Provisional Application No. 62 / 972,194, filed on February 10, 2020; U.S. Provisional Application No. 63 / 022,248, filed on May 8, 2020; U.S. Provisional Application No. 63 / 087,582, filed on October 5, 2020; and U.S. Provisional Application No. 62 / 943,797, filed on December 4, 2019, the entire contents of each of which are hereby incorporated by reference in their entirety for all purposes. Background of the Invention

[0003] Conventional gene therapy involves using DNA to insert desired genetic information into host cells. The DNA introduced into the cells typically integrates to some extent into the genome of one or more transfected cells, allowing for long - term action of the introduced genetic material in the host. While such sustained action can have substantial benefits, the integration of exogenous DNA into the host genome can also have many harmful effects. For example, it is possible that the introduced DNA will insert into an intact gene, resulting in a mutation that blocks or even completely eliminates the function of an endogenous gene. Thus, gene therapy using DNA can lead to impairment of important genetic functions of the treated host, such as, for example, elimination or harmful reduction of the production of essential enzymes or interference with genes that are crucial for the regulation of cell growth, leading to unregulated or cancerous cell proliferation. Additionally, with conventional DNA - based gene therapy, in order to effectively express the desired gene product, it is necessary to include strong promoter sequences, which can also lead to undesirable changes in the regulation of normal gene expression in the cell. It is also possible that DNA - based genetic material will lead to the induction of unwanted anti - DNA antibodies, which in turn can trigger a potentially fatal immune response. Gene therapy methods using viral vectors can also lead to adverse immune responses. In some cases, viral vectors can even integrate into the host genome. Additionally, the production of clinical - grade viral vectors is expensive and time - consuming. Targeted delivery of the introduced genetic material using viral vectors can also be difficult to control. Thus, although gene therapy based on DNA using viral vectors to deliver secreted proteins has been evaluated (U.S. Patent No. 6,066,626; US2004 / 0110709), these methods may be limited for these various reasons.

[0004] Compared with DNA, the use of RNA as a gene therapeutic agent is generally safer because RNA does not involve the risk of stable integration into the genome of transfected cells, thereby eliminating concerns that the introduced genetic material will interfere with the normal operation of essential genes or cause mutations that lead to harmful or carcinogenic effects, and foreign promoter sequences are not required for the effective translation of encoded proteins, again avoiding potentially harmful side effects. In addition, mRNA does not need to enter the nucleus to perform its function, while DNA must overcome this major obstacle.

[0005] Circular RNAs can be used to design and generate stable forms of RNA. The circularization of RNA molecules provides advantages for the study of RNA structure and function, especially in cases where the molecule is prone to folding into an inactive conformation (Wang and Ruffner, 1998). Circular RNAs can also be particularly interesting and useful for in vivo applications, especially in the fields of RNA-based gene expression control and therapeutics, including protein replacement therapy and vaccination.

[0006] Prior to the present invention, there were three main techniques for the in vitro preparation of circular RNAs: the splint-mediated method, the permuted intron-exon method, and the RNA ligase-mediated method. However, the existing methods are limited by the size of the RNA that can be circularized, thus limiting their therapeutic applications. SUMMARY OF THE INVENTION

[0007] The present application provides circular RNAs and delivery vehicles, as well as related compositions and therapeutic methods. The delivery vehicle may comprise, for example, ionizable lipids, PEG-modified lipids, and / or structural lipids to form lipid nanoparticles encapsulating the circular RNA. The circular RNA may comprise Group I intron fragments, spacer regions, IRESs, duplex-forming regions, and / or expression sequences, thereby having characteristics of improved expression, functional stability, low immunogenicity, ease of manufacture, and / or an extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and delivery vehicles are particularly suitable for efficient protein expression in in vivo immune cells. The present application also provides precursor RNAs and materials useful for generating the precursor or circular RNA, the precursor or circular RNA having improved circularization efficiency and / or being compatible with an efficient circular RNA purification method.

[0008] Accordingly, one aspect of the present application provides a pharmaceutical composition comprising: a circular RNA polynucleotide and a delivery vehicle comprising an ionizable lipid represented by formula (1):

[0009]

[0010] Wherein:

[0011] Each n is independently an integer from 2 to 15;

[0012] L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the point of attachment to R1 or R3;

[0013] R1 and R3 are each independently a straight-chain or branched-chain C9-C 20 alkyl or C9-C 20 alkenyl optionally substituted with one or more substituents selected from the group consisting of: oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclic alkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclic)(alkyl)aminoalkyl, heterocyclic, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclic carbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl and alkylsulfonylalkyl; and

[0014] R2 is selected from the group consisting of:

[0015]

[0016] In some embodiments, R1 and R3 are each independently selected from the group consisting of: In some embodiments, R1 and R3 are the same. In some embodiments, R1 and R3 are different.

[0017] In some embodiments, the ionizable lipid of formula (1) is represented by formula (1-1) or formula (1-2):

[0018]

[0019] In some embodiments, the ionizable lipid is selected from the group consisting of:

[0020]

[0021]

[0022] In another aspect, the present application provides a pharmaceutical composition comprising: a circular RNA polynucleotide and a transfer mediator comprising an ionizable lipid represented by formula (2):

[0023]

[0024] Wherein:

[0025] Each n is independently an integer from 1 to 15;

[0026] R1 and R2 are each independently selected from the group consisting of:

[0027]

[0028] And

[0029] R3 is selected from the group consisting of:

[0030]

[0031] In another aspect, the present application provides a pharmaceutical composition comprising: a circular RNA polynucleotide and a transfer mediator comprising an ionizable lipid represented by formula (3):

[0032]

[0033] Wherein:

[0034] X is selected from -O-, -S- or -OC(O)-*, where * indicates the point of attachment to R1;

[0035] R1 is selected from the group consisting of:

[0036]

[0037] And

[0038] R2 is selected from the group consisting of:

[0039]

[0040] In some embodiments, the ionizable lipid of formula (3) is represented by formula (3-1), formula (3-2) or formula (3-3):

[0041]

[0042]

[0043] In another aspect, the present application provides a pharmaceutical composition comprising: a circular RNA polynucleotide and a transfer mediator comprising an ionizable lipid represented by formula (4):

[0044]

[0045] Wherein: each n is independently an integer from 2 to 15; and R2 is defined in formula (1).

[0046] In another aspect, the present application provides a pharmaceutical composition comprising: a circular RNA polynucleotide and a transfer mediator comprising an ionizable lipid selected from Table 10a.

[0047] In some embodiments, the circular RNA comprises a first expression sequence. In some embodiments, the first expression sequence encodes a therapeutic protein. In some embodiments, the first expression sequence encodes a cytokine or a functional fragment thereof. In some embodiments, the first expression sequence encodes a transcription factor. In some embodiments, the first expression sequence encodes an immune checkpoint inhibitor. In some embodiments, the first expression sequence encodes a chimeric antigen receptor (CAR).

[0048] In some embodiments, the circular RNA polynucleotide further comprises a second expression sequence. In some embodiments, the circular RNA polynucleotide further comprises an internal ribosome entry site (IRES).

[0049] In some embodiments, the first and second expression sequences are separated by a ribosome skipping element or a nucleotide sequence encoding a protease cleavage site. In some embodiments, the first expression sequence encodes a first T cell receptor (TCR) chain, and the second expression sequence encodes a second TCR chain.

[0050] In some embodiments, the circular RNA polynucleotide comprises one or more microRNA binding sites. The microRNA binding sites are recognized by microRNAs expressed in the liver. In some embodiments, the microRNA binding sites are recognized by miR-122.

[0051] In some embodiments, the circular RNA polynucleotide comprises a first IRES associated with higher protein expression in human immune cells compared to reference human cells. In some embodiments, the human immune cells are T cells, NK cells, NKT cells, macrophages, or neutrophils. In some embodiments, the reference human cells are hepatocytes.

[0052] In some embodiments, the circular RNA polynucleotide comprises, in the following order: a) a spliced intron fragment of a 3' group I intron fragment, b) an IRES, c) an expression sequence, and d) a spliced intron fragment of a 5' group I intron fragment. In some embodiments, the circular RNA polynucleotide comprises. In some embodiments, the circular RNA polynucleotide comprises a first spacer region before the spliced intron fragment of the 3' group I intron fragment and a second spacer region after the spliced intron fragment of the 5' group I intron fragment. In some embodiments, each of the first and second spacer regions has a length of about 10 to about 60 nucleotides.

[0053] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a 3' group I intron fragment, an IRES, an expression sequence, and a 5' group I intron fragment.

[0054] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a 5' external duplex-forming region, a 3' group I intron fragment, a 5' internal spacer region optionally comprising a 5' internal duplex-forming region, an IRES, an expression sequence, a 3' internal spacer region optionally comprising a 3' internal duplex-forming region, a 5' group I intron fragment, and a 3' external duplex-forming region.

[0055] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a 5' external duplex-forming region, a 5' external spacer region, a 3' group I intron fragment, a 5' internal spacer region optionally comprising a 5' internal duplex-forming region, an IRES, an expression sequence, a 3' internal spacer region optionally comprising a 3' internal duplex-forming region, a 5' group I intron fragment, a 3' external spacer region, and a 3' external duplex-forming region.

[0056] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a 3' group I intron fragment, a 5' internal spacer region comprising a 5' internal duplex-forming region, an IRES, an expression sequence, a 3' internal spacer region comprising a 3' internal duplex-forming region, and a 5' group I intron fragment.

[0057] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a 5' outer duplex-forming region, a 5' outer spacer region, a 3' group I intron fragment, a 5' inner spacer region comprising a 5' inner duplex-forming region, an IRES, an expression sequence, a 3' inner spacer region comprising a 3' inner duplex-forming region, a 5' group I intron fragment, a 3' outer spacer region, and a 3' outer duplex-forming region.

[0058] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a first polyA sequence, a 5' outer duplex-forming region, a 5' outer spacer region, a 3' group I intron fragment, a 5' inner spacer region comprising a 5' inner duplex-forming region, an IRES, an expression sequence, a 3' inner spacer region comprising a 3' inner duplex-forming region, a 5' group I intron fragment, a 3' outer spacer region, a 3' outer duplex-forming region, and a second polyA sequence.

[0059] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a first polyA sequence, a 5' outer spacer region, a 3' group I intron fragment, a 5' inner spacer region comprising a 5' inner duplex-forming region, an IRES, an expression sequence, a 3' inner spacer region comprising a 3' inner duplex-forming region, a 5' group I intron fragment, a 3' outer spacer region, and a second polyA sequence.

[0060] In some embodiments, the circular RNA polynucleotide is prepared by circularization of an RNA polynucleotide that comprises, in the following order: a first polyA sequence, a 5' outer spacer region, a 3' group I intron fragment, a 5' inner spacer region comprising a 5' inner duplex-forming region, an IRES, an expression sequence, a stop codon, a 3' inner spacer region comprising a 3' inner duplex-forming region, a 5' group I intron fragment, a 3' outer spacer region, and a second polyA sequence.

[0061] In some embodiments, at least one of the 3' or 5' inner or outer spacer regions has a length of about 8 to about 60 nucleotides. In some embodiments, each of the 3' and 5' outer duplex-forming regions has a length of about 10 - 50 nucleotides. In some embodiments, each of the 3' and 5' inner duplex-forming regions has a length of about 6 - 30 nucleotides.

[0062] In some embodiments, the IRES is selected from Table 17, or a functional fragment or variant thereof. In some embodiments, the IRES has the sequence of an IRES from the following: 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, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picornavirus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human coxsackievirus B3, Tobacco mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 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, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.Aptamers against 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 naked, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, Tobacco etch virus, Turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picoirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human parechovirus 5, Aichi Virus, Hepatitis A virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, Porcine Pasivirus1, PLV-CHN, PasivirusA, Sicinivirus, Hepatitis virus K, Hepatitis virus A, BVDV1, Border disease virus, BVDV2, CSFV-PK15C, SF573 dicistronic virus, Hubei picornavirus-like virus, CRPV, Apodemus agrarius picornavirus, Caprine crest virus, parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or eIF4G.

[0063] In some embodiments, each of the first and second polyA sequences has a length of about 15-50 nt. In some embodiments, each of the first and second polyA sequences has a length of about 20-25 nt.

[0064] In some embodiments, the circular RNA polynucleotide comprises at least about 80%, at least about 90%, at least about 95% or at least about 99% naturally occurring nucleotides. In some embodiments, the circular RNA polynucleotide consists of naturally occurring nucleotides.

[0065] In some embodiments, the expression sequence is codon-optimized. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one microRNA binding site capable of binding to a microRNA present in a cell, wherein the circular RNA polynucleotide is expressed in the cell. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site capable of being cleaved by an endonuclease present in a cell, wherein the endonuclease is expressed in the cell. In some embodiments, the circular RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site present in an equivalent pre-optimized polynucleotide.

[0066] In some embodiments, the length of the circular RNA polynucleotide is from about 100 nt to about 10,000 nt. In some embodiments, the length of the circular RNA polynucleotide is from about 100 nt to about 15,000 nt. In some embodiments, the circular RNA is more compact than a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide.

[0067] In some embodiments, the duration of the therapeutic effect of the pharmaceutical composition in human cells is greater than or equal to the duration of the therapeutic effect of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. In some embodiments, the reference linear RNA polynucleotide is a linear, unmodified or nucleoside-modified, fully processed mRNA comprising a cap1 structure and a polyA tail having a length of at least 80 nt.

[0068] In some embodiments, the in vivo therapeutic effect duration of the pharmaceutical composition in humans is greater than that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. In some embodiments, the pharmaceutical composition has an in vivo therapeutic effect duration of at least about 10, at least about 20, at least about 30, at least about 40, 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 hours in humans.

[0069] In some embodiments, the functional half-life of the pharmaceutical composition in human cells is greater than or equal to the functional half-life of a predetermined threshold. In some embodiments, the in vivo functional half-life of the pharmaceutical composition in humans is greater than the functional half-life of a predetermined threshold. In some embodiments, the functional half-life is determined by a functional protein assay. In some embodiments, the functional protein assay is an in vitro luciferase assay. In some embodiments, the functional protein assay comprises measuring the level of a protein encoded by the expression sequence of the circular RNA polynucleotide in a patient serum or tissue sample. In some embodiments, the predetermined threshold is the functional half-life of a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. In some embodiments, the pharmaceutical composition has a functional half-life of at least about 20 hours.

[0070] In some embodiments, the pharmaceutical composition comprises a structured lipid and a PEGylated lipid. In some embodiments, the structured lipid binds to C1q and / or promotes the binding of the lipid-containing transfer vehicle to C1q compared to a control transfer vehicle lacking the structured lipid, and / or increases the uptake of the C1q-binding transfer vehicle into immune cells compared to a control transfer vehicle lacking the structured lipid. In some embodiments, the immune cells are T cells, NK cells, NKT cells, macrophages, or neutrophils.

[0071] In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is β-sitosterol. In some embodiments, the structured lipid is not β-sitosterol.

[0072] In some embodiments, the PEGylated lipid is DSPE-PEG, DMG-PEG, or PEG-1. In some embodiments, the PEGylated lipid is DSPE-PEG(2000).

[0073] In some embodiments, the pharmaceutical composition further comprises a helper lipid. In some embodiments, the helper lipid is DSPC or DOPE.

[0074] In some embodiments, the pharmaceutical composition comprises DOPE, cholesterol, and DSPE-PEG.

[0075] In some embodiments, the transfer vehicle comprises from about 0.5% to about 4% PEG-modified lipid on a molar ratio basis. In some embodiments, the transfer vehicle comprises from about 1% to about 2% PEG-modified lipid on a molar ratio basis.

[0076] In some embodiments, the transfer vehicle comprises

[0077] a. An ionizable lipid represented by

[0078]

[0079] b. DOPE,

[0080] c. Cholesterol, and

[0081] d. DSPE-PEG(2000).

[0082] In some embodiments, the molar ratio of ionizable lipid:DSPC:cholesterol:DSPE-PEG(2000) is 62:4:33:1.

[0083] In some embodiments, the transfer vehicle comprises

[0084] a. An ionizable lipid represented by

[0085]

[0086]

[0087]

[0088] b. DOPE,

[0089] c. Cholesterol, and

[0090] d. DSPE-PEG(2000).

[0091] In some embodiments, the molar ratio of ionizable lipid:DSPC:cholesterol:DSPE-PEG(2000) is 50:10:38.5:1.5.

[0092] In some embodiments, the transfer vehicle has a nitrogen:phosphate (N:P) ratio of about 3 to about 6.

[0093] ​​​In some embodiments, the transfer vehicle is capable of binding to APOE. In some embodiments, the transfer vehicle interacts with apolipoprotein E (APOE) less than an equivalent transfer vehicle loaded with a reference linear RNA having the same expression sequence as the circular RNA polynucleotide. In some embodiments, the outer surface of the transfer vehicle is substantially free of APOE binding sites.

[0094] In some embodiments, the transfer vehicle has a diameter of less than about 120 nm. In some embodiments, the transfer vehicle does not form aggregates with a diameter greater than 300 nm.

[0095] In some embodiments, the transfer vehicle has an in vivo half-life of less than about 30 hours.

[0096] In some embodiments, the transfer vehicle is capable of being taken up into cells via the low-density lipoprotein receptor (LDLR). In some embodiments, the transfer vehicle is capable of being taken up into cells independently of the LDLR.

[0097] In some embodiments, the pharmaceutical composition is substantially free of linear RNA.

[0098] In some embodiments, the pharmaceutical composition further comprises a targeting moiety operably linked to the transfer vehicle. In some embodiments, the targeting moiety specifically or indirectly binds to an immune cell antigen. In some embodiments, the immune cell antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of CD2, CD3, CD5, CD7, CD8, CD4, β7 integrin, β2 integrin, and C1q.

[0099] In some embodiments, the pharmaceutical composition further comprises an adapter molecule comprising a transfer vehicle binding moiety and a cell binding moiety, wherein the targeting moiety specifically binds to the transfer vehicle binding moiety and the cell binding moiety specifically binds to a target cell antigen. In some embodiments, the target cell antigen is an immune cell antigen. In some embodiments, the immune cell antigen is a T cell antigen, NK cell, NKT cell, macrophage, or neutrophil. In some embodiments, the T cell antigen is selected from the group consisting of CD2, CD3, CD5, CD7, CD8, CD4, β7 integrin, β2 integrin, CD25, CD39, CD73, A2a receptor, A2b receptor, and C1q. In some embodiments, the immune cell antigen is a macrophage antigen. In some embodiments, the macrophage antigen is selected from the group consisting of mannose receptor, CD206, and C1q.

[0100] In some embodiments, the targeting moiety is a small molecule. In some embodiments, the small molecule binds to an extracellular enzyme on an immune cell, wherein the extracellular enzyme is selected from the group consisting of: CD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. In some embodiments, the small molecule is mannose, lectin, asivicin, biotin, or digoxin.

[0101] In some embodiments, the targeting moiety is a single-chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, small molecule ligand such as folic acid, arginylglycylaspartic acid (RGD), or phenol-soluble modulin α1 peptide (PSMA1), heavy chain variable region, light chain variable region, or a fragment thereof.

[0102] In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than about 2 weeks. In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than about 1 week. In some embodiments, the ionizable lipid has a half-life in the cell membrane of less than about 30 hours. In some embodiments, the half-life of the ionizable lipid in the cell membrane is less than the functional half-life of the circular RNA polynucleotide.

[0103] In another aspect, the present application provides a method of treating or preventing a disease, disorder, or condition, the method comprising administering an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the disease, disorder, or condition is associated with abnormal expression, activity, or localization of a polypeptide selected from Table 27 or 28. In some embodiments, the circular RNA polynucleotide encodes a therapeutic protein. In some embodiments, the expression of the therapeutic protein in the spleen is higher than the expression of the therapeutic protein in the liver. In some embodiments, the expression of the therapeutic protein in the spleen is at least about 2.9-fold that of the expression of the therapeutic protein in the liver. In some embodiments, the therapeutic protein is not expressed at a functional level in the liver. In some embodiments, the therapeutic protein is not expressed at a detectable level in the liver. In some embodiments, the expression of the therapeutic protein in the spleen is at least about 63% of the total therapeutic protein expression.

[0104] In another aspect, the present application provides a linear RNA polynucleotide that, from 5' to 3', comprises a 3'I group intron fragment, an internal ribosome entry site (IRES), an expression sequence, and a 5'I group intron fragment, and further comprises a first spacer region 5' of the 3'I group intron fragment and / or a second spacer region 3' of the 5'I group intron fragment.

[0105] In some embodiments, the linear RNA polynucleotide comprises a first spacer region 5' of the 3' group I intron fragment. In some embodiments, the first spacer region has a length of 10 - 50 nucleotides, optionally 10 - 20 nucleotides, and further optionally about 15 nucleotides. In some embodiments, the first spacer region comprises a polyA sequence.

[0106] In some embodiments, the linear RNA polynucleotide comprises a second spacer region 3' of the 5' group I intron fragment. In some embodiments, the second spacer region has a length of 10 - 50 nucleotides, optionally 10 - 20 nucleotides, and further optionally about 15 nucleotides. In some embodiments, the second spacer region comprises a polyA sequence.

[0107] In some embodiments, the linear RNA polynucleotide further comprises a third spacer region between the 3' group I intron fragment and the IRES. In some embodiments, the third spacer region has a length of about 10 to about 60 nucleotides. In some embodiments, the linear RNA polynucleotide further comprises first and second duplex-forming regions capable of forming a duplex. In some embodiments, each of the first and second duplex-forming regions has a length of about 9 to 19 nucleotides. In some embodiments, each of the first and second duplex-forming regions has a length of about 30 nucleotides.

[0108] In some embodiments, compared to a reference linear RNA polynucleotide, the linear RNA polynucleotide has enhanced expression, cyclization efficiency, functional stability, and / or stability, wherein the reference linear RNA polynucleotide comprises, from 5' to 3', a first polyA sequence, a 5' external spacer region, a 3' group I intron fragment, a 5' internal spacer region comprising a 5' internal duplex-forming region, an IRES, an expression sequence, a stop codon, a 3' internal spacer region comprising a 3' internal duplex-forming region, a 5' group I intron fragment, a 3' external spacer region, and a second polyA sequence.

[0109] In some embodiments, compared to a reference linear RNA polynucleotide, the linear RNA polynucleotide has enhanced expression, cyclization efficiency, functional stability, and / or stability, wherein the reference linear RNA polynucleotide comprises, from 5' to 3', a reference 3' group I intron fragment, a reference IRES, a reference expression sequence, and a reference 5' group I intron fragment, and does not comprise a spacer region 5' of the 3' group I intron fragment or a spacer region 3' of the 5' group I intron fragment. In some embodiments, the expression sequence and the reference expression sequence have the same sequence. In some embodiments, the IRES and the reference IRES have the same sequence.

[0110] In some embodiments, the linear RNA polynucleotide comprises a 3' Anabaena group I intron fragment and a 5' Anabaena group I intron fragment. In some embodiments, the reference RNA polynucleotide comprises a reference 3' Anabaena group I intron fragment and a reference 5' Anabaena group I intron fragment. In some embodiments, the reference 3' Anabaena group I intron fragment and the reference 5' Anabaena group I intron fragment are generated using the L6-5 replacement site. In some embodiments, the 3' Anabaena group I intron fragment and the 5' Anabaena group I intron fragment are not generated using the L6-5 replacement site. In some embodiments, the 3' Anabaena group I intron fragment comprises a sequence selected from SEQ ID NOs: 112-123 and 125-150 or consists of said sequences. In some embodiments, the 5' Anabaena group I intron fragment comprises a corresponding sequence selected from SEQ ID NOs: 73-84 and 86-111. In some embodiments, the 5' Anabaena group I intron fragment comprises a sequence selected from SEQ ID NOs: 73-84 and 86-111 or consists of said sequences. In some embodiments, the 3' Anabaena group I intron fragment comprises a corresponding sequence selected from SEQ ID NOs: 112-124 and 125-150 or consists of said sequences.

[0111] In some embodiments, the IRES comprises a nucleotide sequence selected from SEQ ID NOs: 348-351. In some embodiments, the reference IRES is CVB3. In some embodiments, the IRES is not CVB3. In some embodiments, the IRES comprises a sequence selected from SEQ ID NOs: 1-64 and 66-72.

[0112] In another aspect, the present application discloses a circular RNA polynucleotide produced from the linear RNA disclosed herein.

[0113] In another aspect, the present application discloses a circular RNA which, from 5' to 3', comprises a 3' group I intron fragment, an IRES, an expression sequence, and a 5' group I intron fragment, wherein the IRES comprises a nucleotide sequence selected from SEQ ID NOs: 348-351.

[0114] In some embodiments, the circular RNA polynucleotide further comprises a spacer region between the 3' group I intron fragment and the IRES.

[0115] In some embodiments, the circular RNA polynucleotide further comprises first and second duplex-forming regions capable of forming a duplex. In some embodiments, each of the first and second duplex-forming regions has a length of about 9 to 19 nucleotides. In some embodiments, each of the first and second duplex-forming regions has a length of about 30 nucleotides.

[0116] In some embodiments, the expression sequence has a size of at least about 1,000 nt, at least about 2,000 nt, at least about 3,000 nt, at least about 4,000 nt, or at least about 5,000 nt.

[0117] In some embodiments, the RNA polynucleotide comprises natural nucleotides. In some embodiments, the expression sequence is codon-optimized. In some embodiments, the RNA polynucleotide further comprises a translation termination cassette that includes at least one stop codon in each reading frame. In some embodiments, the translation termination cassette includes at least two stop codons in the reading frame of the expression sequence. In some embodiments, the RNA polynucleotide is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. In some embodiments, the RNA polynucleotide is optimized to lack at least one endonuclease-sensitive site present in an equivalent pre-optimized polynucleotide. In some embodiments, the RNA polynucleotide is optimized to lack at least one RNA editing-sensitive site present in an equivalent pre-optimized polynucleotide.

[0118] In some embodiments, the RNA polynucleotide comprises at least 2 expression sequences. In some embodiments, each expression sequence encodes a different therapeutic protein.

[0119] In some embodiments, the length of the circular RNA polynucleotide disclosed herein is about 100 to 15,000 nucleotides, optionally about 100 to 12,000 nucleotides, and further optionally about 100 to 10,000 nucleotides.

[0120] In some embodiments, the circular RNA polynucleotides disclosed herein have an in vivo therapeutic effect duration of at least about 20 hours in humans. In some embodiments, the circular RNA polynucleotides disclosed herein have a functional half-life of at least about 20 hours. In some embodiments, the in vivo therapeutic effect duration of the circular RNA polynucleotide in human cells is greater than or equal to the in vivo therapeutic effect duration of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the functional half-life of the circular RNA polynucleotide in human cells is greater than or equal to the functional half-life of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the in vivo therapeutic effect duration of the circular RNA polynucleotide in humans is greater than the in vivo therapeutic effect duration of an equivalent linear RNA polynucleotide having the same expression sequence. In some embodiments, the in vivo functional half-life of the circular RNA polynucleotide in humans is greater than the in vivo functional half-life of an equivalent linear RNA polynucleotide having the same expression sequence.

[0121] In another aspect, the present disclosure provides a composition comprising a circular RNA polynucleotide disclosed herein, a nanoparticle, and optionally a targeting moiety operably linked to the nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymeric nanoparticle, or a biodegradable polymeric nanoparticle. In some embodiments, the pharmaceutical composition comprises a targeting moiety, wherein in the absence of cell isolation or purification, the targeting moiety mediates receptor-mediated endocytosis or directly and selectively fuses into the cells of a selected cell population or tissue. In some embodiments, the targeting moiety is an scFv, a nanobody, a peptide, a miniantibody, a polynucleotide aptamer, a heavy chain variable region, a light chain variable region, or a fragment thereof. In some embodiments, less than 1% by weight of the polynucleotide in the composition is double-stranded RNA, a DNA splint, or triphosphorylated RNA. In some embodiments, less than 1% by weight of the polynucleotide and protein in the pharmaceutical composition is double-stranded RNA, a DNA splint, triphosphorylated RNA, a phosphatase protein, a protein ligase, and a capping enzyme.

[0122] In another aspect, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide disclosed herein, a nanoparticle, and optionally a targeting moiety operably linked to the nanoparticle.

[0123] In another aspect, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the targeting moiety is an scfv, a nanobody, a peptide, a minibody, a heavy chain variable region, a light chain variable region, an extracellular domain of a TCR, or a fragment thereof. In some embodiments, the nanoparticle is a lipid nanoparticle, a core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the nanoparticle comprises one or more cationic lipids, ionizable lipids, or poly-β-amino esters. In some embodiments, the nanoparticle comprises one or more non-cationic lipids. In some embodiments, the nanoparticle comprises one or more PEGylated lipids, polyglutamic acid lipids, or hyaluronic acid lipids. In some embodiments, the nanoparticle comprises cholesterol. In some embodiments, the nanoparticle comprises arachidonic acid or oleic acid.

[0124] In some embodiments, the provided pharmaceutical composition comprises a targeting moiety, wherein in the absence of cell selection or purification, the targeting moiety selectively mediates receptor-mediated endocytosis into cells of a selected cell population.

[0125] In some embodiments, the provided nanoparticle comprises more than one circular RNA polynucleotide.

[0126] In another aspect, the present application provides a DNA vector encoding an RNA polynucleotide disclosed herein. In some embodiments, the DNA vector further comprises transcriptional regulatory sequences. In some embodiments, the transcriptional regulatory sequences comprise a promoter and / or an enhancer. In some embodiments, the promoter comprises a T7 promoter. In some embodiments, the DNA vector comprises circular DNA. In some embodiments, the DNA vector comprises linear DNA.

[0127] In another aspect, the present application provides a prokaryotic cell comprising a DNA vector disclosed herein.

[0128] In another aspect, the present application provides a eukaryotic cell comprising a circular RNA polynucleotide disclosed herein. In some embodiments, the eukaryotic cell is a human cell.

[0129] In another aspect, the present application provides a method for generating circular RNA polynucleotides, the method comprising incubating the linear RNA polynucleotides disclosed herein under suitable cyclization conditions. In some embodiments, the method comprises incubating the DNA disclosed herein under suitable transcription conditions. In some embodiments, the DNA is transcribed in vitro. In some embodiments, the suitable conditions include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and RNA polymerase. In some embodiments, the suitable conditions further include guanosine monophosphate (GMP). In some embodiments, the ratio of the GMP concentration to the GTP concentration ranges from about 3:1 to about 15:1, optionally about 4:1, 5:1, or 6:1.

[0130] In another aspect, the present application provides a method for generating circular RNA polynucleotides, the method comprising culturing the prokaryotic cells disclosed herein under suitable conditions for transcribing the DNA in a transcription cell. In some embodiments, the method further comprises purifying the circular RNA polynucleotides. In some embodiments, the circular RNA polynucleotides are purified by negative selection using an affinity oligonucleotide conjugated to a solid surface that hybridizes to the first or second spacer region. In some embodiments, the first or second spacer region comprises a poly-A sequence, and wherein the affinity oligonucleotide is a deoxythymidine oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 depicts luminescence in the supernatant of HEK293 cells ( Figure 1A , 1D and 1E), HepG2 cells ( Figure 1B ), or 1C1C7 ( Figure 1C ) cells 24 hours after transfection with circular RNAs comprising a Gaussia luciferase expression sequence and various IRES sequences.

[0132] Figure 2 depicts luminescence in the supernatant of HEK293 cells ( Figure 2A ), HepG2 cells ( Figure 2B ), or 1C1C7 ( Figure 2C ) cells 24 hours after transfection with circular RNAs comprising a Gaussia luciferase expression sequence and various IRES sequences of different lengths.

[0133] Figure 3 depicts the stability of selected IRES constructs in HepG2 ( Figure 3A ) or 1C1C7 ( Figure 3B ) cells over 3 days as measured by luminescence.

[0134] Figure 4A and 4BDepict protein expression from selected IRES constructs in Jurkat cells as measured by the luminescence of secreted Gaussia luciferase in the supernatant from the cells.

[0135] Figure 5A and 5B Depict the stability of selected IRES constructs in Jurkat cells over 3 days as measured by luminescence.

[0136] Figure 6 depicts a comparison of the 24-hour luminescence ( Figure 6A ) or relative luminescence over 3 days ( Figure 6B ) of modified linear, unpurified circular, or purified circular RNAs encoding Gaussia luciferase.

[0137] Figure 7 depicts the induction of transcripts 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, unpurified circular, or purified circular RNAs.

[0138] Figure 8 depicts a comparison of the luminescence of circular RNAs and modified linear RNAs encoding Gaussia luciferase in human primary monocytes ( Figure 8A ) and macrophages ( Figure 8B and Figure 8C ).

[0139] Figure 9 depicts the relative luminescence over 3 days ( Figure 9A ) or 24-hour luminescence ( Figure 9B ) in the supernatant of primary T cells after transduction with circular RNAs containing Gaussia luciferase expression sequences and different IRES sequences.

[0140] Figure 10 depicts the 24-hour luminescence ( Figure 10A ), or relative luminescence over 3 days ( Figure 10B ) in the supernatant of primary T cells and the 24-hour luminescence ( Figure 10C ) in PBMCs after transduction with circular RNAs or modified linear RNAs containing Gaussia luciferase expression sequences.

[0141] Figure 11 depicts the HPLC chromatograms ( Figure 11A ) and cyclization efficiencies ( Figure 11B ) of RNA constructs with different replacement sites.

[0142] Figure 12 depicts the HPLC chromatograms of RNA constructs with different introns and / or replacement sitesFigure 12A ) and cyclization efficiency ( Figure 12B ).

[0143] Figure 13 depicts the HPLC chromatograms of three RNA constructs with or without homology arms ( Figure 13A ) and cyclization efficiency ( Figure 13B ).

[0144] Figure 14 Depicts the cyclization efficiency of three RNA constructs without homology arms or with homology arms of different lengths and GC contents.

[0145] Figure 15A and 15B Depicts the HPLC chromatograms, which show the contribution of strong homology arms to improving splicing efficiency, the relationship between cyclization efficiency and nicking in selected constructs, and combinations of substitution sites and homology arms that hypothetically exhibit improved cyclization efficiency.

[0146] Figure 16 Shows fluorescence images of T cells electroporated with mock electroporation (left) or circular RNA encoding CAR (right) and co-cultured with Raji cells expressing GFP and firefly luciferase.

[0147] Figure 17 Shows bright field (left), fluorescence (middle), and overlay (right) images of T cells electroporated with mock electroporation (top) or circular RNA encoding CAR (bottom) and co-cultured with Raji cells expressing GFP and firefly luciferase.

[0148] Figure 18 Depicts the specific lysis of Raji target cells by T cells electroporated with mock electroporation or circular RNA encoding different CAR sequences.

[0149] Figure 19 depicts 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 RNA containing the Gaussian luciferase expression sequence and different IRES sequences ( Figure 19A ), and the relative luminescence over 3 days ( Figure 19B ).

[0150] Figure 20 depicts the induction of transcripts of IFN-β1 ( Figure 20A ), RIG-I ( Figure 20B ), IL-2 ( Figure 20C ), IL-6 ( Figure 20D ), IFNγ ( Figure 20E ), and TNFα ( Figure 20F ) in human CD3+ T cells electroporated with modified linear, unpurified circular, or purified circular RNA.

[0151] Figure 21 depicts the specific lysis of Raji target cells by human primary CD3+ T cells electroporated with circRNA encoding CAR as determined by firefly luminescence detection ( Figure 21A ), and IFNγ transcript induction 24 hours after electroporation with circular or linear RNA encoding the CAR sequence at different amounts ( Figure 21B ).

[0152] Figure 22 depicts the specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding CAR at different E:T ratios as determined by detecting firefly luminescence ( Figure 22A and Figure 22B ).

[0153] Figure 23 Depicts the specific lysis of target cells by human CD3+ T cells electroporated with RNA encoding CAR on days 1, 3, 5, and 7 after electroporation.

[0154] Figure 24 Depicts the specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding CD19 or BCMA-targeted CAR.

[0155] Figure 25 Depicts the total flux of organs harvested from CD-1 mice that were administered circular RNA encoding FLuc formulated with 50% lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0156] Figure 26 Shows an image highlighting the luminescence of organs harvested from CD-1 mice that were administered circular RNA encoding FLuc formulated with 50% lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0157] Figure 27 depicts the molecular characterization of lipids 26 and 27 from Table 10a. Figure 27A Shows the proton nuclear magnetic resonance (NMR) spectrum of lipid 26. Figure 27B Shows the retention time of lipid 26 as measured by liquid chromatography-mass spectrometry (LC-MS). Figure 27C Shows the mass spectrum of lipid 26. Figure 27D Shows the proton NMR spectrum of lipid 27. Figure 27E Shows the retention time of lipid 27 as measured by LC-MS. Figure 27F Shows the mass spectrum of lipid 27.

[0158] Figure 28 depicts the molecular characterization of lipid 22-S14 and its synthetic intermediates.Figure 28A Depict the NMR spectrum of 2-(tetradecylthio)ethan-1-ol. Figure 28B Depict the NMR spectrum of 2-(tetradecylthio)ethyl acrylate. Figure 28C Depict the NMR spectrum of bis(2-(tetradecylthio)ethyl) 3,3'-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)dipropionate (lipid 22-S14).

[0159] Figure 29 Depict the NMR spectrum of bis(2-(tetradecylthio)ethyl) 3,3'-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (lipid 93-S14).

[0160] Figure 30 depicts the molecular characterization of octadec-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 54 from Table 10a). Figure 30A Show the proton NMR spectrum of lipid 54. Figure 30B Show the retention time of lipid 54 measured by LC-MS. Figure 30C Show the mass spectrum of lipid 54.

[0161] Figure 31 depicts the molecular characterization of octadec-9-yl 8-((3-(1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (lipid 53 from Table 10a). Figure 31A Show the proton NMR spectrum of lipid 53. Figure 31B Show the retention time of lipid 53 measured by LC-MS. Figure 31C Show the mass spectrum of lipid 53.

[0162] Figure 32A Depict the total flux of spleen and liver harvested from CD-1 mice administered circular RNA encoding firefly luciferase (FLuc) formulated with target ionizable lipid, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 32B Depict the mean radiance of the biodistribution of protein expression.

[0163] Figure 33AImages depicting luminescence highlighting organs harvested from CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 33B Whole body IVIS images of CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0164] Figure 34A Images depicting luminescence highlighting organs harvested from CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 34B Whole body IVIS images of CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0165] Figure 35A Images depicting luminescence highlighting organs harvested from CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 26 from Table 10a, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 35B Whole body IVIS images of CD-1 mice that were administered circular RNAs encoding FLuc formulated with ionizable lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0166] Figure 36 depicts images of luminescence highlighting organs harvested from c57BL / 6J mice that were administered circular RNAs encoding FLuc and encapsulated in lipid 15 from Table 10b ( Figure 36A) Circular RNAs in lipid nanoparticles formed from lipid 53 ( Figure 36B ) from Table 10a or lipid 54 ( Figure 36C ) from Table 10a. PBS was used as a control ( Figure 36D ).

[0167] Figure 37A And 37B depicts the relative luminescence in the lysates of human PBMCs after incubation with test lipid nanoparticles containing circular RNAs encoding firefly luciferase for 24 hours.

[0168] Figure 38 shows the expression of GFP ( Figure 37A ) and CD19 CAR ( Figure 37B ) in human PBMCs after incubation with test lipid nanoparticles containing circular RNAs encoding GFP or CD19 CAR.

[0169] Figure 39 depicts the expression of anti-mouse CD19 CAR in 1C1C7 cells lipotransfected with circular RNAs containing anti-mouse CD19 CAR expression sequences and different IRES sequences.

[0170] Figure 40 shows the cytotoxicity of anti-mouse CD19 CAR against murine T cells. The CD19 CAR is encoded by circular RNA and expressed from the circular RNA, which is electroporated into murine T cells.

[0171] Figure 41 depicts the B cell counts in the peripheral blood ( Figure 40 A and 40B) or spleen ( Figure 40 C) of C57BL / 6J mice injected with test lipid nanoparticles every other day, the lipid nanoparticles encapsulating circular RNAs encoding anti-mouse CD19 CAR.

[0172] Figure 42A And 42B compares the expression levels of anti-human CD19 CAR expressed from circular RNA with those expressed from linear mRNA.

[0173] Figure 43A And 43B compares the cytotoxic effects of anti-human CD19 CAR expressed from circular RNA with those expressed from linear mRNA.

[0174] Figure 44 depicts the cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMACAR) expressed from a single circular RNA in T cells.

[0175] Figure 45A Representative FACS plots showing the frequency of tdTomato expression in various splenic immune cell subsets after treatment with LNPs formed from lipid 27 or 26 from Table 10a or lipid 15 from Table 10b. Figure 45B Shows the quantification (mean + standard deviation, n = 3) of the proportion of myeloid cells, B cells, and T cells expressing tdTomato, corresponding to the proportion of each cell population successfully transfected with Cre circular RNA. Figure 45C Shows the proportion (mean + standard deviation, n = 3) of additional splenic immune cell populations expressing tdTomato (including NK cells, classical monocytes, non-classical monocytes, neutrophils, and dendritic cells) after treatment with lipids 27 and 26.

[0176] Figure 46A Depicts an exemplary RNA construct design with an in-built polyA sequence in the intron. Figure 46B Shows the chromatogram trace of unpurified circular RNA. Figure 46C Shows the chromatogram trace of affinity-purified circular RNA. Figure 46D Shows the immunogenicity of circular RNAs prepared with different IVT conditions and purification methods. (Commercial = commercial IVT mixture; Custom = custom IVT mixture; Aff = affinity purification; Enz = enzyme purification; GMP:GTP ratio = 8, 12.5, or 13.75).

[0177] Figure 47A Depicts an exemplary RNA construct design with a dedicated binding sequence as an alternative to polyA for hybridization purification. Figure 47B Shows the chromatogram trace of unpurified circular RNA. Figure 46C Shows the chromatogram trace of affinity-purified circular RNA.

[0178] Figure 48A Shows the chromatogram trace of unpurified circular RNA encoding dystrophin. Figure 48B Shows the chromatogram trace of enzyme-purified circular RNA encoding dystrophin.

[0179] Figure 49 compares the expression ( Figure 49A ) and stability ( Figure 49B ) of purified circRNAs with different 5' spacer regions between the 3' intron fragment / 5' internal duplex region and the IRES in Jurkat cells. (AC = only A and C are used in the spacer sequence; UC = only U and C are used in the spacer sequence.)

[0180] Figure 50 Shows the luminescence expression level and expression stability of circular RNAs from primary T cells containing the indicated native or modified IRES elements.

[0181] Figure 51 Shows the luminescence expression level and expression stability of circular RNAs from circular RNAs containing the indicated native or modified IRES elements in HepG2 cells.

[0182] Figure 52 Shows the luminescence expression level and expression stability of circular RNAs from circular RNAs containing the indicated native or modified IRES elements in 1C1C7 cells.

[0183] Figure 53 Shows the luminescence expression level and expression stability of circular RNAs from circular RNAs containing an inserted untranslated region (UTR) or a hybrid IRES element in HepG2 cells. "Scr" represents scrambled and is used as a control.

[0184] Figure 54 Shows the luminescence expression level and expression stability of circular RNAs from circular RNAs containing an IRES and a variable stop codon cassette operably linked to a Gaussian luciferase coding sequence in 1C1C7 cells.

[0185] Figure 55 Shows the luminescence expression level and expression stability of circular RNAs from circular RNAs containing an IRES and a variable untranslated region (UTR) inserted before the start codon of the Gaussian luciferase coding sequence in 1C1C7 cells.

[0186] Figure 56 Shows the expression level of human erythropoietin (hEPO) of circular RNAs containing two miR-122 target sites downstream of the hEPO coding sequence in Huh7 cells. Detailed Description

[0187] The present disclosure provides pharmaceutical compositions and delivery vehicles, such as lipid nanoparticles, comprising circular RNAs. The circular RNAs provided herein can be delivered to and / or targeted to cells in a delivery vehicle (e.g., nanoparticle) or a composition comprising the delivery vehicle. In some embodiments, the circular RNAs can also be delivered to a subject in the form of a delivery vehicle or a composition comprising the delivery vehicle. In some embodiments, the delivery vehicle is a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a polymer core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the delivery vehicle comprises one or more ionizable lipids, PEGylated lipids, helper lipids, and / or structural lipids.

[0188] In some embodiments, the transfer vehicle encapsulates circular RNA and comprises an ionizable lipid, a structural lipid, and a PEGylated lipid. In some embodiments, the transfer vehicle encapsulates circular RNA and comprises an ionizable lipid, a structural lipid, a PEGylated lipid, and a helper lipid.

[0189] In some embodiments, the transfer vehicle comprises an ionizable lipid as described herein. In some embodiments, the transfer vehicle comprises an ionizable lipid shown in any of Tables 1-10, 10a, 10b, 11-15, and 15b. In some embodiments, the transfer vehicle comprises an ionizable lipid shown in Table 10a.

[0190] In some embodiments, the RNA in the transfer vehicle is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more circular RNA. In some embodiments, less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of the loaded RNA is on or associated with the outer surface of the transfer vehicle.

[0191] In some embodiments, the transfer vehicle is capable of binding to APOE. In some embodiments, the surface of the transfer vehicle comprises an APOE binding site. In some embodiments, the surface of the transfer vehicle is substantially free of APOE binding sites. In some embodiments, the interaction of the transfer vehicle with APOE is less than that of an equivalent transfer vehicle loaded with linear RNA. In some embodiments, the APOE interaction can be measured by comparing nanoparticle uptake in cells in APO-depleted serum or APO-complemented serum.

[0192] Without wishing to be bound by theory, it is expected that transfer vehicles containing APOE binding sites will deliver circular RNA to the liver more efficiently. Thus, in some embodiments, a transfer vehicle comprising an ionizable lipid as described herein and loaded with circular RNA substantially comprises an APOE binding site on the surface of the transfer vehicle, thereby delivering the circular RNA to the liver at a higher efficiency compared to a transfer vehicle that is substantially lacking an APOE binding site on the surface. In some embodiments, a transfer vehicle comprising an ionizable lipid as described herein and loaded with circular RNA is substantially lacking an APOE binding site on the surface of the transfer vehicle, thereby delivering the circular RNA to the liver at a lower efficiency compared to a transfer vehicle that comprises an APOE binding site on the surface.

[0193] In some embodiments, the transfer vehicle will or is capable of delivering the circular RNA to the spleen. In some embodiments, the circular RNA encodes a therapeutic protein. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the total therapeutic protein expressed in a subject is expressed in the spleen. In some embodiments, more therapeutic protein is expressed in the spleen compared to the liver (e.g., 2-fold, 3-fold, 4-fold or 5-fold more). In some embodiments, the lipid nanoparticle has an ionizable lipid:phosphate ratio of 3-7. In some embodiments, the lipid nanoparticle has an ionizable lipid:phosphate ratio of 4-6. In some embodiments, the lipid nanoparticle has an ionizable lipid:phosphate ratio of 4.5. In some embodiments, the lipid nanoparticle has a nitrogen:phosphate (N:P) ratio of 3-6. In some embodiments, the lipid nanoparticle has an N:P ratio of 5-6. In some embodiments, the lipid nanoparticle has an N:P ratio of 5.7. In some embodiments, the expression of non-secreted proteins can be measured using ELISA and normalized to tissue weight.

[0194] Without wishing to be bound by theory, it is believed that the transfer vehicle described herein protects the encapsulated circular RNA from degradation and provides efficient delivery of the circular RNA to target cells in vivo and in vitro.

[0195] Embodiments of the present disclosure provide lipid compositions described in terms of the respective molar ratios of the components lipids in the formulation. In one embodiment, the mol-% of the ionizable lipid can be from about 10 mol-% to about 80 mol-%. In one embodiment, the mol-% of the ionizable lipid can be from about 20 mol-% to about 70 mol-%. In one embodiment, the mol-% of the ionizable lipid can be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the ionizable lipid can be from about 35 mol-% to about 55 mol-%. In one embodiment, the mol-% of the ionizable lipid can be from about 40 mol-% to about 50 mol-%. In some embodiments, the mol-% of the ionizable lipid in a transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol-%. In certain embodiments, the variability between transfer vehicle batches will be less than 15%, less than 10% or less than 5%.

[0196] In one embodiment, the mol-% of the auxiliary lipid can be from about 1 mol-% to about 50 mol-%. In one embodiment, the mol-% of the auxiliary lipid can be from about 2 mol-% to about 45 mol-%. In one embodiment, the mol-% of the auxiliary lipid can be from about 3 mol-% to about 40 mol-%. In one embodiment, the mol-% of the auxiliary lipid can be from about 4 mol-% to about 35 mol-%. In one embodiment, the mol-% of the auxiliary lipid can be from about 5 mol-% to about 30 mol-%. In one embodiment, the mol-% of the auxiliary lipid can be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the auxiliary lipid in the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol-%.

[0197] In one embodiment, the mol-% of the structural lipid can be from about 10 mol-% to about 80 mol-%. In one embodiment, the mol-% of the structural lipid can be from about 20 mol-% to about 70 mol-%. In one embodiment, the mol-% of the structural lipid can be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the structural lipid can be from about 35 mol-% to about 55 mol-%. In one embodiment, the mol-% of the structural lipid can be from about 40 mol-% to about 50 mol-%. In some embodiments, the mol-% of the structural lipid in the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol-%.

[0198] In one embodiment, the mol-% of the PEG-modified lipid can be from about 0.1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG-modified lipid can be from about 0.2 mol-% to about 5 mol-%. In one embodiment, the mol-% of the PEG-modified lipid can be from about 0.5 mol-% to about 3 mol-%. In one embodiment, the mol-% of the PEG-modified lipid can be from about 1 mol-% to about 2 mol-%. In one embodiment, the mol-% of the PEG-modified lipid can be about 1.5 mol-%. In some embodiments, the mol-% of the PEG-modified lipid in the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol-%.

[0199] Also contemplated are pharmaceutical compositions comprising one or more of the compounds disclosed herein, and in particular, delivery vehicles. In certain embodiments, such delivery vehicles comprise one or more of the PEGylated lipids, ionizable lipids, helper lipids, and / or structural lipids disclosed herein. Also contemplated are delivery vehicles comprising one or more of the compounds disclosed herein and further comprising one or more additional lipids. In certain embodiments, such delivery vehicles are loaded with or otherwise encapsulate circular RNA.

[0200] The delivery vehicles of the invention encapsulate circular RNA. In certain embodiments, the polynucleotides encapsulated by the compounds or pharmaceutical and liposomal compositions of the invention include RNA encoding a protein or enzyme (e.g., a circRNA encoding, for example, phenylalanine hydroxylase (PAH)). The invention contemplates the use of such polynucleotides as therapeutic agents that are capable of being expressed by target cells to thereby contribute to the production (and in some cases, secretion) of a functional enzyme or protein, such as the target cells disclosed in International Application No. PCT / US2010 / 058457 and U.S. Provisional Application No. 61 / 494,881, filed on June 8, 2011, the teachings of which are incorporated herein by reference in their entirety. For example, in certain embodiments, the production of a functional enzyme or protein (e.g., a urea cycle enzyme or an enzyme associated with lysosomal storage disease) that is lacking in a subject can be observed after one or more polynucleotides are expressed in the target cells. As another example, the circular RNA encapsulated by the delivery vehicle can encode one or two polypeptide chains of a T cell receptor protein or can encode a chimeric antigen receptor (CAR).

[0201] Also provided herein are methods of treating a disease in a subject by administering to the subject an effective amount of a composition comprising a circular RNA encoding a functional protein and a delivery vehicle as described herein. In some embodiments, the circular RNA is encapsulated within the delivery vehicle. In certain embodiments, such methods can enhance (e.g., increase) the expression of the polynucleotide and / or increase the production and secretion of a functional polypeptide product in one or more target cells and tissues (e.g., immune cells or hepatocytes). Generally, such methods include contacting the target cells with one or more compounds and / or delivery vehicles that comprise or otherwise encapsulate the circRNA.

[0202] In certain embodiments, transfer vehicles (e.g., lipid nanoparticles) are formulated, in part, based on their ability to facilitate transfection of target cells (e.g., circular RNAs). In another embodiment, transfer vehicles (e.g., lipid nanoparticles) can be selected and / or prepared to optimize delivery of circular RNAs to target cells, tissues, or organs. For example, if the target cells are hepatocytes, or if the target organ is the spleen, the properties of the pharmaceutical and / or liposomal composition (e.g., size, charge, and / or pH) can be optimized to effectively deliver such a composition (e.g., lipid nanoparticles) to the target cells or organ, reduce immune clearance, and / or promote retention in the target cells or organ. Alternatively, if the target tissue is the central nervous system, the selection and preparation of the transfer vehicle must account for its penetration and retention within the blood-brain barrier and / or use alternative means of delivering such a composition (e.g., lipid nanoparticles) directly to such target tissue (e.g., by intracerebrovascular administration). In certain embodiments, the transfer vehicle can be combined with an agent that facilitates transfer of the encapsulating material across the blood-brain barrier (e.g., an agent that disrupts or improves the permeability of the blood-brain barrier and thereby enhances transfer of circular RNAs to target cells). Although the transfer vehicles (e.g., lipid nanoparticles) described herein can facilitate introduction of circRNAs into target cells, addition of polycations (e.g., poly-L-lysine and protamine) as copolymers to one or more lipid nanoparticles, such as those containing pharmaceutical compositions, can also contribute to and in some cases significantly enhance the transfection efficiency of several types of transfer vehicles by 2- to 28-fold in many cell lines in vitro and in vivo (see, N.J. Caplen et al., Gene Ther. 1995; 2:603; S. Li et al., Gene Ther. 1997; 4,891.). In some embodiments, the target cells are immune cells. In some embodiments, the target cells are T cells.

[0203] In certain embodiments, the transfer vehicles (e.g., lipid nanoparticles) described herein are prepared by combining multiple lipid components (e.g., one or more of the compounds disclosed herein) with one or more polymer components. For example, lipid nanoparticles can be prepared using HGT4003, DOPE, cholesterol, and DMG-PEG2000. The lipid nanoparticles can consist of different ratios of additional lipid combinations, including, for example, HGT4001, DOPE, and DMG-PEG2000. The selection of the ionizable lipid, co-lipid, structural lipid, and / or PEGylated lipid that make up the lipid nanoparticles, as well as the relative molar ratios of such lipids to one another, are based on the properties of the selected lipids, the nature of the intended target cells or tissues, and the properties of the material or polynucleotide to be delivered by the lipid nanoparticles. Additional considerations include, for example, the saturation of the alkyl chains, as well as the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipids.

[0204] The transfer vehicles described herein can allow the encapsulated polynucleotides to reach the target cells, or can preferentially allow the encapsulated polynucleotides to reach the target cells or organs on a discriminatory basis (e.g., the transfer vehicle can concentrate in the liver or spleen of a subject to whom such a transfer vehicle has been administered). Alternatively, the transfer vehicle can limit the delivery of the encapsulated polynucleotides to other non-target cells or organs, where the presence of the encapsulated polynucleotides may be undesirable or of limited utility.

[0205] Loading or encapsulating a polynucleotide (e.g., circRNA) into a transfer vehicle can be used to protect the polynucleotide from an environment (e.g., serum) that may contain enzymes or chemicals that degrade such polynucleotides and / or systems or receptors that cause rapid excretion of such polynucleotides. Thus, in some embodiments, the compositions described herein are capable of enhancing the stability of the encapsulated polynucleotide, particularly with respect to the environment to which such polynucleotide will be exposed.

[0206] In certain embodiments, provided herein are vectors for preparing circular RNAs, the vectors comprising a 5' duplex-forming region, a 3' group I intron fragment, an optional first spacer region, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer region, a 5' group I intron fragment, and a 3' duplex-forming region. In some embodiments, these elements are positioned in the vector in the order described above. In some embodiments, the vector further comprises an internal 5' duplex-forming region located between the 3' group I intron fragment and the IRES and an internal 3' duplex-forming region located between the expression sequence and the 5' group I intron fragment. In some embodiments, the internal duplex-forming regions are capable of forming duplexes with each other but not with the external duplex-forming regions. In some embodiments, the internal duplex-forming regions are part of the first and second spacer regions. Additional embodiments include circular RNA polynucleotides, including circular RNA polynucleotides prepared using the vectors provided herein; compositions comprising such circular RNAs; cells comprising such circular RNAs; and methods of using and preparing such vectors, circular RNAs, compositions, and cells.

[0207] In some embodiments, the methods provided herein include administering the circular RNA polynucleotides provided herein into cells for the treatment or production of a useful protein, such as PAH. In some embodiments, due to the resistance of circular RNAs to ribonucleases, the methods facilitate the production of a desired polypeptide with a longer half-life in eukaryotic cells compared to linear RNAs.

[0208] Circular RNA polynucleotides lack free ends required for exonuclease-mediated degradation, rendering them resistant to several RNA degradation mechanisms and allowing for an extended half-life compared to equivalent linear RNAs. Cyclization can allow for stabilization of RNA polynucleotides that typically have a short half-life and can enhance the overall efficacy of exogenous mRNAs in various applications. In one embodiment, the circular RNA polynucleotides provided herein have a half-life in eukaryotic cells (e.g., mammalian cells such as human cells) of at least 20 hours (e.g., at least 80 hours).

[0209] 1. Definitions

[0210] As used herein, the terms “circRNA” or “circular polynucleotide” or “circular RNA” or “oRNA” are used interchangeably and refer to a polynucleotide that forms a circular structure by covalent bonding.

[0211] As used herein, the term “3'I group intron fragment” refers to a sequence having 75% or greater similarity to the 3'-proximal portion of a native group I intron that includes the splice site dinucleotide and optionally a segment of native exon sequence.

[0212] As used herein, the term “5'I group intron fragment” refers to a sequence having 75% or greater similarity to the 5'-proximal portion of a native group I intron that includes the splice site dinucleotide and optionally a segment of native exon sequence.

[0213] As used herein, the term “replacement site” refers to a site in a group I intron where cleavage occurs prior to intron replacement. This cleavage generates 3' and 5' group I intron fragments that are replaced on either side of a segment of precursor RNA to be circularized.

[0214] As used herein, the term “splice site” refers to a dinucleotide that is partially or fully contained within a group I intron and between which the phosphodiester bond is cleaved during RNA cyclization.

[0215] As used herein, the term “therapeutic protein” refers to any protein that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered directly or indirectly to a subject in the form of a translated nucleic acid.

[0216] As used herein, the term "immunogenicity" refers to the potential to induce an immune response to a substance. When the immune system of an organism or a certain type of immune cell is exposed to an immunogenic substance, an immune response can be induced. The term "non-immunogenic" refers to the lack or absence of an immune response to a substance above a detectable threshold. When the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance, no immune response is detected. In some embodiments, when measured by an immunogenicity assay, a non-immunogenic circular polynucleotide as provided herein does not induce an immune response above a predetermined threshold. In some embodiments, when the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic circular polynucleotide as provided herein, no innate immune response is detected. In some embodiments, when the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic circular polynucleotide as provided herein, no adaptive immune response is detected.

[0217] As used herein, the term "cyclization efficiency" refers to a measure of the resulting circular polynucleotide compared to its linear starting material.

[0218] 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 a given amount of transcript encoding the protein or peptide.

[0219] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, modified forms thereof, or analogs thereof. Nucleotides include substances that include purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) as well as pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogs). Nucleotide analogs include nucleotides having modified nucleotides 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 at the exocyclic amine of cytosine, and substitution of 5-bromo-uracil; and 2'-position sugar modifications, including but not limited to sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety as defined herein. Nucleotide analogs are also intended to include nucleotides having bases such as inosine, wybutosine, xanthine; sugars such as 2'-methyl ribose; and non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate, and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0220] 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 1000 bases or up to about 10,000 or more bases), composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), and which can be produced enzymatically or synthetically (e.g., as described in U.S. Patent No. 5,948,902 and references cited therein), which can hybridize to a naturally occurring nucleic acid in a sequence-specific manner analogous to two naturally occurring nucleic acids, e.g., can 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).

[0221] As used herein, the terms "ribonucleic acid" and "RNA" mean a polymer composed of ribonucleotides.

[0222] As used herein, the terms "deoxyribonucleic acid" and "DNA" mean a polymer composed of deoxyribonucleotides.

[0223] "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 of the sample in which it is present (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%). In certain embodiments, a substantially purified component comprises at least 50%, 80%-85% or 90%-95% of the sample. Techniques for purifying target polynucleotides and polypeptides are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation according to density. Generally, a substance is purified when it is present in a sample in greater amounts relative to other components of the sample than it is naturally found.

[0224] As used herein, the terms "duplexed", "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.

[0225] 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 a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule. In some embodiments, nuclease protection assays can be used to functionally characterize unstructured RNA.

[0226] As used herein, "structuring" of RNA refers to an RNA sequence predicted by RNAFold software or a similar prediction tool to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.

[0227] As used herein, two "duplex-forming regions", "homologous arms", or "homologous regions" can be any two regions that are thermodynamically predisposed to cross-pair in a sequence-specific interaction. In some embodiments, the two duplex-forming regions, homologous arms, or homologous regions have a sufficient level of sequence identity with the reverse-complementary sequences of each other to serve as substrates for a hybridization reaction. As used herein, a polynucleotide sequence has "homology" when it is identical or shares sequence identity with a reverse-complementary sequence or "complementary" sequence. The percentage of sequence identity between a homologous region and the reverse-complementary sequence of the corresponding homologous region can be any percentage of sequence identity that permits hybridization to occur. In some embodiments, the internal duplex-forming region of a polynucleotide of the invention is capable of forming a duplex with another internal duplex-forming region and not with an external duplex-forming region.

[0228] A linear nucleic acid molecule is said to have a "5'-end" (5' terminus) and a "3'-end" (3' terminus) because nucleic acid phosphodiester linkages are present at the 5'- and 3'-carbons of the sugar moiety that replaces a mononucleotide. The terminal nucleotide of a polynucleotide is its 5'-terminal nucleotide, at which a new linkage would be a linkage to the 5'-carbon. The terminal nucleotide of a polynucleotide is its 3'-terminal nucleotide, at which a new linkage would be a linkage to the 3'-carbon. As used herein, a terminal nucleotide is a nucleotide located at the terminal position at the 3'- or 5'-end.

[0229] "Transcription" refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. There is no limitation in the present invention regarding the RNA polymerase for transcription. For example, in some embodiments, a T7-type RNA polymerase can be used.

[0230] "Translation" refers to the formation of a polypeptide molecule by ribosomes based on an RNA template.

[0231] It should 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 in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells, or an entire culture of cells; reference to "a polynucleotide" actually includes many copies of the polynucleotide. Unless expressly stated or obvious from context, as used herein, the term "or" is understood to be inclusive. 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, unless defined otherwise herein or in the remainder of the specification below.

[0232] Unless expressly stated or obvious from context, as used herein, the term "about" is understood to be within the normal tolerances in the art, for example within 2 standard deviations of the mean. "About" can be understood to be 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 obvious from context, all numerical values provided herein are modified by the term "about".

[0233] As used herein, the term "encoding" generally refers to any process in which information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first molecule. The second molecule can have a chemical structure that is chemically distinct from that of the first molecule.

[0234] "Co-administering" refers to administering the therapeutic agents provided herein in combination with one or more additional therapeutic agents close enough in time such that the therapeutic agents provided herein enhance the effect of one or more additional therapeutic agents and vice versa.

[0235] As used herein, the terms "treating" and "preventing" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that are recognized by those of ordinary skill in the art as having potential benefit or therapeutic effect. The treatment or prevention provided by the methods disclosed herein can include treating or preventing one or more disorders or symptoms of a disease. In addition, for the purposes of this application, "preventing" can include delaying the onset of a disease or its symptoms or disorders.

[0236] As used herein, the term "expression 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. Exemplary expression sequences encoding a peptide or polypeptide can comprise a plurality of nucleotide triplets, each of which can encode an amino acid and is referred to as a "codon".

[0237] As used herein, "spacer" refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides that separates two other elements along the polynucleotide sequence. The sequence can be defined or can be random. Spacers are typically non-coding. In some embodiments, the spacer includes a duplex-forming region.

[0238] As used herein, "splice site" refers to one or more dinucleotides between which phosphodiester bond cleavage occurs during a splicing reaction. A "5' splice site" refers to the native 5' dinucleotide of an intron (e.g., a group I intron), while a "3' splice site" refers to the native 3' dinucleotide of an intron.

[0239] 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 greater that is capable of initiating translation of a polypeptide in the absence of a typical RNA cap structure. The length of an IRES is typically about 500 nt to about 700 nt.

[0240] As used herein, "miRNA site" refers to a stretch of nucleotides within a polynucleotide that is capable of forming a duplex with at least 8 nucleotides of a native miRNA sequence.

[0241] As used herein, "endonuclease site" refers to a stretch of nucleotides within a polynucleotide that can be recognized and cleaved by an endonuclease protein.

[0242] As used herein, "bicistronic RNA" refers to a polynucleotide that contains two expression sequences encoding two different proteins. These expression sequences can be separated by a nucleotide sequence encoding a cleavable peptide such as a protease cleavage site. They can also be separated by a ribosome skipping element.

[0243] As used herein, the term "ribosome skipping element" refers to a nucleotide sequence encoding a short peptide sequence that is capable of generating two peptide chains from the translation of one RNA molecule. While not wishing to be bound by theory, it is hypothesized that ribosome skipping elements function by (1) terminating the translation of the first peptide chain and reinitiating the translation of the second peptide chain; or (2) cleaving a peptide bond in the peptide sequence encoded by the ribosome skipping element by the intrinsic protease activity of the encoded peptide or by another protease in the environment (e.g., cytosolic).

[0244] As used herein, the term "co-formulation" refers to a nanoparticle formulation comprising two or more nucleic acids or a nucleic acid and other active pharmaceutical substances. Typically, the ratio is equimolar or defined as the amount measured as the ratio of two or more nucleic acids or a nucleic acid and other active pharmaceutical substances.

[0245] As used herein, "delivery vehicle" includes any standard pharmaceutical carrier, diluent, excipient, etc., which is generally intended to be used in conjunction with the administration of bioactive agents including nucleic acids.

[0246] As used herein, the phrase "lipid nanoparticle" refers to a delivery vehicle comprising one or more lipids (e.g., in some embodiments, cationic lipids, non-cationic lipids, and PEGylated lipids).

[0247] As used herein, the phrase "ionizable lipid" refers to any of a variety of lipid species that carry a net positive charge at a selected pH (such as physiological pH 4) and a neutral charge at other pHs (such as physiological pH 7).

[0248] In some embodiments, the lipids (e.g., ionizable lipids) disclosed herein 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 in or adjacent to the subject functional group are broken (e.g., hydrolyzed) or are capable of being broken upon exposure to selected conditions (e.g., upon exposure to enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in a particular embodiment, is a disulfide group capable of being cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional group capable of being cleaved upon exposure to selected biological conditions. For example, a disulfide group can be cleaved enzymatically or by hydrolysis, oxidation, or reduction reactions. Upon cleavage of such a disulfide functional group, one or more functional moieties or groups (e.g., one or more head groups and / or tail groups) bound thereto can be released. 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 or ether group. In some embodiments, the cleavable group is bonded (e.g., by one or more of hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds) to one or more functional moieties or groups (e.g., at least one head group and at least one tail group). In certain embodiments, at least one of the functional moieties or groups is hydrophilic (e.g., a hydrophilic head group comprising one or more of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino, and pyridyl).

[0249] As used herein, the term "hydrophilic" is used to qualitatively denote that a functional group is water-preferred, and generally such groups are water-soluble. For example, compounds are disclosed herein that contain a cleavable disulfide (S—S) functional group bonded 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, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl.

[0250] In certain embodiments, at least one functional group that forms part of the compounds disclosed herein is hydrophobic in nature (e.g., a hydrophobic tail group containing a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used to qualitatively denote that a functional group is water-averse, and generally such groups are insoluble in water. For example, compounds are disclosed herein that contain a cleavable functional group (e.g., a disulfide (S—S) group) bonded 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-C 20 alkyl and / or optionally substituted, variably saturated or unsaturated C6-C 20 acyl.

[0251] The compounds described herein may also contain one or more isotope substitutions. For example, H may be in any isotopic form, including 1 H, 2 H (D or deuterium), and 3 H (or tritium); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O and 18 O; F may be in any isotopic form, including 18 F and 19 F; and so on.

[0252] When describing the present invention, the present invention may include compounds and their pharmaceutically acceptable salts, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions. Unless otherwise indicated, the following terms (if present) have the following meanings. It should also be understood that when described herein, any part of the definitions below may be substituted by a variety of substituents, and each definition is intended to include such substituted parts within the scope as described below. Unless otherwise indicated, the term "substituted" shall be defined as follows. It should be further understood that the terms "groups" and "radicals" are considered interchangeable as used herein.

[0253] When listing a range of values, it is intended to cover every value and sub-range within the stated range. For example, "C 1-6 alkyl" is intended to cover C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0254] In certain embodiments, the compounds disclosed herein comprise, for example, at least one hydrophilic head group and at least one hydrophobic tail group, each bonded to at least one cleavable group, such that such compounds are amphiphilic. As used herein to describe a compound or composition, the term "amphiphilic" refers to the ability to dissolve in both polar (e.g., water) and non-polar (e.g., lipid) environments. For example, in certain embodiments, the compounds disclosed herein comprise at least one lipophilic tail group (e.g., cholesterol or C6-C 20 alkyl) and at least one hydrophilic head group (e.g., imidazole), each bonded to a cleavable group (e.g., a disulfide bond).

[0255] It should be noted that the terms "head group" and "tail group" used to describe the compounds of the present invention and in particular the functional groups that make up such compounds 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., guanidine) is bonded (e.g., by one or more of hydrogen bonding, van der Waals forces, ionic interactions, and covalent bonds) to a cleavable functional group (e.g., a disulfide bond group), which in turn is bonded to a hydrophobic tail group (e.g., cholesterol).

[0256] As used herein, the term "alkyl" refers to straight-chain and branched C1-C 40 hydrocarbons (e.g., C6-C 20 hydrocarbons), and includes both saturated and unsaturated hydrocarbons. In certain embodiments, an alkyl may contain one or more cycloalkyls and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with substituents (e.g., one or more of alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide). In certain embodiments, the alkyls contemplated include (9Z,12Z)-octadeca-9,12-diene. Such as for example "C6-C 20The use of the term “alkyl” having the stated range of carbon atoms is intended to refer to an alkyl group (e.g., straight-chain or branched and including alkenes and alkynes). In some embodiments, the alkyl group has 1 to 10 carbon atoms (“C 1-10 alkyl”). In some embodiments, the alkyl group has 1 to 9 carbon atoms (“C 1-9 alkyl”). In some embodiments, the alkyl group has 1 to 8 carbon atoms (“C 1-8 alkyl”). In some embodiments, the alkyl group has 1 to 7 carbon atoms (“C 1-7 alkyl”). In some embodiments, the alkyl group has 1 to 6 carbon atoms (“C 1-6 alkyl”). In some embodiments, the alkyl group has 1 to 5 carbon atoms (“C 1-5 alkyl”). In some embodiments, the alkyl group has 1 to 4 carbon atoms (“C 1-4 alkyl”). In some embodiments, the alkyl group has 1 to 3 carbon atoms (“C 1-3 alkyl”). In some embodiments, the alkyl group has 1 to 2 carbon atoms (“C 1-2 alkyl”). In some embodiments, the alkyl group has 1 carbon atom (“C1 alkyl”). C 1-6 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[0257] As used herein, “alkenyl” refers to 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) (“C 2-20 alkenyl”). In certain embodiments, the alkenyl group does not contain any triple bonds. In some embodiments, the alkenyl group has 2 to 10 carbon atoms (“C 2-10 alkenyl”). In some embodiments, the alkenyl group has 2 to 9 carbon atoms (“C 2-9 alkenyl”). In some embodiments, the alkenyl group has 2 to 8 carbon atoms (“C 2-8 alkenyl”). In some embodiments, the alkenyl group has 2 to 7 carbon atoms (“C 2-7 alkenyl”). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (“C 2-6 alkenyl”). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (“C 2-5 alkenyl”). In some embodiments, the alkenyl group has 2 to 4 carbon atoms (“C 2-4 alkenyl”). In some embodiments, the alkenyl group has 2 to 3 carbon atoms (“C 2-3"Alkenyl"). In some embodiments, the alkenyl has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). C 2-4 Examples of alkenyls include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. C 2-6 Examples of alkenyls include those mentioned above C 2-4 alkenyls as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Further examples of alkenyls include heptenyl (C7), octenyl (C8), octatrieneyl (C8), etc.

[0258] As used herein, "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) ("C 2-20 alkynyl"). In certain embodiments, the alkynyl does not contain any double bonds. In some embodiments, the alkynyl has 2 to 10 carbon atoms ("C 2-10 alkynyl"). In some embodiments, the alkynyl has 2 to 9 carbon atoms ("C 2-9 alkynyl"). In some embodiments, the alkynyl has 2 to 8 carbon atoms ("C 2-8 alkynyl"). In some embodiments, the alkynyl has 2 to 7 carbon atoms ("C 2-7 alkynyl"). In some embodiments, the alkynyl has 2 to 6 carbon atoms ("C 2-6 alkynyl"). In some embodiments, the alkynyl has 2 to 5 carbon atoms ("C 2-5 alkynyl"). In some embodiments, the alkynyl has 2 to 4 carbon atoms ("C 2-4 alkynyl"). In some embodiments, the alkynyl has 2 to 3 carbon atoms ("C 2-3 alkynyl"). In some embodiments, the alkynyl has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). C 2-4 Examples of alkynyls include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. C 2-6 Examples of alkenyls include those mentioned above C 2-4 alkynyls as well as pentynyl (C5), hexynyl (C6), etc. Further examples of alkynyls include heptynyl (C7), octynyl (C8), etc.

[0259] As used herein, "alkylene", "alkenylene", and "alkynylene" refer respectively to divalent groups of alkyl, alkenyl, and alkynyl. When a range or number of carbons is provided for a particular "alkylene", "alkenylene", or "alkynylene", it is understood that the range or number refers to the range or number of carbons in a linear carbon divalent chain. "Alkylene", "alkenylene", and "alkynylene" may be substituted or unsubstituted with one or more substituents as described herein.

[0260] As used herein, the term "aryl" is an aromatic group having six to ten carbons in the ring portion (e.g., monocyclic, bicyclic, and tricyclic structures). Aryl may optionally be substituted through available carbon atoms and in certain embodiments may include one or more heteroatoms such as oxygen, nitrogen, or sulfur. In some embodiments, aryl has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, aryl has 10 ring carbon atoms ("C 10 aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl).

[0261] As used herein, "heteroaryl" is a group of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in the ring array), having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In a heteroaryl containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, provided that the valence allows. The bicyclic heteroaryl ring system may 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 carbocyclic or heterocyclic groups, wherein the point of attachment is on the heteroaryl ring, and in such cases, the number of ring members continues to indicate 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, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members indicates the number of ring members in the fused (aryl / heteroaryl) ring system. In a bicyclic heteroaryl in which one ring contains no heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment may be on either ring, i.e., the ring carrying the heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl).

[0262] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group having 3-12, 3-8, 4-8, or 4-6 carbon atoms derived from a cycloalkane, referred to herein as, for example, "C 4-8 cycloalkyl". Exemplary cycloalkyls include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane.

[0263] As used herein, "heterocyclic group" or "heterocycle" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclic group"). In a heterocyclic group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, provided that the valence allows. The heterocyclic group may be monocyclic ("monocyclic heterocyclic group") or a fused, bridged, or spiro ring system (such as a bicyclic system ("bicyclic heterocyclic group")), and may be saturated or may be partially unsaturated. The bicyclic ring system of the heterocyclic group may contain one or more heteroatoms in one or both rings. "Heterocyclic group" also includes a ring system in which a heterocyclic group ring as defined above is fused to one or more carbocyclic groups, wherein the point of attachment is on the carbocyclic group or the heterocyclic group ring, or a ring system in which a heterocyclic group ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic group ring, and in such cases, the number of ring members continues to indicate the number of ring members in the heterocyclic group ring system. The terms "heterocycle", "heterocyclic group", "heterocyclic group ring", "heterocyclic moiety", "heterocyclic portion", and "heterocyclic group" are used interchangeably.

[0264] As used herein, "cyano" refers to -CN.

[0265] As used herein, the terms "halo group" and "halogen" refer to atoms selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I). In certain embodiments, the halo group is fluoro or chloro.

[0266] As used herein, the term "alkoxy" refers to an alkyl group attached to another moiety through an oxygen atom (-O(alkyl)). Non-limiting examples include, for example, methoxy, ethoxy, propoxy, and butoxy.

[0267] As used herein, "oxo group" refers to -C=O.

[0268] Generally, the term "substituted", whether or not preceded by the term "optionally", means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced by an admissible substituent, such as a substituent that results in a stable compound upon substitution, such as a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reactions. Unless otherwise specified, 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 the same or different at each position.

[0269] As used herein, "pharmaceutically acceptable salts" refer to those salts that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc. and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. The 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 formed by the reaction of an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by using other methods commonly used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Pharmaceutically acceptable salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide ions, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0270] In typical embodiments, the present invention is intended to cover the compounds disclosed herein, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomeric forms, polymorphs, and prodrugs of such compounds. In some embodiments, the present invention includes pharmaceutically acceptable addition salts, pharmaceutically acceptable esters, solvates (e.g., hydrates) of addition salts, tautomeric forms, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (pure or as racemic or non-racemic mixtures) of the compounds described herein.

[0271] The compounds described herein may contain one or more asymmetric centers and can therefore exist in various isomeric forms (e.g., 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 a mixture of stereoisomers (including racemic mixtures and mixtures enriched in one or more stereoisomers). Isomers can be separated from the mixture by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the 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 page 268 (E.L. Eliel, editor, Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention additionally encompasses the compounds described herein in the form of individual isomers substantially free of other isomers, and alternatively in the form of mixtures of different isomers.

[0272] In certain embodiments, the compounds and delivery vehicles (such as lipid nanoparticles) of which such compounds are components exhibit an 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 include the step of 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 circular RNA encapsulated therein. As used herein, the term "transfect" or "transfection" refers to the intracellular introduction of one or more encapsulating materials (e.g., nucleic acids and / or polynucleotides) into a cell, or preferably into a target cell. The term "transfection efficiency" refers to the relative amount of such encapsulating materials (e.g., polynucleotides) taken up by, introduced into, and / or expressed by the target cells subjected to transfection. In some embodiments, the transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cells after transfection. In some embodiments, the delivery vehicle has a high transfection efficiency. In some embodiments, the delivery vehicle has a transfection efficiency of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0273] As used herein, the term "liposome" generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayers. In certain embodiments, the liposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprising one or more ionizable lipid compounds disclosed herein). Such liposomes can be unilamellar or multilamellar vesicles having a membrane formed of a lipophilic material and an aqueous interior containing the encapsulated circRNA to be delivered to one or more target cells, tissues, and organs. In certain embodiments, the compositions described herein comprise one or more lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that can be used to form the liposomes and lipid nanoparticles under consideration include one or more compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and / or HGT4005). Such liposomes and lipid nanoparticles can also comprise additional ionizable lipids such as C12-200, DLin-KC2-DMA, and / or HGT5001, co-lipids, structural lipids, PEGylated lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLin carbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0274] As used herein, the phrases "non-cationic lipid", "non-cationic co-lipid", and "co-lipid" are used interchangeably and refer to any neutral lipid, zwitterionic lipid, or anionic lipid.

[0275] As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0276] As used herein, the phrase "biodegradable lipid" or "degradable lipid" refers to any of a number of lipid species that decompose in a host environment on the order of minutes, hours, or days, thereby desirably making them less toxic and less likely to accumulate in the host over time. Common modifications to lipids include ester bonds and disulfide bonds, among others, to increase the biodegradability of the lipid.

[0277] As used herein, the phrase "biodegradable PEG lipid" or "degradable PEG lipid" refers to any of a number of lipid species in which the PEG molecule cleaves from the lipid in the host environment on the order of minutes, hours, or days, thereby desirably reducing their immunogenicity. Common modifications to PEG lipids include ester bonds and disulfide bonds, among others, to increase the biodegradability of the lipid.

[0278] In certain embodiments of the invention, a delivery vehicle (e.g., a lipid nanoparticle) is prepared to encapsulate one or more materials or therapeutic agents (e.g., a circRNA). The process of incorporating the desired therapeutic agent (e.g., a circRNA) into the delivery vehicle is referred to herein as "loading" or "encapsulation" (Lasic et al., FEBS Lett., 312:255-258, 1992). The material (e.g., a circRNA) loaded or encapsulated by the delivery vehicle can be located entirely or partially within the internal space of the delivery vehicle, within the bilayer membrane of the delivery vehicle, or associated with the outer surface of the delivery vehicle.

[0279] As used herein, the term "structural lipid" refers to sterols and lipids containing a sterol moiety.

[0280] As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols.

[0281] As used herein, the term "structural lipid" refers to sterols and lipids containing a sterol moiety.

[0282] As used herein, the term "PEG" refers to any polyethylene glycol or other polyalkylene ether polymer.

[0283] As generally defined herein, "PEG-OH lipid" (also referred to herein as "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl groups (-OH) on the lipid.

[0284] As used herein, "phospholipid" is a lipid containing a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains.

[0285] All nucleotide sequences disclosed herein can represent RNA sequences or the corresponding DNA sequences. It should be understood that deoxythymidine (dT or T) in DNA is transcribed as uridine (U) in RNA. Thus, "T" and "U" are used interchangeably herein in nucleotide sequences.

[0286] As used herein, the term "sequence identity" or, for example, a sequence "having 50% identity to" refers to the degree of identity of a sequence on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. Thus, the "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or identical 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 in both sequences 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., the window size), and multiplying the result by 100 to obtain the percent sequence identity. 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 are included, typically where the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0287] 2. Vectors, precursor RNAs, and circular RNAs

[0288] Also provided herein are circular RNAs, precursor RNAs that can be circularized into circular RNAs, and vectors (e.g., DNA vectors) that can be transcribed into precursor RNAs or circular RNAs.

[0289] Two types of spacers have been designed to improve precursor RNA circularization and / or gene expression from circular RNAs. The first type of spacer is an external spacer, i.e., it is present in the precursor RNA but is removed after circularization. Without wishing to be bound by theory, it is expected that the external spacer can improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements from interfering with its folding and function. The second type of spacer is an internal spacer, i.e., it is present in the precursor RNA and remains in the resulting circular RNA. Without wishing to be bound by theory, it is expected that the internal spacer can improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other adjacent sequence elements, particularly adjacent IRESs and coding regions, from interfering with its folding and function. It is also expected that the internal spacer can improve protein expression from IRESs by preventing adjacent sequence elements, particularly intron elements, from hybridizing with sequences within the IRES and inhibiting its ability to fold into its most preferred and active conformation.

[0290] To drive protein expression, the circular RNAs comprise an IRES operably linked to a protein coding sequence. Exemplary IRES sequences are provided in Table 17 below. In some embodiments, the circular RNAs disclosed herein comprise an IRES sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the IRES sequences in Table 17. In some embodiments, the circular RNAs disclosed herein comprise the IRES sequences in Table 17. 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' ends of the IRES, adding a spacer to the 5' of the IRES, modifying the 6 nucleotides (Kozak sequence) 5' of the translation start site, modifying the translation start site substitution, and creating chimeric / hybrid IRES sequences. In some embodiments, the IRES sequences in the circular RNAs disclosed herein comprise one or more of these modifications relative to the native IRES (e.g., the native IRES disclosed in Table 17).

[0291] In certain aspects, the present disclosure provides circular RNA polynucleotides comprising a Group I intron fragment after 3' splicing, an optional first spacer, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer, and a Group I intron fragment after 5' splicing. In some embodiments, these regions are arranged in the stated order. In some embodiments, the circular RNAs are prepared by the methods provided herein or by the vectors provided herein.

[0292] In certain embodiments, transcription of the vectors provided herein (e.g., comprising a 5' homology region, a 3' Group I intron fragment, an optional first spacer, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer, a 5' Group I intron fragment, and a 3' homology region) results in the formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, when incubated in the presence of a guanosine nucleotide or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg 2+ ) the precursor linear RNA polynucleotide circularizes.

[0293] In some embodiments, the vectors and precursor RNA polynucleotides provided herein include a first (5') duplex-forming region and a second (3') duplex-forming region. In certain embodiments, the first and second homologous regions can form perfect or imperfect duplexes. Thus, in certain embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the first and second duplex-forming regions can base pair with each other. In some embodiments, the predicted base pairing of the duplex-forming region with an unintended sequence in the RNA (e.g., a non-duplex-forming region sequence) is less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%). In some embodiments, including such duplex-forming regions at the ends of the precursor RNA strand and adjacent to or very close to Group I intron segments brings the Group I intron segments closer to each other, thereby increasing splicing efficiency. In some embodiments, the length of the duplex-forming region is 3 to 100 nucleotides (e.g., a length of 3-75 nucleotides, a length of 3-50 nucleotides, a length of 20-50 nucleotides, a length of 35-50 nucleotides, a length of 5-25 nucleotides, a length of 9-19 nucleotides). In some embodiments, the length of the duplex-forming region is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In some embodiments, the duplex-forming region has a length of about 9 to about 50 nucleotides. In one embodiment, the duplex-forming region has a length of about 9 to about 19 nucleotides. In some embodiments, the duplex-forming region has a length of about 20 to about 40 nucleotides. In certain embodiments, the duplex-forming region has a length of about 30 nucleotides.

[0294] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include a first (5') and / or a second (3') spacer. In some embodiments, including a spacer between the 3' group I intron fragment and the IRES can protect the secondary structures in those regions by preventing their interaction, thereby enhancing splicing efficiency. In some embodiments, the first spacer (between the 3' group I intron fragment and the IRES) and the second spacer (between the expression sequence and the 5' group I intron fragment) include additional base-pairing regions that are predicted to base-pair with each other rather than with the first and second duplex-forming regions. In some embodiments, such spacer base-pairing brings the group I intron fragments closer to each other, thereby further enhancing splicing efficiency. Additionally, in some embodiments, the combination of base-pairing between the first and second duplex-forming regions, and, separately, the combination of base-pairing between the first and second spacers, promotes the formation of a splicing bubble that contains the group I intron fragments flanking the adjacent base-pairing regions. A typical spacer is a continuous sequence having one or more of the following characteristics: 1) is expected to avoid interfering with proximal structures, such as an IRES, an expression sequence, or an intron; 2) is at least 7 nt and no more than 100 nt in length; 3) is located after and adjacent to the 3' intron fragment and / or before and adjacent to the 5' intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt in length, b) a region at least 5 nt in length that base-pairs with a distal sequence (including another spacer), and c) a structured region at least 7 nt in length that is restricted to the spacer sequence. A spacer can have several regions, including unstructured regions, base-pairing regions, hairpin / structured regions, and combinations thereof. In one embodiment, the spacer has a structured region having 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 includes one or more hairpin structures. In one embodiment, the spacer includes one or more hairpin structures having a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In one embodiment, an additional spacer is present between the 3' group I intron fragment and the IRES. In one embodiment, this additional spacer prevents the structured region of the IRES from interfering with the folding of the 3' group I intron fragment or reduces the extent to which this occurs. In some embodiments, the length of the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides.In some embodiments, the length of the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides. In some embodiments, the length of the 5' spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and / or 25 and 35 nucleotides. In certain embodiments, the length of 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 one embodiment, the 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is a polyAC sequence. In one embodiment, the spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, the spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0295] In certain embodiments, the 3' group I intron fragment is at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to a contiguous sequence of an adjacent exon sequence of the 3'-proximal fragment of a native group I intron, including the 3' splice site dinucleotide and optionally having a length of at least 1 nt (e.g., a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt) and up to the length of the exon. Typically, the 5' group I intron fragment is at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to a contiguous sequence of an adjacent exon sequence of the 5'-proximal fragment of a native group I intron, including the 5' splice site dinucleotide and optionally having a length of at least 1 nt (e.g., a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nt) and up to the length of the exon. As described by Umekage et al. (2012), the outer portions of the 3' group I intron fragment and the 5' group I intron fragment are removed during cyclization, such that the circular RNAs provided herein contain only the portion of the 3' group I intron fragment formed by an optional exon sequence of at least 1 nt in length and the portion of the 5' group I intron fragment formed by an optional exon sequence of at least 1 nt in length, if such sequences are present on the non-circularized precursor RNA. The portion of the 3' group I intron fragment retained by the circular RNA is referred to herein as the spliced 3' group I intron fragment. The portion of the 5' group I intron fragment retained by the circular RNA is referred to herein as the spliced 5' group I intron fragment.

[0296] In certain embodiments, the vectors, precursor RNAs, and circular RNAs provided herein contain an internal ribosome entry site (IRES). Inclusion of an IRES permits 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 ribosome translation initiation complex and facilitates translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22:150-161).

[0297] Multiple IRES sequences are available and include sequences derived from a variety of viruses, such as the leader sequences of picornaviruses derived from, for example, the encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63:1651-1660), the poliovirus leader sequence, the hepatitis A virus leader sequence, 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 foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), the Giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc.

[0298] In some embodiments, the IRES is the IRES sequence of the following viruses: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Arma virilis enterovirus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picornavirus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human coxsackievirus B3, Tobacco mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 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, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, murine Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, murine Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, murine UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, Tobacco etch virus, Turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCVQC64, human kobuvirus E / D, human kobuvirus F, human kobuvirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, salivirus A SH1, salivirus FHB, salivirus NG-J1, human parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, echovirus E14, human parechovirus 5, Aichivirus, hepatitis A virus HA16, Phopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human hepacivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, hepacivirus A 1220, PasivirusA 3, Sapelovirus, Rosavirus B, Bakunsa virus, Tremovirus A, porcine Pasivirus1, PLV-CHN, Pasivirus A, Sicinivirus, hepatitis virus K, hepatitis virus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 dicistrovirus, Hubei picornavirus-like virus, CRPV, salivirus A BN5, salivirus ABN2, salivirus A 02394, salivirus A GUT, salivirus A CH, salivirus A SZ1, salivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer of eIF4G.

[0299] In some embodiments, the polynucleotides herein comprise an expression sequence. In some embodiments, the expression sequence encodes a therapeutic protein.

[0300] In some embodiments, the circular RNA encodes two or more polypeptides. In some embodiments, the circular RNA is a dicistronic RNA. The sequences encoding two or more polypeptides may be separated by a ribosome skipping element or a nucleotide sequence encoding a protease cleavage site. In certain embodiments, the ribosome skipping element encodes the thosea asigna virus 2A peptide (T2A), porcine teschovirus-1 2A peptide (P2A), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), cytoplasmic polyhedrosis virus 2A peptide (BmCPV 2A), or bombyx mori infectious flacherie virus 2A peptide (BmIFV2A).

[0301] In certain embodiments, the vectors provided herein comprise a 3'UTR. In some embodiments, the 3'UTR is from human β-globin, human α-globin, xenopus β-globin, xenopus α-globin, human prolactin, human GAP-43, human eEF1α1, human Tau, human TNFα, dengue virus, hantaan virus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, post-transcriptional regulatory element of woodchuck hepatitis virus, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

[0302] In some embodiments, the vectors provided herein comprise a 5'UTR. In some embodiments, the 5'UTR is from human β-globin, xenopus β-globin, human α-globin, xenopus α-globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or adenovirus tripartite leader sequence.

[0303] In some embodiments, the vectors provided herein comprise a polyA region outside of the 3' and / or 5' group I intron fragment. 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 after circularization. Thus, oligonucleotides that hybridize to the polyA sequence, such as oligo(dT) conjugated to a solid surface (e.g., resin), can be used to separate circular RNA from its precursor RNA. Other sequences can also be placed 5' of the 3' group I intron fragment or 3' of the 5' group I intron fragment, and complementary sequences can be similarly used for circular RNA purification.

[0304] In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein have a length between 300 and 10,000, 400 and 9,000, 500 and 8,000, 600 and 7,000, 700 and 6,000, 800 and 5,000, 900 and 5,000, 1,000 and 5,000, 1,100 and 5,000, 1,200 and 5,000, 1,300 and 5,000, 1,400 and 5,000, and / or 1,500 and 5,000 nucleotides. In some embodiments, the polynucleotide has a length of at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1,000 nt, 1,100 nt, 1,200 nt, 1,300 nt, 1,400 nt, 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, or 5,000 nt. In some embodiments, the polynucleotide has a length not exceeding 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 6,000 nt, 7,000 nt, 8,000 nt, 9,000 nt, or 10,000 nt. In some embodiments, the DNA, linear RNA, and / or circular RNA polynucleotides provided herein have a length of about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1,000 nt, 1,100 nt, 1,200 nt, 1,300 nt, 1,400 nt, 1,500 nt, 2,000 nt, 2,500 nt, 3,000 nt, 3,500 nt, 4,000 nt, 4,500 nt, 5,000 nt, 6,000 nt, 7,000 nt, 8,000 nt, 9,000 nt, or 10,000 nt.

[0305] In some embodiments, vectors are provided herein. In certain embodiments, the vector contains, in the following order, a) a 5' homology region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) an IRES, e) an expression sequence, f) an optional second spacer sequence, g) a 5' group I intron fragment, and h) a 3' homology region. In some embodiments, the vector contains a transcriptional promoter upstream of the 5' homology region. In certain embodiments, the precursor RNA contains, in the following order, a) a polyA sequence, b) an external spacer, c) a 3' group I intron fragment, d) a duplex-forming region, e) an internal spacer, f) an IRES, g) an expression sequence, h) a stop codon cassette, i) an optional internal spacer, j) a duplex-forming region capable of forming a duplex with the duplex-forming region of d, k) a 5' group I intron fragment, l) an external spacer, and m) a polyA sequence.

[0306] In some embodiments, the present disclosure provides precursor RNAs. In certain embodiments, the precursor RNAs are linear RNAs produced by in vitro transcription of the vectors provided herein. In some embodiments, the precursor RNAs comprise, in the following order: a) a 5' homologous region, b) a 3' group I intron fragment, c) an optional first spacer sequence, d) an IRES, e) an expression sequence, f) an optional second spacer sequence, g) a 5' group I intron fragment, and h) a 3' homologous region. The precursor RNAs can be unmodified, partially modified, or fully modified.

[0307] In certain embodiments, the present disclosure provides circular RNAs. In certain embodiments, the circular RNAs are circular RNAs produced by the vectors provided herein. In some embodiments, the circular RNAs are circular RNAs produced by circularization of the precursor RNAs provided herein. In some embodiments, the circular RNAs comprise, in the following order: a) a first spacer sequence, b) an IRES, c) an expression sequence, and d) a second spacer sequence. In some embodiments, the circular RNAs further comprise the portion of the 3' group I intron fragment that is 3' of the 3' splice site. In some embodiments, the circular RNAs further comprise the portion of the 5' group I intron fragment that is 5' of the 5' splice site. In some embodiments, the circular RNAs are at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides in size. The circular RNAs can be unmodified, partially modified, or fully modified.

[0308] In some embodiments, the circular RNAs provided herein have higher functional stability compared to mRNAs comprising the same expression sequence. In some embodiments, the circular RNAs provided herein have higher functional stability compared to mRNAs comprising the same expression sequence, 5moU modification, optimized UTRs, cap, and / or polyA tail.

[0309] In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 hours. In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life of 5 - 80, 10 - 70, 15 - 60, and / or 20 - 50 hours. In some embodiments, the circular RNA polynucleotides provided herein have a functional half-life that is greater than (e.g., at least 1.5-fold, at least 2-fold) the functional half-life of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the functional half-life can be evaluated by detecting functional protein synthesis.

[0310] 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 - 80, 10 - 70, 15 - 60, and / or 20 - 50 hours. In some embodiments, the half-life of the circular RNA polynucleotides provided herein is greater than (e.g., at least 1.5-fold, at least 2-fold) the half-life of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the functional half-life of the circular RNA polynucleotide or its pharmaceutical composition in human cells is greater than or equal to the functional half-life of a predetermined threshold. In some embodiments, the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussia luciferase (GLuc) is measured in the culture medium of human cells (e.g., HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours for 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay, wherein the level of the protein encoded by the expression sequence of the circular RNA polynucleotide is measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours for 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the predetermined threshold is the functional half-life of a reference linear RNA polynucleotide containing the same expression sequence as the circular RNA polynucleotide.

[0311] In some embodiments, the circular RNAs provided herein may have a higher expression value than equivalent linear mRNAs, e.g., a higher expression value 24 hours after administering the RNA to the cells. In some embodiments, the circular RNAs provided herein have a higher expression value compared to mRNAs containing the same expression sequence, 5mU modification, optimized UTRs, cap, and / or polyA tail.

[0312] In some embodiments, when exposed to the immune system of an organism or a certain type of immune cell, the immunogenicity of the circular RNAs provided herein can be lower than that of equivalent mRNAs. In some embodiments, when exposed to the immune system of an organism or a certain type of immune cell, the circular RNAs provided herein are associated with the regulation of cytokine production. For example, in some embodiments, compared to mRNAs containing the same expression sequence, when exposed to the immune system of an organism or a certain type of immune cell, the circular RNAs provided herein are associated with a decrease in the production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα. In some embodiments, compared to mRNAs containing the same expression sequence, when exposed to the immune system of an organism or a certain type of immune cell, the circular RNAs provided herein are associated with less transcriptional induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα. In some embodiments, the immunogenicity of the circular RNAs provided herein is lower than that of mRNAs containing the same expression sequence. In some embodiments, the immunogenicity of the circular RNAs provided herein is lower than that of mRNAs containing the same expression sequence, 5moU modification, optimized UTRs, cap, and / or polyA tail.

[0313] In certain embodiments, the circular RNAs provided herein can be transfected into cells as such, or can be transfected in the form of a DNA vector and transcribed in the cells. Transcription of the circular RNAs from the transfected DNA vector can be carried out by an added polymerase or a polymerase encoded by the nucleic acid transfected into the cells, or preferably by an endogenous polymerase.

[0314] In certain embodiments, the circular RNA polynucleotides provided herein comprise modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleoside is m 5 C (5-methylcytidine). In another embodiment, the modified nucleoside is m 5 U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A (N 6 -methyladenosine). In another embodiment, the modified nucleoside is s 2 U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine). In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine); m 2 A (2-methyladenosine); Am (2’-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i6 A(N 6 -(Isopentenyladenosine); ms 2 i6A(2-Methylthio-N 6 -Isopentenyladenosine); io 6 A(N 6 -(cis-Hydroxyisopentenyladenosine); ms 2 io 6 A(2-Methylthio-N 6 -(cis-Hydroxyisopentenyladenosine); g 6 A(N 6 -Glycylcarbamoyladenosine); t 6 A(N 6 -Threonylcarbamoyladenosine); ms 2 t 6 A(2-Methylthio-N 6 -Threonylcarbamoyladenosine); m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyladenosine); hn 6 A(N 6 -Hydroxy-norvalylcarbamoyladenosine); ms 2 hn 6 A(2-Methylthio-N 6 -Hydroxy-norvalylcarbamoyladenosine); 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(Lysine); 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); m2 Gm(N 2 , 2'-dimethylguanosine); m 2 2Gm(N 2 , N 2 , 2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (under-modified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0 (7-cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G + (archiguanosine); D (dihydrouridine); m 5 Um (5, 2'-O-dimethyluridine); s 4 U (4-thiouridine); m 5 s 2 U (5-methyl-2-thiouridine); s 2 Um (2-thio-2'-O-methyluridine); acp 3 U (3-(3-amino-3-carboxypropyl)uridine); ho 5 U (5-hydroxyuridine); mo 5 U (5-methoxyuridine); cmo 5 U (uridine 5-hydroxyacetate); mcmo 5 U (uridine 5-hydroxyacetate 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 5Um (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-tauromethyluridine); τm 5 s 2 U (5-tauromethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac 6 A(N 6 -acetyladenosine).

[0315] In some embodiments, the modified nucleosides can include compounds selected from the group consisting of: pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyluridine, 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine, 1-tauromethyl-4-thio-uridine, 5-methyl-uridine, 1-methylpseudouridine, 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-methoxypseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, 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-methoxypseudoisocytidine, 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-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In another embodiment, the modification is independently selected from the group consisting of: 5-methylcytosine, pseudouridine, and 1-methylpseudouridine.,

[0316] In some embodiments, the modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.

[0317] In certain embodiments, the polynucleotide can be codon-optimized. A codon-optimized sequence can be a sequence in which the codons in the polynucleotide encoding a polypeptide have been replaced to increase the expression, stability, and / or activity of the polypeptide. Factors affecting codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or gene or synthetic construct bias tables, (ii) variation in the degree of codon bias within an organism, a gene, or a set of genes, (iii) systematic variation of codons (including context), (iv) codon variation according to the tRNA by which it is decoded, (v) codon variation according to GC%, either globally or at one position of the triplet, (vi) variation in similarity to a reference sequence (e.g., a naturally occurring sequence), (vii) variation in codon frequency cut-off, (viii) structural properties of the mRNA transcribed from the DNA sequence, (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based, and / or (x) systematic variation of the codon set for each amino acid. In some embodiments, the codon-optimized polynucleotide can minimize ribozyme collisions and / or limit structural interference between the expression sequence and the IRES.

[0318] In certain embodiments, the circular RNAs provided herein are produced inside cells. In some embodiments, the precursor RNA is transcribed by a phage RNA polymerase in the cytoplasm or by host RNA polymerase II in the nucleus using a DNA template (e.g., in some embodiments, using the vectors provided herein) and then circularized.

[0319] In certain embodiments, the circular RNAs provided herein are injected into an animal (e.g., a human) such that the polypeptide encoded by the circular RNA molecule is expressed in the animal.

[0320] 3. Payload

[0321] In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the proteins listed in the following table.

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the expression sequence encodes a cytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN-β, IL-10, TGF-β, IL-4 or IL-35, or a functional fragment thereof. In some embodiments, the expression sequence encodes an immune checkpoint inhibitor. In some embodiments, the expression sequence encodes an agonist (e.g., a TNFR family member such as CD137L, OX40L, ICOS-L, LIGHT or CD70). In some embodiments, the expression sequence encodes a chimeric antigen receptor. In some embodiments, the expression sequence encodes an inhibitory receptor agonist (e.g., PDL1, PDL2, galectin-9, VISTA, B7H4 or MHCII) or an inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3 or TIM3). In some embodiments, the expression sequence encodes an inhibitory receptor antagonist. In some embodiments, the expression sequence encodes one or more TCR chains (α and β chains or γ and δ chains). In some embodiments, the expression sequence encodes a secreted T cell or immune cell adapter (e.g., a bispecific antibody such as BiTE that targets, e.g., CD3, CD137 or CD28 and a tumor-expressed protein such as CD19, CD20 or BCMA). In some embodiments, the expression sequence encodes a transcription factor (e.g., FOXP3, HELIOS, TOX1 or TOX2). In some embodiments, the expression sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39 / CD73). In some embodiments, the expression sequence encodes for GvHD (e.g., anti-HLA-A2 CAR-Treg).

[0335] In some embodiments, the polynucleotide encodes a protein composed of subunits, where the subunits are encoded by more than one gene. For example, the protein can be a heterodimer, where each chain or subunit of the protein is encoded by a separate gene. It is possible to deliver more than one circRNA molecule in a delivery vehicle, and each circRNA encodes a separate subunit of the protein. Alternatively, a single circRNA can be engineered to encode more than one subunit. In certain embodiments, separate circRNA molecules encoding separate subunits can be administered in separate delivery vehicles.

[0336] 3.1 Cytokines

[0337] Descriptions and / or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-27β, IFNγ, and / or TGFβ1 are provided herein and in the www.uniprot.org database under the following 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).

[0338] 3.2 PD-1 and PD-L1 Antagonists

[0339] In some embodiments, the PD-1 inhibitor is pembrolizumab, pidilizumab, or nivolumab. In some embodiments, nivolumab is described in W02006 / 121168. In some embodiments, pembrolizumab is described in W02009 / 114335. In some embodiments, pidilizumab is described in WO2009 / 101611. Additional anti-PD1 antibodies are described in U.S. Patent No. 8,609,089, US 2010028330, US 20120114649, WO2010 / 027827, and WO2011 / 066342.

[0340] In some embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, BMS-936559, or CK-301.

[0341] Descriptions and / or amino acid sequences of the heavy and light chains of PD-1 and / or PD-L1 antibodies are provided herein and in the www.drugbank.ca database under the following accession numbers: DB09037 (pembrolizumab), DB09035 (nivolumab), DB15383 (pidilizumab), DB11595 (atezolizumab), DB11945 (avelumab), and DB11714 (durvalumab).

[0342] 3.3 T Cell Receptors

[0343] TCRs are described using the international ImMunoGeneTics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. The native α-β heterodimeric TCR has an α-chain and a β-chain. Broadly speaking, each chain can comprise a variable region, a joining region, and a constant region, and the β-chain also typically contains a short diversity region between the variable and joining regions, although this diversity region is usually considered part of the joining region. Each variable region can contain three CDRs (complementary determining regions) embedded in framework sequences, one of which is a hypervariable region called CDR3. There are several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions that are distinguished by their framework, CDR1, and CDR2 sequences and by a partially defined CDR3 sequence. Vα types are designated by unique TRAV numbers in the IMGT nomenclature. Thus, "TRAV21" defines a TCR Vα region with a unique framework and CDR1 and CDR2 sequences and a CDR3 sequence that is partially defined by an amino acid sequence conserved from TCR to TCR but also contains an amino acid sequence that varies from TCR to TCR. In the same way, "TRBV5-1" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences but a CDR3 sequence that is only partially defined.

[0344] The joining regions of TCRs are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions are similarly defined by the IMGT TRAC and TRBC nomenclature.

[0345] The β-chain diversity region is designated by the abbreviation TRBD in the IMGT nomenclature, and as noted above, the tandem TRBD / TRBJ regions are generally considered together as the joining region.

[0346] The unique sequences defined by the IMGT nomenclature are widely known and are available 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 consequent time lag, the information therein sometimes needs to be confirmed by reference to the IMGT database.

[0347] Native TCRs exist as the heterodimers αβ or γδ. However, recombinant TCRs composed of αα or ββ homodimers have previously been shown to bind to peptide MHC molecules. Thus, the TCRs of the present invention can be heterodimeric αβ TCRs or can be αα or ββ homodimeric TCRs.

[0348] For adoptive therapy, an αβ heterodimeric TCR can be transfected, for example, with full-length chains having cytoplasmic and transmembrane domains. In certain embodiments, the TCRs of the invention can have introduced disulfide bonds between residues of the respective constant domains, as described, for example, in WO2006 / 000830.

[0349] The TCRs of the invention, particularly α-β heterodimeric TCRs, can comprise an α-chain TRAC constant domain sequence and / or a β-chain TRBC1 or TRBC2 constant domain sequence. The α- and β-chain constant domain sequences can 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 α- and / or β-chain constant domain sequences can also be modified by substituting a cysteine residue for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, which cysteine forms a disulfide bond between the α- and β-constant domains of the TCR.

[0350] Binding affinity (inversely proportional to the equilibrium constant K D d) and binding half-life (expressed as T1 / 2) can be determined by any suitable method. It should be understood that doubling the affinity of a TCR results in K D being halved. T1 / 2 is calculated as ln 2 divided by the dissociation rate (koff). Thus, doubling T1 / 2 results in koff being halved. The K D and koff values of a TCR are typically measured for the soluble form of the TCR, i.e., those forms that have been truncated to remove cytoplasmic domain residues and transmembrane domain residues. Thus, it should be understood that if the soluble form of a given TCR has the stated characteristics, then the TCR has improved binding affinity and / or binding half-life relative to the parental TCR. Preferably, the binding affinity or binding half-life of a given TCR is measured multiple times (e.g., 3 times or more) using the same assay protocol, and the average of the results is taken.

[0351] Because the TCRs of the invention have utility in adoptive therapy, the invention includes non-naturally occurring and / or purified and / or engineered cells, particularly T cells, that present the TCRs of the invention. There are many methods suitable for transfecting T cells with nucleic acids (such as DNA, cDNA, or RNA) encoding the TCRs of the invention (see, for example, Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCRs of the invention will be suitable for adoptive therapy-based treatment of cancers such as pancreatic cancer and liver cancer. As is known to those of skill in the art, there are many suitable methods for performing adoptive therapy (see, for example, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0352] As is well known in the art, the TCRs of the present invention can be post-translationally modified when expressed by transfected cells. Glycosylation is one such modification, which can include the covalent attachment of an oligosaccharide moiety to defined amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are well-known oligosaccharide attachment sites. The glycosylation state of a particular protein depends on many factors, including the protein sequence, protein conformation, and the availability of certain enzymes. In addition, the glycosylation state (i.e., the type of oligosaccharide, the covalent linkage, and the total number of linkages) can affect protein function. Therefore, it is often necessary to control glycosylation when producing recombinant proteins. The glycosylation of transfected TCRs can be controlled by mutations in the transfected gene (Kuball J et al. (2009), J ExpMed 206(2):463-475). Such mutations are also encompassed by the present invention.

[0353] The TCR can be specific for antigens in the following group: MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, 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 glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, α-actin-4, Bcr-Abl fusion protein, Casp-8, β-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, class I myosin, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, Lage-2, SP17 and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15 (58), CEA, NY-ESO (LAGE), SCP-1, Hom / Mel-40, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,.β.β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein (AFP), 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA242, 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-related protein), TAAL6, TAG72, TLP, and TPS.

[0354] 3.4 Transcription Factors

[0355] Regulatory T cells (Tregs) are important in maintaining homeostasis, controlling the amplitude and duration of inflammatory responses, and preventing autoimmune and allergic responses.

[0356] In general, Tregs are thought to be mainly involved in suppressing immune responses, partly acting as the "self-check" of the immune system to prevent overreaction. In particular, Tregs are involved in maintaining tolerance to self-antigens, harmless substances such as pollen or food, and eliminating autoimmune diseases.

[0357] Tregs are found throughout the body, including but not limited to the gut, skin, lungs, and liver. In addition, Treg cells can also be present in certain 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, and additional information can be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294):1-10, the disclosure of which is hereby incorporated by reference in its entirety.

[0358] Generally, it is known that Tregs require TGF-β and IL-2 for normal activation and development. Tregs that express high levels of the IL-2 receptor (IL-2R) are dependent on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β, both of which are potent immunosuppressive cytokines. In addition, Tregs are known to inhibit the ability of antigen-presenting cells (APCs) to stimulate T cells. One proposed mechanism of APC inhibition is through CTLA-4, which is expressed by Foxp3+ Tregs. It is thought that CTLA-4 can bind to B7 molecules on APCs and block these molecules or remove them by causing internalization, resulting in reduced availability of B7 and an inability to provide sufficient co-stimulation for the immune response. Additional 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 hereby incorporated by reference in its entirety.

[0359] Descriptions and / or amino acid sequences of FOXP3, STAT5B, and / or HELIOS are provided herein and in the www.uniprot.org database under the following accession numbers: Q9BZS1 (FOXP3), P51692 (STAT5b), and / or Q9UKS7 (HELIOS).

[0360] Foxp3

[0361] In some embodiments, the transcription factor is the forkhead box P3 transcription factor (Foxp3). Foxp3 has been shown to be a key regulator of Treg differentiation and activity. In fact, loss-of-function mutations in the Foxp3 gene have been shown to result in the fatal IPEX syndrome (immunodysregulation, polyendocrinopathy, enteropathy, X-linked). Patients with IPEX suffer from severe autoimmune responses, persistent eczema, and colitis. Regulatory T (Treg) cells that express Foxp3 play a key role in limiting intestinal inflammatory responses (Josefowicz, S.Z. et al. Nature, 2012, 482, 395-U1510).

[0362] STAT

[0363] Members of the signal transducer and activator of transcription (STAT) protein family are intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis, and differentiation. They primarily provide Janus kinase (JAK) activation associated with membrane receptors. Dysregulation of this pathway is frequently observed in primary tumors and results in increased angiogenesis, enhanced tumor survival, and immunosuppression. Gene knockout studies have provided evidence that STAT proteins are involved in the development and function of the immune system and play a role in maintaining immune tolerance and tumor surveillance.

[0364] There are 7 identified mammalian STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B), and STAT6.

[0365] Extracellular binding of cytokines or growth factors induces activation of receptor-associated Janus kinases, which phosphorylate specific tyrosine residues within STAT proteins via their SH2 domains, thus promoting dimerization. The phosphorylated dimer is then actively transported to the nucleus via the importin α / β ternary complex. Initially, STAT proteins were described as latent cytoplasmic transcription factors because phosphorylation was thought to be required for nuclear retention. However, unphosphorylated STAT proteins also shuttle between the cytosol and the nucleus and play a role in gene expression. Once STAT reaches the nucleus, it binds to a consensus DNA recognition motif called the γ-activated site (GAS) in the promoter region of cytokine-inducible genes and activates transcription. STAT proteins can be dephosphorylated by nuclear phosphatases, which results in STAT inactivation and subsequent export from the nucleus via the exportin-RanGTP complex.

[0366] In some embodiments, the STAT proteins of the present disclosure can be STAT proteins comprising modifications that regulate their expression levels or activities. In some embodiments, such modifications particularly include mutations that affect STAT dimerization, binding of STAT proteins to signaling partners, STAT protein localization, or STAT protein degradation. In some embodiments, the STAT proteins of the present disclosure have constitutive activity. In some embodiments, the STAT proteins of the present disclosure have constitutive activity due to constitutive dimerization. In some embodiments, the STAT proteins of the present disclosure have constitutive activity due to constitutive phosphorylation, as described by Onishi, M. et al., Mol. Cell. Biol., Vol. 18, No. 7, July 1998, pp. 3871-3879, the entire content of which is incorporated herein by reference.

[0367] 3.5 Chimeric antigen receptor

[0368] Chimeric antigen receptors (CARs or CAR-Ts) are genetically engineered receptors. These engineered receptors can be inserted into immune cells, including T cells, by circular RNAs as described herein and expressed by the immune cells. For CARs, a single receptor can be programmed to recognize a specific antigen and, upon binding to the antigen, activate the immune cells to attack and destroy the cells bearing the antigen. When these antigens are present on tumor cells, the immune cells expressing the CAR can target and kill the tumor cells. In some embodiments, a CAR encoded by a polynucleotide comprises (i) an antigen-binding molecule that specifically binds to a target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activation domain.

[0369] In some embodiments, the orientation of the CAR according to the present disclosure comprises an antigen-binding domain (such as an scFv) in tandem with a co-stimulatory domain and an activation domain. The co-stimulatory domain can comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple co-stimulatory domains can be used in tandem.

[0370] Antigen-binding domain

[0371] By incorporating an antigen-binding molecule that interacts with the targeting antigen, a CAR can be engineered to bind to an antigen (such as a cell surface 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 antibody heavy and light chains 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. The scFv retains the ability of the parental antibody to specifically interact with the target antigen. ScFvs can be used in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain together with other CAR components. Ibid. See also Krause et al., J. Exp. Med., Vol. 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It should be understood that the antigen-binding molecule is generally included within the extracellular portion of the CAR such that it can recognize and bind to the target antigen. Bispecific and multispecific CARs are encompassed within the scope of the present invention and are specific for more than one target.

[0372] In some embodiments, the antigen-binding molecule comprises a single chain in which the heavy chain variable region and the light chain variable region are linked by a linker. In some embodiments, VH is located at the N-terminus of the linker and VL is located at the C-terminus of the linker. In other embodiments, VL is located at the N-terminus of the linker and VH is located at the C-terminus of the linker. In some embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 amino acids.

[0373] 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 anti-carrier protein or other synthetic protein capable of specifically binding to a target protein.

[0374] In some embodiments, the CAR comprises an antigen-binding domain that is specific for an antigen 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 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), prostate enzyme, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macropain factor) subunit beta type 9 (LMP2), glycoprotein 100 (gp100), oncogenic fusion protein (bcr-ab1) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Ab1), tyrosinase, ephrin A type 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 ceramide (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 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, prostaglandin, survival, telomerase, prostate cancer tumor antigen-1, melanoma antigen recognized by T cells 1, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma cell apoptosis inhibitor (ML-IAP), ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosomal protein-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A 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 (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89),Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, αvβθ integrin, αvβ6 integrin, alpha-fetoprotein (AFP), B7-H6, ca-125, CA9, CD44, CD44v7 / 8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), kinase insert domain receptor (KDR), k-light chain, L1 cell adhesion molecule, MUC18, NKG2D, carcinoembryonic antigen (h5T4), tumor / testis antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), hepatitis B surface antigen binding protein (HBsAg), viral capsid antigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, hemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, human metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lymphotropic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, 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.

[0375] Hinge / spacer domain

[0376] In some embodiments, the CARs of the present disclosure include hinge or spacer domains. In some embodiments, the hinge / spacer domain may include a truncated hinge / spacer domain (THD), and the THD domain is a truncated form of the full-length hinge / spacer domain (“CHD”). In some embodiments, the extracellular domain is from or derived from (e.g., includes all or a fragment thereof) ErbB2, glycophorin A (GpA), CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8[T CDlla(IT GAL), CDl lb(IT GAM), CDl lc(ITGAX), 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 (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-delta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), Inducible T cell co-stimulator (ICOS), LFA-1 (CDl la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class I molecule, MHCClass II molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, activating NK cell receptors, Toll ligand receptors, and fragments or combinations thereof. The hinge or spacer domain can be derived from a natural source or a synthetic source.

[0377] 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 the distance between the antigen-binding molecule and the cell membrane surface expressing the CAR. 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 of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof. In some embodiments, the hinge or spacer domain comprises, is from, or is derived from the hinge / spacer of CD8α. In some embodiments, the hinge or spacer domain comprises, is from, or is derived from the hinge / spacer of CD28. In some embodiments, the hinge or spacer domain comprises a fragment of the hinge / spacer of CD8α or a fragment of the hinge / spacer of CD28, wherein the fragment is any fragment that is less than the full hinge / spacer. In some embodiments, the fragment of the CD8α hinge / spacer or the fragment of the CD28 hinge / spacer comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or C-terminus or both of the CD8α hinge / spacer or the CD28 hinge / spacer.

[0378] Transmembrane domain

[0379] The CARs of the present disclosure can also comprise a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain can be designed to be fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified (e.g., by amino acid substitution) to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins so as to minimize interactions with other members of the receptor complex. The transmembrane domain can be derived from a natural source or a synthetic source. In the case where the source is a natural source, the domain can be derived from any membrane-binding protein or transmembrane protein.

[0380] The transmembrane region can be derived from (i.e., 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, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRTAM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptors, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAE, ITGAM, 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; CD11a / CD18), MHC class I molecules, 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; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptors, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncations or combinations thereof.

[0381] In some embodiments, suitable intracellular signaling domains include, but are not limited to, activating 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.

[0382] In some embodiments, the receptor tyrosine kinase can be derived from (e.g., comprise) 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), receptor tyrosine kinase-like orphan receptor 2 (ROR2), muscle-associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage stimulating 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphAl), EPH receptor A2 (EphA2), (EPH receptor A3) EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), 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 proto-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).

[0383] Costimulatory domain

[0384] 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 a preferred embodiment, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3zeta (δ). 4-1BB, CD28, CD3δ may each comprise less than the full-length 4-1BB, CD28, or CD3δ, respectively. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Krause et al. and Finney et al. (supra); Song et al., Blood 119:696-706 (2012); Kalos et al., SciTransl.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).

[0385] In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO: 318 or 320.

[0386] Intracellular signaling domain

[0387] The intracellular (signaling) domain of the engineered T cells disclosed herein can provide signaling to an activation domain, which then activates at least one normal effector function of the immune cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

[0388] In some embodiments, suitable intracellular signaling domains include (e.g., comprise) but are not limited to 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptors, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligands that specifically bind to CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptors, TRANCE / RANKL, VLA1 or VLA-6 or fragments, truncations or combinations thereof.

[0389] CD3 is an element of the T cell receptor on native T cells and has been shown to be an important intracellular activation element in CARs. In some embodiments, the CD3 is CD3δ. In some embodiments, the activation domain comprises an amino acid sequence having 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% identity to the polypeptide sequence of SEQ ID NO: 319.

[0390] 3.6 Trispecific antigen-binding proteins and bispecific antigen-binding proteins

[0391] Disclosed herein are circular RNA polypeptides encoding trispecific antigen-binding proteins (TRITEs), bispecific antigen-binding proteins (BITEs), functional fragments thereof, and pharmaceutical compositions thereof. Also provided herein are recombinant expression vectors that can be used to prepare circular RNAs encoding trispecific antigen-binding proteins or bispecific antigen-binding proteins, and cells comprising the circular RNAs of the present invention. Also provided are methods of using the disclosed trispecific antigen-binding proteins or bispecific antigen-binding proteins for preventing and / or treating liver diseases, disorders, and conditions. The trispecific antigen-binding protein is capable of specifically binding to a target antigen, such as a cancer antigen, as well as CD3, TCR, CD16A, or NKp46, and a liver retention domain or a half-life extending domain, such as a domain that binds human serum albumin (HSA). In some embodiments, a TRITE or BITE is produced in the liver of a patient after administration to the patient in need thereof of a composition comprising the circular RNA polypeptide of the present invention.

[0392] In one aspect, the trispecific antigen-binding protein comprises a domain (A) that specifically binds to CD3, TCR, CD16A, or NKp46, a domain (B) that specifically binds to a half-life extending molecule or a liver retention molecule, and a domain (C) that specifically binds to a target antigen, such as a cancer cell antigen. The three domains in the trispecific antigen-binding protein can be arranged in any order. Thus, the domain order of the trispecific antigen-binding protein is contemplated to be in any of the following orders: (A)-(B)-(C), (A)-(C)-(B), (B)-(A)-(C), (B)-(C)-(A), (C)-(B)-(A), or (C)-(A)-(B).

[0393] In some embodiments, the trispecific antigen-binding protein has a domain order of (A)-(B)-(C). In some embodiments, the trispecific antigen-binding protein has a domain order of (A)-(C)-(B). In some embodiments, the trispecific antigen-binding protein has a domain order of (B)-(A)-(C). In some embodiments, the trispecific antigen-binding protein has a domain order of (B)-(C)-(A). In some embodiments, the trispecific antigen-binding protein has a domain order of (C)-(B)-(A). In some embodiments, the trispecific antigen-binding protein has a domain order of (C)-(A)-(B).

[0394] In one embodiment, the bispecific antigen-binding protein comprises a domain (A) that specifically binds to CD3, TCR, CD16A, or NKp46 and a domain (B) that specifically binds to a target antigen. The two domains in the bispecific antigen-binding protein can be arranged in any order. Thus, the domain order of the bispecific antigen-binding protein can be considered to be: (A)-(B) or (B)-(A).

[0395] The trispecific or bispecific antigen-binding proteins described herein are designed to allow for the specific targeting of cells expressing a target antigen by recruiting cytotoxic T cells or NK cells. This improves efficacy compared to ADCC (antibody-dependent cell-mediated cytotoxicity), which uses full-length antibodies against a single antigen and cannot directly recruit cytotoxic T cells. In contrast, the trispecific or bispecific antigen-binding proteins can crosslink cytotoxic T cells or NK cells with cells expressing the target antigen in a highly specific manner by engaging CD3 molecules specifically expressed on these cells, thereby directing the cytotoxic potential of the recruited T cells or NK cells towards the target cells. The trispecific or bispecific antigen-binding proteins described herein engage cytotoxic T cells by binding to surface-expressed CD3 protein (which forms part of the TCR) or CD16A or NKp46 (which activates NK cells). Simultaneous binding of several trispecific or bispecific antigen-binding proteins to CD3 and the target antigen expressed on a particular cell surface causes T cell activation and mediates the subsequent lysis of cells expressing the particular target antigen. Thus, trispecific or bispecific antigen-binding proteins are expected to exhibit strong, specific, and effective target cell killing. In some embodiments, the trispecific or bispecific antigen-binding proteins described herein stimulate cytotoxic T cells to kill target cells to eliminate pathogenic cells (e.g., tumor cells, virus- or bacteria-infected cells, autoreactive T cells, etc.). In some embodiments, cells are selectively eliminated, thereby reducing the likelihood of toxic side effects. In some embodiments, an anti-41bb or CD137 binding domain is used as a T cell engager.

[0396] Immune cell binding domain

[0397] Specificity of the T cell response is mediated by TCR recognition of antigen presented in the context of the major histocompatibility complex MHC. As part of the TCR, CD3 is a protein complex containing the CD3γ (gamma) chain, CD3δ (delta) chain, and two CD3ε (epsilon) chains present on the cell surface. CD3 associates with the α (alpha) and β (beta) chains of the TCR as well as CD3 zeta (zeta) to form the complete TCR. Clustering of CD3 on T cells (such as by immobilized anti-CD3 antibodies) results in T cell activation, similar to engagement of the T cell receptor, but independent of its clonotypic specificity.

[0398] In one aspect, the bispecific and trispecific proteins described herein comprise domains that specifically bind to CD3. In one aspect, the trispecific proteins described herein comprise domains that specifically bind to human CD3. In some embodiments, the trispecific proteins described herein comprise domains that specifically bind to CD3γ. In some embodiments, the trispecific proteins described herein comprise domains that specifically bind to CD36. In some embodiments, the trispecific proteins described herein comprise domains that specifically bind to CD3ε.

[0399] In other embodiments, the trispecific proteins described herein comprise domains that specifically bind to TCR. In certain cases, the trispecific proteins described herein comprise domains that specifically bind to the α-chain of TCR. In certain cases, the trispecific proteins described herein comprise domains that specifically bind to the β-chain of TCR.

[0400] In some embodiments, the trispecific antigen-binding protein or bispecific antigen-binding protein comprises an NKp46 specific binder. In some embodiments, the trispecific antigen-binding protein or bispecific antigen-binding protein comprises a CD16A specific binder.

[0401] In some embodiments, the CD3, TCR, NKp46 or CD16A binding domains of the antigen-binding protein can be any domain that binds to CD3, TCR, NKp46 or CD16A, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some cases, it is beneficial for the CD3, TCR, NKp46 or CD16A binding domains of the trispecific antigen-binding protein to be derived from the same species in which the trispecific antigen-binding protein will ultimately be used. For example, for use in humans, it can be beneficial for the CD3, TCR, NKp46 or CD16A binding domains of the trispecific antigen-binding protein to comprise human or humanized residues of the antigen-binding domains from antibodies or antibody fragments.

[0402] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD-3, TCR, NKp46 or CD16A binding domain comprises one or more (e.g., all three) light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions 2 (LC CDR2) and light chain complementarity determining regions 3 (LC CDR3) of the humanized or human anti-CD-3, TCR, NKp46 or CD16A binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2) and heavy chain complementarity determining regions 3 (HC CDR3) of the humanized or human anti-CD-3, TCR, NKp46 or CD16A binding domain described herein, e.g., a humanized or human anti-CD-3, TCR, NKp46 or CD16A binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0403] In some embodiments, the humanized or human anti-CD3, TCR, NKp46 or CD16A binding domain comprises a humanized or human heavy chain variable region that is specific for CD3, TCR, NKp46 or CD16A, wherein the heavy chain variable region specific for CD3, TCR, NKp46 or CD16A comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0404] In certain cases, the complementarity determining regions of the heavy and / or light chains are derived from known anti-CD3 antibodies such as muromonab-CD3 (OKT3), ocrelizumab (TRX4), tiprilizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

[0405] In some embodiments, the anti-NKp46 binding domain comprises an antibody or fragment thereof described in U.S. Patent Application 16 / 451051. In some embodiments, the anti-NKp46 binding domain comprises the antibody BAB281, 9E2, 195314 or a fragment thereof.

[0406] In one embodiment, the anti-CD3, TCR, NKp46 or CD16A binding domain is a single-chain variable fragment (scFv) comprising a light chain and a heavy chain containing the amino acid sequences provided herein. In one embodiment, the anti-CD3, TCR, NKp46 or CD16A binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but no more than 30, 20 or 10 modifications (e.g., substitutions), of the amino acid sequence of the light chain variable region provided herein, or a sequence having 95%-99% identity to the amino acid sequence provided herein; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but no more than 30, 20 or 10 modifications (e.g., substitutions), of the amino acid sequence of the heavy chain variable region provided herein, or a sequence having 95%-99% identity to the amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD3 binding domain is an scFv and comprises a light chain variable region of the amino acid sequence described herein attached via an scFv linker to a heavy chain variable region of the amino acid sequence described herein. The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region - scFv linker - heavy chain variable region or heavy chain variable region - scFv linker - light chain variable region.

[0407] In some embodiments, the CD3, TCR, NKp46 or CD16A binding domain of the trispecific antigen-binding protein has an affinity for CD3, TCR, NKp46 or CD16A on cells expressing CD3, TCR, NKp46 or CD16A, with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less or 0.5 nM or less. In some embodiments, the CD3 binding domain of the MSLN trispecific antigen-binding protein has an affinity for CD3ε, γ or δ, with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less or 0.5 nM or less. In other embodiments, the CD3, TCR, NKp46 or CD16A binding domain of the trispecific antigen-binding protein has a low affinity for CD3, TCR, NKp46 or CD16A, i.e., about 100 nM or greater.

[0408] The affinity for binding to CD3, TCR, NKp46 or CD16A can be determined, for example, by the ability of the trispecific antigen-binding protein itself or its CD3, TCR, NKp46 or CD16A binding domain to bind to CD3, TCR, NKp46 or CD16A coated on an assay plate; displayed on the surface of microbial cells; in solution, etc. The binding activity of the trispecific antigen-binding protein of the present disclosure itself or its CD3, TCR, NKp46 or CD16A binding domain to CD3, TCR, NKp46 or CD16A can be measured by immobilizing a ligand (e.g., CD3, TCR, NKp46 or CD16A) or the trispecific antigen-binding protein itself or its CD3, TCR, NKp46 or CD16A binding domain to beads, substrates, cells, etc. Additives can be added in an appropriate buffer, and the binding partners are incubated at a given temperature for a period of time. After washing to remove unbound material, the bound protein can be released with, for example, SDS, a buffer with a high pH, etc., and analyzed, for example, by surface plasmon resonance (SPR).

[0409] In some embodiments, the bispecific antigen-binding protein or bispecific antigen-binding proteins comprise a TCR-binding domain. In some embodiments, the TCR-binding domain is a viral antigen or a fragment thereof. In some embodiments, the viral antigen is from the following families: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV or HIV-III; and other isolates, such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., virus strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, bunyavirus, phlebotomus fever virus, and nairo virus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the pathogen of hepatitis D (thought to be a defective satellite of hepatitis B virus), hepatitis C; Norwalk and related viruses, and astrovirus).

[0410] Linker

[0411] In the trispecific proteins described herein, the domains are connected by internal linkers L1 and L2, where L1 connects the first and second domains of the trispecific protein, and L2 connects the second and third domains of the trispecific protein. In some embodiments, linkers L1 and L2 have optimized lengths and / or amino acid compositions. In some embodiments, linkers L1 and L2 have the same length and amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, internal linker L1 and / or L2 consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain cases, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, internal linker L1 and / or L2 consists of 15, 20, or 25 amino acid residues. In some embodiments, these internal linkers consist of about 3 to about 15, such as 8, 9, or 10 consecutive amino acid residues. With respect to the amino acid composition of internal linkers L1 and L2, peptides are selected that have properties that confer flexibility to the trispecific protein, do not interfere with the binding domains, and resist proteolytic cleavage. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for connecting domains in a trispecific protein include, but are not limited to, (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GGGGS)n, (GGGGG)n, or (GGG)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker L1 and / or L2 is (GGGGS)4 or (GGGGS)3.

[0412] Half-life extension domain

[0413] Domains that extend the half-life of antigen-binding domains are contemplated herein. Contemplating such domains includes, but is not limited to, albumin-binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0414] Human albumin (ALB) is the most abundant protein in plasma, present at approximately 50 mg / ml, and has a half-life of approximately 20 days in humans. ALB is used to maintain plasma pH, promote colloid osmotic pressure, act as a carrier for many metabolites and fatty acids, and serve as the major drug transport protein in plasma.

[0415] Non-covalent association with albumin extends the elimination half-life of short-lived proteins.

[0416] In one aspect, the trispecific proteins described herein include a half-life extension domain, such as a domain that specifically binds to ALB. In some embodiments, the ALB-binding domain of the trispecific antigen-binding protein can be any domain that binds to ALB, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the ALB-binding domain is a single-chain variable fragment (scFv), a single-domain antibody, such as a camelid-derived single-domain antibody specific for HSA, a peptide, a ligand, or the heavy-chain variable domain (VH), light-chain variable domain (VL), and variable domain (VHH) of a small molecule entity. In certain embodiments, the ALB-binding domain is a single-domain antibody. In other embodiments, the HSA-binding domain is a peptide. In other embodiments, the HSA-binding domain is a small molecule. It is contemplated that in some embodiments, the HSA-binding domain of the MSLN trispecific antigen-binding protein is relatively small and does not exceed 25 kD, does not exceed 20 kD, does not exceed 15 kD, or does not exceed 10 kD. In certain instances, if the ALB-binding domain is a peptide or a small molecule entity, it is 5 kD or smaller.

[0417] The half-life extension domain of the trispecific antigen-binding protein provides altered pharmacodynamics and pharmacokinetics of the trispecific antigen-binding protein itself. As described above, the half-life extension domain extends the elimination half-life. The half-life extension domain also alters pharmacodynamic properties, including altering the tissue distribution, penetration, and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue diffusion within the tissue, and enhanced efficacy compared to a protein without a half-life extension binding domain. In one embodiment, the treatment method effectively and efficiently utilizes a reduced amount of the trispecific antigen-binding protein, thereby producing reduced side effects, such as reduced non-tumor cell cytotoxicity.

[0418] Furthermore, the binding affinity of the half-life extension domain can be selected to target a specific elimination half-life in a particular trispecific antigen-binding protein. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As described above, the binding affinity for ALB is determined by known methods such as surface plasmon resonance (SPR).

[0419] Liver retention domain

[0420] This text contemplates domains that permit and promote higher retention of trispecific antigen-binding proteins in the liver. The liver retention domains of trispecific antigen-binding proteins target hepatocyte moieties. In one embodiment, hepatocytes include, but are not limited to, hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells.

[0421] In one embodiment, hepatocytes contain receptors that bind to liver targeting moieties. In one embodiment, liver targeting moieties include, but are not limited to, lactose, cyanuric chloride, cellobiose, polylysine, polyarginine, mannose 6-phosphate, PDGF, human serum albumin, galactoside, galactosamine, linoleic acid, apolipoprotein A-1, acetyl-CKNEKKNIERNNKLKQPP-amide, glycyrrhizin, lactobionic acid, mannose-BSA, BSA, poly-ACO-HAS, KLGR peptide, hyaluronic acid, IFN-α, cRGD peptide, 6-phosphate-HSA, retinol, lactobiotin, galactoside, pullulan, stigmasterol glucoside, asialoseryomucoid, glycyrrhetinic acid / glycyrrhizin, linoleic acid, AMD3100, cleavable hyaluronic acid-glycyrrhetinic acid, hepatitis B virus pre-S1-derived lipoprotein, Apo-A1, or LDL. In one embodiment, hepatocyte receptors include, but are not limited to, galactose receptor, mannose receptor, scavenger receptor, low density lipoprotein receptor, HARE, CD44, IFNα receptor, type VI collagen receptor, 6-phosphate / insulin-like growth factor 2 receptor, platelet-derived growth factor receptor β, RBP receptor, αVβ3 integrin receptor, ASGP receptor, glycyrrhetinic acid / glycyrrhizin receptor, PPAR, heparan sulfate proteoglycan receptor, CXC receptor type 4, glycyrrhetinic acid receptor, HBVP receptor, HDL receptor, scavenger receptor class B member 1 LDL receptor, or combinations thereof.

[0422] Target antigen-binding domain

[0423] The trispecific antigen-binding proteins and bispecific antigen-binding proteins described herein contain domains that bind to a target antigen. The target antigen is involved in a disease, disorder, or affliction (e.g., cancer) and / or is associated with a disease, disorder, or affliction. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is NY-ESO-1, SSX-2, Sp 17, AFP, glypican-3, Gpa33, annexin-A2, WT1, PSMA, Midkine, PRAME, survivin, MUC-1, P53, CEA, RAS, Hsp70, Hsp27, squamous cell carcinoma antigen (SCCA), GP73, TAG-72, or a protein in the MAGE family.

[0424] In some embodiments, the target antigen is an antigen found on non-hepatic tumor cells that have metastasized to the liver.In some embodiments, the bispecific antigen-binding protein or trispecific antigen-binding protein comprises a target antigen-binding domain that is specific for the following group: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2, mesothelin, interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), 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), prostate enzyme, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macropain factor) subunit beta type 9 (LMP2), glycoprotein 100 (gp100), oncogenic fusion protein (bcr-ab1) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Ab1), tyrosinase, ephrin A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97 or CD179a.In some embodiments, the target antigen is an antigen associated with a viral disease, such as a viral antigen. In some embodiments, the target antigen is a hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E antigen.

[0425] The design of the trispecific antigen-binding proteins described herein allows the binding domain for the liver target antigen to be flexible, as the binding domain for the liver target antigen can be any type of binding domain, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the binding domain for the liver target antigen is a single-chain variable fragment (scFv), a single-domain antibody, such as the heavy-chain variable domain (VH), light-chain variable domain (VL), and variable domain (VHH) of a single-domain antibody from camelids. In other embodiments, the binding domain for the liver target antigen is a non-Ig binding domain, i.e., an antibody mimetic, such as an anticalin, affilin, affibody molecule, affimer, affitin, alphabodies, avimer, DARPin, fynomer, kunitz domain peptide, and single-domain antibody. In other embodiments, the binding domain for the liver target antigen is a ligand or peptide that binds or associates with the target antigen.

[0426] 3.7PAH

[0427] In some embodiments, the present invention provides methods and compositions for delivering a circRNA encoding PAH to a subject for the treatment of phenylketonuria (PKU). Suitable PAH circRNAs encode any full-length, fragment, or portion of the PAH protein that can replace the activity of the naturally occurring PAH protein and / or reduce the intensity, severity, and / or frequency of one or more symptoms associated with PKU.

[0428] In some embodiments, the RNA sequences suitable for the present invention include circRNA sequences encoding the human PAH protein.

[0429] In some embodiments, the suitable RNA sequences can be RNA sequences encoding homologs or analogs of human PAH. As used herein, a homolog or analog of the human PAH protein can be a modified human PAH protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type or naturally occurring human PAH protein, while retaining significant PAH protein activity.

[0430] The present invention can be used to treat subjects suffering from or prone to phenylketonuria (PKU). PKU is an autosomal recessive metabolic genetic disorder characterized by mutations in the gene for the liver enzyme phenylalanine hydroxylase (PAH), rendering it non-functional. PAH is necessary for the metabolism of the amino acid phenylalanine (Phe) to the amino acid tyrosine (Tyr). When PAH activity is reduced, phenylalanine accumulates and is converted to phenylpyruvic acid (also known as phenylketone) that can be detected in the urine.

[0431] Phenylalanine is a large neutral amino acid (LNAA). LNAAs compete for transport across the blood-brain barrier (BBB) via large neutral amino acid transporters (LNAATs). Excess Phe in the blood saturates the transporters and tends to reduce the levels of other LNAAs in the brain. Since several of these other amino acids are necessary for protein and neurotransmitter synthesis, Phe accumulation hinders brain development and can lead to mental retardation.

[0432] In addition to hindering brain development, the disease can also present clinically with a variety of symptoms, including seizures, albinism with hyperactivity, developmental delay, rash (eczema), microcephaly, and / or a "mousy" odor in the sweat and urine of infants, which is due to phenylacetic acid, a ketone produced). Untreated children are usually normal at birth but have delayed intellectual and social skills, a significantly lower-than-normal head size, and often exhibit progressive damage to brain function. As children grow and develop, additional symptoms often appear, including hyperactivity, jerking of the arms or legs, EEG abnormalities, rash, tremors, seizures, and severe learning disabilities. However, PKU is usually included in the routine neonatal screening panel in most countries, typically performed 2 - 7 days after birth.

[0433] If PKU is diagnosed early enough, affected newborns can grow with relatively normal brain development but can only manage and control Phe levels through diet or a combination of diet and medication. All PKU patients must adhere to a special diet low in Phe for optimal brain development. The diet requires strict restriction or elimination of Phe-rich foods such as meat, chicken, fish, eggs, nuts, cheese, beans, milk, and other dairy products. Starchy foods such as potatoes, bread, pasta, and corn must also be monitored. Infants can still breastfeed to receive all the benefits of breast milk, but the quantity must also be monitored, and they will require supplementation of missing nutrients. The sweetener aspartame in many diet foods and soft drinks must also be avoided because aspartame contains phenylalanine.

[0434] Throughout life, patients may use supplemental infant formula, pills, or foods with a special formula to obtain amino acids and other essential nutrients that would otherwise be lacking in a low-phenylalanine diet. Some Phe is necessary for the synthesis of many proteins and for proper growth, but its levels must be strictly controlled in patients with PKU. In addition, PKU patients must take tyrosine supplements, which are normally derived from phenylalanine. Other supplements may include fish oil to replace long-chain fatty acids missing from a standard Phe-free diet and to improve neurodevelopment and iron or carnitine. Another potential therapy for PKU is tetrahydrobiopterin (BH4), which is a cofactor for Phe oxidation that can lower blood Phe levels in some patients. Patients who respond to BH4 therapy may also be able to increase the amount of natural protein they can consume.

[0435] In some embodiments, expression of the PAH protein is detectable in the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0436] In some embodiments, administration of the provided composition results in a PAH protein expression level in the liver that is at or above about 100 ng / mg, about 200 ng / mg, about 300 ng / mg, about 400 ng / mg, about 500 ng / mg, about 600 ng / mg, about 700 ng / mg, about 800 ng / mg, about 900 ng / mg, about 1000 ng / mg, about 1200 ng / mg, or about 1400 ng / mg of total protein.

[0437] In some embodiments, the expression of the PAH protein can be detected 1 to 96 hours after administration. For example, in some embodiments, the expression of the PAH protein can be detected 1 to 84 hours, 1 to 72 hours, 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2 to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4 to 84 hours, 4 to 72 hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 96 hours, 6 to 84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 96 hours, 8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 96 hours, 10 to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48 hours, 10 to 36 hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours, 12 to 84 hours, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 hours to 96 hours, 72 hours to 84 hours, or 84 hours to 96 hours after administration. For example, in certain embodiments, the expression of the PAH protein can be detected 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after administration. In some embodiments, the expression of the PAH protein can be detected 1 day to 7 days after administration. For example, in some embodiments, the PAH protein can be detected 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after administration. In some embodiments, the expression of the PAH protein can be detected 1 week to 8 weeks after administration. For example, in some embodiments, the expression of the PAH protein can be detected 1 week, 2 weeks, 3 weeks, and / or 4 weeks after administration. In some embodiments, the expression of the PAH protein can be detected 1 month after administration.

[0438] 3.8 CPS1

[0439] In some embodiments, the present invention provides methods and compositions for delivering a circRNA encoding CPS1 to a subject for treating CPS1 deficiency. Suitable CPS1 circRNAs encode any full-length, fragment, or portion of the CPS1 protein that can replace the activity of the naturally occurring CPS1 protein and / or reduce the intensity, severity, and / or frequency of one or more symptoms associated with CPS1 deficiency.

[0440] In some embodiments, RNA sequences suitable for the present invention include circRNA sequences encoding the human CPS1 protein.

[0441] In some embodiments, suitable RNA sequences can be RNA sequences encoding homologs or analogs of human CPS1. As used herein, a homolog or analog of the human CPS1 protein can be a modified human CPS1 protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type or naturally occurring human CPS1 protein, while retaining significant CPS1 protein activity.

[0442] Carbamoyl phosphate synthetase I (CPS1) catalyzes the conversion of ammonia, bicarbonate, and 2 ATPs to form carbamoyl phosphate in the first step of the urea cycle. It also plays a role in the biosynthesis of arginine, which is in turn a substrate for the biosynthesis of NO, for example in the case of endotoxin shock (see Shoko Tabuchi et al., Regulation of Genes for Inducible Nitric Oxide Synthase and Urea Cycle Enzymes in Rat Liver in Endotoxin Shock, Biochemical and Biophysical Research Communications 268, 221-224 (2000)). CPS 1 should be distinguished from the cytoplasmic enzyme CPS 2, which also plays a role in the urea cycle but processes the substrate glutamine. CPS 1 is known to be located in the mitochondria and is present in large amounts in this form in liver tissue (it accounts for 2%-6% of total liver protein). Its amino acid sequence and gene localization have long been known (see Haraguchi Y. et al., Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I: molecular analysis of hyperammonemia, Gene November 1, 1991; 107(2); 335-340; also see applicant's publication WO03 / 089933A1). Regarding its physiological role, reference can be made to review articles such as H.M. Holder et al., Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product, CMLS, Cell. Mol. Life Sci. 56 (1999) 507-522, and the references cited therein, as well as the introduction to the publication of Mikiko Ozaki et al., Enzyme-Linked Immunosorbent Assay of Carbamoylphosphate Synthetase I: Plasma Enzyme in Rat Experimental Hepatitis and Its Clearance, Enzyme Protein 1994, 95:48:213-221.

[0443] Carbamoyl phosphate synthetase I (CPS1) deficiency is a genetic disorder characterized by mutations in the gene for carbamoyl phosphate synthetase I, which affects its ability to catalyze the synthesis of carbamoyl phosphate from ammonia and bicarbonate. This reaction is the first step in the urea cycle, which is important for removing excess urea from the cell. Defects in the CPS1 protein disrupt the urea cycle and prevent the liver from properly processing excess nitrogen into urea.

[0444] In some embodiments, administration of the provided compositions results in a CPS1 protein expression level in the liver that reaches or is higher than about 100 ng / mg, about 200 ng / mg, about 300 ng / mg, about 400 ng / mg, about 500 ng / mg, about 600 ng / mg, about 700 ng / mg, about 800 ng / mg, about 900 ng / mg, about 1000 ng / mg, about 1200 ng / mg, or about 1400 ng / mg of total protein.

[0445] In some embodiments, the expression of CPS1 protein can be detected 1 to 96 hours after administration. For example, in some embodiments, the expression of CPS1 protein can be detected 1 to 84 hours, 1 to 72 hours, 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2 to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4 to 84 hours, 4 to 72 hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 96 hours, 6 to 84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 96 hours, 8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 96 hours, 10 to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48 hours, 10 to 36 hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours, 12 to 84 hours, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 hours to 96 hours, 72 hours to 84 hours or 84 hours to 96 hours after administration. For example, in certain embodiments, the expression of CPS1 protein can be detected 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and / or 72 hours after administration. In some embodiments, the expression of CPS1 protein can be detected 1 day to 7 days after administration. For example, in some embodiments, the expression of CPS1 protein can be detected 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days after administration. In some embodiments, the expression of CPS1 protein can be detected 1 week to 8 weeks after administration. For example, in some embodiments, the expression of CPS1 protein can be detected 1 week, 2 weeks, 3 weeks and / or 4 weeks after administration. In some embodiments, the expression of CPS1 protein can be detected 1 month after administration.

[0446] In some embodiments, administration of the composition results in a decrease in the ammonia level in the subject as compared to the baseline level prior to treatment. Typically, the baseline level in the subject is measured immediately prior to treatment. Typically, the ammonia level is measured in a biological sample. Suitable biological samples include, for example, whole blood, plasma, serum, urine, or cerebrospinal fluid.

[0447] In some embodiments, administration of the composition results in a decrease in the ammonia level in a biological sample (e.g., a serum, plasma, or urine sample) of at least about 10%, at least about 15%, 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 55%, 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%, or at least about 95% as compared to the baseline level in the subject immediately prior to treatment.

[0448] In some embodiments, administration of the composition provided herein results in a decrease in the ammonia level in plasma or serum as compared to the baseline ammonia level in the subject immediately prior to treatment. In some embodiments, administration of the provided composition results in a decrease in the ammonia level in plasma or serum as compared to the ammonia level in an untreated subject. In some embodiments, administration of the composition results in a decrease in the ammonia level in the plasma or serum of the subject to about 3000 μmol / L or lower, about 2750 μmol / L or lower, about 2500 μmol / L or lower, about 2250 μmol / L or lower, about 2000 μmol / L or lower, about 1750 μmol / L or lower, about 1500 μmol / L or lower, about 1250 μmol / L or lower, about 1000 μmol / L or lower, about 750 μmol / L or lower, about 500 μmol / L or lower, about 250 μmol / L or lower, about 100 μmol / L or lower, or about 50 μmol / L or lower. In a particular embodiment, administration of the composition results in a decrease in the ammonia level in plasma or serum to about 50 μmol / L or lower.

[0449] 3.9 ADAMTS13

[0450] In some embodiments, the present invention provides methods and compositions for delivering a circRNA encoding ADAMTS13 to a subject for the treatment of thrombotic thrombocytopenic purpura (TTP). Suitable ADAMTS13 circRNAs encode any full-length ADAMTS13 protein or a functional fragment or portion thereof that can replace the naturally occurring ADAMTS13 protein and / or reduce the intensity, severity, and / or frequency of one or more symptoms associated with TTP.

[0451] In some embodiments, the RNA sequences of the present invention comprise a circRNA sequence encoding a human ADAMTS13 protein.

[0452] In some embodiments, the RNA sequence can be an RNA sequence encoding a homolog or analog of human ADAMTS13. As used herein, a homolog or analog of the human ADAMTS13 protein can be a modified human ADAMTS13 protein that contains one or more amino acid substitutions, deletions, and / or insertions as compared to the wild-type or naturally occurring human ADAMTS13 protein, while retaining significant ADAMTS13 protein activity.

[0453] The ADAMTS13 enzyme cleaves von Willebrand factor, which in its uncleaved form interacts with platelets and causes them to stick together and adhere to the blood vessel wall, thereby forming a clot. Defects in ADAMTS13 are associated with TTP.

[0454] In some embodiments, administration of the provided composition results in an ADAMTS13 protein expression level in the liver that reaches or is higher than about 100 ng / mg, about 200 ng / mg, about 300 ng / mg, about 400 ng / mg, about 500 ng / mg, about 600 ng / mg, about 700 ng / mg, about 800 ng / mg, about 900 ng / mg, about 1000 ng / mg, about 1200 ng / mg, or about 1400 ng / mg of total protein.

[0455] In some embodiments, the expression of ADAMTS13 protein can be detected 1 to 96 hours after administration. For example, in some embodiments, the expression of ADAMTS13 protein can be detected 1 to 84 hours, 1 to 72 hours, 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2 to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4 to 84 hours, 4 to 72 hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 96 hours, 6 to 84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 96 hours, 8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 96 hours, 10 to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48 hours, 10 to 36 hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours, 12 to 84 hours, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 hours to 96 hours, 72 hours to 84 hours, or 84 hours to 96 hours after administration. For example, in certain embodiments, the expression of ADAMTS13 protein can be detected 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after administration. In some embodiments, the expression of ADAMTS13 protein can be detected 1 day to 7 days after administration. For example, in some embodiments, ADAMTS13 protein can be detected 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after administration. In some embodiments, the expression of ADAMTS13 protein can be detected 1 week to 8 weeks after administration. For example, in some embodiments, ADAMTS13 protein can be detected 1 week, 2 weeks, 3 weeks, and / or 4 weeks after administration.In some embodiments, expression of ADAMTS13 protein can be detected one month after administration.

[0456] In some embodiments, the composition is administered such that the von Willebrand factor (vWF) level in the subject is reduced as compared to the baseline vWF level prior to treatment. Generally, the baseline level in the subject is measured immediately prior to treatment. Generally, the vWF level is measured in a biological sample. Suitable biological samples include, for example, whole blood, plasma, or serum.

[0457] In some embodiments, the composition is administered such that the vWF level in a biological sample taken from the subject is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline vWF level immediately prior to treatment. In some embodiments, the composition is administered such that the plasma vWF level in the subject is reduced to less than about 2000 μM, 1500 μM, 1000 μM, 750 μM, 500 μM, 250 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, or 30 μM.

[0458] In some embodiments, administration of the provided composition reduces the vWF level in a plasma or serum sample taken from the subject as compared to the baseline vWF level immediately prior to treatment. In some embodiments, administration of the provided composition reduces the vWF level in plasma or serum as compared to the vWF level in an untreated subject. In some embodiments, the composition is administered such that the vWF level in plasma or serum is reduced to about 3000 μmol / L or lower, about 2750 μmol / L or lower, about 2500 μmol / L or lower, about 2250 μmol / L or lower, about 2000 μmol / L or lower, about 1750 μmol / L or lower, about 1500 μmol / L or lower, about 1250 μmol / L or lower, about 1000 μmol / L or lower, about 750 μmol / L or lower, about 500 μmol / L or lower, about 250 μmol / L or lower, about 100 μmol / L or lower, or about 50 μmol / L or lower. In a particular embodiment, the composition is administered such that the vWF level in plasma or serum is reduced to about 50 μmol / L or lower.

[0459] 4. Generation of polynucleotides

[0460] The vectors provided herein can be prepared using standard techniques of molecular biology. For example, the 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 generating them from vectors known to contain polynucleotides.

[0461] Based on known sequences, the various elements of the vectors provided herein can also be synthesized rather than cloned. The complete sequences can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311.

[0462] Thus, a specific nucleotide sequence can be obtained from a vector having the desired sequence or, where appropriate, 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. One method of obtaining a nucleotide sequence encoding the desired vector element is to anneal complementary sets of overlapping synthetic oligonucleotides generated in a conventional automated polynucleotide synthesizer, then ligate with a suitable DNA ligase and amplify the ligated nucleotide sequence by PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. In addition, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-directed mutagenesis of pre-existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of nicked oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0463] The precursor RNAs provided herein can be produced by incubating the vectors provided herein under conditions that permit transcription of the precursor RNAs encoded by the vectors. For example, in some embodiments, the precursor RNAs are synthesized by incubating the vectors provided herein with a compatible RNA polymerase under conditions that permit in vitro transcription, the vectors containing an RNA polymerase promoter upstream of their 5' duplex-forming region and / or expression sequence. In some embodiments, the vectors are incubated intracellularly with a bacteriophage RNA polymerase or in the nucleus with a host RNA polymerase II.

[0464] In certain embodiments, methods are provided for producing precursor RNAs by in vitro transcription using the vectors provided herein as templates (e.g., the vectors provided herein having an RNA polymerase promoter upstream of the 5' homologous region).

[0465] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., the circular RNA polynucleotides provided herein) by incubating in the presence of magnesium ions and a guanosine nucleotide or nucleoside at a temperature at which RNA cyclization occurs (e.g., between 20°C and 60°C).

[0466] Accordingly, in certain embodiments, methods for preparing circular RNA are provided herein. In certain embodiments, the method comprises synthesizing a precursor RNA by transcribing (e.g., run-off transcription) using a vector provided herein (e.g., a vector comprising a 5' homology region, a 3' group I intron fragment, a first spacer, an internal ribosome entry site (IRES), an expression sequence, a second spacer, a 5' group I intron fragment, and a 3' homology region in this order) as a template and incubating the resulting precursor RNA in the presence of a divalent cation (e.g., magnesium ion) and GTP such that it cyclizes to form circular RNA. In some embodiments, the precursor RNA disclosed herein is capable of cyclizing in the absence of magnesium ion and GTP and / or without a step of incubating with magnesium ion and GTP. It has been found that circular RNA has reduced immunogenicity relative to the corresponding mRNA, at least in part because mRNA contains an immunogenic 5' cap. When transcribing a DNA vector from certain promoters (e.g., the T7 promoter) to produce precursor RNA, it should be understood that the 5' end of the precursor RNA is G. To reduce the immunogenicity of a circular RNA composition containing a low level of contaminating linear mRNA, an excess of GMP relative to GTP can be provided during transcription such that most transcripts contain 5' GMP, which cannot be capped. Accordingly, in some embodiments, transcription is carried out in the presence of an excess of GMP. In some embodiments, transcription is carried out wherein the ratio of the GMP concentration to the GTP concentration is in the range of about 3:1 to about 15:1, such as about 3:1 to about 10:1, about 3:1 to about 5:1, about 3:1, about 4:1, or about 5:1.

[0467] In some embodiments, compositions comprising circular RNAs have been purified. Circular RNAs 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 sequentially comprises the following steps: 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 proteins, protein ligases, capping enzymes, and / or nicked RNAs compared to unpurified RNA. In some embodiments, the purified composition has lower immunogenicity than the unpurified composition. In some embodiments, immune cells exposed to the purified composition produce less IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα compared to immune cells exposed to the unpurified composition.

[0468] 5. Ionizable lipid

[0469] In certain embodiments disclosed herein, the lipid is an ionizable lipid that can be used as a component of a delivery vehicle to facilitate or enhance the delivery and release of circular RNAs to one or more target cells (e.g., by permeation or fusion with the lipid membrane of such target cells). In certain embodiments, the ionizable lipid comprises one or more cleavable functional groups (e.g., disulfide bonds) that allow, for example, the hydrophilic functional head group to dissociate from the lipophilic functional tail group of the compound (e.g., upon exposure to oxidative, reductive, or acidic conditions), thereby promoting a phase change in the lipid bilayer of one or more target cells.

[0470] In some embodiments, the ionizable lipid is a lipid as described in International Patent Application PCT / US2018 / 058555.

[0471] In some embodiments, the cationic lipid has the following formula:

[0472]

[0473] Wherein:

[0474] R1 and R2 are the same or different and are independently optionally substituted C 10 -C 24 alkyl, optionally substituted C 10 -C 24 alkenyl, optionally substituted C 10 -C 24 alkynyl, or optionally substituted C 10 -C 24 acyl;

[0475] R3 and R4 are the same or different and are independently an optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group or an optionally substituted C2-C6 alkynyl group, or R3 and R4 may be joined to form an optionally substituted heterocycle having 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;

[0476] R5 is absent or present and, when present, is hydrogen or a C1-C6 alkyl group; m, n and p are the same or different and are independently 0 or 1, provided that m, n and p are not all 0 at the same time; q is 0, 1, 2, 3 or 4; and

[0477] Y and Z are the same or different and are independently O, S or NH.

[0478] In one embodiment, R1 and R2 are each linoleyl and the amino lipid is a dilinoleyl amino lipid.

[0479] In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0480] In various other embodiments, the cationic lipid has the following structure:

[0481]

[0482] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0483] R1 and R2 are each independently selected from the group consisting of H and C1-C3 alkyl groups; and

[0484] R3 and R4 are each independently an alkyl group having from about 10 to about 20 carbon atoms, wherein at least one of R3 and R4 contains at least two sites of unsaturation.

[0485] In some embodiments, R3 and R4 are each independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl and eicosadienyl. In one embodiment, both R3 and R4 are linoleyl. In some embodiments, R3 and / or R4 may contain at least three sites of unsaturation (e.g., R3 and / or R4 may be, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl and eicosatrienyl).

[0486] In some embodiments, the cationic lipid has the following structure:

[0487]

[0488] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0489] R1 and R2 are each independently selected from H and C1-C3 alkyl;

[0490] R3 and R4 are each independently an alkyl group having from about 10 to about 20 carbon atoms, wherein at least one of R3 and R4 contains at least two sites of unsaturation.

[0491] In one embodiment, R3 and R4 are the same. For example, in some embodiments, both R3 and R4 are linoleyl (C 18 -alkyl). In another embodiment, R3 and R4 are different. For example, in some embodiments, R3 is tetradecatrienyl (C 14 -alkyl), and R4 is linoleyl (C 18 -alkyl). In a preferred embodiment, the cationic lipid of the present invention is symmetric, i.e., R3 and R4 are the same. In another preferred embodiment, both R3 and R4 contain at least two sites of unsaturation. In some embodiments, R3 and R4 are each independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and eicosadienyl. In one embodiment, both R3 and R4 are linoleyl. In some embodiments, R3 and / or R4 contain at least three sites of unsaturation and are each independently selected from dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and eicosatrienyl.

[0492] In various embodiments, the cationic lipid has the following formula:

[0493]

[0494] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0495] X aa is a D- or L-amino acid residue having the formula -NR N -CR 1 R 2 -C(C=O)-, or a peptide or a peptide of amino acid residues having the formula - {NR N -CR 1 R 2 -C(C=O)} n -, where n is an integer from 2 to 20;

[0496] R 1 is, in each occurrence, independently a substituted or unsubstituted side chain that is non-hydrogen or an amino acid;

[0497] R 2 and R NIndependently at each occurrence, it is hydrogen, an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms or any combination of the foregoing and having 1 to 20 carbon atoms, C (1-5) alkyl, cycloalkyl, cycloalkylalkyl, C (1-5) alkenyl, C (1-5) alkynyl, C (1-5) alkanoyl, C (1-5) alkanoyloxy, C (1-5) alkoxy, C (1-5) alkoxy-C (1-5) alkyl, C (1-5) alkoxy-C (1-5) alkoxy, C (1-5) alkyl-amino-C (1-5) alkyl-, C (1-5) dialkyl-amino-C (1-5) alkyl-, nitro-C (1-5) alkyl, cyano-C (1-5) alkyl, aryl-C (1-5) alkyl, 4-biphenyl-C (1-5) alkyl, carboxyl or hydroxyl;

[0498] Z is -NH-, -O-, -S-, -CH2S-, -CH2S(O)-, or an organic linker consisting of 1 - 40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms (preferably, Z is -NH- or -O-);

[0499] R x and R y are independently (i) a lipophilic tail derived from a lipid (which may be natural or synthetic), such as a phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail optionally contains a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and organic protecting groups; or (iii) a substituted or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) alkenyl, C (3-22) alkynyl, C (3-22) alkoxy or C (6-12) -alkoxyC (3-22) alkyl;

[0500] In some embodiments, one of R x and R y is a lipophilic tail as defined above, and the other is an amino acid terminal group. In some embodiments, both R x and R y are lipophilic tails.

[0501] In some embodiments, R x and R y at least one of which is substituted by one or more biodegradable groups (e.g., -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -S-S-, -C(O)(NR 5 )-, -N(R 5 )C(O)-, -C(S)(NR 5 )-, -N(R 5 )C(O)-, -N(R 5 )C(O)N(R 5 )-, -OC(O)O-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, -OC(O)(CR 3 R 4 )C(O)- or interrupted.

[0502] In some embodiments, R 11 is C2-C8 alkyl or alkenyl.

[0503] In some embodiments, each occurrence of R 5 is independently H or alkyl.

[0504] In some embodiments, each occurrence of R 3 and R 4 is independently H, halogen, OH, alkyl, alkoxy, -NH2, alkylamino or dialkylamino; or R3 and R4 together with the carbon atom to which they are directly attached form cycloalkyl. In some specific embodiments, each occurrence of R 3 and R 4 is independently H or C1-C4 alkyl.

[0505] In some embodiments, R x and R y each independently has one or more carbon-carbon double bonds.

[0506] In some embodiments, the cationic lipid is one of the following:

[0507]

[0508] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0509] R1 and R2 are each independently an alkyl, alkenyl or alkynyl group, each of which may optionally be substituted;

[0510] R3 and R4 are each independently a C1-C6 alkyl group, or R3 and R4 together form an optionally substituted heterocycle.

[0511] Representative useful dilinoleyl amino lipids have the following formula:

[0512]

[0513] wherein n is 0, 1, 2, 3 or 4.

[0514] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (the above DLin-K-DMA, wherein n is 2).

[0515] In one embodiment, the cationic lipid has the following structure:

[0516]

[0517] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0518] R1 and R2 are each independently, upon each occurrence, an optionally substituted C 10 -C 30 alkyl group, an optionally substituted C 10 -C 30 alkenyl group, an optionally substituted C 10 -C 30 alkynyl group or an optionally substituted C 10 -C 30 acyl group;

[0519] R3 is H, an optionally substituted C2-C 10 alkyl group, an optionally substituted C2-C 10 alkenyl group, an optionally substituted C2-C 10 alkynyl group, an alkyl heterocycle, an alkyl phosphate, an alkyl thiophosphate, an alkyl dithiophosphate, an alkyl phosphonate, an alkylamine, a hydroxyalkyl group, an ω-aminoalkyl group, an ω-(substituted) aminoalkyl group, an ω-phosphoalkyl group, an ω-thiophosphoalkyl group, an optionally substituted polyethylene glycol (PEG, mw 100-40K), an optionally substituted mPEG (mw 120-40K), a heteroaryl group or a heterocycle or a linker ligand, for example, in some embodiments, R3 is (CH3)2N(CH2) n -, where n is 1, 2, 3 or 4;

[0520] E is O, S, N(Q), C(O), OC(O), C(O)O, N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O=N, aryl, heteroaryl, cyclic or heterocyclic, such as -C(O)O, where - is the point of attachment to R3; and

[0521] Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphonoalkyl or ω-thiophosphonoalkyl.

[0522] In one specific embodiment, the cationic lipid of embodiment 1, 2, 3, 4 or 5 has the following structure:

[0523]

[0524] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0525] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O=N, aryl, heteroaryl, cyclic or heterocyclic;

[0526] Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphonoalkyl or ω-thiophosphonoalkyl;

[0527] R1 and R2 and R x are each independently, at each occurrence, H, optionally substituted C1-C 10 alkyl, optionally substituted C 10 -C 30 alkyl, optionally substituted C 10 -C 30 alkenyl, optionally substituted C 10 -C 30 alkynyl, optionally substituted C 10 -C 30 acyl or linker-ligand, provided that at least one of R1, R2 and R x is not H;

[0528] R3 is H, optionally substituted C1-C 10 alkyl, optionally substituted C2-C 10 alkenyl, optionally substituted C2-C 10Alkynyl, alkyl heterocycle, alkyl phosphate, alkyl thiophosphate, alkyl dithiophosphate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted) aminoalkyl, ω-phosphoalkyl, ω-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw 100 - 40K), optionally substituted mPEG (mw 120 - 40K), heteroaryl or heterocycle or linker-ligand; and

[0529] n is 0, 1, 2 or 3.

[0530] In one embodiment, the cationic lipid of Embodiment 1, 2, 3, 4 or 5 has the structure of Formula I:

[0531]

[0532] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0533] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, and L 1 or L 2 The other of them is -O(C=O), -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or

[0534] -NR a C(=O)O- or a direct bond;

[0535] R a is H or C1-C 12 alkyl;

[0536] R 1a and R 1bis independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached;

[0537] R 2a and R 2b are independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is attached;

[0538] R 3a and R 3b are independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is attached;

[0539] R 4a and R 4b are independently, at each occurrence, (a) H or C1-C 12 alkyl, or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached;

[0540] R 5 and R 6 are each independently methyl or cycloalkyl;

[0541] R 7 is independently, at each occurrence, H or C1-C 12 alkyl;

[0542] R 8 and R 9 are each independently unsubstituted C1-C 12 alkyl; or R 8 and R 9Together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocycle containing one nitrogen atom;

[0543] a and d are each independently an integer from 0 to 24;

[0544] b and c are each independently an integer from 1 to 24;

[0545] e is 1 or 2; and

[0546] x is 0, 1 or 2.

[0547] In some embodiments of Formula I, L 1 and L 2 are independently -O(C=O)- or -(C=O)O-.

[0548] In certain embodiments of Formula I, R 1a , R 2a , R 3a or R 4a At least one of them is C1-C 12 alkyl, or at least one of L 1 or L 2 is -O(C=O)- or -(C=O)O-. In other embodiments, R 1a and R 1b are not isopropyl when a is 6 or not n-butyl when a is 8.

[0549] In other embodiments of Formula I, R 1a , R 2a , R 3a or R 4a At least one of them is C1-C 12 alkyl, or at least one of L 1 or L 2 is -O(C=O)- or -(C=O)O-; and R 1a and

[0550] R 1b are not isopropyl when a is 6 or not n-butyl when a is 8.

[0551] In other embodiments of Formula I, R 8 and R 9 are each independently unsubstituted C1-C 12 alkyl; or R 8 and R 9 Together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocycle containing one nitrogen atom;

[0552] In certain embodiments of Formula I, L 1 or L 2Any one of them can be -O(C=O)- or a carbon-carbon double bond. L 1 and L 2 can each be -O(C=O)- or can each be a carbon-carbon double bond.

[0553] In some embodiments of Formula I, L 1 or L 2 One of them is -O(C=O)-. In other embodiments, L 1 and L 2 Both are -O(C=O)-.

[0554] In some embodiments of Formula I, L 1 or L 2 One of them is -(C=O)O-. In other embodiments, L 1 and L 2 Both are -(C=O)O-.

[0555] In some other embodiments of Formula I, L 1 or L 2 One of them is a carbon-carbon double bond. In other embodiments, L 1 and L 2 Both are carbon-carbon double bonds.

[0556] In other embodiments of Formula I, L 1 or L 2 One of them is -O(C=O)-, and L 1 or L 2 The other of them is -(C=O)O-. In more embodiments, L 1 or L 2 One of them is -O(C=O)-, and L 1 or L 2 The other of them is a carbon-carbon double bond. In more embodiments, L 1 or L 2 One of them is -(C=O)O-, and L 1 or L 2 The other of them is a carbon-carbon double bond.

[0557] It should be understood that as used throughout the specification, a "carbon-carbon" double bond refers to one of the following structures:

[0558]

[0559] where R a and R b are each independently H or a substituent each time they appear. For example, in some embodiments, R a and R bis independently H, C1-C 12 alkyl or cycloalkyl at each occurrence, such as H or C1-C 12 alkyl.

[0560] In other embodiments, the lipid compound of Formula I has the following formula (Ia):

[0561]

[0562] In other embodiments, the lipid compound of Formula I has the following formula (1b):

[0563]

[0564] In other embodiments, the lipid compound of Formula I has the following formula (Ic):

[0565]

[0566] In certain embodiments of the lipid compound of Formula I, a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or an integer from 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In other embodiments, a is 16.

[0567] In some other embodiments of Formula I, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In other embodiments, b is 16.

[0568] In some further embodiments of Formula I, c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In other embodiments, c is 16.

[0569] In some certain other embodiments of Formula I, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In other embodiments, d is 16.

[0570] In some other various embodiments of Formula I, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0571] The sum of a and b and the sum of c and d in Formula I are factors that can be varied to obtain a lipid of Formula I with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, both the sum of a and b and the sum of c and d are the same integer that can be in the range of 14 to 24. In further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or greater.

[0572] In some embodiments of Formula I, e is 1. In other embodiments, e is 2.

[0573] The R of Formula I 1a 、R 2a 、R 3a and R 4a at the substituents are not particularly limited. In certain embodiments, R 1a 、R 2a, R 3a and R 4a is H each time it appears. In some other embodiments, R 1a , R 2a , R 3a and R 4a at least one of which is C1-C 12 alkyl. In some other embodiments, R 1a , R 2a , R 3a and R 4a at least one of which is C1-C8 alkyl. In some other embodiments, R 1a , R 2a , R 3a and R 4a at least one of which is C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0574] In some embodiments of Formula I, R 1a , R 1b , R 4a and R 4b is C1-C 12 alkyl each time it appears.

[0575] In other embodiments of Formula I, R 1b , R 2b , R 3b and R 4b at least one of which is H, or R 1b , R 2b , R 3b and R 4b is H each time it appears.

[0576] In some embodiments of Formula I, R 1b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached. In the other foregoing embodiments, R 4b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached.

[0577] In the foregoing embodiments, the substituents at R 5 and R 6 of Formula I are not particularly limited. In some embodiments, one or both of R 5 or R 6 is methyl. In some other embodiments, R 5 or R 6One or both of them are cycloalkyl, such as cyclohexyl. In these embodiments, the cycloalkyl may be substituted or unsubstituted. In some other embodiments, the cycloalkyl is substituted with a C1-C 12 alkyl group, such as a tert-butyl group.

[0578] In the foregoing embodiments of Formula I, the substituents at R 7 are not particularly limited. In some embodiments, at least one R 7 is H. In some other embodiments, R 7 is H each time it appears. In some other embodiments, R 7 is a C1-C 12 alkyl group.

[0579] In some other foregoing embodiments of Formula I, one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.

[0580] In some different embodiments of Formula I, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocycle, such as a pyrrolidine ring.

[0581] In some embodiments of Embodiment 3, the first and second cationic lipids are each independently selected from the lipids of Formula I.

[0582] In various different embodiments, the lipid of Formula I has one of the structures listed in Table 1 below.

[0583] Table 1: Representative Lipids of Formula I

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590] In some embodiments, the cationic lipid of Embodiment 1, 2, 3, 4 or 5 has the structure of Formula II:

[0591]

[0592] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0593] L 1 or L 2 one of is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, and L 1 or L 2 the other of is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or

[0594] -NR a C(=O)O- or a direct bond;

[0595] G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond;

[0596] G 2 is -(C=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a - or a direct bond;

[0597] G 3 is C1-C6 alkylene;

[0598] R a is H or C1-C 12 alkyl;

[0599] R 1a and R 1bEach occurrence is independently: (a) H or C1-C 12 alkyl; or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached and the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0600] R 2a and R 2b Each occurrence is independently: (a) H or C1-C 12 alkyl; or (b) R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached and the adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0601] R 3a and R 3b Each occurrence is independently (a): H or C1-C 12 alkyl; or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached and the adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0602] R 4a and R 4b Each occurrence is independently: (a) H or C1-C 12 alkyl; or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached and the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0603] R 5 and R 6 are each independently H or methyl;

[0604] R 7 is C4-C 20 alkyl;

[0605] R 8 and R 9 are each independently C1-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle;

[0606] a, b, c, and d are each independently an integer from 1 to 24; and

[0607] x is 0, 1, or 2.

[0608] In some embodiments of formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond. In other embodiments, G 1 and G 2 are each independently -(C=O)- or a direct bond. In some different embodiments, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond; and G 1 and G 2 are each independently -(C=O)- or a direct bond.

[0609] In some different embodiments of formula (II), L 1 and L 2 are each independently -C(=O), -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR aS (O) x -, or -S(O) x NR a -.

[0610] In any of the foregoing embodiments of formula (II), the lipid compound has one of the following formulas (IIA) or (IIB):

[0611]

[0612] In some embodiments of formula (II), the lipid compound has formula (IIA). In other embodiments, the lipid compound has formula (IIB).

[0613] In any of the foregoing embodiments of formula (II), L 1 or L2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each -O(C=O)-.

[0614] In some different embodiments of formula (II), L 1 or L 2 One of them is -(C=O)O-. For example, in some embodiments, L 1 and L 2 are each -(C=O)O-.

[0615] In different embodiments of formula (II), L 1 or L 2 One of them is a direct bond. As used herein, "direct bond" means that the group (e.g., L 1 or L 2 ) is absent. For example, in some embodiments, L 1 and L 2 are each a direct bond.

[0616] In other different embodiments of formula (II), for at least one occurrence of R 1a and R 1b , R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached.

[0617] In other different embodiments of formula (II), for at least one occurrence of R 4a and R 4b , R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached.

[0618] In more embodiments of formula (II), for at least one occurrence of R 2a and R 2b , R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is attached.

[0619] In other different embodiments of formula (II), for R 3aand R 3b at least one occurrence of, R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is attached.

[0620] In various other embodiments of formula (II), the lipid compound has one of the following formulas (IIC) or (IID):

[0621]

[0622] wherein e, f, g, and h are each independently an integer from 1 to 12.

[0623] In some embodiments of formula (II), the lipid compound has formula (IIC). In other embodiments, the lipid compound has formula (IID).

[0624] In various embodiments of formula (IIC) or (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0625] In certain embodiments of formula (II), a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or an integer from 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In other embodiments, a is 16.

[0626] In some embodiments of formula (II), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In other embodiments, b is 16.

[0627] In some embodiments of formula (II), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In other embodiments, c is 16.

[0628] In certain specific embodiments of formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In other embodiments, d is 16.

[0629] In some embodiments of formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In other embodiments, e is 12.

[0630] In some embodiments of formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In other embodiments, f is 12.

[0631] In some embodiments of formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In other embodiments, g is 12.

[0632] In some embodiments of formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In other embodiments, h is 12.

[0633] In some other various embodiments of formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0634] The sum of a and b and the sum of c and d of formula (II) are factors that can be varied to obtain a lipid with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, both the sum of a and b and the sum of c and d are the same integer that can be in the range of 14 to 24. In further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or greater.

[0635] The R of formula (II) 1a 、R 2a 、R 3a and R 4a The substituents at are not particularly limited. In some embodiments, R 1a, R 2a , R 3a and R 4a is at least one of H. In certain embodiments, R 1a , R 2a , R 3a and R 4a is H each time it appears. In certain other embodiments, R 1a , R 2a , R 3a and R 4a is at least one of C1-C 12 alkyl. In certain other embodiments, R 1a , R 2a , R 3a and R 4a is at least one of C1-C8 alkyl. In certain other embodiments, R 1a , R 2a , R 3a and R 4a is at least one of C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0636] In certain embodiments of formula (II), R 1a , R 1b , R 4a and R 4b is C1-C 12 alkyl each time it appears.

[0637] In other embodiments of formula (II), R 1b , R 2b , R 3b and R 4b is at least one of H, or R 1b , R 2b , R 3b and R 4b is H each time it appears.

[0638] In certain embodiments of formula (II), R 1b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached. In the other foregoing embodiments, R 4b together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached.

[0639] In the foregoing embodiments, R 5 and R6 There are no particular restrictions on the substituents at 5 or R 6 . In certain embodiments, one of R 5 or R 6 is methyl. In other embodiments, each of R

[0640] In the foregoing embodiments, there are no particular restrictions on the substituents at R 7 of formula (II). In certain embodiments, R 7 is C6-C 16 alkyl. In some other embodiments, R 7 is C6-C9 alkyl. In some of these embodiments, R 7 is substituted with -(C═O)OR b , -O(C═O)R b , -C(═O)R b , -OR b , -S(O) x R b , -S-SR b , -C(═O)SR b , -SC(═O)R b , -NR a R b , -NR a C(═O)R b , -C(═O)NR a R b , -NR a C(═O)NR a R b , -OC(═O)NR a R b , -NR a C(═O)OR b , -NR a S(O) x NR a R b , -NR a S(O) x R b or -S(O) x NR a R b , wherein: R a is H or C1-C 12 alkyl; R b is C1-C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 is substituted with -(C═O)OR b or -O(C═O)Rb Substituted.

[0641] In some of the foregoing embodiments of formula (II), R b is a branched C1-C 16 alkyl. For example, in some embodiments, R b has one of the following structures:

[0642]

[0643] In certain other foregoing embodiments of formula (II), R 8 or R 9 is methyl. In other embodiments, R 8 and R 9 are both methyl.

[0644] In some different embodiments of formula (II), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocycle, such as a pyrrolidinyl ring. In some of the foregoing different embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 6-membered heterocycle, such as a piperazinyl ring.

[0645] In certain embodiments of Embodiment 3, the first and second cationic lipids are each independently selected from the lipids of formula II.

[0646] In other embodiments of the foregoing formula (II) lipids, G 3 is a C2-C4 alkylene, such as C3 alkylene. In various different embodiments, the lipid compound has one of the structures listed in Table 2 below

[0647] Table 2: Representative Lipids of Formula (II)

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655] In some other embodiments, the cationic lipid of Embodiment 1, 2, 3, 4 or 5 has the structure of Formula III:

[0656]

[0657] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0658] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-, and L 1 or L 2 The other of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or

[0659] -NR a C(=O)O- or a direct bond;

[0660] G 1 and G 2 Each is independently an unsubstituted C1-C 12 alkylene or C1-C 12 alkenylene;

[0661] G 3 is C1-C 24 alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0662] R a is H or C1-C 12 alkyl;

[0663] R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl;

[0664] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0665] R 4 is C1-C 12 alkyl;

[0666] R 5 is H or C1-C6 alkyl; and

[0667] x is 0, 1 or 2.

[0668] In some of the foregoing embodiments of formula (III), the lipid has one of the following formulas (IIIA) or (IIIB):

[0669]

[0670] wherein:

[0671] A is a 3- to 8-membered cycloalkyl or subcycloalkyl ring;

[0672] R 6 is independently H, OH or C1-C 24 alkyl each time it appears;

[0673] n is an integer in the range of 1 to 15.

[0674] In some of the foregoing embodiments of formula (III), the lipid has formula (IIIA), and in other embodiments, the lipid has formula (IIIB).

[0675] In other embodiments of formula (III), the lipid has one of the following formulas (IIIC) or (IIID):

[0676]

[0677] wherein y and z are each independently an integer in the range of 1 to 12.

[0678] In any of the foregoing embodiments of formula (III), L 1 or L 2One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each -O(C=O)-. In some of any of the foregoing different embodiments, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each -(C=O)O-.

[0679] In some different embodiments of formula (III), the lipid has one of the following formulas (IIIE) or (IIIF):

[0680]

[0681] In some of the foregoing embodiments of formula (III), the lipid has one of the following formulas (IIIG), (IIIH), (IIII), or (IIIJ):

[0682]

[0683] In some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0684] In some other of the foregoing embodiments of formula (III), y and z are each independently integers in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

[0685] In some of the foregoing embodiments of formula (III), R 6 is H. In other of the foregoing embodiments, R 6 is C1-C 24 alkyl. In other embodiments, R 6 is OH.

[0686] In some embodiments of formula (III), G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is a straight-chain C1-C 24 alkylene or a straight-chain C1-C 24 alkenylene.

[0687] In some other of the foregoing embodiments of formula (III), R1 or R 2 or both are C6-C 24 alkenyl. For example, in some embodiments, R 1 and R 2 each independently have the following structure:

[0688]

[0689] wherein:

[0690] R 7a and R 7b are each independently H or C1-C 12 alkyl;

[0691] and

[0692] a is an integer from 2 to 12,

[0693] wherein each selection of R 7a , R 7b and a is such that R 1 and R 2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0694] In some of the foregoing embodiments of formula (III), at least one occurrence of R 7a is H. For example, in some embodiments, R 7a is H at each occurrence. In other different foregoing embodiments, at least one occurrence of R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0695] In different embodiments of formula (III), R 1 or R 2 or both have one of the following structures:

[0696]

[0697] In some of the foregoing embodiments of formula (III), R 3 is OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 . In some embodiments, R 4 is methyl or ethyl.

[0698] In some specific embodiments of Embodiment 3, the first and second cationic lipids are each independently selected from the lipids of Formula III.

[0699] In various different embodiments, the cationic lipid (e.g., cationic lipid, first cationic lipid, second cationic lipid) of any one of the disclosed embodiments of Formula (III) has one of the structures listed in Table 3 below.

[0700] Table 3: Representative Compounds of Formula (III)

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709] In one embodiment, the cationic lipid of any one of Embodiments 1, 2, 3, 4, or 5 has the structure of Formula (IV):

[0710]

[0711] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0712] G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-, and G 1 or G 2Another one of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y , -S-S-, -C(=O)S-, -SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond;

[0713] L is -O(C=O)- each time it appears, where ~ represents a covalent bond with X;

[0714] X is CR a ;

[0715] Z is an alkyl group, a cycloalkyl group or a monovalent moiety containing at least one polar functional group when n is 1; or Z is an alkylene group, a cycloalkylene group or a polyvalent moiety containing at least one polar functional group when n is greater than 1;

[0716] R a is independently H, C1-C 12 alkyl group, C1-C 12 hydroxyalkyl group, C1-C 12 aminoalkyl group, C1-C 12 alkylaminoalkyl group, C1-C 12 alkoxyalkyl group, C1-C 12 alkoxycarbonyl group, C1-C 12 alkylcarbonyloxy group, C1-C 12 alkylcarbonyloxyalkyl group or C1-C 12 alkylcarbonyl;

[0717] R is independently: (a) H or C1-C 12 alkyl group; or (b) R together with the carbon atom to which it is attached and the adjacent R and the carbon atom to which it is attached form a carbon-carbon double bond;

[0718] R 1 and R 2 each have the following structures each time they appear:

[0719]

[0720] a 1 and a 2 are independently integers from 3 to 12 each time they appear;

[0721] b1 and b 2 is independently 0 or 1 each time it appears;

[0722] c 1 and c 2 is independently an integer from 5 to 10 each time it appears;

[0723] d 1 and d 2 is independently an integer from 5 to 10 each time it appears;

[0724] y is independently an integer from 0 to 2 each time it appears; and

[0725] n is an integer from 1 to 6,

[0726] wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted by one or more substituents.

[0727] In some embodiments of formula (IV), G 1 and G 2 are each independently

[0728] -O(C=O)- or -(C=O)O-.

[0729] In other embodiments of formula (IV), X is CH,

[0730] In different embodiments of formula (IV), a 1 +b 1 +c 1 or the sum of a 2 +b 2 +c 2 is an integer from 12 to 26.

[0731] In other embodiments of formula (IV), a 1 and a 2 are independently integers from 3 to 10. For example, in some embodiments, a 1 and a 2 are independently integers from 4 to 9.

[0732] In various embodiments of formula (IV), b 1 and b 2 is 0. In different embodiments, b 1 and b 2 is 1.

[0733] In more embodiments of formula (IV), c 1 、c 2 、d1 and d 2 is independently an integer from 6 to 8.

[0734] In other embodiments of formula (IV), c 1 and c 2 is independently an integer from 6 to 10 each time it appears, and d 1 and d 2 is independently an integer from 6 to 10 each time it appears.

[0735] In other embodiments of formula (IV), c 1 and c 2 is independently an integer from 5 to 9 each time it appears, and d 1 and d 2 is independently an integer from 5 to 9 each time it appears.

[0736] In further embodiments of formula (IV), Z is alkyl, cycloalkyl or, when n is 1, a monovalent moiety containing at least one polar functional group. In other embodiments, Z is alkyl.

[0737] In the various embodiments of formula (IV) above, R is independently each time it appears: (a) H or methyl; or (b) R together with the carbon atom to which it is attached and the adjacent R and the carbon atom to which it is attached form a carbon-carbon double bond. In certain embodiments, each R is H. In other embodiments, at least one R together with the carbon atom to which it is attached and the adjacent R and the carbon atom to which it is attached form a carbon-carbon double bond.

[0738] In other embodiments of the compound of formula (IV), R 1 and R 2 independently have one of the following structures:

[0739]

[0740] In certain embodiments of formula (IV), the compound has one of the following structures:

[0741]

[0742]

[0743]

[0744]

[0745] In different embodiments, the cationic lipid of Embodiment 1, 2, 3, 4 or 5 has the structure of formula (V):

[0746]

[0747] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0748] G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a ))C(=O)-, -C(=O)N(R a ))-, -N(R a ))C(=O)N(R a ))-, -OC(=O)N(R a ))- or -N(R a ))C(=O)O-, and G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, -SC(=O)-, -N(R a ))C(=O)-, -C(=O)N(R a ))-, -N(R a ))C(=O)N(R a ))-, -OC(=O)N(R a ))- or -N(R a ))C(=O)O- or a direct bond;

[0749] L in each occurrence is ~O(C=O)-, where ~ represents a covalent bond to X;

[0750] X is CR a ;

[0751] Z is alkyl, cycloalkyl or a monovalent moiety containing at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety containing at least one polar functional group when n is greater than 1;

[0752] R a in each occurrence is independently H, C1-C 12 alkyl, C1-C 12 hydroxyalkyl, C1-C 12 aminoalkyl, C1-C 12 alkylaminoalkyl, C1-C 12 alkoxyalkyl, C1-C 12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C 12 alkylcarbonyloxyalkyl or C1-C 12 alkylcarbonyl;

[0753] Each occurrence of R is independently: (a) H or C1-C 12 alkyl; or (b) R together with the carbon atom to which it is attached and the adjacent R and the carbon atom to which it is attached form a carbon-carbon double bond;

[0754] R 1 and R 2 Each occurrence respectively has the following structures:

[0755]

[0756] Each occurrence of R' is independently H or C1-C 12 alkyl;

[0757] a 1 and a 2 Each occurrence is independently an integer from 3 to 12;

[0758] b 1 and b 2 Each occurrence is independently 0 or 1;

[0759] c 1 and c 2 Each occurrence is independently an integer from 2 to 12;

[0760] d 1 and d 2 Each occurrence is independently an integer from 2 to 12;

[0761] Each occurrence of y is independently an integer from 0 to 2; and

[0762] n is an integer from 1 to 6,

[0763] wherein a 1 、a 2 、c 1 、c 2 、d 1 and d 2 are selected such that the sum of a 1 +c 1 +d 1 is an integer from 18 to 30, and a 2 +c 2 +d 2The sum is an integer from 18 to 30, and each of the alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl is optionally substituted by one or more substituents.

[0764] In certain embodiments of formula (V), G 1 and G 2 are each independently

[0765] -O(C=O)- or -(C=O)O-.

[0766] In other embodiments of formula (V), X is CH.

[0767] In some embodiments of formula (V), the sum of a 1 + c 1 + d 1 is an integer from 20 to 30, and the sum of a 2 + c 2 + d 2 is an integer from 18 to 30. In other embodiments, the sum of a 1 + c 1 + d 1 is an integer from 20 to 30, and the sum of a 2 + c 2 + d 2 is an integer from 20 to 30. In more embodiments of formula (V), the sum of a 1 + b 1 + c 1 or the sum of a 2 + b 2 + c 2 is an integer from 12 to 26. In other embodiments, a 1 , a 2 , c 1 , c 2 , d 1 and d 2 are selected such that the sum of a 1 + c 1 + d 1 is an integer from 18 to 28, and the sum of a 2 + c 2 + d 2 is an integer from 18 to 28.

[0768] In other embodiments of formula (V), a 1 and a 2 are each independently an integer from 3 to 10, such as an integer from 4 to 9.

[0769] In other embodiments of formula (V), b1 and b 2 is 0. In different embodiments, b 1 and b 2 is 1.

[0770] In certain other embodiments of formula (V), c 1 and c 2 and d 1 and d 2 are independently integers from 6 to 8.

[0771] In different other embodiments of formula (V), Z is an alkyl group or a monovalent moiety containing at least one polar functional group when n is 1; or Z is an alkylene group or a polyvalent moiety containing at least one polar functional group when n is greater than 1.

[0772] In more embodiments of formula (V), Z is an alkyl group, a cycloalkyl group or a monovalent moiety containing at least one polar functional group when n is 1. In other embodiments, Z is an alkyl group.

[0773] In other different embodiments of formula (V), R is independently, each time it appears: (a) H or methyl; or (b) R together with the carbon atom to which it is attached forms a carbon-carbon double bond together with the adjacent R and the carbon atom to which it is attached. For example, in some embodiments, each R is H. In other embodiments, at least one R together with the carbon atom to which it is attached forms a carbon-carbon double bond together with the adjacent R and the carbon atom to which it is attached.

[0774] In more embodiments, each R' is H.

[0775] In certain embodiments of formula (V), a 1 + c 1 + d 1 The sum of is an integer from 20 to 25, and a 2 + c 2 + d 2 The sum of is an integer from 20 to 25.

[0776] In other embodiments of formula (V), R 1 and R 2 independently have one of the following structures:

[0777]

[0778] In more embodiments of formula (V), the compound has one of the following structures:

[0779]

[0780]

[0781]

[0782]

[0783] In any of the foregoing embodiments of formula (IV) or (V), n is 1. In other foregoing embodiments of formula (IV) or (V), n is greater than 1.

[0784] In any of the foregoing embodiments of formula (IV) or (V), Z is a monovalent or polyvalent moiety containing at least one polar functional group. In some embodiments, Z is a monovalent moiety containing at least one polar functional group. In other embodiments...

Claims

1. A pharmaceutical composition, comprising: a. A circular RNA polynucleotide, comprising (i) a 3' spliced intron sequence of a group I intron fragment, (ii) an internal ribosome entry site (IRES), (iii) an expression sequence, and (iv) a 5' spliced intron sequence of a 5' group I intron fragment, and b. A transfer medium comprising an ionizable lipid represented by formula III: or a pharmaceutically acceptable salt thereof, wherein: L 1 or L 2 One of them is -O(C=O)- or -(C=O)O-; G 1 and G 2 each independently is an unsubstituted C1-C 12 alkylene group; G 3 selected from C1-C 12 alkylene and C3-C8 cycloalkylene; R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 selected from H, -OH, -CN, -C(=O)OR 4 and -NHC(=O)R 4 ; and R 4 is methyl or ethyl.

2. The pharmaceutical composition according to claim 1, wherein: Each L 1 and L 2 are both -(C=O)O-; G 3 is a straight-chain C1-C 12 alkylene group; R 1 and R 2 is C6-C 24 alkyl; and / or R 3 selected from -OH, -CN, -C(=O)OR 4 , -OC(=O)R 4 and -NHC(=O)R 4 , wherein R in 4 is methyl or ethyl.

3. The pharmaceutical composition according to claim 1, wherein: Each L 1 and L 2 are both -(C=O)O-; G 3 is a straight-chain C1-C 12 alkylene group; R 1 or R 2 or both are selected from: and R 3 selected from -OH, -CN, -C(=O)OR 4 , -OC(=O)R 4 and -NHC(=O)R 4 , wherein R in 4 is methyl or ethyl.

4. The pharmaceutical composition according to claim 1, wherein the ionizable lipid is 5. The pharmaceutical composition according to claim 1, wherein the expression sequence encodes a chimeric antigen receptor (CAR).

6. The pharmaceutical composition according to claim 1, wherein the circular RNA polynucleotide comprises a 3' spliced group I intron fragment and a 5' spliced group I intron fragment, and further comprises a first spacer region before the 3' spliced group I intron fragment and a second spacer region after the 5' spliced group I intron fragment.

7. The pharmaceutical composition according to claim 6, wherein each of the first spacer region and the second spacer region has a length of 10 to 60 nucleotides.

8. The pharmaceutical composition according to claim 1, wherein the circular RNA polynucleotide comprises a polyA sequence.

9. The pharmaceutical composition according to claim 1, wherein the IRES has a sequence of an IRES selected from the following: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Pseudococcus solenopsis enterovirus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picornavirus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human coxsackievirus B3, Nicotiana tabacum mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 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, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.aptamers against myc, murine Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, murine Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, murine UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobirnavirus, HCV QC64, human kobuvirus E / D, human kobuvirus F, human kobuvirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, salivirus A SH1, salivirus FHB, salivirus NG-J1, human parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, PasivirusA, Pasivirus A 2, echovirus E14, human parechovirus 5, Aichi virus, hepatitis A virus HA16, Phopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human hepacivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, hepacivirus A 1220, Pasivirus A 3, sapelovirus, Rosavirus B, Bakunsa virus, tremovirus A, porcine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, hepatitis virus K, hepatitis virus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 dicistrovirus, Hubei picornavirus-like virus, CRPV, Apodemus agrarius picornavirus, caprine crest virus, parabovirus, salivirus A BN5, salivirus A BN2, salivirus A 02394, salivirus A GUT, salivirus A CH, salivirus A SZ1, salivirus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or eIF4G.

10. The pharmaceutical composition according to claim 1, wherein the circular RNA polynucleotide is prepared by cyclization of a precursor RNA polynucleotide, and the precursor RNA polynucleotide comprises in the following order: (i) a. A 5' external duplex-forming region, b. A 3' spliced group I intron fragment, c. Optionally a 5' internal spacer region comprising a 5' internal duplex-forming region, d. IRES, e. An expression sequence, f. Optionally a 3' internal spacer region comprising a 3' internal duplex-forming region, g. A 5' group I intron fragment, and h. A 3' external duplex-forming region; or (ii) a. A first polyA sequence, b. A 5' external duplex-forming region, c. A 5' external spacer region, d. A 3' group I intron fragment, e. A 5' internal spacer region comprising a 5' internal duplex-forming region, f. IRES, g. An expression sequence, h. A 3' internal spacer region comprising a 3' internal duplex-forming region, i. A 5' group I intron fragment, j. A 3' external spacer region, k. A 3' external duplex-forming region, and l. A second polyA sequence; or (iii) a. A first polyA sequence, b. A 5' external spacer region, c. A 3' group I intron fragment, d. A 5' internal spacer region comprising a 5' internal duplex-forming region, e. IRES, f. An expression sequence, g. A 3' internal spacer region comprising a 3' internal duplex-forming region, h. A 5' group I intron fragment, i. A 3' external spacer region, and j. A second polyA sequence; or (iv) a. A first polyA sequence, b. A 5' external spacer region, c. A 3' group I intron fragment, d. A 5' internal spacer region comprising a 5' internal duplex-forming region, e. IRES, f. Expression sequence, g. Stop codon cassette, h. 3' internal spacer region containing a 3' internal duplex-forming region, i. 5' group I intron fragment, j. 3' external spacer region, and k. Second polyA sequence.

11. The pharmaceutical composition according to claim 10, wherein the expression sequence encodes a CAR.

12. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a targeting moiety operably linked to the transfer vehicle.

13. The pharmaceutical composition according to claim 1, wherein the in vivo therapeutic effect duration of the pharmaceutical composition in humans is greater than that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide.

14. The pharmaceutical composition according to claim 1, wherein the transfer vehicle further comprises at least one of the following: (a) polyethylene glycol (PEG)-lipid, (b) structural lipid, or (c) helper lipid.

15. The pharmaceutical composition according to claim 14, wherein the transfer vehicle further comprises a PEG-lipid and the PEG-lipid is selected from DMG-PEG2000, PEG-c-DOMG, PEG-DMG, PEG-DSPE, DSPE-PEG2000, PEG-DSG, PEG-DAG, PEG-DPPE, PEG-c-DMA, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE.

16. The pharmaceutical composition according to claim 1, wherein the transfer vehicle further comprises a helper lipid and the helper lipid is DSPC or DOPE.

17. The pharmaceutical composition according to claim 1, wherein the transfer vehicle further comprises a structural lipid and the structural lipid is cholesterol.

18. The pharmaceutical composition according to claim 15, wherein the transfer vehicle further comprises a helper lipid, a structural lipid and a PEG-lipid, and wherein the molar ratio of ionizable lipid:helper lipid:structural lipid:PEG-lipid is 16:1:4:1, 62:4:33:1 or 50:10:38.5:1.

5.

19. The pharmaceutical composition according to any one of claims 1-18, wherein the pharmaceutical composition is for delivering a therapeutic agent to a target cell.

20. The pharmaceutical composition according to claim 19, wherein the target cell is an immune cell.

21. The pharmaceutical composition according to claim 20, wherein the immune cell is a T cell, an NK cell or an NKT cell.

22. The pharmaceutical composition according to any one of claims 1-18, wherein the pharmaceutical composition is for treating or preventing a disease.

23. Use of the pharmaceutical composition according to any one of claims 1-18 in the preparation of a medicament for delivering a therapeutic agent to an immune cell, wherein the immune cell is a T cell, an NK cell or an NKT cell.

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