Cyclic RNA compositions and methods

Abstract

Disclosed herein are circular RNAs and transfer mediators, as well as related compositions and methods of treatment. The circular RNA may comprise a Group I intron fragment, a spacer region, an IRES, a duplex forming region, and / or an expression sequence, thereby having characteristics of improved expression, functional stability, low immunogenicity, ease of manufacture, and / or prolonged half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and a transfer vehicle are particularly useful for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials that can be used to produce the precursor or circular RNA, which have improved cyclization efficiency and / or are compatible with an effective circular RNA purification process.

Description

[0001] This application is a divisional application of the invention patent application filed on December 4, 2020, with application number 202080095660.6 and entitled "Circular RNA Composition and Method".

[0002] Cross-references to related applications

[0003] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 943,779, filed December 4, 2019; U.S. Provisional Application No. 62 / 972,194, filed February 10, 2020; U.S. Provisional Application No. 63 / 022,248, filed May 8, 2020; U.S. Provisional Application No. 63 / 087,582, filed October 5, 2020; and U.S. Provisional Application No. 62 / 943,797, filed December 4, 2019, the contents of which are hereby incorporated by reference in their entirety for all purposes. Background Technology

[0004] Conventional gene therapy involves using DNA to insert desired genetic information into host cells. The introduced DNA typically integrates to some extent into the genome of one or more transfected cells, allowing the introduced genetic material to have a long-lasting effect in the host. While such sustained effects can be substantially beneficial, the integration of exogenous DNA into the host genome can also have many detrimental effects. For example, the introduced DNA may insert into an intact gene, leading to mutations that inhibit or even completely eliminate the function of endogenous genes. Therefore, gene therapy using DNA can impair important genetic functions in the treated host, such as eliminating or harmfully reducing the production of essential enzymes or interfering with genes that play a key role in regulating cell growth, leading to unregulated or cancerous cell proliferation. Furthermore, conventional DNA-based gene therapy requires strong promoter sequences to effectively express the desired gene product, which can also lead to undesirable changes in the regulation of normal gene expression in the cell. DNA-based genetic material may also induce unwanted anti-DNA antibodies, which can then trigger a potentially fatal immune response. Gene therapy using viral vectors can also lead to adverse immune responses. In some cases, viral vectors can even integrate into the host genome. Furthermore, 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. Therefore, while DNA-based gene therapies using viral vectors to deliver secreted proteins have been evaluated (US Patent Nos. 6,066,626; US2004 / 0110709), these methods may be limited for these various reasons.

[0005] Compared to DNA, using RNA as a gene therapy agent is generally safer because RNA does not involve the risk of stable integration into the genome of transfected cells. This eliminates concerns that the introduced genetic material will interfere with the normal functioning of essential genes or cause mutations that lead to harmful or carcinogenic effects. Furthermore, the foreign promoter sequence is not required for the effective translation of the encoded protein, again avoiding potentially harmful side effects. In addition, mRNA does not need to enter the cell nucleus to perform its function, while DNA must overcome this major obstacle.

[0006] Circular RNAs can be used to design and produce stable forms of RNA. The circularization of RNA molecules offers advantages for the study of RNA structure and function, especially when the molecule readily folds into an inactive conformation (Wang and Ruffner, 1998). Circular RNAs are also 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.

[0007] Prior to this invention, three main techniques existed for the in vitro preparation of circular RNA: the clip-mediated method, the permuted intron-exon method, and the RNA ligase-mediated method. However, existing methods are limited by the size of the circularizable RNA, thus restricting their therapeutic applications. Summary of the Invention

[0008] This application provides circular RNA and transfer mediators, as well as related compositions and therapeutic methods. The transfer mediators may comprise, for example, ionizable lipids, PEG-modified lipids, and / or structural lipids, thereby forming lipid nanoparticles encapsulating the circular RNA. The circular RNA may comprise group I intron fragments, spacer regions, IRES, double-strand-forming regions, and / or expression sequences, thereby exhibiting improved expression, functional stability, low immunogenicity, ease of manufacture, and / or prolonged half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNA and transfer mediators are particularly suitable for efficient protein expression in immune cells in vivo. This application also provides precursor RNA and materials for generating said precursor or circular RNA, which has improved circularization efficiency and / or compatibility with efficient circular RNA purification methods.

[0009] Therefore, one aspect of this application provides a pharmaceutical composition comprising: a cyclic RNA polynucleotide and a transfer medium comprising an ionizable lipid represented by formula (1):

[0010]

[0011] in:

[0012] Each n is an independent integer from 2 to 15;

[0013] L1 and L3 are each independently -OC(O)-* or -C(O)O-*, where "*" indicates the connection point with R1 or R3;

[0014] R1 and R3 are each independently linear or branched C9-C chains optionally substituted by one or more substituents selected from the group consisting of the following. 20 Alkyl or C9-C 20 Alkenyl: oxoyl, halogen, hydroxyl, 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, alkynylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylsulfonyl and alkylsulfonylalkyl; and

[0015] R2 can be selected from the following groups:

[0016]

[0017] In some implementations, R1 and R3 are each independently selected from the following groups:

[0018] In some implementations, R1 and R3 are the same. In other implementations, R1 and R3 are different.

[0019] In some embodiments, the ionizable lipid of formula (1) is represented by formula (1-1) or formula (1-2):

[0020]

[0021] In some embodiments, the ionizable lipid is selected from the group consisting of:

[0022]

[0023]

[0024] In another aspect, this application provides a pharmaceutical composition comprising: a cyclic RNA polynucleotide and a transfer medium comprising an ionizable lipid represented by formula (2):

[0025]

[0026] in:

[0027] Each n is an independent integer from 1 to 15;

[0028] R1 and R2 are each independently selected from the following groups:

[0029]

[0030]

[0031] and

[0032] R3 can be selected from the following groups:

[0033]

[0034] In another aspect, this application provides a pharmaceutical composition comprising: a cyclic RNA polynucleotide and a transfer medium comprising an ionizable lipid represented by formula (3):

[0035]

[0036] in:

[0037] X is selected from -O-, -S-, or -OC(O)-*, where * indicates the connection point with R1;

[0038] R1 can be selected from the following groups:

[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] In another aspect, this application provides a pharmaceutical composition comprising: a cyclic RNA polynucleotide and a transfer medium comprising an ionizable lipid represented by formula (4):

[0043]

[0044] Where: each n is an integer from 2 to 15 independently; and R2 is defined in equation (1).

[0045] In another aspect, this application provides a pharmaceutical composition comprising: a cyclic RNA polynucleotide and a transfer medium comprising ionizable lipids selected from Table 10a.

[0046] 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).

[0047] 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).

[0048] In some embodiments, the first and second expression sequences are separated by a ribosomal jumping 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.

[0049] In some embodiments, the circular RNA polynucleotide includes one or more microRNA binding sites. These microRNA binding sites are recognized by microRNAs expressed in the liver. In some embodiments, the microRNA binding sites are recognized by miR-122.

[0050] 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.

[0051] In some embodiments, the circular RNA polynucleotide comprises, in the following order: a) a post-splicing intron fragment of the 3'I group intron, b) IRES, c) the expression sequence, and d) a post-splicing intron fragment of the 5'I group intron. In some embodiments, the circular RNA polynucleotide comprises... In some embodiments, the circular RNA polynucleotide comprises a first spacer region preceding the post-splicing intron fragment of the 3'I group intron and a second spacer region following the post-splicing intron fragment of the 5'I group intron. In some embodiments, the first and second spacer regions each have a length of about 10 to about 60 nucleotides.

[0052] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a 3'I intron fragment, an IRES, an expression sequence, and a 5'I intron fragment.

[0053] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a 5' outer double-strand forming region, a 3' I group intron fragment, a 5' internal spacer region optionally comprising a 5' inner double-strand forming region, an IRES, an expression sequence, a 3' internal spacer region optionally comprising a 3' inner double-strand forming region, a 5' I group intron fragment, and a 3' outer double-strand forming region.

[0054] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a 5' outer double-strand forming region, a 5' outer spacer region, a 3' I group intron fragment, a 5' inner spacer region optionally comprising a 5' inner double-strand forming region, an IRES, an expression sequence, a 3' inner spacer region optionally comprising a 3' inner double-strand forming region, a 5' I group intron fragment, a 3' outer spacer region, and a 3' outer double-strand forming region.

[0055] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a 3'I group intron fragment, a 5' internal spacer region containing a 5' internal double-strand forming region, an IRES, an expression sequence, a 3' internal spacer region containing a 3' internal double-strand forming region, and a 5'I group intron fragment.

[0056] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a 5' outer double-strand forming region, a 5' outer spacer region, a 3' I group intron fragment, a 5' inner spacer region containing a 5' inner double-strand forming region, an IRES, an expression sequence, a 3' inner spacer region containing a 3' inner double-strand forming region, a 5' I group intron fragment, a 3' outer spacer region, and a 3' outer double-strand forming region.

[0057] In some embodiments, the circular RNA polynucleotide is prepared by cyclization of an RNA polynucleotide comprising, in the following order: a first polyA sequence, a 5' outer double-strand forming region, a 5' outer spacer region, a 3' I group intron fragment, a 5' inner spacer region containing a 5' inner double-strand forming region, an IRES, an expression sequence, a 3' inner spacer region containing a 3' inner double-strand forming region, a 5' I group intron fragment, a 3' outer spacer region, a 3' outer double-strand forming region, and a second polyA sequence.

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

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

[0060] 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, the 3' and 5' outer double-strand forming regions each have a length of about 10-50 nucleotides. In some embodiments, the 3' and 5' inner double-strand forming regions each have a length of about 6-30 nucleotides.

[0061] In some embodiments, the IRES is selected from Table 17, or a functional fragment or variant thereof. In some embodiments, the IRES has a sequence from the following IRES: Taura syndrome virus, triatomine virus, Theiler's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, rice constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stali enterovirus, Kashmir bee virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, glass leafhopper 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, Tea geometrid moth microRNA virus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human Coxsackievirus B3, Tobacco mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black bee queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorosis and ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennae and legs, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, tobacco etch virus, turnip shrunken virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, small disegmented RNA virus (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 paraenteric virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Paraenterovirus 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 Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepatitis Virus K, Hepatitis Virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Bicistronic Virus, Hubei MicroRNA Virus-like Virus, CRPV, Striped Field Mouse MicroRNA Virus, Goat Ridge 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 aptamers.

[0062] In some embodiments, the first and second polyA sequences each have a length of about 15-50 nt. In some embodiments, the first and second polyA sequences each have a length of about 20-25 nt.

[0063] In some embodiments, the circular RNA polynucleotide contains 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 is composed of naturally occurring nucleotides.

[0064] In some embodiments, the expressed 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 microRNA present in the 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 the 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.

[0065] 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.

[0066] In some embodiments, the duration of therapeutic effect of the pharmaceutical composition in human cells is greater than or equal to the duration of 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 of at least 80 nt in length.

[0067] In some embodiments, the duration of therapeutic effect of the pharmaceutical composition in humans is greater than the duration of 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 pharmaceutical composition has a duration of therapeutic effect in humans 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.

[0068] In some embodiments, the functional half-life of the pharmaceutical composition in human cells is greater than or equal to a predetermined threshold functional half-life. In some embodiments, the in vivo functional half-life of the pharmaceutical composition in humans is greater than a predetermined threshold functional half-life. 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 includes measuring the level of a protein encoded by the expression sequence of the circular RNA polynucleotide in a patient's serum or tissue sample. 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. In some embodiments, the pharmaceutical composition has a functional half-life of at least about 20 hours.

[0069] In some embodiments, the pharmaceutical composition comprises a structural lipid and a PEG-modified lipid. In some embodiments, the structural lipid binds to C1q and / or promotes the binding of the lipid-containing transfer medium to C1q compared to a control transfer medium lacking the structural lipid, and / or increases the uptake of the C1q-bound transfer medium into immune cells compared to a control transfer medium lacking the structural lipid. In some embodiments, the immune cells are T cells, NK cells, NKT cells, macrophages, or neutrophils.

[0070] In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is β-sitosterol. In some embodiments, the structural lipid is not β-sitosterol.

[0071] In some embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, or PEG-1. In some embodiments, the PEG-modified lipid is DSPE-PEG(2000).

[0072] In some embodiments, the pharmaceutical composition further comprises an auxiliary lipid. In some embodiments, the auxiliary lipid is DSPC or DOPE.

[0073] In some embodiments, the pharmaceutical composition comprises DOPE, cholesterol, and DSPE-PEG.

[0074] In some embodiments, the transfer medium comprises about 0.5% to about 4% PEG-modified lipids by molar ratio. In some embodiments, the transfer medium comprises about 1% to about 2% PEG-modified lipids by molar ratio.

[0075] In some embodiments, the transfer medium comprises

[0076] a. by

[0077]

[0078] The ionizable lipids represented

[0079] b.DOPE,

[0080] c. Cholesterol, and

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

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

[0083] In some embodiments, the transfer medium comprises

[0084] a. by

[0085]

[0086] The ionizable lipids represented

[0087] b.DOPE,

[0088] c. Cholesterol, and

[0089] d.DSPE-PEG(2000).

[0090] In some implementations, the molar ratio of ionizable lipids:DSPC:cholesterol:DSPE-PEG(2000) is 50:10:38.5:1.5.

[0091] In some embodiments, the transfer medium has a nitrogen:phosphate (N:P) ratio of about 3 to about 6.

[0092] In some embodiments, the transfer medium is formulated for endosome release of the circular RNA polynucleotide.

[0093] In some embodiments, the transfer medium is capable of binding to APOE. In some embodiments, the transfer medium interacts with apolipoprotein E (APOE) less than an equivalent transfer medium 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 medium is substantially free of APOE binding sites.

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

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

[0096] In some embodiments, the transfer mediator can be taken up into cells via low-density lipoprotein receptor (LDLR). In some embodiments, the transfer mediator can be taken up into cells independently of 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 portion operatively linked to the transfer medium. In some embodiments, the targeting portion 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 a connective molecule comprising a transfer-mediator binding portion and a cell-binding portion, wherein the targeting portion specifically binds to the transfer-mediator binding portion and the cell-binding portion 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 portion 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, acevitine, biotin, or digoxin.

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

[0102] In some embodiments, the ionizable lipid has a half-life of less than about 2 weeks in the cell membrane. In some embodiments, the ionizable lipid has a half-life of less than about 1 week in the cell membrane. In some embodiments, the ionizable lipid has a half-life of less than about 30 hours in the cell membrane. 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, this application provides a method for treating or preventing a disease, symptom, or disorder, the method comprising administering an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the disease, symptom, or disorder is associated with abnormal expression, activity, or localization of peptides selected from Tables 27 or 28. In some embodiments, the circular RNA polynucleotide encodes a therapeutic protein. In some embodiments, the therapeutic protein is expressed in the spleen at a higher level than in the liver. In some embodiments, the therapeutic protein is expressed in the spleen at at least about 2.9 times the therapeutic protein is expressed 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 therapeutic protein is expressed in the spleen at at least about 63% of the total therapeutic protein expression.

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

[0105] In some embodiments, the linear RNA polynucleotide is contained in a first spacer region of the 3'I group intron fragment 5'. 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 contains a poly-A sequence.

[0106] In some embodiments, the linear RNA polynucleotide is contained in a second spacer region at the 3' of the 5'I group 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 contains a poly-A sequence.

[0107] In some embodiments, the linear RNA polynucleotide further includes a third spacer region between the 3'I group intron fragment and 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 includes first and second duplex forming regions capable of forming duplexes. 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, the linear RNA polynucleotide has enhanced expression, cyclization efficiency, functional stability, and / or stability compared to a reference linear RNA polynucleotide, wherein the reference linear RNA polynucleotide comprises, from 5' to 3', a first polyA sequence, a 5' outer spacer region, a 3' I group intron fragment, a 5' inner spacer region containing a 5' inner double-strand forming region, IRES, an expression sequence, a stop codon, a 3' inner spacer region containing a 3' inner double-strand forming region, a 5' I group intron fragment, a 3' outer spacer region, and a second polyA sequence.

[0109] In some embodiments, the linear RNA polynucleotide has enhanced expression, cyclization efficiency, functional stability, and / or stability compared to a reference linear RNA polynucleotide, wherein the reference linear RNA polynucleotide comprises, from 5' to 3', a reference 3' I intron fragment, a reference IRES, a reference expression sequence, and a reference 5' I intron fragment, and does not include the spacer region at 5' of the 3' I intron fragment or the spacer region at 3' of the 5' 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 an L6-5 substitution site. In some embodiments, the 3' Anabaena group I intron fragment and the 5' Anabaena group I intron fragment are not generated using an L6-5 substitution site. In some embodiments, the 3' Anabaena group I intron fragment comprises or consists of a sequence selected from SEQ ID NO: 112-123 and 125-150. In some embodiments, the 5' Anabaena group I intron fragment comprises a corresponding sequence selected from SEQ ID NO: 73-84 and 86-111. In some embodiments, the 5' Anabaena Group I intron fragment comprises or consists of sequences selected from or composed of said sequences. In some embodiments, the 3' Anabaena Group I intron fragment comprises or consists of corresponding sequences selected from or composed of said sequences.

[0111] In some embodiments, the IRES comprises a nucleotide sequence selected from SEQ ID NO: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 NO:1-64 and 66-72.

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

[0113] In another aspect, this application discloses a circular RNA comprising, from 5' to 3', a 3'I group intron fragment, an IRES, an expression sequence, and a 5'I group intron fragment, wherein the IRES comprises a nucleotide sequence selected from SEQ ID NO:348-351.

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

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

[0116] In some embodiments, the expressed 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 a natural nucleotide. In some embodiments, the expressed sequence is codon-optimized. In some embodiments, the RNA polynucleotide further comprises a translation termination cassette containing at least one stop codon in each reading frame. In some embodiments, the translation termination cassette contains at least two stop codons in the reading frames of the expressed 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 two expression sequences. In some embodiments, each expression sequence encodes a different therapeutic protein.

[0119] In some embodiments, the circular RNA polynucleotide disclosed herein is about 100 to 15,000 nucleotides in length, 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 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 therapeutic duration of the circular RNA polynucleotide in human cells is greater than or equal to the therapeutic duration of an equivalent linear RNA polynucleotide containing 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 containing the same expression sequence. In some embodiments, the in vivo therapeutic duration of the circular RNA polynucleotide in humans is greater than the in vivo therapeutic 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, this disclosure provides a composition comprising the circular RNA polynucleotide disclosed herein, nanoparticles, and optionally a targeting portion operatively linked to the nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, core-shell nanoparticles, biodegradable nanoparticles, biodegradable lipid nanoparticles, polymer nanoparticles, or biodegradable polymer nanoparticles. In some embodiments, the pharmaceutical composition comprises a targeting portion wherein, without cell isolation or purification, the targeting portion mediates receptor-mediated endocytosis or direct selective fusion into cells of a selected cell population or tissue. In some embodiments, the targeting portion is an scFv, nanobody, peptide, microantibody, polynucleotide aptamer, heavy chain variable region, light chain variable region, or fragment thereof. In some embodiments, less than 1% by weight of the polynucleotide in the composition is double-stranded RNA, DNA clipping, or triphosphorylated RNA. In some embodiments, less than 1% by weight of the polynucleotide and protein in the pharmaceutical composition are double-stranded RNA, DNA clipping, triphosphorylated RNA, phosphatase protein, protein ligase, and capping enzyme.

[0122] In another aspect, this disclosure provides a method of treating a subject in need, the method comprising administering a therapeutically effective amount of a composition comprising the circular RNA polynucleotide disclosed herein, nanoparticles, and optionally a targeting portion operatively linked to said nanoparticles.

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

[0124] In some embodiments, the provided pharmaceutical composition comprises a targeting portion, wherein the targeting portion selectively mediates receptor-mediated endocytosis into cells of a selected cell population without cell selection or purification.

[0125] In some implementations, the provided nanoparticles contain more than one type of cyclic RNA polynucleotide.

[0126] In another aspect, this application provides a DNA vector encoding the RNA polynucleotides disclosed herein. In some embodiments, the DNA vector further comprises a transcriptional regulatory sequence. In some embodiments, the transcriptional regulatory sequence comprises 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, this application provides a prokaryotic cell comprising the DNA vector disclosed herein.

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

[0129] In another aspect, this 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), guanine triphosphate (GTP), cytosine triphosphate (CTP), uridine triphosphate (UTP), and RNA polymerase. In some embodiments, the suitable conditions further include guanine monophosphate (GMP). In some embodiments, the ratio of GMP concentration to GTP concentration is in the range of about 3:1 to about 15:1, optionally about 4:1, 5:1, or 6:1.

[0130] In another aspect, this application provides a method for producing circular RNA polynucleotides, the method comprising culturing prokaryotic cells disclosed herein under suitable conditions for transcribing DNA in cells. 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 affinity oligonucleotides conjugated to a solid surface that hybridize with the first or second spacer region. In some embodiments, the first or second spacer region comprises a poly-A sequence, and wherein said affinity oligonucleotide is a deoxythymidine oligonucleotide. Attached Figure Description

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

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

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

[0134] Figure 4A and 4BProtein expression of selected IRES constructs from Jurkat cells was depicted as measured by the luminescence of Gaussian luciferase secreted from the cell supernatant.

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

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

[0137] Figure 7 depicts the effect of electroporation of Jurkat cells with modified linear, unpurified circular, or purified circular RNA followed by IFNγ (...). Figure 7A ), IL-6 Figure 7B ), IL-2 Figure 7C ), RIG-I ( Figure 7D ), IFN-β1 ( Figure 7E ) and TNFα Figure 7F Transcriptional induction.

[0138] Figure 8 depicts a primary human monocyte ( Figure 8A ) and macrophages ( Figure 8B and Figure 8C Comparison of luminescence between circular RNA encoding Gaussian luciferase and modified linear RNA.

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

[0140] Figure 10 depicts the 24-hour luminescence in the supernatant of primary T cells transduced with circular RNA containing Gaussian luciferase expression sequences or modified linear RNA. Figure 10A ), or relative luminescence within 3 days ( Figure 10B ) and 24-hour luminescence in PBMC ( Figure 10C ).

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

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

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

[0144] Figure 14 The circularization efficiency of three RNA constructs with no homologous arms or homologous arms of different lengths and GC contents was depicted.

[0145] Figure 15A and 15B HPLC chromatograms were plotted to show the contribution of strong homologous arms to improved splicing efficiency, the relationship between cyclization efficiency and nick in selected constructs, and the combination of substitution sites and homologous arms that hypothesize to improve cyclization efficiency.

[0146] Figure 16 The images show fluorescent images of T cells simulated by electroporation (left) or by electroporation with circular RNA encoding CAR (right) and co-cultured with Raji cells expressing GFP and firefly luciferase.

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

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

[0149] Figure 19 depicts the luminescence in the supernatant of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction with linear or circular RNA containing Gaussian luciferase expression sequences and different IRES sequences. Figure 19A ), and relative luminescence over 3 days ( Figure 19B ).

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

[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 bioluminescence detection. Figure 21A ), and IFNγ transcript-induced 24 hours after electroporation with different amounts of circular or linear RNA encoding CAR sequences ( Figure 21B ).

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

[0153] Figure 23 The study depicts the specific lysis of target cells by human CD3+ T cells electroporated with CAR-encoding RNA 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 targeting CAR.

[0155] Figure 25 The total throughput of organs harvested from CD-1 mice, which were given circular RNA encoding FLuc and formulated with 50% lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG and 38.5% cholesterol, was depicted.

[0156] Figure 26 The image shows a glowing organ harvested from CD-1 mice, which were given circular RNA encoding FLuc and 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 The proton nuclear magnetic resonance (NMR) spectrum of lipid 26 is shown. Figure 27B The retention time of lipid 26 is shown as measured by liquid chromatography-mass spectrometry (LC-MS). Figure 27C The mass spectrum of lipid 26 is shown. Figure 27D The proton NMR spectrum of lipid 27 is shown. Figure 27E The retention time of lipid 27 is shown as measured by LC-MS. Figure 27F The mass spectrum of lipid 27 is shown.

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

[0159] Figure 29 NMR spectra of bis(2-(tetradecylthio)ethyl)3,3'-((3-(1H-imidazol-1-yl)propyl)azaalkyldiyl)dipropionate (lipid 93-S14) were depicted.

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

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

[0162] Figure 32A The total flux of spleen and liver harvested from CD-1 mice was depicted, which were given circular RNA encoding firefly luciferase (FLuc) and formulated with target ionizable lipids, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1. Figure 32B Mean radiance that depicts the biological distribution of protein expression.

[0163] Figure 33AImages depicting the glowing of organs harvested from CD-1 mice given circular RNA encoding FLuc and formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in 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 given circular RNA encoding FLuc and formulated with ionizable lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0164] Figure 34A Images depicting the glowing of organs harvested from CD-1 mice given circular RNA encoding FLuc and formulated with ionizable lipids 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in 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 given circular RNA encoding FLuc formulated with ionizable lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0165] Figure 35A Images depicting the glowing of organs harvested from CD-1 mice given circular RNA encoding FLuc and formulated with ionizable lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) from Table 10a in 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 given circular RNA encoding FLuc and formulated with ionizable lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (AvantiPolar Lipids Inc.) in a weight ratio of 16:1:4:1 or a molar ratio of 62:4:33:1.

[0166] Figure 36 depicts a luminescent image highlighting an organ harvested from c57BL / 6J mice, which were given an FLuc-encoded protein encapsulated in lipid 15 from Table 10b. Figure 36A), lipids 53 from Table 10a Figure 36B ) or lipids 54 from Table 10a ( Figure 36C Circular RNA in lipid nanoparticles formed. PBS was used as a control. Figure 36D ).

[0167] Figure 37A and 37B The relative luminescence in human PBMC lysates was depicted after incubation for 24 hours with test lipid nanoparticles containing circular RNA encoding firefly luciferase.

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

[0169] Figure 39 The expression of anti-mouse CD19 CAR in 1C1C7 cells transfected with circular RNA lipids containing anti-mouse CD19 CAR expression sequences and different IRES sequences was depicted.

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

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

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

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

[0174] Figure 44 The cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMACAR) expressed from single circular RNAs in T cells was depicted.

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

[0176] Figure 46A An exemplary RNA construct design is depicted with a built-in polyA sequence in an intron. Figure 46B The chromatographic trace of unpurified circular RNA is shown. Figure 46C The chromatographic traces of affinity-purified circular RNA are shown. Figure 46D Immunogenicity of circular RNA prepared using different IVT conditions and purification methods is shown. (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 An exemplary RNA construct design is described, featuring a dedicated binding sequence as an alternative to polyA for hybridization purification. Figure 47B The chromatographic trace of unpurified circular RNA is shown. Figure 47C The chromatographic traces of affinity-purified circular RNA are shown.

[0178] Figure 48A The chromatographic trace of unpurified circular RNA encoding dystrophin is shown. Figure 48B The chromatographic trace of the enzyme-purified circular RNA encoding dystrophin is shown.

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

[0180] Figure 50The luminescent expression levels and stability of circular RNAs containing the indicated original or modified IRES elements are shown in primary T cells.

[0181] Figure 51 The luminescent expression levels and expression stability of circular RNAs containing the indicated original or modified IRES elements are shown in HepG2 cells.

[0182] Figure 52 The luminescent expression levels and expression stability of circular RNAs containing the indicated original or modified IRES elements are shown in 1C1C7 cells.

[0183] Figure 53 The luminescent expression levels and stability of circular RNA from IRES elements containing inserted untranslated regions (UTRs) or hybrid IRES elements are shown in HepG2 cells. “Scr” indicates disordered expression and is used as a control.

[0184] Figure 54 The luminescent expression levels and expression stability of circular RNAs containing IRES operatively linked to a Gaussian luciferase coding sequence and a variable stop codon box are shown in 1C1C7 cells.

[0185] Figure 55 The luminescent expression levels and expression stability of circular RNAs containing IRES and a variable untranslated region (UTR) inserted before the start codon of the Gaussian luciferase coding sequence are shown in 1C1C7 cells.

[0186] Figure 56 This study shows the expression level of human erythropoietin (hEPO) in Huh7 cells derived from a circular RNA containing two miR-122 target sites downstream of the hEPO coding sequence. Detailed Implementation

[0187] This document provides pharmaceutical compositions and transfer mediators, such as lipid nanoparticles, comprising circular RNA. The circular RNAs provided herein can be delivered to and / or targeted to cells in a transfer mediator (e.g., nanoparticles) or a composition comprising a transfer mediator. In some embodiments, the circular RNA may also be delivered to a subject in the form of a transfer mediator or a composition comprising a transfer mediator. In some embodiments, the transfer mediator is a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a polymeric core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the transfer mediator comprises one or more ionizable lipids, PEG-modified lipids, accessory lipids, and / or structural lipids.

[0188] In some embodiments, the transfer medium encapsulates circular RNA and comprises ionizable lipids, structural lipids, and PEG-modified lipids.

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

[0190] In some embodiments, the RNA in the transfer medium 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 medium.

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

[0192] Without being bound by theory, it is anticipated that transfer media containing APOE binding sites will deliver circular RNA more efficiently to the liver. Therefore, in some embodiments, transfer media comprising the ionizable lipids described herein and loaded with circular RNA substantially contain APOE binding sites on the surface of the transfer media, thereby delivering the circular RNA to the liver with higher efficiency compared to transfer media substantially lacking APOE binding sites on their surface. In some embodiments, transfer media comprising the ionizable lipids described herein and loaded with circular RNA substantially lack APOE binding sites on the surface of the transfer media, thereby delivering the circular RNA to the liver with lower efficiency compared to transfer media containing APOE binding sites on their surface.

[0193] In some embodiments, the transfer medium delivers or is capable of delivering 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 the subject is expressed in the spleen. In some embodiments, more therapeutic protein is expressed in the spleen than in the liver (e.g., 2-, 3-, 4-, or 5-fold more). In some embodiments, the lipid nanoparticles have an ionizable lipid:phosphate ratio of 3-7. In some embodiments, the lipid nanoparticles have an ionizable lipid:phosphate ratio of 4-6. In some embodiments, the lipid nanoparticles have an ionizable lipid:phosphate ratio of 4.5. In some embodiments, the lipid nanoparticles have a nitrogen:phosphate (N:P) ratio of 3-6. In some embodiments, the lipid nanoparticles have an N:P ratio of 5-6. In some embodiments, the lipid nanoparticles have an N:P ratio of 5.7. In some embodiments, the expression of non-secretory proteins can be measured using an ELISA, normalized to tissue weight.

[0194] Without being bound by theory, it is believed that the transfer medium described in this article 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 this disclosure provide lipid compositions described according to the corresponding molar ratios of component lipids in a formulation. In one embodiment, the mol-% of ionizable lipids can be from about 10 mol-% to about 80 mol-%. In one embodiment, the mol-% of ionizable lipids can be from about 20 mol-% to about 70 mol-%. In one embodiment, the mol-% of ionizable lipids can be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of ionizable lipids can be from about 35 mol-% to about 55 mol-%. In one embodiment, the mol-% of ionizable lipids can be from about 40 mol-% to about 50 mol-%. In some embodiments, the mol-% of ionizable lipids in a batch of transfer medium will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of a target mol-%. In some embodiments, the batch-to-batch variability of the transfer medium will be less than 15%, less than 10%, or less than 5%.

[0196] In one embodiment, the mol-% of the assisting lipid can be from about 1 mol-% to about 50 mol-%. In one embodiment, the mol-% of the assisting lipid can be from about 2 mol-% to about 45 mol-%. In one embodiment, the mol-% of the assisting lipid can be from about 3 mol-% to about 40 mol-%. In one embodiment, the mol-% of the assisting lipid can be from about 4 mol-% to about 35 mol-%. In one embodiment, the mol-% of the assisting lipid can be from about 5 mol-% to about 30 mol-%. In one embodiment, the mol-% of the assisting lipid can be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the assisting lipid in the transfer medium 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 medium 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 from about 1.5 mol%. In some embodiments, the mol% of the PEG-modified lipid in the transfer medium batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol%.

[0199] Pharmaceutical compositions comprising one or more of the compounds disclosed herein, and particularly transfer media, are also considered. In some embodiments, such transfer media comprise one or more of the PEG-modified lipids, ionizable lipids, accessory lipids, and / or structural lipids disclosed herein. Transfer media comprising one or more of the compounds disclosed herein and further comprising one or more additional lipids are also considered. In some embodiments, such transfer media are loaded with or otherwise encapsulated with circular RNA.

[0200] The transfer mediators of the present invention encapsulate circular RNA. In some embodiments, the polynucleotides encapsulated by the compounds or pharmaceutical and liposome compositions of the present invention comprise RNA encoding proteins or enzymes (e.g., circRNA encoding, for example, phenylalanine hydroxylase (PAH)). The present invention contemplates the use of such polynucleotides as therapeutic agents capable of being expressed by target cells to thereby contribute to the production (and in some cases, secretion) of functional enzymes or proteins, such as those disclosed, for example, in International Application No. PCT / US2010 / 058457 and U.S. Provisional Application No. 61 / 494,881, filed June 8, 2011, the doctrines of which are incorporated herein by reference in their entirety. For example, in some embodiments, the production of functional enzymes or proteins (e.g., urea cycle enzymes or enzymes associated with lysosomal storage diseases) lacking in the subject can be observed after expression of one or more polynucleotides in target cells. As another example, the circular RNA encapsulated by the transfer mediator may encapsulate one or two polypeptide chains of a T-cell receptor protein or encapsulate a chimeric antigen receptor (CAR).

[0201] This document also provides a method for treating a subject's disease by administering to a subject an effective amount of a composition comprising a circular RNA encoding a functional protein and the transfer medium described herein. In some embodiments, the circular RNA is encapsulated within a transfer medium. In some embodiments, such methods may enhance (e.g., increase) the expression of polynucleotides and / or increase the production and secretion of functional polypeptide products in one or more target cells and tissues (e.g., immune cells or hepatocytes). Typically, such methods involve contacting target cells with one or more compounds comprising or otherwise encapsulating circRNA and / or a transfer medium.

[0202] In some embodiments, the transfer medium (e.g., lipid nanoparticles) is formulated in part based on its ability to promote transfection of target cells (e.g., circular RNA). In another embodiment, the transfer medium (e.g., lipid nanoparticles) may be selected and / or prepared to optimize the delivery of circular RNA to target cells, tissues, or organs. For example, if the target cells are hepatocytes, or if the target organ is the spleen, the properties (e.g., size, charge, and / or pH) of the drug and / or liposome composition may be optimized to efficiently deliver such a composition (e.g., lipid nanoparticles) to the target cells or organ, reducing immune clearance and / or promoting retention in the target cells or organ. Alternatively, if the target tissue is the central nervous system, the selection and preparation of the transfer medium must take into account its penetration and retention within the blood-brain barrier and / or the use of alternative methods for directly delivering such compositions (e.g., lipid nanoparticles) to such target tissue (e.g., via intravascular cerebrovascular administration). In some embodiments, the transfer medium may be combined with an agent that promotes the transfer of encapsulation materials across the blood-brain barrier (e.g., an agent that disrupts or improves the permeability of the blood-brain barrier and thereby enhances the transfer of circular RNA to target cells). While the transfer mediums described herein (e.g., lipid nanoparticles) can promote the introduction of circRNA into target cells, the addition of polycations (e.g., poly-L-lysine and protamine) as copolymers to one or more lipid nanoparticles, such as those containing a pharmaceutical composition, can also help, and in some cases significantly enhance, transfection efficiency of several types of transfer mediums by 2–28-fold in vitro and in vivo in many cell lines (see, NJ Caplen et al., Gene Ther. 1995; 2:603; S. Li et al., Gene Ther. 1997; 4,891.). In some embodiments, the target cells are immune cells. In some embodiments, the target cells are T cells.

[0203] In some embodiments, the transfer mediators described herein (e.g., lipid nanoparticles) are prepared by combining multiple lipid components (e.g., one or more compounds disclosed herein) with one or more polymer components. For example, lipid nanoparticles can be prepared using HGT4003, DOPE, cholesterol, and DMG-PEG2000. Lipid nanoparticles may consist of additional lipid combinations in varying proportions, including, for example, HGT4001, DOPE, and DMG-PEG2000. The selection of ionizable lipids, accessory lipids, structural lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, and the relative molar ratios of such lipids to each other, is 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 chain, and the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipids.

[0204] The transfer mediators described herein may allow encapsulated polynucleotides to reach target cells, or may preferentially allow encapsulated polynucleotides to reach target cells or organs on a discriminative basis (e.g., the transfer mediator may be concentrated in the liver or spleen of a subject who has been administered such a transfer mediator). Alternatively, the transfer mediator may restrict the delivery of 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 polynucleotides (e.g., circRNA) into transfer media can protect polynucleotides from environments (e.g., serum) that may contain enzymes or chemicals that degrade such polynucleotides and / or cause rapid excretion of such polynucleotides. Therefore, in some embodiments, the compositions described herein are capable of enhancing the stability of the encapsulated polynucleotides, particularly with respect to the environments in which such polynucleotides will be exposed.

[0206] In some embodiments, this document provides a vector for preparing circular RNA, the vector comprising a 5' double-strand forming region, a 3' I-group intron fragment, an optional first spacer region, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer region, the 5' I-group intron fragment, and the 3' double-strand 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' double-strand forming region located between the 3' I-group intron fragment and the IRES, and an internal 3' double-strand forming region located between the expression sequence and the 5' I-group intron fragment. In some embodiments, the internal double-strand forming regions are capable of forming double strands with each other but not with the external double-strand forming regions. In some embodiments, the internal double-strand forming regions are part of the first and second spacer regions. Further embodiments include circular RNA polynucleotides, including circular RNA polynucleotides prepared using the vectors provided herein; compositions containing such circular RNA; cells containing such circular RNA; and methods for 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 therapeutic purposes or to produce useful proteins, such as PAHs. In some embodiments, the methods are advantageous for producing desired peptides with a longer half-life compared to linear RNA within eukaryotic cells due to the resistance of the circular RNA to ribonucleases.

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

[0209] 1. Definition

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

[0211] As used herein, the term “3' I group intron fragment” refers to a sequence that has 75% or higher similarity to the 3' proximal end of a natural group I intron, which contains a splice site dinucleotide and an optional segment of a natural exon sequence.

[0212] As used herein, the term “5' group I intron fragment” refers to a sequence that has 75% or higher similarity to the 5' proximal end of a natural group I intron, which contains a splice site dinucleotide and an optional segment of a natural exon sequence.

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

[0214] As used herein, the term "splicing site" refers to a dinucleotide that is partially or completely contained within a group I intron and whose phosphodiester bond is cleaved during RNA circularization.

[0215] As used herein, the term "therapeutic protein" means any protein that, when administered directly or indirectly to a subject in the form of translated nucleic acid, has therapeutic, diagnostic, and / or preventative effects and / or induces the desired biological and / or pharmacological activity.

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

[0217] As used herein, the term "cyclization efficiency" refers to a measurement of the cyclic polynucleotide obtained compared to its linear starting material.

[0218] As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide produced from ribonucleotide transcripts. In some embodiments, translation efficiency may be expressed as the amount of protein or peptide produced per given amount of transcript encoding a protein or peptide.

[0219] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, their modified forms, or analogs. Nucleotides include substances comprising purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) and pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogs). Nucleotide analogs include nucleotides having modified chemical structures in their bases, sugars, and / or phosphate groups, including but not limited to 5'-position pyrimidine modification, 8'-position purine modification, modification at the extracyclic amine of cytosine, and substitution of 5-bromo-uracil; and 2'-position sugar modification, including but not limited to sugar-modified ribonucleotides, wherein the 2'-OH is substituted by a group such as H, OR, R, a halogen, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety as defined herein. Nucleotide analogs are also intended to include nucleotides with bases, such as inosine, piracetam, and xanthine; sugars, such as 2'-methylribose; non-natural phosphodiester bonds, such as methylphosphonates, thiophosphates, and peptide bonds. Nucleotide analogs include 5-methoxyuridine, 1-methylpseuuridine, and 6-methyladenosine.

[0220] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe polymers of any length (e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or up to about 10,000 or more bases) composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and which can be enzymatically or synthetically produced (e.g., as described in U.S. Patent No. 5,948,902 and the references cited therein) that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids, for example, by participating in Watson-Crick base-pairing interactions. Naturally occurring nucleic acids consist of nucleotides including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0221] As used in this article, the terms “ribonucleic acid” and “RNA” refer to polymers composed of ribonucleotides.

[0222] As used in this article, the terms “deoxyribonucleic acid” and “DNA” refer to polymers composed of deoxyribonucleotides.

[0223] "Separated" or "purified" generally refers to the separation of a substance (e.g., in some embodiments, a compound, polynucleotide, protein, polypeptide, polynucleotide composition, or polypeptide composition) such that the substance constitutes a significant percentage (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, typically up to about 90%-100%) of the sample. In some embodiments, the substantially purified component constitutes 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 density-based sedimentation. Generally, a substance is purified when it is present in a sample in a greater amount than its naturally occurring components relative to other components of the sample.

[0224] As used herein, the terms "double-stranded," "double-stranded," or "hybrid" refer to nucleic acids formed by the hybridization of two single strands of nucleic acids 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” 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 in this article, “structuring” of RNA refers to an RNA sequence 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.

[0227] As used herein, two “double-strand forming regions,” “homologous arms,” or “homologous regions” can be any two regions thermodynamically inclined to cross-pair in sequence-specific interactions. In some embodiments, two double-strand forming regions, homologous arms, or homologous regions have a sufficient level of sequence identity with each other’s anticomplementary sequence to serve as substrates for hybridization. As used herein, a polynucleotide sequence is “homological” when it is identical to or shares sequence identity with its anticomplementary or “complementary” sequence. The percentage of sequence identity between a homologous region and its corresponding anticomplementary sequence can be any percentage of sequence identity that allows hybridization to occur. In some embodiments, the inner double-strand forming regions of the polynucleotides of the present invention are capable of forming double strands with another inner double-strand forming region but not with an outer double-strand forming region.

[0228] Linear nucleic acid molecules are described as having a “5’-terminus” (5’ end) and a “3’-terminus” (3’ end) because nucleic acid phosphodiester bonds are present at the 5’ and 3’ carbons of the sugar moiety that replaces the mononucleotide. The terminal nucleotide of a polynucleotide is its 5’-terminal nucleotide, at which a new bond would be a bond to the 5’ carbon. The terminal nucleotide of a polynucleotide is its 3’-terminal nucleotide, at which a new bond would be a bond to the 3’ carbon. As used herein, a terminal nucleotide is a nucleotide located at the 3’- or 5’-terminal end.

[0229] "Transcription" refers to the process of forming or synthesizing RNA molecules using DNA molecules as templates via RNA polymerase. This invention does not limit the RNA polymerase used for transcription. For example, in some embodiments, T7 RNA polymerase may be used.

[0230] "Translation" refers to the formation of polypeptide molecules from 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, the singular forms “a / an” and “the” include plural referents unless otherwise expressly indicated. Thus, for example, a reference to “a cell” includes a combination of two or more cells, or an entire culture of cells; a reference to “polynucleotide” actually includes many copies of said polynucleotide. Unless expressly stated or obvious from the context, the term “or” is understood to be inclusive as used herein. 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 pertains, unless otherwise defined herein or in the remainder of the specification.

[0232] Unless explicitly stated or obvious from the context, as used herein, the term “about” should be understood as falling within the normal tolerance range in the field, such as within two standard deviations of the mean. “About” can be understood as 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 the context, all numerical values ​​provided herein are modified by the term “about”.

[0233] As used herein, the term "encoding" refers to any process in which information in a polymerized macromolecule is used to guide the production of a second molecule that differs from the first molecule. The second molecule may have a chemical structure that differs from that of the first molecule.

[0234] "Co-administration" means combining the therapeutic agent provided herein with one or more additional therapeutic agents at sufficiently close time intervals, such that the therapeutic agent provided herein enhances the effect of one or more additional therapeutic agents, or vice versa.

[0235] As used herein, the terms “treatment” and “prevention”, and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, and those skilled in the art will consider that said treatment or prevention has potential benefit or therapeutic effect. Treatment or prevention provided by the methods disclosed herein may include treatment or prevention of one or more ailments or symptoms of a disease. Furthermore, for the purposes of this document, “prevention” may include delaying the onset of a disease or its symptoms or ailments.

[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. An exemplary expression sequence encoding a peptide or polypeptide may comprise multiple nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon".

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

[0238] As used herein, a “splicing site” refers to one or more dinucleotides in which a phosphodiester bond is cleaved during a splicing reaction. A “5’ splicing site” refers to the native 5’ dinucleotide of an intron (e.g., a group I intron), while a “3’ splicing 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 larger that is capable of initiating the translation of a polypeptide without a typical RNA cap structure. IRES are typically about 500 nt to about 700 nt in length.

[0240] As used in this article, a "miRNA site" refers to a segment of nucleotides within a polynucleotide that can form a double helix with at least eight nucleotides of the natural miRNA sequence.

[0241] As used in this article, "endonuclease site" refers to a segment of nucleotides within a polynucleotide that can be recognized and cleaved by endonuclease proteins.

[0242] As used in this article, "bicistronic RNA" refers to a polynucleotide containing two expression sequences encoding two different proteins. These expression sequences can be separated by nucleotide sequences encoding cleavable peptides such as protease cleavage sites. They can also be separated by ribosomal jumping elements.

[0243] As used herein, the term "ribosomal jumping element" refers to a nucleotide sequence that encodes a short peptide sequence capable of producing two peptide chains from the translation of an RNA molecule. While not wishing to be bound by theory, it is assumed that the ribosomal jumping element functions by (1) terminating the translation of the first peptide chain and restarting the translation of the second peptide chain; or (2) by the intrinsic protease activity of the peptide encoding the peptide or by another protease in the environment (e.g., cytosol) cleaving the peptide bonds in the peptide sequence encoded by the ribosomal jumping element.

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

[0245] As used in this article, “transfer medium” includes any standard drug carrier, diluent, excipient, etc., which is generally intended for use in conjunction with the administration of bioactive agents, including nucleic acids.

[0246] As used herein, the phrase "lipid nanoparticles" refers to a transfer medium comprising one or more lipids (e.g., in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified 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 (e.g., physiological pH 4) and a neutral charge at other pH (e.g., physiological pH 7).

[0248] In some embodiments, the lipids disclosed herein (e.g., ionizable lipids) comprise one or more cleavable groups. The terms “cleavable” and “cleavable” are used herein to mean that one or more chemical bonds (e.g., one or more of covalent, hydrogen, van der Waals, and / or ionic interactions) between atoms in or adjacent to the subject functional group are broken (e.g., hydrolyzed) or can be broken upon exposure to selected conditions (e.g., upon exposure to enzymatic conditions). In some embodiments, the cleavable group is a disulfide functional group, and in certain embodiments, a disulfide functional group capable of being cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In some embodiments, the cleavable group is an ester functional group capable of being cleaved upon exposure to selected biological conditions. For example, a disulfide functional group can be cleaved by enzymatic reactions or by hydrolysis, oxidation, or reduction. Upon cleavage of such a disulfide functional group, one or more functional moieties or groups (e.g., one or more head groups and / or tail groups) bound thereto may be released. Exemplary cleavable groups may include, but are not limited to, disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl esters and aryl esters). In some embodiments, the cleavable group is not an ester or ether group. In some embodiments, the cleavable group is bonded (e.g., via 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 some 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 groups).

[0249] As used herein, the term "hydrophilic" is used qualitatively to indicate that the functional group is water-preferred, and typically such groups are water-soluble. For example, this document discloses compounds comprising a cleavable disulfide bond (S-S) functional group bonded to one or more hydrophilic groups (e.g., hydrophilic head groups), wherein such hydrophilic groups comprise 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 some embodiments, at least one functional group constituting a portion of the compounds disclosed herein is inherently hydrophobic (e.g., containing a hydrophobic tail of a naturally occurring lipid such as cholesterol). As used herein, the term "hydrophobic" is used qualitatively to indicate that the functional group is water-repellent, and typically such groups are insoluble in water. For example, compounds comprising a cleavable functional group (e.g., a disulfide (S-S) group) bonded to one or more hydrophobic groups are disclosed herein, wherein such hydrophobic groups contain one or more naturally occurring lipids such as cholesterol, and / or optionally substituted, variable-saturated or unsaturated C6-C groups. 20 Alkyl and / or optionally substituted, variable-saturated or unsaturated C6-C 20 Acyl group.

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

[0252] When describing this invention, it may include compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions. Unless otherwise stated, the following terms (if present) shall have the following meanings. It should also be understood that, as described herein, any portion defined below may be substituted with various substituents, and each definition is intended to include such substituted portions within the scope described below. Unless otherwise stated, the term “substituted” shall be defined as follows. It should be further understood that the terms “groups” and “radicals” are considered interchangeable when used herein.

[0253] When listing a range of values, the intention is to cover every value within that range and its subranges. For example, "C 1-6 The term "alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, and C6 alkyl groups. 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 some 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, thereby making such compounds amphiphilic. As used herein to describe compounds or compositions, the term "amphiphilic" refers to the ability to dissolve in both polar (e.g., water) and nonpolar (e.g., lipid) environments. For example, in some embodiments, the compounds disclosed herein comprise at least one lipophilic tail group (e.g., cholesterol or C6-C...). 20 An alkyl group and at least one hydrophilic head group (e.g., imidazole) are 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 particularly the functional groups constituting 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 some embodiments, a hydrophilic head group (e.g., guanidine) is bonded (e.g., via one or more of hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds) to a cleavable functional group (e.g., a disulfide 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), including both saturated and unsaturated hydrocarbons. In some embodiments, the alkyl group may comprise one or more cyclic alkyl groups 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, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). In some embodiments, the alkyl group considered includes (9Z,12Z)-octadec-9,12-diene. Such as, for example, "C6-C 20The use of the name “C” is intended to refer to alkyl groups (e.g., straight-chain or branched and including alkenes and alkyl groups) having the range of carbon atoms described. In some embodiments, the alkyl group has 1 to 10 carbon atoms (“C”). 1-10 Alkyl group (“C”). In some embodiments, the alkyl group has 1 to 9 carbon atoms (“C”). 1-9 Alkyl group). In some embodiments, the alkyl group has 1 to 8 carbon atoms (“C1”). 1-8 Alkyl group). In some embodiments, the alkyl group has 1 to 7 carbon atoms (“C1”). 1-7 Alkyl group). In some embodiments, the alkyl group has 1 to 6 carbon atoms (“C6”). 1-6 Alkyl group (“C”). In some embodiments, the alkyl group has 1 to 5 carbon atoms (“C”). 1-5 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 4 carbon atoms (“C”). 1-4 Alkyl group). In some embodiments, the alkyl group has 1 to 3 carbon atoms (“C1”). 1-3 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 2 carbon atoms (“C”). 1-2 Alkyl group (“C1 alkyl”). In some embodiments, the alkyl group has one carbon atom (“C1 alkyl”). 1-6 Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, etc.

[0257] As used herein, “alkenyl” refers to a straight-chain or branched hydrocarbon group (“C”) 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). 2-20 Alkenyl group (“Alkenyl”). In some 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 group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 9 carbon atoms (“C”). 2-9 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 8 carbon atoms (“C”). 2-8 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 7 carbon atoms (“C”). 2-7 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (“C”). 2-6 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (“C”). 2-5 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 4 carbon atoms (“C”). 2-4 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 3 carbon atoms (“C”). 2-3The alkenyl group (“C2-alkenyl”) has two carbon atoms in some embodiments. The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). 2-4 Examples of alkenyl groups include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. 2-6 Examples of alkenyl groups include the C group mentioned above. 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), and hexenyl (C6). Other examples of alkenyl groups include heptenyl (C7), octenyl (C8), and octtrienyl (C8).

[0258] As used herein, “alkynyl” refers to a straight-chain or branched hydrocarbon group (“C”) 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). 2-20 The alkynyl group (“Alynyl”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 10 carbon atoms (“C”). 2-10 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 9 carbon atoms (“C”). 2-9 The alkynyl group (“acetylenic”) has 2 to 8 carbon atoms in some embodiments. 2-8 The alkynyl group (“acetylation”) has 2 to 7 carbon atoms in some embodiments. 2-7 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 6 carbon atoms (“C”). 2-6 The alkynyl group (“H”) has 2 to 5 carbon atoms in some embodiments. 2-5 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 4 carbon atoms (“C”). 2-4 The alkynyl group (“C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 3 carbon atoms (“C”). 2-3 The alkynyl group (“C2-alkynyl”) is present in some embodiments. One or more carbon-carbon triple bonds may be internal (as in 2-butynyl) or terminal (as in 1-butynyl). 2-4 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), and 2-butynyl (C4). 2-6 Examples of alkenyl groups include the C group mentioned above. 2-4 Alkynyl groups include pentynyl (C5), hexynyl (C6), etc. Other examples of alkynyl groups include heptynyl (C7), octynyl (C8), etc.

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

[0260] As used herein, the term "aryl" refers to an aromatic group (e.g., monocyclic, bicyclic, and tricyclic structures) containing six to ten carbon atoms in its ring moiety. The aryl group may optionally be substituted with available carbon atoms and, in some embodiments, may include one or more heteroatoms, such as oxygen, nitrogen, or sulfur. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl).

[0261] As used herein, “heteroaryl” refers to 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 a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In heteroaryls containing one or more nitrogen atoms, the linkage can be a carbon or nitrogen atom, provided the valence allows. Heteroaryl bicyclic ring systems 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 with one or more carbocyclic or heterocyclic groups, wherein the linkage 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 with one or more aryl groups, wherein the linking point is on an 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 bicyclic heteroaryl groups (e.g., indolyl, quinolinyl, carbazolyl, etc.) where one ring does not contain a heteroatom, the linking point can be on either ring, i.e., the ring carrying the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

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

[0263] As used herein, "heterocyclic group" or "heterocycle" refers to a group having a 3- to 10-membered nonaromatic ring system having a ring carbon atom 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 groups"). In heterocyclic groups containing one or more nitrogen atoms, the linking point can be a carbon or nitrogen atom, provided the valence allows. Heterocyclic groups can be monocyclic ("monocyclic heterocyclic group") or fused, bridged, or spirocyclic systems (such as bicyclic systems ("bicyclic heterocyclic group")), and can be saturated or partially unsaturated. Heterocyclic bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclic group" also includes ring systems in which a heterocyclic ring as defined above is fused with one or more carbocyclic groups, wherein the connection point is on the carbocyclic or heterocyclic ring; or ring systems in which a heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the connection point is on the heterocyclic ring. In such cases, the number of ring members continues to indicate the number of ring members in the heterocyclic ring system. The terms "heterocycle," "heterocyclic group," "heterocyclic ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic group" are used interchangeably.

[0264] As used in this article, "cyano" refers to -CN.

[0265] As used herein, the terms "halogen" and "halogen" refer to an atom selected from fluorine (fluorinated, -F), chlorine (chlorinated, -Cl), bromine (brominated, -Br), and iodine (iodinated, -I). In some embodiments, the halogen is fluorinated or chlorinated.

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

[0267] As used in this article, "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 atom present on a group (e.g., a carbon or nitrogen atom) is replaced by a permissible substituent, such as a substituent that, upon substitution, yields a stable compound, such as a compound that is not spontaneously transformed, for example, by rearrangement, cyclization, elimination, or other reactions. Unless otherwise stated, a "substituted" group has substituents at one or more substituted positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position.

[0269] As used herein, “pharmaceutically acceptable salt” means that, to the extent of reasonable medical judgment, it is suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this 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 amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Pharmaceutically acceptable salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N. + (C 1-4 Alkyl 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 from counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0270] In typical embodiments, the present invention is intended to cover the compounds disclosed herein, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomers, 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, tautomers, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (pure or as racemic or non-racemic mixtures).

[0271] The compounds described herein may contain one or more asymmetric centers and therefore can exist in a variety of 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 mixtures of stereoisomers (including racemic mixtures and mixtures rich in one or more stereoisomers). Isomers can be separated from mixtures 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 preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, editor, Univ. of Notre Dame Press, Notre Dame, IN 1972). This invention further covers compounds described herein in their single isomer form, which is substantially free of other isomers, and alternatively, in mixtures of different isomers.

[0272] In some embodiments, compounds and transfer mediators (e.g., lipid nanoparticles) in which such compounds are components exhibit enhanced (e.g., increased) transfection capabilities of one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods typically involve contacting one or more target cells with the compounds and / or pharmaceutical compositions disclosed herein to transfect the one or more target cells with circular RNA encapsulated therein. As used herein, the term “transfect” or “transfection” refers to the introduction of one or more encapsulating materials (e.g., nucleic acids and / or polynucleotides) into cells, or preferably into target cells. The term “transfection efficiency” refers to the relative amount of such encapsulating material (e.g., polynucleotides) absorbed, introduced into, and / or expressed by the transfected target cells. In some embodiments, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cells after transfection. In some embodiments, the transfer mediator has high transfection efficiency. In some implementations, the transfer medium 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 some embodiments, liposomes are lipid nanoparticles (e.g., lipid nanoparticles comprising one or more ionizable lipid compounds disclosed herein). Such liposomes can be monolayer or multilayer vesicles having a membrane formed of a lipophilic material and an aqueous interior containing encapsulated circRNA to be delivered to one or more target cells, tissues, and organs. In some 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 of the consideration include one or more compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and / or HGT4005). These liposomes and lipid nanoparticles may also contain additional ionizable lipids, such as C12-200, DLin-KC2-DMA and / or HGT5001, accessory lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0274] As used herein, the phrases “non-cationic lipids,” “non-cationic auxiliary lipids,” and “auxiliary lipids” are used interchangeably and refer to any neutral, zwitterionic, or anionic lipids.

[0275] As used in this article, the phrase “anionic lipid” refers to any of the many types of lipids that carry a net negative charge at a selected pH, such as physiological pH.

[0276] As used herein, the phrase "biodegradable lipids" or "degradable lipids" refers to any of a variety of lipid types that break down in the host environment on the order of minutes, hours, or days, thus ideally making them less toxic and less likely to accumulate in the host over time. Common modifications to lipids include ester bonds and disulfide bonds to increase their biodegradability.

[0277] As used herein, the phrase "biodegradable PEG lipids" or "degradable PEG lipids" refers to any of many types of lipids in which PEG molecules cleave from lipids in the host environment on a scale of minutes, hours, or days, thereby ideally reducing their immunogenicity. Common modifications to PEG lipids include ester bonds and disulfide bonds to increase the biodegradability of the lipids.

[0278] In some embodiments of the invention, transfer media (e.g., lipid nanoparticles) are prepared to encapsulate one or more materials or therapeutic agents (e.g., circRNA). The process of incorporating the desired therapeutic agent (e.g., circRNA) into the transfer media is referred to herein as “loading” or “encapsulation” (Lasic et al., FEBS Lett., 312:255-258, 1992). The material (e.g., circRNA) loaded or encapsulated by the transfer media may be wholly or partially located within the internal space of the transfer media, within the bilayer membrane of the transfer media, or associated with the outer surface of the transfer media.

[0279] As used in this article, the term “structural lipid” refers to sterols and lipids containing sterol moieties.

[0280] As defined in this article, "sterols" are a subgroup of steroids consisting of steroids.

[0281] As used in this article, the term “structural lipid” refers to sterols and lipids containing sterol moieties.

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

[0283] As generally defined herein, “PEG-OH lipids” (also referred to herein as “hydroxy-PEGylated lipids”) are PEGylated lipids having one or more hydroxyl groups (-OH) on them.

[0284] As used in this article, "phospholipid" is a lipid that comprises a phosphate ester moiety and one or more carbon chains, such as unsaturated fatty acid chains.

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

[0286] As used herein, the expression “sequence identity” or, for example, “a sequence that is 50% identical to…” refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. Therefore, the “sequence identity percentage” can be calculated as follows: compare two best-aligned sequences within a comparison window; determine the number of positions in the two sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appears to obtain the number of matching positions; divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size); and multiply the result by 100 to obtain the sequence identity percentage. This includes nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any reference sequence described herein, wherein the polypeptide variant typically retains at least one biological activity of the reference polypeptide.

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

[0288] This article also provides information on circular RNA, precursor RNA that can be circularized into circular RNA, and vectors (e.g., DNA vectors) that can be transcribed into precursor RNA or circular RNA.

[0289] Two types of spacers have been designed to improve precursor RNA circularization and / or gene expression from circular RNA. The first type is the external spacer, which is present in the precursor RNA but removed after circularization. While not wishing to be bound by theory, external spacers are expected to improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing interference from other adjacent sequence elements in its folding and function. The second type is the internal spacer, which is present in the precursor RNA and retained in the resulting circular RNA. While not wishing to be bound by theory, internal spacers are expected to improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing interference from other adjacent sequence elements, particularly adjacent IRES and coding regions, in its folding and function. Internal spacers are also expected to improve protein expression from IRES by preventing hybridization of adjacent sequence elements, particularly intron elements, with sequences within the IRES and inhibiting their ability to fold into their optimal and active conformation.

[0290] To drive protein expression, the circular RNA includes an IRES operatively linked to a protein-coding sequence. Exemplary IRES sequences are provided in Table 17 below. In some embodiments, the circular RNAs disclosed herein include 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 include the IRES sequences in Table 17. Modifications to the IRES and auxiliary 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 region to the 5' end of the IRES, modifying the 6 nucleotides (Kozak sequence) at the 5' of the translation initiation site, substituting for modifications to the translation initiation site, and creating chimeric / hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNAs disclosed herein includes one or more of these modifications relative to the native IRES (e.g., the native IRES disclosed in Table 17).

[0291] In some respects, this document provides a circular RNA polynucleotide comprising a 3' post-splicing group I intron fragment, an optional first spacer region, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer region, and a 5' post-splicing group I intron fragment. In some embodiments, these regions are arranged in the order described herein. In some embodiments, the circular RNA is prepared by the methods provided herein or by the vectors provided herein.

[0292] In some embodiments, transcription of the vector provided herein (e.g., comprising a 5' homologous region, a 3' I-group intron fragment, an optional first spacer region, an internal ribosome entry site (IRES), an expression sequence, an optional second spacer region, a 5' I-group intron fragment, and a 3' homologous region) results in the formation of a circularizable precursor linear RNA polynucleotide. In some embodiments, transcription occurs when guanosine nucleotides or nucleosides (e.g., GTP) and divalent cations (e.g., Mg) are involved. 2+ When incubated in the presence of ), the previously linear RNA polynucleotides were cyclized.

[0293] In some embodiments, the vector and precursor RNA polynucleotide provided herein comprise a first (5') double-strand-forming region and a second (3') double-strand-forming region. In some embodiments, the first and second homologous regions may form perfect or imperfect double strands. Thus, in some embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the first and second double-strand-forming regions are base-paired with each other. In some embodiments, the predicted base pairing between the double-strand-forming region and an unexpected sequence in the RNA (e.g., a non-double-strand-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 a double-strand-forming region at the end of the precursor RNA strand and placing it adjacent to or very close to Group I intron fragments brings the Group I intron fragments closer together, thereby increasing splicing efficiency. In some embodiments, the length of the double-strand-forming region is 3 to 100 nucleotides (e.g., 3-75 nucleotides, 3-50 nucleotides, 20-50 nucleotides, 35-50 nucleotides, 5-25 nucleotides, or 9-19 nucleotides). In some embodiments, the length of the double-strand-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 double-strand-forming region has a length of about 9 to about 50 nucleotides. In one embodiment, the double-strand-forming region has a length of about 9 to about 19 nucleotides. In some embodiments, the double-strand-forming region has a length of about 20 to about 40 nucleotides. In some embodiments, the double-strand-forming region has a length of about 30 nucleotides.

[0294] In some embodiments, the vectors, precursor RNAs, and circular RNAs provided herein include first (5') and / or second (3') spacer regions. In some embodiments, including a spacer region between the 3' group I intron fragment and the IRES can improve splicing efficiency by protecting secondary structures in those regions from interaction. In some embodiments, the first spacer region (between the 3' group I intron fragment and the IRES) and the second spacer region (between the expressed sequence and the 5' group I intron fragment) include additional base-pairing regions that are predicted to pair with each other rather than with the first and second duplex forming regions. In some embodiments, such spacer region base pairing brings the group I intron fragments closer together, thereby further improving splicing efficiency. Furthermore, in some embodiments, combinations of base pairings between the first and second duplex forming regions, and combinations of base pairings between the first and second spacer regions individually, promote the formation of splice bubbles containing group I intron fragments flanked by adjacent base-pairing regions. A typical spacer region is a contiguous sequence having one or more of the following characteristics: 1) expected to avoid interference with proximal structures, such as IRES, expressed sequences, or introns; 2) a length of at least 7 nt and no more than 100 nt; 3) located after and adjacent to a 3' intron and / or before and adjacent to a 5' intron; and 4) containing one or more of the following: a) an unstructured region at least 5 nt long, b) a region at least 5 nt long that pairs with the bases of the distal sequence (including another spacer region), and c) a structured region at least 7 nt long whose extent is confined to the spacer region sequence. A spacer region may have several regions, including unstructured regions, base-pairing regions, hairpin / structured regions, and combinations thereof. In one embodiment, the spacer region has a structured region with a high GC content. In one embodiment, a region within the spacer region pairs with a region within the same spacer region. In one embodiment, a region within the spacer region pairs with a region within another spacer region. In one embodiment, the spacer region contains one or more hairpin structures. In one embodiment, the spacer region comprises 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 region exists between the 3'I group intron fragment and the IRES. In one embodiment, this additional spacer region prevents the structured regions of the IRES from interfering with the folding of the 3'I group intron fragment or reduces the extent to which this occurs. In some embodiments, the length of the 5' spacer region 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 does not exceed 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 some 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 polyacrylamide (AC) sequence. In one embodiment, the spacer contains about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyacrylamide. In another embodiment, the spacer contains about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

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

[0296] In some embodiments, the vectors, precursor RNAs, and circular RNAs provided herein contain internal ribosome entry sites (IRES). The presence of an IRES allows translation from the circular RNA of one or more open reading frames (e.g., open reading frames forming the expression sequence). The IRES element attracts the eukaryotic ribosome translation initiation complex and facilitates translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161).

[0297] A variety of IRES sequences are available and include sequences derived from a variety of viruses, such as leader sequences of microRNA viruses derived from the UTR of encephalomyocarditis virus (EMCV) (Jang et al. J.Virol. (1989) 63:1651-1660), polio virus leader sequences, hepatitis A virus leader sequences, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), Giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc.

[0298] In some implementations, the IRES is the IRES sequence of the following viruses: Taura syndrome virus, stag beetle virus, Tiller's encephalomyelitis virus, simian virus 40, red imported fire ant virus 1, rice constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, P. stearnivirus, Kashmir bee virus, human rhinovirus 2, glass leafhopper virus-1, human immunodeficiency virus type 1, Himetobi virus. P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Tea geometrid moth microRNA virus-like virus, Encephalomyocarditis virus, Drosophila C virus, Human Coxsackievirus B3, Tobacco mosaic virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black bee queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorosis and ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennae and legs, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine scamper, Drosophila ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, hairless Drosophila, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, tobacco etch virus, turnip shrunken virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, small disegmented RNA virus, HCVQC64, human cosavirus E / D, human cosavirus F, human cosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, rosavirus A SH1, rosavirus FHB, rosavirus NG-J1, human paraenteric orphan virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A. Pasivirus A, Pasivirus A 2. Echovirus E14, Human Paraenterovirus 5. Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Hepatic Virus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Hepatic Virus A 1220, Pasivirus A 3. Sapellovirus, Rosavirus B, Bakunsa Virus, Tremor Virus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepatitis Virus K, Hepatitis Virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Bicistronic Virus, Hubei MicroRNA Virus-like Virus, CRPV, Salivirus A BN5, Salivirus ABN2, Salivirus A 02394, Salivirus A Aptamers of GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0299] In some embodiments, the polynucleotides described herein contain 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 bicistronic RNA. The sequences encoding two or more polypeptides can be separated by ribosomal jumping elements or nucleotide sequences encoding protease cleavage sites. In some embodiments, the ribosomal jumping elements encode thosea-asigna virus 2A peptide (T2A), porcine swine fluvirus-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 silkworm fibrosis virus 2A peptide (BmIFV 2A).

[0301] In some embodiments, the vectors provided herein contain a 3'UTR. In some embodiments, the 3'UTR is derived from human β-globin, human α-globin, Xenopus β-globin, Xenopus α-globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFα, dengue virus, Hantavirus small mRNA, Bunyavirus small mRNA, turnip mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human microtubules, hibiscus chlorotic rinsgspot virus, marmot hepatitis virus post-translational regulatory element, Sindbis virus, turnip shrunken virus, tobacco etched virus, or Venezuelan equine encephalitis virus.

[0302] In some embodiments, the vector provided herein contains a 5'UTR. In some embodiments, the 5'UTR is derived from a triple leader sequence of human β-globin, Xenopus β-globin, human α-globin, Xenopus α-globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip shrinkage virus, or adenovirus.

[0303] In some embodiments, the vector provided herein comprises a polyA region outside the 3' and / or 5' I group intron fragments. In some embodiments, the polyA region is at least 15, 30, or 60 nucleotides long. In some embodiments, one or both polyA regions are 15-50 nucleotides long. In some embodiments, one or both polyA regions are 20-25 nucleotides long. The polyA sequence is removed after circularization. Therefore, oligonucleotides hybridizing with the polyA sequence, such as deoxythymidine oligonucleotides (oligo(dT)) conjugated to a solid surface (e.g., resin), can be used to separate circular RNA from its precursor RNA. Other sequences may also be set at the 5' of the 3' I group intron fragment or the 3' of the 5' I group intron fragment, and complementary sequences can be similarly used for circular RNA purification.

[0304] In some embodiments, the lengths of the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and / or circular RNA polynucleotides provided herein are between 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and / or 1500 and 5000 nucleotides. In some embodiments, the length of the polynucleotide is at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt. In some embodiments, the length of the polynucleotide is not more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt. In some implementations, the DNA, linear RNA, and / or circular RNA polynucleotides provided herein are approximately 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt.

[0305] In some embodiments, a vector is provided herein. In some embodiments, the vector comprises, in the following order: a) a 5' homologous region, b) a 3' I intron fragment, c) an optional first spacer region sequence, d) an IRES, e) an expression sequence, f) an optional second spacer region sequence, g) a 5' I intron fragment, and h) a 3' homologous region. In some embodiments, the vector comprises a transcription promoter located upstream of the 5' homologous region. In some embodiments, the precursor RNA comprises, in the following order: a) a polyA sequence, b) an outer spacer region, c) a 3' I intron fragment, d) a duplex forming region, e) an inner spacer region, f) an IRES, g) an expression sequence, h) a stop codon box, i) an optional inner spacer region, j) a duplex forming region capable of forming a duplex with the duplex forming region of d), k) a 5' I intron fragment, l) an outer spacer region, and m) a polyA sequence.

[0306] In some embodiments, a precursor RNA is provided herein. In some embodiments, the precursor RNA is a linear RNA generated by in vitro transcription using a vector provided herein. In some embodiments, the precursor RNA comprises, in the following order: a) a 5' homologous region, b) a 3' I-group intron fragment, c) an optional first spacer region sequence, d) IRES, e) an expression sequence, f) an optional second spacer region sequence, g) a 5' I-group intron fragment, and h) a 3' homologous region. The precursor RNA may be unmodified, partially modified, or fully modified.

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

[0308] In some embodiments, the circular RNAs provided herein exhibit higher functional stability compared to mRNAs containing the same expression sequence. In some embodiments, the circular RNAs provided herein exhibit higher functional stability compared to mRNAs containing the same expression sequence, 5 molU modification, optimized UTR, cap, and / or polyA tail.

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

[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 times, at least 2 times) 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 a predetermined threshold functional half-life. 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 Gaussian luciferase (GLuc) is measured in a culture medium of human cells (e.g., HepG2) expressing a circular RNA polynucleotide at 1, 2, 6, 7, or 14 days, at 1, 2, 6, 12, or 24 hours. 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 a patient serum or tissue sample at 1, 2, 6, 12, or 24 hours, at 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, a 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 higher expression levels than equivalent linear mRNAs, for example, higher expression levels 24 hours after RNA administration to cells. In some embodiments, the circular RNAs provided herein have higher expression levels compared to mRNAs containing the same expression sequence, 5 molU modification, optimized UTR, cap, and / or polyA tail.

[0312] In some embodiments, the immunogenicity of the circular RNA provided herein may be lower than that of the equivalent mRNA when exposed to the immune system or a certain type of immune cells of an organism. In some embodiments, the circular RNA provided herein is associated with the regulation of cytokine production when exposed to the immune system or a certain type of immune cells of an organism. For example, in some embodiments, compared to mRNA containing the same expression sequence, the circular RNA provided herein is associated with reduced production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to the immune system or a certain type of immune cells of an organism. In some embodiments, compared to mRNA containing the same expression sequence, the circular RNA provided herein is associated with less transcriptional induction of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα when exposed to the immune system or a certain type of immune cells of an organism. In some embodiments, the immunogenicity of the circular RNA provided herein is lower than that of mRNA containing the same expression sequence. In some implementations, the circular RNAs provided herein have lower immunogenicity than mRNAs containing the same expression sequence, 5 molU modification, optimized UTR, cap and / or poly-A tail.

[0313] In some embodiments, the circular RNA provided herein can be transfected into cells as is, or it can be transfected into a DNA vector and transcribed in cells. Transcription of the circular RNA from the transfected DNA vector can be performed by an added polymerase or a polymerase encoded by the nucleic acid transfected into the cell, or preferably by an endogenous polymerase.

[0314] In some embodiments, the circular RNA polynucleotides provided herein comprise modified RNA nucleotides and / or modified nucleosides. In some embodiments, the modified nucleosides are 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 -Isopentenyl adenosine); ms 2 i6A(2-methylthio-N) 6 (Isopentenyl adenosine); io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A(2-methylthio-N) 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A(N 6 -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 -hydroxyn-valinecarbamoyladenosine); ms 2 hn 6 A(2-methylthio-N) 6 -hydroxyvaline carbamoyl adenosine); Ar(p)(2'-O-ribosyl adenosine (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 (huaitin); o2yW (peroxyhuaitin); OHyW (hydroxyhuaitin); OHyW* (undermodified hydroxyhuaitin); imG (huaoside); mimG (methylhuaoside); Q (pigmentin); oQ (epoxypigmentin); galQ (galactosylpigmentin); manQ (mannosylpigmentin); preQ0 (7-cyano-7-deazoguanosine); preQ1 (7-aminomethyl-7-deazoguanosine); G + (Ancient purine); 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-hydroxyacetic acid); mcmo 5 U (uridine 5-hydroxyacetic acid methyl ester); chm 5 U(5-(carboxyhydroxymethyl)uridine)); mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U(5-methoxycarbonylmethyluridine); mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine); nm 5 S 2 U(5-aminomethyl-2-thiouridine); mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine); ncm 5 U(5-carbamoylmethyluridine); ncm 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-Taurine methyluridine); τm 5 s 2 U (5-Tauratemethyl-2-thiouridine); imG-14 (4-Demethylwoyoside); imG2 (Isowoyoside); or ac 6 A(N 6 -acetyl adenosine.

[0315] In some embodiments, the modified nucleoside may include compounds selected from the group consisting of: pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurate methyluridine, 1-taurate methylpseudouridine, 5-taurate methyl-2-thiouridine, 1-taurate methyl-4-thiouridine, 5-methyluridine, 1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazo-pseudouridine, 2- Thio-1-methyl-1-deazo-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deazo-pseudoisocytidine, 1-methyl-1-deazo-pseudoisocytidine Nitrogen-pseudoisocytidine, zabran, 5-aza-zabran, 5-methyl-zabran, 5-aza-2-thio-zabran, 2-thio-zabran, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-aminopurine, 7-deadenine-8-aza-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-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-methyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, inosine, 1-methyl-inosine, woyoside, woyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-deazo-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-pseuuridine, N6-methyladenosine, and / or pseudouridine. In some embodiments, such modified ribonucleosides provide additional stability and resistance to immune activation.

[0317] In certain embodiments, the polynucleotide may be codon-optimized. A codon-optimized sequence may be a sequence in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variations in codon bias between two or more organisms or genes or synthetic constructs of bias tables; (ii) variations in the degree of codon bias within an organism, a gene, or a group of genes; (iii) systematic variation of codons (including background); (iv) codon variation according to its decoding tRNA; (v) codon variation according to GC% (whether overall or at a position in a triplet); (vi) variations in similarity to a reference sequence (e.g., a naturally occurring sequence); (vii) variations in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a 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 implementations, codon-optimized polynucleotides can minimize ribozyme collisions and / or structural interference between the restricted expression sequence and the IRES.

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

[0319] In some embodiments, the circular RNA provided herein is 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 expressed sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the proteins listed in the table below.

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] In some embodiments, a sequence encoding a therapeutic protein is expressed. In some embodiments, a sequence encoding a cytokine, such as 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, is expressed. In some embodiments, a sequence encoding an immune checkpoint inhibitor is expressed. In some embodiments, a sequence encoding an agonist (e.g., a member of the TNFR family, such as CD137L, OX40L, ICOSL, LIGHT, or CD70) is expressed. In some embodiments, a sequence encoding a chimeric antigen receptor is expressed. In some embodiments, a sequence encoding an inhibitory receptor agonist (e.g., PDL1, PDL2, galactolectin-9, VISTA, B7H4, or MHCII) or an inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3, or TIM3) is expressed. In some embodiments, a sequence encoding an inhibitory receptor antagonist is expressed. In some embodiments, the expressed sequence encodes one or more TCR chains (α and β chains or γ and δ chains). In some embodiments, the expressed sequence encodes a secreted T cell or immune cell connective (e.g., a bispecific antibody such as BiTE targeting, for example, CD3, CD137, or CD28, and tumor-expressed proteins such as CD19, CD20, or BCMA). In some embodiments, the expressed sequence encodes a transcription factor (e.g., FOXP3, HELIOS, TOX1, or TOX2). In some embodiments, the expressed sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39 / CD73). In some embodiments, the expressed sequence encodes GvHD (e.g., an anti-HLA-A2 CAR-Treg).

[0335] In some embodiments, the polynucleotide encodes a protein composed of subunits, which are encoded by more than one gene. For example, the protein may be a heterodimer, wherein 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 transfer medium, and each circRNA encodes a separate subunit of the protein. Alternatively, a single circRNA may be engineered to encode more than one subunit. In some embodiments, a separate circRNA molecule encoding a separate subunit may be administered in a separate transfer medium.

[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 in this article 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, pildizumab, or nivolumab. Nivolumab is described in WO2006 / 121168 in some embodiments. Pembrolizumab is described in WO2009 / 114335 in some embodiments. Pildizumab is described in WO2009 / 101611 in some embodiments. Other anti-PD1 antibodies are described in U.S. Patent Nos. 8,609,089, US2010028330, US20120114649, WO2010 / 027827, and WO2011 / 066342.

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

[0341] Descriptions of the heavy and light chains and / or amino acid sequences of PD-1 and / or PD-L1 antibodies are available in this article and in the www.drugbank.ca database under the following accessions: DB09037 (pembrolizumab), DB09035 (nivolumab), DB15383 (pildizumab), DB11595 (atezil), DB11945 (averumab), and DB11714 (dvorumab).

[0342] 3.3 T cell receptor

[0343] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. Natural α-β heterodimer TCRs have α and β chains. Broadly speaking, each chain may contain a variable region, a linker region, and a constant region, and the β chain typically also contains a short region of diversity between the variable and linker regions, but this region of diversity is usually considered part of the linker region. Each variable region may contain three CDRs (complementarity-determining regions) embedded in the frame sequence, one of which is a hypervariable region called CDR3. Several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions exist, distinguished by their frame, CDR1, and CDR2 sequences and by a partially defined CDR3 sequence. The Vα type is indicated by a unique TRAV number in the IMGT nomenclature. Therefore, “TRAV21” defines a TCR Vα region with a unique frame and CDR1 and CDR2 sequences as well as a CDR3 sequence, wherein the CDR3 sequence is partially defined by the amino acid sequence retained from TCR to TCR, but also includes the amino acid sequence that changes from TCR to TCR. Similarly, “TRBV5-1” defines a TCR Vβ region with a unique frame and CDR1 and CDR2 sequences, but only partially defined by the CDR3 sequence.

[0344] The connection region of the TCR is defined similarly to the unique IMGT TRAJ and TRBJ nomenclature, and the constant region is defined similarly to the IMGTTRAC and TRBC nomenclature.

[0345] The β-chain diversity region is abbreviated as TRBD in the IMGT nomenclature, and as previously mentioned, cascaded TRBD / TRBJ regions are usually considered together as connection regions.

[0346] The unique sequences defined by the IMGT nomenclature are widely known and available to those working in the TCR field. For example, they can be found in the IMGT public database. Sequences defined by the IMGT nomenclature are also disclosed in “Tcell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, but due to its publication date and the resulting time lag, the information therein sometimes requires confirmation by referring to the IMGT database.

[0347] Natural TCRs exist as heterodimers in the form of αβ or γδ. However, recombinant TCRs composed of αα or ββ homodimers have previously been shown to bind to peptide MHC molecules. Therefore, the TCRs of the present invention can be heterodimer αβ TCRs or αα or ββ homodimer TCRs.

[0348] For use in adoptive therapy, the αβ heterodimeric TCR can, for example, be transfected into a full-length chain having a cytoplasmic domain and a transmembrane domain. In some embodiments, the TCR of the present invention may have introduced disulfide bonds between residues of the respective constant domains, as described, for example, in WO 2006 / 000830.

[0349] The TCR of the present invention, particularly the α-β heterodimer TCR, may 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 may be modified by truncation or substitution to eliminate 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 may also be modified by substituting Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 with cysteine ​​residues that form a disulfide bond between the α and β constant domains of the TCR.

[0350] Combining affinity (with equilibrium constant K) D The inversely proportional (inversely proportional) binding half-life (expressed as T1 / 2) can be determined by any suitable method. It should be understood that doubling the affinity of the TCR results in K... D Halved. T1 / 2 is calculated as ln 2 divided by the dissociation rate (koff). Therefore, doubling T1 / 2 results in koff being halved. K of TCR D Koff values ​​are typically measured for the soluble form of a TCR, i.e., those truncated to remove cytoplasmic and transmembrane domain residues. Therefore, it should be understood that if a soluble form of a given TCR has the aforementioned characteristics, then that TCR has improved binding affinity and / or binding half-life to its parent TCR. Preferably, the binding affinity or binding half-life of a given TCR is measured multiple times (e.g., three or more times) using the same assay protocol, and the results are averaged.

[0351] Because the TCR of the present invention is effective in adoptive therapy, the present invention includes non-naturally occurring and / or purified and / or engineered cells, particularly T cells, that present the TCR of the present invention. Numerous methods are suitable for transfecting T cells with nucleic acids (such as DNA, cDNA, or RNA) encoding the TCR of the present invention (see, for example, Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCR of the present invention will be suitable for adoptive therapy-based treatment of cancers such as pancreatic and liver cancer. As is known to those skilled in the art, many suitable methods exist 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-translational modified when expressed by transfected cells. Glycosylation is such a modification, which may include the covalent linking of an oligosaccharide moiety to a defined amino acid in the TCR chain. For example, asparagine residues or serine / threonine residues are well known oligosaccharide linking sites. The glycosylation state of a particular protein depends on many factors, including protein sequence, protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation state (i.e., oligosaccharide type, covalent bonds, and total number of links) can affect protein function. Therefore, glycosylation control is often required when producing recombinant proteins. 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 covered in this invention.

[0353] TCRs are specific for antigens in the following groups: 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, 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, h sp70-2, KIAAO205, Mart2, Mum-2 and 3, neo-PAP, type I myosin, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triose phosphate 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) antigen 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.- 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, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclic protein 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 magnitude and duration of inflammatory responses, and preventing autoimmune and allergic responses.

[0356] Generally, Tregs are thought to primarily participate in suppressing immune responses, partially acting as a "self-check" mechanism 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] Treg cells are distributed throughout the body, including but not limited to the intestines, skin, lungs, and liver. Furthermore, Treg cells can also exist 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 known or suspected Treg cell populations possesses one or more unique characteristics, and further information can be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294):1-10, the contents of which are hereby incorporated in their entirety.

[0358] Typically, Tregs are known to require TGF-β and IL-2 for normal activation and development. Tregs expressing high levels of the IL-2 receptor (IL-2R) depend on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β, both of which are potent immunosuppressive cytokines. Furthermore, Tregs are known to suppress the ability of antigen-presenting cells (APCs) to stimulate T cells. One proposed mechanism of APC suppression is through CTLA-4, which is expressed by Foxp3+ Tregs. It is believed that CTLA-4 can bind to B7 molecules on APCs and block or remove these molecules by inducing internalization, thereby reducing B7 availability and failing to provide adequate co-stimulation for the immune response. Further discussions on 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, differentiation, and function, 2013, Frontiers in Immunol., 4(253):1-11, and the published content of the aforementioned literature is hereby incorporated in its entirety.

[0359] Descriptions and / or amino acid sequences of FOXP3, STAT5B, and / or HELIOS are provided in this paper 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 implementations, 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 cause fatal IPEX syndrome (immune dysregulation, multiple endocrine disorders, enteropathy, X-linked). IPEX patients suffer from severe autoimmune responses, persistent eczema, and colitis. Regulatory T (Treg) cells expressing Foxp3 play a crucial role in limiting intestinal inflammatory responses (Josefowicz, SZ et al., Nature, 2012, 482, 395-U1510).

[0362] STAT

[0363] Members of the signal transduction factor and transcription activator (STAT) protein family are intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis, and differentiation. They primarily provide activation of membrane receptor-associated Janus kinase (JAK). Dysregulation of this pathway is frequently observed in primary tumors and leads to 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 seven identified mammalian STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B) and STATE.

[0365] Extracellular binding of cytokines or growth factors induces activation of receptor-associated Janus kinases, which promote dimerization by phosphorylating specific tyrosine residues within STAT proteins via their SH2 domain. The phosphorylated dimers are then actively transported to the nucleus via the α / β ternary complex of the export protein. Initially, STAT proteins were described as potential cytoplasmic transcription factors because phosphorylation was thought essential for nuclear retention. However, unphosphorylated STAT proteins also shuttle between the cytosol and nucleus and play a role in gene expression. Once STAT reaches the nucleus, it binds to a shared DNA recognition motif called the γ-activation site (GAS) in the promoter region of cytokine-inducible genes and activates transcription. STAT proteins can be dephosphorylated by ribophosphatases, leading to STAT inactivation and subsequent transport out of the nucleus via the efflux protein-RanGTP complex.

[0366] In some embodiments, the STAT protein of this disclosure may be a STAT protein containing modifications that regulate its expression level or activity. In some embodiments, such modifications particularly include mutations affecting STAT dimerization, STAT protein binding to signal transduction counterparts, STAT protein localization, or STAT protein degradation. In some embodiments, the STAT protein of this disclosure has constitutive activity. In some embodiments, the STAT protein of this disclosure has constitutive activity due to constitutive dimerization. In some embodiments, the STAT protein of this disclosure has constitutive activity due to constitutive phosphorylation, as described in Onishi, M. et al., Mol. Cell. Biol., July 1998, Vol. 18, No. 7, pp. 3871-3879, the entire contents of which are 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 and expressed by immune cells, including T cells, via circular RNA as described herein. With a CAR, a single receptor can be programmed to recognize a specific antigen and, upon binding to the antigen, activate the immune cell to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, CAR-expressing immune cells 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 the 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 this disclosure includes an antigen-binding domain (such as scFv) in tandem with a co-stimulatory domain and an activation domain. The co-stimulatory domain may include one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple co-stimulatory domains may be used in tandem.

[0370] Antigen-binding domain

[0371] CARs can be engineered to bind to antigens (e.g., cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. In some embodiments, the antigen-binding molecule is an antibody fragment, such as one or more single-chain antibody fragments (scFvs). scFvs are single-chain antibody fragments having variable regions of linked heavy and light chains of antibody. See U.S. Patents 7,741,465 and 6,319,494 and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. scFvs retain the ability of the parent 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 along 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 antigen-binding molecules are typically contained within the extracellular portion of the CAR, enabling it to recognize and bind to the target antigen. Bispecific and multispecific CARs are covered within the scope of this invention, exhibiting specificity for more than one target.

[0372] In some embodiments, the antigen-binding molecule comprises a single chain, wherein 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 DARPin. In some embodiments, the antigen-binding molecule comprises an anticarrier protein or other synthetic protein capable of specifically binding to a target protein.

[0374] In some implementations, the CAR includes an antigen-binding domain specific to antigens 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), and 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 α-2, mesothelin, interleukin-11 receptor α (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 β (PDGFR-β), stage-specific embryonic antigen 4 (SSEA-4), CD20, folate receptor α, HER2, HER3, mucin 1, cell surface-associated protein (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostate enzymes, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), liver glycoprotein B2, fibroblast activation protein The following proteins are listed: white alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macroprotein factor) subunit β9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein (bcr-ab1) composed of breakpoint cluster region (BCR) and Abelson mouse leukemia virus oncogene homologue 1 (Ab1), tyrosinase, liver ligand A receptor 2 (EphA2), fucose GM1, sialylated Lewis adhesion molecule (sLe), ganglioside GM3, and transglutaminase 5 (TGS5). High molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-associated (TEM7R), tight junction protein 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1).The hexasaccharide moiety of globoH ceramide (GloboH), mammary differentiation antigen (NY-BR-1), urolytic protein 2 (UPK2), hepatitis A virus cell receptor 1 (HAVCR1), adrenaline receptor β3 (ADRB3), pan-connector protein 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCRγ alternating 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), spermin 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-associated antigen 1, tumor protein p53 (p53), p53 mutant, prostaglandins, survival, telomerase, prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma cell apoptosis inhibitor (ML-IAP), ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosamine transferase V (NA17), pairing box protein Pax-3 (PAX3), androgen receptor, cell cycle protein B1, v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-associated protein 2 (TRP-2), cytochrome P450 CYP1B1, CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 (SART3) recognized by T cells, pairing box protein Pax-5 (PAX5), pro-acromial protein-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal glycoside 1 (RU1), renal glycoside 2 (RU2), lentinan, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxyl esterase, 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 molecular-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), phosphatidylinositol proteoglycan-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 cells, etc. Glycoprotein-2 (EGP-2), Epithelial Glycoprotein-40 (EGP-40), ErbB4, Epithelial Tumor Antigen (ETA), Folate-Binding Protein (FBP), Kinase Insertion Domain Receptor (KDR), κ-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 λ-like Peptide 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 The antigens include 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 lymphotrophic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, Kaposi's sarcoma-associated herpesvirus (KSHV) lysate nuclear antigen, and KSHV latent nuclear antigen. In some embodiments, the antigen-binding domain includes SEQ ID NO: 321 and / or 322.

[0375] Hinge / Gap Zone Structural Domain

[0376] In some embodiments, the CAR of this disclosure comprises a hinge or spacer region domain. In some embodiments, the hinge / spacer region domain may comprise a truncated hinge / spacer region domain (THD), which is a truncated form of the complete hinge / spacer region domain (“CHD”). In some embodiments, the extracellular domain is derived from or originates from (e.g., comprising all or fragments thereof) ErbB2, blood group glycoprotein A (GpA), CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8[T CD1a (IT GAL), CD1b (IT GAM), CD1c (IT GAX), CD1d (IT GAL), CD1d (IT GAL), CD11c (IT GAL), CD1d ...GAD), 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), CD6 6d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1) , CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR 2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 ( CD3-ζ), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2 D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulatory factor (ICOS), LFA-1 (CD11a / 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 1 molecules, MHCClass 2 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, NK cell-activating receptors, Toll ligand receptors, and fragments or combinations thereof. Hinge or spacer domains may be of natural or synthetic origin.

[0377] In some embodiments, the hinge or spacer region domain is located between the antigen-binding molecule (e.g., scFv) and the transmembrane domain. In this orientation, the hinge / spacer region domain provides the distance between the antigen-binding molecule and the cell membrane surface expressing the CAR. In some embodiments, the hinge or spacer region domain is derived from or originates from immunoglobulins. In some embodiments, the hinge or spacer region domain is selected from the hinge / spacer regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof. In some embodiments, the hinge or spacer region domain comprises, is derived from, or originates from the hinge / spacer region of CD8α. In some embodiments, the hinge or spacer region domain comprises, is derived from, or originates from the hinge / spacer region of CD28. In some embodiments, the hinge or spacer region domain comprises a fragment of the hinge / spacer region of CD8α or a fragment of the hinge / spacer region of CD28, wherein said fragment is any fragment smaller than a complete hinge / spacer region. In some embodiments, the fragment of the CD8α hinge / spacer region or the fragment of the CD28 hinge / spacer region comprises an amino acid sequence excluding the N-terminus or C-terminus or both of the CD8α hinge / spacer region or the CD28 hinge / spacer region.

[0378] Transmembrane domain

[0379] The CAR disclosed herein may also include a transmembrane domain and / or an intracellular signaling domain. The transmembrane domain may be designed to fuse with an extracellular domain of the CAR. It may similarly fuse with an intracellular domain of the CAR. In some embodiments, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain may be selected or modified (e.g., by amino acid substitution) to prevent such domains from binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. The transmembrane domain may be derived from a natural or synthetic source. In the case of a natural source, the domain may be derived from any membrane-binding or transmembrane protein.

[0380] The transmembrane region may originate from (i.e., contain) receptor tyrosine kinases (e.g., ErbB2), blood group glycoprotein A (GpA), 4-1BB / CD137, activated 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, CDS, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IE-2Rβ, IE-2Rγ, IE-7Rα, inducible T cell co-stimulatory factor (ICOS), integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, ligands that specifically bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments thereof, truncated or combined.

[0381] In some implementations, suitable intracellular signal transduction domains include, but are not limited to, activation receptors for macrophages / myeloid cells 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 may be derived from (e.g., including) the insulin receptor (InsR), insulin-like growth factor I receptor (IGF1R), insulin receptor-associated receptor (IRR), platelet-derived growth factor receptor α (PDGFRa), platelet-derived growth factor receptor β (PDGFRfi), KIT proto-oncogene receptor tyrosine kinase (Kit), colony-stimulating factor 1 receptor (CSFR), fms-associated tyrosine kinase 3 (FLT3), fms-associated tyrosine kinase 1 (VEGFR-1), kinase insertion domain receptor (VEGFR-2), fms-associated tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FG... FR1), 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 receptor 1 (Ron), AXL receptor tyrosine kinase (Axl) ), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase 1 (TIE1) with immunoglobulin-like and EGF-like domains, TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), (EPH receptor A3)EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphA10), EPH receptor B1 (EphB1), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret proto-oncogene (Ret), receptor-like tyrosine kinase (RYK), discoid domain receptor tyrosine kinase 1 (DDR1), discoid domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis-associated 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] Co-stimulatory domain

[0384] In some embodiments, the CAR includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain includes 4-1BB (CD137), CD28, or both, and / or an intracellular T cell signaling domain. In a preferred embodiment, the co-stimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3zeta (ζ). 4-1BB, CD28, and CD3ζ may each contain less than a complete 4-1BB, CD28, or CD3ζ. Chimeric antigen receptors may be incorporated into the co-stimulatory (signaling) domain to increase their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Krause et al. and Finney et al. (ibid.); 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 implementations, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:318 or 320.

[0386] Intracellular signal transduction domains

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

[0388] In some implementations, suitable intracellular signal transduction domains include (e.g., contain) but are not limited to 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulatory factor (ICOS), integrin, 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; CD1-1a / CD18), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments thereof, truncated or combined.

[0389] CD3 is a component of the T cell receptor on natural T cells and has been shown to be an important intracellular activating element in CARs. In some embodiments, CD3 is CD3ζ. 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 with the polypeptide sequence of SEQ ID NO:319.

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

[0391] This document discloses circular RNA polypeptides encoding trispecific antigen-binding proteins (TRITE), bispecific antigen-binding proteins (BITE), functional fragments thereof, and pharmaceutical compositions thereof. It also provides recombinant expression vectors for preparing circular RNAs encoding trispecific or bispecific antigen-binding proteins, and cells comprising the circular RNAs of the present invention. Methods for preventing and / or treating liver diseases, disorders, and conditions using the disclosed trispecific or bispecific antigen-binding proteins are also provided. Trispecific antigen-binding proteins are capable of specifically binding to target antigens, such as cancer antigens, and CD3, TCR, CD16A, or NKp46, as well as liver-retaining domains or half-life-extending domains, such as domains binding human serum albumin (HSA). In some embodiments, TRITE or BITE is generated in the liver of a patient after administration of a composition comprising the circular RNA polypeptides of the present invention to a patient in need.

[0392] In one aspect, a trispecific antigen-binding protein comprises a domain (A) that specifically binds to CD3, TCR, CD16A, or NKp46, a domain (B) that specifically binds to half-life-extending molecules or liver-preserving molecules, and a domain (C) that specifically binds to a target antigen, such as a cancer cell antigen. The three domains in a trispecific antigen-binding protein can be arranged in any order. Therefore, consider a trispecific antigen-binding protein with a domain sequence of any of the following: (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 (A)-(B)-(C) domain sequence. In some embodiments, the trispecific antigen-binding protein has a (A)-(C)-(B) domain sequence. In some embodiments, the trispecific antigen-binding protein has a (B)-(A)-(C) domain sequence. In some embodiments, the trispecific antigen-binding protein has a (B)-(C)-(A) domain sequence. In some embodiments, the trispecific antigen-binding protein has a (C)-(A)-(B) domain sequence.

[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 the target antigen. The two domains in the bispecific antigen-binding protein can be arranged in any order. Therefore, the domain order of a bispecific antigen-binding protein can be (A)-(B) or (B)-(A).

[0395] The trispecific or bispecific antigen-binding proteins described herein are engineered to allow specific targeting of cells expressing target antigens 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, by binding to CD3 molecules specifically expressed on these cells, trispecific or bispecific antigen-binding proteins enable cytotoxic T cells or NK cells to crosslink with cells expressing target antigens in a highly specific manner, thereby guiding recruited T cells or NK cells toward the cytotoxic potential of the target cells. The trispecific or bispecific antigen-binding proteins described herein bind to cytotoxic T cells by binding to surface-expressed CD3 proteins (which form part of the TCR) or CD16A or NKp46 (which activate NK cells). The simultaneous binding of several trispecific or bispecific antigen-binding proteins to CD3 and target antigens expressed on specific cell surfaces induces T cell activation and mediates subsequent lysis of cells expressing specific target antigens. Therefore, 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 bacterial-infected cells, autoreactive T cells, etc.). In some embodiments, cells are selectively eliminated, thereby reducing the possibility of toxic side effects. In some embodiments, anti-41bb or CD137 binding domains are used as T cell adaptors.

[0396] Immune cell binding domain

[0397] The specificity of T cell responses is mediated by TCR recognition of antigens (displayed against the background of the major histocompatibility complex, MHC). As part of the TCR, CD3 is a protein complex comprising the CD3γ (γ) chain, CD3δ (δ) chain, and two CD3ε (ε) chains present on the cell surface. CD3 associates with the α (α) and β (β) chains of the TCR, as well as CD3 zeta (ζ), to form the complete TCR. Clustering of CD3 on T cells (e.g., through immobilization of anti-CD3 antibodies) leads to T cell activation, similar to T cell receptor conjugation, but independent of its clonal typical specificity.

[0398] In one aspect, the bispecific and trispecific proteins described herein include a domain that specifically binds to CD3. In one aspect, the trispecific proteins described herein include a domain that specifically binds to human CD3. In some embodiments, the trispecific proteins described herein include a domain that specifically binds to CD3γ. In some embodiments, the trispecific proteins described herein include a domain that specifically binds to CD36. In some embodiments, the trispecific proteins described herein include a domain that specifically binds to CD3ε.

[0399] In other embodiments, the trispecific protein described herein includes a domain that specifically binds to the TCR. In some cases, the trispecific protein described herein includes a domain that specifically binds to the α-chain of the TCR. In some cases, the trispecific protein described herein includes a domain that specifically binds to the β-chain of the 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 domain of the antigen-binding protein can be any domain that binds to CD3, TCR, NKp46, or CD16A, including but not limited to domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some cases, it is advantageous for the CD3, TCR, NKp46, or CD16A binding domain to originate from the same species in which the trispecific antigen-binding protein will ultimately be used. For example, for use in humans, it may be advantageous for the CD3, TCR, NKp46, or CD16A binding domain of the trispecific antigen-binding protein to contain human or humanized residues of the antigen-binding domain derived from an antibody or antibody fragment.

[0402] Therefore, in one respect, the antigen-binding domain contains humanized or human antibody or antibody fragment, or mouse antibody or antibody fragment. In one embodiment, the humanized or 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), 2 (LC CDR2), and 3 (LC CDR3) of the humanized or 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), 2 (HC CDR2), and 3 (HC CDR3) of the humanized or anti-CD-3, TCR, NKp46, or CD16A binding domain described herein, for example, a humanized or 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 implementations, the humanized or human anti-CD3, TCR, NKp46, or CD16A binding domain includes a humanized or human heavy chain variable region that is specific to CD3, TCR, NKp46, or CD16A, wherein the heavy chain variable region that is specific to CD3, TCR, NKp46, or CD16A includes a human or non-human heavy chain CDR in the human heavy chain framework region.

[0404] In some cases, the complementarity-determining regions of the heavy and / or light chains are derived from known anti-CD3 antibodies, such as moromuzumab-CD3 (OKT3), osimertzumab (TRX4), teplezumab (MGA031), vesizumab (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 a fragment thereof as described in U.S. Patent Application 16 / 451051. In some embodiments, the anti-NKp46 binding domain comprises antibodies BAB281, 9E2, 195314, or fragments thereof.

[0406] In one embodiment, the anti-CD3, TCR, NKp46, or CD16A binding domain is a single-chain variable fragment (scFv) comprising the amino acid sequence provided herein and the light chain. 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 not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95%-99% identity with 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 not more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95%-99% identity with the amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD3 binding domain is an scFv, and a light chain variable region comprising the amino acid sequence described herein is attached to a heavy chain variable region comprising the amino acid sequence described herein via an scFv linker. 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 implementations, 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 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 domains to bind to CD3, TCR, NKp46, or CD16A coated on an assay plate; displayed on the surface of microbial cells; or in solution. The binding activity of the trispecific antigen-binding protein of this disclosure or its CD3, TCR, NKp46, or CD16A binding domains to CD3, TCR, NKp46, or CD16A can be determined by immobilizing the ligand (e.g., CD3, TCR, NKp46, or CD16A) or the trispecific antigen-binding protein itself or its CD3, TCR, NKp46, or CD16A binding domains to beads, substrates, cells, etc. Additives can be added to a suitable buffer, and the binding pair can be incubated at a given temperature for a period of time. After washing to remove unbound material, the bound proteins can be released using, for example, SDS, a buffer with a high pH, ​​and analyzed, for example, by surface plasmon resonance (SPR).

[0409] In some embodiments, the bispecific antigen-binding protein or bispecific antigen-binding protein includes a TCR-binding domain. In some embodiments, the TCR-binding domain is a viral antigen or a fragment thereof. In some implementations, the viral antigens are derived 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., viral strains that cause gastroenteritis); Clonorchiviridae (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, Sandfly virus, and Nairovirus); Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g., reovirus, circovirus, and rotavirus); BiRNAviridae; Hepatoviridae (hepatitis B virus); Parvoviridae (parvovirus); Papillomavirusidae (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 (considered a defective satellite of hepatitis B virus), hepatitis C; Norwalk and related viruses, and astroviruses).

[0410] connector

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

[0412] Half-life extension domain

[0413] This article considers domains that extend the half-life of antigen-binding domains. Such domains include, but are not limited to, albumin-binding domains, Fc domains, small molecule domains, and other half-life-extending domains known in the art.

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

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

[0416] In one aspect, the trispecific proteins described herein include a half-life-extending 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 derived 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 camel-derived single-domain antibody specific for HSA, a peptide, a ligand, or a heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of a small molecule entity. In some 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. Considering in some embodiments, the HSA-binding domain of the MSLN trispecific antigen-binding protein is relatively small and does not exceed 25 kD, 20 kD, 15 kD, or 10 kD. In some cases, if the ALB binding domain is a peptide or a small molecule, it is 5 kDa or smaller.

[0417] The half-life extension domain of a 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 changing 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, intra-tissue diffusion, and enhanced efficacy compared to proteins without the half-life extension 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 cytotoxicity to non-tumor cells.

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

[0419] Liver-preserving domain

[0420] This article considers domains that allow and promote greater retention of trispecific antigen-binding proteins in the liver. The liver-retaining domains of trispecific antigen-binding proteins target hepatocyte portions. In one embodiment, hepatocytes include, but are not limited to, hepatocytes, hepatic stellate cells, and hepatic sinusoidal endothelial cells.

[0421] In one embodiment, hepatocytes contain receptors that bind to the liver-targeting moiety. In one embodiment, the liver-targeting moiety includes, but is 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, lactose-biotin, galactoside, pullulan, daidzein, desialylated serum mucoprotein, 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 receptors, mannose receptors, scavenger receptors, low-density lipoprotein receptors, HARE, CD44, IFNα receptors, type VI collagen receptors, 6-phosphate / insulin-like growth factor 2 receptors, platelet-derived growth factor receptor β, RBP receptors, αVβ3 integrin receptors, ASGP receptors, glycyrrhetinic acid / glycyrrhizin receptors, PPAR, heparan sulfate glycosaminoglycan receptors, CXC receptor type 4, glycyrrhetinic acid receptors, HBVP receptors, HDL receptors, scavenger receptor class B member 1 LDL receptors, or combinations thereof.

[0422] Target antigen binding domain

[0423] The trispecific and bispecific antigen-binding proteins described herein include a domain that binds to a target antigen. The target antigen is involved in and / or associated with a disease, condition, or disorder (e.g., cancer). In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is a protein from the NY-ESO-1, SSX-2, Sp17, AFP, phosphatidylinositol proteoglycan-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 the MAGE family.

[0424] In some implementations, the target antigen is an antigen found on non-hepatic tumor cells that have already metastasized to the liver.In some embodiments, bispecific or trispecific antigen-binding proteins include target antigen-binding domains specific to the following groups: 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), and 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 α-2, mesothelin, interleukin-11 receptor α (IL-11Ra), prostate stem cell antigen (P SCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor β (PDGFR-β), stage-specific embryonic antigen 4 (SSEA-4), CD20, folate receptor α, HER2, HER3, mucin 1, cell surface-associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostate enzymes, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), liver glycoprotein B2, fibroblasts Cellular activation protein α (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macroprotein factor) subunit β-9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein (bcr-ab1) composed of breakpoint cluster region (BCR) and Abelson mouse leukemia virus oncogene homologue 1 (Ab1), tyrosinase, liver glycoside A receptor 2 (EphA2), fucose GM1, sialylated Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase Aminoaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-associated (TEM7R), tight junction protein 6 (CLDN6), tight junction protein 18.2 (CLDN18.2), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97 or CD179a.In some implementations, the target antigen is an antigen associated with a viral disease, such as a viral antigen. In some implementations, the target antigen is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E antigens.

[0425] The design of the trispecific antigen-binding protein described herein allows for flexibility in the binding domain of the liver target antigen, as the binding domain can be of any type, including but not limited to domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding domain of 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 camel-derived single-domain antibody. In other embodiments, the binding domain of the liver target antigen is a non-Ig-binding domain, i.e., antibody mimics, such as anticarrier proteins, affilin, affinity molecules, affimier, affitin, alphabodies, avimer, DARPin, fynomer, kunitz domain peptides, and single-domain antibodies. In other embodiments, the binding domain of the liver target antigen is a ligand or peptide that binds to or associates with the target antigen.

[0426] 3.7 PAH

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

[0428] In some embodiments, the RNA sequence suitable for use in this invention includes a circRNA sequence encoding a human PAH protein.

[0429] In some implementations, a suitable RNA sequence may be an RNA sequence encoding a homolog or analog of human PAH. As used herein, a homolog or analog of human PAH protein may be a modified human PAH protein containing one or more amino acid substitutions, deletions, and / or insertions compared to wild-type or naturally occurring human PAH protein, while retaining significant PAH protein activity.

[0430] This invention can be used to treat subjects who have or are susceptible to phenylketonuria (PKU). PKU is an autosomal recessive metabolic disorder characterized by mutations in the gene for the liver enzyme phenylalanine hydroxylase (PAH), rendering it nonfunctional. PAH is essential 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 into phenylpyruvic acid (also known as phenyl ketone), which can be detected in 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 essential for the synthesis of proteins and neurotransmitters, Phe accumulation can hinder brain development and lead to intellectual disability.

[0432] Besides hindering brain development, the condition can present with a variety of clinical symptoms, including seizures, albinism-related ADHD, developmental delays, rashes (eczema), microcephaly, and / or a "musty" odor in the baby's sweat and urine (due to phenylacetic acid, a ketone produced by this process). Untreated children are often normal at birth but have delayed intellectual and social skills, a significantly smaller than normal head size, and frequently exhibit progressive impairment of brain function. As the child grows and develops, additional symptoms often appear, including hyperactivity, tachycardia of the arms or legs, abnormal EEG, rashes, tremors, seizures, and severe learning disabilities. However, PKU is usually included in routine newborn screening programs in most countries and is typically performed 2–7 days after birth.

[0433] If PKU is diagnosed early enough, affected newborns can grow up with relatively normal brain development, but Phe levels can only be managed and controlled through diet or a combination of diet and medication. All PKU patients must adhere to a special low-Phe diet for optimal brain development. This 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 be monitored. Infants can still receive all the benefits of breast milk through breastfeeding, but the amount must be monitored, and any missing nutrients will need to be supplemented. The sweetener aspartame, found in many weight-loss foods and soft drinks, must also be avoided because it contains phenylalanine.

[0434] Throughout life, patients can obtain amino acids and other essential nutrients that would otherwise be lacking in a low-phenylalanine diet by supplementing with infant formula, pills, or specially formulated foods. Some Phe is essential for the synthesis of many proteins and for proper growth, but its levels must be strictly controlled in PKU patients. In addition, PKU patients must take tyrosine supplements, which are typically derived from phenylalanine. Other supplements may include fish oil to replace the long-chain fatty acids missing from a standard Phe-free diet and to improve neurodevelopment and iron or carnitine levels. Another potential treatment for PKU is tetrahydrobiopterin (BH4), 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 implementations, PAH protein expression 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 PAH protein expression levels in the liver reaching or exceeding 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 implementations, PAH protein expression can be detected 1 to 96 hours after administration. For example, in some implementations, PAH protein expression can be detected at the following timeframes after administration: 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 hours. Up 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 Up 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 PAH protein expression can be detected at 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 to 96 hours, 72 to 84 hours, or 84 to 96 hours. For example, in some embodiments, PAH protein expression can be detected at 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after administration. In some embodiments, PAH protein expression can be detected from 1 to 7 days after administration. For example, in some embodiments, PAH protein can be detected 1, 2, 3, 4, 5, 6, and / or 7 days after application. In some embodiments, PAH protein expression can be detected from 1 to 8 weeks after application. For example, in some embodiments, PAH protein expression can be detected 1, 2, 3, and / or 4 weeks after application. In some embodiments, PAH protein expression can be detected one month after application.

[0438] 3.8CPS1

[0439] In some embodiments, the present invention provides methods and compositions for delivering a circRNA encoding CPS1 to a subject to treat CPS1 deficiency. A suitable CPS1 circRNA encodes any full-length, fragment, or portion of the CPS1 protein, which may displace naturally occurring CPS1 protein activity and / or reduce the intensity, severity, and / or frequency of one or more symptoms associated with CPS1 deficiency.

[0440] In some embodiments, the RNA sequence suitable for use in this invention includes a circRNA sequence encoding the human CPS1 protein.

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

[0442] Carbamoyl phosphate synthase I (CPS1) catalyzes the conversion of ammonia, bicarbonate, and 2ATP to form carbamoyl phosphate in the first step of the urea cycle. It also plays a role in the biosynthesis of arginine, which is a substrate for the biosynthesis of NO, for example, in cases 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)). CPS1 should be distinguished from the cytoplasmic enzyme CPS2, which also plays a role in the urea cycle but processes the substrate glutamine. CPS1 is known to be located in mitochondria and is present in large quantities in liver tissue in this form (accounting for 2%–6% of total liver protein). Its amino acid sequence and gene location are already known (see Haraguchi Y. et al., Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I:molecular analysis of hyperammonemia, Gene 1 November 1991; 107(2); 335-340; see also the applicant’s publication WO 03 / 089933A1). For information on its physiological effects, please refer to review articles such as HMHolder 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 mentioned therein, as well as the introduction to the publication by 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 synthase I (CPS1) deficiency is a genetic disorder characterized by mutations in the gene for carbamoyl phosphate synthase I, thereby affecting its ability to catalyze the synthesis of carbamoyl phosphate from ammonia and bicarbonate. This reaction is the first step in the urea cycle and is essential for removing excess urea from cells. A defect in the CPS1 protein disrupts the urea cycle and prevents the liver from properly processing excess nitrogen into urea.

[0444] In some embodiments, administration of the provided composition results in CPS1 protein expression levels in the liver reaching or exceeding 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 implementations, CPS1 protein expression can be detected 1 to 96 hours after administration. For example, in some implementations, expression is detected at 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, and 4 hours after administration. Up 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 Up 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. CPS1 protein expression can be detected at 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 to 96 hours, 72 to 84 hours, or 84 to 96 hours. For example, in some embodiments, CPS1 protein expression can be detected at 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after administration. In some embodiments, CPS1 protein expression can be detected from 1 to 7 days after administration. For example, in some embodiments, CPS1 protein can be detected 1, 2, 3, 4, 5, 6, and / or 7 days after administration. In some embodiments, CPS1 protein expression can be detected from 1 to 8 weeks after administration. For example, in some embodiments, CPS1 protein can be detected 1, 2, 3, and / or 4 weeks after administration. In some embodiments, CPS1 protein expression can be detected one month after administration.

[0446] In some implementations, administration of the composition reduces ammonia levels in the subject compared to baseline levels before treatment. Typically, baseline levels in the subject are measured immediately before treatment. Ammonia levels are usually 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 reduction of ammonia levels in a biological sample (e.g., serum, plasma, or urine sample) by 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% compared to baseline levels in the subject immediately prior to treatment.

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

[0449] 3.9ADAMTS13

[0450] In some embodiments, the present invention provides methods and compositions for delivering a circRNA encoding ADAMTS13 to a subject to treat thrombotic thrombocytopenic purpura (TTP). A suitable ADAMTS13 circRNA encodes any full-length ADAMTS13 protein or a functional fragment or portion thereof, which may 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 sequence of the present invention comprises a circRNA sequence encoding the human ADAMTS13 protein.

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

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

[0454] In some embodiments, administration of the provided composition results in ADAMTS13 protein expression levels in the liver reaching or exceeding approximately 100 ng / mg, approximately 200 ng / mg, approximately 300 ng / mg, approximately 400 ng / mg, approximately 500 ng / mg, approximately 600 ng / mg, approximately 700 ng / mg, approximately 800 ng / mg, approximately 900 ng / mg, approximately 1000 ng / mg, approximately 1200 ng / mg, or approximately 1400 ng / mg of total protein.

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

[0456] In some embodiments, administration of the composition results in a reduction in von Willebrand factor (vWF) levels in the subject compared to baseline vWR levels prior to treatment. Typically, baseline levels in the subject are measured immediately prior to treatment. vWF levels are typically measured in a biological sample. Suitable biological samples include, for example, whole blood, plasma, or serum.

[0457] In some embodiments, administration of the composition results in a reduction of vWF levels in biological samples taken from the subject by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline vWF levels immediately prior to treatment. In some embodiments, administration of the composition results in a reduction of plasma vWF levels in the subject 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 results in a reduction of vWF levels in plasma or serum samples taken from a subject compared to baseline vWF levels immediately prior to treatment. In some embodiments, administration of the provided composition results in a reduction of vWF levels in plasma or serum compared to vWF levels in untreated subjects. In some embodiments, administration of the composition results in a reduction of vWF levels in plasma or serum to about 3000 μmol / L or less, about 2750 μmol / L or less, about 2500 μmol / L or less, about 2250 μmol / L or less, about 2000 μmol / L or less, about 1750 μmol / L or less, about 1500 μmol / L or less, about 1250 μmol / L or less, about 1000 μmol / L or less, about 750 μmol / L or less, about 500 μmol / L or less, about 250 μmol / L or less, about 100 μmol / L or less, or about 50 μmol / L or less. In one particular embodiment, administration of the composition reduces vWF levels in plasma or serum to about 50 μmol / L or lower.

[0459] 4. Production of polynucleotides

[0460] The vectors provided in this article can be prepared using standard molecular biology techniques. For example, the various elements of the vectors provided in this article 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, various elements of the vectors provided in this paper can also be synthesized, rather than cloned. The complete sequence can be assembled from overlapping oligonucleotides prepared and assembled into a complete sequence using standard methods. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311.

[0462] Therefore, specific nucleotide sequences can be obtained from vectors having the desired sequence, or, where appropriate, synthesized completely or partially using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR). One method for obtaining the nucleotide sequence encoding the desired vector element is to anneal the complementary sets of overlapping synthetic oligonucleotides produced in a conventional automated polynucleotide synthesizer, then ligate them 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-guided synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-guided 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 RNA provided herein can be generated by incubating the vector provided herein under conditions that allow transcription of the precursor RNA encoded by the vector. For example, in some embodiments, the precursor RNA is synthesized by incubating the vector provided herein with a compatible RNA polymerase under conditions that allow in vitro transcription, said vector containing an RNA polymerase promoter upstream of its 5' double-strand forming region and / or expression sequence. In some embodiments, the vector is incubated intracellularly by phage RNA polymerase or in the nucleus by host RNA polymerase II.

[0464] In some implementations, this document provides a method for generating precursor RNA by in vitro transcription using a vector provided herein as a template (e.g., a vector provided herein having an RNA polymerase promoter located upstream of the 5' homologous region).

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

[0466] Therefore, in some embodiments, methods for preparing circular RNA are provided herein. In some embodiments, the method comprises synthesizing precursor RNA by transcribing (e.g., run-off transcription) using a vector provided herein (e.g., a vector comprising, in the following order: a 5' homologous region, a 3' I group intron fragment, a first spacer region, an internal ribosome entry site (IRES), an expression sequence, a second spacer region, a 5' I group intron fragment, and a 3' homologous region) as a template and incubating the resulting precursor RNA in the presence of a divalent cation (e.g., magnesium ions) and GTP to circularize it into circular RNA. In some embodiments, the precursor RNA disclosed herein is capable of circularization in the absence of magnesium ions and GTP and / or without the step of incubation with magnesium ions and GTP. Circular RNA has been found to have reduced immunogenicity relative to the corresponding mRNA, at least in part because the mRNA contains an immunogenic 5' cap. When transcribed from 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 circular RNA compositions containing low levels of contaminating linear mRNA, an excess of GMP relative to GTP can be provided during transcription so that most transcripts contain 5' GMP, which cannot be capped. Therefore, in some embodiments, transcription is performed in the presence of excess GMP. In some embodiments, transcription is performed where the ratio of GMP concentration to GTP concentration is in the range of about 3:1 to about 15:1, for example, 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, the composition containing circular RNA has been purified. Circular RNA can be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification includes one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification sequentially includes the following steps: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification includes reversed-phase HPLC. In some embodiments, the purified composition contains less double-stranded RNA, DNA clips, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes, and / or nicked RNA 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 lipids

[0469] In some embodiments disclosed herein, the lipids are ionizable lipids that can be used as components of transfer mediators to promote or enhance the delivery and release of circular RNA to one or more target cells (e.g., by permeation or fusion with the lipid membrane of such target cells). In some embodiments, the ionizable lipids contain one or more cleavable functional groups (e.g., disulfide bonds) that allow, for example, a hydrophilic functional head group to dissociate from a lipophilic functional tail group of the compound (e.g., upon exposure to oxidative, reducing, or acidic conditions), thereby promoting a phase transition in the lipid bilayer of one or more target cells.

[0470] In some embodiments, the ionizable lipid is the lipid described in international patent application PCT / US2018 / 058555.

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

[0472]

[0473] in:

[0474] R1 and R2 may be the same or different, and are independently optional substitutions of 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 group;

[0475] R3 and R4 may be the same or different, and independently may be optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 ynyl, or R3 and R4 may be linked to form a heterocycle with 4 to 6 carbon atoms and 1 or 2 optional substituted heteroatoms selected from nitrogen and oxygen.

[0476] R5 may or may not exist, and when present it is hydrogen or a C1-C6 alkyl group; m, n, and p may be the same or different, and independently 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and

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

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

[0479] In one embodiment, the amino lipid is a dilinoleylamino 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 about 10 to about 20 carbon atoms, wherein at least one of R3 and R4 contains at least two unsaturated sites.

[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 unsaturated sites (e.g., R3 and / or R4 may be, for example, dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and eicosadienyl).

[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 groups;

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

[0491] In one implementation, R3 and R4 are the same; for example, in some implementations, both R3 and R4 are linoleyl (C)-based. 18 -alkyl). In another embodiment, R3 and R4 are different; for example, in some embodiments, R3 is tetradecanetrienyl (C 14 -alkyl), and R4 is linoleyl (C 18 -alkyl). In a preferred embodiment, the cationic lipid of the present invention is symmetrical, i.e., R3 and R4 are identical. In another preferred embodiment, both R3 and R4 contain at least two unsaturated sites. 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 unsaturated sites and are each independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and eicosadienyl.

[0492] In various embodiments, cationic lipids have the following formula:

[0493]

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

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

[0496] R 1 Each time it appears, it is independently a non-hydrogen or amino acid substituted or unsubstituted side chain;

[0497] R 2 and R NEach time it appears, it is independently hydrogen, an organic group composed of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms or any combination thereof, and having 1 to 20 carbon atoms; C (1-5) Alkyl, cycloalkyl, cycloalkylalkyl, C (1-5) alkenyl, C (1-5) alkynyl group, C (1-5) Alkyl group, C (1-5) Alkyloxy, 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 groups;

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

[0499] R x and R y Independently, it is (i) a lipophilic tail derived from lipids (which may be naturally occurring or synthetic), such as phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelins, cerebrosides, or gangliosides, wherein said 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 group, C (3-22) Alkoxy or C (6-12) -alkoxy C (3-22) alkyl;

[0500] In some implementation schemes, R x and R y One of them is the lipophilic tail as defined above, and the other is the terminal amino acid group. In some embodiments, R x and R y Both have lipophilic tails.

[0501] In some implementation schemes, R x and R y At least one of them is contaminated with one or more biodegradable groups (e.g., -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -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 Interruption.

[0502] In some implementation schemes, R 11 It is a C2-C8 alkyl or alkenyl group.

[0503] In some implementation schemes, R 5 Each occurrence of is independently H or alkyl.

[0504] In some implementation schemes, R 3 and R 4 Each occurrence of R3 is independently H, halogen, OH, alkyl, alkoxy, -NH2, alkylamino, or dialkylamino; or R3 and R4 together with the carbon atom directly attached to them form a cycloalkyl group. In some specific embodiments, R3... 3 and R 4 Each occurrence of it is independently of H or C1-C4 alkyl.

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

[0506] In some implementations, 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 alkyl, alkenyl, or alkynyl, and each may be optionally substituted;

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

[0511] Representative useful dilinylamino lipids have the following formula:

[0512]

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

[0514] In one embodiment, the cationic lipid is DLin-K-DMA. In another embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, where 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 optional substitutions of C each time they appear. 10 -C 30 Alkyl, optionally substituted C 10 -C 30 alkenyl, optionally substituted C 10 -C 30 alkynyl or optionally substituted C 10 -C 30 Acyl group;

[0519] R3 is H, an optional substituted C2-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-C 10 Alkyne, alkyl heterocyclic, alkyl phosphate, alkyl thiophosphate, alkyl dithiophosphate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl, ω-thiophosphate alkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl or heterocyclic or 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 junction point with R3; and

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

[0522] In one specific embodiment, the cationic lipids of embodiments 1, 2, 3, 4, or 5 have 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-phosphoalkyl, or ω-thiophosphoalkyl;

[0527] R1 and R2 and R x Each occurrence is independently H, or an 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 group, optionally substituted C 10 -C 30 Acyl or linker-ligand, conditions are R1, R2 and R x At least one of them is not H;

[0528] R3 is H, or an optional substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-C 10Alkyne, alkyl heterocyclic, alkyl phosphate, alkyl thiophosphate, alkyl dithiophosphate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl, ω-thiophosphate alkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl or heterocyclic 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 -, -SS-, -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 two are -O(C=O), -(C=O)O-, -C(=O)-, -O-, and -S(O). x -, -SS-, -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 direct bond;

[0535] R a Is it H or C1-C? 12 alkyl;

[0536] R 1a and R 1bEach occurrence is independently (a)H or C1-C. 12 Alkyl, or (b)R 1a Is it H or C1-C? 12 Alkyl, and R 1b Together with the carbon atom it bonds to, and the adjacent R 1b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0537] R 2a and R 2b Each occurrence is independently (a)H or C1-C. 12 Alkyl, or (b)R 2a Is it H or C1-C? 12 Alkyl, and R 2b Together with the carbon atom it bonds to, and the adjacent R 2b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0538] R 3a and R 3b Each occurrence is independently (a)H or C1-C. 12 Alkyl, or (b)R 3a Is it H or C1-C? 12 Alkyl, and R 3b Together with the carbon atom it bonds to, and the adjacent R 3b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0539] R 4a and R 4b Each occurrence is independently (a)H or C1-C. 12 Alkyl, or (b)R 4a Is it H or C1-C? 12 Alkyl, and R 4b Together with the carbon atom it bonds to, and the adjacent R 4b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0540] R 5 and R 6 Each is independently either methyl or cycloalkyl;

[0541] R 7 Each occurrence is independently H or Cl-C. 12 alkyl;

[0542] R 8 and R 9 Each is an unsubstituted C1-C. 12 Alkyl; or R 8 and R 9Together with the nitrogen atoms they are attached to, they form 5, 6, or 7-membered heterocycles containing one nitrogen atom;

[0543] a and d are each independent integers from 0 to 24;

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

[0545] e is 1 or 2; and

[0546] x is 0, 1, or 2.

[0547] In some implementations of Formula I, L 1 and L 2 Independently, it is -O(C=O)- or -(C=O)O-.

[0548] In some embodiments of Formula I, R 1a R 2a R 3a or R 4a At least one of them is C1-C 12 Alkyl, or L 1 or L 2 At least one of them is -O(C=O)- or -(C=O)O-. In other embodiments, R 1a and R 1b It is 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 L 1 or L 2 At least one of them is -O(C=O)- or -(C=O)O-; and R 1a and

[0550] R 1b It is 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 Each is an unsubstituted C1-C. 12 Alkyl; or R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 5, 6, or 7-membered heterocycles containing one nitrogen atom;

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

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

[0554] In some implementations of Formula I, L 1 or L 2 One of them is -(C=O)O-. In other implementations, 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 one is -(C=O)O-. In more implementations, L 1 or L 2 One of them is -O(C=O)-, and L 1 or L 2 The other 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 one is a carbon-carbon double bond.

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

[0558]

[0559] Where R a and R b It is either H or a substituent, occurring independently each time. For example, in some embodiments, R... a and R bEach occurrence is independently H, C1-C 12 Alkyl or cycloalkyl, 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 some embodiments of the lipid compound of Formula I, a, b, c, and d are each independently an integer from 2 to 12 or from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or from 5 to 9. In some 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 other embodiments of Formula I, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more 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] In Formula I, the sums of a and b, and the sums of c and d, are factors that can be varied to obtain the lipid of Formula I with the desired properties. In one embodiment, a and b are chosen such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer in the range of 14 to 24. In other embodiments, the sums of a and b, and the sums of c and d, are the same. For example, in some embodiments, the sums of a and b, and the sums of c and d, are both the same integer in the range of 14 to 24. In many more embodiments, a, b, c, and d are chosen such that the sum of a and b, and the sum of c and d, is 12 or greater.

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

[0573] R of Formula I 1a R 2a R 3a and R 4a There are no particular restrictions on the substituents at the location. In some embodiments, R 1a R 2aR 3a and R 4a It is H each time it appears. In some other implementations, R 1a R 2a R 3a and R 4a At least one of them is C1-C 12 Alkyl group. In some other embodiments, R 1a R 2a R 3a and R 4a At least one of them is a C1-C8 alkyl group. In some other embodiments, R 1a R 2a R 3a and R 4a At least one of them is a C1-C6 alkyl group. In some of the foregoing embodiments, the C1-C8 alkyl group 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 Each occurrence is C1-C 12 alkyl.

[0575] In other embodiments of Formula I, R 1b R 2b R 3b and R 4b At least one of them is H, or R. 1b R 2b R 3b and R 4b It appears as H each time.

[0576] In some embodiments of Formula I, R 1b Together with the carbon atom it bonds to, and the adjacent R 1b Together with the carbon atom it bonds to, it forms a carbon-carbon double bond. In the other embodiments described above, R 4b Together with the carbon atom it bonds to, and the adjacent R 4b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0577] In the aforementioned implementation scheme, R of Formula I 5 and R 6 There are no particular restrictions on the substituents at the location. In some embodiments, R 5 or R 6 One or both of them are methyl. In some other embodiments, R 5 or R 6One or both of them are cycloalkyl, such as cyclohexyl. In these embodiments, the cycloalkyl group may be substituted or unsubstituted. In some other embodiments, the cycloalkyl group is C1-C2. 12 Alkyl groups, such as tert-butyl substituted groups.

[0578] In the aforementioned implementation scheme of Formula I, R 7 There are no particular limitations on the substituents at the location. In some embodiments, at least one R 7 It is H. In some other implementations, R 7 It is H each time it appears. In some other implementations, R 7 It is C1-C 12 alkyl.

[0579] In some other of the foregoing embodiments of Formula I, R 8 or R 9 One of them is methyl. In other embodiments, R 8 and R 9 Both are methyl groups.

[0580] In some different implementations of Formula I, R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 5, 6, or 7-membered heterocycles. In some of the aforementioned embodiments, R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 5-membered heterocycles, such as pyrrolidinyl rings.

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

[0582] In various implementations, 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 lipids of embodiments 1, 2, 3, 4, or 5 have 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 them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -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 two are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). x -, -SS-, -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 direct bond;

[0595] G 1 It is a C1-C2 alkylene group, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)-

[0596] Or a direct key;

[0597] G 2 Is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a -or direct key;

[0598] G 3 It is a C1-C6 alkylene group;

[0599] R a Is it H or C1-C? 12 alkyl;

[0600] R 1aand R 1b Each time it appears independently, it is: (a) H or C1-C 12 Alkyl; or (b)R 1a Is it H or C1-C? 12 Alkyl, and R 1b Together with the carbon atom it bonds to, and the adjacent R 1b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0601] R 2a and R 2b Each time it appears independently, it is: (a) H or C1-C 12 Alkyl; or (b)R 2a Is it H or C1-C? 12 Alkyl, and R 2b Together with the carbon atom it bonds to, and the adjacent R 2b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0602] R 3a and R 3b Each time it appears independently, it is (a): H or C1-C 12 Alkyl; or (b)R 3a Is it H or C1-C? 12 Alkyl, and R 3b Together with the carbon atom it bonds to, and the adjacent R 3b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0603] R 4a and R 4b Each time it appears independently, it is: (a) H or C1-C 12 Alkyl; or (b)R 4a Is it H or C1-C? 12 Alkyl, and R 4b Together with the carbon atom it bonds to, and the adjacent R 4b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond;

[0604] R 5 and R 6 Each is independently either H or methyl;

[0605] R 7 It is C4-C 20 alkyl;

[0606] R 8 and R 9 Each is independently C1-C 12 Alkyl; or R 8 and R 9Together with the nitrogen atoms they are attached to, they form 5, 6, or 7-membered heterocycles;

[0607] a, b, c, and d are each an independent integer from 1 to 24; and

[0608] x is 0, 1, or 2.

[0609] In some implementations of formula (II), L 1 and L 2 Each is independently -O(C=O)-, -(C=O)O-, or a direct bond. In other implementations, G 1 and G 2 Each is independently either a -(C=O)- or a direct bond. In some different implementations, L 1 and L 2 Each is independently -O(C=O)-, -(C=O)O-, or a direct bond; and G 1 and G 2 Each is independently either a -(C=O)- or a direct bond.

[0610] In some different implementations of formula (II), L 1 and L 2 Each of these can be independently -C (=O), -O-, or -S (O). x -, -SS-, -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 -

[0611] In other aforementioned embodiments of formula (II), the lipid compound has one of the following formulas (IIA) or (IIB):

[0612]

[0613] In some embodiments of formula (II), the lipid compound has formula (IIA). In other embodiments, the lipid compound has formula (IIB).

[0614] In any of the foregoing embodiments of formula (II), L 1 or L 2 One of them is -O (C=O)-. For example, in some implementations, L 1 and L 2 Each is -O(C=O)-.

[0615] In some different implementations of formula (II), L 1 or L 2 One of them is -(C=O)O-. For example, in some implementations, L 1 and L 2 Each is -(C=O)O-.

[0616] In different implementations of formula (II), L 1 or L 2 One of them is a direct bond. As used in this article, a "direct bond" refers to a group (e.g., L...). 1 or L 2 ) does not exist. For example, in some implementations, L 1 and L 2 Each is a direct key.

[0617] In other different embodiments of equation (II), for R 1a and R 1b R appears at least once. 1a Is it H or C1-C? 12 Alkyl, and R 1b Together with the carbon atom it bonds to, and the adjacent R 1b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0618] In other different embodiments of equation (II), for R 4a and R 4b R appears at least once. 4a Is it H or C1-C? 12 Alkyl, and R 4b Together with the carbon atom it bonds to, and the adjacent R 4b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0619] In further embodiments of formula (II), for R 2a and R 2b R appears at least once. 2a Is it H or C1-C? 12 Alkyl, and R 2b Together with the carbon atom it bonds to, and the adjacent R 2b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0620] In other different embodiments of equation (II), for R 3a and R 3b R appears at least once. 3a Is it H or C1-C? 12 Alkyl, and R 3b Together with the carbon atom it bonds to, and the adjacent R 3b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0621] In various other embodiments of formula (II), the lipid compound has one of the following formulas (IIC) or (IID):

[0622]

[0623] Where e, f, g, and h are each an integer from 1 to 12.

[0624] In some embodiments of formula (II), the lipid compound has formula (IIC). In other embodiments, the lipid compound has formula (IID).

[0625] In various implementations of formula (IIC) or (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0626] In some embodiments of formula (II), a, b, c, and d are each independently an integer from 2 to 12 or from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or from 5 to 9. In some 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.

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

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

[0629] In some embodiments of formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In many embodiments, d is 3. In other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In many embodiments, d is 7. In other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In many embodiments, d is 11. In other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In many embodiments, d is 15. In other embodiments, d is 16.

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

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

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

[0633] In some embodiments of formula (II), h is 1. In other embodiments, e is 2. In many more embodiments, h is 3. In other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In many more embodiments, h is 7. In other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In many more embodiments, h is 11. In other embodiments, h is 12.

[0634] In some other various embodiments of equation (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.

[0635] The sums of a and b, and the sums of c and d in formula (II) are factors that can be changed to obtain lipids with desired properties. In one embodiment, a and b are chosen such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer in the range of 14 to 24. In other embodiments, the sums of a and b, and the sums of c and d are the same. For example, in some embodiments, the sums of a and b, and the sums of c and d, are both the same integer in the range of 14 to 24. In many more embodiments, a, b, c, and d are chosen such that the sum of a and b, and the sum of c and d, is 12 or greater.

[0636] R in equation (II) 1a R 2a R 3a and R 4a The substituents at the position are not particularly limited. In some embodiments, R 1aR 2a R 3a and R 4a At least one of them is H. In some implementations, R 1a R 2a R 3a and R 4a It is H each time it appears. In some other implementations, R 1a R 2a R 3a and R 4a At least one of them is C1-C 12 Alkyl group. In some other embodiments, R 1a R 2a R 3a and R 4a At least one of them is a C1-C8 alkyl group. In some other embodiments, R 1a R 2a R 3a and R 4a At least one of them is a C1-C6 alkyl group. In some of the foregoing embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0637] In some embodiments of formula (II), R 1a R 1b R 4a and R 4b Each occurrence is C1-C 12 alkyl.

[0638] In other embodiments of formula (II), R 1b R 2b R 3b and R 4b At least one of them is H, or R. 1b R 2b R 3b and R 4b It appears as H each time.

[0639] In some embodiments of formula (II), R 1b Together with the carbon atom it bonds to, and the adjacent R 1b Together with the carbon atom it bonds to, it forms a carbon-carbon double bond. In the other embodiments described above, R 4b Together with the carbon atom it bonds to, and the adjacent R 4b Together with the carbon atom it bonds with, it forms a carbon-carbon double bond.

[0640] In the aforementioned implementation scheme, R in formula (II) 5 and R6 There are no particular restrictions on the substituents at the location. In some embodiments, R 5 or R 6 One of them is methyl. In other embodiments, R 5 or R 6 Each is a methyl group.

[0641] In the aforementioned implementation scheme, R in formula (II) 7 There are no particular restrictions on the substituents at the location. In some embodiments, R 7 It is C6-C 16 Alkyl group. In some other embodiments, R 7 It is a C6-C9 alkyl group. In some of these embodiments, R 7 -(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 , where: R a Is it H or C1-C? 12 Alkyl; R b It is C1-C 15 Alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 -(C=O)OR b or -O(C=O)Rb replace.

[0642] In some of the aforementioned embodiments of formula (II), R b It is a branch C1-C 16 Alkyl group. For example, in some embodiments, R b It has one of the following structures:

[0643]

[0644] In some other of the foregoing embodiments of formula (II), R 8 or R 9 One of them is methyl. In other embodiments, R 8 and R 9 Both are methyl groups.

[0645] In some different implementations of formula (II), R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 5, 6, or 7-membered heterocycles. In some of the aforementioned embodiments, R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 5-membered heterocycles, such as pyrrolidinyl rings. In some of the different embodiments described above, R 8 and R 9 Together with the nitrogen atoms they are attached to, they form 6-membered heterocycles, such as piperazine rings.

[0646] In some embodiments of embodiment 3, the first and second cationic lipids are each independently selected from lipids of formula II.

[0647] In other embodiments of the lipid of formula (II) described above, G 3 It is a C2-C4 alkylene, such as a C3 alkylene. In various embodiments, the lipid compound has one of the structures listed in Table 2 below.

[0648] Table 2: Representative lipids of formula (II)

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656] In some other embodiments, the cationic lipids of embodiments 1, 2, 3, 4, or 5 have the structure of formula III:

[0657]

[0658] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0659] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -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 two are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or

[0660] -NR a C(=O)O- or direct bond;

[0661] G 1 and G 2 Each is an unsubstituted C1-C. 12 Alkylene or C1-C 12 alkenyl;

[0662] G 3 It is C1-C 24 Alkylene, C1-C 24 C3-C8 cycloalkylene, C3-C8 cycloalkylene;

[0663] R a Is it H or C1-C? 12 alkyl;

[0664] R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl;

[0665] R 3 Is it H or OR? 5 CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0666] R 4 It is C1-C 12 alkyl;

[0667] R 5 It is an H or C1-C6 alkyl group; and

[0668] x is 0, 1, or 2.

[0669] In some of the foregoing embodiments of formula (III), the lipid has one of the following formulas (IIIA) or (IIIB):

[0670]

[0671] in:

[0672] A is a 3- to 8-membered cycloalkyl or cyclohexane ring;

[0673] R 6 Each time it appears, it is independently H, OH, or Cl-C. 24 alkyl;

[0674] n is an integer in the range of 1 to 15.

[0675] In some of the foregoing embodiments of formula (III), the lipid has formula (IIIA), and in other embodiments, the lipid has formula (IIIB).

[0676] In other embodiments of formula (III), the lipid has one of the following formulas (IIIC) or (IIID):

[0677]

[0678] Where y and z are each an integer in the range of 1 to 12.

[0679] In any of the foregoing embodiments of formula (III), L 1 or L 2One of them is -O (C=O)-. For example, in some implementations, L 1 and L 2 Each is -O (C = O)-. In any of the aforementioned different implementations, L 1 and L 2 Each is independently -(C=O)O- or -O(C=O)-. For example, in some implementations, L 1 and L 2 Each is -(C=O)O-.

[0680] In some different embodiments of formula (III), the lipid has one of the following formulas (IIIE) or (IIIF):

[0681]

[0682] In some of the foregoing embodiments of formula (III), the lipid has one of the following formulas (IIIG), (IIIH), (IIII), or (IIIJ):

[0683]

[0684] In some of the foregoing embodiments of equation (III), n is an integer from 2 to 12, for example, an integer in the range of 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.

[0685] In some other of the foregoing embodiments of equation (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

[0686] In some of the aforementioned embodiments of formula (III), R 6 It is H. In other of the aforementioned embodiments, R 6 It is C1-C 24 Alkyl group. In other embodiments, R 6 It is OH.

[0687] In some implementations of formula (III), G 3 Not replaced. In other implementations, G3 is replaced. In various different implementations, G... 3 It is a straight-chain C1-C 24 Alkylene or straight-chain C1-C 24 Alkenyl group.

[0688] 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 of them independently has the following structure:

[0689]

[0690] in:

[0691] R 7a and R 7b Each occurrence is independently H or Cl-C. 12 alkyl;

[0692] and

[0693] a is an integer from 2 to 12.

[0694] Each of them chooses R. 7a R 7b and a to make R 1 and R 2 Each contains 6 to 20 carbon atoms independently. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0695] In some of the aforementioned embodiments of formula (III), R 7a The at least one occurrence of is H. For example, in some implementations, R 7a It is H each time it appears. In the other different implementations mentioned above, R... 7b The presence of at least one of the elements is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0696] In different implementations of formula (III), R 1 or R 2 Or both have one of the following structures:

[0697]

[0698] In some of the aforementioned embodiments of formula (III), R 3 It is OH, CN, -C(=O)OR 4 -OC(=O)R 4 or -NHC(=O)R 4 In some implementations, R 4 It is methyl or ethyl.

[0699] In some specific embodiments of implementation scheme 3, the first and second cationic lipids are each independently selected from lipids of formula III.

[0700] In various different embodiments, the cationic lipids (e.g., cationic lipids, first cationic lipids, second cationic lipids) of any of the disclosed embodiments of formula (III) have one of the structures listed in Table 3 below.

[0701] Table 3: Representative compounds of formula (III)

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710] In one embodiment, the cationic lipid of any one of embodiments 1, 2, 3, 4, or 5 has the structure of formula (IV):

[0711]

[0712] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0713] G 1 or G 2 One of them appears each time as -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -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 2The other one appears each time as -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y , -SS-, -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 direct bond;

[0714] L is -O (C = O)- each time it appears, where ~ represents a covalent bond with X;

[0715] X is CR a ;

[0716] Z is an alkyl, cycloalkyl, or, when n is 1, a monovalent portion containing at least one polar functional group; or Z is an alkylene, cycloalkylene, or, when n is greater than 1, a polyvalent portion containing at least one polar functional group.

[0717] R a 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-C 12 Alkyl carbonyloxy group, C1-C 12 alkyl carbonyl oxyalkyl or C1-C 12 Alkyl carbonyl;

[0718] R is independent each time it appears: (a) H or C1-C 12 Alkyl group; or (b) R together with the carbon atom to which it is bonded, together with the adjacent R and the carbon atom to which it is bonded, forms a carbon-carbon double bond;

[0719] R 1 and R 2 Each occurrence has the following structure:

[0720]

[0721] a 1 and a 2 Each occurrence is an integer from 3 to 12 independently;

[0722] b1 and b 2 It is 0 or 1 independently each time it appears;

[0723] c 1 and c 2 Each occurrence is an integer between 5 and 10 independently;

[0724] d 1 and d 2 Each occurrence is an integer between 5 and 10 independently;

[0725] y is an integer from 0 to 2 each time it appears; and

[0726] n is an integer from 1 to 6.

[0727] Each of the alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl groups may optionally be substituted by one or more substituents.

[0728] In some implementations of formula (IV), G 1 and G 2 Each independently is

[0729] -O(C=O)- or -(C=O)O-.

[0730] In other embodiments of formula (IV), X is CH,

[0731] In different implementations of formula (IV), a 1 +b 1 +c 1 The sum or a 2 +b 2 +c 2 The sum is an integer between 12 and 26.

[0732] In other embodiments of formula (IV), a 1 and a 2 Independently, it is an integer from 3 to 10. For example, in some implementations, a 1 and a 2 Independently, it is an integer from 4 to 9.

[0733] In various implementations of formula (IV), b 1 and b 2 It is 0. In different implementation schemes, b 1 and b 2 It is 1.

[0734] In further embodiments of formula (IV), c 1 c 2 d1 and d 2 Independently, it is an integer from 6 to 8.

[0735] In other embodiments of formula (IV), c 1 and c 2 Each occurrence is an independent integer from 6 to 10, and d 1 and d 2 Each occurrence is an integer between 6 and 10.

[0736] In other embodiments of formula (IV), c 1 and c 2 Each occurrence is an independent integer from 5 to 9, and d 1 and d 2 Each occurrence is an integer from 5 to 9.

[0737] In further embodiments of formula (IV), Z is an alkyl, cycloalkyl, or, when n is 1, a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is an alkyl group.

[0738] In various embodiments of formula (IV) above, R is independently: (a) H or methyl; or (b) R, together with the carbon atom it is bonded to, forms a carbon-carbon double bond with the adjacent R and the carbon atom it is bonded to. In some embodiments, each R is H. In other embodiments, at least one R, together with the carbon atom it is bonded to, forms a carbon-carbon double bond with the adjacent R and the carbon atom it is bonded to.

[0739] In other embodiments of the compound of formula (IV), R 1 and R 2 Independently possessing one of the following structures:

[0740]

[0741] In some embodiments of formula (IV), the compound has one of the following structures:

[0742]

[0743]

[0744]

[0745]

[0746] In different implementation schemes, the cationic lipids of implementation schemes 1, 2, 3, 4, or 5 have the structure of formula (V):

[0747]

[0748] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0749] G 1 or G 2 One of them appears each time as -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -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 The other one appears each time as -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -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 direct bond;

[0750] L appears as ~O(C=O)- each time, where ~ represents a covalent bond with X;

[0751] X is CR a ;

[0752] Z is an alkyl, cycloalkyl, or, when n is 1, a monovalent portion containing at least one polar functional group; or Z is an alkylene, cycloalkylene, or, when n is greater than 1, a polyvalent portion containing at least one polar functional group.

[0753] R a 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 Alkyl carbonyloxy group, C1-C 12 alkyl carbonyl oxyalkyl or C1-C 12 Alkyl carbonyl;

[0754] R is independent each time it appears: (a) H or C1-C 12 Alkyl group; or (b) R together with the carbon atom to which it is bonded, together with the adjacent R and the carbon atom to which it is bonded, forms a carbon-carbon double bond;

[0755] R 1 and R 2 Each occurrence has the following structure:

[0756]

[0757] R' is independently H or C1-C each time it appears. 12 alkyl;

[0758] a 1 and a 2 Each occurrence is an integer from 3 to 12 independently;

[0759] b 1 and b 2 It is 0 or 1 independently each time it appears;

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

[0761] d 1 and d 2 Each occurrence is an integer from 2 to 12 independently;

[0762] y is an integer from 0 to 2 each time it appears; and

[0763] n is an integer from 1 to 6.

[0764] Among them, choose a 1 a 2 c 1 c 2 d 1 and d 2 make a 1 +c 1 +d 1 The sum is an integer between 18 and 30, and a 2 +c 2 +d 2The sum is an integer from 18 to 30, and each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl is optionally substituted by one or more substituents.

[0765] In some implementations of formula (V), G 1 and G 2 Each independently is

[0766] -O(C=O)- or -(C=O)O-.

[0767] In other embodiments of formula (V), X is CH.

[0768] In some implementations of formula (V), a 1 +c 1 +d 1 The sum is an integer between 20 and 30, and a 2 +c 2 +d 2 The sum is an integer between 18 and 30. In other implementations, a 1 +c 1 +d 1 The sum is an integer between 20 and 30, and a 2 +c 2 +d 2 The sum is an integer between 20 and 30. In further implementations of equation (V), a 1 +b 1 +c 1 The sum or a 2 +b 2 +c 2 The sum is an integer between 12 and 26. In other implementations, choose a. 1 a 2 c 1 c 2 d 1 and d 2 make a 1 +c 1 +d 1 The sum is an integer between 18 and 28, and a 2 +c 2 +d 2 The sum is an integer between 18 and 28.

[0769] In other embodiments of formula (V), a 1 and a 2 Independently, it is an integer from 3 to 10, such as an integer from 4 to 9.

[0770] In other embodiments of formula (V), b1 and b 2 It is 0. In different implementation schemes, b 1 and b 2 It is 1.

[0771] In some other embodiments of equation (V), c 1 c 2 d 1 and d 2 Independently, it is an integer from 6 to 8.

[0772] In different other embodiments of formula (V), Z is an alkyl group or, when n is 1, a monovalent portion containing at least one polar functional group; or Z is an alkylene group or, when n is greater than 1, a polyvalent portion containing at least one polar functional group.

[0773] In further embodiments of formula (V), Z is an alkyl, cycloalkyl, or, when n is 1, a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is an alkyl group.

[0774] In other different embodiments of formula (V), R is independently: (a) H or methyl; or (b) R, together with the carbon atom it is bonded to, forms a carbon-carbon double bond with the adjacent R and the carbon atom it is bonded to. For example, in some embodiments, each R is H. In other embodiments, at least one R, together with the carbon atom it is bonded to, forms a carbon-carbon double bond with the adjacent R and the carbon atom it is bonded to.

[0775] In more implementations, each R' is H.

[0776] In some implementations of formula (V), a 1 +c 1 +d 1 The sum is an integer between 20 and 25, and a 2 +c 2 +d 2 The sum is an integer between 20 and 25.

[0777] In other embodiments of formula (V), R 1 and R 2 Independently possessing one of the following structures:

[0778]

[0779] In further embodiments of formula (V), the compound has one of the following structures:

[0780]

[0781]

[0782]

[0783]

[0784] 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.

[0785] In any further of the foregoing embodiments of formula (IV) or (V), Z is a monovalent or multivalent portion comprising at least one polar functional group. In some embodiments, Z is a monovalent portion comprising at least one polar functional group. In other embodiments, Z is a multivalent portion comprising at least one polar functional group.

[0786] In any of the foregoing embodiments of formula (IV) or (V), the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylamine, heterocyclic, or heteroaryl functional group.

[0787] In any of the foregoing embodiments of formula (IV) or (V), Z is hydroxyl, hydro...

Claims

1. A pharmaceutical composition comprising: a. a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (1): wherein: each n is independently an integer from 2-15; each of L1and L3is independently -OC(O)-* or -C(O)O-*, where "*" indicates a point of attachment to R1or R3; R1and R3are each independently straight or branched chain C9-C 20 alkyl or C9-C 20 alkenyl: oxo, halo, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and R2is selected from the group consisting of:

2. The pharmaceutical composition of claim 1, wherein each of R1and R3is independently selected from the group consisting of:

3. The pharmaceutical composition of claim 1 or 2, wherein R1and R3are the same.

4. The pharmaceutical composition of claim 1 or 2, wherein R1and R3are different.

5. The pharmaceutical composition of any one of claims 1-4, wherein the ionizable lipid is represented by Formula (1-1) or Formula (1-2):

6. The pharmaceutical composition of any one of claims 1-5, wherein the ionizable lipid is selected from the group consisting of:

7. A pharmaceutical composition comprising: a. a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (2): wherein: each n is independently an integer from 1-15; each of R1and R2is independently selected from the group consisting of: and R3is selected from the group consisting of:

8. A pharmaceutical composition comprising: a. a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (3): wherein: X is selected from -O-, -S-, or -OC(O)-*, where * indicates a point of attachment to R1; R1is selected from the group consisting of: and R2is selected from the group consisting of:

9. The pharmaceutical composition of claim 8, wherein the ionizable lipid is represented by Formula (3-1), Formula (3-2), or Formula (3-3):

10. A pharmaceutical composition comprising: a. a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (4): wherein: each n is independently an integer from 2-15; and R2is as defined in claim 2.

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